Patent application title: COMPOSITIONS OF HUMANIZED NOTCH FUSION PROTEINS AND METHODS OF TREATMENT
Inventors:
Jan Kitajewski (Ridgewood, NJ, US)
Carrie Shawber (Township Of Washington, NJ, US)
Assignees:
THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK
IPC8 Class: AC07K14705FI
USPC Class:
4241341
Class name: Immunoglobulin, antiserum, antibody, or antibody fragment, except conjugate or complex of the same with nonimmunoglobulin material structurally-modified antibody, immunoglobulin, or fragment thereof (e.g., chimeric, humanized, cdr-grafted, mutated, etc.) antibody, immunoglobulin, or fragment thereof fused via peptide linkage to nonimmunoglobulin protein, polypeptide, or fragment thereof (i.e., antibody or immunoglobulin fusion protein or polypeptide)
Publication date: 2016-04-28
Patent application number: 20160115217
Abstract:
This invention provides a fusion protein comprising a signal peptide, an
extracellular domain of human Notch receptor protein and an Fc portion of
an antibody bound thereto. This invention also provides a method for
treating a subject having a tumor, a method for inhibiting angiogenesis
in a subject, a method for treating a subject having ovarian cancer, and
a method for treating a subject having a metabolic disorder, comprising
administering to the subject an amount of the above fusion protein
effective to treat the subject. This invention further provides uses of
the above fusion protein for the preparation of a pharmaceutical
composition for the treatment of a subject having a tumor, for inhibiting
angiogenesis in a subject, for treating a subject having ovarian cancer,
and for treating a subject having a metabolic disorder.Claims:
1-101. (canceled)
102. A fusion protein the sequence of which (a) is identical to the sequence of a portion of the extracellular domain of a human Notch4 receptor followed by (b) a sequence identical to the sequence of an Fc portion of an antibody, wherein the portion of the extracellular domain of the human Notch4 receptor is selected from the group consisting of EGF-like repeats 1-29, 1-13, EGF-like repeats 1-23, EGF-like repeats 9-23, EGF-like repeats 9-29, EGF-like repeats 13-23 and EGF-like repeats 21-29, wherein (b) is located to the carboxy terminal side of (a), and wherein (b) is attached to (a) either directly or by means of a linker sequence.
103. The fusion protein of claim 102, wherein the Fc portion of the antibody is the Fc portion of a human antibody.
104. The fusion protein of claim 102, portion of the extracellular domain of the human Notch4 receptor protein is EGF-like repeats 1-29.
105. The fusion protein of claim 102, portion of the extracellular domain of the human Notch4 receptor protein is EGF-like repeats 1-13.
106. The fusion protein of claim 102, portion of the extracellular domain of the human Notch4 receptor protein is EGF-like repeats 1-23.
107. The fusion protein of claim 102, portion of the extracellular domain of the human Notch4 receptor protein is EGF-like repeats 9-23.
108. The fusion protein of claim 102, portion of the extracellular domain of the human Notch4 receptor protein is EGF-like repeats 9-29.
109. The fusion protein of claim 102, portion of the extracellular domain of the human Notch4 receptor protein is EGF-like repeats 13-23.
110. The fusion protein of claim 102, portion of the extracellular domain of the human Notch4 receptor protein is EGF-like repeats 21-29.
111. The fusion protein of claim 102, wherein (b) is attached to (a) by means of a linker sequence.
112. The fusion protein of claim 102, wherein (b) is attached directly to (a).
113. A fusion protein the sequence of which (a) is identical to the sequence of a signal peptide followed by a sequence identical to the sequence of a portion of the extracellular domain of a human Notch4 receptor followed by (b) a sequence identical to the sequence of an Fc portion of an antibody, wherein the portion of the extracellular domain of the human Notch4 receptor is selected from the group consisting of EGF-like repeats 1-29, 1-13, EGF-like repeats 1-23, EGF-like repeats 9-23, EGF-like repeats 9-29, EGF-like repeats 13-23 and EGF-like repeats 21-29, wherein (b) is located to the carboxy terminal side of (a), and wherein (b) is attached to (a) either directly or by means of a linker sequence.
114. The fusion protein of claim 113, the sequence of which is set forth in any of SEQ ID NOs: 78-88.
115. The fusion protein of claim 114, wherein the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in any of SEQ ID NOs: 93-103.
116. The fusion protein of claim 113, wherein the signal peptide is the signal peptide of human Notch1 receptor protein, human Notch2 receptor protein, human Notch3 receptor protein, human Notch4 receptor protein, or an IgG Heavy Chain.
117. The fusion protein of claim 113, wherein (b) is attached to (a) by means of a linker sequence.
118. The fusion protein of claim 113, wherein (b) is attached directly to (a).
119. A method for treating a subject having a tumor, ovarian cancer, metabolic disorder, or vascular proliferative retinopathy comprising administering to the subject an amount of the fusion protein of claim 102 effective to treat the subject having the tumor, ovarian cancer, a metabolic disorder, or vascular proliferative retinopathy.
120. The method of claim 119, wherein the metabolic disorder is diabetes, obesity, atherosclerosis, ischemia, stroke, or cardiovascular disease.
121. The method of claim 119, wherein the vascular proliferative retinopathy is diabetic retinopathy, macular defernation or retinopathy of prematurity.
122. A method for inhibiting angiogenesis, physiological lymphangiogenesis or pathological lymphangiogenesis, tumor metastasis, growth of a secondary tumor, or blood vessel cooption, in a subject comprising administering to the subject an amount of the fusion protein of claim 102 effective to inhibit angiogenesis physiological lymphangiogenesis or pathological lymphangiogenesis, tumor metastasis, growth of a secondary tumor, or blood vessel cooption in the subject.
123. The method of claim 122, wherein the pathological lymphangiogenesis is tumor lymphangiogenesis or lymph node metastasis.
124. The method of claim 122, wherein the metastasis occurs via a blood vessel, the lymphatic vasculature or a lymph node.
125. A method of treating cancer in a subject comprising administering to the subject the fusion protein of claim 102 and one or more of (i) an inhibitor of Vascular Endothelial Growth Factor (VEGF), (ii) an inhibitor of Platelet Derived Growth Factor (PDGF), (iii) an inhibitor of HER2/neu, (iv) a VEGF receptor inhibitor, or (v) a PDGF receptor antagonist, each in an amount effective to treat the cancer in the subject.
126. The method of claim 125, wherein the inhibitor of VEGF is an inhibitor of VEGF-A, PGIF, VEGF-B, VEGF-C, or VEGF-D.
127. The method of claim 125, wherein the inhibitor of Platelet Derived Growth Factors is an inhibitor of PDGF-A or an inhibitor of PDGF-B
128. The method of claim 125, wherein the VEGF receptor inhibitor is a VEGFR-1 inhibitor, VEGFR-2 inhibitor, or VEGFR-3 inhibitor.
129. The method of claim 125, wherein the PDGF receptor antagonist is a PDGF Receptor-B antagonist.
130. A method for treating a subject having a tumor, ovarian cancer, metabolic disorder, or vascular proliferative retinopathy comprising administering to the subject an amount of a Notch1 fusion protein effective to treat the subject having the tumor, ovarian cancer, a metabolic disorder, or vascular proliferative retinopathy, wherein the sequence of the Notch1 fusion protein (a) is identical to the sequence of a portion of the extracellular domain of a human Notch1 receptor followed by (b) a sequence identical to the sequence of an Fc portion of an antibody, wherein the portion of the extracellular domain of the human Notch1 receptor is selected from the group consisting of EGF-like repeats 1-13, EGF-like repeats 1-24, EGF-like repeats 9-23, EGF-like repeats 9-36, EGF-like repeats 13-24 and EGF-like repeats 25-36, wherein (b) is located to the carboxy terminal side of (a), and wherein (b) is attached to (a) either directly or by means of a linker sequence.
131. The method of claim 130, wherein the metabolic disorder is diabetes, obesity, atherosclerosis, ischemia, stroke, or cardiovascular disease.
132. The method of claim 130, wherein the vascular proliferative retinopathy is diabetic retinopathy, macular defernation or retinopathy of prematurity.
133. A method for inhibiting angiogenesis, physiological lymphangiogenesis or pathological lymphangiogenesis, tumor metastasis, growth of a secondary tumor, or blood vessel cooption, in a subject comprising administering to the subject an amount of a Notch1 fusion protein effective to inhibit angiogenesis physiological lymphangiogenesis or pathological lymphangiogenesis, tumor metastasis, growth of a secondary tumor, or blood vessel cooption in the subject, wherein the sequence of the Notch1 fusion protein is (a) identical to the sequence of a portion of the extracellular domain of a human Notch1 receptor followed by (b) a sequence identical to the sequence of an Fc portion of an antibody, wherein the portion of the extracellular domain of the human Notch1 receptor is selected from the group consisting of EGF-like repeats 1-13, EGF-like repeats 1-24, EGF-like repeats 9-23, EGF-like repeats 9-36, EGF-like repeats 13-24 and EGF-like repeats 25-36, wherein (b) is located to the carboxy terminal side of (a), and wherein (b) is attached to (a) either directly or by means of a linker sequence.
134. The method of claim 133, wherein the pathological lymphangiogenesis is tumor lymphangiogenesis or lymph node metastasis.
135. The method of claim 133, wherein the metastasis occurs via a blood vessel, the lymphatic vasculature or a lymph node.
136. A method of treating cancer in a subject comprising administering to the subject a Notch1 fusion protein and one or more of (i) an inhibitor of Vascular Endothelial Growth Factor (VEGF), (ii) an inhibitor of Platelet Derived Growth Factor (PDGF), (iii) an inhibitor of HER2/neu, (iv) a VEGF receptor inhibitor, or (v) a PDGF receptor antagonist, each in an amount effective to treat the cancer in the subject, wherein the sequence of the Notch1 fusion protein is (a) identical to the sequence of a portion of the extracellular domain of a human Notch1 receptor followed by (b) a sequence identical to the sequence of an Fc portion of an antibody, wherein the portion of the extracellular domain of the human Notch1 receptor is selected from the group consisting of EGF-like repeats 1-13, EGF-like repeats 1-24, EGF-like repeats 9-23, EGF-like repeats 9-36, EGF-like repeats 13-24 and EGF-like repeats 25-36, wherein (b) is located to the carboxy terminal side of (a), and wherein (b) is attached to (a) either directly or by means of a linker sequence.
137. The method of claim 136, wherein the inhibitor of VEGF is an inhibitor of VEGF-A, PGIF, VEGF-B, VEGF-C, or VEGF-D.
138. The method of claim 136, wherein the inhibitor of Platelet Derived Growth Factors is an inhibitor of PDGF-A or an inhibitor of PDGF-B
139. The method of claim 136, wherein the VEGF receptor inhibitor is a VEGFR-1 inhibitor, VEGFR-2 inhibitor, or VEGFR-3 inhibitor.
140. The method of claim 136, wherein the PDGF receptor antagonist is a PDGF Receptor-B antagonist.
141. A composition comprising the fusion protein of claim 102 and a VEGF receptor inhibitor.
142. The composition of claim 141, wherein the VEGF receptor inhibitor is a VEGFR-1 inhibitor, VEGFR-2 inhibitor, or VEGFR-3 inhibitor.
143. A composition comprising a Notch1 fusion protein and a VEGF receptor inhibitor, wherein the sequence of the Notch1 fusion protein is (a) identical to the sequence of a portion of the extracellular domain of a human Notch1 receptor followed by (b) a sequence identical to the sequence of an Fr portion of an antibody, wherein the portion of the extracellular domain of the human Notch1 receptor is selected from the group consisting of EGF-like repeats 1-13, EGF-like repeats 1-24, EGF-like repeats 9-23, EGF-like repeats 9-36, EGF-like repeats 13-24 and EGF-like repeats 25-36, wherein (b) is located to the carboxy terminal side of (a), and wherein (b) is attached to (a) either directly or by means of a linker sequence.
144. The composition of claim 143, wherein the VEGF receptor inhibitor is a VEGFR-1 inhibitor, VEGFR-2 inhibitor, or VEGFR-3 inhibitor.
Description:
[0001] This application is a continuation of U.S. Ser. No. 12/733,329,
filed Jul. 12, 2010, now allowed, which is a §371 national stage of
PCT International Application No. PCT/US2008/010045, filed Aug. 22, 2008,
which claims the benefit of U.S. Provisional Patent Application No.
60/966,052, filed Aug. 23, 2007, the entire contents of each of which are
hereby incorporated by reference into this application.
[0003] This application incorporates-by-reference nucleotide and/or amino acid sequences which are present in the file named "150903_0575_77764-AA-PCT-US Substitute Sequence Listing_AHC.txt", which is 402 kilobytes in size, and which was created Sep. 3, 2015 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, which is contained in the text file filed Sep. 3, 2015 as part of this application.
[0004] Throughout this application, various publications are referenced by arabic numbers within parentheses or by author and publication date within parentheses. Full citations for these publications may be found at the end of the specification. The disclosures of these publications are hereby incorporated by reference into this application to describe more fully the art to which this invention pertains.
BACKGROUND OF THE INVENTION
[0005] Vascular Development
[0006] During mammalian embryogenesis, formation of the vascular system is an early and essential process. In the embryo, vascular development initiates with the pluripotent hemangioblast derived from the paraxial and lateral plate mesoderm. The hemangioblast has the potential to differentiate into either a hematopoietic progenitor or an endothelial cell progenitor, known as the angioblast.
[0007] Vascular development begins with a process known as vasculogenesis whereby angioblasts differentiate into endothelial cells and migrate together to form the primitive vascular plexus. This initial vascular network consists of vessels that are homogenous in size and made up wholly of endothelial cells. The vascular plexus is then remodeled via angiogenesis.
[0008] Angiogenesis involves the sprouting of new vessels, the migration of these vessels into avascular regions, and the recruitment of accessory cells, pericytes and smooth muscle cells (Gale and Yancopoulos, 1999). The smooth muscle cells that differentiate and form the contractile vessel walls originate from multiple progenitors including neural crest cells, mesenchymal cells and even endothelial cells (Owens, 1995). In adults, angiogenesis is involved in follicular development, wound healing, and pathological processes such as tumor angiogenesis and heart disease.
[0009] The Notch Family and Notch Ligands
[0010] Studies of Drosophila, C. Elegans, zebrafish and mammals have demonstrated that the Notch pathway is an evolutionarily conserved signaling mechanism that functions to modulate numerous cell-fate decisions. Notch signaling is required for the proper patterning of cells originating from all three germ layers. Depending on the cellular context, Notch signaling may both inhibit and induce differentiation, induce proliferation, and promote cell survival (Artavanis-Tsakonas et al., 1995; Lewis, 1998; Weinmaster, 1997). In Drosophila, a single Notch protein is activated by two ligands, Serrate and Delta. In mammals these families have been expanded to four Notch genes (Notch1, Notch2, Notch3 and Notch4) and five ligands, 2 Serrate-like (Jagged1-2) and 3 Delta (Dll, 3, 4) (Bettenhausen et al., 1995; Dunwoodie et al., 1997; Gallahan and Callahan, 1997; Lardelli et al., 1994; Lindsell et al., 1995; Shawber et al., 1996a; Shutter et al., 2000a; Uyttendaele et al., 1996; Weinmaster et al., 1992; Weinmaster et al., 1991). During embryogenesis, Notch receptors and ligands are expressed in dynamic spatial and temporal patterns. However, it is not known if all ligands activate all receptors.
[0011] Notch Signaling and Function
[0012] Notch signaling influences many different types of cell-fate decisions by providing inhibitory, inductive or proliferative signals depending on the environmental context (reviewed in Artavanis-Tsakonas et al., 1995; Greenwald, 1998; Robey, 1997; Vervoort et al., 1997). This pleiotropic function suggests that Notch modulates multiple signaling pathways in a spatio-temporal manner.
[0013] Consistent with Notch regulating cell-fate decisions, both the receptors and ligands are cell surface proteins with single transmembrane domains (FIG. 1). The regulatory extracellular domain of Notch proteins consists largely of tandemly arranged EGF-like repeats that are required for ligand binding (Artavanis-Tsakonas et al., 1995; Weinmaster, 1998). C-terminal to the EGF-like repeats are an additional three cysteine-rich repeats, designated the LIN12/Notch repeats (LNR) (Greenwald, 1994). Downstream of the LNR lies the proteolytic cleavage sequence (RXRR) that is recognized by a furin-like convertase. For Notch1, cleavage at this site yields a 180 kilodalton extracellular peptide and a 120 kilodalton intracellular peptide that are held together to generate a heterodimeric receptor at the cell surface (Blaumueller et al., 1997; Kopan et al., 1996; Logeat et al., 1998).
[0014] The intracellular domain of Notch (NotchICD, FIG. 1) rescues loss-of-function Notch phenotypes indicating that this form of Notch signals constitutively (Fortini and Artavanis-Tsakonas, 1993; Lyman and Young, 1993; Rebay et al., 1993; Struhl et al., 1993).
[0015] The cytoplasmic domain of Notch contains three identifiable domains: the RAM domain, the ankyrin repeat domain and the C-terminal PEST domain (FIG. 1). Upon ligand-activation Notch undergoes two additional proteolytic cleavages which results in the release of the cytoplasmic domain (Weinmaster, 1998). This Notch peptide translocates to the nucleus and interacts with transcriptional repressors known as CSL (CBF, Su (H), Lag-2) and converts it to transcriptional activator. The CSL/Notch interaction is dependent on the presence of the RAM domain of Notch; while, transcriptional activity also requires the presence of the ankyrin repeats (Hsieh et al., 1996; Hsieh et al., 1997; Roehl et al., 1996; Tamura et al., 1995; Wettstein et al., 1997). Both in vivo and in vitro studies indicate that the HES and Hey genes are the direct targets of Notch/CSL-dependent signaling (Bailey and Posakony, 1995; Eastman et al., 1997; Henderson et al., 2001; Jarriault et al., 1995; Nakagawa et al., 2000; Wettstein et al., 1997). The HES and Hey genes are bHLH transcriptional repressor that bind DNA at N-boxes (Nakagawa et al., 2000; Sasai et al., 1992; Tietze et al., 1992). Notch has also been proposed to signal by a CSL-independent pathway. In fact, expression of just the ankyrin repeat domain is necessary and sufficient for some forms of Notch signaling (Lieber et al., 1993; Matsuno et al., 1997; Shawber et al., 1996b).
[0016] Finally, the PEST domain has been implicated in protein turnover by a SEL-10/ubiquitin-dependent pathway (Greenwald, 1994; Oberg et al., 2001; Rogers et al., 1986; Wu et al., 1998; Wu et al., 2001). Similar to the receptors, the extracellular domain of the Notch ligands also consist mostly of tandemly arranged EGF-like repeats (FIG. 1). Upstream of these repeats is a divergent EGF-like repeat known as the DSL (Delta, Serrate, Lag-2) that is required for ligand binding and activation of the receptors (Artavanis-Tsakonas et al., 1995).
[0017] Notch Signaling and Vascular Development
[0018] Although many of the genes that function to induce vasculogenesis and angiogenesis have been identified, little is known about how cell-fate decisions are specified during vascular development. A number of observations suggest that the Notch signaling pathway may play a role in cell fate determination and patterning of the vascular system.
[0019] Notch1, Notch4, Jagged1 and Dll4 are all expressed in the developing vasculature, while Notch3 is expressed in the accessory smooth muscle cells (Krebs et al., 2000; Shutter et al., 2000b; Uyttendaele et al., 1996; Villa et al., 2001; Xue et al., 1999). Mice lacking Jagged1 are embryonic lethal and have severe vascular defects (Xue et al., 1999). Mice nullizygous for Notch1 are embryonic lethal and die of severe neuronal defects, but also have defects in angiogenesis (Krebs et al., 2000; Swiatek et al., 1994). Mice lacking Notch4 are born and appear to be normal, but embryos that have lost both Notch1 and Notch4 die at E9.5 of severe hemorrhaging and vascular patterning defects indicating Notch1 and Notch4 may be functionally redundant during vascular development (Krebs et al., 2000). Exogenous expression of an activated form of Notch4 in endothelium also resulted in vascular defects similar to those seen for the double Notch1/Notch4 nullizygous mice, suggesting that appropriate levels of Notch signaling is critical for proper development of the embryonic vasculature (Uyttendaele et al., 2001).
[0020] Taken together, the data from mice mutant for Notch/Notch signaling components uncover several processes dependent on Notch including vascular remodeling, arterial venous specification, vascular smooth muscle cell recruitment and heart/heart outflow vessel development.
[0021] Recent experiments have implicated Notch signaling in arterial/venous endothelial cell specification. In situ analysis of E13.5 embryos found that Notch1, Notch3, Notch4, D14, Jagged1 and Jagged2 expression was restricted to the arteries and absent in the veins (Villa et al., 2001). Consistent with expression data, disruption of Notch signaling in Zebrafish was associated with loss of the arterial marker ephrinB2; while, ectopic expression of an activated form of Notch lead to a loss in the venous cell marker EphB4 within the dorsal aorta (Lawson et al., 2001). These data suggest that Notch signaling may help to specify arterial and venous cell fates during angiogenesis.
[0022] Taken together, the data from mice mutant for Notch/Notch signaling components uncover several processes dependent on Notch including vascular remodeling, arterial venous specification, vascular smooth muscle cell recruitment and heart/heart outflow vessel development.
[0023] Notch signaling has also been suggested to function in the adult vascular system. In humans, missense mutations in the extracellular domain of Notch3 correlate with the development of the degenerative vascular disease, CADASIL (Caronti et al., 1998; Desmond et al., 1998; Joutel et al., 2000; Joutel et al., 1996). In a wound healing model, an increase in Jagged1 expression was observed at the regenerating endothelial wound edge, suggesting Notch signaling may function during processes of adult angiogenesis (Lindner et al., 2001). Taken together these data support Notch signaling functions at a number of critical steps during vascular development: vasculogenesis, vascular patterning/angiogenesis, and arterial/venous specification. However, the molecular mechanism(s) by which the Notch signaling pathways influence these different steps has yet to be elucidated.
[0024] Significance
[0025] Shimizu et al. (J. Biol. Chem. 274(46): 32961-32969 (1999)) describe the use of Notch1ECD/Fc, Notch2ECD/Fc and Notch3ECD/Fc in binding studies. However, Shimizu et al. do not mention the use of such proteins for inhibiting angiogenesis.
[0026] U.S. Pat. No. 6,379,925 issued Apr. 30, 2002 to Kitajewski et al. describes murine Notch4. However, it does not describe Notch-based fusion proteins as set forth in the subject application.
[0027] Notch proteins play key roles in developmental decisions involving the vasculature, the hematopoietic system, and the nervous system. As such, an understanding of their function is key to understanding how cell-fate decisions and commitment are controlled during development and in adult tissues. To date, several reports on Notch or Notch ligand gene disruptions have described vascular phenotypes providing emphasis that this pathway is a fundamental part of the machinery that guides vascular development. Aberrant Notch activity has been linked to human pathologies; including both cancer and vascular disorders (CADASIL). The analysis of Notch in tumor angiogenesis has only recently begun; however, our discovery of potential downstream targets of Notch suggests a role in pathological processes associated with angiogenesis. For instance, VEGFR-3 has been linked to both tumor angiogenesis and tumor lymphangiogenesis. The expression or function of several other potential Notch targets has also been linked to tumor angiogenesis; including ephrinB2, Id3, Angiopoietin 1, and PDGF-B. Insights on the role of these targets in Notch gene function will clearly facilitate future analysis of Notch in human pathologies.
SUMMARY OF THE INVENTION
[0028] This invention provides a fusion protein comprising a signal peptide, an extracellular domain of human Notch receptor protein and an Fc portion of an antibody bound thereto.
[0029] This invention provides a method for treating a subject having a tumor comprising administering to the subject an amount of the above fusion protein effective to treat the subject, thereby treating the subject having a tumor.
[0030] This invention provides a method for inhibiting angiogenesis in a subject comprising administering to the subject an amount of the above fusion protein effective to inhibit angiogenesis in the subject, thereby inhibiting angiogenesis in the subject.
[0031] This invention provides a method for treating a subject having ovarian cancer comprising administering to the subject an amount of the above fusion protein effective to treat the subject, thereby treating the subject having ovarian cancer.
[0032] This invention provides a method for treating a subject having a metabolic disorder comprising administering to the subject an amount of the above fusion protein effective to treat the subject, thereby treating the subject having a metabolic disorder.
[0033] This invention provides use of the above fusion protein for the preparation of a pharmaceutical composition for the treatment of a subject having a tumor.
[0034] This invention provides use of the above fusion protein for the preparation of a pharmaceutical composition for inhibiting angiogenesis in a subject.
[0035] This invention provides use of the above fusion protein for the preparation of a pharmaceutical composition for treating a subject having ovarian cancer.
[0036] This invention provides use of the above fusion protein for the preparation of a pharmaceutical composition for for treating a subject having a metabolic disorder.
[0037] This invention provides a method for inhibiting physiological lymphangiogenesis or pathological lymphangionesis in a subject comprising administering to the subject an amount of the above fusion protein effective to inhibit physiological lymphangiogenesis or pathological lymphangionesis in the subject.
[0038] This invention provides a method of inhibiting tumor metastasis in a subject comprising administering to the subject an amount of the above fusion protein effective to inhibit tumor metastasis in the subject.
[0039] This invention provides a method of inhibiting growth of a secondary tumor in a subject comprising administering to the subject an amount of the above fusion protein of effective to inhibit growth of the secondary tumor in the subject.
[0040] This invention provides a method of inhibiting blood vessel cooption by a tumor in subject comprising administering to the subject an amount of the above fusion protein effective to inhibit blood vessel cooption by a tumor in the subject.
[0041] This invention provides a method of treating cancer in a subject comprising administering to the subject the above fusion protein and an inhibitor of Vascular Endothelial Growth Factor (VEGF), VEGF-A, P1GF, VEGF-B, VEGF-C, orVEGF-D, each in an amount effective to treat the cancer in the subject.
[0042] This invention provides a method of treating cancer in a subject comprising administering to the subject the above fusion protein and a VEGF receptor antagonist, a VEGFR-1 antagonist, a VEGFR-2 antagonist or a VEGFR-3 antagonist, each in an amount effective to treat the cancer in the subject.
[0043] This invention provides a method of treating cancer in a subject comprising administering to the subject the above fusion protein and an inhibitor of Platelet Derived Growth Factor (PDGF), PDGF-A or PDGF-B, each in an amount effective to treat the cancer in the subject.
[0044] This invention provides a method of treating cancer in a subject comprising administering to the subject the above fusion protein and a PDGF receptor antagonist, each in an amount effective to treat the cancer in the subject.
[0045] This invention provides a method of treating cancer in a subject comprising administering to the subject the above fusion protein and an inhibitor of HER2/neu, each in an amount effective to treat the cancer in the subject.
[0046] This invention provides a method of treating vascular proliferative retinopathy comprising administering to the subject the above fusion protein in an emount effective to treat the vascular proliferative retinopathy.
BRIEF DESCRIPTION OF THE FIGURES
[0047] FIG. 1
[0048] This Figure shows the schematic structure of Notch and Notch ligands: Notch1, Notch2, Notch3, Notch4, Jagged-1, Jagged-2, Delta-like 1, Delta-like 3, Delta-like 4.
[0049] FIG. 2
[0050] This Figure shows the schematic design of Notch-based fusion proteins (NotchECD/Fc). The extracellular domain of Notch1, Notch2, Notch3, or Notch4 containing the EGF-repeats is fused to the Fc portion of an antibody.
[0051] FIG. 3
[0052] This Figure shows a co-culture assay for testing the activity of Notch-based fusion proteins. Notch and Notch responsive transcriptional reporters are expressed in a "Notch-responsive" cell, HeLa. Notch ligands, Jagged-1, Delta-like 1, or Delta-like 4 are expressed in a "ligand-presenting" cell, 293. Expression is mediated by transfection of individual cell populations, cells are co-cultured, and then assayed for Notch-dependent reporter activity.
[0053] FIG. 4
[0054] This Figure shows the inhibitory activity of Notch-based fusion protein against activation of Notch signaling by interaction between Notch and Notch ligand. Induction of Notch signaling was detected by co-cultivating both Notch1- and 3 types of Notch ligand-expressing cells and these inductions were inhibited by co-transfection of Notch-based fusion protein-expressing vector into Notch1-expressing cells. Therefore, Notch-based fusion proteins can be used as Notch inhibitor based on inhibition of interaction between Notch and Notch ligand.
[0055] FIG. 5
[0056] This Figure shows the expression of Notch1-based fusion protein (Notch1ECD/Fc) in 293. Panel A: expression in cell lystates (lys) or secreted into media (sup). Panel B: expression in 293 lysates of NECD/Fcs, as listed.
[0057] FIG. 6
[0058] This Figure shows activation of Notch signaling in HUVEC infected with adenoviral-encoding VEGF-165. Activation of Notch signaling can be detected by using CBF1 promoter activity. Transcriptional activity of CBF1 promoter is activated by binding of Notch-IC to CBF1. We measured CBF1 promoter activity in HUVEC which was infected with adenovirus-encoding VEGF-165 at different MOI. Induction of CBF1 promoter was clearly detected in Ad-VEGF-infected HUVEC, compared to Ad-LacZ-infected cells in the MOI dependent manner. This data showed overexpression of VEGF could activate Notch signaling in HUVEC.
[0059] FIG. 7
[0060] This Figure shows the effect of Notch-based fusion proteins on VEGF-induced activation of Notch signaling. Co-infection of Ad-Notch-based fusion protein with Ad-VEGF clearly reduced activation of CBF1 promoter activity induced by Ad-VEGF infection alone. In the case of infection at 40 MOI for each adenovirus in panel A, 60% inhibition at 24 hour and 90% inhibition at 48 hour after reporter gene transfection was detected. This inhibitory activity of Notch trap was dependent on MOI of Ad-Notch-based fusion protein.
[0061] FIG. 8
[0062] This Figure shows an experiment in which we evaluated the effect of Notch-based fusion proteins on induction of budding by overexpressed VEGF-165 in HUVEC. When Ad-VEGF-infected HUVEC were cultured on type collagen gel for 8 days, budding was induced into collagen gel. This induction of budding by overexpressed VEGF was clearly inhibited by coinfection of adenoviral-encoding Notch-based fusion proteins. Ad-Notch-based fusion protein itself had less effect on morphology.
[0063] FIG. 9
[0064] This Figure shows the result of counting buds per field under microscope. Ad-VEGF-infection into HUVEC increased the number of buds depending on used MOI. Even though a half MOI of Notch-based fusion protein was used, compared to Ad-VEGF, Ad-VEGF-induced budding was clearly inhibited. These data suggested that VEGF induced budding of HUVEC through activation of Notch signaling and Notch-based fusion protein could inhibit VEGF-induced budding.
[0065] FIG. 10
[0066] This Figure shows the amino acid sequence of the extracellular domain of the rat Notch1 protein (SEQ ID NO:1) and a linker sequence (SEQ ID NO:2).
[0067] FIG. 11
[0068] This Figure shows the amino acid sequence of the extracellular domain of the rat Notch2 protein (SEQ ID NO:3) and a linker sequence (SEQ ID NO:2).
[0069] FIG. 12
[0070] This Figure shows the amino acid sequence of the extracellular domain of the mouse Notch3 protein (SEQ ID NO:4).
[0071] FIG. 13
[0072] This Figure shows the amino acid sequence of the extracellular domain of the mouse Notch4 protein (SEQ ID NO:5) and a linker sequence (SEQ ID NO:2).
[0073] FIG. 14A and FIG. 14B
[0074] These Figures shows the nucleic acid sequence of the extracellular domain of the rat Notch1 gene (SEQ ID NO:6).
[0075] FIG. 15A and FIG. 15B
[0076] These Figures shows the nucleic acid sequence of the extracellular domain of the rat Notch2 gene (SEQ ID NO:7).
[0077] FIG. 16A and FIG. 16B
[0078] These Figures shows the nucleic acid sequence of the extracellular domain of the mouse Notch3 gene (SEQ ID NO:8).
[0079] FIG. 17A and FIG. 17B
[0080] These Figures shows the nucleic acid sequence of the extracellular domain of the mouse Notch4 gene (SEQ ID NO:9) and the nucleic acid sequence (SEQ ID NO:10) and the amino acid sequence (SEQ ID NO:2) of a linker sequence.
[0081] FIG. 18A and FIG. 18B
[0082] These Figures shows the nucleic acid sequence of the extracellular domain of the human Notch1 gene (SEQ ID NO:11).
[0083] FIG. 19A and FIG. 19B
[0084] These Figures shows the nucleic acid sequence of the extracellular domain of the human Notch2 gene (SEQ ID NO:12).
[0085] FIG. 20A and FIG. 20B
[0086] These Figures shows the nucleic acid sequence of the extracellular domain of the human Notch3 gene (SEQ ID NO:13).
[0087] FIG. 21A and FIG. 21B
[0088] These Figures shows the nucleic acid sequence of the extracellular domain of the human Notch4 gene (SEQ ID NO:14).
[0089] FIGS. 22A-22I
[0090] These Figures show that VEGF activates Notch signaling to induce HUVEC budding. HUVEC were transduced with Ad-VEGF at 40 MOI (FIGS. 22A, 22H, 22I) or 20 MOI (FIGS. 22C, 22G). Ad-LacZ was co-transduced to HUVEC to make the same total amount of adenovirus 60 MOI (FIG. 22G), 80 MOI (FIG. 22A) and 100 MOI (FIGS. 22H, 22I). FIG. 22A shows RT-PCR analysis of Notch and Notch ligand expression. Numbers show PCR cycles. FIG. 22B shows the effect of transduced VEGF on CSL reporter activity. FIG. 22C shows the effect of SU5416 on CSL reporter activity transactivated by Ad-VEGF. FIG. 22D shows the construct of Notch decoy (N1ECDFc). FIG. 22E shows secretion of N1ECDFc from HUVEC trasduced with Ad-N1ECDFc. FIG. 22F shows the effect of N1ECDFc against ligand-induced CSL reporter activity in a co-culture assay (quadrature: (-); .box-solid.: 0.33 ng pHyTC-N1ECDFc; .box-solid.: 0.67 ng pHyTC-N1ECDFc). FIG. 22G-FIG. 22I show the effect of N1ECDFc against Ad-VEGF-transduced HUVEC. Notch signaling was activated with transduction of Ad-VEGF in HUVEC in the absence or presence of co-transduction of Ad-N1ECDFc at indicated dosage. FIG. 22G shows the effect of N1ECDFc on CSL reporter activity transactivated by Ad-VEGF. FIG. 22H shows inhibition of budding of Ad-VEGF-transduced HUVEC with co-transduction of Ad-N1ECDFc at 40 MOI. FIG. 22I shows quantification of the effect of N1ECDFc on budding of Ad-VEGF-transduced HUVEC (quadrature: bud; .box-solid.: cell number).
[0091] FIGS. 23A-23J
[0092] These Figures show that Notch signaling up-regulates Flt1 expression to induce HUVEC budding. HUVEC were transduced with either Ad-LacZ or Ad-N1IC at 40 MOI. FIGS. 23A-23C show the effect of inhibitors for receptor tyrosine kinases on Notch-induced HUVEC budding. FIG. 23A is a photograph of budding of Ad-N1IC-transduced HUVEC treated with PD166866, ZD1893 at 1 μM and SU5416 at 0.5 μM. FIG. 23B shows quantification of the effect of inhibitors at 1 μM (quadrature: bud; .box-solid.: cell number). FIG. 23C shows dose-dependency of the effect of SU5416 (quadrature: bud; .box-solid.: cell number). FIGS. 23D-E show induction of Flt-1 expression in Ad-N1IC-transduced HUVEC. FIG. 23D shows RT-PCR analysis of Flt-1 mRNA expression. FIG. 23E shows W.B. analysis of Flt-1 protein expression. FIG. 23F-G show promotion of Notch-induced HUVEC budding with P1GF stimulation. Ad-N1IC-transduced HUVEC were cultured on collagen gel with SFM, instead of complete medium, in the absence or presence of 50 ng/ml P1GF. FIG. 23F shows P1GF-induced budding of Ad-N1IC-transducec HUVEC (arrow head: buds with single filopodia; arrow: buds with multiple filopodia). FIG. 23G shows the quantification of the effect of P1GF on budding of Ad-N1IC-transduced HUVEC (quadrature: multi; .box-solid.: total). FIG. 23H-FIG. 23I show the effect of Flt-1 siRNA transfection on Flt1 expression. Ad-N1IC-transduced HUVEC were transfected with 200 pmol of either control (CT) or Flt-1 siRNA. FIG. 23H shows the reduction of Flt-1 mRNA expression. FIG. 23I shows the reduction of Flt-1 protein expression. FIG. 23J shows the effect of Flt-1 siRNA transfection on Notch-induced HUVEC budding. Ad-N1IC-transduced HUVEC were transfected with either 100 or 200 pmol of siRNA and cultured on collagen gel for 2 days.
[0093] FIGS. 24A-24E
[0094] These Figures show that VEGF regulates gelatinase activity via Notch signaling by up-regulation of both MMP-9 and MT1-MMP. FIGS. 24A-B show gelatin zymography analysis of MMP-9 and MMP-2 activity stimulated by VEGF in HUVEC. FIG. 24A shows the effect of N1ECDFc on MMP-9 activity. Transduced HUVEC were cultured on fibrin gel on the indicated day (i.e. D2, D4, D6, D8). Similar results were also obtained by using collagen gel, although induction of MMP-9 was stronger on fibrin gel than collagen gel (data not shown). FIG. 24B shows the effect of N1ECDFc on MMP-2 activity. HUVEC were transduced with Ad-N1ECDFc at the indicated doses and condition medium was collected from HUVEC cultured on collagen gel at day 4. FIGS. 24C-D show up-regulation of MMP-9 and MT1-MMP with Notch signaling. HUVEC were transduced with either Ad-LacZ or Ad-N1IC at 40 MOI. Numbers show PCR cycles. FIG. 24C shows RT-PCR analysis of the effect of Notch signaling on expression of MMP-9 and MMP-2. FIG. 24D shows the induction of MT1-MMP expression of both transcript and protein with Notch signaling. FIG. 24E shows RT-PCR analysis of MMP-9 and MT1-MMP expression in Ad-VEGF-HUVEC with co-transduction of Ad-N1ECDFc. HUVEC were transduced with Ad-VEGF in the absence or presence of co-transduction of Ad-N1ECDFc at 40 MOI each. Ad-LacZ was co-transduced to make the same total amount of adenovirus at 80 MOI.
[0095] FIGS. 25A-25D
[0096] These Figures show the role of Notch signaling in VEGF-dependent in vivo angiogenesis. FIGS. 25A-25D show inhibition of VEGF-induced angiogenesis with N1ECDFc in mouse DAS assay. Representative photographs are shown. FIG. 25A show subcutaneous induced angiogenesis with 293/VEGF transfectant versus 293/VEGF also expressing Notch decoy (Notch-based fusion protein) N1ECDFc. FIG. 25B shows the quantitation of degree of vascularization induced by 293/VEGF in control versus 293 expressing Notch decoy (Notch-based fusion protein)--N1ECDFc. FIG. 25C shows subcutaneous induced angiogenesis with Ad-LacZ infected MDA-MB-231 cells versus Ad-N1ECDFc (Notch-based fusion protein) infected MDA-MB-231 cells. MDA-MB-231 breast cancer cells produce VEGF (data not shown). FIG. 25D shows quantitation of degree of vascularization induced by Ad-LacZ infected MDA-MB-231 cells versus Ad-N1ECDFc (Notch-based fusion protein) infected MDA-MB-231 cells.
[0097] FIG. 26A and FIG. 26B
[0098] These Figures show proliferation of Ad-VEGF165-transduced HUVEC. HUVEC were transduced with Ad-VEGF165 at the indicated dosages. Ad-LacZ was also co-infected to make the same total amount of adenovirus at a MOI of 40 pfu/cell. HUVEC were suspended in SFM supplemented with 1% FBS and then plated at 1×104 cells/well in 24-well multi-well plates with 0.4 ml of medium. After 4 days, cell numbers were determined using the CCK-8 kit and the results are indicated as the ratio of cell numbers determined to the number of control cells, which were transduced with Ad-GFP at a MOI of 40 pfu/cell. FIG. 26A shows the effect of transduced VEGF on proliferation. FIG. 26B shows the inhibitory effect of SU5416. Ad-VEGF-transduced HUVEC were treated with SU5416 at the indicated dosages.
[0099] FIG. 27A and FIG. 27B
[0100] These Figures show the induction of HUVEC buds on type I collagen gel. HUVEC were transduced with either Ad-VEGF165 or AD-N1IC at the indicated dosages. Ad-LacZ was also co-infected to make the same total amount of adenovirus at a MOI of 40 pfu/cell. Transduced. HUVEC were cultured on collagen gel with complete medium. The amount of budding was evaluated under microscopy at day 7.
[0101] FIG. 28A and FIG. 28B
[0102] These Figures show the effect of alteration of Notch signaling on cell proliferation. The cells were transduced with the indicated adenoviruses. Ad-GFP was also co-infected to make the same total amount of adenovirus at a MOI of 60 pfu/cell. After 4 days, cell numbers were determined using the CCK-8 kit and results are indicated as the ratio of cell numbers determined to the number of control cells, which were transduced with AD-GFP at MOI of 60 pfu/cell. FIG. 28A shows the effect of transduced N1IC and Notch fusion protein on the proliferation of HUVEC. Transduced HUVEC were suspended in complete medium and then plated at 1×104 cells/well in 24-well multiwell plates with 0.4 ml of indicated medium (quadrature: Ad-N1IC; .box-solid.: Ad-N1ECDFc). FIG. 28B shows the effect of Notch fusion protein on proliferation of KP1/VEGF transfectants. Transduced KP1/VEGF transfectants were suspended in RPMI1640 medium and then plated at 2×104 cells/well in 24-well multiwell plates with 0.5 ml of medium.
[0103] FIG. 29
[0104] This Figure shows the RT-PCR analysis of induction of PIGF expression in Ad-N1IC-transduced HUVEC. HUVEC were infected with either Ad-LacZ or Ad-N1IC at a MOI of 40 pfu/cell. Total RNA was isolated from transduced HUVEC cultured on collagen gel for 5 days with complete medium.
[0105] FIGS. 30A-30C
[0106] These Figures show inhibition of budding of either Ad-N1IC- or Ad-VEGF-transduced HUVEC with Flk-1 siRNA transfection. FIG. 30A shows reduction of Flk-1 mRNA and protein expression in Ad-VEGF-HUVEC with transfection of 200 pmol Flk-1 siRNA. Ad-VEGF-HUVEC at a MOI of 40 pfu/cell were transfected with 200 pmol of either control (CT) or Flk-1 siRNA. Total RNA was isolated 48 hours after transfection. Total cell lysate was collected from serum starved cells with SFM for 48 hours after transfection. FIG. 30B and FIG. 30C show the inhibitory effect of Flk-1 siRNA transfection on either VEGF or Notch-induced HUVEC buds. Either Ad-N1IC- or Ad-VEGF-HUVEC at a MOI of 40 pfu/cell were transfected with 200 pmol of siRNA as indicated and cultured on collagen gel for 5 days. FIG. 30B shows the effect of Flk-1 siRNA transfection on HUVEC buds (quadrature: Ad-VEGF; .box-solid.: Ad-N1IC). FIG. 30C shows quantification of the inhibitory effect of Flk-1 siRNA transfection.
[0107] FIG. 31A and FIG. 31B
[0108] These Figures show inhibition of budding of Ad-N1IC-transduced HUVEC with treatment of matrix metallo-proteinase inhibitor GM6001. Either Ad-LacZ or Ad-N1IC-HUVEC at a MOI of 40 pfu/cell were cultured on collagen gel for 5 days in the absence or presence of GM6001 at 50 μm. FIG. 31A shows the effect of GM6001 on Notch-induced HUVEC buds. FIG. 31B shows quantification of the inhibitory effect of GM6001.
[0109] FIGS. 32A-32D
[0110] These Figures show that Notch1 decoy inhibits activation of Notch signaling stimulated by Notch ligands. FIG. 32A shows a schematic of Notch1 decoy (N1ECDFc) and Western blotting to detect secreted Notch1 decoy in conditioned medium. HUVECs transduced with adenovirus coding Notch1 decoy (Ad-N1ECDFc) at indicated m.o.i. FIG. 32B shows that Notch1 decoy inhibits ligand-induced CSL reporter activity in co-culture signaling assay. Activation of Notch signaling was measured in HeLa cells expressing Notch1 co-cultured with 293 cells expressing Notch ligands. Data is shown as mean+SD. FIG. 32C shows ectopic expression of Notch4 induces the morphogenesis of HUVECs cultured on fibrin gel. HUVECs were transduced with adenovirus encoding Notch4 (Ad-Notch4) at 30 m.o.i. and Ad-GFP at 10 m.o.i, to mark infected cells. Two days later, HUVECs were co-cultured with stable HUVEC transfectants on fibrin gel and morphological changes were documented using fluorescence microscopy. Notch 4 induces cell extensions (upper left, white arrows) and treatment with 200 nM compound E blocks Notch4-induced extensions (upper right). Notch1 decoy expression blocks Notch4-induced cell extensions. Adenovirus-transduced HUVECs were co-cultured on fibrin gels with stable HUVEC transfectants expressing either Fc (lower left) or Notch1 decoy (lower right) and photographed two days later. Bar=200 μm. FIG. 32D shows quantification of effect of Notch signal inhibition on Notch4-induced extensions. Reduction in sprouting was statistically significant after treatment with compound E and transduction of N1ECDFc (p<0.0001, both; data is shown as mean+SD).
[0111] FIGS. 33A-33D
[0112] These Figures show that FGF4 induces the expression of Notch ligands in murine mammary carcinoma Mm5MT cells. Stable Mm5MT transfectants generated by retroviral gene transfer. FIG. 33A shows quantitative RT-PCR analysis of the expression of Notch ligands showing induction of Jagged1 and Dll1 in Mm5MT transfectants expressing FGF4 (Mm5MT-FGF4), compared to mock transfectants (Mm5MT-X). FIG. 33B shows Jagged1 protein is elevated in Mm5MT-FGF4 versus Mm5MT-X, as determined by western blotting. FIG. 33C shows reduction of Notch ligand expression in Mm5MT-FGF4 cells with PD166866, an inhibitor of FGF receptor kinase. FIG. 33DB shows immunohistochemical analysis of Jagged1 staining in Mm5MT transfectants. Bar=50 μm.
[0113] FIGS. 34A-34C
[0114] These Figures show that Notch1 decoy inhibits angiogenesis and subcutaneous tumor growth of Mm5MT-FGF4 tumors in mice. FIG. 34A shows tumor volumes of Mm5MT-FGF4-X and Mm5MT-FGF4-Fc differ significantly from Mm5MT-FGF4-N1ECDFc transfectants in mice (day 21, P=0.037 and P=0.008, Mm5MT-FGF4-X and Mm5MT-FGF4-Fc versus Mm5MT-FGF4-N1ECDFc respectively; data is shown as mean+SD). FIG. 34B shows immunohistochemical analysis of neovessels with CD31 staining within tumors of Mm5MT-FGF4 transfectants. Upper panels, Bar=100 μm, Lower panels, Bar=50 μm. FIG. 34C shows quantitative analysis demonstrated a reduction in CD31(+) neovessels in Mm5MT-FGF4-N1ECDFc transfectants as compared to Fc or mock-transfected tumors (P<0.001 for both Mm5MT-FGF4-X and Mm5MT-FGF4-Fc versus Mm5MT-FGF4-N1ECDFc; data is shown as mean+SD). Xenografts were harvested 22 days after inoculation and stained with anti-CD31 antibody.
[0115] FIGS. 35A-35D
[0116] These Figures show Notch1 decoy expression disrupts angiogenesis and impairs tumor viability in human NGP xenografts. We have previously reported that these human neuroblastoma xenografts in mice have a mature, hierarchical vasculature that is relatively resistant to VEGF blockade (16). To determine whether Notch receptor activation contributed to NGP angiogenesis, we transfected NGP cells with the Notch1 decoy construct, which did not affect their ability to grow in culture (data not shown). There was, however, a marked decrease in tumor viability in vivo (FIG. 35A) TUNEL=red fluorescence, erythrocytes=green fluorescence, Bar=100 μm), with (FIG. 35B) significantly increased tumor cell apoptosis (P=0.0002, TUNEL-positive cells in NGP-N1ECDFc vs. NGP-LacZ tumors), and (FIG. 35C) increased intratumoral hemorrhage (p<0.0001, quantitation of parenchymal erythrocyte signal). In addition, the tumor vessel networks in NGP-N1ECDFc xenografts appeared to have been physically disrupted as compared to NGP-LacZ controls, with (FIG. 35D) immunostaining for ECs and VMCs (using anti-CD31 and aSMA antibodies, respectively) demonstrating lack of continuity of these vascular cell layers, (Bar=50 μm). Individual vascular cells appeared detached from one another. Taken together, these results suggest that Notch1 decoy expression disrupted the ability of ECs and VMCs to form stable vascular conduits, causing vessel breakdown, hemorrhage, and ischemia of tumor tissues.
[0117] FIG. 36
[0118] This Figure shows that SKOV3 tumor cells programmed expressing the Notch1 decoy block the growth of ovarian cancer xenografts.
[0119] FIG. 37
[0120] This Figure shows regulation of myoblast differentiation by Foxo and Notch. C2C12 cells were immunostained with anti-Myosin antibody (green) and DAPI (blue). See text for panel description. Each experiment was repeated≧six times.
[0121] FIGS. 38A-38C
[0122] These Figures show the quantitative analysis of C2C12 differentiation. FIG. 38A shows Western blotting analysis of myosin expression in C2C12 cells. FIG. 38B shows morphometric analysis of Myosin-positive cells. Results from differentiation experiments were analyzed by scoring the number of Myosin-immunostained cells as percentage of all DAPI-positive cells. FIG. 38C shows DBD-Foxo1ADA reporter gene assays. We carried out reporter gene assays using the canonical Foxo1-responsive Igfbp1 promoter (left panel) and the Hes1 promoter (right panel) in cells co-transfected with Foxo1-ADA or DBD-Foxo1ADA. Western blot (inset) demonstrates that expression levels of the two proteins are similar. An asterisk indicates P<0.01 by ANOVA.
[0123] FIGS. 39A-39E
[0124] These Figures show Foxo1 co-immunoprecipitates with Cs1. FIG. 39A, Co-immunoprecipitation of endogenous Foxo1 and Cs1 in C2C12 cells co-cultured with LacZ- (denoted by the "-" sign) or Jagged1-expressing HEK293 cells (denoted by the "+" sign). FIG. 39B-FIG. 39C, Co-immunoprecipitation experiments in C2C12 cells co-transfected with FLAG-Cs1 and HA-Foxo1. FIG. 39D-FIG. 39E, Co-immunoprecipitation experiments in C2C12 cells co-transfected with FLAG-Cs1 and the truncated mutant Myc- or HA-tagged Δ256 Foxo1.
[0125] FIGS. 40A-40D
[0126] These Figures show that Foxo1 binds directly to Cs1. FIG. 40A, GST pull-down assays of GST-Foxo1 fusion protein with Cs1 immunoprecipitated from HEK293 cells. FIG. 40B-FIG. 40C, Binding of GST-Foxo1 and GST-FLAG-Cs1 in a cell-free system and mapping of the Cs1 interaction domain. Full-length and truncated fragments of GST-Foxo1 and GST-FLAG/Cs1 were purified from bacteria and co-incubated. Thereafter, Cs1 was isolated using anti-FLAG antibody, and the immunoprecipitate was analyzed by immunoblotting with anti-Foxo1 or anti-FLAG antibodies. FIG. 40D, Hes1 promoter ChIP spanning the Cs1 binding site in C2C12 cells to detect endogenous Foxo1, Cs1 and Notch1 (Endog) or following transduction with Foxo1-ADA (Foxo1-ADA) during myoblast differentiation. Input represents DNA extracted from chromatin prior to immunoprecipitation. Hes1 (semiquantitative RT-PCR) and Myosin (Western blot) expression corresponding to each time point are shown. Day 0 is defined as the time when cells are serum-deprived to induce myoblast fusion. Abbreviations: IP: immunoprecipitation; IB: immunoblotting; TCL: total cellular lysate.
[0127] FIGS. 41A-41B
[0128] These Figures show that Foxo1 regulates Notch-induced Hes1, Hes5 and Hey1 expression. FIG. 41A, Hes1, Hes5 and Hey1 expression measured by semiquantitative RT-PCR in C2C12 cells transduced with Foxo1-ADA or Notch1-IC following transfection of Gfp, Foxo1 or Cs1 siRNA as indicated. FIG. 41B, Hes1 reporter gene assays in HEK293 cells transduced with Foxo1-ADA, Notch1-IC, Foxo1 siRNA, GFP siRNA or control plasmid. We measured luciferase activity and normalized it by β-galactosidase activity. The data represent arbitrary units relative to control empty vector.
[0129] FIGS. 42A-42F
[0130] These Figures show that Foxo1 is required for Notch binding to the Hes1 promoter and activation of Hes1 target genes.
[0131] FIG. 42A, ChIP assays of endogenous Foxo1 and Notch1 in C2C12 cells co-cultured with LacZ- (denoted by a "-" sign) or or Jagged1-expressing HEK293 cells (denoted by a "+" sign) in the absence (lanes 1-2) and presence (lanes 3-4) of Cs1 siRNA. FIG. 42B, ChIP assays of endogenous Notch1 in co-culture system in the absence (lanes 1-2) and presence (lanes 3-4) of Foxo1 siRNA. c, Hes1 promoter assays following co-culture in the absence and presence of Foxo1 or Gfp siRNA. FIG. 42D, ChIP assays of Ncor and Smrt and Maml1 binding to Hes1 in the co-culture system in the absence (lanes 1-2) and presence (lanes 3-4) of Foxo1 siRNA. FIG. 42E, Expression of MyoD, Myf5 and β-actin in C2C12 cells by semiquantitative RT-PCR. FIG. 42F, Model of Foxo1 and Notch regulation of Hes1 promoter.
[0132] FIGS. 43A-43D
[0133] These Figures show conditional ablation of Foxo1 in skeletal muscle.
[0134] FIG. 43A, Western blot analysis of Foxo1 and Foxo4 expression levels in various muscle types. FIG. 43B, Metachromatic and immunohistochemical analysis of soleus and plantaris muscle from Myog-Foxo1 mice and control (lox/lox) littermates. FIG. 43C, Gene expression analysis of Myog-Foxo1 (sold bars) and control mice (empty bars); TropC: troponin-C; TropT: troponin-T; Mlc: myosin light chain; Myog: Myogenin; Mck: muscle-type creatine kinase. Data are means±SEM of three independent measurements (n=6 for each genotype). An asterisk indicates P<0.05 by ANOVA. FIG. 43D, Treadmill performance test in 8 week-old Myog-Foxo1 mice and lox/lox littermates (n=6 for each genotype). An asterisk indicates P<0.05 by ANOVA.
[0135] FIG. 44
[0136] This Figure shows the efficiency of adenoviral transduction in C2C12 cells. We transduced cells with HA-Foxo1-ADA or HA-Notch1-IC adenovirus and performed immunohistochemistry with anti-HA antibody (red) and DAPI (blue).
[0137] FIG. 45
[0138] This Figure shows inhibition of transfected Foxo1 expression. We tested the ability of Foxo1 siRNA to inhibit expression of endogenous (left panel) and transfected (right panel) Foxo1 following adenoviral transduction.
[0139] FIG. 46
[0140] This Figure shows the specificity of Foxo1 siRNA. Western blot analysis of Foxo1, Foxo3 and Foxo4 expression in C2C12 cells transfected with Foxo1 siRNA.
[0141] FIG. 47
[0142] This Figure shows that Foxo1-ADA and Notch1-IC do not affect cell proliferation. We transduced C2C12 cells with LacZ, Foxo1-ADA or Notch1-IC adenovirus, performed immunohistochemistry with anti-Ki67 antibody and DAPI and quantitated the Ki67 labeling index as percentage of Ki67-positive cells by counting at least 1,000 cells.
[0143] FIG. 48
[0144] This Figure shows siRNA-resistant Foxo1-ADA. Western blot of Foxo1-ADA and siRNA-resistant Foxo1-ADA in cells transfected with Foxo1 siRNA.
[0145] FIG. 49
[0146] This Figure shows the specificity of Foxo1-Cs1 co-immunoprecipitation. Following co-transfection with Foxo3 or Foxo4 expression vectors, we performed co-immunoprecipitation experiments with Cs1.
[0147] FIG. 50
[0148] This Figure shows Hes1 promoter assays. We used a synthetic Hes1 reporter gene containing four tandem repeats of the Cs1 binding site in promoter assays with Foxo1 and Notch1-IC in C2C12 cells.
[0149] FIG. 51
[0150] This Figure shows inhibition of Cs1 expression by siRNA. We measured Cs1 levels by western blot following transfection of C2C12 cells with Cs1 siRNA at different concentrations.
[0151] FIG. 52
[0152] This Figure shows a schematization of eleven formulations of human Notch1 decoys.
[0153] FIG. 53
[0154] This Figure shows signal sequence analysis of Notch 1 to determine where Notch1 signal peptide ends. The prediction results of analysis utilizing the Signall 3.0 Server provided online by the Technical Univeristy of Denmark are shown. The results predict a major site of cleavage located between alanine 18 (A19) and A19 and a minor site of cleavage between A19 and Arginine 20 (R20). These two cleavage sites are indicated by the "/" in amino acid sequence 1-20 of human Notch 1: MPPLLAPLLCLALLPALA/A/R (SEQ ID NO:15) The nucleotide sequence encoding amino acids 1-23 of human Notch1 is atgccgccgc tcctggcgcc cctgctctgc ctggcgctgc tgcccgcgct cgccgcacga ggcccgcga (SEQ ID NO:16).
[0155] FIG. 54
[0156] This Figure shows signal sequence analysis of human Hc to determine where human Hc signal peptide ends. MWGWKCLLFWAVLVTATLCTA/R (SEQ ID NO: 17). The prediction results of analysis utilizing the SignalIP 3.0 Server provided online by the Technical University of Denmark are shown above. These results predict a major site of cleavage located between alanine 21 (A21) and arginine 22 (R22). This cleavage site is indicated by the "/" in amino acid sequence 1-22 of human Hc (SEQ ID NO:17) provided above. The nucleotide sequence encoding amino acids 1-22 of human Hc is atgtggggct ggaagtgcct cctcttctgg gctgtgctgg tcacagccac tctctgcact gccagg (SEQ ID NO:18).
[0157] FIG. 55
[0158] This Figure shows the human Notch1/Fc fusion sequence for all constructs that end after EGF Repeat 36 of human Notch1.
[0159] FIG. 56
[0160] This Figure shows the human Notch1/Fc fusion sequence for all constructs that end after EGF Repeat 13 of human Notch1.
[0161] FIG. 57
[0162] This Figure shows the human Notch1/Fc fusion sequence for all constructs that end after EGF Repeat 23 of human Notch1.
[0163] FIG. 58
[0164] This Figure shows the human Notch1/Fc fusion sequence for all constructs that end after EGF Repeat 24 of human Notch1.
[0165] FIG. 59A and FIG. 59B
[0166] This Figure shows the full-length amino acid (aa) sequence of human Notch1, consisting of aa residue 1 (M=methionine) to aa residue 2555 (K=lysine) (SEQ ID NO: 52). The signal peptide and first 36 EGF-like repeat domains are present in aa 1-1433 of this sequence. Amino acids 1-1433, or a subset of these aa, were utilized for the design of the human Notch1 decoy proteins, described in the ensuing sections. The amino acids encompassing EGF-repeats 1-36 are underlined.
[0167] FIG. 60
[0168] This Figure shows the human Fc amino acid sequence utilized to generate the Fc tag on Notch1 decoy proteins (SEQ ID NO: 53). The 237 amino acids of human Fc were fused at the C-terminus of all Notch1 decoy constructs, just downstream of Notch1 EGF-like repeats. This region of human Fc allows for detection and purification of the Notch decoys and serves to stabilize the secreted human Notch1-human Fc fusion proteins.
[0169] FIG. 61
[0170] This Figure shows the amino acid sequence of h-Notch1.sup.(1-36) decoy protein (SEQ ID NO: 54). h-Notch1.sup.(1-36) decoy protein consists of the following three components: (1) human Notch1 signal sequence consisting of amino acids 1-23 of human Notch1, followed by (2) amino acids encoding the EGF-like repeats 1-36 of human Notch1 consisting of amino acids 24-1433 followed by (3) amino acids 1434-1670 that contain the human HC tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 1670 amino acids.
[0171] FIG. 62
[0172] This Figure shows the amino acid sequence of h-Notch1.sup.(1-13) decoy protein (SEQ ID NO: 55). h-Notch1.sup.(1-13) decoy protein consists of the following three components: (1) human Notch1 signal sequence consisting of amino acids 1-23 of human Notch1, followed by (2) amino acids encoding the EGF-like repeats 1-13 of human Notch1 consisting of amino acids 24-531 followed by (3) amino acids 532-768 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 768 amino acids.
[0173] FIG. 63
[0174] This Figure shows the amino acid sequence of h-Notch1.sup.(1-24) decoy protein (SEQ ID NO: 56). h-Notch1.sup.(1-24) decoy protein consists of the following three components: (1) human Notch1 signal sequence consisting of amino acids 1-23 of human Notch1, followed by (2) amino acids encoding the EGF-like repeats 1-24 of human Notch1 consisting of amino acids 24-948 followed by (3) amino acids 949-1185 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 1185 amino acids.
[0175] FIG. 64
[0176] This Figure shows the amino acid sequence of h-spNNotch1.sup.(9-23) decoy protein (SEQ ID NO: 57). h-spNNotch1.sup.(9-23) decoy protein consists of the following three components: (1) human Notch1 signal sequence consisting of amino acids 1-23 of human Notch1, followed by (2) amino acids encoding the EGF-like repeats 9-23 of human Notch1 consisting of amino acids 24-594 followed by (3) amino acids 595-831 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 829 amino acids.
[0177] FIG. 65
[0178] This Figure shows the amino acid sequence of h-spHCNotch1.sup.(9-23) decoy protein (SEQ ID NO: 58). h-spHCNotch1.sup.(9-23) decoy protein consists of the following three components: (1) human HC signal sequence consisting of amino acids 1-22 of human HC, followed by (2) amino acids encoding the EGF-like repeats 9-23 of human Notch1 consisting of amino acids 23-593 followed by (3) amino acids 594-830 that contain the human HC tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 829 amino acids.
[0179] FIG. 66
[0180] This Figure shows the amino acid sequence of h-spNNotch1.sup.(9-36) decoy protein (SEQ ID NO: 59). The abbreviation spN denotes that the human Notch1 signal peptide is used in this formulation. h-spNNotch1.sup.(9-36) decoy protein consists of the following three components: (1) human Notch1 signal sequence consisting of amino acids 1-23 of human Notch1, followed by (2) amino acids encoding the EGF-like repeats 9-36 of human Notch1 consisting of amino acids 24-1118 followed by (3) amino acids 1119-1355 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 1355 amino acids.
[0181] FIG. 67
[0182] This Figure shows the amino acid sequence of h-spHCNotch1.sup.(9-36) decoy protein (SEQ ID NO: 60). The abbreviation spHC denotes that the human HC signal peptide is used in this formulation. h-spHCNotch1.sup.(9-36) decoy protein consists of the following three components: (1) human HC signal sequence consisting of amino acids 1-22 of human HC, followed by (2) amino acids encoding the EGF-like repeats 9-36 of human Notch1 consisting of amino acids 23-1117 followed by (3) amino acids 1118-1354 that contain the human HC tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 1354 amino acids.
[0183] FIG. 68
[0184] This Figure shows the amino acid sequence of h-spNNotch1.sup.(13-29) decoy protein (SEQ ID NO: 61). The abbreviation spN denotes that the human Notch1 signal peptide is used in this formulation. h-spNNotch1.sup.(13-24) decoy protein consists of the following three components: (1) human Notch1 signal sequence consisting of amino acids 1-23 of human Notch1, followed by (2) amino acids encoding the EGF-like repeats 13-24 of human Notch1 consisting of amino acids 24-478 followed by (3) amino acids 479-715 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 715 amino acids.
[0185] FIG. 69
[0186] This Figure shows the amino acid sequence of h-spHCNotch1.sup.(13-29) decoy protein (SEQ ID NO: 62). The abbreviation spHC denotes that the human HC signal peptide is used in this formulation. h-spHCNotch1.sup.(13-24) decoy protein consists of the following three components: (1) human HC signal sequence consisting of amino acids 1-22 of human HC, followed by (2) amino acids encoding the EGF-like repeats 13-24 of human Notch1 consisting of amino acids 23-477 followed by (3) amino acids 478-714 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 714 amino acids.
[0187] FIG. 70
[0188] This Figure shows the amino acid sequence of h-spNNotch1.sup.(25-36) decoy protein (SEQ ID NO: 63). The abbreviation spN denotes that the human Notch1 signal peptide is used in this formulation. h-spNNotch1.sup.(25-36) decoy protein consists of the following three components: (1) human Notch1 signal sequence consisting of amino acids 1-23 of human Notch1, followed by (2) amino acids encoding the EGF-like repeats 25-36 of human Notch1 consisting of amino acids 24-508 followed by (3) amino acids 509-745 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 745 amino acids.
[0189] FIG. 71
[0190] This Figure shows the amino acid sequence of h-spHCNotch1.sup.(25-36) decoy protein (SEQ ID NO: 64). The abbreviation spHC denotes that the human HC signal peptide is used in this formulation. h-spHCNotch1.sup.(25-36) decoy protein consists of the following three components: (1) human HC signal sequence consisting of amino acids 1-22 of human HC, followed by (2) amino acids encoding the EGF-like repeats 25-36 of human Notch1 consisting of amino acids 23-507 followed by (3) amino acids 508-744 that contain the human HC tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 744 amino acids.
[0191] FIG. 72A and FIG. 72B
[0192] This Figure shows the nucleic acid sequence which encodes h-Notch1.sup.(1-36) decoy protein set forth in FIG. 61 (SEQ ID NO: 65).
[0193] FIG. 73
[0194] This Figure shows the nucleic acid sequence which encodes h-Notch1.sup.(1-13) decoy protein set forth in FIG. 62 (SEQ ID NO: 66).
[0195] FIG. 74A and FIG. 74B
[0196] This Figure shows the nucleic acid sequence which encodes h-Notch1.sup.(1-24) decoy protein set forth in FIG. 63 (SEQ ID NO: 67).
[0197] FIG. 75
[0198] This Figure shows the nucleic acid sequence which encodes h-spNNotch1.sup.(9-23) decoy protein set forth in FIG. 64 (SEQ ID NO: 68).
[0199] FIG. 76
[0200] This Figure shows the nucleic acid sequence which encodes h-spHCNotch1.sup.(9-23) decoy protein set forth in FIG. 65 (SEQ ID NO: 69).
[0201] FIGS. 77A and 77B
[0202] This Figure shows the nucleic acid sequence which encodes h-spNNotch1.sup.(9-36) decoy protein set forth in FIG. 66 (SEQ ID NO: 70).
[0203] FIGS. 78A and 78B
[0204] This Figure shows the nucleic acid sequence which encodes h-spHCNotch1.sup.(9-36) decoy protein set forth in FIG. 67 (SEQ ID NO: 71).
[0205] FIG. 79
[0206] This Figure shows the nucleic acid sequence which encodes h-spNNotch1.sup.(13-24) decoy protein set forth in FIG. 68 (SEQ ID NO: 72).
[0207] FIG. 80
[0208] This Figure shows the nucleic acid sequence which encodes h-spHCNotch1.sup.(13-24) decoy protein set forth in FIG. 69 (SEQ ID NO: 73).
[0209] FIG. 81
[0210] This Figure shows the nucleic acid sequence which encodes h-spNNotch1.sup.(25-36) decoy protein set forth in FIG. 70 (SEQ ID NO: 74).
[0211] FIG. 82
[0212] This Figure shows the nucleic acid sequence which encodes h-spHCNotch1.sup.(25-36) decoy protein set forth in FIG. 71 (SEQ ID NO: 75).
[0213] FIG. 83
[0214] This Figure shows the full-length amino acid (aa) sequence of human Notch4, consisting of aa 1 (M=methionine) to aa 2003 (K=lysine) (SEQ ID NO: 76). The signal peptide and first 29 EGF-like repeat domains are present in aa 1-1174 of this sequence. Amino acids 1-1174, or a subset of these aa, were utilized for the design of the human Notch4 decoy proteins, described in the ensuing sections. The amino acids encompassing EGF-repeats 1-29 are underlined.
[0215] FIG. 84
[0216] This Figure shows the Human Fc sequence utilized to generate the Fc tag on Notch4 decoy proteins (SEQ ID NO: 77). The 237 amino acids of human Fc, shown here, were fused at the C-terminus of all Notch4 decoy constructs, just downstream of Notch4 EGF-like repeats. This region of human Fc allows for detection and purification of the Notch decoys and serves to stabilize the secreted human Notch4-human Fc fusion proteins.
[0217] FIG. 85
[0218] This Figure shows the amino acid sequence of h-Notch4.sup.(1-29) decoy protein (SEQ ID NO: 78). h-Notch4.sup.(1-29) decoy protein consists of the following three components: (1) human Notch4 signal sequence consisting of amino acids 1-27 of human Notch4, followed by (2) amino acids encoding the EGF-like repeats 1-29 of human Notch4 consisting of amino acids 28-1173 followed by (3) amino acids 1174-1410 that contain the human HC tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 1410 amino acids.
[0219] FIG. 86
[0220] This Figure shows the amino acid sequence of h-Notch4.sup.(1-13) decoy protein (SEQ ID NO: 79). h-Notch4.sup.(1-13) decoy protein consists of the following three components: (1) human Notch4 signal sequence consisting of amino acids 1-27 of human Notch4, followed by (2) amino acids encoding the EGF-like repeats 1-13 of human Notch4 consisting of amino acids 28-554 followed by (3) amino acids 555-791 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 791 amino acids.
[0221] FIG. 87
[0222] This Figure shows the amino acid sequence of h-Notch4.sup.(1-23) decoy protein (SEQ ID NO: 80). h-Notch4.sup.(1-23) decoy protein consists of the following three components: (1) human Notch4 signal sequence consisting of amino acids 1-27 of human Notch4, followed by (2) amino acids encoding the EGF-like repeats 1-23 of human Notch4 consisting of amino acids 28-933 followed by (3) amino acids 934-1170 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 1170 amino acids.
[0223] FIG. 88
[0224] This Figure shows the amino acid sequence of h-spNNotch4.sup.(9-23) decoy protein (SEQ ID NO: 81). The abbreviation spN denotes that the human Notch4 signal peptide is used in this formulation. h-spNNotch4.sup.(9-23) decoy protein consists of the following three components: (1) human Notch4 signal sequence consisting of amino acids 1-27 of human Notch4, followed by (2) amino acids encoding the EGF-like repeats 9-23 of human Notch4 consisting of amino acids 28-602 followed by (3) amino acids 603-839 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 839 amino acids.
[0225] FIG. 89
[0226] This Figure shows the amino acid sequence of h-spHCNotch4.sup.(9-23) decoy protein (SEQ ID NO: 82). The abbreviation spHC denotes that the human HC signal peptide is used in this formulation. h-spHCNotch4.sup.(9-23) decoy protein consists of the following three components: (1) human HC signal sequence consisting of amino acids 1-22 of human HC, followed by (2) amino acids encoding the EGF-like repeats 9-23 of human Notch4 consisting of amino acids 23-597 followed by (3) amino acids 598-834 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 834 amino acids.
[0227] FIG. 90
[0228] This Figure shows the amino acid sequence of h-spNNotch4.sup.(9-29) decoy protein (SEQ ID NO: 83). The abbreviation spN denotes that the human Notch4 signal peptide is used in this formulation. h-spNNotch4.sup.(9-29) decoy protein consists of the following three components: (1) human Notch4 signal sequence consisting of amino acids 1-27 of human Notch4, followed by (2) amino acids encoding the EGF-like repeats 9-29 of human Notch4 consisting of amino acids 28-843 followed by (3) amino acids 844-1080 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 1080 amino acids.
[0229] FIG. 91
[0230] This Figure shows the amino acid sequence of h-spHCNotch4.sup.(9-29) decoy protein (SEQ ID NO: 84). The abbreviation spHC denotes that the human HC signal peptide is used in this formulation. h-spHCNotch4.sup.(9-29) decoy protein consists of the following three components: (1) human HC signal sequence consisting of amino acids 1-22 of human HC, followed by (2) amino acids encoding the EGF-like repeats 9-29 of human Notch4 consisting of amino acids 23-838 followed by (3) amino acids 839-1075 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 1075 amino acids.
[0231] FIG. 92
[0232] This Figure shows the amino acid sequence of h-spNNotch4.sup.(13-23) decoy protein (SEQ ID NO: 85). The abbreviation spN denotes that the human Notch4 signal peptide is used in this formulation. h-spNNotch4.sup.(13-23) decoy protein consists of the following three components: (1) human Notch4 signal sequence consisting of amino acids 1-27 of human Notch4, followed by (2) amino acids encoding the EGF-like repeats 13-23 of human Notch4 consisting of amino acids 28-444 followed by (3) amino acids 445-681 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 681 amino acids.
[0233] FIG. 93
[0234] This Figure shows the amino acid sequence of h-spHCNotch4.sup.(13-23) decoy protein (SEQ ID NO: 86). The abbreviation spHC denotes that the human HC signal peptide is used in this formulation. h-spHCNotch4.sup.(13-23) decoy protein consists of the following three components: (1) human HC signal sequence consisting of amino acids 1-22 of human HC, followed by (2) amino acids encoding the EGF-like repeats 13-23 of human Notch4 consisting of amino acids 23-439 followed by (3) amino acids 440-676 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 676 amino acids.
[0235] FIG. 94
[0236] This Figure shows the amino acid sequence of h-spNNotch4.sup.(21-29) decoy protein (SEQ ID NO: 87). The abbreviation spN denotes that the human Notch4 signal peptide is used in this formulation. h-spNNotch4 (21-29) decoy protein consists of the following three components: (1) human Notch4 signal sequence consisting of amino acids 1-27 of human Notch4, followed by (2) amino acids encoding the EGF-like repeats 21-29 of human Notch4 consisting of amino acids 28-392 followed by (3) amino acids 393-629 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 629 amino acids.
[0237] FIG. 95
[0238] This Figure shows the amino acid sequence of h-spHCNotch4.sup.(21-29) decoy protein (SEQ ID NO: 88). The abbreviation spHC denotes that the human HC signal peptide is used in this formulation. h-spHCNotch4.sup.(21-29) decoy protein consists of the following three components: (1) human HC signal sequence consisting of amino acids 1-22 of human HC, followed by (2) amino acids encoding the EGF-like repeats 21-29 of human Notch4 consisting of amino acids 23-387 followed by (3) amino acids 388-624 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 624 amino acids.
[0239] FIG. 96A and FIG. 96B
[0240] These Figures shows the nucleic acid sequence of human Notch4 (SEQ ID NO: 89).
[0241] FIG. 97
[0242] This Figure shows human Notch4 signal peptide sequence (SEQ ID NO: 90). The underlined sequence encodes Signal Peptide.
[0243] FIG. 98
[0244] This Figure shows the nucleic acid sequence of human HC signal peptide (nt 1-66) (SEQ ID NO: 91).
[0245] FIG. 99
[0246] This Figure shows the nucleic acid sequence of the human FC Tag (SEQ ID NO: 92).
[0247] FIG. 100A and FIG. 100B
[0248] These Figures shows the nucleic acid sequence which encodes h-Notch4.sup.(1-29) decoy protein (SEQ ID NO: 93). Human Notch4 decoy (EGF like repeats 1-29) [nt 1-3522].
[0249] FIG. 101
[0250] This Figure shows the nucleic acid sequence which encodes h-Notch4.sup.(1-13) decoy protein (SEQ ID NO: 94). Human Notch4 decoy (EGF like repeats 1-13) [nt 1-1662].
[0251] FIG. 102A and FIG. 102B
[0252] These Figures show the nucleic acid sequence which encodes h-Notch4.sup.(1-23) decoy protein (SEQ ID NO: 95). Human Notch4 decoy (EGF like repeats 1-23) [nt1-2799].
[0253] FIG. 103
[0254] This Figure shows the nucleic acid sequence which encodes h-spNNotch4.sup.(9-29) decoy protein (SEQ ID NO: 96). Human Notch4 decoy (EGF like repeats 9-29) [nt 1-81, 1075-3522].
[0255] FIG. 104
[0256] This Figure shows the nucleic acid sequence which encodes h-spHCNotch4.sup.(9-29) decoy protein (SEQ ID NO: 97). Human Notch4 decoy (EGF like repeats 9-29) [nt 1075-3522] & HC Signal Peptide [nt 1-66].
[0257] FIG. 105
[0258] This Figure shows the nucleic acid sequence which encodes h-spNNotch4.sup.(9-23) decoy protein (SEQ ID NO: 98). Human Notch4 decoy (EGF like repeats 9-23) [nt 1-81, 1075-2799].
[0259] FIG. 106
[0260] This Figure shows the nucleic acid sequence which encodes h-spHCNotch4.sup.(9-23) decoy protein (SEQ ID NO: 99). Human Notch4 decoy (EGF like repeats 9-23) [nt 1075-2799] & HC Signal Peptide [nt 1-66].
[0261] FIG. 107
[0262] This Figure shows the nucleic acid sequence which encodes h-spNNotch4.sup.(13-23) decoy protein (SEQ ID NO: 100). Human Notch4 decoy (EGF like repeats 13-23) [nt 1-81, 1549-2799].
[0263] FIG. 108
[0264] This Figure shows the nucleic acid sequence which encodes h-spHCNotch4.sup.(13-23) decoy protein (SEQ ID NO: 101). Human Notch4 decoy (EGF like repeats 13-23) [nt 1549-2799] & HC Signal Peptide [nt 1-66].
[0265] FIG. 109
[0266] This Figure shows the nucleic acid sequence which encodes h-spNNotch4.sup.(21-29) decoy protein (SEQ ID NO: 102). Human Notch4 decoy (EGF like repeats 21-29) [nt 1-81, 2428-3522].
[0267] FIG. 110
[0268] This Figure shows the nucleic acid sequence which encodes h-spHCNotch4.sup.(21-29) decoy protein (SEQ ID NO: 103). Human Notch4 decoy (EGF like repeats 21-29) [nt 2428-3522] & HC Signal Peptide [nt 1-66].
[0269] FIG. 111
[0270] This Figure shows a schematization of eleven formulations of human Notch4 decoys.
[0271] FIG. 112
[0272] This Figure shows signal sequence analysis of Notch 4 to determine where Notch4 signal peptide ends.
[0273] FIG. 113
[0274] This Figure shows the human Notch4/Fc fusion sequence for all constructs that end after EGF Repeat 29 of human Notch4.
[0275] FIG. 114
[0276] This Figure shows the human Notch4/Fc fusion sequence for all constructs that end after EGF Repeat 13 of human Notch4.
[0277] FIG. 115
[0278] This Figure shows the human Notch4/Fc fusion sequence for all constructs that end after EGF Repeat 23 of human Notch4.
[0279] FIG. 116A and FIG. 116B
[0280] These Figures show Notch4 insufficiency reduces circulating glucose and insulin levels. We are currently analyzing the blood glucose and insulin levels of Notch4 (N4) mutant mice. At weaning (P21), multiple litters generated from N4+/- matings have either been fed a normal diet or a high fat diet consisting of 45% of calories from fat. At 5 months of age, blood was drawn and glucose and insulin levels determined from random fed mice or mice after 6 hours of fasting. In females, reduction in N4 alleles correlated with a significant decrease in circulating glucose levels independent of the diet. In contrast, N4 insufficiency was associated with a decrease in glucose levels in only males fed a normal diet. Insulin levels of all females were unaffected (data not shown). This may be due to female mice being genetically protected against insulin resistance, and thus the metabolic abnormalities are exquisitely mild. On the normal diet, there were no differences in the insulin levels of the male mice. However, abalation of N4 correlated with a significant decrease in the random fed insulin levels of male mice fed a high fat diet. A similar trend was observed for N4 knockout males that had been fasted 6 hours. Thus, loss of N4 correlated with a significant decrease in blood glucose levels in both males and females fed a normal diet. In females, this reduction of glucose levels, but not insulin levels was observed in N4 mutant females fed a high fat diet. In contrast, glucose levels were unchanged in N4 knockout males, whereas insulin levels were reduced. These results are consistent with Notch4 insufficiency protecting against genetic and environmental forms of hyperglycemia due to disrupted insulin signaling.
[0281] FIG. 117
[0282] This Figure shows loss of Notch4 expression suppressed weight gain in mice fed a high fat diet.
[0283] FIG. 118
[0284] This Figure shows Rat Notch1 decoy is present in murine serum. The stability of the rat Notch1 decoy formulation in the mammalian blood stream was tested. Western blot analysis demonstrates that the full-length protein can be expressed in mice and is present at detectable levels with little evidence of degradation.
[0285] FIG. 119
[0286] This Figure shows Human Notch 1 decoy (n-Notch(1-36) decoy) and rat Notch1 decoy block mouse mammary tumor growth. The growth curve presented here demonstrates that either Rat Notch1 decoy or human Notch1 decoy reduced the growth of tumor xenografts in nude mice.
[0287] FIG. 120
[0288] This Figure shows rat Notch1 decoy inhibits SKNEP1 metastasis to lung tissue. SKNEP1 Ewings Sarcoma cells were programmed to express control Fc protein or rat Notch1 decoy s1 (sort 2) or rat Notch1 decoy s4 (sort 4). These SKNEP1 cell lines were orthotopically implanted into kidney of nude mice. After 6 weeks of tumor growth, metastasis to lung was assessed histologically. SKNEP1 cells expressing Rat Notch1 decoy showed fewer lungs that were positive for metastasis. We conclude that expression of the rat Notch1 decoy in nude mice diminishes the capacity of SKNEP1 cells to metastasize to lung.
[0289] FIG. 121:
[0290] This figure shoes Notch1 and Notch4 are co-expressed with VEGFR-3 and LYVE-1 in lymphatics of mouse skin. The expression of Notch1 and Notch4 was analyzed in the vasculature of mouse P4 dorsal skin. At this time point, the dermal lymphatics are actively remodeling into the lymphatic capillaries near the surface and collecting ducts in the lower dermal layers. 5 μm cross-sections of skin were co-stained with antibodies against Notch1 or Notch4 (red), and PECAM, VEGFR-3 or LYVE-1 (green). Notch1 and Notch4 share an overlapping pattern of expression with the blood and lymphatic endothelial cell marker, PECAM (upper panels). Notch1 and Notch4 were co-expressed with both VEGFR-3 (middle panels) and LYVE-1 in the dermal vasculature (lower panels). This expression pattern demonstrates that Notch1 and Notch4 are expressed and may function in the lymphatic vessels of the neonatal dermis.
[0291] FIG. 122
[0292] This Figure shows dermal lymphatic capillaries are altered in Notch4 homozygous knockout mice. We examined the dermal lymphatics of P4 mice. Sections of wildtype and Notch4 nullizygous were immunostained with antibodies against PECAM and LYVE-1 (green). Analysis of PECAM staining appeared similar between mutant and wildtype skin (upper panels). In contrast, LYVE-1-positive vessels in the dermis of Notch4 mutants had a different morphology than that of wildtype (middle panels). Notch4 mutant LYVE-1 vessels were often dilated and LYVE-1 staining was discontinuous (lower panels). These results suggest that Notch4 signaling may be involved in remodeling of the lymphatic vascular plexus.
[0293] FIGS. 123A-123C
[0294] These Figures show loss of Notch4 correlates with reduced LYVE1 expression in murine dermal lymphatics. Notch4 heterozygous (N4+/-) mice were mated and the dorsal skin of the resulting pups removed and embedded 14 days postnatally. Cross-sections of skin were immunostained for the endothelial cell marker, PECAM (data not shown). FIG. 123A shows cross-sections of skin were immunostained for the lymphatic endothelial cell marker, LYVE1. Five areas for each were captured by microscopy and PECAM and LYVE1 staining quantitated using imaging software. FIG. 123B shows PECAM expression was reduced approximately 25% in the N4-/- dermis compared to wild-type (WT) dermis. FIG. 123C shows LYVE-1 staining was more affected than the PECAM with LYVE1 staining decreased nearly 50% in N4-/- relative to WT mice. There was also a reduction in the intensity of the LYVE1 staining in the N4-/- lymphatics relative to the WT.
[0295] FIG. 124
[0296] This Figure shows Notch1 and Notch4 are expressed in human breast cancer lymphatic vessels. We performed double immunohistochemistry with antibodies against VEGFR-3 or LYVE-1 (green) and Notch1 or Notch4 (red) of human breast cancers. Notch1 and Notch4 were expressed in the extratumoral blood and lymphatic endothelium of human micropapillary breast carcinomas. To determine if Notch1 signaling was activated within the tumoral lymphatic endothelium, we double stained with an antibody against podoplanin (green) and N1Val (red; Cell Signaling), an antibody that specifically detects the activated Notch1 peptide. Expression of the activated Notch1 peptide was observed in most (white arrows) but not all (yellow arrows) of the lymphatic endothelial nuclei (lower panel). These results demonstrate that Notch1 was actively signaling in the pathological lymphatic vessels.
DETAILED DESCRIPTION OF THE INVENTION
Terms
[0297] As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below.
[0298] "Administering" may be effected or performed using any of the methods known to one skilled in the art. The methods comprise, for example, intralesional, intramuscular, subcutaneous, intravenous, intraperitoneal, liposome-mediated, transmucosal, intestinal, topical, nasal, oral, anal, ocular or otic means of delivery.
[0299] "Affixed" shall mean attached by any means. In one embodiment, affixed means attached by a covalent bond. In another embodiment, affixed means attached non-covalently.
[0300] "Amino acid," "amino acid residue" and "residue" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide or peptide. The amino acid can be, for example, a naturally occurring amino acid or an analog of a natural amino acid that can function in a manner similar to that of the naturally occurring amino acid.
[0301] "Antibody" shall include, without limitation, (a) an immunoglobulin molecule comprising two heavy chains and two light chains and which recognizes an antigen; (b) a polyclonal or monoclonal immunoglobulin molecule; and (c) a monovalent or divalent fragment thereof. Immunoglobulin molecules may derive from any of the commonly known classes, including but not limited to IgA, secretory IgA, IgG, IgE and IgM. IgG subclasses are well known to those in the art and include, but are not limited to, human IgG1, IgG2, IgG3 and IgG4. Antibodies can be both naturally occurring and non-naturally occurring. Furthermore, antibodies include chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. Antibodies may be human or nonhuman. Nonhuman antibodies may be humanized by recombinant methods to reduce their immunogenicity in humans. Antibody fragments include, without limitation, Fab and Fc fragments. The "Fc portion of an antibody", in one embodiment, is a crystallizable fragment obtained by papain digestion of immunoglobulin that consists of the C-terminal half of two heavy chains linked by disulfide bonds and known as the "effector region" of the immunoglobulin. In another embodiment, "Fc portion of an antibody" means all, or substantially all, of one C-terminal half of a heavy chain.
[0302] "Humanized", with respect to an antibody, means an antibody wherein some, most or all of the amino acids outside the CDR region are replaced with corresponding amino acids derived from a human immunoglobulin molecule. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not abrogate the ability of the antibody to bind a given antigen. Suitable human immunoglobulin molecules include, without limitation, IgG1, IgG2, IgG3, IgG4, IgA and IgM molecules. Various publications describe how to make humanized antibodies, e.g., U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089 and 5,693,761, and PCT International Publication No. WO 90/07861.
[0303] As used herein, the term "composition", as in pharmaceutical composition, is intended to encompass a product comprising the active ingredient(s) and the inert ingredient(s) that make up the carrier, as well as any product which results, directly or indirectly from combination, complexation, or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients.
[0304] As used herein, "effective amount" refers to an amount which is capable of treating a subject having a tumor, a disease or a disorder. Accordingly, the effective amount will vary with the subject being treated, as well as the condition to be treated. A person of ordinary skill in the art can perform routine titration experiments to determine such sufficient amount. The effective amount of a compound will vary depending on the subject and upon the particular route of administration used. Based upon the compound, the amount can be delivered continuously, such as by continuous pump, or at periodic intervals (for example, on one or more separate occasions). Desired time intervals of multiple amounts of a particular compound can be determined without undue experimentation by one skilled in the art. In one embodiment, the effective amount is between about 1 μg/kg-10 mg/kg. In another embodiment, the effective amount is beteen about 10 μg/kg-1 mg/kg. In a further embodiment, the effective amount is 100 μg/kg.
[0305] "Extracellular domain" as used in connection with Notch receptor protein means all or a portion of Notch which (i) exists extracellularly (i.e. exists neither as a transmembrane portion or an intracellular portion) and (ii) binds to extracellular ligands to which intact Notch receptor protein binds. The extracellular domain of Notch may optionally include a signal peptide. "Extracellular domain", "ECD" and "Ectodomain" are synonymous.
[0306] "Half-life-increasing moiety" means a moiety which, when operably affixed to a second moiety, increases the in vivo half-life of the second moiety. Half-life-increasing moieties include, for example, Fc portions of antibodies, glycosylation tags (i.e. glycosylated polypeptides), polyethylene glycol (PEG), polypeptides having PEG affixed thereto, and lipid-modified polypeptides.
[0307] "Inhibiting" the onset of a disorder or undesirable biological process shall mean either lessening the likelihood of the disorder's or process' onset, or preventing the onset of the disorder or process entirely. In the preferred embodiment, inhibiting the onset of a disorder or process means preventing its onset entirely.
[0308] "Notch", "Notch protein", and "Notch receptor protein" are synonymous. In addition, the terms "Notch-based fusion protein" and "Notch decoy" are synonymous. The following Notch amino acid sequences are known and hereby incorporated by reference: Notch1 (Genbank accession no. S18188 (rat)); Notch2 (Genbank accession no. NP_077334 (rat)); Notch3 (Genbank accession no. Q61982 (mouse)); and Notch4 (Genbank accession no. T09059 (mouse)). The following Notch nucleic acid sequences are known and hereby incorporated by reference: Notch1 (Genbank accession no. XM_342392 (rat) and NM_017617 (human)); Notch2 (Genbank accession no. NM_024358 (rat), M99437 (human and AF308601 (human)); Notch3 (Genbank accession no. NM_008716 (mouse) and XM_009303 (human)); and Notch4 (Genbank accession no. NM_010929 (mouse) and NM_004557 (human)).
[0309] The terms "nucleic acid", "polynucleotide" and "nucleic acid sequence" are used interchangeably herein, and each refers to a polymer of deoxyribonucleotides and/or ribonucleotides. The deoxyribonucleotides and ribonucleotides can be naturally occurring or synthetic analogues thereof. "Nucleic acid" shall mean any nucleic acid, including, without limitation, DNA, RNA and hybrids thereof. The nucleic acid bases that form nucleic acid molecules can be the bases A, C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well known in the art, and are exemplified in PCR Systems, Reagents and Consumables (Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems, Inc., Branchburg, N.J., USA). Nucleic acids include, without limitation, anti-sense molecules and catalytic nucleic acid molecules such as ribozymes and DNAzymes. Nucleic acids also include nucleic acids coding for peptide analogs, fragments or derivatives which differ from the naturally-occurring forms in terms of the identity of one or more amino acid residues (deletion analogs containing less than all of the specified residues; substitution analogs wherein one or more residues are replaced by one or more residues; and addition analogs, wherein one or more resides are added to a terminal or medial portion of the peptide) which share some or all of the properties of the naturally-occurring forms.
[0310] "Operably affixed" means, with respect to a first moiety affixed to a second moiety, affixed in a manner permitting the first moiety to function (e.g. binding properties) as it would were it not so affixed.
[0311] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein, and each means a polymer of amino acid residues. The amino acid residues can be naturally occurring or chemical analogues thereof. Polypeptides, peptides and proteins can also include modifications such as glycosylation, lipid attachment, sulfation, hydroxylation, and ADP-ribosylation.
[0312] As used herein, "pharmaceutically acceptable carrier" means that the carrier is compatible with the other ingredients of the formulation and is not deleterious to the recipient thereof, and encompasses any of the standard pharmaceutically accepted carriers. Such carriers include, for example, 0.01-0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions and suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases, and the like.
[0313] "Subject" shall mean any organism including, without limitation, a mammal such as a mouse, a rat, a dog, a guinea pig, a ferret, a rabbit and a primate. In the preferred embodiment, the subject is a human being.
[0314] "Treating" means either slowing, stopping or reversing the progression of a disease or disorder. As used herein, "treating" also means the amelioration of symptoms associated with the disease or disorder. Diseases include, but are not limited to, Tumor Angiogenesis, Atherosclerosis, Wound Healing, Macular degeneration, Retinopathy of Prematurity, Pre-eclampsia, Diabetic retinopathy, Ischemia, Stroke, Cardiovascular Disease, Psoriasis, lymphedema, tumorigenesis and tumor lymphangiogenesis.
[0315] Angiogenesis is encountered during wound healing processes, the female menstrual cycle and endometrial remodeling, as well as during embryonic development and organ growth. In the pathological setting, angiogenesis plays an important role in different diseases like rheumatoid arthritis, psoriasis, macular degeneration, diabetic retinopathy, and tumor growth.
[0316] There has been considerable evidence in vivo, including clinical observations, that abnormal angiogenesis is implicated in a number of disease conditions, which include rheumatoid arthritis, inflammation, cancer, psoriasis, degenerative eye conditions and others.
[0317] Other diseases for use of Notch fusion proteins are metabolic disorders such as, but not limited to, Diabetes, Obesity, Prediabetic state, Atherosclerosis, Ischemia, Stroke, Cardiovascular Disease, Regulating expression of Insulin, and Regulating the function of Insulin.
[0318] The use of Notch fusion proteins is also indicated for Metabolic Syndrome refers to a combination of medical disorders that increases the risk to a person for cardiovascular disease and diabetes. Other known names referring to such syndrome is syndrome X, insulin resistance syndrome, Reaven's syndrome. Several features of the syndromes include: fasting hyperglycemia, high blood pressure, central obesity (also known as visceral obesity), decreased High Density Lipoprotein (LDL), elevated triglycerides, elevated uric acid levels. Fasting hyperglycemia, listed above, includes diabetes mellitus type 2 or impaired fasting glucocse and impaired glucose tolerance or insulin resistance. In addition to metabolic syndrome, the Notch decoy may have indications for pre-diabetic states.
[0319] Units, prefixes and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acid sequences are written left to right in 5' to 3' orientation and amino acid sequences are written left to right in amino- to carboxy-terminal orientation. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
[0320] The following abbreviations are used herein: ECD: extracellular domain; IC: intracellular domain; NECD/Fc: Notch-based fusion protein; N1: Notch1; N2: Notch2; N3: Notch3; N4: Notch4; Dll: Delta-like; EC: endothelial cells; FGF: fibroblast growth factor; FGFR: fibroblast growth factor receptor; HUVEC: human umbilical vein endothelial cell; m.o.i.: multiplicity of infection; VMC: vascular mural cells; VEGF: vascular endothelial cell growth factor; VEGFR: vascular endothelial cell growth factor receptor; sp: signal peptide; PDGF: platelet derived growth factor; PDGFR: platelet derived growth factor receptor; P1GF: placental growth factor.
EMBODIMENTS OF THE INVENTION
[0321] This invention provides a fusion protein comprising a signal peptide, an extracellular domain of human Notch receptor protein and an Fc portion of an antibody bound thereto.
[0322] In a first embodiment of the fusion protein, the Notch receptor protein is Notch1 receptor protein. In one embodiment, the extracellular domain of Notch1 receptor protein comprises EGF-like repeats 1-36. In another embodiment, the extracellular domain of Notch1 receptor protein comprises EGF-like repeats 1-13. In another embodiment, the extracellular domain of Notch1 receptor protein comprises EGF-like repeats 1-24. In another embodiment, the extracellular domain of Notch1 receptor protein comprises EGF-like repeats 9-23. In another embodiment, the extracellular domain of Notch1 receptor protein comprises EGF-like repeats 9-36. In another embodiment, the extracellular domain of Notch1 receptor protein comprises EGF-like repeats 13-24. In a further embodiment, the extracellular domain of Notch1 receptor protein comprises EGF-like repeats 25-36.
[0323] In a second embodiment of the fusion protein, the Notch receptor protein is Notch1 receptor protein.
[0324] In a third embodiment of the fusion protein, the Notch receptor protein is Notch3 receptor protein.
[0325] In a fourth embodiment of the fusion protein, the Notch receptor protein is Notch4 receptor protein. In one embodiment, the extracellular domain of Notch4 receptor protein comprises EGF-like repeats 1-29. In another embodiment, the extracellular domain of Notch4 receptor protein comprises EGF-like repeats 1-13. In another embodiment, the extracellular domain of Notch4 receptor protein comprises EGF-like repeats 1-23. In another embodiment, the extracellular domain of Notch4 receptor protein comprises EGF-like repeats 9-23. In another embodiment, the extracellular domain of Notch4 receptor protein comprises EGF-like repeats 9-29. In another embodiment, the extracellular domain of Notch4 receptor protein comprises EGF-like repeats 13-23. In a further embodiment, the extracellular domain of Notch4 receptor protein comprises EGF-like repeats 21-29.
[0326] In one embodiment of the fusion protein, the Fc portion of the antibody is the Fc portion of a human antibody.
[0327] In one embodiment of the fusion protein, the signal peptide is the signal peptide of Notch1, Notch2, Notch3, Notch4, or the Hc (HC; Heavy Chain) portion of an antibody.
[0328] In one embodiment, the fusion protein comprises consecutive amino acids, the sequence of which is set forth in SEQ ID NO: 54. In another embodiment, the fusion protein comprises consecutive amino acids, the sequence of which is set forth in SEQ ID NO: 55. In another embodiment, the fusion protein comprises consecutive amino acids, the sequence of which is set forth in SEQ ID NO: 56. In another embodiment, the fusion protein comprises consecutive amino acids, the sequence of which is set forth in SEQ ID NO: 57. In another embodiment, the fusion protein comprises consecutive amino acids, the sequence of which is set forth in SEQ ID NO: 58. In another embodiment, the fusion protein comprises consecutive amino acids, the sequence of which is set forth in SEQ ID NO: 59. In another embodiment, the fusion protein comprises consecutive amino acids, the sequence of which is set forth in SEQ ID NO: 60. In another embodiment, the fusion protein comprises consecutive amino acids, the sequence of which is set forth in SEQ ID NO: 61. In another embodiment, the fusion protein comprises consecutive amino acids, the sequence of which is set forth in SEQ ID NO: 62. In another embodiment, the fusion protein comprises consecutive amino acids, the sequence of which is set forth in SEQ ID NO: 63. In a further embodiment, the fusion protein comprises consecutive amino acids, the sequence of which is set forth in SEQ ID NO: 64.
[0329] In one embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 65. In another embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 66. In another embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 67. In another embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 68. In another embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 69. In another embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 70. In another embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 71. In another embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 72. In another embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 73. In another embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 74. In a further embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 75.
[0330] In one embodiment, the fusion protein comprises consecutive amino acids, the sequence of which is set forth in SEQ ID NO: 78. In another embodiment, the fusion protein comprises consecutive amino acids, the sequence of which is set forth in SEQ ID NO: 79. In another embodiment, the fusion protein comprises consecutive amino acids, the sequence of which is set forth in SEQ ID NO: 80. In another embodiment, the fusion protein comprises consecutive amino acids, the sequence of which is set forth in SEQ ID NO: 81. In another embodiment, the fusion protein comprises consecutive amino acids, the sequence of which is set forth in SEQ ID NO: 82. In another embodiment, the fusion protein comprises consecutive amino acids, the sequence of which is set forth in SEQ ID NO: 83. In another embodiment, the fusion protein comprises consecutive amino acids, the sequence of which is set forth in SEQ ID NO: 84. In another embodiment, the fusion protein comprises consecutive amino acids, the sequence of which is set forth in SEQ ID NO: 85. In another embodiment, the fusion protein comprises consecutive amino acids, the sequence of which is set forth in SEQ ID NO: 86. In another embodiment, the fusion protein comprises consecutive amino acids, the sequence of which is set forth in SEQ ID NO: 87. In another embodiment, the fusion protein comprises consecutive amino acids, the sequence of which is set forth in SEQ ID NO: 88.
[0331] In one embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 89. In another embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 90. In another embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 91. In another embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 92. In another embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 93. In another embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 94. In another embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 95. In another embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 96. In another embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 97. In another embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 98. In another embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 99. In another embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 100. In another embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 101. In another embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 102. In another embodiment, the fusion protein is encoded by consecutive nucleotides, the sequence of which is set forth in SEQ ID NO: 103.
[0332] This invention provides a method for treating a subject having a tumor comprising administering to the subject an amount of the above fusion protein effective to treat the subject, thereby treating the subject having a tumor.
[0333] This invention provides a method for inhibiting angiogenesis in a subject comprising administering to the subject an amount of the above fusion protein effective to inhibit angiogenesis in the subject, thereby inhibiting angiogenesis in the subject.
[0334] This invention provides a method for treating a subject having ovarian cancer comprising administering to the subject an amount of the above fusion protein effective to treat the subject, thereby treating the subject having ovarian cancer.
[0335] This invention provides a method for treating a subject having a metabolic disorder comprising administering to the subject an amount of the above fusion protein effective to treat the subject, thereby treating the subject having a metabolic disorder. In one embodiment, the metabolic disorder is diabetes, obesity, atherosclerosis, ischemia, stroke, or cardiovascular disease.
[0336] This invention provides use of the above fusion protein for the preparation of a pharmaceutical composition for the treatment of a subject having a tumor.
[0337] This invention provides use of the above fusion protein for the preparation of a pharmaceutical composition for inhibiting angiogenesis in a subject.
[0338] This invention provides use of the above fusion protein for the preparation of a pharmaceutical composition for treating a subject having ovarian cancer.
[0339] This invention provides use of the above fusion protein for the preparation of a pharmaceutical composition for for treating a subject having a metabolic disorder. In one embodiment, the metabolic disorder is diabetes, obesity, atherosclerosis, ischemia, stroke, or cardiovascular disease.
[0340] This invention provides a method for inhibiting physiological lymphangiogenesis or pathological lymphangionesis in a subject comprising administering to the subject an amount of the above fusion protein effective to inhibit physiological lymphangiogenesis or pathological lymphangionesis in the subject. In one embodiment the pathological lymphangiogenesis is tumor lymphangiogenesis or lymph node metastasis that may be dependent on tumor lymphangiogenesis.
[0341] This invention provides method of inhibiting tumor metastasis in a subject comprising administering to the subject an amount of the above fusion effective to inhibit tumor metastasis in the subject. In on embodiment, the metastasis occurs via a blood vessel, the lymphatic vasculature or a lymph node. Tumor metastasis is the spread of cancer from one organ to another non-adjacent organ.
[0342] This invention provides a method of inhibiting growth of a secondary tumor in a subject comprising administering to the subject an amount of the above fusion protein effective to inhibit growth of the secondary tumor in the subject. Inhibition may also be of the tumor angiogenesis associated with the secondary or metastatic tumor. In one embodiment the secondary tumor growth is inhibited by inhibition of angiogenesis associated with the secondary tumor.
[0343] This invention provides a method of inhibiting blood vessel cooption by a tumor in subject comprising administering to the subject an amount of the above fusion protein effective to inhibit blood vessel cooption by a tumor in the subject. The process of vessel coopion is a process whereby tumor cells associate with pre-existing vessels and growth with assistance of coopted vessels. This growth of tumors on coopted vessels may be in the absence of, precede, or be in conjunction with tumor angiogenesis.
[0344] This invention provides a method of treating cancer in a subject comprising administering to the subject the above fusion protein and an inhibitor of Vascular Endothelial Growth Factor (VEGF), each in an amount effective to treat the cancer in the subject. In one embodiment the inhibitor of VEGF is an inhibitor of VEGF-A, an inhibitor of P1GF, an inhibitor of VEGF-B, an inhibitor of VEGF-C, or an inhibitor of VEGF-D. Examples of VEGF-inhibitors include, but are not limited to, bevacizumab, PTK787, Bay43-9006, SU11248, AG013676, ZD6474, VEGF-trap and Anti-VEGFR2. Examples of such inhibitors are more fully described in Ferrara et al., (2004) Nature Reviews Drug Discovery, Vol. 3:391-400 and Ellis et al. (2008) Nature Reviews Cancer Vol 8:579-591, the contents of each of which are hereby incorporated by reference.
[0345] This invention provides a method of treating cancer in a subject comprising administering to the subject the above fusion protein and an inhibitor of VEGFR-1, VEGFR-2 or VEGFR-3, each in an amount effective to treat the cancer in the subject. In one embodiment, the inhibitor targets one or more of the VEGFR.
[0346] This invention provides a method of treating cancer in a subject comprising administering to the subject the above fusion protein and an inhibitor of Platelet Derived Growth Factor (PDGF), each in an amount effective to treat the cancer in the subject. In on embodiment the inhibitor of Platelet Derived Growth Factors is an inhibitor of PDGF-A or an inhibitor of PDGF-B
[0347] This invention provides a method of treating cancer in a subject comprising administering to the subject the above fusion protein and a PDGF receptor antagonist, each in an amount effective to treat the cancer in the subject. In one embodiment the PDGF receptor antagonist is a PDGF Receptor-B antagonist.
[0348] This invention provides a method of treating cancer in a subject comprising administering to the subject the above fusion protein and an inhibitor of HER2/neu, each in an amount effective to treat the cancer in the subject.
[0349] This invention provides a method of treating vascular proliferative retinopathy comprising administering to the subject the above fusion protein in an emount effective to treat the vascular proliferative retinopathy.
[0350] 101. The method of claim 100, wherein the vascular proliferative retinopathy is diabetic retinopathy, macular defernation or retinopathy of prematurity
[0351] This invention also provides a first method for treating a subject having a tumor comprising administering to the subject an effective amount of a composition of matter comprising the extracellular domain of a Notch receptor protein operably affixed to a half-life-increasing moiety, so as to thereby treat the subject.
[0352] This invention also provides a second method for inhibiting angiogenesis in a subject comprising administering to the subject an effective amount of a composition of matter comprising the extracellular domain of a Notch receptor protein operably affixed to a half-life-increasing moiety, so as to thereby inhibit angiogenesis in the subject.
[0353] In a first embodiment of the above methods, the Notch receptor protein is Notch1 receptor protein. In one embodiment, the Notch1 receptor protein is human Notch1 receptor protein. In another embodiment, the half-life-increasing moiety is an Fc portion of an antibody. In another embodiment, the Fc portion of the antibody is the Fc portion of a human antibody. In a further embodiment, the extracellular domain and the half-life-increasing moiety are within the same polypeptide chain.
[0354] In a second embodiment of the above methods, the Notch receptor protein is Notch2 receptor protein. In one embodiment, the Notch2 receptor protein is human Notch2 receptor protein. In another embodiment, the half-life-increasing moiety is an Fc portion of an antibody. In another embodiment, the Fc portion of the antibody is the Fc portion of a human antibody. In a further embodiment, the extracellular domain and the half-life-increasing moiety are within the same polypeptide chain.
[0355] In a third embodiment of the above methods, the Notch receptor protein is Notch3 receptor protein. In one embodiment, the Notch3 receptor protein is human Notch3 receptor protein. In another embodiment, the half-life-increasing moiety is an Fc portion of an antibody. In another embodiment, the Fc portion of the antibody is the Fc portion of a human antibody. In a further embodiment, the extracellular domain and the half-life-increasing moiety are within the same polypeptide chain.
[0356] In a fourth embodiment of the above methods, the Notch receptor protein is Notch4 receptor protein. In one embodiment, the Notch4 receptor protein is human Notch4 receptor protein. In another embodiment, the half-life-increasing moiety is an Fc portion of an antibody. In another embodiment, the Fc portion of the antibody is the Fc portion of a human antibody. In a further embodiment, the extracellular domain and the half-life-increasing moiety are within the same polypeptide chain.
[0357] In a fifth embodiment of the above methods, the subject is a mammal. In one embodiment, the mammal is a human.
[0358] In a sixth embodiment of the above methods, the angiogenesis is tumor angiogenesis.
[0359] In a further embodiment of the second method, the subject has a tumor. In another embodiment, the subject is afflicted with a pathologic vascular hyperplasia. In one embodiment, the pathologic vascular hyperplasia is a benign hemagioma. In a further embodiment, the subject is afflicted with a lymphatic vascular proliferative disease.
[0360] This invention provides a first composition of matter comprising the extracellular domain of Notch4 receptor protein operably affixed to a half-life-increasing moiety. In one embodiment, the extracellular domain is covalently bound to the half-life-increasing moiety. In another embodiment, the extracellular domain and the half-life-increasing moiety are within the same polypeptide chain.
[0361] This invention also provides a second composition of matter comprising the extracellular domain of Notch4 receptor protein operably affixed to a half-life-increasing moiety and a pharmaceutically acceptable carrier.
[0362] This invention further provides an article of manufacture comprising (i) a packaging material having therein a composition of matter comprising the extracellular domain of a Notch receptor protein operably affixed to a half-life-increasing moiety and (ii) a label indicating that the composition is intended for use in treating a subject having a tumor or other disorder treatable by inhibiting angiogenesis in the subject.
[0363] In a first embodiment of the above article, the Notch receptor protein is Notch1 receptor protein. In one embodiment, the Notch1 receptor protein is human Notch1 receptor protein. In another embodiment, the half-life-increasing moiety is an Fc portion of an antibody. In another embodiment, the Fc portion of the antibody is the Fc portion of a human antibody. In a further embodiment, the extracellular domain and the Half-life-increasing moiety are within the same polypeptide chain.
[0364] In a second embodiment of the above article, the Notch receptor protein is Notch2 receptor protein. In one embodiment, the Notch2 receptor protein is human Notch2 receptor protein. In another embodiment, the half-life-increasing moiety is an Fc portion of an antibody. In another embodiment, the Fc portion of the antibody is the Fc portion of a human antibody. In a further embodiment, the extracellular domain and the Half-life-increasing moiety are within the same polypeptide chain.
[0365] In a third embodiment of the above article, the Notch receptor protein is Notch3 receptor protein. In one embodiment, the Notch3 receptor protein is human Notch3 receptor protein. In another embodiment, the half-life-increasing moiety is an Fc portion of an antibody. In another embodiment, the Fc portion of the antibody is the Fc portion of a human antibody. In a further embodiment, the extracellular domain and the Half-life-increasing moiety are within the same polypeptide chain.
[0366] In a fourth embodiment of the above article, the Notch receptor protein is Notch4 receptor protein. In one embodiment, the Notch4 receptor protein is human Notch4 receptor protein. In another embodiment, the half-life-increasing moiety is an Fc portion of an antibody. In another embodiment, the Fc portion of the antibody is the Fc portion of a human antibody. In a further embodiment, the extracellular domain and the Half-life-increasing moiety are within the same polypeptide chain.
[0367] In another embodiment of the above article, the composition is admixed with a pharmaceutical carrier. In a final embodiment, the subject is a human.
[0368] This invention provides a replicable vector which encodes a polypeptide comprising the extracellular domain of a Notch4 receptor protein operably affixed to a half-life-increasing moiety. In one embodiment, the half-life-increasing moiety is an Fc portion of an antibody. In another embodiment, the vector includes, without limitation, a plasmid, a cosmid, a retrovirus, an adenovirus, a lambda phage or a YAC.
[0369] This invention also provides a host vector system which comprises a replicable vector which encodes a polypeptide comprising the extracellular domain of a Notch receptor protein operably affixed to a half-life-increasing moiety and a suitable host cell. In one embodiment, the host cell is a eukaryotic cell. In another embodiment, the eukaryotic cell is a CHO cell. In a another embodiment, the eukaryotic cell is a HeLa cell. In a further embodiment, the host cell is a bacterial cell.
[0370] Finally, this invention provides a third method of producing a polypeptide which comprises growing a host vector system which comprises a replicable vector which encodes a polypeptide comprising the extracellular domain of a Notch receptor protein operably affixed to a half-life-increasing moiety and a suitable host cell under conditions permitting production of the polypeptide, and recovering the polypeptide so produced.
[0371] This invention is illustrated in the Experimental Details section which follows. This section is set forth to aid in an understanding of the invention but is not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow thereafter.
EXPERIMENTAL DETAILS
First Series of Experiments
Human Notch1 Fusion Proteins (Notch Decoys)
[0372] The Notch1 decoys are assembled using sequences encoding a signal peptide, a portion of the Notch1 extracellular domain encompassing all or a subset of the EGF-like repeat domains, and a portion of the human Fc protein (amino acids 1-237). The complete full-length sequence of human Notch1 is provided in FIG. 59.
[0373] The signal peptides utilized are either the native Notch1 signal peptide or the human Hc signal peptide, each fused to a region of Notch1. The signal peptide allows for secretion of the Notch decoy proteins.
[0374] The Notch1 extracellular domains used are designed to bind to Notch ligands and consist of all or a subset of the 36 EGF-like repeat domains of the human Notch1 protein.
[0375] The Fc tag is fused to the C-terminus of a given EGF-like repeat of human Notch1 and serves to allow for purification, detection, and stabilization of the Notch1 decoy proteins.
[0376] The overall design of the human Notch1 decoys, eleven formulations, is to encode for; (1) a signal peptide to allow for secretion of Notch1 decoy proteins into the extracellular media of eukaryotic cells that are used to produce the proteins, (2) a portion of the extracellular domain of all or a portion of the EGF-like repeats of human Notch1 to allow for association with Notch ligands, and (3) a portion of the human Fc protein to allow for detection.
[0377] The following eleven formulations of human Notch1 decoys will be described and are schematized in FIG. 52.
1) h-Notch1.sup.(1-36) decoy (N1-1 of FIG. 52) 2) h-Notch1.sup.(1-13) decoy (N1-2 of FIG. 52) 3) h-Notch1.sup.(1-24) decoy (N1-3 of FIG. 52) 4) h-spNNotch1.sup.(9-23) decoy (N1-4 of FIG. 52) 5) h-spHCNotch1.sup.(9-23) decoy (N1-5 of FIG. 52) 6) h-spNNotch1.sup.(9-36) decoy (N1-6 of FIG. 52) 7) h-spHCNotch1.sup.(9-36) decoy (N1-7 of FIG. 52) 8) h-spNNotch1.sup.(13-24) decoy (N1-8 of FIG. 52) 9) h-spHCNotch1.sup.(13-24) decoy (N1-9 of FIG. 52) 10) h-spNNotch1.sup.(25-36) decoy (N1-10 of FIG. 52) 11) h-spHCNotch1.sup.(25-36) decoy (N1-11 of FIG. 52)
[0378] Human Notch1 Sequence
[0379] The full-length amino acid (aa) sequence of human Notch1, consisting of aa residue 1 (M=methionine) to aa residue 2555 (K=lysine) is set forth in FIG. 59. The signal peptide and first 36 EGF-like repeat domains are present in aa 1-1433 of this sequence. Amino acids 1-1433, or a subset of these aa, were utilized for the design of the human Notch1 decoy proteins, described in the ensuing sections. The amino acids encompassing EGF-repeats 1-36 are underlined.
[0380] Human Fc Sequence Utilized to Generate the Fc Tag on Notch1 Decoy Proteins
[0381] The 237 amino acids of human Fc, shown in FIG. 60, were fused at the C-terminus of all Notch1 decoy constructs, just downstream of Notch1 EGF-like repeats. This region of human Fc allows for detection and purification of the Notch decoys and serves to stabilize the secreted human Notch1-human Fc fusion proteins.
[0382] Signal Peptides Utilized in Notch1 Decoy Proteins
[0383] Two distinct signal peptide sequences were incorporated into the design of the human Notch1 decoy proteins. The first is the human Notch1 signal peptide that is predicted to encompass amino acids 1-20 of human Notch1. This determination was made using the Signal IP 3.0 Server program provided by the Technical University of Denmark. The second is the human Hc signal peptide that is predicted to encompass amino acids 1-22 of human IgG heavy chain (HC) signal peptide.
[0384] 1. Human Notch1 Signal Peptide (aa 1-20)
TABLE-US-00001 (SEQ ID NO: 16) MPPLLAPLLCLALLPALA/A/R
[0385] Amino acid sequence of the predicted human Notch1 signal peptide is schematized in FIG. 53. The prediction results of analysis utilizing the SignalIP 3.0 Server provided online by the Technical University of Denmark are shown in FIG. 53. These results predict a major site of cleavage located between alanine 18 (A18) and alanine 19 (A19) and a minor site of cleavage between A19 and Arginine 20 (R20). These two cleavage sites are indicated by the "/" in amino acid sequence 1-20 of human Notch1, provided above.
[0386] 2. Human Notch1 Signal Peptide Fusion Peptide (Aa 1-23) Utilized in Notch1 Decoys that Utilize this Signal Sequence.
[0387] In order to make sure that the Notch1 signal peptide is utilized efficiently three additional amino acids beyond the predicted minor site of cleavage are provided in the human Notch1 decoys. Thus the amino acid sequence utilized in the human Notch1 decoy formulation, that incorporate a Notch1 signal peptide, contains glycine-proline-arginine (GPR-bold/underlined) between the sites of predicted signal peptide cleavage and the Notch1 EGF-like repeats as shown below.
TABLE-US-00002 (SEQ ID NO: 130) MPPLLAPLLCLALLPALAARGPR
[0388] 3. Human HC Signal Peptide (aa 1-22)
[0389] The amino acid sequence of the predicted human Hc signal peptide is
TABLE-US-00003 (SEQ ID NO: 18) MWGWKCLLFWAVLVTATLCTA/R
[0390] The prediction results of analysis utilizing the SignalIP 3.0 Server provided online by the Technical University of Denmark are shown above. These results predict a major site of cleavage located between alanine 21 (A21) and arginine 22 (22). This cleavage site is indicated by the "/" in amino acid sequence 1-22 of human Hc provided above.
[0391] h-Notch1.sup.(1-36) Decoy
[0392] h-Notch1.sup.(1-36) decoy denotes the human Notch1 decoy that encompass EGF-like repeats 1-36 of Notch1 (N1-1 of FIG. 52).
[0393] h-Notch1.sup.(1-36) decoy protein which is set forth in FIG. 61 consists of the following three components:
[0394] (1) human Notch1 signal sequence consisting of amino acids 1-23 of human Notch1, followed by (2) amino acids encoding the EGF-like repeats 1-36 of human Notch1 consisting of amino acids 24-1433 followed by (3) amino acids 1434-1670 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 1670 amino acids.
[0395] h-Notch1.sup.(1-13) Decoy
[0396] h-Notch1.sup.(1-13) decoy denotes the human Notch1 decoy that encompass EGF-like repeats 1-13 of Notch1 (N1-2 of FIG. 52).
[0397] h-Notch1.sup.(1-13) decoy protein which is set forth in FIG. 62 consists of the following three components:
[0398] (1) human Notch1 signal sequence consisting of amino acids 1-23 of human Notch1, followed by (2) amino acids encoding the EGF-like repeats 1-13 of human Notch1 consisting of amino acids 24-531 followed by (3) amino acids 532-768 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 768 amino acids.
[0399] h-Notch1.sup.(1-24) Decoy
[0400] h-Notch1.sup.(1-24) decoy denotes the human Notch1 decoy that encompass EGF-like repeats 1-24 of Notch1 (N1-3 of FIG. 52).
[0401] h-Notch1.sup.(1-24) decoy protein which is set forth in FIG. 63 consists of the following three components:
[0402] (1) human Notch1 signal sequence consisting of amino acids 1-23 of human Notch1, followed by (2) amino acids encoding the EGF-like repeats 1-24 of human Notch1 consisting of amino acids 24-948 followed by (3) amino acids 949-1185 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 1185 amino acids.
[0403] h-spNNotch1.sup.(9-23) Decoy
[0404] h-spNNotch1.sup.(9-23) decoy denotes the human Notch1 decoy that encompass EGF-like repeats 9-23 of Notch1 (N1-4 of FIG. 52). The abbreviation spN denotes that the human Notch1 signal peptide is used in this formulation.
[0405] h-spNNotch1.sup.(9-23) decoy protein which is set forth in FIG. 64 consists of the following three components:
[0406] (1) human Notch1 signal sequence consisting of amino acids 1-23 of human Notch1, followed by (2) amino acids encoding the EGF-like repeats 9-23 of human Notch1 consisting of amino acids 24-593 followed by (3) amino acids 594-830 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 830 amino acids.
[0407] h-spHCNotch1.sup.(9-23) Decoy
[0408] h-spHCNotch1.sup.(9-23) decoy denotes the human Notch1 decoy that encompass EGF-like repeats 9-23 of Notch1 (N1-5 of FIG. 52). The abbreviation spHC denotes that the human Hc signal peptide is used in this formulation.
[0409] h-spHCNotch1.sup.(9-23) decoy protein which is set forth in FIG. 65 consists of the following three components:
[0410] (1) human Hc signal sequence consisting of amino acids 1-22 of human Hc, followed by (2) amino acids encoding the EGF-like repeats 9-23 of human Notch1 consisting of amino acids 23-592 followed by (3) amino acids 593-829 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 829 amino acids.
[0411] h-spNNotch1.sup.(9-36) Decoy
[0412] h-spNNotch1.sup.(9-36) decoy denotes the human Notch1 decoy that encompass EGF-like repeats 9-36 of Notch1 (N1-6 of FIG. 52). The abbreviation spN denotes that the human Notch1 signal peptide is used in this formulation.
[0413] h-spNNotch1.sup.(9-36) decoy protein which is set forth in FIG. 66 consists of the following three components:
[0414] (1) human Notch1 signal sequence consisting of amino acids 1-23 of human Notch1, followed by (2) amino acids encoding the EGF-like repeats 9-36 of human Notch1 consisting of amino acids 24-1118 followed by (3) amino acids 1119-1355 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 1355 amino acids.
[0415] h-spHCNotch1.sup.(9-36) Decoy
[0416] h-spHCNotch1.sup.(9-36) decoy denotes the human Notch1 decoy that encompass EGF-like repeats 9-36 of Notch1 (N1-7 of FIG. 52). The abbreviation spHC denotes that the human Hc signal peptide is used in this formulation.
[0417] h-spHCNotch1.sup.(9-36) decoy protein which is set forth in FIG. 67 consists of the following three components:
[0418] (1) human Hc signal sequence consisting of amino acids 1-22 of human Hc, followed by (2) amino acids encoding the EGF-like repeats 9-36 of human Notch1 consisting of amino acids 23-1117 followed by (3) amino acids 1118-1354 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 1354 amino acids.
[0419] h-spNNotch1.sup.(13-29) Decoy
[0420] h-spNNotch1.sup.(13-24) decoy denotes the human Notch1 decoy that encompass EGF-like repeats 13-24 of Notch1 (N1-8 of FIG. 52). The abbreviation spN denotes that the human Notch1 signal peptide is used in this formulation.
[0421] h-spNNotch1.sup.(13-24) decoy protein which is set forth in FIG. 68 consists of the following three components:
[0422] (1) human Notch1 signal sequence consisting of amino acids 1-23 of human Notch1, followed by (2) amino acids encoding the EGF-like repeats 13-24 of human Notch1 consisting of amino acids 24-478 followed by (3) amino acids 479-715 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 715 amino acids.
[0423] h-spHCNotch1.sup.(13-24) Decoy
[0424] h-spHCNotch1.sup.(13-24) decoy denotes the human Notch1 decoy that encompass EGF-like repeats 13-24 of Notch1 (N1-9 of FIG. 52). The abbreviation spHc denotes that the human Hc signal peptide is used in this formulation.
[0425] h-spHCNotch1.sup.(13-24) decoy protein which is set forth in FIG. 69 consists of the following three components:
[0426] (1) human Hc signal sequence consisting of amino acids 1-22 of human Hc, followed by (2) amino acids encoding the EGF-like repeats 13-24 of human Notch1 consisting of amino acids 23-477 followed by (3) amino acids 478-714 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 714 amino acids.
[0427] h-spNNotch1.sup.(25-36) Decoy
[0428] h-spNNotch1.sup.(25-36) decoy denotes the human Notch1 decoy that encompass EGF-like repeats 25-36 of Notch1 (N1-10 of FIG. 52). The abbreviation spN denotes that the human Notch1 signal peptide is used in this formulation.
[0429] h-spNNotch1.sup.(25-36) decoy protein which is set forth in FIG. 70 consists of the following three components:
[0430] (1) human Notch1 signal sequence consisting of amino acids 1-23 of human Notch1, followed by (2) amino acids encoding the EGF-like repeats 25-36 of human Notch1 consisting of amino acids 24-508 followed by (3) amino acids 509-745 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 745 amino acids.
[0431] h-spHCNotch1.sup.(25-36) Decoy
[0432] h-spHCNotch1.sup.(25-36) decoy denotes the human Notch1 decoy that encompass EGF-like repeats 25-36 of Notch1 (N1-11 of FIG. 52). The abbreviation spHC denotes that the human Hc signal peptide is used in this formulation.
[0433] h-spHCNotch1.sup.(25-36) decoy protein which is set forth in FIG. 71 consists of the following three components:
[0434] (1) human Hc signal sequence consisting of amino acids 1-22 of human Hc, followed by (2) amino acids encoding the EGF-like repeats 25-36 of human Notch1 consisting of amino acids 23-507 followed by (3) amino acids 508-744 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 744 amino acids.
[0435] Methods
[0436] Construction of Human Notch1 Decoys
[0437] Total RNA from human umbilical venous endothelial cells (HUVEC) was used to generate the human Notch1 decoy variants. Total RNA was reverse transcribed with M-MLV reverse transcriptase and either random hexamer primers or a Notch1 decoy specific primer. The synthesized cDNA was then amplified with Notch1 decoy specific upstream (sense) and downstream (antisense) primers.
[0438] The downstream primer encodes either BamHI or BglII restriction site at the 5' end that will ligate with the BglII site in the Fc sequence to generate an in frame human Notch1/Fc chimera.
[0439] In the case of Notch1 decoys that generate the fusion after nucleotide sequence encoding EGF-like repeat 36, a BglII site will be generated to create the fusion site and this fusion sequence is provided (Notch1, FIG. 4). This applies to formulations; h-Notch1.sup.(1-36) decoy, h-spNNotch1.sup.(9-36) decoy, h-spHCNotch1.sup.(9-36) decoy, h-spNNotch1.sup.(26-36) decoy, h-spHCNotch1.sup.(25-36) decoy.
[0440] In the case of Notch1 decoys that generate the fusion after nucleotide sequence encoding EGF-like repeat 13, a BamHI site will be generated to create the fusion site and this fusion sequence is provided (Notch1, FIG. 5). This applies to formulation h-Notch1.sup.(1-13) decoy.
[0441] In the case of Notch1 decoys that generate the fusion after nucleotide sequence encoding EGF-like repeat 23, a BglII site will be generated to create the fusion site and this fusion sequence is provided (Notch1, FIG. 6). This applies to formulations h-spNNotch1.sup.(9-23) decoy, h-spHCNotch1.sup.(9-23) decoy.
[0442] In the case of Notch1 decoys that generate the fusion after nucleotide sequence encoding EGF-like repeat 24, a BglII site will be generated to create the fusion site and this fusion sequence is provided (Notch1, FIG. 7). This applies to formulations h-Notch1.sup.(1-24) decoy, h-spNNotch1.sup.(13-24) decoy, h-spHCNOtCh1.sup.(13-24) decoy.
[0443] The amplified PCR product is sublconed into pBluescript SK II Fc to generate the different human Notch1/Fc chimeras. The human Notch1/Fc decoy sequences are then shuttled into mammalian expression vectors (pAd-lox, pCCL, pcDNA3) for expression and purification of human Notch1 decoy proteins.
[0444] Human Notch4 Fusion Proteins (Notch Decoys)
[0445] The Notch4 decoys are assembled using sequences encoding a signal peptide, a portion of the Notch4 extracellular domain encompassing all or a subset of the EGF-like repeat domains, and a portion of the human Fc protein (amino acids 1-237). The complete full-length sequence of human Notch4 is provided in FIG. 83.
[0446] The signal peptides utilized are either the native Notch4 signal peptide or the human Hc signal peptide, each fused to a region of Notch4. The signal peptide allows for secretion of the Notch decoy proteins.
[0447] The Notch4 extracellular domains used are designed to bind to Notch ligands and consist of all or a subset of the 29 EGF-like repeat domains of the human Notch4 protein
[0448] The Fc tag is fused to the C-terminus of a given EGF-like repeat of human Notch4 and serves to allow for purification, detection, and stabilization of the Notch4 decoy proteins.
[0449] The overall design of the human Notch4 decoys, eleven formulations, is to encode for; (1) a signal peptide to allow for secretion of Notch4 decoy proteins into the extracellular media of eukaryotic cells that are used to produce the proteins, (2) a portion of the extracellular domain of all or a portion of the EGF-like repeats of human Notch4 to allow for association with Notch ligands, and (3) a portion of the human Fc protein to allow for detection.
[0450] The following eleven formulations of human Notch4 decoys will be described and are schematized in FIG. 111.
1) h-Notch4.sup.(1-29) decoy (N4-1 of FIG. 111) 2) h-Notch4.sup.(1-13) decoy (N4-2 of FIG. 111) 3) h-Notch4.sup.(1-23) decoy (N4-3 of FIG. 111) 4) h-spNNotch4.sup.(9-23) decoy (N4-4 of FIG. 111) 5) h-spHCNotch4.sup.(9-23) decoy (N4-5 of FIG. 111) 6) h-spNNotch4.sup.(9-29) decoy (N4-6 of FIG. 111) 7) h-spHCNotch4.sup.(9-29) decoy (N4-7 of FIG. 111) 8) h-spNNotch4.sup.(13-23) decoy (N4-8 of FIG. 111) 9) h-spHCNotch4.sup.(13-23) decoy (N4-9 of FIG. 111) 10) h-spNNotch4.sup.(21-29) decoy (N4-10 of FIG. 111) 11) h-spHCNotch4.sup.(21-29) decoy (N4-11 of FIG. 111)
[0451] Human Notch4 Sequence
[0452] The full-length amino acid (aa) sequence of human Notch4, consisting of aa residue 1 (M=methionine) to aa residue 2003 (K=lysine) is set forth in FIG. 83. The signal peptide and first 29 EGF-like repeat domains are present in aa 1-1174 of this sequence. Amino acids 1-1174, or a subset of these aa, were utilized for the design of the human Notch4 decoy proteins, described in the ensuing sections. The amino acids encompassing EGF-repeats 1-29 are underlined.
[0453] Human Fc Sequence Utilized to Generate the Fc Tag on Notch4 Decoy Proteins
[0454] The 237 amino acids of human Fc, shown in FIG. 84, were fused at the C-terminus of all Notch4 decoy constructs, just downstream of Notch4 EGF-like repeats. This region of human Fc allows for detection and purification of the Notch decoys and serves to stabilize the secreted human Notch4-human Fc fusion proteins.
[0455] Signal Peptides Utilized in Notch4 Decoy Proteins
[0456] Two distinct signal peptide sequences were incorporated into the design of the human Notch4 decoy proteins. The first is the human Notch4 signal peptide that is predicted to encompass amino acids 1-24 of human Notch4. This determination was made using the Signal IP 3.0 Server program provided by the Technical University of Denmark. The second is the human Hc signal peptide that is predicted to encompass amino acids 1-22 of human Hc.
[0457] 1. Human Notch4 Signal Peptide (aa 1-24)
TABLE-US-00004 (SEQ ID NO: 104) MQPPSLLLLLLLLLLLCVSVVRP/R
[0458] Amino acid sequence of the predicted human Notch4 signal peptide is schematized in FIG. 112. The prediction results of analysis utilizing the SignalIP 3.0 Server provided online by the Technical University of Denmark are shown in FIG. 112. These results predict a site of cleavage located between proline 23 (A23) and Arginine 24 (R24). The cleavage site is indicated by the "/" in amino acid sequence 1-24 of human Notch4, provided above.
[0459] 2. Human Notch4 Signal Peptide Fusion Peptide (aa 1-27) Utilized in Notch4 Decoys that Utilize this Signal Sequence
[0460] In order to make sure that the Notch4 signal peptide is utilized efficiently three additional amino acids beyond the predicted minor site of cleavage are provided in the human Notch4 decoys. Thus the amino acid sequence utilized in the human Notch4 decoy formulation, that incorporate a Notch4 signal peptide, contains glycine-proline-arginine (GLL-bold/underlined) between the sites of predicted signal peptide cleavage and the Notch4 EGF-like repeats.
TABLE-US-00005 (SEQ ID NO: 131) MQPPSLLLLLLLLLLLCVSVVRPRGLL
[0461] 3. Human HC Signal Peptide (aa 1-22)
[0462] The amino acid sequence of the predicted human Hc signal peptide is
TABLE-US-00006 (SEQ ID NO: 17) MWGWKCLLFWAVLVTATLCTA/R
[0463] The prediction results of analysis utilizing the SignalIP 3.0 Server provided online by the Technical University of Denmark are shown above. These results predict a major site of cleavage located between alanine 21 (A21) and arginine 22 (22). This cleavage sites is indicated by the "/" in amino acid sequence 1-22 of human Hc provided above.
[0464] h-Notch4.sup.(1-29) Decoy
[0465] h-Notch4.sup.(1-29) decoy denotes the human Notch4 decoy that encompasses EGF-like repeats 1-29 of Notch4 (N4-1 of FIG. 111).
[0466] h-Notch4.sup.(1-29) decoy protein consists of the following three components:
[0467] (1) human Notch4 signal sequence consisting of amino acids 1-27 of human Notch4, followed by (2) amino acids encoding the EGF-like repeats 1-29 of human Notch4 consisting of amino acids 28-1173 followed by (3) amino acids 1174-1410 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 1410 amino acids.
[0468] h-Notch4.sup.(1-13) Decoy
[0469] h-Notch4.sup.(1-13) decoy denotes the human Notch4 decoy that encompasses EGF-like repeats 1-13 of Notch4 (N4-2 of FIG. 111).
[0470] h-Notch4.sup.(1-13) decoy protein consists of the following three components:
[0471] (1) human Notch4 signal sequence consisting of amino acids 1-27 of human Notch4, followed by (2) amino acids encoding the EGF-like repeats 1-13 of human Notch4 consisting of amino acids 28-554 followed by (3) amino acids 555-791 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 791 amino acids.
[0472] h-Notch4.sup.(1-23) Decoy
[0473] h-Notch4.sup.(1-23) decoy denotes the human Notch4 decoy that encompasses EGF-like repeats 1-23 of Notch4 (N4-3 of FIG. 111).
[0474] h-Notch4.sup.(1-23) decoy protein consists of the following three components:
[0475] (1) human Notch4 signal sequence consisting of amino acids 1-27 of human Notch4, followed by (2) amino acids encoding the EGF-like repeats 1-23 of human Notch4 consisting of amino acids 28-933 followed by (3) amino acids 934-1170 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 1170 amino acids.
[0476] h-spNNotch4.sup.(9-23) Decoy
[0477] h-spNNotch4.sup.(9-23) decoy denotes the human Notch4 decoy that encompasses EGF-like repeats 9-23 of Notch4 (N4-4 of FIG. 111). The abbreviation spN denotes that the human Notch4 signal peptide is used in this formulation.
[0478] h-spNNotch4.sup.(9-23) decoy protein consists of the following three components:
[0479] (1) human Notch4 signal sequence consisting of amino acids 1-27 of human Notch4, followed by (2) amino acids encoding the EGF-like repeats 9-23 of human Notch4 consisting of amino acids 28-602 followed by (3) amino acids 603-839 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 839 amino acids.
[0480] h-spHCNotch4.sup.(9-23) Decoy
[0481] h-spHCNotch4.sup.(9-23) decoy denotes the human Notch4 decoy that encompasses EGF-like repeats 9-23 of Notch4 (N4-5 of FIG. 111). The abbreviation spHC denotes that the human Hc signal peptide is used in this formulation.
[0482] h-spHCNotch4.sup.(9-23) decoy protein consists of the following three components:
[0483] (1) human Hc signal sequence consisting of amino acids 1-22 of human Hc, followed by (2) amino acids encoding the EGF-like repeats 9-23 of human Notch4 consisting of amino acids 23-597 followed by (3) amino acids 598-834 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 834 amino acids.
[0484] h-sp.sub.NNotch4.sup.(9-29) Decoy
[0485] h-spNNotch4.sup.(9-29) decoy denotes the human Notch4 decoy that encompasses EGF-like repeats 9-29 of Notch4 (N4-6 of FIG. 111). The abbreviation spN denotes that the human Notch4 signal peptide is used in this formulation.
[0486] h-spNNotch4.sup.(9-29) decoy protein consists of the following three components:
[0487] (1) human Notch4 signal sequence consisting of amino acids 1-27 of human Notch4, followed by (2) amino acids encoding the EGF-like repeats 9-29 of human Notch4 consisting of amino acids 28-843 followed by (3) amino acids 844-1080 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 1080 amino acids.
[0488] h-spHCNotch4.sup.(9-29) Decoy
[0489] h-spHCNotch4.sup.(9-29) decoy denotes the human Notch4 decoy that encompasses EGF-like repeats 9-29 of Notch4 (N4-7 of FIG. 111). The abbreviation spHC denotes that the human Hc signal peptide is used in this formulation.
[0490] h-spHCNotch4.sup.(9-29) decoy protein consists of the following three components:
[0491] (1) human Hc signal sequence consisting of amino acids 1-22 of human Hc, followed by (2) amino acids encoding the EGF-like repeats 9-29 of human Notch4 consisting of amino acids 23-838 followed by (3) amino acids 839-1075 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 1075 amino acids.
[0492] h-spNNotch4.sup.(13-23) Decoy
[0493] h-spNNotch4.sup.(13-23) decoy denotes the human Notch4 decoy that encompasses EGF-like repeats 13-23 of Notch4 (N4-8 of FIG. 111). The abbreviation spN denotes that the human Notch4 signal peptide is used in this formulation.
[0494] h-spNNotch4.sup.(13-23) decoy protein consists of the following three components:
[0495] (1) human Notch4 signal sequence consisting of amino acids 1-27 of human Notch4, followed by (2) amino acids encoding the EGF-like repeats 13-23 of human Notch4 consisting of amino acids 28-444 followed by (3) amino acids 445-681 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 681 amino acids.
[0496] h-spHCNotch4.sup.(13-23) Decoy
[0497] h-spHCNotch4.sup.(13-23) decoy denotes the human Notch4 decoy that encompasses EGF-like repeats 13-23 of Notch4 (N4-9 of FIG. 111). The abbreviation spHC denotes that the human Hc signal peptide is used in this formulation.
[0498] h-spHCNotch4.sup.(13-23) decoy protein consists of the following three components:
[0499] (1) human Hc signal sequence consisting of amino acids 1-22 of human Hc, followed by (2) amino acids encoding the EGF-like repeats 13-23 of human Notch4 consisting of amino acids 23-439 followed by (3) amino acids 440-676 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 676 amino acids.
[0500] h-spNNotch4.sup.(21-29) Decoy
[0501] h-spNNotch4.sup.(21-29) decoy denotes the human Notch4 decoy that encompasses EGF-like repeats 21-29 of Notch4 (N4-10 of FIG. 111). The abbreviation spN denotes that the human Notch4 signal peptide is used in this formulation.
[0502] h-spNNotch4.sup.(21-29) decoy protein consists of the following three components:
[0503] (1) human Notch4 signal sequence consisting of amino acids 1-27 of human Notch4, followed by (2) amino acids encoding the EGF-like repeats 21-29 of human Notch4 consisting of amino acids 28-392 followed by (3) amino acids 393-629 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 629 amino acids.
[0504] h-spHCNotch4.sup.(21-29) Decoy
[0505] h-spHCNotch4.sup.(21-29) decoy denotes the human Notch4 decoy that encompass EGF-like repeats 21-29 of Notch4 (N4-11 of FIG. 111). The abbreviation spHC denotes that the human Hc signal peptide is used in this formulation.
[0506] h-spHCNotch4.sup.(21-29) decoy protein consists of the following three components:
[0507] (1) human Hc signal sequence consisting of amino acids 1-22 of human Hc, followed by (2) amino acids encoding the EGF-like repeats 21-29 of human Notch4 consisting of amino acids 23-387 followed by (3) amino acids 388-624 that contain the human Fc tag. The predicted signal peptide sequence is underlined and the human Fc tag is underlined and italicized. This formulation contains 624 amino acids.
[0508] Methods
[0509] Construction of Human Notch4 Decoys
[0510] Total RNA from human umbilical venous endothelial cells (HUVEC) was used to generate the human Notch4 decoy variants. Total RNA was reverse transcribed with M-MLV reverse transcriptase and either random hexamer primers or a Notch4 decoy specific primer. The synthesized cDNA was then amplified with Notch4 decoy specific upstream (sense) and downstream (antisense) primers.
[0511] The downstream primer encodes either BamHI or BglII restriction site at the 5' end that will ligate with the BglII site in the Fc sequence to generate an in frame human Notch4/Fc chimera.
[0512] In the case of Notch4 decoys that generate the fusion after nucleotide sequence encoding EGF-like repeat 29, a BglII site will be generated to create the fusion site and this fusion sequence is provided (Notch4, FIG. 113).
[0513] This applies to formulations; h-Notch4.sup.(1-29) decoy, h-spNNotch4.sup.(9-29) decoy, h-spHCNotch4.sup.(9-29) decoy, h-spNNotch4.sup.(21-29) decoy, h-spHCNotch4.sup.(21-29) decoy.
[0514] In the case of Notch4 decoys that generate the fusion after nucleotide sequence encoding EGF-like repeat 13, a BamHI site will be generated to create the fusion site and this fusion sequence is provided (Notch4, FIG. 114). This applies to formulation h-Notch4.sup.(1-12) decoy.
[0515] In the case of Notch4 decoys that generate the fusion after nucleotide sequence encoding EGF-like repeat 23, a BglII site will be generated to create the fusion site and this fusion sequence is provided (Notch4, FIG. 115). This applies to formulations h-Notch4.sup.(1-23) decoy, h-spNNotch4.sup.(9-23) decoy, h-spHCNotch4.sup.(9-23) decoy, h-spNNotch4.sup.(13-23) decoy, h-spHCNotch4.sup.(13-23) decoy.
[0516] The amplified PCR product is sublconed into pBluescript SK II Fc to generate the different human Notch4/Fc chimeras. The human Notch4/Fc decoy sequences are then shuttled into mammalian expression vectors (pAd-lox, pCCL) for expression and purification of human Notch4 decoy proteins.
Second Series of Experiments
Materials & Methods
[0517] Plasmid Constructs
[0518] Adenovirus constructs encoding LacZ, full-length Notch4, or the activated form of Notch4/int3 have been previously described (Shawber et al., 2003). An activated form of Notch1 cDNA fused in frame with 6 myc tags (Kopan et al., 1994) was cloned into the adenovirus expression vector, pAd-lox. Both VEGF165 and N1ECDFc was also cloned into the pAd-lox. Adenoviral stocks were generated and titered as previously described (Hardy et al., 1997). The retroviral expression vector pHyTc encoding either LacZ, the activated form of Notch4/int3, J1, Dll1 and Dll4 have been previously described (Uyttendaele et al., 2000, Shawber et al., 2003, Das et al., 2004 in print). Plasmids encoding the intracellular domain of Notch1 (bp 5479-7833, Genbank accession # X57405) and the extracellular domain of Dll4 (bp 1-1545, Genbank accession # AF253468, provided by Chiron) fused in frame with a myc/His tag, were engineered into pHyTC.
[0519] Notch1ECD, Notch2ECD, Notch3ECD and Notch4ECD are engineered using the Fc sequences contained in the plasmid pCMX-sFR1-IgG using the methods set forth in Clin. Exp. Immunol. (1992) 87(1):105-110 to create the Notch-based fusion proteins, i.e. Notch1ECD/Fc, Notch2ECD/Fc, Notch3ECD/Fc and Notch4ECD/Fc.
[0520] Adenoviral Gene Transfer
[0521] 7.5×105 cells of HUVEC at passage 3 were seeded into type I collagen-coated 6 well plates on the day before adenoviral infection. Adenoviral infection with Ad-lacZ, Ad-VEGF165 or Ad-N1ECDFc was performed at indicated m.o.i., and incubated at 37° C. for 1 hr with occasional swirling of plates.
[0522] Luciferase Reporter Assays
[0523] To determine ligand-induced Notch signaling, co-culture assays were performed using HeLa and 293-derived Bosc cells. Transient transfections were performed by calcium phosphate precipitation. Hela cells plated 1-day prior in 10-cm plates at 1.5×106 were transfected with 333 ng of pBOS Notch1, 333 ng pGA981-6, and 83 ng pLNC lacZ with either 666 ng pCMV-Fc or pHyTC-N1ECDFc (333 ng for x1, 666 ng for x2). Bosc cells plated 1-day prior in 10-cm plates at 4×106 were transfected with either 680 ng pHyTc-Jagged1, pHyTc-Dll1, pHyTc-Dll4, or pHyTc-x (empty vector). One day after transfection, the cells were co-cultured in triplicate (HeLa:Bosc, 1:2) on 12-well plates for 24 hours. Cells were harvested and luciferase activity was determined 2-days post-transfection using the Enhanced Luciferase assay kit (BD PharMingen), and (3-galactosidase activity was determined using the Galacto-Light Plus kit (PE Biosystems). All assays were performed in a Berthold dual-injection luminometer.
[0524] To determine VEGF-induced Notch signaling, HUVEC which were infected with adenovirus were used. HUVEC plated 1-day prior in 6 well plates at 8.0×105 were infected with either Ad-LacZ as control or Ad-VEGF at indicated m.o.i. in the presence or absence of Ad-N1ECD/Fc. Two days after infection, infected HUVEC were re-seeded into 24-well plate at 1.5×105 cell in triplicate and cultured for 24 hours, and then transfected with 12.5 ng pRL-SV40 (Promega) and 137.5 ng pGA981-6 using Effectene transfection reagent (Qiagen). Cells were harvested either 1 or 2 days post-transfection and luciferase activity was determined by using the Dual-Luciferase® Reporter Assay System (Promega).
[0525] Sprouting Assay
[0526] For making collagen gels, an ice-cold solution of porcine type I collagen (Nitta gelatin, Tokyo, Japan) was mixed with 10×RPMI1640 medium and neutralization buffer at the ratio of 8:1:1. 400 μl aliquots of collagen gel were then added to 24-well plates and allowed to gel for at least 1 hour at 37° C. Following adenoviral infection (above), HUVEC was harvested and plated at 1.3×105 cells per well onto the top of the collagen gel in 24-well plates in 0.8 ml of EGM2 medium. HUVEC became nearly confluent 48 hours after plating. After seeding, medium was changed every 2 days for 1 week. sprouting was observed and photographs taken after 8 days with an Olympus digital camera mounted to a microscope. For quantification of the number of sprouts, 5 fields per each well were randomly selected and sprouting was counted under microscopy in a blind manner by two investigators.
Results and Discussion
[0527] NOTCHECD/Fc Fusion Proteins Function as Antagonists of Notch
[0528] Notch Antagonists-NotchECD/Fc Fusion Proteins
[0529] We have made several Notch antagonists (FIG. 2). Our strategy was to fuse the coding sequence of Notch EGF repeats in the Extracellular Domain (ECD) to the human or mouse Fc domain. This design makes a secreted protein without signaling function but which retains the ligand-binding domain and thus should bind to and inhibit ligand function. We refer to these proteins as "NotchECD/Fc" and all four Notch1-4ECD/Fcs have been made. The Fc domain facilitates affinity purification and protein detection by immunoblotting or immunohistochemistry.
[0530] Testing Notch Antagonists
[0531] An in vitro co-culture system (FIG. 3) with ligands expressed on one cell and Notch receptor activation scored in another cell was used to measure transcriptional activation of the Notch pathway. We used this co-culture assay to show that Notch1ECD/Fc functions to block ligand-dependent Notch signaling (FIG. 4). The N1ECD/Fc expression vector was co-transfected at different ratios with full-length Notch1 and the CSL-luciferase reporter in HeLa cells, followed by co-culture with ligand expressing 293 cells. We observed that activation of Notch1 signaling by Notch ligands was reduced by N1ECD/Fc expression. This effect displayed concentration-dependency; a 2:1 ratio of N1ECD/Fc to Notch1 was more effective in inhibiting signaling than a 1:1 ratio. Notch1ECD/Fc could block signaling mediated by Jagged1, Delta-like 1 or Delta-like 4.
[0532] Expressing and Purifying Notch Antagonists We have made CHO and HeLa cell lines expressing NotchECD/FCs using retroviral vectors for the purpose of protein purification. N1ECD/Fc proteins are secreted (FIG. 5); as shown in conditioned media collected from HeLa-NotchECD/Fc lines and purified with Protein-A (pA) agarose. The pA purified sample (Sup) and whole cell lysates (Lys) were immunoblotted with α-Fc antibody (FIG. 5, panel A) demonstrating that N1ECD/Fc is secreted into the media. Adenovirus vectors for NotchECD/Fc were used to infect HeLa cells and lysates from these cells were immunoblotted with α-Fc antibodies demonstrating that they express NotchECD/Fc(1, 2, 3, 4) proteins (FIG. 5, panel B). We are currently purifying N1ECD/Fc from CHO cell conditioned media using pA-affinity chromatography.
[0533] Defining Angiogenic Inhibition Using Notch Fusion Proteins
[0534] Activation of Notch Signaling can be Detected by Using CBF1 Promoter Activity
[0535] One can measure Notch signaling function by measuring transcriptional activity of CBF1 promoter, which is activated by binding of Notch-IC to CBF1. We measured CBF1 promoter activity in HUVEC which was infected with adenovirus encoding VEGF-165 at different MOI (FIG. 6). Induction of CBF1 promoter was clearly detected in Ad-VEGF-infected HUVEC, compared to Ad-LacZ-infected cells in the MOI dependent manner. This data showed overexpression of VEGF could activate Notch signaling in HUVEC. Thus VEGF induced Notch signaling activity.
[0536] We asked whether Notch fusion proteins could block VEGF-induced activation of Notch signaling. Co-infection of Ad-Notch fusion protein with Ad-VEGF clearly reduced activation of CBF1 promoter activity induced by Ad-VEGF infection alone (FIG. 7). In the case of infection at 40 MOI for each adenovirus in FIG. 7 (panel A), 60% inhibition at 24 hr and 90% inhibition at 48 hr after reporter gene transfection were detected also the inhibitory activity of Notch decoy was dependent on MOI of Ad-Notch fusion protein.
[0537] Notch Fusion Proteins Block Initiation of Angiogenic Sprouting Induced by VEGF
[0538] In this experiment, we evaluated the effect of Notch decoy on induction of budding (initiation of sprouting) by over-expressed VEGF-165 in HUVEC. When Ad-VEGF-infected HUVEC were cultured on type collagen gel for 8 days, budding was induced into collagen gel. This induction of budding by overexpressed VEGF was clearly inhibited by coinfection of adenoviral encoding Notch fusion protein (FIG. 8). Ad-Notch fusion protein itself had less effect on morphology.
[0539] In FIG. 9 we counted buds per field using the microscope. Ad-VEGF-infection into HUVEC increased the number of buds depending on the MOI used. Ad-VEGF-induced budding was clearly inhibited. These data suggest that VEGF induced budding of HUVEC through activation of Notch signaling and that the Notch fusion protein could inhibit VEGF-induced budding.
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Third Series of Experiments
VEGF Initiates Angiogenesis Via an Activation of Notch Signaling
[0601] Both the VEGF and Notch signaling pathways are critical for vascular development. Here we show that VEGF activates Notch signaling to initiate angiogenesis. VEGF increased the expression of Delta4 and Notch4 causing Notch signal activation and inducing filopodia in cultured primary endothelial cells. Studies using VEGF Receptor inhibitors show that Notch signal activation in turn enhances VEGF action by inducing VEGFR-1 (Flt-1) expression. Other elements of VEGF action, including the induction of MMP-9 and MT1-MMP, are mediated by Notch. Using in vivo assays to model VEGF-induced skin neovascularization, we found that a secreted Notch inhibitor (Notch-based fusion protein) blocks VEGF-induced neo-vascularization and induction of VEGFR-1 expression. Thus, Notch signaling is requisite for angiogenesis regulated by VEGF, likely at the level of initiation.
[0602] VEGF is a key regulator of angiogenesis progression consisting of multiple processes, such as degradation of ECM, budding (filopodia formation), proliferation, survival, and migration of endothelial cells. Although most of the steps might be co-operated with downstream molecules of VEGF signaling, it is not known how these steps are coordinately regulated to result in more complex morphogenetic events, such as angiogenic sprouting. Notch signaling is an evolutionarily conserved signaling mechanism that functions to regulate cell fate decisions (1). Upon binding by a ligand, such as Jagged and Delta-like, the cytoplasmic domain of Notch (NotchIC) is released by presenilin/γ-secretase, translocates to the nucleus, interacts with the transcriptional repressor CSL (CBF1/Su(H)/lag2), and converts it to a transcriptional activator (1). Roles of Notch signaling in vascular development were suggested by studies of mice with targeted mutation (2). Since Notch activation within the endothelium also disrupts vascular remodeling, proper Notch signaling is essential for vascular development (3). Although relevance of Notch to VEGF signaling is suggested (4-6), it is still unclear how Notch signaling has a role in VEGF-regulated angiogenesis and whether Notch signaling participates in physiological and pathological angiogenesis in the adult vasculature.
[0603] HUVEC (Human Umbilical Vein Endothelial cells) growth are dependent on VEGF (FIGS. 26A and 26B) and differentiation-related biological responses, such as sprouting, and can be evaluated at an early stage (7). At first, we examined whether adenovirally transduced VEGF induced both Notch and Notch ligand expression in HUVEC cultured with complete medium containing bFGF (FIG. 22A), as reported (5). RT-PCR analysis showed that both D14 and Notch4 mRNA was up-regulated in adenovirally-transduced VEGF HUVEC (Ad-VEGF-HUVEC), compared to adenovirally-transduced LacZ HUVEC (Ad-LacZ-HUVEC) (FIG. 22A). Transduced VEGF did not appear to induce Jagged1 and Notch1 expression. Transduced-VEGF also activated Notch signaling in a dose-dependent manner by measuring CSL-luciferase reporter activity (FIG. 22B), which was transactivated with Notch signaling (8). Notch signaling was activated at a higher dosage of Ad-VEGF, compared to proliferation (FIG. 26A). Since SU5416, which is an inhibitor of VEGFR kinases, decreased VEGF-induced CSL-luciferase reporter activity (FIG. 22C), VEGF induced Notch signaling through activation of receptor kinase. Since Notch mutants lacking both transmembrane and cytoplasmic domains functioned as dominant negative inhibitors against Notch signaling (9), we made a Notch-based fusion protein or decoy (N1ECDFc) to inhibit Notch signaling (FIG. 22D). Western blotting analysis of conditioned medium of Ad-N1ECDFc-transduced HUVEC (Ad-N1ECDFc-HUVEC) demonstrated that N1ECDFc was expressed and secreted well (FIG. 22E). By using a co-culture assay, in which Bosc cells expressing Notch ligands (either J1, Dll or D14) activated Notch signaling in HeLa cells expressing Notch1 compared to control Bosc cells, we determined inhibition of Notch signaling with transfection of a N1ECDFc-expression plasmid (FIG. 22F). Then, we examined whether N1ECDFc inhibited activation of Notch signaling by transduced VEGF in HUVEC (FIG. 22G). Co-transduction of Ad-N1ECDFc with Ad-VEGF into HUVEC clearly decreased CSL luciferase activity induced by VEGF. Gerhardt et al. reported that VEGF controlled angiogenesis in the early postnatal retina by guiding filopodia extension at the tips of the vascular sprouts (10). During angiogenic sprouting, the formation of a specialized endothelial cell making filopodia projections among quiescent endothelial cells, might be one of the early events. Here we mean formation of a single endothelial cell making filopodia protrusions as budding. Budding of the primary endothelial cells is induced by cultivating them 3-dimensionally on either fibrin or collagen gel (11). In the case where Ad-VEGF-HUVEC were cultured on collagen gel with complete medium, transduced-HUVEC made filopodia extensions into the collagen gel for 5 days (FIG. 22H) and the number of buds was increased in a dose-dependent manner (FIG. 27A). Activation of Notch signaling by adenovirus encoding the activated form of Notch4 (Ad-Notch4/int3) induced HUVEC budding (12) and that of Notch1 (Ad-N1IC) also induced HUVEC budding (FIGS. 23A & 27B). Since both VEGF and Notch signaling induce HUVEC budding, we examined whether N1ECDFc inhibited VEGF-induced HUVEC budding (FIG. 22H-I). Budding of Ad-VEGF-HUVEC was clearly inhibited by co-transduction of Ad-N1ECDFc. Neither Ad-LacZ or Ad-N1ECDFc-transduced HUVEC formed buds (FIG. 22H). N1ECDFc inhibited VEGF-induced HUVEC budding without affecting cell number (FIG. 22I). Transduced-N1ECDFc did not clearly alter proliferation of HUVEC, while that of Ad-N1IC-transduced HUVEC was inhibited in a dose-dependent manner (FIG. 28A), consistent with the inhibitory efficacy of Notch signaling against endothelial proliferation (13).
[0604] To test whether Notch signaling is down-stream of VEGF, we evaluated three distinct inhibitors for receptor tyrosine kinases, including VEGFR on N1IC-induced HUVEC budding, because three growth factors existed in complete medium (FIG. 23A-C). At a concentration of 1 each compound showed selective inhibition against each kinase (data not shown). Neither PD166866 or ZD1893 affected budding of Ad-N1IC-HUVEC, while SU5416 clearly inhibited it (FIG. 23A-B). SU5416 selectively inhibited budding of Ad-N1IC-HUVEC with less reduction of viability at lower concentrations (FIG. 23C). Since Taylor et al. reported that Notch down-regulated Flk1/KDR/VEGFR2 expression (14), it was unlikely that Notch co-operated with Flk1 to promote budding. Thus, we examined whether activation of Notch signaling affected Flt1/VEGFR1 expression in HUVEC, because SU5416 inhibits both Flt1 and Flk1 kinase activity (15). RT-PCR analysis demonstrated that expression of Flt1 mRNA was up-regulated in Ad-N1IC-HUVEC, while expression of endothelial cell maker, CD31 mRNA, was not compared to that in Ad-LacZ-HUVEC (FIG. 23D). Western blotting analysis also showed that expression of Flt1 protein was up-regulated in Ad-N1IC-HUVEC (FIG. 23E). Thus, we examined whether P1GF, which is a selective ligand for Flt1, promoted budding of HUVEC in which Flt1 was up-regulated via activation of Notch signaling (FIG. 23F-G). P1GF increased the number of Ad-N1IC-HUVEC buds by 150%, compared to the absence of P1GF (FIG. 23F). Moreover, P1GF increased HUVEC buds containing multiple filopodia by 250% (FIG. 23G). While reduction of Flt1 expression using small interfering RNA (siRNA) for Flt1 inhibited budding of Ad-N1IC-HUVEC (FIG. 23J), transfection of which selectively decreased expression of Flt1 mRNA (FIG. 23H) and that of Flt1 protein (FIG. 23I). Although reduction of Flk1 expression with Flk1 siRNA also inhibited budding of Ad-N1IC-HUVEC (FIG. 30B), the inhibitory efficacy of Flk1 siRNA was less than that of Flt1 siRNA (FIG. 23J). Effects of Flk1 siRNA were more effective on budding of Ad-VEGF-HUVEC than that of Ad-N1IC-HUVEC (FIG. 30B-C). Transfection with Flt1 siRNA inhibited budding of both Ad-N1IC- and Ad-VEGF-HUVEC to a similar extent (data not shown).
[0605] Several studies demonstrated that VEGF regulated gelatinase activities in endothelial cells and the significance of gelatinase activity like MMP-2 and MMP-9 has been firmly established to induce angiogenic sprouting (16). We examined whether VEGF regulated gelatinase activity via Notch signaling in HUVEC.
[0606] In Gelatin zymography, conditioned medium of Ad-VEGF-HUVEC showed both induction and activation of MMP9, which started to be detected at day 6 (FIG. 24A) and activation of MMP2, which was detected at day 4 (FIG. 24B), compared to those of Ad-LacZ-HUVEC. Co-transduction of Ad-N1ECDFc with Ad-VEGF showed inhibition of both induction and activation of MMP9 (FIG. 24A) and an activation of MMP2 (FIG. 24B). RT-PCR analysis demonstrated that expression of MMP9 mRNA was up-regulated in Ad-N1IC-HUVEC, but expression of MMP2 mRNA was decreased in Ad-N1IC-HUVEC (FIG. 24C). Since induction of MMP2 activity was not detected in gelatin zymography (FIG. 24B), this result was a likely consequence. While expression of MT1-MMP, which is able to activate MMP2 at the cell surface (17), was up-regulated at both the transcript and protein levels in Ad-N1IC-HUVEC (FIG. 24D). As VEGF can regulate both gelatinase and MT1-MMP expression (16), RT-PCR analysis demonstrated that both MMP9 and MT1-MMP were up-regulated in Ad-VEGF-HUVEC, compared to Ad-LacZ-HUVEC and this induction was inhibited with co-transduction of Ad-N1ECDFc (FIG. 24E). Ad-N1ECDFc infection alone did not affect expression of either MMP9 or MT1-MMP in Ad-LacZ infected HUVEC (data not shown). Requisition of MMPs for angiogenic sprouting has been established by synthetic MMP inhibitors (16). GM6001 is one broad inhibitor against MMPs including MMP2, MMP9 and MT1-MMP (18). GM6001 clearly decreased budding of Ad-N1IC-HUVEC on both collagen (FIG. 31A-B) and fibrin gel (data not shown).
[0607] In the mouse Dorsa Air Sac (DAS) assay (19), stable transfectant of 293 cells over-expressing VEGF121 (293/VEGF) significantly induced in vivo angiogenesis (FIG. 25A, left panel). This VEGF-induced angiogenesis was clearly inhibited by coexpression of N1ECDFc, compared to 293/VEGF alone (FIG. 25A). Vessel density was measured and an index of angiogenesis given in FIG. 25B, demonstrating the 293/VEGF induced angiogenesis is inhibited by co-expression of 293/N1ECDFc (FIG. 25B).
[0608] Also, in the mouse Dorsa Air Sac (DAS) assay (19), the human breast cancer cell line, MDA-MB-231 significantly induced in vivo angiogenesis, presumably via the secretion of VEGF (FIG. 25C, left panel). This VEGF-induced angiogenesis was clearly inhibited by adenovirus mediated expression of N1ECDFc, compared to adenovirus expressing LacZ. (FIG. 25C). Vessel density was measured and an index of angiogenesis given in FIG. 25D, demonstrating the MDA-MB-231 induced angiogenesis is inhibited by expression of N1ECDFc.
[0609] Flk1 is a major positive signal transducer for angiogenesis through its strong tyrosine kinase activity in the embryo, while Flt1 is thought to be a negative signal transducer for angiogenesis. However, a positive role for Flt-1 was demonstrated in adult mice, as in vivo growth of LLC over-expressing P1GF2 was severely compromised in mice lacking the cytoplasmic Flt-1 kinase domain (20). Notch might function to alter VEGF signaling by inducing Flt-1 signaling and moderate Flk-1 signaling either to induce filopodia extension or potentiate angiogenic sprouting, since P1GF/Flt-1 signaling altered the phospholyration site of Flk-1 and potentiated ischemic myocardial angiogenesis (21). Interestingly, Notch signaling also up-regulated P1GF expression (FIG. 29). However, continuous activation of Notch signaling inhibits formation of multi-cellular lumen-containing angiogenic sprouts; as previously reported (22). Notch signaling should be turned off after budding/filopodia formation and transient activation of the Notch pathway might be required. In a transgenic mouse model of pancreatic beta-cell carcinogenesis (Rip1Tag2 mice) in which tumor angiogenesis is VEGF dependent, the level of VEGF expression is not increased, but mobilization of extracellular VEGF stored in the matrix to VEGF receptors occurs. MMP-9 is responsible for this mobilization and tumor progression was inhibited in Rip1Tag23MMP-9-null double-transgenic mice (23). Notch up-regulated MMP-9 expression and might increase local VEGF level at the site for angiogenic sprouting. While Notch also up-regulates MT1-MMP expression, extracellular MMP-2 might be targeted to the cell membrane of Notch-activated endothelial cells. Notch might determine the site for angiogenic sprouting by regulating gelatinase activity and VEGF concentration. Since endothelial MMP-9 was regulated by Flt-1 in lung specific metastasis (20), Flt-1 might participate in induction of MMP-9 indirectly.
REFERENCES CITED IN THIRD SERIES OF EXPERIMENTS
[0610] 1. Artavanis-Tsakonas S, Rand M D, Lake R J. Notch Signaling: Cell Fate Control and Signal Integration in Development. Science 1999; 284(5415):770-776.
[0611] 2. Shawber C J, J. K. Notch function in the vasculature: insights from zebrafish, mouse and man. Bioessays. 2004; 26(3):225-34.
[0612] 3. Uyttendaele H, Ho J, Rossant J, J. K. Vascular patterning defects associated with expression of activated Notch4 in embryonic endothelium. Proc Natl Acad Sci USA. 2001; 98(10):5643-8.
[0613] 4. Lawson N D, Vogel A M, B M. W. sonic hedgehog and vascular endothelial growth factor act upstream of the Notch pathway during arterial endothelial differentiation. Dev Cell 2002; 3(1):127-36.
[0614] 5. Liu Z J, Shirakawa T, Li Y, Soma A, Oka M, Dotto G P, et al. Regulation of Notch1 and Dll4 by vascular endothelial growth factor in arterial endothelial cells: implications for modulating arteriogenesis and angiogenesis. Mol Cell Biol. 2003; 23(1):14-25.
[0615] 6. Gale N W, Dominguez M G, Noguera I, Pan L, Hughes V, Valenzuela D M, et al. Haploinsufficiency of delta-like 4 ligand results in embryonic lethality due to major defects in arterial and vascular development. Proc Natl Acad Sci USA. 2004; 101(45):5949-54.
[0616] 7. Montesano R, L. O. Phorbol esters induce angiogenesis in vitro from large-vessel endothelial cells. J Cell Physiol. 1987; 130(2):284-91.
[0617] 8. Jarriault S, Brou C, Logeat F, Schroeter E H, Kopan R, A. I. Signalling downstream of activated mammalian Notch. Nature. 1995; 377(6547):355-8.
[0618] 9. Small D, Kovalenko D, Kacer D, Liaw L, Landriscina M, Di Serio C, et al. Soluble Jagged 1 represses the function of its transmembrane form to induce the formation of the Src-dependent chord-like phenotype. J Biol Chem 2001; 276(34):32022-30.
[0619] 10. Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A, Abramsson A, et al. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol 2003; 161(6):1163-77.
[0620] 11. Koolwijk P, van Erck M G, de Vree W J, Vermeer M A, Weich H A, Hanemaaijer R, et al. Cooperative effect of TNFalpha, bFGF, and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix. Role of urokinase activity. J Cell Biol 1996; 132(6):1177-88.
[0621] 12. Das I, Craig C, Funahashi Y, Jung K M, Kim T W, Byers R, et al. Notch oncoproteins depend on gamma-secretase/presenilin activity for processing and function. J Biol Chem 2004; 279(29):30771-80.
[0622] 13. Noseda M, Chang L, McLean G, Grim J E, Clurman B E, Smith L L, et al. Notch activation induces endothelial cell cycle arrest and participates in contact inhibition: role of p21Cip1 repression. Mol Cell Biol 2004; 24(20):8813-22.
[0623] 14. Taylor K L, Henderson A M, C C. H. Notch activation during endothelial cell network formation in vitro targets the basic HLH transcription factor HESR-1 and downregulates VEGFR-2/KDR expression. Microvasc Res 2002; 64(3):372-83.
[0624] 15. Itokawa T, Nokihara H, Nishioka Y, Sone S, Iwamoto Y, Yamada Y, et al. Antiangiogenic effect by SU5416 is partly attributable to inhibition of Flt-1 receptor signaling. Mol Cancer Ther 2002; 1(5):295-302.
[0625] 16. Pepper M S. Role of the matrix metalloproteinase and plasminogen activator-plasmin systems in angiogenesis. Arterioscler Thromb Vasc Biol 2001; 21(7):1104-17.
[0626] 17. Seiki M, Koshikawa N, I. Y. Role of pericellular proteolysis by membrane-type 1 matrix metalloproteinase in cancer invasion and angiogenesis. Cancer Metastasis Rev 2003; 22(2-3):129-43.
[0627] 18. Yamamoto M, Tsujishita H, Hori N, Ohishi Y, Inoue S, Ikeda S, et al. Inhibition of membrane-type 1 matrix metalloproteinase by hydroxamate inhibitors: an examination of the subsite pocket. J Med Chem 1998; 41(8):1209-17.
[0628] 19. Funahashi Y, Wakabayashi T, Semba T, Sonoda J, Kitoh K, K. Y. Establishment of a quantitative mouse dorsal air sac model and its application to evaluate a new angiogenesis inhibitor. Oncol Res. 1999; 11(7):319-29.
[0629] 20. Hiratsuka S, Nakamura K, Iwai S, Murakami M, Itoh T, Kijima H, et al. MMP9 induction by vascular endothelial growth factor receptor-1 is involved in lung-specific metastasis. Cancer Cell 2002; 2(4):289-300.
[0630] 21. Autiero M, Waltenberger J, Communi D, Kranz A, Moons L, Lambrechts D, et al. Role of P1GF in the intra- and intermolecular cross talk between the VEGF receptors Flt1 and Flk1. Nat Med 2003; 9(7):936-43.
[0631] 22. Leong K G, Hu X L L, Noseda M, Larrivee B, Hull C, Hood L, et al. Activated Notch4 inhibits angiogenesis: role of beta 1-integrin activation. Mol Cell Biol 2002; 22(8):2830-41.
[0632] 23. Bergers G, Brekken R, McMahon G, Vu T H, Itoh T, Tamaki K, et al. Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat Cell Biol 2000; 2(10):737-44.
Fourth Series of Experiments
A Novel Construct Derived from the Notch1 Ectodomain Inhibits Notch Signaling, Endothelial Morphogenesis, and Tumor Angiogenesis
[0633] Notch signaling is required for vascular development, but also functions in tumor angiogenesis. Inhibition of vascular endothelial growth factor (VEGF) is a validated anti-angiogenic therapy, and VEGF can induce both Notch and Notch ligand Delta-like 4 (Dll4) expression in endothelial cells (EC). Although Dll4 inhibition can restrict tumor growth and disrupt neovasculature, the effect of inhibiting Notch receptor function on tumor angiogenesis has yet to be defined. In this study, we generated a soluble fusion protein of the Notch1 receptor (N1ECDFc, or Notch1 decoy) to block this pathway, and assessed its effect on angiogenesis in vitro and in vivo. Notch1 decoy expression reduced signaling stimulated by the binding of three distinct Notch ligands to Notch1, and also inhibited morphogenesis of EC overexpressing Notch4. We tested the effects of Notch1 decoy expression on tumor angiogenesis using two models: mouse mammary tumor Mm5MT cells overexpressing fibroblast growth factor 4 (Mm5MT-FGF4), and NGP human neuroblastoma cells. Exogenously expressed FGF4 induced the expression of Notch ligands Jagged1 and Delta-like 1 (Dll1) in Mm5MT-FGF4 cells, and Jagged1 was expressed in Mm5MT-FGF4 xenografts. Overexpression of Notch1 decoy did not affect tumorigenicity of Mm5MT-FGF4 cells in vitro, but restricted Mm5MT-FGF4 xenograft growth in mice, while markedly impairing neoangiogenesis. Similarly, Notch1 decoy expression did not affect NGP cells in vitro but disrupted vessels and decreased tumor viability in NGP xenografts. These results strongly suggest that Notch receptor signaling is required for tumor neoangiogenesis, and provides a new target for tumor therapy.
[0634] Angiogenesis is exquisitely regulated by multiple signal pathways, including VEGFs, fibroblast growth factors (FGFs), and hepatocyte growth factor (HGF). Among these, VEGF critically influences almost all steps of angiogenesis, including endothelial proliferation, survival, and tube formation (1). Consistent with this protean role, VEGF inhibitors reduce angiogenesis in preclinical models, and have been clinically validated as cancer therapy (2). Despite this established efficacy, different tumor types exhibit widely varying susceptibility to VEGF blockade (2). The underlying reasons for this variability are not clear. One possibility is that alternative signals rescue tumor vasculature, allowing for perfusion despite VEGF inhibition. Identification of such pathways is therefore of clear therapeutic importance.
[0635] The highly conserved Notch gene family encodes transmembrane receptors (Notch1, -2, -3, -4) and ligands (Jagged1, -2; Delta-like or Dll1, -3, -4), also transmembrane proteins. Upon ligand binding, the Notch cytoplasmic domain (NotchIC) is released by presenilin/γ-secretase (3). Notch signaling defects produce severe vascular defects in embryos (4), with haploinsufficiency of Dll4 causing lethality. The potential role of Notch signaling in tumor angiogenesis has thus excited much recent interest. Mice transgenic for a Dll4-reporter construct demonstrate expression in tumor endothelial cells (EC) (5), and increased Dll4 expression has been detected in human cancers (6, 7). Two recent reports confirm that this role is critical by demonstrating that Dll4 blockade suppresses growth and perfusion in experimental tumors (8, 9). Intriguingly, in these studies Dll4 inhibition disorganized tumor vasculature rather than simply preventing vessel proliferation, suggesting that Dll4 is required for functional vessel assembly.
[0636] Recent data indicate that Notch receptors also function in tumor vasculature. For example, in head and neck squamous cell carcinoma (HNSCC) HGF was recently shown to up-regulate expression of Jagged1 on tumor cells, but not on endothelium. Increased Jagged1 expression activated Notch signaling in neighboring EC, stimulating tumor angiogenesis and growth in mice (10). Thus, these data suggest that there are at least two distinct mechanisms for activating Notch signaling in tumor EC.
[0637] In these studies, we evaluated the role of Notch receptor activation in angiogenesis using a novel soluble construct based on the extracellular domain of Notch1 (N1ECDFc, or Notch1 decoy). In vitro, Notch1 decoy inhibited both ligand-induced activation of Notch signaling and morphogenesis of EC adenovirally over-expressing Notch4. In vivo, Notch1 decoy expression delayed growth of murine Mm5MT xenografts in which Jagged1 expression was up-regulated by transduction of FGF4, and disrupted vasculature and tumor viability in NGP neuroblastoma tumors. These data support a requirement for Notch receptor function during tumor neoangiogenesis, and suggest that inhibition of this pathway may provide an effective new anti-tumor strategy.
[0638] Materials and Methods
[0639] Reagents and Expression Vectors
[0640] Compound E was purchased from Calbiochem (San Diego, Calif.), and PD166866 from Eisai (Tokyo, Japan). Notch1 decoy (N1ECDFc) encodes the rat Notch1 ectodomain (bp 241-4229, Genbank accession #X57405) fused in frame to human IgG Fc. Retroviral pHyTC-Jagged1, -Dll1, -Dll4, and pBos-Notch1 have been described (11). Notch1 decoy and Fc were engineered into retroviral vector pHyTCX, and mouse FGF4 engineered into pQNCX. Adenoviral constructs encoding LacZ and mouse Notch4 and pAdlox-GFP have been described (12)(13).
[0641] HUVECs, Adenoviral, and Retroviral Infections
[0642] HUVECs were isolated as described (14) and mouse mammary carcinoma Mm5MT obtained (ATCC, Manassas, Va.). We used adenovirus at indicated multiplicity of infection (m.o.i.) and retroviral supernatants from GP2-293 cells (BD Biosciences, Bedford, Mass.) for infection. HUVECs were selected using 304 μg/ml hygromycinB (Invitrogen, Carlsbad, Calif.) and Mm5MT-FGF4 selected in 1 mg/ml G418 (Gibco-Invitrogen, Grand Island, N.Y.), with double transfectants in 300 μg/ml hygromycinB.
[0643] Western Blotting
[0644] Ad-N1ECDFc-transduced HUVEC were cultured in endothelium serum-free medium (GIBCO-Invitrogen) 48 h, and Mm5MT-FGF4 transfectants in DMEM. Western blots were performed using anti-human Fc (Pierce, Rockford, Ill.).
[0645] Quantitative RT-PCR
[0646] Mm5MT transfectants were cultured 7 days with vehicle or 1 μM PD166866 (inhibitor of FGF receptor-kinase), total RNA isolated (RNeasy mini-kit, Qiagen, Valencia, Calif.), and first-strand cDNA synthesized (SuperScript® First-Strand Synthesis System, Invitrogen). Quantitative RT-PCR for β-actin, FGF4, Jagged1, Dll1, and Dll4 (SYBER Green PCR Master Mix, 7300 Real Time PCR; Applied Biosystems, Foster City, Calif.) was performed in triplicate and values normalized for β-actin. Values are shown for fold induction compared to controls (primer sequences available on request).
[0647] Co-Culture Signaling Assay
[0648] Notch1 decoy inhibition of ligand-induced signaling was performed as described (11). HeLa cells were transfected with 333 ng pBOS-Notch1, 333 ng pGA981-6, and 83 ng pLNC-LacZ with either 666 ng pCMV-Fc or pHyTC-N1ECDFc (333 ng for x1, 666 ng for x2). 293 cells were transfected with 680 ng pHyTc-Jagged1, pHyTc-Dll1, pHyTc-Dll4, or pHyTc-X (empty vector). Cells were harvested, luciferase activity determined 48 h post-transfection (Enhanced Luciferase assay kit, BD PharMingen, San Diego, Calif.), and β-galactosidase activity determined (Galacto-Light Plus kit, Applied Biosystems). Assays were performed in triplicate.
[0649] Endothelial Co-Culture Morphogenesis Assay
[0650] HUVEC morphogenesis was assessed as described (11), modified by adding co-culturing of Ad-Notch4 transduced HUVEC with Notch1 decoy- or Fc-HUVEC transfectants. Ad-GFP at 10 m.o.i. was co-transduced in HUVECs with Ad-LacZ or Ad-Notch4 at 30 m.o.i and 48 h later seeded on fibrin gels (24-well plates, 1.5×104 cells/well). Stable HUVEC-mock (HUVEC-X), HUVEC-Fc, or HUVEC-N1ECDFc transfectants were seeded at 1.35×105 cells/well, and vehicle or 200 nM compound E added 3 h later. Seven days later, HUVEC morphogenesis was calculated as the number of GFP-positive cells with processes compared to total GFP-positive cells/field.
[0651] Mm5MT Tumor Model
[0652] 6-8 week-old female C3H mice (Taconic, Hudson, N.Y.) underwent subcutaneous implantation of 106 Mm5MT transfectants (N=10 each). Tumor diameters were measured with calipers, and volume calculated (length (mm)×width (mm)2×1/2). Tumors were harvested at day 22 and analyzed. Experiments were performed thrice.
[0653] Immunohistochemistry
[0654] 5 μm fresh-frozen Mm5MT tissue sections were immunostained (15) (see supplemental data for antibody list). CD31 quantitation was performed using an Eclipse E800 microscope and ImagePro Plus v.4.01 (Silver spring, Md.). 20 different fields/slide were measured, and density ratios calculated as (area of specific staining)/(total area, each field). Data is shown as the ratio of the mean of average density ratios of each Mm5MT transfectant to Mm5MT mock-transfectant.
[0655] NGP Tumor Model
[0656] The NGP tumor model has previously been described in detail (16). NGP cells were transfected with LacZ or N1ECDFc, as above, and 106 NGP-LacZ or NGP-N1ECDFc cells implanted intrarenally in 4-6 week old NCR nude mice (Taconic, Germantown, N.Y.; NGP-LacZ n=11, NGP-N1ECDFc n=13). At 6 weeks, tumors were harvested for analysis. 5 uM paraffin-embedded sections were immunostained for CD-31/PECAM and α-smooth muscle actin (αSMA). To detect apoptosis (TUNEL assay) we used the Apoptag Red in situ Kit (Chemicon). Signal was quantified by photographing 20-23 randomly selected fields of each tissue, excluding areas of normal kidney. Each frame was photographed in both red (TUNEL signal) and green channels. Using Adobe Photoshop, green channel signals were subtracted, in order to eliminate erythrocyte autofluorescence. A uniform red-channel threshold was arbitrarily selected, and total signal area measured in 4 NGP-N1ECDFc and 3 NGP-LacZ tumors. Erythrocyte quantification was performed similarly.
[0657] Statistical Analysis
[0658] Significance in quantitative studies was assessed using Tukey-Kramer tests (CD31 quantitation) and Kruskal-Wallis analysis (all others).
[0659] Results
[0660] Notch1 Decoy Inhibits Ligand-Induced Notch Signaling in Cells Expressing Notch1
[0661] Notch1 decoy is based on the ectodomain of Notch1 fused to human IgG Fc and is secreted, as determined by blotting of media conditioned by adenovirus Notch1 decoy (Ad-N1ECDFc)-infected HUVEC (FIG. 32A). We assessed Notch1 decoy activity using co-culture signaling assays (11). 293 cells expressing Notch ligands (Jagged1, Dll1, Dll4) activated Notch signaling when cultured with HeLa cells expressing Notch1, measured by CSL-luciferase reporter activity (FIG. 32B). Expression of Notch1 decoy in either HeLa (FIG. 32B) or 293 cells (data not shown) blocked Notch1 signaling in co-culture assays, indicating that Notch1 decoy prevented activation by Jagged1, Dll1, or Dll4.
[0662] Notch1 Decoy Blocks Morphogenesis of HUVEC Induced by Notch4
[0663] Notch4 expression induced cellular extensions from HUVECs cultured on fibrin gels (FIG. 32C), resembling morphologic changes induced by VEGF and FGF2 (17, 18). Fibrin can induce Jagged1 in EC (19). We tested the hypothesis that such extensions reflect endogenous Notch ligand activation of Notch4 transduced in HUVECs using either Compound E (CE), a (-secretase inhibitor (GSI), or Notch1 decoy. Compared to vehicle, treatment with 200 nM CE clearly inhibited extensions in Notch4-HUVECs, (FIG. 32C upper panels, 32D). Reduction in sprouting was significant (FIG. 32D, p<0.0001 for both compound E treatment and N1ECDFc transduction; data shown as mean±SD). Notch4+Notch1 decoy co-expression in HUVECs similarly blocked endothelial extensions relative to Notch4+Fc control (FIG. 32C, lower panels; 32D). Collectively, these data indicate that Notch receptor activation is both necessary and sufficient to induce HUVEC extensions in this assay, and that the Notch1 decoy functions similarly to GSI, further validating its activity as a Notch receptor inhibitor.
[0664] FGF4 Induced the Expression of Notch Ligands, Jagged1 and Dill, in Mouse Mammary Tumor Mm5MT Cells
[0665] Overexpression of FGF4 in Mm5MT cells promoted tumorigenicity in clonogenic and xenograft assays (data not shown). Since HGF/MAPK signaling induced Jagged1 expression in HNSCC (10), we asked whether FGF4 would stimulate expression of Notch ligands in Mm5MT cells. We detected up-regulation of Jagged1 and Dll1 in Mm5MT-FGF4 transfectants using quantitative PCR (Dll4 expression was unaltered) (FIG. 33A). The FGFR-kinase inhibitor PD166866 suppressed induction of both Jagged1 and Dll1 in Mm5MT-FGF4 transfectants (FIG. 33C) indicating that FGF4-induced Jagged1 and Dll1 expression requires FGFR signaling. Immunoblotting confirmed up-regulation of Jagged1 protein in Mm5MT-FGF4 cells (FIG. 33B), and immunostaining demonstrated strikingly increased Jagged1 in Mm5MT-FGF4 tumors (FIG. 33D). In addition, Notch4 was detected in Mm5MT-FGF4 tumor endothelium (not shown).
[0666] Notch1 Decoy Expression Inhibited Angiogenesis and Growth of Mm5MT-FGF4 Tumors in Mice
[0667] We hypothesized that Mm5MT-FGF4 tumors expressing Jagged1 promote angiogenesis by signaling via endothelial Notch receptors. Thus, we evaluated the effect of Notch1 decoy expression on Mm5MT-FGF4 xenograft growth in mice. Tumorigenicity of Mm5MT-FGF4 stably overexpressing either Fc or Notch1 decoy was unaltered by clonogenic assay (data not shown). However, Mm5MT-FGF4-N1ECDFc xenograft growth was significantly delayed as compared to both Mm5MT-FGF4 mock- and Fc-transfectants, suggesting that Notch inhibition had impaired a critical element in tumorigenesis (FIG. 34A). Immunostaining for the endothelial marker CD31/PECAN demonstrated marked inhibition of angiogenesis in Mm5MT-FGF4-N1ECDFc tumors (FIG. 34B). Consistent with a requirement for Notch in vessel assembly, EC appeared as detached solitary cells or small clusters, with few organized vessels detected. Quantitative analysis of anti-CD31 staining demonstrated a 58% decrease in microvessel density in Notch1 decoy-expressing tumors (P<0.001 for both Mm5MT-FGF4-X and Mm5MT-FGF4-Fc versus Mm5MT-FGF4-N1ECDFc; data shown as mean±SD; FIG. 34C).
[0668] Notch1 Decoy Expression Disrupted Angiogenesis in Human NGP Neuroblastoma Xenografts
[0669] NGP xenografts in mice form a mature hierarchical vasculature that is comparatively resistant to VEGF blockade (16). To determine whether Notch receptor activation contributed to NGP angiogenesis, we transfected NGP cells with N1ECDFc, as above. Similar to results observed with Mm5MT-FGF4-N1ECDFc cells, growth of NGP-N1ECDFc cells in culture was unaffected by transfection (not shown). However, xenograft viability was strikingly impaired (FIG. 35A), with significantly increased tumor cell apoptosis (P=0.0002, TUNEL-positive cells in NGP-N1ECDFc vs. NGP-LacZ tumors, FIG. 35B). Intratumoral hemorrhage was significantly increased in NGP-N1ECDFc tumors, suggesting that vessels were physically disrupted (P<0.0001, FIG. 36C). Immunostaining for the vascular basement membrane component collagen IV indicated an overall decrease in vasculature, with diminished branching, although remaining collagen sleeves appeared smooth and intact (not shown). However, immunostaining for EC and vascular mural cells (VMC) (using anti-CD31 and anti-αSMA antibodies, respectively) demonstrated disorder of these normally contiguous cell layers. Individual vascular cells appeared irregular, and were erratically detached from one another, with loss of vessel continuity (FIG. 35D). Taken together, these results suggest that Notch1 decoy expression disrupted endothelial and VMC interactions in tumor vasculature, leading to instability, hemorrhage, and defective perfusion of tumor tissues.
DISCUSSION
[0670] Recent reports confirm the critical role of the Notch ligand Dll4 in angiogenesis, and demonstrate that Dll4 blockade can effectively repress tumor growth by disrupting vasculature (8, 9). In this study, we show that blockade of Notch receptor function using a novel construct derived from the Notch1 ectodomain also effectively reduces tumor perfusion, although its effects on vasculature are distinct. The Notch1 decoy inhibited signaling induced by the binding of ligands Jagged1, Dll1 and Dll4 to Notch1. Consistent with a role for Notch receptor activation in neoangiogenesis, overexpression of Notch4 induced endothelial cell extensions, which could be prevented by blocking Notch signaling with either Notch1 decoy or GSI. Although Notch1 decoy did not inhibit tumor cell growth in vitro, expression of Notch1 decoy inhibited growth and angiogenesis of Mm5MT-FGF4 xenografts, in which Jagged1 expression is up-regulated. Similarly, Notch1 decoy expression had no effect on NGP tumor cell proliferation in vitro, but disrupted tumor vessels and viability in vivo.
[0671] Notch4 overexpression in HUVECs was sufficient to induce endothelial extensions on fibrin gel without exogenous expression of Notch ligands. Since fibrin is known to induce Jagged1 expression in EC, and thus may have functioned to promote HUVEC expression of Jagged1 in this assay, we speculate that this caused activation of Notch4. In HeLa co-culture signaling assays, the Notch1 decoy inhibited signaling via ligand-Notch1 receptor interaction. We were unable to similarly evaluate Notch4 activity as Notch4 was poorly processed and presented in HeLa cells (not shown). However, processed Notch4 is found on HUVECs after adenoviral Notch4 transduction (not shown), indicating that Notch1 decoy can block ligand-induced Notch4 activation.
[0672] The multiple roles recently demonstrated for Notch signaling in tumorigenesis increase the attractiveness of this pathway as a potential target for cancer therapy. While Notch activation is likely to function directly in malignant transformation in human cancers (20, 21), it may also be required for angiogenesis (8, 9). Interestingly, Notch ligand induction can be regulated by growth factor signals. For example, Jagged1 is induced in tumor cells by HGF (10), and Dll4 induced in EC by VEGF (22). Here we show that FGF4 can similarly stimulate Jagged1 and Dll1 expression in murine Mm5MT cells. Notch1 decoy reduced Mm5MT-FGF4 tumor growth and angiogenesis in vivo but did not affect tumorigenicity in vitro. Thus, these results suggest that Notch receptor activation in Mm5MT vasculature rather than tumor cells is required for neoplastic growth in this system.
[0673] While both Mm5MT-FGF4 and NGP xenografts displayed striking disorder of tumor vasculature after Notch1 decoy expression, the differences in vascular phenotype observed in these models suggest that tumor-specific patterns of Notch function may fine-tune vessel assembly. Mm5MT-FGF4 tumors proliferate rapidly, and develop dense, erratic endothelial networks relatively devoid of recruited VMC. These immature vascular beds express Dll4 (data not shown) and are extensively ablated by Notch1 decoy expression, leaving small clusters or individual EC isolated in tumor parenchyma. Consistent with this initial failure to form vessel networks, decoy-expressing tumor remnants are not necrotic and are significantly smaller than controls. In contrast, NGP tumors develop a mature vascular plexus, with near-uniform coverage of endothelium by VMC. NGP vessels express Notch1 and relatively little Dll4 (not shown). Notch1 decoy expression in NGP tumors causes intratumoral hemorrhage and necrosis, with loss of vessel continuity, suggesting that perfused vessels become unstable after some degree of tumor growth has occurred.
[0674] Collectively, these data provide support for a model in which Notch signaling controls multiple aspects of tumor angiogenesis. While Notch activation is broadly required for neoangiogenesis, individual Notch proteins may differentially regulate vascular remodeling. Our results confirm the importance of Notch ligand-receptor interactions in tumor vasculature, and suggest that perturbing Notch receptor function may provide a novel and effective means of disrupting tumor angiogenesis.
REFERENCES FOR FOURTH SERIES OF EXPERIMENTS
[0675] 1. Ferrara, N. Vascular Endothelial Growth Factor: Basic Science and Clinical Progress. Endocr. Rev., 25: 581-611, 2004.
[0676] 2. Jain, R., Duda, D., Clark, J., and Loeffler, J. Lessons from phase III clinical trials on anti-VEGF therapy for cancer. Nat Clin Pract Oncol. 2006 January; 3(1):24-40, 3: 24-40, 2006.
[0677] 3. Kopan, R. Notch: a membrane-bound transcription factor. J Cell Sci., 115: 1095-1097, 2002.
[0678] 4. Shawber, C. and Kitajewski, J. Notch function in the vasculature: insights from zebrafish, mouse and man. Bioessays., 26: 225-234, 2004.
[0679] 5. Gale, N. W., Dominguez, M. G., Noguera, I., Pan, L., Hughes, V., Valenzuela, D. M., Murphy, A. J., Adams, N. C., Lin, H. C., Holash, J., Thurston, G., and Yancopoulos, G. D. Haploinsufficiency of delta-like 4 ligand results in embryonic lethality due to major defects in arterial and vascular development. Proc Natl Acad Sci 101: 15949-15954, 2004.
[0680] 6. Patel, N. S., Li, J.-L., Generali, D., Poulsom, R., Cranston, D. W., and Harris, A. L. Up-regulation of Delta-like 4 Ligand in Human Tumor Vasculature and the Role of Basal Expression in Endothelial Cell Function. Cancer Res., 65: 8690-8697, 2005.
[0681] 7. Patel, N. S., Dobbie, M. S., Rochester, M., Steers, G., Poulsom, R., Le Monnier, K., Cranston, D. W., Li, J.-L., and Harris, A. L. Up-Regulation of Endothelial Delta-like 4 Expression Correlates with Vessel Maturation in Bladder Cancer. Clin Cancer Res, 12: 4836-4844, 2006.
[0682] 8. Ridgway, J., Zhang, G., Wu, Y., Stawicki, S., Liang, W. C., Chanthery, Y., Kowalski, J., Watts, R. J., Callahan, C., Kasman, I., Singh, M., Chien, M., Tan, C., Hongo, J. A., de Sauvage, F., Plowman, G., and Yan, M. Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis. Nature, 444: 1083-1087, 2006.
[0683] 9. Noguera-Troise, I., Daly, C., Papadopoulos, N. J., Coetzee, S., Boland, P., Gale, N. W., Lin, H. C., Yancopoulos, G. D., and Thurston, G. Blockade of Dll4 inhibits tumour growth by promoting non-productive angiogenesis. Nature, 444: 1032-1037, 2006.
[0684] 10. Zeng, Q., Li, S., Chepeha, D. B., Giordano, T. J., Li, J., Zhang, H., Polverini, P. J., Nor, J., Kitajewski, J., and Wang, C.-Y. Crosstalk between tumor and endothelial cells promotes tumor angiogenesis by MAPK activation of Notch signaling. Cancer Cell, 8: 13-23, 2005.
[0685] 11. Das, I., Craig, C., Funahashi, Y., Jung, K.-M., Kim, T.-W., Byers, R., Weng, A. P., Kutok, J. L., Aster, J. C., and Kitajewski, J. Notch Oncoproteins Depend on {gamma}-Secretase/Presenilin Activity for Processing and Function. J Biol Chem., 279: 30771-30780, 2004.
[0686] 12. Shawber, C. J., Das, I., Francisco, E., and Kitajewski, J. A. N. Notch Signaling in Primary Endothelial Cells. Ann NY Acad Sci, 995: 162-170, 2003.
[0687] 13. Hardy, S., Kitamura, M., Harris-Stansil, T., Dai, Y., and Phipps, M. L. Construction of adenovirus vectors through Cre-lox recombination. J Virol., 71: 1842-1849, 1997.
[0688] 14. Jaffe, E., Nachman, R., Becker, C., and Minick, C. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest., 52: 2745-2756, 1973.
[0689] 15. Vorontchikhina, M. A., Zimmermann, R. C., Shawber, C. J., Tang, H., and Kitajewski, J. Unique patterns of Notch1, Notch4 and Jagged1 expression in ovarian vessels during folliculogenesis and corpus luteum formation. Gene Expression Patterns, 5: 701-709, 2005.
[0690] 16. Kim, E. S., Serur, A., Huang, J., Manley, C. A., McCrudden, K. W., Frischer, J. S., Soffer, S. Z., Ring, L., New, T., Zabski, S., Rudge, J. S., Holash, J., Yancopoulos, G. D., Kandel, J. J., and Yamashiro, D. J. Potent VEGF blockade causes regression of coopted vessels in a model of neuroblastoma. Proc Natl Acad Sci USA, 99: 11399-11404, 2002.
[0691] 17. Montesano R and L., O. Phorbol esters induce angiogenesis in vitro from large-vessel endothelial cells. J Cell Physiol., 130: 284-291, 1987.
[0692] 18. Koolwijk P, van Erck M G, de Vree W J, Vermeer M A, Weich H A, Hanemaaijer R, and V W., v. H. Cooperative effect of TNFalpha, bFGF, and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix. Role of urokinase activity. J Cell Biol, 132: 1177-1188, 1996.
[0693] 19. Zimrin, A. B., Pepper, M. S., McMahon, G. A., Nguyen, F., Montesano, R., and Maciag, T. An Antisense Oligonucleotide to the Notch Ligand Jagged Enhances Fibroblast Growth Factor-induced Angiogenesis in Vitro. J Biol Chem, 271: 32499-32502, 1996.
[0694] 20. Nickoloff, B., Osborne, B., and L., M. Notch signaling as a therapeutic target in cancer: a new approach to the development of cell fate modifying agents. Oncogene, 22: 6598-6608, 2003.
[0695] 21. Radtke, F. and Raj, K. THE ROLE OF NOTCH IN TUMORIGENESIS: ONCOGENE OR TUMOUR SUPPRESSOR? Nature Reviews Cancer, 3: 756-767, 2003.
[0696] 22. Liu, Z.-J., Shirakawa, T., Li, Y., Soma, A., Oka, M., Dotto, G. P., Fairman, R. M., Velazquez, O. C., and Herlyn, M. Regulation of Notch1 and Dll4 by Vascular Endothelial Growth Factor in Arterial Endothelial Cells: Implications for Modulating Arteriogenesis and Angiogenesis. Mol Cell Biol 23: 14-25, 2003.
Fifth Series of Experiments
Notch as a Therapeutic Target in Ovarian Cancer
[0697] Epithelial ovarian carcinoma is the leading cause of death from gynecological cancer in the U.S. Patients suffer as a result of our inability to diagnose the disease at an early stage, and from the lack of novel therapeutics directed against this tumor. Vascular endothelial growth factor (VEGF) is produced by malignant epithelial ovarian cancer cells, which, in turn, promotes tumor angiogenesis that nourishes the tumor [1]. Recent clinical trials demonstrate that targeting VEGF for therapeutic intervention can improve outcome, in some cases [2]. Thus, anti-angiogenic therapeutics have been validated as an approach to treat ovarian cancer, but more work needs to be done to build on these gains. We have identified the Notch signaling pathway as a novel angiogenic pathway in both physiological and pathological angiogenesis [3, 4]. Notch is a cell surface receptor (FIG. 1--Notch1) that promotes cell fate determination, cell survival and proliferation. Our lab has developed a Notch inhibitor, called the Notch decoy (schematized in FIG. 1), which is a fusion protein comprising the Notch1 extra-cellular domain fused to an Fc tag (N1ECDFc) which can inhibit ligand-dependent Notch signaling (data not shown). We have demonstrated that the Notch decoy can block VEGF-induced angiogenesis and tumor angiogenesis in a Wilms Tumor mouse model (data not shown). We generated human ovarian cancer cells SKOV3 that over-express the Notch decoy, SKOV3-N1ECDFc. We found that expression of Notch decoy in SKOV3 cell significantly inhibited the growth of tumor when xenografted into mice (FIG. 36). Thus, the Notch decoy blocks ovarian cancer growth. We hypothesize that this inhibition is targeting tumor angiogenesis. Further, we have surveyed human ovarian cancer samples and have found that several Notch ligands and Notch receptors are highly expressed in tumor vessels associated with ovarian cancers, thus supporting the hypothesis that one can target Notch in human ovarian tumor vessels and ultimately block their growth.
[0698] This study will test the hypothesis that Notch signaling promotes tumor angiogenesis in ovarian cancer. In addition, we hypothesize that inhibition of Notch, utilizing a "Notch decoy," will block tumor growth. We will determine if Notch decoy action is against tumor vessels or growth of tumor cells or both. The overall goal is to establish Notch decoy as a therapeutic for treatment of ovarian cancer.
[0699] Specific Aims:
[0700] Specific Aim 1: Determine if Notch decoy expression blocks tumor xenograft growth of SKOV3 or OVCAR3 cells and define whether Notch decoy is targeting tumor angiogenesis.
[0701] Specific Aim 2: Modify the Notch decoy design to make versions with increased efficacy/stability.
[0702] Specific Aim 3: Use purified Notch decoys to block ovarian cancer tumor xenografts in mice, also in combination with standard chemotherapeutic agents.
[0703] Research Strategy:
[0704] Specific Aim 1:
[0705] Determine if Notch decoy blocks tumor xenograft growth of SKOV3 or OVCAR3 cells and define whether Notch decoy is targeting tumor angiogenesis.
[0706] Our preliminary results demonstrate that SKOV3 tumor cells programmed expressing the Notch1 decoy block the growth of ovarian cancer xenografts (FIG. 36). This finding will be replicated in another ovarian cancer cell line, OVCAR3. The Notch decoy may inhibit growth of tumor cells, tumor vessels or both. To determine if Notch decoy targets tumor cells, we will determine if ovarian cancer lines expressing Notch decoy grow more poorly in soft-agar assays. To determine if the decoy is targeting tumor angiogenesis, we will evaluate xenografted tumors for evidence of such inhibition. Measures of tumor angiogenesis for control xenografts will include microvessel density assessment, proliferative index of tumor endothelial cells, and imaging tumor vasculature via lectin perfusion. Measures of inhibition of tumor angiogenesis in Notch decoy expressing xenografts will include assessment of apoptotic endothelial cells, reduction of tumor microvasculature and reduced ascites accumulation.
[0707] Specific Aim 2:
[0708] Modify the Notch decoy design to make versions with increased efficacy/stability.
[0709] The current Notch1 decoy, although effective as a Notch blocking agent, may not be readily amenable to purification, as it is a relatively large protein. We propose to develop variants of Notch1 decoy that encompass smaller parts of the extracellular domain. These variants will be screened as inhibitors of Notch signaling in cell culture-based assays. Inhibitory variants will also be screened for: their efficiency of secretion from producing cells, their stability once secreted, their solubility, and the ease of purification via the Fc affinity tag. Purified variants will be injected into mice to evaluate potential toxicity.
[0710] Specific Aim 3:
[0711] Use purified Notch decoys to block ovarian cancer tumor xenografts in mice, also in combination with standard chemotherapeutic agents.
[0712] The purified decoys, from specific Aim 2, will be employed, either as single agents, or in combination with chemotherapy, to evaluate their efficacy against ovarian cancer xenografts (SKOV3/OVCAR3). Using the purified Notch decoy as a single agent, we will attempt to replicate the block to tumor xenograft growth seen in experiments of specific Aim 1 and determine the optimal concentration for inhibition. To accomplish this end, we will determine the optimal dose, dosing schedule, and duration of treatment needed to block SKOV3/OVCAR3 xenograft growth in mice. Next, we will use the Notch decoy in combination with paclitaxol. An optimal dose of Notch decoy will be used with or without paclitaxol to determine efficacy in blocking xenograft growth.
[0713] Impact:
[0714] This study is aimed at directly advancing the treatment of ovarian cancer. Clinical studies have recently established the success of treating ovarian cancers with VEGF blocking agents. Few new therapeutic agents have been developed to supplement chemotherapeutic treatment. Despite this success, targeting of VEGF to block tumor growth may only limit tumor growth to an extent. It may also lead to new tumor vessels growing that are resistant to VEGF blockade. We propose to target an alternative tumor angiogenic pathway implicated in ovarian cancer, the Notch signaling pathway. This study will advance treatment by developing a novel therapeutic entity, the Notch decoy. Our preliminary studies already establish that Notch decoy can block ovarian cancer growth in a mouse model. Thus, the impact of development of the Notch decoy as a therapeutic entity may be highly significant.
[0715] Innovation:
[0716] Our recent development of the Notch decoy presents a novel and previously untested paradigm to treat ovarian cancer. No other published study has yet implicated this pathway in the treatment of ovarian cancer, yet our preliminary evidence defines this as a key area for exploration. In addition, despite the recent success of VEGF-blockade as a new therapeutic approach for treatment of ovarian cancer, we feel that this approach may need to be supplemented by inhibition of other angiogenic pathways, such as Notch.
REFERENCES FOR FIFTH SERIES OF EXPERIMENTS
[0717] 1. Abu-Jawdeh, G. M., et al., Strong expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in ovarian borderline and malignant neoplasms. Lab Invest, 1996. 74(6): p. 1105-15.
[0718] 2. Monk, B. J., et al., Activity of bevacizumab (rhuMAB VEGF) in advanced refractory epithelial ovarian cancer. Gynecol Oncol, 2005. 96(3): p. 902-5.
[0719] 3. Shawber, C., J. J. Kandel, and J. Kitajewski, Notch: cell fate determination from vascular development to human vasculopathy. Drug Discovery Today: Disease Models, 2004. 1(3): p. 351-8.
[0720] 4. Zeng, Q., et al., Crosstalk between tumor and endothelial cells promotes tumor angiogenesis by MAPK activation of Notch signaling. Cancer Cell, 2005. 8(1): p. 13-23.
Sixth Series of Experiments
A Foxo/Notch Pathway Controls Myogenic Differentiation and Fiber Type Specification
[0721] Foxo transcription factors govern metabolism and cellular differentiation. Unlike Foxo-dependent metabolic pathways and target genes, the mechanisms by which these proteins regulate differentiation have not been explored. Activation of Notch signaling mimics the effects of Foxo gain-of-function on cellular differentiation. Using muscle differentiation as a model system, we show that Foxo physically and functionally interacts with Notch by promoting co-repressor clearance from the Notch effector Cs1, leading to activation of Notch target genes. Inhibition of myoblast differentiation by constitutively active Foxo1 is partly rescued by inhibition of Notch signaling, while Foxo1 loss-of-function precludes Notch inhibition of myogenesis and increases MyoD expression. Accordingly, conditional Foxo1 ablation in skeletal muscle results in increased formation of MyoD-containing (fast-twitch) muscle fibers and altered fiber type distribution at the expense of Myogenin-containing (slow-twitch) fibers. Notch/Foxo1 cooperation may integrate environmental cues through Notch with metabolic cues through Foxo1 to regulate progenitor cell maintenance and differentiation.
[0722] A central question in regenerative medicine is to understand how highly specialized cell types arise from undifferentiated stem or progenitor cells (1). Germane to this issue is how biochemical signals engendered by microenvironmental and endocrine/nutritional cues are transcriptionally integrated to activate cellular differentiation processes.
[0723] The O subfamily of forkhead (Fox) proteins regulates hormonal, nutrient and stress responses to promote cell survival and metabolism. The ability to fine-tune Foxo transcription is essential to control these cellular functions, and is largely dependent on post-transcriptional modifications, including phosphorylation and acetylation (2). In addition to their role in terminally differentiated cells, Foxo proteins have also been implicated in myoblast (3), pre-adipocyte (4) and endothelial cell differentiation (5). Moreover, Foxo4 regulates vascular smooth muscle cells differentiation through interactions with Myocardin (6). Foxo3 knockout mice display premature ovarian failure, consistent with a role for this gene in ovarian follicle maturation (7). The mechanisms by which Foxo proteins control cellular differentiation remain unclear, and recent conditional ablation studies are consistent with a significant degree of functional overlap amongst the three Foxo isoforms in the hematopoietic lineage (8, 9).
[0724] The Notch pathway plays an important role in neural, vascular, muscular and endocrine differentiation during embryogenesis (10). Upon ligand-induced cleavage, the intracellular domain of the Notch receptor translocates to the nucleus, where it interacts with the DNA binding protein Cs1, changing its transcriptional properties from a suppressor to an activator of transcription (11). Cs1 targets include the Hairy and Enhancer of split (Hes, Hey) genes. Hes1 controls gut endoderm (12), pre-adipocyte (13) and neurogenic differentiation (14). Active Notch signaling, or gain of Notch1 receptor function, inhibits differentiation of C2C12 and 10T/2 myoblasts by suppressing MyoD transcription (15-21).
[0725] It is noteworthy that Foxo1 gain-of-function (3-5) phenocopies Notch1 activation (13, 17, 22, 23) in every cellular differentiation context. Moreover, Foxo1 ablation (24) phenocopies Notch1 ablation (25) in mice. Despite these intriguing similarities, Foxo and Notch signal through two seemingly distinct mechanisms, the phosphatidylinositol-3-kinase pathway (Foxo), and the Hes/Hey pathway (Notch). In this study, we show that Foxo physically and functionally interacts with Notch by promoting co-repressor clearance from Cs1, thus controlling the myogenic program.
[0726] Myogenic precursors arise from mesodermal stem cells (26) and are converted into myotubes by a multi-step process culminating in the expression of myogenic transcription factors of the MRF family (MyoD, Myogenin, MRF4 and Myf5) (27). Myogenic transcription factors heterodimerize with E proteins and promote expression of muscle-specific genes, acting in close coordination with myocyte-specific MEF2 enhancer factors (28).
[0727] Adult muscle is a heterogeneous tissue, primarily defined by its myofiber content (29). Different myosin heavy chain (MyHC) sub-types characterize different myofibers. Type I fibers express primarily slow-twitch MyHC, whereas type II fibers express fast-twitch MyHC (29). The process of fiber-type specification is controlled at multiple steps. First, there appears to be heterogeneity among myogenic precursor cells, and evidence from avian embryo cross-transplantation experiments indicates that early precursors contribute primarily to slow muscle fibers, and later precursors to fast fibers (29). Post-natally, fiber type specification is also affected by cell autonomous factors, including innervation and endocrine/nutritional cues (28). The Foxo co-activator Pgc1α plays a critical role in promoting the formation of slow-twitch fibers (30), and recent data have also implicated the Foxo deacetylase Sirt1 in this process (31). Using conditional mutagenesis in mice, we show that Foxo1's role in suppressing MyoD-dependent myogenesis in C2C12 cells is mirrored by an increase of MyoD-containing myofibers in Foxo1-deficient skeletal muscle, consistent with a key function in myoblast lineage specification.
[0728] Results
[0729] Interaction of Foxo1 and Notch Signaling in C2C12 Differentiation
[0730] To understand whether Notch and Foxo interact to control muscle development, we used a cellular differentiation model. C2C12 cells undergo myogenic conversion and myotube fusion upon growth factor withdrawal, a process associated with Foxo1 nuclear translocation (3). Accordingly, transduction of adenovirus encoding a constitutively active Foxo1 mutant (Foxo1-ADA)(4) blocked the effect of serum withdrawal to induce C2C12 differentiation, as reflected by inhibition of myoblast fusion (FIG. 37A-37C). Conversely, Foxo1 inhibition by siRNA did not affect these processes (FIG. 37D). Similarly, constitutively active Notch (Notch1-IC) phenocopied Foxo1-ADA in blocking myoblast differentiation (FIG. 37E). Virtually all cells became transduced with the adenoviruses (FIG. 44). Foxo1 siRNA effectively suppressed expression of both endogenous Foxo1 and transfected FLAG-Foxo1 (FIG. 45) in a dose-dependent manner, without affecting control proteins or other Foxo isoforms (FIG. 46). Neither Foxo1-ADA nor Notch1-IC affected C2C12 proliferation (FIG. 47).
[0731] We asked whether we could preempt the effect of Foxo1-ADA by inhibition of endogenous Notch signaling. To this end, we used a truncated Notch1 receptor lacking the transmembrane anchor and intracellular domain, which acts as a decoy receptor by binding Notch ligands (32, 33) (Y. F. and J. K., unpublished observation). The decoy did not affect C2C12's ability to undergo differentiation in response to growth factor withdrawal (FIG. 37F), but partly rescued Foxo1-ADA inhibition of myoblast differentiation (FIG. 37G). As an alternative probe to block Notch signaling, the presenilin inhibitor (PSI) Compound E (34) also rescued Foxo1-ADA inhibition of myoblast differentiation (FIG. 37H).
[0732] To examine the effect of Foxo1 on Notch signaling, we co-transfected Foxo1 siRNA and Notch-IC. Foxo1 siRNA rescued inhibition of myoblast differentiation and Myosin expression by Notch1-IC (FIG. 37I), while control siRNA had no effect (data not shown). To rule out non-specific effects of Foxo1 siRNA on myoblast differentiation, we generated a siRNA-resistant Foxo1-ADA (FIG. 48). Foxo1 siRNA reversed the effects of Foxo1-ADA (FIG. 37J), but failed to rescue inhibition of C2C12 differentiation caused by siRNA-resistant Foxo1-ADA (FIG. 37K). We present a quantitative analysis of these data in FIG. 38A, showing that Foxo1 and Notch1-IC decreased myosin levels by >80%, while Notch decoy and Foxo1 siRNA restored them to ˜70% of fully differentiated cells. We obtained similar data by performing a morphometric analysis of Myosin-positive cells (FIG. 38B). These data indicate that Foxo1 is required for the effect of Notch on myoblast differentiation.
[0733] We next tested whether Foxo1 affects differentiation via its transcriptional function. To this end, we generated a DNA Binding Deficient mutant in the backbone of the ADA mutant, by replacement of N208A and H212R (DBD-Foxo1ADA) (6, 35). We confirmed that this mutant is unable to bind DNA by measuring Igfbp1 promoter activity, a canonical Foxo1 target. Foxo1-ADA increased Igfbp1 promoter activity by 10-fold, whereas DBD-Foxo1ADA was unable to do so (FIG. 38C, left panel). Surprisingly, this mutant was as effective as the DNA binding-competent Foxo1-ADA at inhibiting differentiation (FIG. 37L). These data indicate that Foxo1 controls differentiation independently of its ability to bind DNA in a sequence-specific manner.
[0734] Foxo1 Binds to Cs1 and is Recruited to the Hes1 Promoter
[0735] Notch1-IC binds to and co-activates Cs1 to promote Hes and Hey expression (11). Based on the results with the DBD-Foxo1ADA mutant, we tested whether Foxo1 interacts with Cs1 in a Notch-dependent manner using co-culture of C2C12 cells expressing Notch1 receptor with HEK293 cells expressing the Notch ligand Jagged1 (denoted by the "+" sign), or LacZ as a negative control (denoted by the "-" sign). We provide several lines of evidence that Foxo1 and Cs1 interact in cultured cells. We detected endogenous Foxo1 in endogenous Cs1 immunoprecipitates, and the co-immunoprecipitation was significantly enhanced by activation of Notch signaling (FIG. 39A). To confirm the specificity of the interaction, we expressed HA-tagged Foxo1 and FLAG-tagged Cs1 in C2C12 cells. Following immunoprecipitation with anti-HA (Foxo1) antiserum, we detected FLAG-Cs1 in immunoblots (FIG. 39B). Conversely, following immunoprecipitation with anti-FLAG (Cs1) antiserum, we detected HA-Foxo1 in immunoblots (FIG. 39C). The ability to co-immunoprecipitate with Cs1 appears to be specific to Foxo1, as we failed to detect other Foxo isoforms in Cs1 immunoprecipitates (FIG. 42). A truncated Foxo1 mutant (Δ256, encoding amino acids 1-256)(36) retained the ability to interact with Cs1. We detected FLAG-Cs1 in Myc-Δ256 immunoprecipitates (FIG. 39D), and HA-Δ256 in FLAG-Cs1 immunoprecipitates (FIG. 39E), indicating that Cs1 interacts with the Foxo1 N-terminal domain.
[0736] To determine whether this is a direct protein-protein interaction and map the interaction domain(s), we first carried out pull-down assays with affinity-purified GST-Foxo1 produced in bacteria and FLAG-Cs1 expressed in HEK293 cells. We detected Cs1 association with full-length and N-terminal Foxo1 (amino acids 1-300), but not with C-terminal Foxo1 (aa. 290-655) or GST (FIG. 40A). We next mapped the Cs1 domain that interacts with Foxo1 using a cell-free system with GST-Foxo1 and GST-Flag/Cs1 purified from bacterial cultures. Again, we recovered full-length (1-655) and N-terminal (1-300), but not C-terminal (290-655) Foxo1 in Cs1 immunoprecipitates. Conversely, N-terminal Foxo1 interacts with N-terminal Cs1 (FIG. 40B).
[0737] We used Cs1 deletion mutants to map the Foxo1-binding domain in Cs1. These studies indicate that Foxo1 binds to a domain encompassing amino acids 172-279 (FIG. 40C), which is contained within the Cs1 NTD domain (37) (FIG. 40C, diagram). Interestingly, this domain is required for DNA and corepressor binding, but does not contribute to Notch binding (38, 39).
[0738] Cs1 binds to a consensus sequence in the Hes1 promoter (40), which thus provides a useful readout assay of the Foxo/Cs1 interaction. If the latter were required to regulate C2C12 differentiation, three predictions should be met: (a) Foxo1 should be detected in chromatin immunoprecipitation assays (ChIP) spanning the Cs1 element in the Hes1 promoter, (b) the interaction should be differentiation-dependent and (c) inhibition of differentiation by Foxo1-ADA should be accompanied by constitutive binding to the Cs1 element in the Hes1 promoter. FIG. 40D demonstrates that all predictions are fulfilled. First, we performed ChIPs using primers spanning the Cs1 binding site of Hes1 in differentiating C2C12 cells. We detected endogenous Foxo1, Notch1 and Cs1 in immunoprecipitates from undifferentiated cells (FIG. 40D Endog lanes, Day 0). As the PCR-amplified sequence contains no forkhead binding sites, we conclude that Foxo1 binds to this DNA fragment via Cs1. Moreover, binding of both Foxo1 and Notch1 decreased as cells became differentiated (day 1 and 2). When we transduced cells with constitutively nuclear Foxo1-ADA, differentiation was inhibited (FIG. 37C) and the mutant Foxo1 was persistently bound to the Hes1 promoter, as were Cs1 and Notch1 (FIG. 40D, Foxo1-ADA lanes).
[0739] We next analyzed Hes1 expression. The prediction was that Hes1 levels should correlate with occupancy of the Hes1 promoter by Foxo1 and Notch1. Indeed, Hes1 mRNA expression declined as Foxo1 and Notch1 binding to Cs1 decreased, while Myosin protein levels increased (FIG. 40D). To rule out a direct effect of Foxo1 on Cs1 transcription, we carried out reporter gene assays with the Cs1 promoter. Foxo1 failed to activate expression of a Cs1 reporter gene, despite the presence of ten repeats of a forkhead binding site in the Cs1 promoter (41) (Data not shown). Moreover, Cs1 expression was unaffected in C2C12 cells expressing Foxo1-ADA (not shown). These data indicate that Foxo1 regulates Notch-dependent differentiation via protein/protein interactions with Cs1.
[0740] Foxo1 is Required for Notch Induction of Hes and Hey Genes Via Cs1
[0741] We examined the ability of Foxo1-ADA to promote expression of endogenous Hes1, Hes5 and Hey1 in C2C12 cells. Both Foxo1-ADA and Notch1-IC increased the expression of the three genes, while Foxo1 siRNA inhibited Hes1, Hes5 and Hey1 expression induced by Notch1-IC (FIG. 41A). Foxo1 siRNA had no effect on Hes1, Hes5 and Hey1 expression in growth factor-deprived cells (FIG. 41A).
[0742] We focused the next set of experiments on Hes1, as a prototypical Notch target gene. We tested Foxo1's ability to regulate Hes1 transcription using reporter assays with the Hes1 promoter, as well as measurements of Hes1 expression. Foxo1-ADA and Notch1-IC induced Hes1 promoter activity by 1.8- and 2.5-fold, respectively. Co-transfection of Foxo1-ADA with Notch1-IC caused a 2.5-fold increase (FIG. 41B). Co-transfection of Foxo1 siRNA suppressed Notch-induced Hes1 activity in a dose-dependent manner, while control siRNA had no effect (FIG. 41B). We obtained similar results with a synthetic Hes1 reporter containing four tandem repeats of the Cs1 binding motif (FIG. 50). Moreover, the DBD-Foxo1ADA was able to induce Hes1 reporter gene activity to an even greater extent than Foxo1-ADA, confirming that direct DNA binding is not required for Foxo1 activation of Hes1 (FIG. 38C, right panel).
[0743] The failure of Notch1-IC to induce Hes1 expression in cells expressing Foxo1 siRNA suggests that Foxo1 is required for Cs1/Notch interaction. Thus, we investigated the binding of Foxo1 and Notch1 to the Hes1 promoter in a co-culture system. We co-cultured C2C12 cells expressing Notch1 with HEK293 cells expressing the Notch ligand Jagged1 to induce activation of endogenous Notch signaling. Co-culture in the presence of Jagged1-expressing cells increased endogenous Foxo1 (FIG. 42A, lanes 1-2) and Notch1 binding to the Hes1 promoter in ChIP assays (FIG. 42A, b, lanes 1-2) (42). These data are consistent with the observation that Foxo1 co-immunoprecipitation with Cs1 increased upon co-culture (FIG. 39A). To test whether Foxo1 binding to the Hes1 promoter is Cs1-dependent, we inhibited Cs1 expression with siRNA (FIG. 51). Transfection of Cs1 siRNA inhibited both Foxo1 and Notch1 binding to Hes1 promoter (FIG. 42A, lanes 3-4), indicating that they are Cs1-dependent. Moreover, Foxo1-ADA failed to induce Hes1 expression in the presence of Cs1 siRNA (FIG. 42A, lane 5). The results of ChIP experiments were corroborated by Hes1 promoter assays. Expression of Jagged1 or Notch1 alone had no effect on Hes1 activity, but co-culturing yielded a 3.7-fold increase in Hes1 reporter gene activity (FIG. 42C). Foxo1 siRNA abolished Notch binding to the Hes1 promoter in ChIP assays (FIG. 42B, lanes 3-4) and induction of Hes1 promoter activity (FIG. 42C). These results suggest that Foxo1 is required for binding of Notch1 to the Hes1 promoter, and provide a mechanism whereby inhibition of Foxo1 expression restores differentiation of myoblasts expressing. Notch1-IC. The ability of Foxo1 siRNA to inhibit Notch induction of Hes1 in a co-culture system rules out the possibility that the effects observed in differentiation experiments with Notch1-IC are due to non-physiologic activation of Notch signaling by the truncated intracellular Notch1 mutant (15).
[0744] Foxo1 Promotes Corepressor Clearance and Maml1 Binding to Cs1
[0745] To clarify the molecular mechanism of Foxo1-dependent activation of Hes1 expression, we investigated corepressor/coactivator exchange at the Hes1 promoter. Activation of Notch cleared the corepressors Ncor and Smrt (43) and recruited the coactivator Maml1 (42) to the Hes1 promoter. Foxo1 siRNA prevented Notch-induced corepressor exchange (FIG. 42D). These data are consistent with the observation that Foxo1 binds to the region 172-279 of Cs1 (FIG. 40C), which has been shown to contain the Ncor/Smrt binding sites (38, 39).
[0746] To demonstrate that the observed changes in the transcriptional complex result in changes in Hes1 activity, we investigated expression of Hes1 target genes involved in myogenesis. Hes1 has been proposed to suppress myoblast differentiation by inhibiting the bHLH transcription factor MyoD, without affecting Myf5 (16, 17). Expression analyses revealed that Notch1-IC or Foxo1-ADA suppressed MyoD, while Myf5 was unaffected. Notch decoy or Foxo1 siRNA partly restored MyoD expression (FIG. 42E).
[0747] Altered Fiber Type Composition in Skeletal Muscle Lacking Foxo1
[0748] Based on the cellular data, we undertook to probe Foxo1 function in muscle differentiation in vivo using conditional gene inactivation. The predicted outcome of this experiment is accelerated differentiation of MyoD-containing, but not Myf5-containing myoblasts. Because MyoD is the predominant myogenic factor in fast fibers, while Myogenin is predominant factor in slow fibers (44), the removal of Foxo/Notch inhibition on MyoD expression should result in increased formation of fast fibers, potentially at the expense of slow fibers.
[0749] There are three Foxo isoforms in mice: Foxo1, 3, and 4 (8, 9). The latter is predominant in most muscle types (45) except soleus, where Foxo1 is the most abundant (FIG. 43A). Coincidentally, soleus is also physiologically enriched in slow-twitch fibers, and thus allowed us to readily test our hypothesis. We inactivated Foxo1 expression in skeletal muscle by crossing mice homozygous for a floxed Foxo1 allele with Myogenin-cre transgenics. mRNA analysis indicated that the knockout occurred as planned (data not shown). Histological analyses revealed a reduction of type I (slow-twitch) fibers in soleus of Myog-Foxo1 mice, while type II fiber-enriched muscles were unaffected (FIG. 43B). Consistent with the histological findings, expression of type I fiber markers decreased, while type II fiber markers increased in Myog-Foxo1 mice (FIG. 43C). We then analyzed expression of the myogenic transcription factors MyoD, Myf5 and Myogenin. MyoD is the predominant factor in fast fibers, and Myogenin in slow fibers (44). Consistent with the histopathology, we found a twofold increase in MyoD expression and ˜80% decrease in Myogenin, while Myf5 expression was unchanged (FIG. 43C). Moreover, expression the Foxo1 coactivator Pgc1α, which regulates type I fiber determination (30) was unchanged, indicating that the phenotype of Myog-Foxo1 mice cannot be accounted for by decreased Foxo1-dependent Pgc1α transcription (FIG. 43C) (46). As a functional correlate of the observed fiber type switch, we examined running performance on a treadmill. Indeed, Myog-Foxo1 mice displayed reduced running capacity, as predicted from the reduction in type I (endurance) fibers (FIG. 43D).
[0750] Finally, to determine whether these changes reflected developmental alterations in fiber-type specification, as opposed to adaptive or cell-nonautonomous factors, we determined MyoD expression in Foxo1 (24) and Notch1 knockout (25) embryos at E9.5. In Foxo1.sup.-/- embryos, MyoD levels increased 3.1±1.1 fold, and in Notch1.sup.-/- embryos 7.3±2.9 fold compared to controls (P<0.05 in both mutants vs. wildtype, n=4). The increase in MyoD expression observed in vivo is consistent with the physical and functional interactions between Foxo1 and Notch at this key signaling nexus in myoblast differentiation. Thus, we propose that the fiber-type switch in Myog-Foxo1 mice is the result of accelerated differentiation of MyoD-containing myoblasts during embryonic development.
DISCUSSION
[0751] This study provides biochemical, cellular and genetic evidence that Foxo and Notch pathways cooperate in the regulation of muscle differentiation. The data reveal a novel mode of Foxo1 action to promote corepressor exchange at the Hes1 promoter via direct binding to the Cs1 NTD region (FIG. 42F). We propose that Foxo1 binding to this domain stabilizes the Notch/Cs1 complex and promotes corepressor clearance and Maml1 recruitment, consistent with the proposed role of NTD from structural studies (37). The findings also provide a mechanism by which two major biochemical pathways, the phosphoinositol-3-kinase/Akt pathway and the Notch/Hes pathway, converge in a synergistic manner to control cellular differentiation in vivo.
[0752] The proposed role for Foxo1 is independent of its transcriptional function, and involves a direct interaction with Cs1. While our studies have focused on Hes-1 as a prototypical effector of Notch1 signaling, our data should not be construed to indicate that Hes-1 is the sole mediator of the Notch/Foxo interaction. For example, we have observed a similar Foxo/Notch epistasis in the differentiation of pre-adipocytes, PC-12, and HUVECs, suggesting that Foxo interacts with Notch in multiple cell contexts (data not shown). We propose that Notch/Foxo cooperation integrates environmental cues through Notch with metabolic cues through Foxo1 to regulate progenitor cell maintenance and differentiation. This two-tiered mechanism allows committed progenitor cells in various tissues to avoid differentiation in response to developmental cues (Notch) when Foxo1 is active, i.e., in the absence of growth factors. These cells would then persist in a dormant state in adult tissues, where they can terminally differentiate in response to a combination of Notch ligand and hormonal/nutritional cues leading to Foxo1 inhibition. This interpretation is consistent with the fiber-type switch observed in Foxo1-deficient muscle, an observation that appears to position Foxo1 as a fate decider within the myogenic lineage, as opposed to an inducer of the myogenic program. It remains to be seen whether other Foxo and Notch isoforms also interact and how they contribute to this process.
[0753] The demonstration that Foxo1 is a coregulator of gene expression provides a potential explanation for the protean functions of this transcription factor. An interesting question emerging from our studies is how the switch from one function to the other is effected, and how the complex post-translational modifications of Foxo1 in response to growth factors, hormones and nutrients impinge on this process. The findings have broad implications for the pathophysiology of disease processes that involve Foxo1 signaling. A potential implication of our observation is the ability to explore the use of agents that inhibit Notch signaling (47) as a treatment of metabolic disorders characterized by excessive Foxo function (48).
[0754] Materials and Methods
[0755] Animal Generation and Analysis
[0756] Myogenin-cre (49) and Foxo1flox mice have been described (9). The wild-type, null and Foxo1flox alleles were detected using PCR with primers 5'-GCT TAG AGC AGA GAT GTT CTC ACA TT-3', 5'-CCA GAG TCT TTG TAT CAG GCA AAT AA-3' and 5'-CAA GTC CAT TAA TTC AGC ACA TTG A-3'. Prior to the treadmill performance test, mice were trained for 2 days (Columbus Instruments). The test was performed at 15 m/min for the first 30 min, followed by 1 m/min increases at 10 min intervals until exhaustion. Skeletal muscle samples were quickly frozen in OCT matrix, and 7 μm serial sections were obtained. Muscle fibers were typed using metachromatic ATPase (50) or immunostaining with anti-skeletal slow myosin (Sigma). For embryonic studies, we set up timed matings of heterozygous Foxo1 (24) or Notch1 (25) mice and recovered embryos at E9.5. mRNA was isolated from whole embryos and real-time RT-PCR was performed as described below.
[0757] Viral Expression Studies
[0758] C2C12 cells were differentiated as described (3, 4). Foxo1-ADA, Notch1-IC, Jagged1, Cs1 and Notch decoy adenoviral and mammalian expression vectors have been described (36, 51). We generated retroviruses expressing Foxo1-ADA and Notch1-IC using the pQCXIH vector. To generate Notch decoy (pAdlox Notch1ECD-Fc), the extracellular domain of Notch1 (bp 241-4229, GenBank accession # X57405) was fused in frame with human IgG Fc tag and cloned into pAdlox. Retroviral supernatant was produced from cells transiently co-transfected with pVSV-G vector and designated pQCXIH vector into GP2-293 cells (BD Bioscience). To generate the DNA binding-deficient Foxo1, we replaced N208 and H212 with alanine and arginine, respectively, using QuikChange Mutagenesis Kit (Stratagene). The mutations were then cloned in the backbone of the Foxo1-ADA mutant.
[0759] Luciferase Assay and Co-Culture Assay
[0760] We transfected HEK293 cells with Hes1-luciferase (-194 to 160 from transcription start site) (Hes1/pGL2 basic), Synthetic Hes1-luciferase (containing a 4×Cs1 binding site, 4×Cs1/pGL2 basic) or Cs1-luciferase (-1536 to 22, Cs1/pGL2 basic) reporter genes along with pCMV5, pCMV5-Foxo1-ADA, pQNC-Notch1-IC, pHyTc Notch decoy or Foxo1 siRNA, We used plasmid pRSV-β-galactosidase as a control of transfection efficiency (51). For co-culture assay, we expressed Notch1 in C2C12 cells and Jagged1 or LacZ in HEK293 cells by transfection. We then harvested HEK293 cells and seeded them on C2C12 cells. After 1 hr incubation, we used the co-cultured cells for experiments.
[0761] Western Blotting and Immunoprecipitation
[0762] We performed these assays according to standard techniques using anti-Myosin (MF-20), anti-HA (12CA5, Boehringer Mannheim), anti-FLAG (M2, Sigma), anti-Foxo1 (H128 and N20, Santa Cruz), anti-Notch1 (C-20, Santa-Cruz), anti-Cs1 (Chemicon and Santa-Cruz), anti-NcoR (Santa-Cruz), anti-SMRT (Santa-Cruz) or anti-MAML1 (Chemicon) antibodies. For Foxo/Cs1 co-immunoprecipitation, we used purified nuclear fractions (52). Because Cs1 migrates close to IgG heavy chain on SDS-PAGE, we used dimethylpyrimilidate (DMP from Pierce) to cross-link antibodies to Protein A beads and avoid IgG contamination of eluted protein complexes (52).
[0763] Chromatin Immunoprecipitation Assays
[0764] We performed ChIP assay in C2C12 cells as described previously (4) and in co-cultured cells as described by Fryer (42). The primer pairs employed to amplify the Cs1 binding site of the Hes1 promoter are: 5'-GCAAAGCCCAGAGGAAAGAGTTAG-3' and 5'-AGGAGAGAGGTAGACAGGGGATTC-3'.
[0765] siRNA Transfection and siRNA-Resistant Foxo1
[0766] The Foxo1-specific siRNA sequence is 5'-ACGGAGGATTGAACCAGTATA-3'. The Cs1 specific siRNA sequence is 5'-TAGGGAAGCTATGCGAAATTA-3'. siRNA was transfected using lipofectamine-plus reagent (Invitrogen). We generated siRNA-resistant Foxo1 by replacing three residues (underlined) in the sequence 5'-ACGGCGGTCTGAACCAGTATA-3'. Primer sequences employed for real-time RT-PCR studies are available on request.
[0767] Recombinant Proteins and Interaction Assays
[0768] We generated GST-FLAG-Cs1 encompassing amino acids 1-527, 1-279, 1-172 and 279-527 fragments by cloning into pGEX6P-1. GST-Foxo1 constructs have been described (53). Following bacterial culture and IPTG induction, we purified GST fusion proteins and incubated them together. Thereafter, we isolated GST-FLAG/Cs1 by immunoprecipitation with anti-FLAG antibody, washed the immune pellets extensively and performed immunoblot with anti-Foxo1 antiserum.
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Seventh Series of Experiments
[0822] Diabetic patients often develop obesity and vascular pathologies. The molecular mechanisms that contribute to diabetic complications remain to be elucidated. In the past two years, I have been evaluating Notch4 knockout mice for postnatal defects. These analyses revealed that Notch4 mutant mice develop hallmarks of diabetes: 1) early onset obesity as seen by a dramatic increase in subcutaneous fat, and 2) reduced pericyte content in retinal vasculature reminiscent of diabetic retinopathy. We have found that Notch and Foxo1, a transcriptional regulator of insulin signaling, cooperate to regulate adipogenesis and angiogenesis. Mice deficient for Notch1, Notch1/Notch4, or Foxo1 die in utero with angiogenic defects. These data lead us to hypothesize that dysregulated Notch signaling contributes to diabetic obesity and vasculopathologies. The proposal objective is to examine this hypothesis and define the roles of Notch and insulin signaling interactions in adipogenesis and angiogenesis. Mouse models will be used to alter Notch, Foxo1, and insulin receptor activity via genetic manipulation. Adipogenesis and metabolic dysfunction will be evaluated in Notch4 and insulin receptor knockout mice and embryonic fibroblasts derived from these mice. Embryonic and retinal angiogenesis will be evaluated in mice haploinsufficient for Notch1, Notch4 and/or Foxo1. Finally, the function of Notch and Foxo1 signaling in proliferative retinopathy will be evaluated in a hypoxia-driven retinal angiogenesis mouse model. My career goals are to become an independent scientific investigator in the field of diabetic research.
[0823] Evaluation of Notch Function in Metabolism.
[0824] We have found that Notch and Foxo1, a transcriptional regulator of Insulin signaling, form a transcriptional complex with CSL to regulate cell fate decision. Loss of one allele of Foxo1 rescues hyperglycemia and hyperinsulinemia in insulin receptor haploinsufficient mice. Similarly, loss of Notch4 in mice correlated with lower blood glucose levels as compared to wildtype littermates. Foxo1 and Notch4 share an overlapping expression pattern in the β-cells of the adult murine pancreas, while Notch1 is expressed in both the α- and β-cells. In this aim, we will further characterize the metabolism of Notch mutant mice. We will also determine if defects are present in the pancreases of Notch and Notch/Foxo1 mutant mice.
[0825] Evaluation of Notch4 Function in Adipogenesis.
[0826] We have found that Notch4 knockout mice have larger adipose tissue depots. In the skin, Notch4 is expressed in both the adipocytes and vessels. Notch4 may regulate adipogenesis by either a cell autonomous or cell nonautonomous mechanism. In the cell autonomous model, Notch functions in the adipocyte to regulate differentiation from committed preadipocytes in the stromavascular fraction. Alternatively, Notch may regulate angiogenesis within adipose tissue, which then affects adipogenesis. We have established that Notch and Foxo1, cooperate to inhibit hormone-induced adipogenesis of cultured fibroblasts. In insulin receptor mutant mice, adipogenesis was perturbed and differentiation was partially rescued by inhibiting Foxo1. However, it is unknown whether abnormalities of Notch function can affect insulin-dependent adipocyte differentiation and function in vivo. To begin to address this question, we will further. characterize the adipose phenotype in Notch mutant mice with a focus on the subcutaneous and visceral adipose depots. We will then determine if Notch4 insufficiency rescues the subcutaneous adipose phenotype in Insr mutant mice. Finally, we will evaluate Notch function in adipocyte differentiation of embryonic fibroblasts derived from Notch4 and Insr deficient mice. Our goal is to determine the role of Notch and Insulin signaling interactions in adipocyte differentiation.
[0827] Diabetes and Obesity
[0828] Obesity is a primary risk factor for insulin resistance, hyperglycemia and the development of type 2 diabetes {Eckel, 2005 #769}. It is also associated with cardiovascular dysfunction. Adipose tissue has an important metabolic function in storing triacylglycerol in times of energy excess and releasing free fatty acids and glycerol in times of energy deprivation. In addition, adipocytes regulate metabolic homeostasis by producing a number of bioactive substances, termed adipokines. Adipokines consist of hormone cytokines, growth factors, and other bioactive compounds. Theses include leptin, tumor necrosis factor alpha, angiotensin II, interleukin-6, interleukin-1, adiponectin, resistin, and prostaglandins. These secreted factors play a major role in regulating both metabolic and vascular biology and as such have been proposed to be the connection between insulin resistance and cardiovascular disease.
[0829] The development of obesity appears to be regulated by insulin signaling and dependent on angiogenesis. In adipocytes, insulin signaling induces Vascular Endothelial Growth Factor (VEGF) {Mick, 2002 #767}. VEGF is a potent inducer of angiogenesis that can promote endothelial cell proliferation, migration, and differentiation as well as vessel wall leakiness {Yancopoulos, 2000 #65}. In mouse models of obesity, antagonists of VEGF disrupt not only angiogenesis, but prevent adipogenesis {Rupnick, 2002 #766; Fukumura, 2003 #768}. Thus in adipose tissue, there is a reciprocal paracrine regulation of adipocyte differentiation and angiogenesis.
[0830] Notch Function in Adipogenesis
[0831] A role for Notch in adipocyte differentiation is just beginning to be elucidated. Using in vitro assays, Notch signaling has been shown to both promote and inhibit hormone-induced adipogenesis of fibroblasts. In stromal cell lines, Notch perturbed osteoblast differentiation that then led to an increase in adipocyte differentiation {Sciaudone, 2003 #772}. In contrast, both ligand-mediated Notch signaling, or ectopic expression of a constitutively active Notch1 inhibited adipogenesis of 3T3-L1 fibroblasts {Garces, 1997 #177; Ross, 2004 #773}. Similarly, overexpression of the Notch target gene, HES1 inhibited adipocyte differentiation of fibroblasts {Ross, 2004 #773}. Disrupting HES1 expression with siRNA also blocked fibroblast adipocyte differentiation. Thus, both inhibition and activation of Notch perturbs adipogenesis suggesting adipogenesis is dosage sensitive to Notch signaling.
[0832] Notch-mediated inhibition of fibroblast adipogenesis correlated with loss of the adipocyte-specific genes C/EBPα and PPARγquadrature Adipocyte differentiation of fibroblasts in which Notch signaling was activated was rescued by the expression either C/EBPα or PPARγquadrature, suggesting that Notch inhibits adipogenesis by suppressing the expression of these two genes. Consistent with the fibroblast data, retinoic acid induced adipogenesis was slightly enhanced in Notch1 knockout embryoid bodies {Nichols, 2004 #770}. Finally, Notch1 expression was found to be reduced in adipose tissues isolated from insulin-resistant patients relative to insulin-sensitive subjects suggesting a role for Notch in diabetes related adipocyte differentiation {Yang, 2003 #777}.
[0833] Vascular Complications in Diabetes
[0834] Diabetic patients display multiple vascular complications, including arterial hypertension, strokes, ischemia, retinopathy, atherosclerosis and heart attacks. However, little is known as to the molecular mechanisms contributing to diabetic vascular complications. Blindness is one of such complications that has a vascular origin but is poorly understood. Within 20 years of diagnosis, a quarter of diabetics develop proliferative retinopathy leading to blindness. Diabetic retinopathy initiates with an increase in vascular permeability, thickening of the basement membrane, and loss of pericytes in the retinal microvasculature followed by a proliferative phase of neovascularization {Cukiernik, 2004 #749}. Vascular growth factor (VEGF) has been implicated in the development of diabetic proliferative retinopathy. VEGF signaling can promote endothelial cell proliferation, migration, differentiation and vessel wall leakiness {Yancopoulos, 2000 #65}. In mouse models of type I and type II diabetes, the hypoxia sensing transcription factor, HIF-1α, and VEGF are found to be increased within the eyes {Kondo, 2004 #744} suggesting that hypoxia is an initiating event in the development of diabetic retinopathy. The induction of VEGF is likely mediated by HIF-1α, as VEGF is a direct transcriptional target of HIF-1α {Yancopoulos, 2000'#65}. Supporting the role for VEGF in diabetic retinopathy, ectopic expression of VEGF in primate eyes leads to rapid development of proliferative retinopathy and macular edema {Lebherz, 2005 #748}. In a diabetic rat model, subcutaneous injection of the VEGF receptor signaling inhibitor SU5416 suppresses VEGF induced retinal microvascular permeability and vasoconstriction {Cukiernik, 2004 #749}. Finally, an intravitreal injection of an inhibitor of all three VEGFRs, PTK/ZK, reduces retinal neovascularization in a hypoxic mouse model {Maier, 2005 #750}. Thus, dysregulation of VEGF signaling plays a critical role in the development of proliferative retinopathy and may also contribute to other diabetic vascular complications.
[0835] Evaluation of Notch Function in Metabolism.
[0836] We have found that Notch and Foxo1, a transcriptional regulator of Insulin signaling, form a transcriptional complex with CSL to regulate cell fate decisions. Loss of one allele of Foxo1 rescues hyperglycemia and hyperinsulinemia in insulin signaling deficient mice (Nakae 2002 ng). In mice, loss of N4 correlated with a significant decrease in blood glucose levels compare to wildtype littermates. Like Foxo1, Notch4 may oppose insulin signaling in regulating the metabolism. Circulating glucose and insulin levels are regulated by both the glucose producing tissues, such as liver and the insulin producing islet β-cells of the pancreas. Increased Foxo1 signaling in the β-cells of Foxo1 transgenic leads to quadrature-cell failure and the development of diabetes (Nakea 2002 ng). Foxo1 and Notch4 share an overlapping expression pattern in the β-cells of the adult murine pancreata, while Notch1 is expressed in both the α- and β-cells. Thus, Notch4 and/or Notch1 may have a function in the endocrine cells of the pancreatic islets. In this aim, we will further characterize the metabolism and pancreata of Notch mutant mice. Notch mutant mice will be crossed with L2 Ttr-Insr.sup.-/- mice (FIG. 21), and used to determine if N4 and/or N1 insufficiency suppresses the diabetic and pancreatic defects observed in this Insr deficient background.
[0837] Evaluation of Notch Function in Adipogenesis.
[0838] We have found that N4 knockout mice have dermal adipose hypertrophy. This adipose tissue phenotype in N4 mutant mice may arise from a cell autonomous defect in the adipocytes or from a non-cell autonomous angiogenic defect. In contrast to N4 nullizygous mice, Insr deficient mice display adipose tissue hypotrophy {Cinti, 1998 #774; Kitamura, 2004 #745} (Okamoto JCI 2004). Ectopic expression of dominant negative Foxo1 restores adipogenesis of Insr.sup.-/- embryonic fibroblasts {Nakae, 2003 #765}. We found that Notch and Foxo1 cooperate to inhibit hormone-induced adipogenesis of fibroblasts. Since Foxo1 functions in epistasis with insulin signaling (FIG. 3), Notch may also have an opposing function to insulin signaling in adipogenesis. Consistent with adipose cell autonomous function for Notch4, Notch4 is expressed within the subcutaneous adipocytes. Thus, we will evaluate Notch function adipogenesis of embryonic fibroblasts derived from Notch and Notch; Insr deficient mice. We will further characterize the subcutaneous fat defect and evaluate the visceral fat depot in Notch mutant mice. Finally, we will determine if Notch4 insufficiency rescues the dermal adipose tissue defect in Insr mutant mice.
[0839] In mouse models of obesity, antagonists of VEGF block both angiogenesis and adipogenesis {Rupnick, 2002 #766; Fukumura, 2003 #768}, indicating that there is reciprocal regulation of adipogenesis and angiogenesis. Since N4 nullizygous mice also display defects in retinal angiogenesis, the observed increase in subcutaneous adipose tissue may arise from endothelial cell dysfunction. Therefore, we will also determine if there are differences in the adipose tissue vasculature of Notch mutant mice.
Eighth Series of Experiments
Rat Notch1 Decoy Present in Murine Serum
[0840] The stability of rat Notch1 decoy formulation in the mammalian blood stream was tested. As shown in FIG. 118, Notch decoys are stable in the mammalian circulatory system.
[0841] Nude mice were injected with control adenovirus or adenovirus expressing rat Notch1 decoy (rN1 decoy). 2 weeks after injection, serum was collected and 4 microliters were evaluated by Western blot analysis. This analysis demonstrates that the full-length rat Notch1 decoy protein (see arrow in FIG. 118) can be expressed in mice and is present at detectable levels with little evidence of degradation.
Ninth Series of Experiments
Human Notch1 Decoy (h-Notch (1-36) Decoy) and Rat Notch 1 Decoy Block Mouse Mammary Tumor Growth
[0842] The activity of the human Notch1 decoy and the Rat Notch 1 decoy was compared against the growth of the mammary tumor cell line, Mm5MT-FGF4. As shown in FIG. 119, both hNotch1 decoy and rNaotch1 decoy reduce the growth rate of Mm5Mt-FGF4.
[0843] We developed a tumor model which utilized Mm5MT-FGF4 cells grown in nude mice. In this experiment, 2×105 Mm5MT-FGF4 cells were implanted into nude mice and four days later adenovirus encoding Fc control, rat Notch1 decoy or human Notch1 decoy was injected into ocular vein. Notch decoys are produced by adenovirus infected liver of mice and secreted into the bloodstream (example in FIG. 118). The growth curve presented in FIG. 119 demonstrates that either Rat Notch1 decoy or human Notch1 decoy reduced the growth of tumor xenografts in nude mice.
[0844] Rat Notch1 Decoy Inhibits SKNEP1 Metastasis to Lung Tissue
[0845] Notch1 decoys can block metastasis in mouse model. We have tested the activity of the rat Notch1 decoy against tumor growth and metastasis of Ewing's Sarcoma cell line, SKNEP. In this tumor model, SKNEP tumor cells are orthotopically implanted into the kidney where the tumor grows and then metastasized to the lung. Expression of rat Notch1 decoy in SKNEP tumor cells reduced tumor growth and metastasis to lung, as shown in FIG. 120.
[0846] SKNEP1 Ewings Sarcoma cells were programmed to express control Fc protein or rat Notch1 decoy s1 (sort 2) or rat Notch1 decoy s4 (sort 4). These SKNEP1 cell lines were orthotopically implanted into kidney of nude mice. After 6 weeks of tumor growth, metastasis to lung was assessed histologically. SKNEP1 cells expressing Rat Notch1 decoy showed fewer lungs that were positive for metastasis. We conclude that expression of the rat Notch1 decoy in nude mice diminishes the capacity of SKNEP1 cells to metastasize to lung.
Tenth Series of Experiments
Notch1 and Notch4 are Co-Expressed with VEGFR-3 and LYVE-1 in Lumphatics of Mouse Skin
[0847] Notch1 and Notch4 are Expressed in Lymphatics of Mouse Skin
[0848] We analyzed the expression of Notch1 and Notch4 in the vasculature of mouse P4 dorsal skin. At this time point, the dermal lymphatics are actively remodeling into the lymphatic capillaries near the surface and collecting ducts in the lower dermal layers. 5 μm cross-sections of skin were co-stained with antibodies against Notch1 or Notch4 (red), and PECAM, VEGFR-3 or LYVE-1 (green). Notch1 and Notch4 share an overlapping pattern of expression with the blood and lymphatic endothelial cell marker, PECAM (upper panels, FIG. 121). Notch1 and Notch4 were co-expressed with both VEGFR-3 (middle panels, FIG. 121) and LYVE-1 in the dermal vasculature (lower panels, FIG. 121). This expression pattern demonstrates that Notch1 and Notch4 are expressed and may function in the lymphatic vessels of the neonatal dermis.
[0849] Dermal Lymphatic Capillaries are Altered in Notch4 Mutant Mice
[0850] We examined the dermal lymphatics of P4 mice. Sections of wildtype and Notch4 nullizygous were immunostained with antibodies against PECAM and LYVE-1 (green). Analysis of PECAM staining appeared similar between mutant and wildtype skin (upper panels, FIG. 122). In contrast, LYVE-1-positive vessels in the dermis of Notch4 mutants had a different morphology than that of wildtype (middle panels, FIG. 122). Notch4 mutant LYVE-1 vessels were often dilated and LYVE-1 staining was discontinuous (lower panels, FIG. 122). These results suggest that Notch4 signaling may be involved in remodeling of the lymphatic vascular plexus.
[0851] Loss of Notch4 Results in Reduced LYVE-1 Positive Vessels
[0852] Notch4 heterozygous (N4.sup.+/-) mice were mated and the dorsal skin of the resulting pups removed and embedded 14 days postnatally. The results are set forth in FIG. 123. Cros-sections of skin were immunostained for the endothelial cell marker, PECAM (data not shown), or the lymphatic endothelial cell marker, LYVE1 (A). Five areas for each were captured by microscopy and PECAM and LYVE1 staining quantitated using imaging software (B, C). PECAM expression was reduced approximately 25% in the N4.sup.-/- dermis compared to wild-type (WT) dermis (B). LYVE-1 staining was more affected than the PECAM with LYVE1 staining decreased nearly 50% in N4.sup.-/- relative to WT mice (C). There was also a reduction in the intensity of the LYVE1 staining in the N4.sup.-/- lymphatics relative to the WT (A).
[0853] Loss of Notch4 function in mice disrupts development of dermal lymphatic's suggesting a role in lymphangiogenesis.
[0854] Notch1 and Notch4 Expressed in Human Breast Cancer
[0855] We performed double immunohistochemistry with antibodies against VEGFR-3 or LYVE-1 (green) and Notch1 or Notch4 (red) of human breast cancers. The results are set forth in FIG. 124. Notch1 and Notch4 were expressed in the extratumoral blood and lymphatic endothelium of human micropapillary breast carcinomas. To determine if Notch1 signaling was activated within the tumoral lymphatic endothelium, we double stained with an antibody against podoplanin (green) and N1Val (red; Cell Signaling), an antibody that specifically detects the activated Notch1 peptide. Expression of the activated Notch1 peptide was observed in most (white arrows) but not all (yellow arrows) of the lymphatic endothelial nuclei(lower panel). These results demonstrate that Notch1 was actively signaling in the pathological lymphatic vessels. These results also demonstrate that Notch1 and Notch4 may function in tumor lymphangiogenesis.
Sequence CWU
1
1
13111433PRTRattus norvegicus 1Met Pro Arg Leu Leu Ala Pro Leu Leu Cys Leu
Thr Leu Leu Pro Ala 1 5 10
15 Leu Ala Ala Arg Gly Leu Arg Cys Ser Gln Pro Ser Gly Thr Cys Leu
20 25 30 Asn Gly
Gly Arg Cys Glu Val Ala Asn Gly Thr Glu Ala Cys Val Cys 35
40 45 Ser Gly Ala Phe Val Gly Gln
Arg Cys Gln Asp Pro Ser Pro Cys Leu 50 55
60 Ser Thr Pro Cys Lys Asn Ala Gly Thr Cys Tyr Val
Val Asp His Gly 65 70 75
80 Gly Ile Val Asp Tyr Ala Cys Ser Cys Pro Leu Gly Phe Ser Gly Pro
85 90 95 Leu Cys Leu
Thr Pro Leu Ala Asn Ala Cys Leu Ala Asn Pro Cys Arg 100
105 110 Asn Gly Gly Thr Cys Asp Leu Leu
Thr Leu Thr Glu Tyr Lys Cys Arg 115 120
125 Cys Pro Pro Gly Trp Ser Gly Lys Ser Cys Gln Gln Ala
Asp Pro Cys 130 135 140
Ala Ser Asn Pro Cys Ala Asn Gly Gly Gln Cys Leu Pro Phe Glu Ser 145
150 155 160 Ser Tyr Ile Cys
Gly Cys Pro Pro Gly Phe His Gly Pro Thr Cys Arg 165
170 175 Gln Asp Val Asn Glu Cys Ser Gln Asn
Pro Gly Leu Cys Arg His Gly 180 185
190 Gly Thr Cys His Asn Glu Ile Gly Ser Tyr Arg Cys Ala Cys
Arg Ala 195 200 205
Thr His Thr Gly Pro His Cys Glu Leu Pro Tyr Val Pro Cys Ser Pro 210
215 220 Ser Pro Cys Gln Asn
Gly Gly Thr Cys Arg Pro Thr Gly Asp Thr Thr 225 230
235 240 His Glu Cys Ala Cys Leu Pro Gly Phe Ala
Gly Gln Asn Cys Glu Glu 245 250
255 Asn Val Asp Asp Cys Pro Gly Asn Asn Cys Lys Asn Gly Gly Ala
Cys 260 265 270 Val
Asp Gly Val Asn Thr Tyr Asn Cys Arg Cys Pro Pro Glu Trp Thr 275
280 285 Gly Gln Tyr Cys Thr Glu
Asp Val Asp Glu Cys Gln Leu Met Pro Asn 290 295
300 Ala Cys Gln Asn Ala Gly Thr Cys His Asn Ser
His Gly Gly Tyr Asn 305 310 315
320 Cys Val Cys Val Asn Gly Trp Thr Gly Glu Asp Cys Ser Asp Asn Ile
325 330 335 Asp Asp
Cys Ala Ser Ala Ala Cys Phe Gln Gly Ala Thr Cys His Asp 340
345 350 Arg Val Ala Ser Phe Tyr Cys
Glu Cys Pro His Gly Arg Thr Gly Leu 355 360
365 Leu Cys His Leu Asn Asp Ala Cys Ile Ser Asn Pro
Cys Asn Glu Gly 370 375 380
Ser Asn Cys Asp Thr Asn Pro Val Asn Gly Lys Ala Ile Cys Thr Cys 385
390 395 400 Pro Arg Gly
Tyr Thr Gly Pro Ala Cys Ser Gln Asp Val Asp Glu Cys 405
410 415 Ala Leu Gly Ala Asn Pro Cys Glu
His Ala Gly Lys Cys Leu Asn Thr 420 425
430 Leu Gly Ser Phe Glu Cys Gln Cys Leu Gln Gly Tyr Thr
Gly Pro Arg 435 440 445
Cys Glu Ile Asp Val Asn Glu Cys Ile Ser Asn Pro Cys Gln Asn Asp 450
455 460 Ala Thr Cys Leu
Asp Gln Ile Gly Glu Phe Gln Cys Ile Cys Met Pro 465 470
475 480 Gly Tyr Glu Gly Val Tyr Cys Glu Ile
Asn Thr Asp Glu Cys Ala Ser 485 490
495 Ser Pro Cys Leu His Asn Gly Arg Cys Val Asp Lys Ile Asn
Glu Phe 500 505 510
Leu Cys Gln Cys Pro Lys Gly Phe Ser Gly His Leu Cys Gln Tyr Asp
515 520 525 Val Asp Glu Cys
Ala Ser Thr Pro Cys Lys Asn Gly Ala Lys Cys Leu 530
535 540 Asp Gly Pro Asn Thr Tyr Thr Cys
Val Cys Thr Glu Gly Tyr Thr Gly 545 550
555 560 Thr His Cys Glu Val Asp Ile Asp Glu Cys Asp Pro
Asp Pro Cys His 565 570
575 Ile Gly Leu Cys Lys Asp Gly Val Ala Thr Phe Thr Cys Leu Cys Gln
580 585 590 Pro Gly Tyr
Thr Gly His His Cys Glu Thr Asn Ile Asn Glu Cys His 595
600 605 Ser Gln Pro Cys Arg His Gly Gly
Thr Cys Gln Asp Arg Asp Asn Tyr 610 615
620 Tyr Leu Cys Leu Cys Leu Lys Gly Thr Thr Gly Pro Asn
Cys Glu Ile 625 630 635
640 Asn Leu Asp Asp Cys Ala Ser Asn Pro Cys Asp Ser Gly Thr Cys Leu
645 650 655 Asp Lys Ile Asp
Gly Tyr Glu Cys Ala Cys Glu Pro Gly Tyr Thr Gly 660
665 670 Ser Met Cys Asn Val Asn Ile Asp Glu
Cys Ala Gly Ser Pro Cys His 675 680
685 Asn Gly Gly Thr Cys Glu Asp Gly Ile Ala Gly Phe Thr Cys
Arg Cys 690 695 700
Pro Glu Gly Tyr His Asp Pro Thr Cys Leu Ser Glu Val Asn Glu Cys 705
710 715 720 Asn Ser Asn Pro Cys
Ile His Gly Ala Cys Arg Asp Gly Leu Asn Gly 725
730 735 Tyr Lys Cys Asp Cys Ala Pro Gly Trp Ser
Gly Thr Asn Cys Asp Ile 740 745
750 Asn Asn Asn Glu Cys Glu Ser Asn Pro Cys Val Asn Gly Gly Thr
Cys 755 760 765 Lys
Asp Met Thr Ser Gly Tyr Val Cys Thr Cys Arg Glu Gly Phe Ser 770
775 780 Gly Pro Asn Cys Gln Thr
Asn Ile Asn Glu Cys Ala Ser Asn Pro Cys 785 790
795 800 Leu Asn Gln Gly Thr Cys Ile Asp Asp Val Ala
Gly Tyr Lys Cys Asn 805 810
815 Cys Pro Leu Pro Tyr Thr Gly Ala Thr Cys Glu Val Val Leu Ala Pro
820 825 830 Cys Ala
Thr Ser Pro Cys Lys Asn Ser Gly Val Cys Lys Glu Ser Glu 835
840 845 Asp Tyr Glu Ser Phe Ser Cys
Val Cys Pro Thr Gly Trp Gln Gly Gln 850 855
860 Thr Cys Glu Ile Asp Ile Asn Glu Cys Val Lys Ser
Pro Cys Arg His 865 870 875
880 Gly Ala Ser Cys Gln Asn Thr Asn Gly Ser Tyr Arg Cys Leu Cys Gln
885 890 895 Ala Gly Tyr
Thr Gly Arg Asn Cys Glu Ser Asp Ile Asp Asp Cys Arg 900
905 910 Pro Asn Pro Cys His Asn Gly Gly
Ser Cys Thr Asp Gly Val Asn Ala 915 920
925 Ala Phe Cys Asp Cys Leu Pro Gly Phe Gln Gly Ala Phe
Cys Glu Glu 930 935 940
Asp Ile Asn Glu Cys Ala Thr Asn Pro Cys Gln Asn Gly Ala Asn Cys 945
950 955 960 Thr Asp Cys Val
Asp Ser Tyr Thr Cys Thr Cys Pro Thr Gly Phe Asn 965
970 975 Gly Ile His Cys Glu Asn Asn Thr Pro
Asp Cys Thr Glu Ser Ser Cys 980 985
990 Phe Asn Gly Gly Thr Cys Val Asp Gly Ile Asn Ser Phe
Thr Cys Leu 995 1000 1005
Cys Pro Pro Gly Phe Thr Gly Ser Tyr Cys Gln Tyr Asp Val Asn
1010 1015 1020 Glu Cys Asp
Ser Arg Pro Cys Leu His Gly Gly Thr Cys Gln Asp 1025
1030 1035 Ser Tyr Gly Thr Tyr Lys Cys Thr
Cys Pro Gln Gly Tyr Thr Gly 1040 1045
1050 Leu Asn Cys Gln Asn Leu Val Arg Trp Cys Asp Ser Ala
Pro Cys 1055 1060 1065
Lys Asn Gly Gly Lys Cys Trp Gln Thr Asn Thr Gln Tyr His Cys 1070
1075 1080 Glu Cys Arg Ser Gly
Trp Thr Gly Phe Asn Cys Asp Val Leu Ser 1085 1090
1095 Val Ser Cys Glu Val Ala Ala Gln Lys Arg
Gly Ile Asp Val Thr 1100 1105 1110
Leu Leu Cys Gln His Gly Gly Leu Cys Val Asp Glu Glu Asp Lys
1115 1120 1125 His Tyr
Cys His Cys Gln Ala Gly Tyr Thr Gly Ser Tyr Cys Glu 1130
1135 1140 Asp Glu Val Asp Glu Cys Ser
Pro Asn Pro Cys Gln Asn Gly Ala 1145 1150
1155 Thr Cys Thr Asp Tyr Leu Gly Gly Phe Ser Cys Lys
Cys Val Ala 1160 1165 1170
Gly Tyr His Gly Ser Asn Cys Ser Glu Glu Ile Asn Glu Cys Leu 1175
1180 1185 Ser Gln Pro Cys Gln
Asn Gly Gly Thr Cys Ile Asp Leu Thr Asn 1190 1195
1200 Thr Tyr Lys Cys Ser Cys Pro Arg Gly Thr
Gln Gly Val His Cys 1205 1210 1215
Glu Ile Asn Val Asp Asp Cys His Pro Pro Leu Asp Pro Ala Ser
1220 1225 1230 Arg Ser
Pro Lys Cys Phe Asn Asn Gly Thr Cys Val Asp Gln Val 1235
1240 1245 Gly Gly Tyr Thr Cys Thr Cys
Pro Pro Gly Phe Val Gly Glu Arg 1250 1255
1260 Cys Glu Gly Asp Val Asn Glu Cys Leu Ser Asn Pro
Cys Asp Pro 1265 1270 1275
Arg Gly Thr Gln Asn Cys Val Gln Arg Val Asn Asp Phe His Cys 1280
1285 1290 Glu Cys Arg Ala Gly
His Thr Gly Arg Arg Cys Glu Ser Val Ile 1295 1300
1305 Asn Gly Cys Arg Gly Lys Pro Cys Arg Asn
Gly Gly Val Cys Ala 1310 1315 1320
Val Ala Ser Asn Thr Ala Arg Gly Phe Ile Cys Arg Cys Pro Ala
1325 1330 1335 Arg Phe
Glu Gly Ala Thr Cys Glu Asn Asp Ala Arg Thr Cys Gly 1340
1345 1350 Ser Leu Arg Cys Leu Asn Gly
Gly Thr Cys Ile Ser Gly Pro Arg 1355 1360
1365 Ser Pro Thr Cys Leu Cys Leu Gly Ser Phe Thr Gly
Pro Glu Cys 1370 1375 1380
Gln Phe Pro Ala Ser Ser Pro Cys Val Gly Ser Asn Pro Cys Tyr 1385
1390 1395 Asn Gln Gly Thr Cys
Glu Pro Thr Ser Glu Ser Pro Phe Tyr Arg 1400 1405
1410 Cys Leu Cys Pro Ala Lys Phe Asn Gly Leu
Leu Cys His Ile Leu 1415 1420 1425
Asp Tyr Ser Phe Thr 1430 25PRTRattus norvegius
2Asp Leu Gly Pro Gly 1 5 31419PRTRattus norvegicus 3Met
Pro Ala Leu Arg Pro Ala Ala Leu Arg Ala Leu Leu Trp Leu Trp 1
5 10 15 Leu Cys Gly Ala Gly Pro
Ala His Ala Leu Gln Cys Arg Gly Gly Gln 20
25 30 Glu Pro Cys Val Asn Glu Gly Thr Cys Val
Thr Tyr His Asn Gly Thr 35 40
45 Gly Tyr Cys Arg Cys Pro Glu Gly Phe Leu Gly Glu Tyr Cys
Gln His 50 55 60
Arg Asp Pro Cys Glu Lys Asn Arg Cys Gln Asn Gly Gly Thr Cys Val 65
70 75 80 Thr Gln Ala Met Leu
Gly Lys Ala Thr Cys Arg Cys Ala Pro Gly Phe 85
90 95 Thr Gly Glu Asp Cys Gln Tyr Ser Thr Ser
His Pro Cys Phe Val Ser 100 105
110 Arg Pro Cys Gln Asn Gly Gly Thr Cys His Met Leu Ser Trp Asp
Thr 115 120 125 Tyr
Glu Cys Thr Cys Gln Val Gly Phe Thr Gly Lys Gln Cys Gln Trp 130
135 140 Thr Asp Val Cys Leu Ser
His Pro Cys Glu Asn Gly Ser Thr Cys Ser 145 150
155 160 Ser Val Ala Asn Gln Phe Ser Cys Arg Cys Pro
Ala Gly Ile Thr Gly 165 170
175 Gln Lys Cys Asp Ala Asp Ile Asn Glu Cys Asp Ile Pro Gly Arg Cys
180 185 190 Gln His
Gly Gly Thr Cys Leu Asn Leu Pro Gly Ser Tyr Arg Cys Gln 195
200 205 Cys Pro Gln Arg Phe Thr Gly
Gln His Cys Asp Ser Pro Tyr Val Pro 210 215
220 Cys Ala Pro Ser Pro Cys Val Asn Gly Gly Thr Cys
Arg Gln Thr Gly 225 230 235
240 Asp Phe Thr Ser Glu Cys His Cys Leu Pro Gly Phe Glu Gly Ser Asn
245 250 255 Cys Glu Arg
Asn Ile Asp Asp Cys Pro Asn His Lys Cys Gln Asn Gly 260
265 270 Gly Val Cys Val Asp Gly Val Asn
Thr Tyr Asn Cys Arg Cys Pro Pro 275 280
285 Gln Trp Thr Gly Gln Phe Cys Thr Glu Asp Val Asp Glu
Cys Leu Leu 290 295 300
Gln Pro Asn Ala Cys Gln Asn Gly Gly Thr Cys Thr Asn Arg Asn Gly 305
310 315 320 Gly Tyr Gly Cys
Val Cys Val Asn Gly Trp Ser Gly Asp Asp Cys Ser 325
330 335 Glu Asn Ile Asp Asp Cys Ala Phe Ala
Ser Cys Thr Pro Gly Ser Thr 340 345
350 Cys Ile Asp Arg Val Ala Ser Phe Ser Cys Leu Cys Pro Glu
Gly Lys 355 360 365
Ala Gly Leu Leu Cys His Leu Asp Asp Ala Cys Ile Ser Asn Pro Cys 370
375 380 His Lys Gly Ala Leu
Cys Asp Thr Asn Pro Leu Asn Gly Gln Tyr Ile 385 390
395 400 Cys Thr Cys Pro Gln Ala Tyr Lys Gly Ala
Asp Cys Thr Glu Asp Val 405 410
415 Asp Glu Cys Ala Met Ala Asn Ser Asn Pro Cys Glu His Ala Gly
Lys 420 425 430 Cys
Val Asn Thr Asp Gly Ala Phe His Cys Glu Cys Leu Lys Gly Tyr 435
440 445 Ala Gly Pro Arg Cys Glu
Met Asp Ile Asn Glu Cys His Ser Asp Pro 450 455
460 Cys Gln Asn Asp Ala Thr Cys Leu Asp Lys Ile
Gly Gly Phe Thr Cys 465 470 475
480 Leu Cys Met Pro Gly Phe Lys Gly Val His Cys Glu Leu Glu Val Asn
485 490 495 Glu Cys
Gln Ser Asn Pro Cys Val Asn Asn Gly Gln Cys Val Asp Lys 500
505 510 Val Asn Arg Phe Gln Cys Leu
Cys Pro Pro Gly Phe Thr Gly Pro Val 515 520
525 Cys Gln Ile Asp Ile Asp Asp Cys Ser Ser Thr Pro
Cys Leu Asn Gly 530 535 540
Ala Lys Cys Ile Asp His Pro Asn Gly Tyr Glu Cys Gln Cys Ala Thr 545
550 555 560 Gly Phe Thr
Gly Thr Leu Cys Asp Glu Asn Ile Asp Asn Cys Asp Pro 565
570 575 Asp Pro Cys His His Gly Gln Cys
Gln Asp Gly Ile Asp Ser Tyr Thr 580 585
590 Cys Ile Cys Asn Pro Gly Tyr Met Gly Ala Ile Cys Ser
Asp Gln Ile 595 600 605
Asp Glu Cys Tyr Ser Ser Pro Cys Leu Asn Asp Gly Arg Cys Ile Asp 610
615 620 Leu Val Asn Gly
Tyr Gln Cys Asn Cys Gln Pro Gly Thr Ser Gly Leu 625 630
635 640 Asn Cys Glu Ile Asn Phe Asp Asp Cys
Ala Ser Asn Pro Cys Leu His 645 650
655 Gly Ala Cys Val Asp Gly Ile Asn Arg Tyr Ser Cys Val Cys
Ser Pro 660 665 670
Gly Phe Thr Gly Gln Arg Cys Asn Ile Asp Ile Asp Glu Cys Ala Ser
675 680 685 Asn Pro Cys Arg
Lys Asp Ala Thr Cys Ile Asn Asp Val Asn Gly Phe 690
695 700 Arg Cys Met Cys Pro Glu Gly Pro
His His Pro Ser Cys Tyr Ser Gln 705 710
715 720 Val Asn Glu Cys Leu Ser Ser Pro Cys Ile His Gly
Asn Cys Thr Gly 725 730
735 Gly Leu Ser Gly Tyr Lys Cys Leu Cys Asp Ala Gly Trp Val Gly Ile
740 745 750 Asn Cys Glu
Val Asp Lys Asn Glu Cys Leu Ser Asn Pro Cys Gln Asn 755
760 765 Gly Gly Thr Cys Asn Asn Leu Val
Asn Gly Tyr Arg Cys Thr Cys Lys 770 775
780 Lys Gly Phe Lys Gly Tyr Asn Cys Gln Val Asn Ile Asp
Glu Cys Ala 785 790 795
800 Ser Asn Pro Cys Leu Asn Gln Gly Thr Cys Leu Asp Asp Val Ser Gly
805 810 815 Tyr Thr Cys His
Cys Met Leu Pro Tyr Thr Gly Lys Asn Cys Gln Thr 820
825 830 Val Leu Ala Pro Cys Ser Pro Asn Pro
Cys Glu Asn Ala Ala Val Cys 835 840
845 Lys Glu Ala Pro Asn Phe Glu Ser Phe Thr Cys Leu Cys Ala
Pro Gly 850 855 860
Trp Gln Gly Gln Arg Cys Thr Val Asp Val Asp Glu Cys Val Ser Lys 865
870 875 880 Pro Cys Met Asn Asn
Gly Ile Cys His Asn Thr Gln Gly Ser Tyr Met 885
890 895 Cys Glu Cys Pro Pro Gly Phe Ser Gly Met
Asp Cys Glu Glu Asp Ile 900 905
910 Asn Asp Cys Leu Ala Asn Pro Cys Gln Asn Gly Gly Ser Cys Val
Asp 915 920 925 Lys
Val Asn Thr Phe Ser Cys Leu Cys Leu Pro Gly Phe Val Gly Asp 930
935 940 Lys Cys Gln Thr Asp Met
Asn Glu Cys Leu Ser Glu Pro Cys Lys Asn 945 950
955 960 Gly Gly Thr Cys Ser Asp Tyr Val Asn Ser Tyr
Thr Cys Thr Cys Pro 965 970
975 Ala Gly Phe His Gly Val His Cys Glu Asn Asn Ile Asp Glu Cys Thr
980 985 990 Glu Ser
Ser Cys Phe Asn Gly Gly Thr Cys Val Asp Gly Ile Asn Ser 995
1000 1005 Phe Ser Cys Leu Cys
Pro Val Gly Phe Thr Gly Pro Phe Cys Leu 1010 1015
1020 His Asp Ile Asn Glu Cys Ser Ser Asn Pro
Cys Leu Asn Ser Gly 1025 1030 1035
Thr Cys Val Asp Gly Leu Gly Thr Tyr Arg Cys Thr Cys Pro Leu
1040 1045 1050 Gly Tyr
Thr Gly Lys Asn Cys Gln Thr Leu Val Asn Leu Cys Ser 1055
1060 1065 Pro Ser Pro Cys Lys Asn Lys
Gly Thr Cys Ala Gln Glu Lys Ala 1070 1075
1080 Arg Pro Arg Cys Leu Cys Pro Pro Gly Trp Asp Gly
Ala Tyr Cys 1085 1090 1095
Asp Val Leu Asn Val Ser Cys Lys Ala Ala Ala Leu Gln Lys Gly 1100
1105 1110 Val Pro Val Glu His
Leu Cys Gln His Ser Gly Ile Cys Ile Asn 1115 1120
1125 Ala Gly Asn Thr His His Cys Gln Cys Pro
Leu Gly Tyr Thr Gly 1130 1135 1140
Ser Tyr Cys Glu Glu Gln Leu Asp Glu Cys Ala Ser Asn Pro Cys
1145 1150 1155 Gln His
Gly Ala Thr Cys Ser Asp Phe Ile Gly Gly Tyr Arg Cys 1160
1165 1170 Glu Cys Val Pro Gly Tyr Gln
Gly Val Asn Cys Glu Tyr Glu Val 1175 1180
1185 Asp Glu Cys Gln Asn Gln Pro Cys Gln Asn Gly Gly
Thr Cys Ile 1190 1195 1200
Asp Leu Val Asn His Phe Lys Cys Ser Cys Pro Pro Gly Thr Arg 1205
1210 1215 Gly Leu Leu Cys Glu
Glu Asn Ile Asp Asp Cys Ala Gly Ala Pro 1220 1225
1230 His Cys Leu Asn Gly Gly Gln Cys Val Asp
Arg Ile Gly Gly Tyr 1235 1240 1245
Ser Cys Arg Cys Leu Pro Gly Phe Ala Gly Glu Arg Cys Glu Gly
1250 1255 1260 Asp Ile
Asn Glu Cys Leu Ser Asn Pro Cys Ser Ser Glu Gly Ser 1265
1270 1275 Leu Asp Cys Ile Gln Leu Lys
Asn Asn Tyr Gln Cys Val Cys Arg 1280 1285
1290 Ser Ala Phe Thr Gly Arg His Cys Glu Thr Phe Leu
Asp Val Cys 1295 1300 1305
Pro Gln Lys Pro Cys Leu Asn Gly Gly Thr Cys Ala Val Ala Ser 1310
1315 1320 Asn Val Pro Asp Gly
Phe Ile Cys Arg Cys Pro Pro Gly Phe Ser 1325 1330
1335 Gly Ala Arg Cys Gln Ser Ser Cys Gly Gln
Val Lys Cys Arg Arg 1340 1345 1350
Gly Glu Gln Cys Val His Thr Ala Ser Gly Pro His Cys Phe Cys
1355 1360 1365 Pro Asn
His Lys Asp Cys Glu Ser Gly Cys Ala Ser Asn Pro Cys 1370
1375 1380 Gln His Gly Gly Thr Cys Tyr
Pro Gln Arg Gln Pro Pro Tyr Tyr 1385 1390
1395 Ser Cys Arg Cys Ser Pro Pro Phe Trp Gly Ser His
Cys Glu Ser 1400 1405 1410
Tyr Thr Ala Pro Thr Ser 1415 41379PRTMus musculus
4Met Gly Leu Gly Ala Arg Gly Arg Arg Arg Arg Arg Arg Leu Met Ala 1
5 10 15 Leu Pro Pro Pro
Pro Pro Pro Met Arg Ala Leu Pro Leu Leu Leu Leu 20
25 30 Leu Ala Gly Leu Gly Ala Ala Ala Pro
Pro Cys Leu Asp Gly Ser Pro 35 40
45 Cys Ala Asn Gly Gly Arg Cys Thr His Gln Gln Pro Ser Leu
Glu Ala 50 55 60
Ala Cys Leu Cys Leu Pro Gly Trp Val Gly Glu Arg Cys Gln Leu Glu 65
70 75 80 Asp Pro Cys His Ser
Gly Pro Cys Ala Gly Arg Gly Val Cys Gln Ser 85
90 95 Ser Val Val Ala Gly Thr Ala Arg Phe Ser
Cys Arg Cys Leu Arg Gly 100 105
110 Phe Gln Gly Pro Asp Cys Ser Gln Pro Asp Pro Cys Val Ser Arg
Pro 115 120 125 Cys
Val His Gly Ala Pro Cys Ser Val Gly Pro Asp Gly Arg Phe Ala 130
135 140 Cys Ala Cys Pro Pro Gly
Tyr Gln Gly Gln Ser Cys Gln Ser Asp Ile 145 150
155 160 Asp Glu Cys Arg Ser Gly Thr Thr Cys Arg His
Gly Gly Thr Cys Leu 165 170
175 Asn Thr Pro Gly Ser Phe Arg Cys Gln Cys Pro Leu Gly Tyr Thr Gly
180 185 190 Leu Leu
Cys Glu Asn Pro Val Val Pro Cys Ala Pro Ser Pro Cys Arg 195
200 205 Asn Gly Gly Thr Cys Arg Gln
Ser Ser Asp Val Thr Tyr Asp Cys Ala 210 215
220 Cys Leu Pro Gly Phe Glu Gly Gln Asn Cys Glu Val
Asn Val Asp Asp 225 230 235
240 Cys Pro Gly His Arg Cys Leu Asn Gly Gly Thr Cys Val Asp Gly Val
245 250 255 Asn Thr Tyr
Asn Cys Gln Cys Pro Pro Glu Trp Thr Gly Gln Phe Cys 260
265 270 Thr Glu Asp Val Asp Glu Cys Gln
Leu Gln Pro Asn Ala Cys His Asn 275 280
285 Gly Gly Thr Cys Phe Asn Leu Leu Gly Gly His Ser Cys
Val Cys Val 290 295 300
Asn Gly Trp Thr Gly Glu Ser Cys Ser Gln Asn Ile Asp Asp Cys Ala 305
310 315 320 Thr Ala Val Cys
Phe His Gly Ala Thr Cys His Asp Arg Val Ala Ser 325
330 335 Phe Tyr Cys Ala Cys Pro Met Gly Lys
Thr Gly Leu Leu Cys His Leu 340 345
350 Asp Asp Ala Cys Val Ser Asn Pro Cys His Glu Asp Ala Ile
Cys Asp 355 360 365
Thr Asn Pro Val Ser Gly Arg Ala Ile Cys Thr Cys Pro Pro Gly Phe 370
375 380 Thr Gly Gly Ala Cys
Asp Gln Asp Val Asp Glu Cys Ser Ile Gly Ala 385 390
395 400 Asn Pro Cys Glu His Leu Gly Arg Cys Val
Asn Thr Gln Gly Ser Phe 405 410
415 Leu Cys Gln Cys Gly Arg Gly Tyr Thr Gly Pro Arg Cys Glu Thr
Asp 420 425 430 Val
Asn Glu Cys Leu Ser Gly Pro Cys Arg Asn Gln Ala Thr Cys Leu 435
440 445 Asp Arg Ile Gly Gln Phe
Thr Cys Ile Cys Met Ala Gly Phe Thr Gly 450 455
460 Thr Tyr Cys Glu Val Asp Ile Asp Glu Cys Gln
Ser Ser Pro Cys Val 465 470 475
480 Asn Gly Gly Val Cys Lys Asp Arg Val Asn Gly Phe Ser Cys Thr Cys
485 490 495 Pro Ser
Gly Phe Ser Gly Ser Met Cys Gln Leu Asp Val Asp Glu Cys 500
505 510 Ala Ser Thr Pro Cys Arg Asn
Gly Ala Lys Cys Val Asp Gln Pro Asp 515 520
525 Gly Tyr Glu Cys Arg Cys Ala Glu Gly Phe Glu Gly
Thr Leu Cys Glu 530 535 540
Arg Asn Val Asp Asp Cys Ser Pro Asp Pro Cys His His Gly Arg Cys 545
550 555 560 Val Asp Gly
Ile Ala Ser Phe Ser Cys Ala Cys Ala Pro Gly Tyr Thr 565
570 575 Gly Ile Arg Cys Glu Ser Gln Val
Asp Glu Cys Arg Ser Gln Pro Cys 580 585
590 Arg Tyr Gly Gly Lys Cys Leu Asp Leu Val Asp Lys Tyr
Leu Cys Arg 595 600 605
Cys Pro Pro Gly Thr Thr Gly Val Asn Cys Glu Val Asn Ile Asp Asp 610
615 620 Cys Ala Ser Asn
Pro Cys Thr Phe Gly Val Cys Arg Asp Gly Ile Asn 625 630
635 640 Arg Tyr Asp Cys Val Cys Gln Pro Gly
Phe Thr Gly Pro Leu Cys Asn 645 650
655 Val Glu Ile Asn Glu Cys Ala Ser Ser Pro Cys Gly Glu Gly
Gly Ser 660 665 670
Cys Val Asp Gly Glu Asn Gly Phe His Cys Leu Cys Pro Pro Gly Ser
675 680 685 Leu Pro Pro Leu
Cys Leu Pro Ala Asn His Pro Cys Ala His Lys Pro 690
695 700 Cys Ser His Gly Val Cys His Asp
Ala Pro Gly Gly Phe Arg Cys Val 705 710
715 720 Cys Glu Pro Gly Trp Ser Gly Pro Arg Cys Ser Gln
Ser Leu Ala Pro 725 730
735 Asp Ala Cys Glu Ser Gln Pro Cys Gln Ala Gly Gly Thr Cys Thr Ser
740 745 750 Asp Gly Ile
Gly Phe Arg Cys Thr Cys Ala Pro Gly Phe Gln Gly His 755
760 765 Gln Cys Glu Val Leu Ser Pro Cys
Thr Pro Ser Leu Cys Glu His Gly 770 775
780 Gly His Cys Glu Ser Asp Pro Asp Arg Leu Thr Val Cys
Ser Cys Pro 785 790 795
800 Pro Gly Trp Gln Gly Pro Arg Cys Gln Gln Asp Val Asp Glu Cys Ala
805 810 815 Gly Ala Ser Pro
Cys Gly Pro His Gly Thr Cys Thr Asn Leu Pro Gly 820
825 830 Asn Phe Arg Cys Ile Cys His Arg Gly
Tyr Thr Gly Pro Phe Cys Asp 835 840
845 Gln Asp Ile Asp Asp Cys Asp Pro Asn Pro Cys Leu His Gly
Gly Ser 850 855 860
Cys Gln Asp Gly Val Gly Ser Phe Ser Cys Ser Cys Leu Asp Gly Phe 865
870 875 880 Ala Gly Pro Arg Cys
Ala Arg Asp Val Asp Glu Cys Leu Ser Ser Pro 885
890 895 Cys Gly Pro Gly Thr Cys Thr Asp His Val
Ala Ser Phe Thr Cys Ala 900 905
910 Cys Pro Pro Gly Tyr Gly Gly Phe His Cys Glu Ile Asp Leu Pro
Asp 915 920 925 Cys
Ser Pro Ser Ser Cys Phe Asn Gly Gly Thr Cys Val Asp Gly Val 930
935 940 Ser Ser Phe Ser Cys Leu
Cys Arg Pro Gly Tyr Thr Gly Thr His Cys 945 950
955 960 Gln Tyr Glu Ala Asp Pro Cys Phe Ser Arg Pro
Cys Leu His Gly Gly 965 970
975 Ile Cys Asn Pro Thr His Pro Gly Phe Glu Cys Thr Cys Arg Glu Gly
980 985 990 Phe Thr
Gly Ser Gln Cys Gln Asn Pro Val Asp Trp Cys Ser Gln Ala 995
1000 1005 Pro Cys Gln Asn Gly
Gly Arg Cys Val Gln Thr Gly Ala Tyr Cys 1010 1015
1020 Ile Cys Pro Pro Gly Trp Ser Gly Arg Leu
Cys Asp Ile Gln Ser 1025 1030 1035
Leu Pro Cys Thr Glu Ala Ala Ala Gln Met Gly Val Arg Leu Glu
1040 1045 1050 Gln Leu
Cys Gln Glu Gly Gly Lys Cys Ile Asp Lys Gly Arg Ser 1055
1060 1065 His Tyr Cys Val Cys Pro Glu
Gly Arg Thr Gly Ser His Cys Glu 1070 1075
1080 His Glu Val Asp Pro Cys Thr Ala Gln Pro Cys Gln
His Gly Gly 1085 1090 1095
Thr Cys Arg Gly Tyr Met Gly Gly Tyr Val Cys Glu Cys Pro Ala 1100
1105 1110 Gly Tyr Ala Gly Asp
Ser Cys Glu Asp Asn Ile Asp Glu Cys Ala 1115 1120
1125 Ser Gln Pro Cys Gln Asn Gly Gly Ser Cys
Ile Asp Leu Val Ala 1130 1135 1140
Arg Tyr Leu Cys Ser Cys Pro Pro Gly Thr Leu Gly Val Leu Cys
1145 1150 1155 Glu Ile
Asn Glu Asp Asp Cys Asp Leu Gly Pro Ser Leu Asp Ser 1160
1165 1170 Gly Val Gln Cys Leu His Asn
Gly Thr Cys Val Asp Leu Val Gly 1175 1180
1185 Gly Phe Arg Cys Asn Cys Pro Pro Gly Tyr Thr Gly
Leu His Cys 1190 1195 1200
Glu Ala Asp Ile Asn Glu Cys Arg Pro Gly Ala Cys His Ala Ala 1205
1210 1215 His Thr Arg Asp Cys
Leu Gln Asp Pro Gly Gly His Phe Arg Cys 1220 1225
1230 Val Cys His Pro Gly Phe Thr Gly Pro Arg
Cys Gln Ile Ala Leu 1235 1240 1245
Ser Pro Cys Glu Ser Gln Pro Cys Gln His Gly Gly Gln Cys Arg
1250 1255 1260 His Ser
Leu Gly Arg Gly Gly Gly Leu Thr Phe Thr Cys His Cys 1265
1270 1275 Val Pro Pro Phe Trp Gly Leu
Arg Cys Glu Arg Val Ala Arg Ser 1280 1285
1290 Cys Arg Glu Leu Gln Cys Pro Val Gly Ile Pro Cys
Gln Gln Thr 1295 1300 1305
Ala Arg Gly Pro Arg Cys Ala Cys Pro Pro Gly Leu Ser Gly Pro 1310
1315 1320 Ser Cys Arg Val Ser
Arg Ala Ser Pro Ser Gly Ala Thr Asn Ala 1325 1330
1335 Ser Cys Ala Ser Ala Pro Cys Leu His Gly
Gly Ser Cys Leu Pro 1340 1345 1350
Val Gln Ser Val Pro Phe Phe Arg Cys Val Cys Ala Pro Gly Trp
1355 1360 1365 Gly Gly
Pro Arg Cys Glu Thr Pro Ser Ala Ala 1370 1375
51170PRTMus musculus 5Met Gln Pro Gln Leu Leu Leu Leu Leu Leu
Leu Pro Leu Asn Phe Pro 1 5 10
15 Val Ile Leu Thr Arg Glu Leu Leu Cys Gly Gly Ser Pro Glu Pro
Cys 20 25 30 Ala
Asn Gly Gly Thr Cys Leu Arg Leu Ser Arg Gly Gln Gly Ile Cys 35
40 45 Gln Cys Ala Pro Gly Phe
Leu Gly Glu Thr Cys Gln Phe Pro Asp Pro 50 55
60 Cys Arg Asp Thr Gln Leu Cys Lys Asn Gly Gly
Ser Cys Gln Ala Leu 65 70 75
80 Leu Pro Thr Pro Pro Ser Ser Arg Ser Pro Thr Ser Pro Leu Thr Pro
85 90 95 His Phe
Ser Cys Thr Cys Pro Ser Gly Phe Thr Gly Asp Arg Cys Gln 100
105 110 Thr His Leu Glu Glu Leu Cys
Pro Pro Ser Phe Cys Ser Asn Gly Gly 115 120
125 His Cys Tyr Val Gln Ala Ser Gly Arg Pro Gln Cys
Ser Cys Glu Pro 130 135 140
Gly Trp Thr Gly Glu Gln Cys Gln Leu Arg Asp Phe Cys Ser Ala Asn 145
150 155 160 Pro Cys Ala
Asn Gly Gly Val Cys Leu Ala Thr Tyr Pro Gln Ile Gln 165
170 175 Cys Arg Cys Pro Pro Gly Phe Glu
Gly His Thr Cys Glu Arg Asp Ile 180 185
190 Asn Glu Cys Phe Leu Glu Pro Gly Pro Cys Pro Gln Gly
Thr Ser Cys 195 200 205
His Asn Thr Leu Gly Ser Tyr Gln Cys Leu Cys Pro Val Gly Gln Glu 210
215 220 Gly Pro Gln Cys
Lys Leu Arg Lys Gly Ala Cys Pro Pro Gly Ser Cys 225 230
235 240 Leu Asn Gly Gly Thr Cys Gln Leu Val
Pro Glu Gly His Ser Thr Phe 245 250
255 His Leu Cys Leu Cys Pro Pro Gly Phe Thr Gly Leu Asp Cys
Glu Met 260 265 270
Asn Pro Asp Asp Cys Val Arg His Gln Cys Gln Asn Gly Ala Thr Cys
275 280 285 Leu Asp Gly Leu
Asp Thr Tyr Thr Cys Leu Cys Pro Lys Thr Trp Lys 290
295 300 Gly Trp Asp Cys Ser Glu Asp Ile
Asp Glu Cys Glu Ala Arg Gly Pro 305 310
315 320 Pro Arg Cys Arg Asn Gly Gly Thr Cys Gln Asn Thr
Ala Gly Ser Phe 325 330
335 His Cys Val Cys Val Ser Gly Trp Gly Gly Ala Gly Cys Glu Glu Asn
340 345 350 Leu Asp Asp
Cys Ala Ala Ala Thr Cys Ala Pro Gly Ser Thr Cys Ile 355
360 365 Asp Arg Val Gly Ser Phe Ser Cys
Leu Cys Pro Pro Gly Arg Thr Gly 370 375
380 Leu Leu Cys His Leu Glu Asp Met Cys Leu Ser Gln Pro
Cys His Val 385 390 395
400 Asn Ala Gln Cys Ser Thr Asn Pro Leu Thr Gly Ser Thr Leu Cys Ile
405 410 415 Cys Gln Pro Gly
Tyr Ser Gly Ser Thr Cys His Gln Asp Leu Asp Glu 420
425 430 Cys Gln Met Ala Gln Gln Gly Pro Ser
Pro Cys Glu His Gly Gly Ser 435 440
445 Cys Ile Asn Thr Pro Gly Ser Phe Asn Cys Leu Cys Leu Pro
Gly Tyr 450 455 460
Thr Gly Ser Arg Cys Glu Ala Asp His Asn Glu Cys Leu Ser Gln Pro 465
470 475 480 Cys His Pro Gly Ser
Thr Cys Leu Asp Leu Leu Ala Thr Phe His Cys 485
490 495 Leu Cys Pro Pro Gly Leu Glu Gly Arg Leu
Cys Glu Val Glu Val Asn 500 505
510 Glu Cys Thr Ser Asn Pro Cys Leu Asn Gln Ala Ala Cys His Asp
Leu 515 520 525 Leu
Asn Gly Phe Gln Cys Leu Cys Leu Pro Gly Phe Thr Gly Ala Arg 530
535 540 Cys Glu Lys Asp Met Asp
Glu Cys Ser Ser Thr Pro Cys Ala Asn Gly 545 550
555 560 Gly Arg Cys Arg Asp Gln Pro Gly Ala Phe Tyr
Cys Glu Cys Leu Pro 565 570
575 Gly Phe Glu Gly Pro His Cys Glu Lys Glu Val Asp Glu Cys Leu Ser
580 585 590 Asp Pro
Cys Pro Val Gly Ala Ser Cys Leu Asp Leu Pro Gly Ala Phe 595
600 605 Phe Cys Leu Cys Arg Pro Gly
Phe Thr Gly Gln Leu Cys Glu Val Pro 610 615
620 Leu Cys Thr Pro Asn Met Cys Gln Pro Gly Gln Gln
Cys Gln Gly Gln 625 630 635
640 Glu His Arg Ala Pro Cys Leu Cys Pro Asp Gly Ser Pro Gly Cys Val
645 650 655 Pro Ala Glu
Asp Asn Cys Pro Cys His His Gly His Cys Gln Arg Ser 660
665 670 Leu Cys Val Cys Asp Glu Gly Trp
Thr Gly Pro Glu Cys Glu Thr Glu 675 680
685 Leu Gly Gly Cys Ile Ser Thr Pro Cys Ala His Gly Gly
Thr Cys His 690 695 700
Pro Gln Pro Ser Gly Tyr Asn Cys Thr Cys Pro Ala Gly Tyr Met Gly 705
710 715 720 Leu Thr Cys Ser
Glu Glu Val Thr Ala Cys His Ser Gly Pro Cys Leu 725
730 735 Asn Gly Gly Ser Cys Ser Ile Arg Pro
Glu Gly Tyr Ser Cys Thr Cys 740 745
750 Leu Pro Ser His Thr Gly Arg His Cys Gln Thr Ala Val Asp
His Cys 755 760 765
Val Ser Ala Ser Cys Leu Asn Gly Gly Thr Cys Val Asn Lys Pro Gly 770
775 780 Thr Phe Phe Cys Leu
Cys Ala Thr Gly Phe Gln Gly Leu His Cys Glu 785 790
795 800 Glu Lys Thr Asn Pro Ser Cys Ala Asp Ser
Pro Cys Arg Asn Lys Ala 805 810
815 Thr Cys Gln Asp Thr Pro Arg Gly Ala Arg Cys Leu Cys Ser Pro
Gly 820 825 830 Tyr
Thr Gly Ser Ser Cys Gln Thr Leu Ile Asp Leu Cys Ala Arg Lys 835
840 845 Pro Cys Pro His Thr Ala
Arg Cys Leu Gln Ser Gly Pro Ser Phe Gln 850 855
860 Cys Leu Cys Leu Gln Gly Trp Thr Gly Ala Leu
Cys Asp Phe Pro Leu 865 870 875
880 Ser Cys Gln Lys Ala Ala Met Ser Gln Gly Ile Glu Ile Ser Gly Leu
885 890 895 Cys Gln
Asn Gly Gly Leu Cys Ile Asp Thr Gly Ser Ser Tyr Phe Cys 900
905 910 Arg Cys Pro Pro Gly Phe Gln
Gly Lys Leu Cys Gln Asp Asn Val Asn 915 920
925 Pro Cys Glu Pro Asn Pro Cys His His Gly Ser Thr
Cys Val Pro Gln 930 935 940
Pro Ser Gly Tyr Val Cys Gln Cys Ala Pro Gly Tyr Glu Gly Gln Asn 945
950 955 960 Cys Ser Lys
Val Leu Asp Ala Cys Gln Ser Gln Pro Cys His Asn His 965
970 975 Gly Thr Cys Thr Ser Arg Pro Gly
Gly Phe His Cys Ala Cys Pro Pro 980 985
990 Gly Phe Val Gly Leu Arg Cys Glu Gly Asp Val Asp
Glu Cys Leu Asp 995 1000 1005
Arg Pro Cys His Pro Ser Gly Thr Ala Ala Cys His Ser Leu Ala
1010 1015 1020 Asn Ala Phe
Tyr Cys Gln Cys Leu Pro Gly His Thr Gly Gln Arg 1025
1030 1035 Cys Glu Val Glu Met Asp Leu Cys
Gln Ser Gln Pro Cys Ser Asn 1040 1045
1050 Gly Gly Ser Cys Glu Ile Thr Thr Gly Pro Pro Pro Gly
Phe Thr 1055 1060 1065
Cys His Cys Pro Lys Gly Phe Glu Gly Pro Thr Cys Ser His Lys 1070
1075 1080 Ala Leu Ser Cys Gly
Ile His His Cys His Asn Gly Gly Leu Cys 1085 1090
1095 Leu Pro Ser Pro Lys Pro Gly Ser Pro Pro
Leu Cys Ala Cys Leu 1100 1105 1110
Ser Gly Phe Gly Gly Pro Asp Cys Leu Thr Pro Pro Ala Pro Pro
1115 1120 1125 Gly Cys
Gly Pro Pro Ser Pro Cys Leu His Asn Gly Thr Cys Thr 1130
1135 1140 Glu Thr Pro Gly Leu Gly Asn
Pro Gly Phe Gln Cys Thr Cys Pro 1145 1150
1155 Pro Asp Ser Pro Gly Pro Arg Cys Gln Arg Pro Gly
1160 1165 1170 64293DNARattus
norvegicus 6atgacaggct tccagggctg ccaggccctg ctgcatctgg ccaaggccgt
ggttcgcttg 60agatgctccc agccaagtgg gacctgcctg aatggaggga ggtgcgaagt
ggccaacggc 120actgaagcct gtgtctgcag cggagcgttc gtgggccagc gatgccagga
ccccagccct 180tgcctcagca caccatgtaa gaatgctgga acgtgctatg ttgtggacca
tggcggcatc 240gtggactatg cctgcagttg ccccctgggt ttctctgggc ccctctgcct
gacacctctg 300gccaatgcct gcctggccaa cccctgccgc aacgggggga cctgtgacct
gctcactctc 360acagaataca agtgccggtg cccgccaggg tggtcaggaa agtcatgtca
gcaagccgac 420ccctgtgcct ccaacccctg tgccaatggt ggccagtgcc tgccctttga
gtcttcatac 480atctgtggct gcccgcccgg cttccatggc cccacctgca gacaagatgt
taacgagtgc 540agccagaacc ctgggttgtg ccgtcatggc ggcacgtgcc acaatgagat
tggctcctat 600cgctgtgcct gccgtgccac ccacactggt ccccactgcg agctgcccta
cgtgccctgc 660agcccctcac cctgccagaa cggaggcacc tgccgcccta cgggggacac
cacccacgag 720tgtgcctgcc tgccaggctt tgctggacag aactgtgaag aaaatgtgga
tgactgccca 780ggaaacaact gcaagaacgg gggtgcctgt gtggacggtg tgaataccta
caattgccgc 840tgcccaccgg agtggacagg tcagtactgc acagaggatg tggacgagtg
tcagctcatg 900cccaacgcct gccagaatgg cggaacctgc cacaactccc acggtggcta
caactgcgtg 960tgtgtcaatg gctggactgg tgaggactgc agtgagaaca ttgatgactg
tgccagtgcc 1020gcctgttttc agggtgccac ctgccatgac cgtgtggctt ccttctactg
cgagtgtcca 1080catgggcgca caggcctgct gtgccacctg aacgatgcgt gtatcagcaa
cccctgcaac 1140gagggctcca actgcgacac caaccctgtc aacggcaagg ccatctgcac
ttgcccctcg 1200gggtacacgg ggccagcctg cagccaggac gtggatgagt gcgctctagg
tgccaacccg 1260tgtgagcacg cgggcaagtg cctcaacaca ctgggctctt tcgagtgtca
gtgtctacag 1320ggctacactg ggccccgctg tgagattgat gtcaacgagt gcatctccaa
cccatgtcag 1380aatgatgcca cgtgcctgga ccagattggg gagtttcagt gtatatgtat
gccaggttat 1440gagggtgtat actgtgagat caacacggac gagtgtgcca gcagcccctg
tctacacaat 1500ggccgctgcg tggacaagat caacgagttc ctgtgtcagt gtcccaaagg
cttcagcggg 1560cacctgtgcc agtatgacgt ggatgagtgc gccagcacac catgcaagaa
cggcgccaag 1620tgcctggatg ggcccaacac ctacacctgc gtgtgcacag aaggttacac
ggggacccac 1680tgcgaggtgg acattgacga gtgtgaccct gacccctgtc actatggttt
gtgcaaggat 1740ggtgtggcca cctttacctg cctctgccag ccaggctaca caggccatca
ctgtgagacc 1800aacattaatg agtgtcacag ccagccgtgc cgccatggcg gcacctgcca
ggaccgtgac 1860aactactacc tctgcttatg cctcaagggg accacaggac ccaactgtga
gatcaatctg 1920gatgactgtg cgagcaaccc ctgtgactct ggcacgtgtc tggacaagat
cgatggctac 1980gagtgtgcgt gcgagccagg ctacacaggg agcatgtgta atgtcaacat
tgacgaatgt 2040gcgggcagcc cctgccacaa cgggggcacc tgtgaggatg gcatcgccgg
cttcacttgc 2100cgctgccccg agggctacca cgaccctacg tgcctgtctg aggtcaacga
gtgcaacagt 2160aacccctgca tccatggagc ttgccgggat ggcctcaatg gatacaaatg
tgactgtgcc 2220cctgggtgga gtgggacaaa ctgtgacatc aacaacaatg agtgtgagtc
caacccttgt 2280gtcaacggtg gcacctgcaa agacatgacc agtggctacg tatgcacctg
ccgagaaggc 2340ttcagtggcc ctaactgcca gaccaacatt aacgaatgtg cttccaaccc
ctgcctgaac 2400cagggcacct gcattgatga tgtcgctggg tacaaatgca actgccctct
gccctataca 2460ggagccacat gtgaggtggt gttggcccca tgtgccacca gcccctgcaa
aaacagtggg 2520gtatgcaagg agtctgagga ctatgagagc ttttcctgtg tctgtcccac
aggctggcaa 2580ggtcaaacct gcgagatcga catcaatgag tgtgtgaaaa gcccgtgtcg
ccatggtgcc 2640tcttgccaga acaccaatgg cagctaccgc tgcctctgcc aggctggcta
cacgggtcgc 2700aactgcgaga gtgacatcga tgactgccga cccaacccat gtcacaacgg
gggttcctgc 2760actgacgggg tcaacgcggc cttctgcgac tgcctgcccg gcttccaggg
tgccttctgt 2820gaggaggaca tcaacgaatg cgccagcaat ccatgccaaa atggcgccaa
ctgcactgac 2880tgcgtggaca gctacacgtg cacctgcccc acgggcttca atggcatcca
ttgcgagaac 2940aacacacctg actgtaccga gagctcctgt ttcaatggtg gcacctgtgt
ggatggtatc 3000aactccttca cctgtctgtg cccacctggc ttcacgggca gctactgcca
gtatgacgtc 3060aatgagtgtg actcacggcc ctgtctgcat ggtggcacct gccaagacag
ctatggtacc 3120tataagtgta cctgcccaca gggctacact ggtctcaact gccagaacct
tgtgcgctgg 3180tgtgactcag ctccctgcaa gaatggcggc aagtgctggc agaccaacac
acagtaccac 3240tgcgagtgcc gcagcggctg gactggcttc aactgcgacg tgctcagtgt
gtcctgcgag 3300gtggctgcac agaagcgagg catcgatgtc actctcctat gccagcacgg
agggctctgt 3360gtggatgagg aagacaagca ttactgccac tgccaggcag gatacacggg
cagctactgt 3420gaggacgagg tggacgagtg ctcacctaat ccctgccaga acggagccac
ctgcactgac 3480tatctcggtg gcttttcctg caagtgtgtg gctgggtacc atggctctaa
ctgctctgag 3540gagatcaacg agtgcctatc ccaaccctgc cagaatgggg gtacctgcat
tgatctgacc 3600aacacctaca agtgctcctg ccccaggggc acacagggtg tacactgtga
gatcaacgtc 3660gatgactgcc atcctcccct agaccctgct tcccgaagcc ccaaatgctt
caataatggc 3720acctgcgtgg accaggtggg tggctatacc tgcacctgcc cgccaggctt
cgtcggggag 3780cggtgcgagg gcgatgtcaa tgagtgtctc tccaacccct gtgacccacg
tggcacccag 3840aactgcgtgc agcgtgttaa tgacttccac tgcgagtgcc gggctggcca
cactggacgc 3900cgctgtgagt cggtcattaa tggctgcagg ggcaaaccat gcaggaatgg
aggtgtctgt 3960gctgtggcct ccaacaccgc ccgtggattc atctgtaggt gccctgcggg
cttcgagggt 4020gccacttgtg aaaatgacgc ccgcacttgt ggcagtttgc gctgcctcaa
cggtggtacg 4080tgcatctcag gcccacgcag tcccacctgc ctatgcctgg gctccttcac
tggccctgaa 4140tgccagttcc cagccagcag cccctgtgtg ggtagcaacc cctgctacaa
tcagggcacc 4200tgtgagccca catccgagag ccctttctac cgctgtctat gccctgccaa
attcaacggg 4260ctgctgtgcc acatcctgga ctacagcttc aca
429374257DNARattus norvegicus 7atgcccgctc tgcgtcccgc
cgcgctgcgg gcgctgctgt ggctctggct gtgcggcgcg 60ggccccgcgc acgctttgca
gtgtcgaggt ggtcaagagc cctgtgtaaa tgaggggacc 120tgtgttacct accacaacgg
cacaggctac tgccgatgtc cagagggctt cttgggagaa 180tattgtcaac atcgagaccc
ttgtgagaag aaccgctgtc agaatggtgg tacttgtgtg 240acgcaggcca tgttgggaaa
agccacctgt cgatgtgctc cagggttcac aggggaggac 300tgccaatact cgacctctca
cccctgtttt gtttcccgcc cctgtcagaa tggaggtacc 360tgccacatgc tcagctggga
cacctatgag tgcacctgtc aagttggctt cacaggaaag 420cagtgtcagt ggacagatgt
ctgtctgtct catccctgtg aaaatggaag cacctgtagc 480tctgtggcca accagttctc
ctgcagatgt cctgcaggca tcacaggcca gaagtgtgac 540gccgacatca atgaatgtga
cattccagga cgctgccaac atggtggcac ctgcctcaac 600cttcctgggt cctaccgatg
ccaatgccct cagcggttca caggccagca ctgtgacagc 660ccttacgtgc cctgtgcacc
ctcaccctgc gtcaatggag gcacctgccg tcagactgga 720gacttcactt ctgaatgcca
ttgcctgcca ggctttgaag ggagcaactg cgagcggaat 780atcgacgact gccctaacca
caagtgtcag aatggagggg tgtgtgtgga tggcgtcaat 840acttacaact gccgctgccc
ccctcagtgg actgggcagt tctgcacaga agacgtggat 900gagtgtctgc tgcagcccaa
tgcttgtcag aatggaggca cttgcaccaa ccgcaacgga 960ggctacggct gcgtgtgcgt
gaacggctgg agtggggatg actgcagcga gaacatcgat 1020gactgtgcct tcgcttcctg
cacgccaggc tccacctgta ttgaccgtgt ggcctccttc 1080tcctgccttt gtccagaggg
aaaggcaggg ctcctgtgtc atctggatga tgcctgtatc 1140agcaaccctt gtcacaaggg
ggcgctgtgt gataccaacc ccctgaatgg gcagtacatt 1200tgcacctgcc cacaggcgta
caagggcgct gactgcacag aagacgtgga tgagtgtgct 1260atggccaaca gtaacccttg
tgagcatgca ggaaagtgtg tgaatacaga tggcgccttc 1320cactgcgagt gtctgaaggg
ctacgcaggg cctcgctgtg agatggacat caacgagtgt 1380cactcagacc cctgtcagaa
cgacgccacc tgcctggata agattggagg cttcacctgt 1440ctctgcatgc cgggtttcaa
aggtgtgcat tgtgaactgg aggtgaatga atgccagagc 1500aacccgtgtg taaacaatgg
gcagtgtgtg gacaaagtca atcgcttcca gtgtctgtgt 1560ccccctggtt tcacaggacc
agtgtgccag atcgacattg acgactgctc cagtactccc 1620tgcctgaatg gggccaagtg
catcgatcac ccgaatggct atgaatgcca gtgtgccaca 1680ggattcactg gcacactgtg
tgatgagaac atcgacaact gtgacccgga tccttgccac 1740catggccagt gccaggatgg
gattgactcc tacacctgca tctgcaaccc cgggtacatg 1800ggagccatct gtagtgacca
gattgatgaa tgctacagca gcccctgcct gaatgatgga 1860cgctgcatcg acctggtgaa
cggctaccag tgcaactgcc aaccgggtac ctcaggcctt 1920aattgtgaaa ttaattttga
tgactgtgcc agcaaccctt gtctgcacgg agcctgtgtg 1980gacggcatca accgttacag
ttgtgtgtgc tctccgggat tcacagggca gaggtgcaac 2040atagacattg atgagtgtgc
ctccaacccc tgtcgcaagg atgcgacgtg catcaatgac 2100gtgaatggtt tccggtgtat
gtgccctgag ggaccacacc atcccagctg ctactcacag 2160gtgaacgagt gtttgagcag
tccctgcatc catggaaact gtactggagg tctcagtggc 2220tataagtgcc tctgcgatgc
aggctgggtt ggtatcaact gcgaagtgga caaaaatgag 2280tgtctttcta acccgtgcca
gaatggaggg acatgtaata acctggtgaa tggctacagg 2340tgtacatgca agaaggggtt
caaaggctat aactgccagg tgaacataga tgagtgtgcc 2400tcgaacccgt gtctgaacca
agggacctgc ctcgatgacg tcagtggcta cacctgccac 2460tgcatgctgc cttacacagg
caagaattgt caaacggtgt tggcgccctg ctcccctaac 2520ccgtgtgaga acgctgcagt
ttgtaaagag gcacccaact ttgagagctt cacctgcctg 2580tgtgcccctg gctggcaagg
tcagcgctgt acagttgacg ttgatgagtg tgtctccaag 2640ccgtgtatga acaatggcat
ctgccataat actcagggca gctacatgtg cgagtgccct 2700cccggcttca gtggtatgga
ctgtgaggag gacatcaatg actgccttgc caacccctgc 2760cagaacggag gctcctgtgt
ggacaaagtg aacaccttct cctgcctgtg ccttcctggc 2820ttcgtagggg acaagtgcca
aacagacatg aatgaatgtc tgagcgagcc ctgtaagaat 2880ggggggacct gctctgacta
cgtcaacagc tacacctgca cgtgccctgc gggcttccat 2940ggagtccact gtgaaaacaa
catcgatgag tgcactgaga gctcctgttt caatggcggc 3000acgtgtgttg atgggatcaa
ctctttctct tgcttatgcc ctgtgggttt cactggtccc 3060ttctgcctcc atgatatcaa
tgagtgcagc tctaacccgt gcctgaattc gggaacgtgt 3120gttgatggcc tgggtaccta
ccgatgcacc tgtcccttgg gctacactgg gaaaaactgt 3180cagaccctgg tgaacctctg
cagcccctct ccatgtaaaa acaaaggaac ttgtgctcag 3240gaaaaggcaa ggccacgctg
cctgtgtccg cctggatggg atggcgcata ctgtgatgtg 3300ctcaatgtgt cctgtaaggc
ggcagccttg cagaaaggag tacctgttga acacttgtgc 3360cagcactcgg gtatctgtat
caatgctggc aacacgcatc actgccagtg ccccctgggc 3420tacacgggga gctactgcga
ggaacagctt gacgagtgtg cgtccaatcc atgccagcat 3480ggtgccacct gcagtgactt
catcggagga tacagatgtg agtgtgttcc agggtatcag 3540ggtgtcaact gtgagtatga
agtggacgag tgccagaacc agccctgtca gaacggaggc 3600acctgcatcg acctcgtgaa
ccatttcaag tgctcgtgcc caccaggcac ccggggcctg 3660ctttgtgaag agaacattga
tgactgtgct ggggcccccc actgccttaa tggtggccag 3720tgtgtggacc ggattggagg
ctacagttgt cgctgtttgc ctggctttgc tggggagcgg 3780tgtgaggggg acatcaatga
atgcctgtcc aatccttgca gctcagaggg cagcctggac 3840tgcattcagc tcaaaaataa
ctaccagtgt gtctgccgca gcgccttcac aggccgacac 3900tgcgaaacct tcctagatgt
gtgtccccag aagccttgcc tgaatggagg gacttgtgct 3960gtggctagca acgtgcctga
tggcttcatt tgtcgttgtc ccccagggtt ctccggggca 4020agatgccaga gcagctgtgg
acaagtgaag tgcagaagag gggagcagtg tgtgcacacc 4080gcctcgggac cccactgctt
ctgcccgaac cacaaggact gcgagtcagg ttgcgctagt 4140aacccctgcc agcacggagg
cacctgctac cctcagcgcc agcctcctta ctactcttgc 4200cgctgctccc caccgttctg
gggcagccac tgcgagagct acacagcccc caccagc 425784137DNAMus musculus
8atggggctgg gggcccgggg ccgccgccgc cgtcgtcgcc tgatggcctt gccaccgcca
60ccaccgccca tgcgggcgct gcccctgctg ctgctgctag cggggctggg ggctgcagca
120cccccttgtc tggatggaag cccatgtgca aatggaggtc ggtgcaccca ccagcagccc
180tccctggagg ctgcttgcct gtgcctgcca ggctgggtgg gtgagcggtg ccagctggaa
240gacccttgcc actcaggccc ttgtgctggc cgaggcgttt gccagagttc agtggtggcg
300ggcaccgccc gattctcctg tcgttgtctc cgtggcttcc aaggcccaga ctgctcccag
360ccagacccct gcgtcagcag gccctgtgtt catggtgccc cctgctcagt ggggccggat
420ggccgatttg cctgtgcctg cccacctggc taccagggtc aaagctgcca aagtgacata
480gatgagtgcc gatctggtac aacttgccgt catggtggta cctgtctcaa tacacctgga
540tccttccgct gccagtgtcc tcttggttat acagggctgc tgtgtgagaa ccccgtagtg
600ccctgtgccc cttccccgtg tcgtaatggt ggcacctgta ggcagagcag tgatgtcaca
660tatgactgtg cttgccttcc tggcttcgag ggccagaact gtgaagtcaa cgtggatgac
720tgtcctggac atcggtgtct caatggggga acgtgtgtag acggtgtcaa tacttacaac
780tgccagtgcc ctccggagtg gacaggccag ttctgtacag aagatgtgga tgagtgtcag
840ctgcagccca atgcctgcca caatgggggt acctgcttca acctactggg tggccacagc
900tgtgtatgtg tcaatggctg gacgggtgag agctgcagtc agaatatcga tgactgtgct
960acagccgtgt gtttccatgg ggccacctgc catgaccgtg tggcctcttt ctactgtgcc
1020tgccctatgg ggaagacagg cctcttgtgt catctggatg atgcatgtgt cagcaacccc
1080tgccatgagg atgctatctg tgacacaaac cctgtgagtg gccgggccat ctgcacctgc
1140ccacctggct tcactggagg ggcatgtgac caggatgtgg atgagtgctc gattggtgcc
1200aacccctgtg aacatttggg tcggtgtgtg aatacacagg gctcattctt gtgccaatgt
1260ggccgtggct atactggacc tcgctgtgag actgatgtca atgagtgtct ctccgggccc
1320tgccgcaacc aggccacgtg tcttgaccga attggccagt ttacttgcat ctgcatggca
1380ggcttcacag ggacctactg tgaggtggac atcgacgaat gtcagagcag cccatgtgtc
1440aatggtggtg tctgcaagga cagagtcaat ggcttcagct gcacctgccc atcaggattc
1500agtgggtcca tgtgtcagct ggatgtggat gagtgtgcaa gcactccctg ccggaatggt
1560gccaagtgtg tggaccagcc tgacggctat gagtgtcgct gtgcagaggg ctttgagggc
1620actttgtgtg agcgaaacgt ggatgactgc tctccggatc cctgccacca cgggcgctgt
1680gtcgatggca ttgctagctt ctcgtgtgct tgtgccccag gctatacggg catacgctgt
1740gagagccagg tggatgagtg ccgcagccag ccctgtcgat atgggggcaa atgtctagac
1800ttggtggaca agtacctctg ccgttgtcct cccggaacca caggtgtgaa ctgtgaagtc
1860aacattgatg actgtgccag taacccctgt acctttggag tttgccgtga tggcatcaac
1920cgttatgact gtgtctgtca gcctggattc acagggcccc tctgcaacgt ggagatcaat
1980gagtgtgcat ccagcccatg tggagagggt ggctcctgtg tggatgggga aaatggcttc
2040cactgcctct gtccacctgg ctccctgcct ccactttgcc tacctgcgaa ccatccctgt
2100gcccacaagc cctgtagtca tggagtctgc catgatgcac caggcgggtt ccgctgtgtt
2160tgtgagcccg ggtggagtgg ccctcgctgt agccagagcc tggctccaga tgcctgtgag
2220tcccagccct gccaggctgg tggcacctgc accagtgatg gaataggctt tcgctgcacc
2280tgtgcccctg gattccaggg ccatcagtgt gaggtgctgt ccccctgtac tccaagcctc
2340tgtgagcacg gaggccactg tgagtctgac cctgaccggc tgactgtctg ttcctgtccc
2400ccaggctggc aaggcccacg atgccagcag gatgtggatg aatgtgccgg tgcctcaccc
2460tgcggccccc atggtacctg caccaacctg ccagggaatt tcaggtgcat ctgccacagg
2520ggatacactg gccccttctg tgatcaagac attgacgact gtgaccccaa cccgtgcctc
2580catggtggct cctgccagga tggcgtgggc tccttttcct gttcttgcct cgacggcttt
2640gctggtcctc gctgtgcccg agatgtggac gaatgtctga gcagcccctg tggccctggc
2700acctgtactg atcacgtggc ctccttcacc tgtgcctgtc cacctggtta tggaggcttc
2760cactgtgaga ttgacttgcc ggactgcagc cccagttcct gcttcaatgg agggacctgt
2820gtggatggcg tgagctcctt cagctgtctg tgtcgccccg gctacacagg cacacactgc
2880caatacgagg ctgacccctg cttttcccgg ccctgtctgc acgggggcat ctgcaacccc
2940acccacccag gatttgaatg cacctgccgg gagggcttca ctgggagtca gtgtcagaac
3000ccagtggact ggtgcagcca ggcaccctgt cagaatgggg gtcgctgtgt ccagactggg
3060gcttactgca tttgtccacc tggatggagt ggccgcctgt gcgacataca aagcctgccc
3120tgcacggagg ccgcagccca gatgggggtg aggttggagc agctgtgtca ggaaggtgga
3180aagtgcatag acaagggccg ctcccactac tgtgtgtgtc cagagggccg tacgggtagt
3240cactgtgaac acgaggtgga tccctgcacg gcccagcctt gccagcacgg gggcacttgc
3300cgtggttaca tggggggcta tgtgtgtgag tgtccagctg gctatgctgg tgacagttgt
3360gaggataata tagatgagtg tgcttcccag ccctgccaga acggaggctc ctgtatcgat
3420cttgtggccc gctatctctg ttcctgtccc cctggcacac tgggagttct ctgtgagatc
3480aatgaggacg actgtgacct aggcccatcc ttggactcag gcgttcagtg cctacacaat
3540ggcacctgtg tggacctggt gggtggcttc cgctgtaact gtcccccagg atacacaggt
3600ctgcactgtg aggcagacat caatgagtgt cgcccgggtg cctgccatgc agcgcatact
3660cgggactgcc tacaagatcc aggtgggcat ttccgctgcg tctgccatcc tggcttcaca
3720gggcctcgct gtcagattgc tctgtccccc tgtgagtccc agccatgtca gcatggaggc
3780cagtgccgtc acagcctagg ccgtggaggt gggctgacct tcacctgtca ctgtgtcccg
3840ccattctggg gtctgcgttg tgagcgggtg gcacgctctt gccgagagct gcagtgccca
3900gtgggtatcc catgccagca gacagcccgt ggaccacgct gcgcttgtcc tccggggctg
3960tccgggccct cctgccgggt ttctagggcg tcaccctcag gagctactaa cgccagctgc
4020gcctctgccc cttgtctgca tgggggctca tgcctacctg tacagagtgt ccctttcttc
4080cgctgtgtgt gcgctccggg ctggggcggc ccgcgttgtg agaccccttc cgcagcc
413793510DNAMus musculus 9atgcagcccc agttgctgct gctgctgctc ttgccactca
atttccctgt catcctgacc 60agagagcttc tgtgtggagg atccccagag ccctgtgcca
acggaggcac ctgcctgagg 120ctatctcagg gacaagggat ctgccagtgt gcccctggat
ttctgggtga gacttgccag 180tttcctgacc cctgcaggga tacccaactc tgcaagaatg
gtggcagctg ccaagccctg 240ctccccacac ccccaagctc ccgtagtcct acttctccac
tgacccctca cttctcctgc 300acctgcccct ctggcttcac cggtgatcga tgccaaaccc
atctggaaga gctctgtcca 360ccttctttct gttccaacgg gggtcactgc tatgttcagg
cctcaggccg cccacagtgc 420tcctgcgagc ctgggtggac aggtgagcaa tgccagctcc
gagacttctg ctcagccaac 480ccctgtgcca acggaggcgt gtgcctggcc acataccccc
agatccagtg ccgctgtcca 540cctgggttcg agggtcacac ctgtgaacgc gacatcaacg
agtgcttcct ggagccggga 600ccctgccctc agggcacctc ctgccataac accttgggtt
cctaccagtg tctctgccct 660gtggggcagg aaggtcccca gtgcaagctc aggaagggag
cctgccctcc tggaagctgt 720ctcaatgggg gcacctgcca gctggtccca gagggacact
ccacctttca tctctgcctc 780tgtcccccag gtttcacggg gctggactgt gagatgaacc
cagatgactg tgtcaggcac 840cagtgtcaga acggggccac ctgtctggat gggctggata
cctacacctg cctctgcccc 900aagacatgga agggctggga ctgctctgaa gatatagatg
aatgtgaagc ccggggtccc 960cctcgctgca ggaacggtgg cacctgccag aacacagctg
gcagctttca ctgtgtgtgc 1020gtgagtggct ggggcggtgc aggttgtgag gagaacctgg
atgactgtgc agctgccacc 1080tgtgccccgg gatccacctg catcgaccgt gtgggctctt
tctcctgcct ctgcccacct 1140ggacgcacag gcctcctgtg ccacctggaa gacatgtgtt
tgagtcagcc gtgccacgtg 1200aatgcccagt gcagcaccaa ccctctgaca ggctccaccc
tctgcatatg ccagcctggc 1260tactcaggat ccacctgtca ccaagatctg gatgagtgcc
aaatggccca gcaaggaccc 1320agtccctgcg aacatggcgg ctcctgcatc aacacccctg
gctccttcaa ctgcctctgc 1380ctgcctggtt acacgggctc ccgctgtgaa gctgaccaca
atgagtgcct gtcacagccc 1440tgccacccag gcagcacctg cctggacctg cttgcaacct
tccactgcct ctgcccacca 1500ggcttggaag ggaggctctg tgaggtggag gtcaatgagt
gcacctctaa tccctgcctg 1560aaccaagctg cctgccatga cctgctcaac ggcttccagt
gcctctgcct tcctggattc 1620accggcgccc gatgtgagaa agacatggac gagtgtagca
gcaccccctg tgccaatggg 1680gggcgctgcc gagaccagcc tggagccttc tactgcgagt
gtctcccagg ctttgaaggg 1740ccacactgtg agaaagaagt ggacgaatgt ctgagtgacc
cctgccccgt gggagccagc 1800tgccttgatc tccccggagc attcttctgc ctctgccgtc
ctggtttcac aggtcaactt 1860tgtgaggttc ccttgtgcac ccccaacatg tgccaacctg
gacagcaatg ccaaggtcag 1920gaacacagag ccccctgcct ctgccctgac ggaagtcctg
gctgtgttcc tgccgaggac 1980aactgcccct gtcaccatgg ccattgccag agatccttgt
gtgtgtgtga tgagggctgg 2040actggaccag aatgcgagac agaactgggt ggctgcatct
ccacaccctg tgcccatggg 2100gggacctgcc acccacagcc gtctggctac aactgtacct
gccctgcagg ctacatgggg 2160ttgacctgta gtgaggaggt gacagcttgt cactcagggc
cctgtctcaa tggtggctct 2220tgcagcatcc gtcctgaggg ctattcctgc acctgccttc
caagtcacac aggtcgccac 2280tgccagactg ccgtggacca ctgtgtgtct gcctcgtgcc
tcaatggggg tacctgtgtg 2340aacaagcctg gcactttctt ctgcctctgt gccactggct
tccaggggct gcactgtgag 2400gagaagacta accccagctg tgcagacagc ccctgcagga
acaaggcaac ctgccaagac 2460acacctcgag gggcccgctg cctctgcagc cctggctata
caggaagcag ctgccagact 2520ctgatagact tgtgtgcccg gaagccctgt ccacacactg
ctcgatgcct ccagagtggg 2580ccctcgttcc agtgcctgtg cctccaggga tggacagggg
ctctctgtga cttcccactg 2640tcctgccaga tggccgcaat gagccaaggc atagagatct
ctggcctgtg ccagaatgga 2700ggcctctgta ttgacacggg ctcctcctat ttctgccgct
gccctcctgg attccaaggc 2760aagttatgcc aggataatat gaacccctgc gagcccaatc
cctgccatca cgggtctacc 2820tgtgtgcctc agcccagtgg ctatgtctgc cagtgtgccc
caggctatga gggacagaac 2880tgctcaaaag tacttgaagc ttgtcagtcc cagccctgcc
acaaccacgg aacctgtacc 2940tccaggcctg gaggcttcca ctgtgcctgc cctccaggct
tcgtgggact gcgctgtgag 3000ggagatgtgg atgagtgtct ggaccggccc tgtcacccct
cgggcactgc agcttgccac 3060tctttagcca acgccttcta ctgccagtgt ctgcctgggc
acacaggcca gcggtgtgag 3120gtggagatgg acctctgtca gagccaaccc tgctccaatg
gaggatcctg tgagatcaca 3180acagggccac cccctggctt cacctgtcac tgccccaagg
gttttgaagg ccccacctgc 3240agccacaaag ccctttcctg cggcatccat cactgccaca
atggaggcct atgtctgccc 3300tcccctaagc cagggtcacc accactctgt gcctgcctca
gtggttttgg gggccctgac 3360tgtctgacac ctccagctcc accgggctgc ggtcccccct
caccctgcct gcacaatggt 3420acctgcactg agacccctgg gttgggcaac ccgggctttc
aatgcacctg ccctcctgac 3480tctccagggc cccggtgtca aaggccaggg
35101015DNAMus musculus 10gatctgggcc cgggc
15114299DNAHomo sapiens
11atgccgccgc tcctggcgcc cctgctctgc ctggcgctgc tgcccgcgct cgccgcacga
60ggcccgcgat gctcccagcc cggtgagacc tgcctgaatg gcgggaagtg tgaagcggcc
120aatggcacgg aggcctgcgt ctgtggcggg gccttcgtgg gcccgcgatg ccaggacccc
180aacccgtgcc tcagcacccc ctgcaagaac gccgggacat gccacgtggt ggaccgcaga
240ggcgtggcag actatgcctg cagctgtgcc ctgggcttct ctgggcccct ctgcctgaca
300cccctggaca atgcctgcct caccaacccc tgccgcaacg ggggcacctg cgacctgctc
360acgctgacgg agtacaagtg ccgctgcccg cccggctggt cagggaaatc gtgccagcag
420gctgacccgt gcgcctccaa cccctgcgcc aacggtggcc agtgcctgcc cttcgaggcc
480tcctacatct gccactgccc acccagcttc catggcccca cctgccggca ggatgtcaac
540gagtgtggcc agaagcccgg gctttgccgc cacggaggca cctgccacaa cgaggtcggc
600tcctaccgct gcgtctgccg cgccacccac actggcccca actgcgagcg gccctacgtg
660ccctgcagcc cctcgccctg ccagaacggg ggcacctgcc gccccacggg cgacgtcacc
720cacgagtgtg cctgcctgcc aggcttcacc ggccagaact gtgaggaaaa tatcgacgat
780tgtccaggaa acaactgcaa gaacgggggt gcctgtgtgg acggcgtgaa cacctacaac
840tgccgctgcc cgccagagtg gacaggtcag tactgtaccg aggatgtgga cgagtgccag
900ctgatgccaa atgcctgcca gaacggcggg acctgccaca acacccacgg tggctacaac
960tgcgtgtgtg tcaacggctg gactggtgag gactgcagcg agaacattga tgactgtgcc
1020agcgccgcct gcttccacgg cgccacctgc catgaccgtg tggcctcctt ctactgcgag
1080tgtccccatg gccgcacagg tctgctgtgc cacctcaacg acgcatgcat cagcaacccc
1140tgtaacgagg gctccaactg cgacaccaac cctgtcaatg gcaaggccat ctgcacctgc
1200ccctcggggt acacgggccc ggcctgcagc caggacgtgg atgagtgctc gctgggtgcc
1260aacccctgcg agcatgcggg caagtgcatc aacacgctgg gctccttcga gtgccagtgt
1320ctgcagggct acacgggccc ccgatgcgag atcgacgtca acgagtgcgt ctcgaacccg
1380tgccagaacg acgccacctg cctggaccag attggggagt tccagtgcat ctgcatgccc
1440ggctacgagg gtgtgcactg cgaggtcaac acagacgagt gtgccagcag cccctgcctg
1500cacaatggcc gctgcctgga caagatcaat gagttccagt gcgagtgccc cacgggcttc
1560actgggcatc tgtgccagta cgatgtggac gagtgtgcca gcaccccctg caagaatggt
1620gccaagtgcc tggacggacc caacacttac acctgtgtgt gcacggaagg gtacacgggg
1680acgcactgcg aggtggacat cgatgagtgc gaccccgacc cctgccacta cggctcctgc
1740aaggacggcg tcgccacctt cacctgcctc tgccgcccag gctacacggg ccaccactgc
1800gagaccaaca tcaacgagtg ctccagccag ccctgccgcc acgggggcac ctgccaggac
1860cgcgacaacg cctacctctg cttctgcctg aaggggacca caggacccaa ctgcgagatc
1920aacctggatg actgtgccag cagcccctgc gactcgggca cctgtctgga caagatcgat
1980ggctacgagt gtgcctgtga gccgggctac acagggagca tgtgtaacat caacatcgat
2040gagtgtgcgg gcaacccctg ccacaacggg ggcacctgcg aggacggcat caatggcttc
2100acctgccgct gccccgaggg ctaccacgac cccacctgcc tgtctgaggt caatgagtgc
2160aacagcaacc cctgcgtcca cggggcctgc cgggacagcc tcaacgggta caagtgcgac
2220tgtgaccctg ggtggagtgg gaccaactgt gacatcaaca acaatgagtg tgaatccaac
2280ccttgtgtca acggcggcac ctgcaaagac atgaccagtg gctacgtgtg cacctgccgg
2340gagggcttca gcggtcccaa ctgccagacc aacatcaacg agtgtgcgtc caacccatgt
2400ctgaaccagg gcacgtgtat tgacgacgtt gccgggtaca agtgcaactg cctgctgccc
2460tacacaggtg ccacgtgtga ggtggtgctg gccccgtgtg cccccagccc ctgcagaaac
2520ggcggggagt gcaggcaatc cgaggactat gagagcttct cctgtgtctg ccccacgggc
2580tggcaagcag ggcagacctg tgaggtcgac atcaacgagt gcgttctgag cccgtgccgg
2640cacggcgcat cctgccagaa cacccacggc ggctaccgct gccactgcca ggccggctac
2700agtgggcgca actgcgagac cgacatcgac gactgccggc ccaacccgtg tcacaacggg
2760ggctcctgca cagacggcat caacacggcc ttctgcgact gcctgcccgg cttccggggc
2820actttctgtg aggaggacat caacgagtgt gccagtgacc cctgccgcaa cggggccaac
2880tgcacggact gcgtggacag ctacacgtgc acctgccccg caggcttcag cgggatccac
2940tgtgagaaca acacgcctga ctgcacagag agctcctgct tcaacggtgg cacctgcgtg
3000gacggcatca actcgttcac ctgcctgtgt ccacccggct tcacgggcag ctactgccag
3060cacgatgtca atgagtgcga ctcacagccc tgcctgcatg gcggcacctg tcaggacggc
3120tgcggctcct acaggtgcac ctgcccccag ggctacactg gccccaactg ccagaacctt
3180gtgcactggt gtgactcctc gccctgcaag aacggcggca aatgctggca gacccacacc
3240cagtaccgct gcgagtgccc cagcggctgg accggccttt actgcgacgt gcccagcgtg
3300tcctgtgagg tggctgcgca gcgacaaggt gttgacgttg cccgcctgtg ccagcatgga
3360gggctctgtg tggacgcggg caacacgcac cactgccgct gccaggcggg ctacacaggc
3420agctactgtg aggacctggt ggacgagtgc tcacccagcc cctgccagaa cggggccacc
3480tgcacggact acctgggcgg ctactcctgc aagtgcgtgg ccggctacca cggggtgaac
3540tgctctgagg agatcgacga gtgcctctcc cacccctgcc agaacggggg cacctgcctc
3600gacctcccca acacctacaa gtgctcctgc ccacggggca ctcagggtgt gcactgtgag
3660atcaacgtgg acgactgcaa tccccccgtt gaccccgtgt cccggagccc caagtgcttt
3720aacaacggca cctgcgtgga ccaggtgggc ggctacagct gcacctgccc gccgggcttc
3780gtgggtgagc gctgtgaggg ggatgtcaac gagtgcctgt ccaatccctg cgacgcccgt
3840ggcacccaga actgcgtgca gcgcgtcaat gacttccact gcgagtgccg tgctggtcac
3900accgggcgcc gctgcgagtc cgtcatcaat ggctgcaaag gcaagccctg caagaatggg
3960ggcacctgcg ccgtggcctc caacaccgcc cgcgggttca tctgcaagtg ccctgcgggc
4020ttcgagggcg ccacgtgtga gaatgacgct cgtacctgcg gcagcctgcg ctgcctcaac
4080ggcggcacat gcatctccgg cccgcgcagc cccacctgcc tgtgcctggg ccccttcacg
4140ggccccgaat gccagttccc ggccagcagc ccctgcctgg gcggcaaccc ctgctacaac
4200caggggacct gtgagcccac atccgagagc cccttctacc gttgcctgtg ccccgccaaa
4260ttcaacgggc tcttgtgcca catcctggac tacagcttc
4299124213DNAHomo sapiens 12tcatctggaa ttatgcccgc cctgcgcccc gctctgctgt
gggcgctgct ggcgctctgg 60ctgtgctgcg cggcccccgc gcatgcattg cagtgtcgag
atggctatga accctgtgta 120aatgaaggaa tgtgtgttac ctaccacaat ggcacaggat
actgcaaatg tccagaaggc 180ttcttggggg aatattgtca acatcgagac ccctgtgaga
agaaccgctg ccagaatggt 240gggacttgtg tggcccaggc catgctgggg aaagccacgt
gccgatgtgc ctcagggttt 300acaggagagg actgccagta ctcaacatct catccatgct
ttgtgtctcg accctgcctg 360aatggcggca catgccatat gctcagccgg gatacctatg
agtgcacctg tcaagtcggg 420tttacaggta aggagtgcca atggacggat gcctgcctgt
ctcatccctg tgcaaatgga 480agtacctgta ccactgtggc caaccagttc tcctgcaaat
gcctcacagg cttcacaggg 540cagaaatgtg agactgatgt caatgagtgt gacattccag
gacactgcca gcatggtggc 600acctgcctca acctgcctgg ttcctaccag tgccagtgcc
ctcagggctt cacaggccag 660tactgtgaca gcctgtatgt gccctgtgca ccctcacctt
gtgtcaatgg aggcacctgt 720cggcagactg gtgacttcac ttttgagtgc aactgccttc
caggttttga agggagcacc 780tgtgagagga atattgatga ctgccctaac cacaggtgtc
agaatggagg ggtttgtgtg 840gatggggtca acacttacaa ctgccgctgt cccccacaat
ggacaggaca gttctgcaca 900gaggatgtgg atgaatgcct gctgcagccc aatgcctgtc
aaaatggggg cacctgtgcc 960aaccgcaatg gaggctatgg ctgtgtatgt gtcaacggct
ggagtggaga tgactgcagt 1020gagaacattg atgattgtgc cttcgcctcc tgtactccag
gctccacctg catcgaccgt 1080gtggcctcct tctcttgcat gtgcccagag gggaaggcag
gtctcctgtg tcatctggat 1140gatgcatgca tcagcaatcc ttgccacaag ggggcactgt
gtgacaccaa ccccctaaat 1200gggcaatata tttgcacctg cccacaaggc tacaaagggg
ctgactgcac agaagatgtg 1260gatgaatgtg ccatggccaa tagcaatcct tgtgagcatg
caggaaaatg tgtgaacacg 1320gatggcgcct tccactgtga gtgtctgaag ggttatgcag
gacctcgttg tgagatggac 1380atcaatgagt gccattcaga cccctgccag aatgatgcta
cctgtctgga taagattgga 1440ggcttcacat gtctgtgcat gccaggtttc aaaggtgtgc
attgtgaatt agaaataaat 1500gaatgtcaga gcaacccttg tgtgaacaat gggcagtgtg
tggataaagt caatcgtttc 1560cagtgcctgt gtcctcctgg tttcactggg ccagtttgcc
agattgatat tgatgactgt 1620tccagtactc cgtgtctgaa tggggcaaag tgtatcgatc
acccgaatgg ctatgaatgc 1680cagtgtgcca caggtttcac tggtgtgttg tgtgaggaga
acattgacaa ctgtgacccc 1740gatccttgcc accatggtca gtgtcaggat ggtattgatt
cctacacctg catctgcaat 1800cccgggtaca tgggcgccat ctgcagtgac cagattgatg
aatgttacag cagcccttgc 1860ctgaacgatg gtcgctgcat tgacctggtc aatggctacc
agtgcaactg ccagccaggc 1920acgtcagggg ttaattgtga aattaatttt gatgactgtg
caagtaaccc ttgtatccat 1980ggaatctgta tggatggcat taatcgctac agttgtgtct
gctcaccagg attcacaggg 2040cagagatgta acattgacat tgatgagtgt gcctccaatc
cctgtcgcaa gggtgcaaca 2100tgtatcaacg gtgtgaatgg tttccgctgt atatgccccg
agggacccca tcaccccagc 2160tgctactcac aggtgaacga atgcctgagc aatccctgca
tccatggaaa ctgtactgga 2220ggtctcagtg gatataagtg tctctgtgat gcaggctggg
ttggcatcaa ctgtgaagtg 2280gacaaaaatg aatgcctttc gaatccatgc cagaatggag
gaacttgtga caatctggtg 2340aatggataca ggtgtacttg caagaagggc tttaaaggct
ataactgcca ggtgaatatt 2400gatgaatgtg cctcaaatcc atgcctgaac caaggaacct
gctttgatga cataagtggc 2460tacacttgcc actgtgtgct gccatacaca ggcaagaatt
gtcagacagt attggctccc 2520tgttccccaa acccttgtga gaatgctgct gtttgcaaag
agtcaccaaa ttttgagagt 2580tatacttgct tgtgtgctcc tggctggcaa ggtcagcggt
gtaccattga cattgacgag 2640tgtatctcca agccctgcat gaaccatggt ctctgccata
acacccaggg cagctacatg 2700tgtgaatgtc caccaggctt cagtggtatg gactgtgagg
aggacattga tgactgcctt 2760gccaatcctt gccagaatgg aggttcctgt atggatggag
tgaatacttt ctcctgcctc 2820tgccttccgg gtttcactgg ggataagtgc cagacagaca
tgaatgagtg tctgagtgaa 2880ccctgtaaga atggagggac ctgctctgac tacgtcaaca
gttacacttg caagtgccag 2940gcaggatttg atggagtcca ttgtgagaac aacatcaatg
agtgcactga gagctcctgt 3000ttcaatggtg gcacatgtgt tgatgggatt aactccttct
cttgcttgtg ccctgtgggt 3060ttcactggat ccttctgcct ccatgagatc aatgaatgca
gctctcatcc atgcctgaat 3120gagggaacgt gtgttgatgg cctgggtacc taccgctgca
gctgccccct gggctacact 3180gggaaaaact gtcagaccct ggtgaatctc tgcagtcggt
ctccatgtaa aaacaaaggt 3240acttgtgttc agaaaaaagc agagtcccag tgcctatgtc
catctggatg ggctggtgcc 3300tattgtgacg tgcccaatgt ctcttgtgac atagcagcct
ccaggagagg tgtgcttgtt 3360gaacacttgt gccagcactc aggtgtctgc atcaatgctg
gcaacacgca ttactgtcag 3420tgccccctgg gctatactgg gagctactgt gaggagcaac
tcgatgagtg tgcgtccaac 3480ccctgccagc acggggcaac atgcagtgac ttcattggtg
gatacagatg cgagtgtgtc 3540ccaggctatc agggtgtcaa ctgtgagtat gaagtggatg
agtgccagaa tcagccctgc 3600cagaatggag gcacctgtat tgaccttgtg aaccatttca
agtgctcttg cccaccaggc 3660actcggggcc tactctgtga agagaacatt gatgactgtg
cccggggtcc ccattgcctt 3720aatggtggtc agtgcatgga taggattgga ggctacagtt
gtcgctgctt gcctggcttt 3780gctggggagc gttgtgaggg agacatcaac gagtgcctct
ccaacccctg cagctctgag 3840ggcagcctgg actgtataca gctcaccaat gactacctgt
gtgtttgccg tagtgccttt 3900actggccggc actgtgaaac cttcgtcgat gtgtgtcccc
agatgccctg cctgaatgga 3960gggacttgtg ctgtggccag taacatgcct gatggtttca
tttgccgttg tcccccggga 4020ttttccgggg caaggtgcca gagcagctgt ggacaagtga
aatgtaggaa gggggagcag 4080tgtgtgcaca ccgcctctgg accccgctgc ttctgcccca
gtccccggga ctgcgagtca 4140ggctgtgcca gtagcccctg ccagcacggg ggcagctgcc
accctcagcg ccagcctcct 4200tattactcct gcc
4213133974DNAHomo sapiens 13atggggccgg gggcccgtgg
ccgccgccgc cgccgtcgcc cgatgtcgcc gccaccgcca 60ccgccacccg tgcgggcgct
gcccctgctg ctgctgctag cggggccggg ggctgcagcc 120cccccttgcc tggacggaag
cccgtgtgca aatggaggtc gttgcaccca gctgccctcc 180cgggaggctg cctgcctgtg
cccgcctggc tgggtgggtg agcggtgtca gctggaggac 240ccctgtcact caggcccctg
tgctggccgt ggtgtctgcc agagttcagt ggtggctggc 300accgcccgat tctcatgccg
gtgcccccgt ggcttccgag gccctgactg ctccctgcca 360gatccctgcc tcagcagccc
ttgtgcccac ggtgcccgct gctcagtggg gcccgatgga 420cgcttcctct gctcctgccc
acctggctac cagggccgca gctgccgaag cgacgtggat 480gagtgccggg tgggtgagcc
ctgccgccat ggtggcacct gcctcaacac acctggctcc 540ttccgctgcc agtgtccagc
tggctacaca gggccactat gtgagaaccc cgcggtgccc 600tgtgcaccct caccatgccg
taacgggggc acctgcaggc agagtggcga cctcacttac 660gactgtgcct gtcttcctgg
gtttgagggt cagaattgtg aagtgaacgt ggacgactgt 720ccaggacacc gatgtctcaa
tggggggaca tgcgtggatg gcgtcaacac ctataactgc 780cagtgccctc ctgagtggac
aggccagttc tgcacggagg acgtggatga gtgtcagctg 840cagcccaacg cctgccacaa
tgggggtacc tgcttcaaca cgctgggtgg ccacagctgc 900gtgtgtgtca atggctggac
aggcgagagc tgcagtcaga atatcgatga ctgtgccaca 960gccgtgtgct tccatggggc
cacctgccat gaccgcgtgg cttctttcta ctgtgcctgc 1020cccatgggca agactggcct
cctgtgtcac ctggatgacg cctgtgtcag caacccctgc 1080cacgaggatg ctatctgtga
cacaaatccg gtgaacggcc gggccatttg cacctgtcct 1140cccggcttca cgggtggggc
atgtgaccag gatgtggacg agtgctctat cggcgccaac 1200ccctgcgagc acttgggcag
gtgcgtgaac acgcagggct ccttcctgtg ccagtgcggt 1260cgtggctaca ctggacctcg
ctgtgagacc gatgtcaacg agtgtctgtc ggggccctgc 1320cgaaaccagg ccacgtgcct
cgaccgcata ggccagttca cctgtatctg tatggcaggc 1380ttcacaggaa cctattgcga
ggtggacatt gacgagtgtc agagtagccc ctgtgtcaac 1440ggtggggtct gcaaggaccg
agtcaatggc ttcagctgca cctgcccctc gggcttcagc 1500ggctccacgt gtcagctgga
cgtggacgaa tgcgccagca cgccctgcag gaatggcgcc 1560aaatgcgtgg accagcccga
tggctacgag tgccgctgtg ccgagggctt tgagggcacg 1620ctgtgtgatc gcaacgtgga
cgactgctcc cctgacccat gccaccatgg tcgctgcgtg 1680gatggcatcg ccagcttctc
atgtgcctgt gctcctggct acacgggcac acgctgcgag 1740agccaggtgg acgaatgccg
cagccagccc tgccgccatg gcggcaaatg cctagacctg 1800gtggacaagt acctctgccg
ctgcccttct gggaccacag gtgtgaactg cgaagtgaac 1860attgacgact gtgccagcaa
cccctgcacc tttggagtct gccgtgatgg catcaaccgc 1920tacgactgtg tctgccaacc
tggcttcaca gggccccttt gtaacgtgga gatcaatgag 1980tgtgcttcca gcccatgcgg
cgagggaggt tcctgtgtgg atggggaaaa tggcttccgc 2040tgcctctgcc cgcctggctc
cttgccccca ctctgcctcc ccccgagcca tccctgtgcc 2100catgagccct gcagtcacgg
catctgctat gatgcacctg gcgggttccg ctgtgtgtgt 2160gagcctggct ggagtggccc
ccgctgcagc cagagcctgg cccgagacgc ctgtgagtcc 2220cagccgtgca gggccggtgg
gacatgcagc agcgatggaa tgggtttcca ctgcacctgc 2280ccgcctggtg tccagggacg
tcagtgtgaa ctcctctccc cctgcacccc gaacccctgt 2340gagcatgggg gccgctgcga
gtctgcccct ggccagctgc ctgtctgctc ctgcccccag 2400ggctggcaag gcccacgatg
ccagcaggat gtggacgagt gtgctggccc cgcaccctgt 2460ggccctcatg gtatctgcac
caacctggca gggagtttca gctgcacctg ccatggaggg 2520tacactggcc cttcctgcga
tcaggacatc aatgactgtg accccaaccc atgcctgaac 2580ggtggctcgt gccaagacgg
cgtgggctcc ttttcctgct cctgcctccc tggtttcgcc 2640ggcccacgat gcgcccgcga
tgtggatgag tgcctgagca acccctgcgg cccgggcacc 2700tgtaccgacc acgtggcctc
cttcacctgc acctgcccgc caggctacgg aggcttccac 2760tgcgaacagg acctgcccga
ctgcagcccc agctcctgct tcaatggcgg gacctgtgtg 2820gacggcgtga actcgttcag
ctgcctgtgc cgtcccggct acacaggagc ccactgccaa 2880catgaggcag acccctgcct
ctcgcggccc tgcctacacg ggggcgtctg cagcgccgcc 2940caccctggct tccgctgcac
ctgcctcgag agcttcacgg gcccgcagtg ccagacgctg 3000gtggattggt gcagccgcca
gccttgtcaa aacgggggtc gctgcgtcca gactggggcc 3060tattgccttt gtccccctgg
atggagcgga cgcctctgtg acatccgaag cttgccctgc 3120agggaggccg cagcccagat
cggggtgcgg ctggagcagc tgtgtcaggc gggtgggcag 3180tgtgtggatg aagacagctc
ccactactgc gtgtgcccag agggccgtac tggtagccac 3240tgtgagcagg aggtggaccc
ctgcttggcc cagccctgcc agcatggggg gacctgccgt 3300ggctatatgg ggggctacat
gtgtgagtgt cttcctggct acaatggtga taactgtgag 3360gacgacgtgg acgagtgtgc
ctcccagccc tgccagcacg ggggttcatg cattgacctc 3420gtggcccgct atctctgctc
ctgtccccca ggaacgctgg gggtgctctg cgagattaat 3480gaggatgact gcggcccagg
cccaccgctg gactcagggc cccggtgcct acacaatggc 3540acctgcgtgg acctggtggg
tggtttccgc tgcacctgtc ccccaggata cactggtttg 3600cgctgcgagg cagacatcaa
tgagtgtcgc tcaggtgcct gccacgcggc acacacccgg 3660gactgcctgc aggacccagg
cggaggtttc cgttgccttt gtcatgctgg cttctcaggt 3720cctcgctgtc agactgtcct
gtctccctgc gagtcccagc catgccagca tggaggccag 3780tgccgtccta gcccgggtcc
tgggggtggg ctgaccttca cctgtcactg tgcccagccg 3840ttctggggtc cgcgttgcga
gcgggtggcg cgctcctgcc gggagctgca gtgcccggtg 3900ggcgtcccat gccagcagac
gccccgcggg ccgcgctgcg cctgcccccc agggttgtcg 3960ggaccctcct gccg
3974143522DNAHomo sapiens
14atgcagcccc cttcactgct gctgctgctg ctgctgctgc tgctgctatg tgtctcagtg
60gtcagaccca gagggctgct gtgtgggagt ttcccagaac cctgtgccaa tggaggcacc
120tgcctgagcc tgtctctggg acaagggacc tgccagtgtg cccctggctt cctgggtgag
180acgtgccagt ttcctgaccc ctgccagaac gcccagctct gccaaaatgg aggcagctgc
240caagccctgc ttcccgctcc cctagggctc cccagctctc cctctccatt gacacccagc
300ttcttgtgca cttgcctccc tggcttcact ggtgagagat gccaggccaa gcttgaagac
360ccttgtcctc cctccttctg ttccaaaagg ggccgctgcc acatccaggc ctcgggccgc
420ccacagtgct cctgcatgcc tggatggaca ggtgagcagt gccagcttcg ggacttctgt
480tcagccaacc catgtgttaa tggaggggtg tgtctggcca cataccccca gatccagtgc
540cactgcccac cgggcttcga gggccatgcc tgtgaacgtg atgtcaacga gtgcttccag
600gacccaggac cctgccccaa aggcacctcc tgccataaca ccctgggctc cttccagtgc
660ctctgccctg tggggcagga gggtccacgt tgtgagctgc gggcaggacc ctgccctcct
720aggggctgtt cgaatggggg cacctgccag ctgatgccag agaaagactc cacctttcac
780ctctgcctct gtcccccagg tttcataggc ccagactgtg aggtgaatcc agacaactgt
840gtcagccacc agtgtcagaa tgggggcact tgccaggatg ggctggacac ctacacctgc
900ctctgcccag aaacctggac aggctgggac tgctccgaag atgtggatga gtgtgagacc
960cagggtcccc ctcactgcag aaacgggggc acctgccaga actctgctgg tagctttcac
1020tgcgtgtgtg tgagtggctg gggcggcaca agctgtgagg agaacctgga tgactgtatt
1080gctgccacct gtgccccggg atccacctgc attgaccggg tgggctcttt ctcctgcctc
1140tgcccacctg gacgcacagg actcctgtgc cacttggaag acatgtgtct gagccagccg
1200tgccatgggg atgcccaatg cagcaccaac cccctcacag gctccacact ctgcctgtgt
1260cagcctggct attcggggcc cacctgccac caggacctgg acgagtgtct gatggcccag
1320caaggcccaa gtccctgtga acatggcggt tcctgcctca acactcctgg ctccttcaac
1380tgcctctgtc cacctggcta cacaggctcc cgttgtgagg ctgatcacaa tgagtgcctc
1440tcccagccct gccacccagg aagcacctgt ctggacctac ttgccacctt ccactgcctc
1500tgcccgccag gcttagaagg gcagctctgt gaggtggaga ccaacgagtg tgcctcagct
1560ccctgcctga accacgcgga ttgccatgac ctgctcaacg gcttccagtg catctgcctg
1620cctggattct ccggcacccg atgtgaggag gatatcgatg agtgcagaag ctctccctgt
1680gccaatggtg ggcagtgcca ggaccagcct ggagccttcc actgcaagtg tctcccaggc
1740tttgaagggc cacgctgtca aacagaggtg gatgagtgcc tgagtgaccc atgtcccgtt
1800ggagccagct gccttgatct tccaggagcc ttcttttgcc tctgcccctc tggtttcaca
1860ggccagctct gtgaggttcc cctgtgtgct cccaacctgt gccagcccaa gcagatatgt
1920aaggaccaga aagacaaggc caactgcctc tgtcctgatg gaagccctgg ctgtgcccca
1980cctgaggaca actgcacctg ccaccacggg cactgccaga gatcctcatg tgtgtgtgac
2040gtgggttgga cggggccaga gtgtgaggca gagctagggg gctgcatctc tgcaccctgt
2100gcccatgggg ggacctgcta cccccagccc tctggctaca actgcacctg ccctacaggc
2160tacacaggac ccacctgtag tgaggagatg acagcttgtc actcagggcc atgtctcaat
2220ggcggctcct gcaaccctag ccctggaggc tactactgca cctgccctcc aagccacaca
2280gggccccagt gccaaaccag cactgactac tgtgtgtctg ccccgtgctt caatgggggt
2340acctgtgtga acaggcctgg caccttctcc tgcctctgtg ccatgggctt ccagggcccg
2400cgctgtgagg gaaagctccg ccccagctgt gcagacagcc cctgtaggaa tagggcaacc
2460tgccaggaca gccctcaggg tccccgctgc ctctgcccca ctggctacac cggaggcagc
2520tgccagactc tgatggactt atgtgcccag aagccctgcc cacgcaattc ccactgcctc
2580cagactgggc cctccttcca ctgcttgtgc ctccagggat ggaccgggcc tctctgcaac
2640cttccactgt cctcctgcca gaaggctgca ctgagccaag gcatagacgt ctcttccctt
2700tgccacaatg gaggcctctg tgtcgacagc ggcccctcct atttctgcca ctgcccccct
2760ggattccaag gcagcctgtg ccaggatcac gtgaacccat gtgagtccag gccttgccag
2820aacggggcca cctgcatggc ccagcccagt gggtatctct gccagtgtgc cccaggctac
2880gatggacaga actgctcaaa ggaactcgat gcttgtcagt cccaaccctg tcacaaccat
2940ggaacctgta ctcccaaacc tggaggattc cactgtgcct gccctccagg ctttgtgggg
3000ctacgctgtg agggagacgt ggacgagtgt ctggaccagc cctgccaccc cacaggcact
3060gcagcctgcc actctctggc caatgccttc tactgccagt gtctgcctgg acacacaggc
3120cagtggtgtg aggtggagat agacccctgc cacagccaac cctgctttca tggagggacc
3180tgtgaggcca cagcaggatc acccctgggt ttcatctgcc actgccccaa gggttttgaa
3240ggccccacct gcagccacag ggccccttcc tgcggcttcc atcactgcca ccacggaggc
3300ctgtgtctgc cctcccctaa gccaggcttc ccaccacgct gtgcctgcct cagtggctat
3360gggggtcctg actgcctgac cccaccagct cctaaaggct gtggccctcc ctccccatgc
3420ctatacaatg gcagctgctc agagaccacg ggcttggggg gcccaggctt tcgatgctcc
3480tgccctcaca gctctccagg gccccggtgt cagaaacccg ga
35221520PRTHomo sapiens 15Met Pro Pro Leu Leu Ala Pro Leu Leu Cys Leu Ala
Leu Leu Pro Ala 1 5 10
15 Leu Ala Ala Arg 20 1669DNAHomo sapiens 16atgccgccgc
tcctggcgcc cctgctctgc ctggcgctgc tgcccgcgct cgccgcacga 60ggcccgcga
691722PRTHomo
sapiens 17Met Trp Gly Trp Lys Cys Leu Leu Phe Trp Ala Val Leu Val Thr Ala
1 5 10 15 Thr Leu
Cys Thr Ala Arg 20 1866DNAHomo sapiens 18atgtggggct
ggaagtgcct cctcttctgg gctgtgctgg tcacagccac tctctgcact 60gccagg
661912DNAHomo
sapiens 19tacagcttcg gg
122012DNAHomo sapiens 20cccgaagctg ta
12214PRTHomo sapiens 21Tyr Ser Phe Gly 1
2217DNAHomo sapiens 22tacagcttcg gagatct
172317DNAHomo sapiens 23agatctccga agctgta
172424DNAHomo sapiens
24tacagcttcg gagatctggg cccg
242524DNAHomo sapiens 25cgggcccaga tctccgaagc tgta
24268PRTHomo sapiens 26Tyr Ser Phe Gly Asp Leu Gly
Pro 1 5 2712DNAHomo sapiens 27gatgtggacg ag
122812DNAHomo
sapiens 28ctcgtcctca tc
12294PRTHomo sapiens 29Asp Val Asp Glu 1 3017DNAHomo
sapiens 30gatgtggacg aggatcc
173117DNAHomo sapiens 31ggatcctcgt cctcatc
173224DNAHomo sapiens 32gatgtggacg aggatctggg
cccg 243324DNAHomo sapiens
33cgggcccaga tcctcgtcct catc
24348PRTHomo sapiens 34Asp Val Asp Glu Asp Leu Gly Pro 1 5
3518DNAHomo sapiens 35gagaccgaca tcgacgac
183618DNAHomo sapiens 36gtcgtcgatg
tcggtctc 18376PRTHomo
sapiens 37Glu Thr Asp Ile Asp Asp 1 5 3817DNAHomo
sapiens 38gagaccgaca tagatct
173917DNAHomo sapiens 39agatctatgt cggtctc
17404PRTHomo sapiens 40Glu Thr Asp Ile 1
4124DNAHomo sapiens 41gagaccgaca tagatctggg cccg
244224DNAHomo sapiens 42cgggcccaga tctatgtcgg
tctc 24438PRTHomo sapiens
43Glu Thr Asp Ile Asp Leu Gly Pro 1 5
4412DNAHomo sapiens 44gacatcaacg ag
124512DNAHomo sapiens 45ctcgttgatg tc
12464PRTHomo sapiens 46Asp Ile Asn
Glu 1 4717DNAHomo sapiens 47gacatcaacg aggatcc
174817DNAHomo sapiens 48ggatcctcgt
tgatgtc 174924DNAHomo
sapiens 49gacatcaacg aggatctggg cccg
245024DNAHomo sapiens 50cgggcccaga tcctcgttga tgtc
24518PRTHomo sapiens 51Asp Ile Asn Glu Asp Leu
Gly Pro 1 5 522555PRTHomo sapiens 52Met Pro
Pro Leu Leu Ala Pro Leu Leu Cys Leu Ala Leu Leu Pro Ala 1 5
10 15 Leu Ala Ala Arg Gly Pro Arg
Cys Ser Gln Pro Gly Glu Thr Cys Leu 20 25
30 Asn Gly Gly Lys Cys Glu Ala Ala Asn Gly Thr Glu
Ala Cys Val Cys 35 40 45
Gly Gly Ala Phe Val Gly Pro Arg Cys Gln Asp Pro Asn Pro Cys Leu
50 55 60 Ser Thr Pro
Cys Lys Asn Ala Gly Thr Cys His Val Val Asp Arg Arg 65
70 75 80 Gly Val Ala Asp Tyr Ala Cys
Ser Cys Ala Leu Gly Phe Ser Gly Pro 85
90 95 Leu Cys Leu Thr Pro Leu Asp Asn Ala Cys Leu
Thr Asn Pro Cys Arg 100 105
110 Asn Gly Gly Thr Cys Asp Leu Leu Thr Leu Thr Glu Tyr Lys Cys
Arg 115 120 125 Cys
Pro Pro Gly Trp Ser Gly Lys Ser Cys Gln Gln Ala Asp Pro Cys 130
135 140 Ala Ser Asn Pro Cys Ala
Asn Gly Gly Gln Cys Leu Pro Phe Glu Ala 145 150
155 160 Ser Tyr Ile Cys His Cys Pro Pro Ser Phe His
Gly Pro Thr Cys Arg 165 170
175 Gln Asp Val Asn Glu Cys Gly Gln Lys Pro Gly Leu Cys Arg His Gly
180 185 190 Gly Thr
Cys His Asn Glu Val Gly Ser Tyr Arg Cys Val Cys Arg Ala 195
200 205 Thr His Thr Gly Pro Asn Cys
Glu Arg Pro Tyr Val Pro Cys Ser Pro 210 215
220 Ser Pro Cys Gln Asn Gly Gly Thr Cys Arg Pro Thr
Gly Asp Val Thr 225 230 235
240 His Glu Cys Ala Cys Leu Pro Gly Phe Thr Gly Gln Asn Cys Glu Glu
245 250 255 Asn Ile Asp
Asp Cys Pro Gly Asn Asn Cys Lys Asn Gly Gly Ala Cys 260
265 270 Val Asp Gly Val Asn Thr Tyr Asn
Cys Arg Cys Pro Pro Glu Trp Thr 275 280
285 Gly Gln Tyr Cys Thr Glu Asp Val Asp Glu Cys Gln Leu
Met Pro Asn 290 295 300
Ala Cys Gln Asn Gly Gly Thr Cys His Asn Thr His Gly Gly Tyr Asn 305
310 315 320 Cys Val Cys Val
Asn Gly Trp Thr Gly Glu Asp Cys Ser Glu Asn Ile 325
330 335 Asp Asp Cys Ala Ser Ala Ala Cys Phe
His Gly Ala Thr Cys His Asp 340 345
350 Arg Val Ala Ser Phe Tyr Cys Glu Cys Pro His Gly Arg Thr
Gly Leu 355 360 365
Leu Cys His Leu Asn Asp Ala Cys Ile Ser Asn Pro Cys Asn Glu Gly 370
375 380 Ser Asn Cys Asp Thr
Asn Pro Val Asn Gly Lys Ala Ile Cys Thr Cys 385 390
395 400 Pro Ser Gly Tyr Thr Gly Pro Ala Cys Ser
Gln Asp Val Asp Glu Cys 405 410
415 Ser Leu Gly Ala Asn Pro Cys Glu His Ala Gly Lys Cys Ile Asn
Thr 420 425 430 Leu
Gly Ser Phe Glu Cys Gln Cys Leu Gln Gly Tyr Thr Gly Pro Arg 435
440 445 Cys Glu Ile Asp Val Asn
Glu Cys Val Ser Asn Pro Cys Gln Asn Asp 450 455
460 Ala Thr Cys Leu Asp Gln Ile Gly Glu Phe Gln
Cys Ile Cys Met Pro 465 470 475
480 Gly Tyr Glu Gly Val His Cys Glu Val Asn Thr Asp Glu Cys Ala Ser
485 490 495 Ser Pro
Cys Leu His Asn Gly Arg Cys Leu Asp Lys Ile Asn Glu Phe 500
505 510 Gln Cys Glu Cys Pro Thr Gly
Phe Thr Gly His Leu Cys Gln Tyr Asp 515 520
525 Val Asp Glu Cys Ala Ser Thr Pro Cys Lys Asn Gly
Ala Lys Cys Leu 530 535 540
Asp Gly Pro Asn Thr Tyr Thr Cys Val Cys Thr Glu Gly Tyr Thr Gly 545
550 555 560 Thr His Cys
Glu Val Asp Ile Asp Glu Cys Asp Pro Asp Pro Cys His 565
570 575 Tyr Gly Ser Cys Lys Asp Gly Val
Ala Thr Phe Thr Cys Leu Cys Arg 580 585
590 Pro Gly Tyr Thr Gly His His Cys Glu Thr Asn Ile Asn
Glu Cys Ser 595 600 605
Ser Gln Pro Cys Arg His Gly Gly Thr Cys Gln Asp Arg Asp Asn Ala 610
615 620 Tyr Leu Cys Phe
Cys Leu Lys Gly Thr Thr Gly Pro Asn Cys Glu Ile 625 630
635 640 Asn Leu Asp Asp Cys Ala Ser Ser Pro
Cys Asp Ser Gly Thr Cys Leu 645 650
655 Asp Lys Ile Asp Gly Tyr Glu Cys Ala Cys Glu Pro Gly Tyr
Thr Gly 660 665 670
Ser Met Cys Asn Ile Asn Ile Asp Glu Cys Ala Gly Asn Pro Cys His
675 680 685 Asn Gly Gly Thr
Cys Glu Asp Gly Ile Asn Gly Phe Thr Cys Arg Cys 690
695 700 Pro Glu Gly Tyr His Asp Pro Thr
Cys Leu Ser Glu Val Asn Glu Cys 705 710
715 720 Asn Ser Asn Pro Cys Val His Gly Ala Cys Arg Asp
Ser Leu Asn Gly 725 730
735 Tyr Lys Cys Asp Cys Asp Pro Gly Trp Ser Gly Thr Asn Cys Asp Ile
740 745 750 Asn Asn Asn
Glu Cys Glu Ser Asn Pro Cys Val Asn Gly Gly Thr Cys 755
760 765 Lys Asp Met Thr Ser Gly Tyr Val
Cys Thr Cys Arg Glu Gly Phe Ser 770 775
780 Gly Pro Asn Cys Gln Thr Asn Ile Asn Glu Cys Ala Ser
Asn Pro Cys 785 790 795
800 Leu Asn Gln Gly Thr Cys Ile Asp Asp Val Ala Gly Tyr Lys Cys Asn
805 810 815 Cys Leu Leu Pro
Tyr Thr Gly Ala Thr Cys Glu Val Val Leu Ala Pro 820
825 830 Cys Ala Pro Ser Pro Cys Arg Asn Gly
Gly Glu Cys Arg Gln Ser Glu 835 840
845 Asp Tyr Glu Ser Phe Ser Cys Val Cys Pro Thr Gly Trp Gln
Gly Gln 850 855 860
Thr Cys Glu Val Asp Ile Asn Glu Cys Val Leu Ser Pro Cys Arg His 865
870 875 880 Gly Ala Ser Cys Gln
Asn Thr His Gly Gly Tyr Arg Cys His Cys Gln 885
890 895 Ala Gly Tyr Ser Gly Arg Asn Cys Glu Thr
Asp Ile Asp Asp Cys Arg 900 905
910 Pro Asn Pro Cys His Asn Gly Gly Ser Cys Thr Asp Gly Ile Asn
Thr 915 920 925 Ala
Phe Cys Asp Cys Leu Pro Gly Phe Arg Gly Thr Phe Cys Glu Glu 930
935 940 Asp Ile Asn Glu Cys Ala
Ser Asp Pro Cys Arg Asn Gly Ala Asn Cys 945 950
955 960 Thr Asp Cys Val Asp Ser Tyr Thr Cys Thr Cys
Pro Ala Gly Phe Ser 965 970
975 Gly Ile His Cys Glu Asn Asn Thr Pro Asp Cys Thr Glu Ser Ser Cys
980 985 990 Phe Asn
Gly Gly Thr Cys Val Asp Gly Ile Asn Ser Phe Thr Cys Leu 995
1000 1005 Cys Pro Pro Gly Phe
Thr Gly Ser Tyr Cys Gln His Asp Val Asn 1010 1015
1020 Glu Cys Asp Ser Gln Pro Cys Leu His Gly
Gly Thr Cys Gln Asp 1025 1030 1035
Gly Cys Gly Ser Tyr Arg Cys Thr Cys Pro Gln Gly Tyr Thr Gly
1040 1045 1050 Pro Asn
Cys Gln Asn Leu Val His Trp Cys Asp Ser Ser Pro Cys 1055
1060 1065 Lys Asn Gly Gly Lys Cys Trp
Gln Thr His Thr Gln Tyr Arg Cys 1070 1075
1080 Glu Cys Pro Ser Gly Trp Thr Gly Leu Tyr Cys Asp
Val Pro Ser 1085 1090 1095
Val Ser Cys Glu Val Ala Ala Gln Arg Gln Gly Val Asp Val Ala 1100
1105 1110 Arg Leu Cys Gln His
Gly Gly Leu Cys Val Asp Ala Gly Asn Thr 1115 1120
1125 His His Cys Arg Cys Gln Ala Gly Tyr Thr
Gly Ser Tyr Cys Glu 1130 1135 1140
Asp Leu Val Asp Glu Cys Ser Pro Ser Pro Cys Gln Asn Gly Ala
1145 1150 1155 Thr Cys
Thr Asp Tyr Leu Gly Gly Tyr Ser Cys Lys Cys Val Ala 1160
1165 1170 Gly Tyr His Gly Val Asn Cys
Ser Glu Glu Ile Asp Glu Cys Leu 1175 1180
1185 Ser His Pro Cys Gln Asn Gly Gly Thr Cys Leu Asp
Leu Pro Asn 1190 1195 1200
Thr Tyr Lys Cys Ser Cys Pro Arg Gly Thr Gln Gly Val His Cys 1205
1210 1215 Glu Ile Asn Val Asp
Asp Cys Asn Pro Pro Val Asp Pro Val Ser 1220 1225
1230 Arg Ser Pro Lys Cys Phe Asn Asn Gly Thr
Cys Val Asp Gln Val 1235 1240 1245
Gly Gly Tyr Ser Cys Thr Cys Pro Pro Gly Phe Val Gly Glu Arg
1250 1255 1260 Cys Glu
Gly Asp Val Asn Glu Cys Leu Ser Asn Pro Cys Asp Ala 1265
1270 1275 Arg Gly Thr Gln Asn Cys Val
Gln Arg Val Asn Asp Phe His Cys 1280 1285
1290 Glu Cys Arg Ala Gly His Thr Gly Arg Arg Cys Glu
Ser Val Ile 1295 1300 1305
Asn Gly Cys Lys Gly Lys Pro Cys Lys Asn Gly Gly Thr Cys Ala 1310
1315 1320 Val Ala Ser Asn Thr
Ala Arg Gly Phe Ile Cys Lys Cys Pro Ala 1325 1330
1335 Gly Phe Glu Gly Ala Thr Cys Glu Asn Asp
Ala Arg Thr Cys Gly 1340 1345 1350
Ser Leu Arg Cys Leu Asn Gly Gly Thr Cys Ile Ser Gly Pro Arg
1355 1360 1365 Ser Pro
Thr Cys Leu Cys Leu Gly Pro Phe Thr Gly Pro Glu Cys 1370
1375 1380 Gln Phe Pro Ala Ser Ser Pro
Cys Leu Gly Gly Asn Pro Cys Tyr 1385 1390
1395 Asn Gln Gly Thr Cys Glu Pro Thr Ser Glu Ser Pro
Phe Tyr Arg 1400 1405 1410
Cys Leu Cys Pro Ala Lys Phe Asn Gly Leu Leu Cys His Ile Leu 1415
1420 1425 Asp Tyr Ser Phe Gly
Gly Gly Ala Gly Arg Asp Ile Pro Pro Pro 1430 1435
1440 Leu Ile Glu Glu Ala Cys Glu Leu Pro Glu
Cys Gln Glu Asp Ala 1445 1450 1455
Gly Asn Lys Val Cys Ser Leu Gln Cys Asn Asn His Ala Cys Gly
1460 1465 1470 Trp Asp
Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys 1475
1480 1485 Asn Cys Thr Gln Ser Leu Gln
Cys Trp Lys Tyr Phe Ser Asp Gly 1490 1495
1500 His Cys Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu
Phe Asp Gly 1505 1510 1515
Phe Asp Cys Gln Arg Ala Glu Gly Gln Cys Asn Pro Leu Tyr Asp 1520
1525 1530 Gln Tyr Cys Lys Asp
His Phe Ser Asp Gly His Cys Asp Gln Gly 1535 1540
1545 Cys Asn Ser Ala Glu Cys Glu Trp Asp Gly
Leu Asp Cys Ala Glu 1550 1555 1560
His Val Pro Glu Arg Leu Ala Ala Gly Thr Leu Val Val Val Val
1565 1570 1575 Leu Met
Pro Pro Glu Gln Leu Arg Asn Ser Ser Phe His Phe Leu 1580
1585 1590 Arg Glu Leu Ser Arg Val Leu
His Thr Asn Val Val Phe Lys Arg 1595 1600
1605 Asp Ala His Gly Gln Gln Met Ile Phe Pro Tyr Tyr
Gly Arg Glu 1610 1615 1620
Glu Glu Leu Arg Lys His Pro Ile Lys Arg Ala Ala Glu Gly Trp 1625
1630 1635 Ala Ala Pro Asp Ala
Leu Leu Gly Gln Val Lys Ala Ser Leu Leu 1640 1645
1650 Pro Gly Gly Ser Glu Gly Gly Arg Arg Arg
Arg Glu Leu Asp Pro 1655 1660 1665
Met Asp Val Arg Gly Ser Ile Val Tyr Leu Glu Ile Asp Asn Arg
1670 1675 1680 Gln Cys
Val Gln Ala Ser Ser Gln Cys Phe Gln Ser Ala Thr Asp 1685
1690 1695 Val Ala Ala Phe Leu Gly Ala
Leu Ala Ser Leu Gly Ser Leu Asn 1700 1705
1710 Ile Pro Tyr Lys Ile Glu Ala Val Gln Ser Glu Thr
Val Glu Pro 1715 1720 1725
Pro Pro Pro Ala Gln Leu His Phe Met Tyr Val Ala Ala Ala Ala 1730
1735 1740 Phe Val Leu Leu Phe
Phe Val Gly Cys Gly Val Leu Leu Ser Arg 1745 1750
1755 Lys Arg Arg Arg Gln His Gly Gln Leu Trp
Phe Pro Glu Gly Phe 1760 1765 1770
Lys Val Ser Glu Ala Ser Lys Lys Lys Arg Arg Glu Pro Leu Gly
1775 1780 1785 Glu Asp
Ser Val Gly Leu Lys Pro Leu Lys Asn Ala Ser Asp Gly 1790
1795 1800 Ala Leu Met Asp Asp Asn Gln
Asn Glu Trp Gly Asp Glu Asp Leu 1805 1810
1815 Glu Thr Lys Lys Phe Arg Phe Glu Glu Pro Val Val
Leu Pro Asp 1820 1825 1830
Leu Asp Asp Gln Thr Asp His Arg Gln Trp Thr Gln Gln His Leu 1835
1840 1845 Asp Ala Ala Asp Leu
Arg Met Ser Ala Met Ala Pro Thr Pro Pro 1850 1855
1860 Gln Gly Glu Val Asp Ala Asp Cys Met Asp
Val Asn Val Arg Gly 1865 1870 1875
Pro Asp Gly Phe Thr Pro Leu Met Ile Ala Ser Cys Ser Gly Gly
1880 1885 1890 Gly Leu
Glu Thr Gly Asn Ser Glu Glu Glu Glu Asp Ala Pro Ala 1895
1900 1905 Val Ile Ser Asp Phe Ile Tyr
Gln Gly Ala Ser Leu His Asn Gln 1910 1915
1920 Thr Asp Arg Thr Gly Glu Thr Ala Leu His Leu Ala
Ala Arg Tyr 1925 1930 1935
Ser Arg Ser Asp Ala Ala Lys Arg Leu Leu Glu Ala Ser Ala Asp 1940
1945 1950 Ala Asn Ile Gln Asp
Asn Met Gly Arg Thr Pro Leu His Ala Ala 1955 1960
1965 Val Ser Ala Asp Ala Gln Gly Val Phe Gln
Ile Leu Ile Arg Asn 1970 1975 1980
Arg Ala Thr Asp Leu Asp Ala Arg Met His Asp Gly Thr Thr Pro
1985 1990 1995 Leu Ile
Leu Ala Ala Arg Leu Ala Val Glu Gly Met Leu Glu Asp 2000
2005 2010 Leu Ile Asn Ser His Ala Asp
Val Asn Ala Val Asp Asp Leu Gly 2015 2020
2025 Lys Ser Ala Leu His Trp Ala Ala Ala Val Asn Asn
Val Asp Ala 2030 2035 2040
Ala Val Val Leu Leu Lys Asn Gly Ala Asn Lys Asp Met Gln Asn 2045
2050 2055 Asn Arg Glu Glu Thr
Pro Leu Phe Leu Ala Ala Arg Glu Gly Ser 2060 2065
2070 Tyr Glu Thr Ala Lys Val Leu Leu Asp His
Phe Ala Asn Arg Asp 2075 2080 2085
Ile Thr Asp His Met Asp Arg Leu Pro Arg Asp Ile Ala Gln Glu
2090 2095 2100 Arg Met
His His Asp Ile Val Arg Leu Leu Asp Glu Tyr Asn Leu 2105
2110 2115 Val Arg Ser Pro Gln Leu His
Gly Ala Pro Leu Gly Gly Thr Pro 2120 2125
2130 Thr Leu Ser Pro Pro Leu Cys Ser Pro Asn Gly Tyr
Leu Gly Ser 2135 2140 2145
Leu Lys Pro Gly Val Gln Gly Lys Lys Val Arg Lys Pro Ser Ser 2150
2155 2160 Lys Gly Leu Ala Cys
Gly Ser Lys Glu Ala Lys Asp Leu Lys Ala 2165 2170
2175 Arg Arg Lys Lys Ser Gln Asp Gly Lys Gly
Cys Leu Leu Asp Ser 2180 2185 2190
Ser Gly Met Leu Ser Pro Val Asp Ser Leu Glu Ser Pro His Gly
2195 2200 2205 Tyr Leu
Ser Asp Val Ala Ser Pro Pro Leu Leu Pro Ser Pro Phe 2210
2215 2220 Gln Gln Ser Pro Ser Val Pro
Leu Asn His Leu Pro Gly Met Pro 2225 2230
2235 Asp Thr His Leu Gly Ile Gly His Leu Asn Val Ala
Ala Lys Pro 2240 2245 2250
Glu Met Ala Ala Leu Gly Gly Gly Gly Arg Leu Ala Phe Glu Thr 2255
2260 2265 Gly Pro Pro Arg Leu
Ser His Leu Pro Val Ala Ser Gly Thr Ser 2270 2275
2280 Thr Val Leu Gly Ser Ser Ser Gly Gly Ala
Leu Asn Phe Thr Val 2285 2290 2295
Gly Gly Ser Thr Ser Leu Asn Gly Gln Cys Glu Trp Leu Ser Arg
2300 2305 2310 Leu Gln
Ser Gly Met Val Pro Asn Gln Tyr Asn Pro Leu Arg Gly 2315
2320 2325 Ser Val Ala Pro Gly Pro Leu
Ser Thr Gln Ala Pro Ser Leu Gln 2330 2335
2340 His Gly Met Val Gly Pro Leu His Ser Ser Leu Ala
Ala Ser Ala 2345 2350 2355
Leu Ser Gln Met Met Ser Tyr Gln Gly Leu Pro Ser Thr Arg Leu 2360
2365 2370 Ala Thr Gln Pro His
Leu Val Gln Thr Gln Gln Val Gln Pro Gln 2375 2380
2385 Asn Leu Gln Met Gln Gln Gln Asn Leu Gln
Pro Ala Asn Ile Gln 2390 2395 2400
Gln Gln Gln Ser Leu Gln Pro Pro Pro Pro Pro Pro Gln Pro His
2405 2410 2415 Leu Gly
Val Ser Ser Ala Ala Ser Gly His Leu Gly Arg Ser Phe 2420
2425 2430 Leu Ser Gly Glu Pro Ser Gln
Ala Asp Val Gln Pro Leu Gly Pro 2435 2440
2445 Ser Ser Leu Ala Val His Thr Ile Leu Pro Gln Glu
Ser Pro Ala 2450 2455 2460
Leu Pro Thr Ser Leu Pro Ser Ser Leu Val Pro Pro Val Thr Ala 2465
2470 2475 Ala Gln Phe Leu Thr
Pro Pro Ser Gln His Ser Tyr Ser Ser Pro 2480 2485
2490 Val Asp Asn Thr Pro Ser His Gln Leu Gln
Val Pro Glu His Pro 2495 2500 2505
Phe Leu Thr Pro Ser Pro Glu Ser Pro Asp Gln Trp Ser Ser Ser
2510 2515 2520 Ser Pro
His Ser Asn Val Ser Asp Trp Ser Glu Gly Val Ser Ser 2525
2530 2535 Pro Pro Thr Ser Met Gln Ser
Gln Ile Ala Arg Ile Pro Glu Ala 2540 2545
2550 Phe Lys 2555 53237PRTHomo sapiens 53Asp Leu
Gly Pro Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 1 5
10 15 Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu 20 25
30 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu 35 40 45
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
50 55 60 Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 65
70 75 80 Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu 85
90 95 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys 100 105
110 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys 115 120 125 Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 130
135 140 Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys 145 150
155 160 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln 165 170
175 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
180 185 190 Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 195
200 205 Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn 210 215
220 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 225 230 235 541670PRTHomo
sapiens 54Met Pro Pro Leu Leu Ala Pro Leu Leu Cys Leu Ala Leu Leu Pro Ala
1 5 10 15 Leu Ala
Ala Arg Gly Pro Arg Cys Ser Gln Pro Gly Glu Thr Cys Leu 20
25 30 Asn Gly Gly Lys Cys Glu Ala
Ala Asn Gly Thr Glu Ala Cys Val Cys 35 40
45 Gly Gly Ala Phe Val Gly Pro Arg Cys Gln Asp Pro
Asn Pro Cys Leu 50 55 60
Ser Thr Pro Cys Lys Asn Ala Gly Thr Cys His Val Val Asp Arg Arg 65
70 75 80 Gly Val Ala
Asp Tyr Ala Cys Ser Cys Ala Leu Gly Phe Ser Gly Pro 85
90 95 Leu Cys Leu Thr Pro Leu Asp Asn
Ala Cys Leu Thr Asn Pro Cys Arg 100 105
110 Asn Gly Gly Thr Cys Asp Leu Leu Thr Leu Thr Glu Tyr
Lys Cys Arg 115 120 125
Cys Pro Pro Gly Trp Ser Gly Lys Ser Cys Gln Gln Ala Asp Pro Cys 130
135 140 Ala Ser Asn Pro
Cys Ala Asn Gly Gly Gln Cys Leu Pro Phe Glu Ala 145 150
155 160 Ser Tyr Ile Cys His Cys Pro Pro Ser
Phe His Gly Pro Thr Cys Arg 165 170
175 Gln Asp Val Asn Glu Cys Gly Gln Lys Pro Gly Leu Cys Arg
His Gly 180 185 190
Gly Thr Cys His Asn Glu Val Gly Ser Tyr Arg Cys Val Cys Arg Ala
195 200 205 Thr His Thr Gly
Pro Asn Cys Glu Arg Pro Tyr Val Pro Cys Ser Pro 210
215 220 Ser Pro Cys Gln Asn Gly Gly Thr
Cys Arg Pro Thr Gly Asp Val Thr 225 230
235 240 His Glu Cys Ala Cys Leu Pro Gly Phe Thr Gly Gln
Asn Cys Glu Glu 245 250
255 Asn Ile Asp Asp Cys Pro Gly Asn Asn Cys Lys Asn Gly Gly Ala Cys
260 265 270 Val Asp Gly
Val Asn Thr Tyr Asn Cys Arg Cys Pro Pro Glu Trp Thr 275
280 285 Gly Gln Tyr Cys Thr Glu Asp Val
Asp Glu Cys Gln Leu Met Pro Asn 290 295
300 Ala Cys Gln Asn Gly Gly Thr Cys His Asn Thr His Gly
Gly Tyr Asn 305 310 315
320 Cys Val Cys Val Asn Gly Trp Thr Gly Glu Asp Cys Ser Glu Asn Ile
325 330 335 Asp Asp Cys Ala
Ser Ala Ala Cys Phe His Gly Ala Thr Cys His Asp 340
345 350 Arg Val Ala Ser Phe Tyr Cys Glu Cys
Pro His Gly Arg Thr Gly Leu 355 360
365 Leu Cys His Leu Asn Asp Ala Cys Ile Ser Asn Pro Cys Asn
Glu Gly 370 375 380
Ser Asn Cys Asp Thr Asn Pro Val Asn Gly Lys Ala Ile Cys Thr Cys 385
390 395 400 Pro Ser Gly Tyr Thr
Gly Pro Ala Cys Ser Gln Asp Val Asp Glu Cys 405
410 415 Ser Leu Gly Ala Asn Pro Cys Glu His Ala
Gly Lys Cys Ile Asn Thr 420 425
430 Leu Gly Ser Phe Glu Cys Gln Cys Leu Gln Gly Tyr Thr Gly Pro
Arg 435 440 445 Cys
Glu Ile Asp Val Asn Glu Cys Val Ser Asn Pro Cys Gln Asn Asp 450
455 460 Ala Thr Cys Leu Asp Gln
Ile Gly Glu Phe Gln Cys Ile Cys Met Pro 465 470
475 480 Gly Tyr Glu Gly Val His Cys Glu Val Asn Thr
Asp Glu Cys Ala Ser 485 490
495 Ser Pro Cys Leu His Asn Gly Arg Cys Leu Asp Lys Ile Asn Glu Phe
500 505 510 Gln Cys
Glu Cys Pro Thr Gly Phe Thr Gly His Leu Cys Gln Tyr Asp 515
520 525 Val Asp Glu Cys Ala Ser Thr
Pro Cys Lys Asn Gly Ala Lys Cys Leu 530 535
540 Asp Gly Pro Asn Thr Tyr Thr Cys Val Cys Thr Glu
Gly Tyr Thr Gly 545 550 555
560 Thr His Cys Glu Val Asp Ile Asp Glu Cys Asp Pro Asp Pro Cys His
565 570 575 Tyr Gly Ser
Cys Lys Asp Gly Val Ala Thr Phe Thr Cys Leu Cys Arg 580
585 590 Pro Gly Tyr Thr Gly His His Cys
Glu Thr Asn Ile Asn Glu Cys Ser 595 600
605 Ser Gln Pro Cys Arg His Gly Gly Thr Cys Gln Asp Arg
Asp Asn Ala 610 615 620
Tyr Leu Cys Phe Cys Leu Lys Gly Thr Thr Gly Pro Asn Cys Glu Ile 625
630 635 640 Asn Leu Asp Asp
Cys Ala Ser Ser Pro Cys Asp Ser Gly Thr Cys Leu 645
650 655 Asp Lys Ile Asp Gly Tyr Glu Cys Ala
Cys Glu Pro Gly Tyr Thr Gly 660 665
670 Ser Met Cys Asn Ile Asn Ile Asp Glu Cys Ala Gly Asn Pro
Cys His 675 680 685
Asn Gly Gly Thr Cys Glu Asp Gly Ile Asn Gly Phe Thr Cys Arg Cys 690
695 700 Pro Glu Gly Tyr His
Asp Pro Thr Cys Leu Ser Glu Val Asn Glu Cys 705 710
715 720 Asn Ser Asn Pro Cys Val His Gly Ala Cys
Arg Asp Ser Leu Asn Gly 725 730
735 Tyr Lys Cys Asp Cys Asp Pro Gly Trp Ser Gly Thr Asn Cys Asp
Ile 740 745 750 Asn
Asn Asn Glu Cys Glu Ser Asn Pro Cys Val Asn Gly Gly Thr Cys 755
760 765 Lys Asp Met Thr Ser Gly
Tyr Val Cys Thr Cys Arg Glu Gly Phe Ser 770 775
780 Gly Pro Asn Cys Gln Thr Asn Ile Asn Glu Cys
Ala Ser Asn Pro Cys 785 790 795
800 Leu Asn Gln Gly Thr Cys Ile Asp Asp Val Ala Gly Tyr Lys Cys Asn
805 810 815 Cys Leu
Leu Pro Tyr Thr Gly Ala Thr Cys Glu Val Val Leu Ala Pro 820
825 830 Cys Ala Pro Ser Pro Cys Arg
Asn Gly Gly Glu Cys Arg Gln Ser Glu 835 840
845 Asp Tyr Glu Ser Phe Ser Cys Val Cys Pro Thr Gly
Trp Gln Gly Gln 850 855 860
Thr Cys Glu Val Asp Ile Asn Glu Cys Val Leu Ser Pro Cys Arg His 865
870 875 880 Gly Ala Ser
Cys Gln Asn Thr His Gly Gly Tyr Arg Cys His Cys Gln 885
890 895 Ala Gly Tyr Ser Gly Arg Asn Cys
Glu Thr Asp Ile Asp Asp Cys Arg 900 905
910 Pro Asn Pro Cys His Asn Gly Gly Ser Cys Thr Asp Gly
Ile Asn Thr 915 920 925
Ala Phe Cys Asp Cys Leu Pro Gly Phe Arg Gly Thr Phe Cys Glu Glu 930
935 940 Asp Ile Asn Glu
Cys Ala Ser Asp Pro Cys Arg Asn Gly Ala Asn Cys 945 950
955 960 Thr Asp Cys Val Asp Ser Tyr Thr Cys
Thr Cys Pro Ala Gly Phe Ser 965 970
975 Gly Ile His Cys Glu Asn Asn Thr Pro Asp Cys Thr Glu Ser
Ser Cys 980 985 990
Phe Asn Gly Gly Thr Cys Val Asp Gly Ile Asn Ser Phe Thr Cys Leu
995 1000 1005 Cys Pro Pro
Gly Phe Thr Gly Ser Tyr Cys Gln His Asp Val Asn 1010
1015 1020 Glu Cys Asp Ser Gln Pro Cys Leu
His Gly Gly Thr Cys Gln Asp 1025 1030
1035 Gly Cys Gly Ser Tyr Arg Cys Thr Cys Pro Gln Gly Tyr
Thr Gly 1040 1045 1050
Pro Asn Cys Gln Asn Leu Val His Trp Cys Asp Ser Ser Pro Cys 1055
1060 1065 Lys Asn Gly Gly Lys
Cys Trp Gln Thr His Thr Gln Tyr Arg Cys 1070 1075
1080 Glu Cys Pro Ser Gly Trp Thr Gly Leu Tyr
Cys Asp Val Pro Ser 1085 1090 1095
Val Ser Cys Glu Val Ala Ala Gln Arg Gln Gly Val Asp Val Ala
1100 1105 1110 Arg Leu
Cys Gln His Gly Gly Leu Cys Val Asp Ala Gly Asn Thr 1115
1120 1125 His His Cys Arg Cys Gln Ala
Gly Tyr Thr Gly Ser Tyr Cys Glu 1130 1135
1140 Asp Leu Val Asp Glu Cys Ser Pro Ser Pro Cys Gln
Asn Gly Ala 1145 1150 1155
Thr Cys Thr Asp Tyr Leu Gly Gly Tyr Ser Cys Lys Cys Val Ala 1160
1165 1170 Gly Tyr His Gly Val
Asn Cys Ser Glu Glu Ile Asp Glu Cys Leu 1175 1180
1185 Ser His Pro Cys Gln Asn Gly Gly Thr Cys
Leu Asp Leu Pro Asn 1190 1195 1200
Thr Tyr Lys Cys Ser Cys Pro Arg Gly Thr Gln Gly Val His Cys
1205 1210 1215 Glu Ile
Asn Val Asp Asp Cys Asn Pro Pro Val Asp Pro Val Ser 1220
1225 1230 Arg Ser Pro Lys Cys Phe Asn
Asn Gly Thr Cys Val Asp Gln Val 1235 1240
1245 Gly Gly Tyr Ser Cys Thr Cys Pro Pro Gly Phe Val
Gly Glu Arg 1250 1255 1260
Cys Glu Gly Asp Val Asn Glu Cys Leu Ser Asn Pro Cys Asp Ala 1265
1270 1275 Arg Gly Thr Gln Asn
Cys Val Gln Arg Val Asn Asp Phe His Cys 1280 1285
1290 Glu Cys Arg Ala Gly His Thr Gly Arg Arg
Cys Glu Ser Val Ile 1295 1300 1305
Asn Gly Cys Lys Gly Lys Pro Cys Lys Asn Gly Gly Thr Cys Ala
1310 1315 1320 Val Ala
Ser Asn Thr Ala Arg Gly Phe Ile Cys Lys Cys Pro Ala 1325
1330 1335 Gly Phe Glu Gly Ala Thr Cys
Glu Asn Asp Ala Arg Thr Cys Gly 1340 1345
1350 Ser Leu Arg Cys Leu Asn Gly Gly Thr Cys Ile Ser
Gly Pro Arg 1355 1360 1365
Ser Pro Thr Cys Leu Cys Leu Gly Pro Phe Thr Gly Pro Glu Cys 1370
1375 1380 Gln Phe Pro Ala Ser
Ser Pro Cys Leu Gly Gly Asn Pro Cys Tyr 1385 1390
1395 Asn Gln Gly Thr Cys Glu Pro Thr Ser Glu
Ser Pro Phe Tyr Arg 1400 1405 1410
Cys Leu Cys Pro Ala Lys Phe Asn Gly Leu Leu Cys His Ile Leu
1415 1420 1425 Asp Tyr
Ser Phe Gly Asp Leu Gly Pro Gly Glu Pro Lys Ser Cys 1430
1435 1440 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu 1445 1450
1455 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr 1460 1465 1470
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 1475
1480 1485 Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 1490 1495
1500 Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln 1505 1510 1515
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
1520 1525 1530 Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 1535
1540 1545 Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys 1550 1555
1560 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg 1565 1570 1575
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 1580
1585 1590 Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 1595 1600
1605 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser 1610 1615 1620
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
1625 1630 1635 Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 1640
1645 1650 Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro 1655 1660
1665 Gly Lys 1670 55768PRTHomo sapiens 55Met Pro
Pro Leu Leu Ala Pro Leu Leu Cys Leu Ala Leu Leu Pro Ala 1 5
10 15 Leu Ala Ala Arg Gly Pro Arg
Cys Ser Gln Pro Gly Glu Thr Cys Leu 20 25
30 Asn Gly Gly Lys Cys Glu Ala Ala Asn Gly Thr Glu
Ala Cys Val Cys 35 40 45
Gly Gly Ala Phe Val Gly Pro Arg Cys Gln Asp Pro Asn Pro Cys Leu
50 55 60 Ser Thr Pro
Cys Lys Asn Ala Gly Thr Cys His Val Val Asp Arg Arg 65
70 75 80 Gly Val Ala Asp Tyr Ala Cys
Ser Cys Ala Leu Gly Phe Ser Gly Pro 85
90 95 Leu Cys Leu Thr Pro Leu Asp Asn Ala Cys Leu
Thr Asn Pro Cys Arg 100 105
110 Asn Gly Gly Thr Cys Asp Leu Leu Thr Leu Thr Glu Tyr Lys Cys
Arg 115 120 125 Cys
Pro Pro Gly Trp Ser Gly Lys Ser Cys Gln Gln Ala Asp Pro Cys 130
135 140 Ala Ser Asn Pro Cys Ala
Asn Gly Gly Gln Cys Leu Pro Phe Glu Ala 145 150
155 160 Ser Tyr Ile Cys His Cys Pro Pro Ser Phe His
Gly Pro Thr Cys Arg 165 170
175 Gln Asp Val Asn Glu Cys Gly Gln Lys Pro Gly Leu Cys Arg His Gly
180 185 190 Gly Thr
Cys His Asn Glu Val Gly Ser Tyr Arg Cys Val Cys Arg Ala 195
200 205 Thr His Thr Gly Pro Asn Cys
Glu Arg Pro Tyr Val Pro Cys Ser Pro 210 215
220 Ser Pro Cys Gln Asn Gly Gly Thr Cys Arg Pro Thr
Gly Asp Val Thr 225 230 235
240 His Glu Cys Ala Cys Leu Pro Gly Phe Thr Gly Gln Asn Cys Glu Glu
245 250 255 Asn Ile Asp
Asp Cys Pro Gly Asn Asn Cys Lys Asn Gly Gly Ala Cys 260
265 270 Val Asp Gly Val Asn Thr Tyr Asn
Cys Arg Cys Pro Pro Glu Trp Thr 275 280
285 Gly Gln Tyr Cys Thr Glu Asp Val Asp Glu Cys Gln Leu
Met Pro Asn 290 295 300
Ala Cys Gln Asn Gly Gly Thr Cys His Asn Thr His Gly Gly Tyr Asn 305
310 315 320 Cys Val Cys Val
Asn Gly Trp Thr Gly Glu Asp Cys Ser Glu Asn Ile 325
330 335 Asp Asp Cys Ala Ser Ala Ala Cys Phe
His Gly Ala Thr Cys His Asp 340 345
350 Arg Val Ala Ser Phe Tyr Cys Glu Cys Pro His Gly Arg Thr
Gly Leu 355 360 365
Leu Cys His Leu Asn Asp Ala Cys Ile Ser Asn Pro Cys Asn Glu Gly 370
375 380 Ser Asn Cys Asp Thr
Asn Pro Val Asn Gly Lys Ala Ile Cys Thr Cys 385 390
395 400 Pro Ser Gly Tyr Thr Gly Pro Ala Cys Ser
Gln Asp Val Asp Glu Cys 405 410
415 Ser Leu Gly Ala Asn Pro Cys Glu His Ala Gly Lys Cys Ile Asn
Thr 420 425 430 Leu
Gly Ser Phe Glu Cys Gln Cys Leu Gln Gly Tyr Thr Gly Pro Arg 435
440 445 Cys Glu Ile Asp Val Asn
Glu Cys Val Ser Asn Pro Cys Gln Asn Asp 450 455
460 Ala Thr Cys Leu Asp Gln Ile Gly Glu Phe Gln
Cys Ile Cys Met Pro 465 470 475
480 Gly Tyr Glu Gly Val His Cys Glu Val Asn Thr Asp Glu Cys Ala Ser
485 490 495 Ser Pro
Cys Leu His Asn Gly Arg Cys Leu Asp Lys Ile Asn Glu Phe 500
505 510 Gln Cys Glu Cys Pro Thr Gly
Phe Thr Gly His Leu Cys Gln Tyr Asp 515 520
525 Val Asp Glu Asp Leu Gly Pro Gly Glu Pro Lys Ser
Cys Asp Lys Thr 530 535 540
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 545
550 555 560 Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 565
570 575 Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro 580 585
590 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala 595 600 605
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 610
615 620 Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 625 630
635 640 Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr 645 650
655 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu 660 665 670
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
675 680 685 Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 690
695 700 Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp 705 710
715 720 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser 725 730
735 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
740 745 750 Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 755
760 765 561185PRTHomo sapiens 56Met Pro
Pro Leu Leu Ala Pro Leu Leu Cys Leu Ala Leu Leu Pro Ala 1 5
10 15 Leu Ala Ala Arg Gly Pro Arg
Cys Ser Gln Pro Gly Glu Thr Cys Leu 20 25
30 Asn Gly Gly Lys Cys Glu Ala Ala Asn Gly Thr Glu
Ala Cys Val Cys 35 40 45
Gly Gly Ala Phe Val Gly Pro Arg Cys Gln Asp Pro Asn Pro Cys Leu
50 55 60 Ser Thr Pro
Cys Lys Asn Ala Gly Thr Cys His Val Val Asp Arg Arg 65
70 75 80 Gly Val Ala Asp Tyr Ala Cys
Ser Cys Ala Leu Gly Phe Ser Gly Pro 85
90 95 Leu Cys Leu Thr Pro Leu Asp Asn Ala Cys Leu
Thr Asn Pro Cys Arg 100 105
110 Asn Gly Gly Thr Cys Asp Leu Leu Thr Leu Thr Glu Tyr Lys Cys
Arg 115 120 125 Cys
Pro Pro Gly Trp Ser Gly Lys Ser Cys Gln Gln Ala Asp Pro Cys 130
135 140 Ala Ser Asn Pro Cys Ala
Asn Gly Gly Gln Cys Leu Pro Phe Glu Ala 145 150
155 160 Ser Tyr Ile Cys His Cys Pro Pro Ser Phe His
Gly Pro Thr Cys Arg 165 170
175 Gln Asp Val Asn Glu Cys Gly Gln Lys Pro Gly Leu Cys Arg His Gly
180 185 190 Gly Thr
Cys His Asn Glu Val Gly Ser Tyr Arg Cys Val Cys Arg Ala 195
200 205 Thr His Thr Gly Pro Asn Cys
Glu Arg Pro Tyr Val Pro Cys Ser Pro 210 215
220 Ser Pro Cys Gln Asn Gly Gly Thr Cys Arg Pro Thr
Gly Asp Val Thr 225 230 235
240 His Glu Cys Ala Cys Leu Pro Gly Phe Thr Gly Gln Asn Cys Glu Glu
245 250 255 Asn Ile Asp
Asp Cys Pro Gly Asn Asn Cys Lys Asn Gly Gly Ala Cys 260
265 270 Val Asp Gly Val Asn Thr Tyr Asn
Cys Arg Cys Pro Pro Glu Trp Thr 275 280
285 Gly Gln Tyr Cys Thr Glu Asp Val Asp Glu Cys Gln Leu
Met Pro Asn 290 295 300
Ala Cys Gln Asn Gly Gly Thr Cys His Asn Thr His Gly Gly Tyr Asn 305
310 315 320 Cys Val Cys Val
Asn Gly Trp Thr Gly Glu Asp Cys Ser Glu Asn Ile 325
330 335 Asp Asp Cys Ala Ser Ala Ala Cys Phe
His Gly Ala Thr Cys His Asp 340 345
350 Arg Val Ala Ser Phe Tyr Cys Glu Cys Pro His Gly Arg Thr
Gly Leu 355 360 365
Leu Cys His Leu Asn Asp Ala Cys Ile Ser Asn Pro Cys Asn Glu Gly 370
375 380 Ser Asn Cys Asp Thr
Asn Pro Val Asn Gly Lys Ala Ile Cys Thr Cys 385 390
395 400 Pro Ser Gly Tyr Thr Gly Pro Ala Cys Ser
Gln Asp Val Asp Glu Cys 405 410
415 Ser Leu Gly Ala Asn Pro Cys Glu His Ala Gly Lys Cys Ile Asn
Thr 420 425 430 Leu
Gly Ser Phe Glu Cys Gln Cys Leu Gln Gly Tyr Thr Gly Pro Arg 435
440 445 Cys Glu Ile Asp Val Asn
Glu Cys Val Ser Asn Pro Cys Gln Asn Asp 450 455
460 Ala Thr Cys Leu Asp Gln Ile Gly Glu Phe Gln
Cys Ile Cys Met Pro 465 470 475
480 Gly Tyr Glu Gly Val His Cys Glu Val Asn Thr Asp Glu Cys Ala Ser
485 490 495 Ser Pro
Cys Leu His Asn Gly Arg Cys Leu Asp Lys Ile Asn Glu Phe 500
505 510 Gln Cys Glu Cys Pro Thr Gly
Phe Thr Gly His Leu Cys Gln Tyr Asp 515 520
525 Val Asp Glu Cys Ala Ser Thr Pro Cys Lys Asn Gly
Ala Lys Cys Leu 530 535 540
Asp Gly Pro Asn Thr Tyr Thr Cys Val Cys Thr Glu Gly Tyr Thr Gly 545
550 555 560 Thr His Cys
Glu Val Asp Ile Asp Glu Cys Asp Pro Asp Pro Cys His 565
570 575 Tyr Gly Ser Cys Lys Asp Gly Val
Ala Thr Phe Thr Cys Leu Cys Arg 580 585
590 Pro Gly Tyr Thr Gly His His Cys Glu Thr Asn Ile Asn
Glu Cys Ser 595 600 605
Ser Gln Pro Cys Arg His Gly Gly Thr Cys Gln Asp Arg Asp Asn Ala 610
615 620 Tyr Leu Cys Phe
Cys Leu Lys Gly Thr Thr Gly Pro Asn Cys Glu Ile 625 630
635 640 Asn Leu Asp Asp Cys Ala Ser Ser Pro
Cys Asp Ser Gly Thr Cys Leu 645 650
655 Asp Lys Ile Asp Gly Tyr Glu Cys Ala Cys Glu Pro Gly Tyr
Thr Gly 660 665 670
Ser Met Cys Asn Ile Asn Ile Asp Glu Cys Ala Gly Asn Pro Cys His
675 680 685 Asn Gly Gly Thr
Cys Glu Asp Gly Ile Asn Gly Phe Thr Cys Arg Cys 690
695 700 Pro Glu Gly Tyr His Asp Pro Thr
Cys Leu Ser Glu Val Asn Glu Cys 705 710
715 720 Asn Ser Asn Pro Cys Val His Gly Ala Cys Arg Asp
Ser Leu Asn Gly 725 730
735 Tyr Lys Cys Asp Cys Asp Pro Gly Trp Ser Gly Thr Asn Cys Asp Ile
740 745 750 Asn Asn Asn
Glu Cys Glu Ser Asn Pro Cys Val Asn Gly Gly Thr Cys 755
760 765 Lys Asp Met Thr Ser Gly Tyr Val
Cys Thr Cys Arg Glu Gly Phe Ser 770 775
780 Gly Pro Asn Cys Gln Thr Asn Ile Asn Glu Cys Ala Ser
Asn Pro Cys 785 790 795
800 Leu Asn Gln Gly Thr Cys Ile Asp Asp Val Ala Gly Tyr Lys Cys Asn
805 810 815 Cys Leu Leu Pro
Tyr Thr Gly Ala Thr Cys Glu Val Val Leu Ala Pro 820
825 830 Cys Ala Pro Ser Pro Cys Arg Asn Gly
Gly Glu Cys Arg Gln Ser Glu 835 840
845 Asp Tyr Glu Ser Phe Ser Cys Val Cys Pro Thr Gly Trp Gln
Gly Gln 850 855 860
Thr Cys Glu Val Asp Ile Asn Glu Cys Val Leu Ser Pro Cys Arg His 865
870 875 880 Gly Ala Ser Cys Gln
Asn Thr His Gly Gly Tyr Arg Cys His Cys Gln 885
890 895 Ala Gly Tyr Ser Gly Arg Asn Cys Glu Thr
Asp Ile Asp Asp Cys Arg 900 905
910 Pro Asn Pro Cys His Asn Gly Gly Ser Cys Thr Asp Gly Ile Asn
Thr 915 920 925 Ala
Phe Cys Asp Cys Leu Pro Gly Phe Arg Gly Thr Phe Cys Glu Glu 930
935 940 Asp Ile Asn Glu Asp Leu
Gly Pro Gly Glu Pro Lys Ser Cys Asp Lys 945 950
955 960 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro 965 970
975 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
980 985 990 Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 995
1000 1005 Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His 1010 1015
1020 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr 1025 1030 1035
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
1040 1045 1050 Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 1055
1060 1065 Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu 1070 1075
1080 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys 1085 1090 1095
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 1100
1105 1110 Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 1115 1120
1125 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 1130 1135 1140
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
1145 1150 1155 Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 1160
1165 1170 Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 1175 1180 1185
57830PRTHomo sapiens 57Met Pro Pro Leu Leu Ala Pro Leu Leu Cys Leu Ala
Leu Leu Pro Ala 1 5 10
15 Leu Ala Ala Arg Gly Pro Arg Cys Ala Ser Ala Ala Cys Phe His Gly
20 25 30 Ala Thr Cys
His Asp Arg Val Ala Ser Phe Tyr Cys Glu Cys Pro His 35
40 45 Gly Arg Thr Gly Leu Leu Cys His
Leu Asn Asp Ala Cys Ile Ser Asn 50 55
60 Pro Cys Asn Glu Gly Ser Asn Cys Asp Thr Asn Pro Val
Asn Gly Lys 65 70 75
80 Ala Ile Cys Thr Cys Pro Ser Gly Tyr Thr Gly Pro Ala Cys Ser Gln
85 90 95 Asp Val Asp Glu
Cys Ser Leu Gly Ala Asn Pro Cys Glu His Ala Gly 100
105 110 Lys Cys Ile Asn Thr Leu Gly Ser Phe
Glu Cys Gln Cys Leu Gln Gly 115 120
125 Tyr Thr Gly Pro Arg Cys Glu Ile Asp Val Asn Glu Cys Val
Ser Asn 130 135 140
Pro Cys Gln Asn Asp Ala Thr Cys Leu Asp Gln Ile Gly Glu Phe Gln 145
150 155 160 Cys Ile Cys Met Pro
Gly Tyr Glu Gly Val His Cys Glu Val Asn Thr 165
170 175 Asp Glu Cys Ala Ser Ser Pro Cys Leu His
Asn Gly Arg Cys Leu Asp 180 185
190 Lys Ile Asn Glu Phe Gln Cys Glu Cys Pro Thr Gly Phe Thr Gly
His 195 200 205 Leu
Cys Gln Tyr Asp Val Asp Glu Cys Ala Ser Thr Pro Cys Lys Asn 210
215 220 Gly Ala Lys Cys Leu Asp
Gly Pro Asn Thr Tyr Thr Cys Val Cys Thr 225 230
235 240 Glu Gly Tyr Thr Gly Thr His Cys Glu Val Asp
Ile Asp Glu Cys Asp 245 250
255 Pro Asp Pro Cys His Tyr Gly Ser Cys Lys Asp Gly Val Ala Thr Phe
260 265 270 Thr Cys
Leu Cys Arg Pro Gly Tyr Thr Gly His His Cys Glu Thr Asn 275
280 285 Ile Asn Glu Cys Ser Ser Gln
Pro Cys Arg His Gly Gly Thr Cys Gln 290 295
300 Asp Arg Asp Asn Ala Tyr Leu Cys Phe Cys Leu Lys
Gly Thr Thr Gly 305 310 315
320 Pro Asn Cys Glu Ile Asn Leu Asp Asp Cys Ala Ser Ser Pro Cys Asp
325 330 335 Ser Gly Thr
Cys Leu Asp Lys Ile Asp Gly Tyr Glu Cys Ala Cys Glu 340
345 350 Pro Gly Tyr Thr Gly Ser Met Cys
Asn Ile Asn Ile Asp Glu Cys Ala 355 360
365 Gly Asn Pro Cys His Asn Gly Gly Thr Cys Glu Asp Gly
Ile Asn Gly 370 375 380
Phe Thr Cys Arg Cys Pro Glu Gly Tyr His Asp Pro Thr Cys Leu Ser 385
390 395 400 Glu Val Asn Glu
Cys Asn Ser Asn Pro Cys Val His Gly Ala Cys Arg 405
410 415 Asp Ser Leu Asn Gly Tyr Lys Cys Asp
Cys Asp Pro Gly Trp Ser Gly 420 425
430 Thr Asn Cys Asp Ile Asn Asn Asn Glu Cys Glu Ser Asn Pro
Cys Val 435 440 445
Asn Gly Gly Thr Cys Lys Asp Met Thr Ser Gly Tyr Val Cys Thr Cys 450
455 460 Arg Glu Gly Phe Ser
Gly Pro Asn Cys Gln Thr Asn Ile Asn Glu Cys 465 470
475 480 Ala Ser Asn Pro Cys Leu Asn Gln Gly Thr
Cys Ile Asp Asp Val Ala 485 490
495 Gly Tyr Lys Cys Asn Cys Leu Leu Pro Tyr Thr Gly Ala Thr Cys
Glu 500 505 510 Val
Val Leu Ala Pro Cys Ala Pro Ser Pro Cys Arg Asn Gly Gly Glu 515
520 525 Cys Arg Gln Ser Glu Asp
Tyr Glu Ser Phe Ser Cys Val Cys Pro Thr 530 535
540 Gly Trp Gln Gly Gln Thr Cys Glu Val Asp Ile
Asn Glu Cys Val Leu 545 550 555
560 Ser Pro Cys Arg His Gly Ala Ser Cys Gln Asn Thr His Gly Gly Tyr
565 570 575 Arg Cys
His Cys Gln Ala Gly Tyr Ser Gly Arg Asn Cys Glu Thr Asp 580
585 590 Ile Asp Leu Gly Pro Gly Glu
Pro Lys Ser Cys Asp Lys Thr His Thr 595 600
605 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe 610 615 620
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 625
630 635 640 Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 645
650 655 Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr 660 665
670 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val 675 680 685
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 690
695 700 Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 705 710
715 720 Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro 725 730
735 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val 740 745 750
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
755 760 765 Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 770
775 780 Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp 785 790
795 800 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His 805 810
815 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
820 825 830 58829PRTHomo sapiens 58Met
Trp Gly Trp Lys Cys Leu Leu Phe Trp Ala Val Leu Val Thr Ala 1
5 10 15 Thr Leu Cys Thr Ala Arg
Cys Ala Ser Ala Ala Cys Phe His Gly Ala 20
25 30 Thr Cys His Asp Arg Val Ala Ser Phe Tyr
Cys Glu Cys Pro His Gly 35 40
45 Arg Thr Gly Leu Leu Cys His Leu Asn Asp Ala Cys Ile Ser
Asn Pro 50 55 60
Cys Asn Glu Gly Ser Asn Cys Asp Thr Asn Pro Val Asn Gly Lys Ala 65
70 75 80 Ile Cys Thr Cys Pro
Ser Gly Tyr Thr Gly Pro Ala Cys Ser Gln Asp 85
90 95 Val Asp Glu Cys Ser Leu Gly Ala Asn Pro
Cys Glu His Ala Gly Lys 100 105
110 Cys Ile Asn Thr Leu Gly Ser Phe Glu Cys Gln Cys Leu Gln Gly
Tyr 115 120 125 Thr
Gly Pro Arg Cys Glu Ile Asp Val Asn Glu Cys Val Ser Asn Pro 130
135 140 Cys Gln Asn Asp Ala Thr
Cys Leu Asp Gln Ile Gly Glu Phe Gln Cys 145 150
155 160 Ile Cys Met Pro Gly Tyr Glu Gly Val His Cys
Glu Val Asn Thr Asp 165 170
175 Glu Cys Ala Ser Ser Pro Cys Leu His Asn Gly Arg Cys Leu Asp Lys
180 185 190 Ile Asn
Glu Phe Gln Cys Glu Cys Pro Thr Gly Phe Thr Gly His Leu 195
200 205 Cys Gln Tyr Asp Val Asp Glu
Cys Ala Ser Thr Pro Cys Lys Asn Gly 210 215
220 Ala Lys Cys Leu Asp Gly Pro Asn Thr Tyr Thr Cys
Val Cys Thr Glu 225 230 235
240 Gly Tyr Thr Gly Thr His Cys Glu Val Asp Ile Asp Glu Cys Asp Pro
245 250 255 Asp Pro Cys
His Tyr Gly Ser Cys Lys Asp Gly Val Ala Thr Phe Thr 260
265 270 Cys Leu Cys Arg Pro Gly Tyr Thr
Gly His His Cys Glu Thr Asn Ile 275 280
285 Asn Glu Cys Ser Ser Gln Pro Cys Arg His Gly Gly Thr
Cys Gln Asp 290 295 300
Arg Asp Asn Ala Tyr Leu Cys Phe Cys Leu Lys Gly Thr Thr Gly Pro 305
310 315 320 Asn Cys Glu Ile
Asn Leu Asp Asp Cys Ala Ser Ser Pro Cys Asp Ser 325
330 335 Gly Thr Cys Leu Asp Lys Ile Asp Gly
Tyr Glu Cys Ala Cys Glu Pro 340 345
350 Gly Tyr Thr Gly Ser Met Cys Asn Ile Asn Ile Asp Glu Cys
Ala Gly 355 360 365
Asn Pro Cys His Asn Gly Gly Thr Cys Glu Asp Gly Ile Asn Gly Phe 370
375 380 Thr Cys Arg Cys Pro
Glu Gly Tyr His Asp Pro Thr Cys Leu Ser Glu 385 390
395 400 Val Asn Glu Cys Asn Ser Asn Pro Cys Val
His Gly Ala Cys Arg Asp 405 410
415 Ser Leu Asn Gly Tyr Lys Cys Asp Cys Asp Pro Gly Trp Ser Gly
Thr 420 425 430 Asn
Cys Asp Ile Asn Asn Asn Glu Cys Glu Ser Asn Pro Cys Val Asn 435
440 445 Gly Gly Thr Cys Lys Asp
Met Thr Ser Gly Tyr Val Cys Thr Cys Arg 450 455
460 Glu Gly Phe Ser Gly Pro Asn Cys Gln Thr Asn
Ile Asn Glu Cys Ala 465 470 475
480 Ser Asn Pro Cys Leu Asn Gln Gly Thr Cys Ile Asp Asp Val Ala Gly
485 490 495 Tyr Lys
Cys Asn Cys Leu Leu Pro Tyr Thr Gly Ala Thr Cys Glu Val 500
505 510 Val Leu Ala Pro Cys Ala Pro
Ser Pro Cys Arg Asn Gly Gly Glu Cys 515 520
525 Arg Gln Ser Glu Asp Tyr Glu Ser Phe Ser Cys Val
Cys Pro Thr Gly 530 535 540
Trp Gln Gly Gln Thr Cys Glu Val Asp Ile Asn Glu Cys Val Leu Ser 545
550 555 560 Pro Cys Arg
His Gly Ala Ser Cys Gln Asn Thr His Gly Gly Tyr Arg 565
570 575 Cys His Cys Gln Ala Gly Tyr Ser
Gly Arg Asn Cys Glu Thr Asp Ile 580 585
590 Asp Leu Gly Pro Gly Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys 595 600 605
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 610
615 620 Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 625 630
635 640 Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys 645 650
655 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys 660 665 670
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
675 680 685 Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 690
695 700 Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys 705 710
715 720 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser 725 730
735 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
740 745 750 Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 755
760 765 Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly 770 775
780 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln 785 790 795
800 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
805 810 815 His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 820 825
591355PRTHomo sapiens 59Met Pro Pro Leu Leu Ala Pro Leu Leu
Cys Leu Ala Leu Leu Pro Ala 1 5 10
15 Leu Ala Ala Arg Gly Pro Arg Cys Ala Ser Ala Ala Cys Phe
His Gly 20 25 30
Ala Thr Cys His Asp Arg Val Ala Ser Phe Tyr Cys Glu Cys Pro His
35 40 45 Gly Arg Thr Gly
Leu Leu Cys His Leu Asn Asp Ala Cys Ile Ser Asn 50
55 60 Pro Cys Asn Glu Gly Ser Asn Cys
Asp Thr Asn Pro Val Asn Gly Lys 65 70
75 80 Ala Ile Cys Thr Cys Pro Ser Gly Tyr Thr Gly Pro
Ala Cys Ser Gln 85 90
95 Asp Val Asp Glu Cys Ser Leu Gly Ala Asn Pro Cys Glu His Ala Gly
100 105 110 Lys Cys Ile
Asn Thr Leu Gly Ser Phe Glu Cys Gln Cys Leu Gln Gly 115
120 125 Tyr Thr Gly Pro Arg Cys Glu Ile
Asp Val Asn Glu Cys Val Ser Asn 130 135
140 Pro Cys Gln Asn Asp Ala Thr Cys Leu Asp Gln Ile Gly
Glu Phe Gln 145 150 155
160 Cys Ile Cys Met Pro Gly Tyr Glu Gly Val His Cys Glu Val Asn Thr
165 170 175 Asp Glu Cys Ala
Ser Ser Pro Cys Leu His Asn Gly Arg Cys Leu Asp 180
185 190 Lys Ile Asn Glu Phe Gln Cys Glu Cys
Pro Thr Gly Phe Thr Gly His 195 200
205 Leu Cys Gln Tyr Asp Val Asp Glu Cys Ala Ser Thr Pro Cys
Lys Asn 210 215 220
Gly Ala Lys Cys Leu Asp Gly Pro Asn Thr Tyr Thr Cys Val Cys Thr 225
230 235 240 Glu Gly Tyr Thr Gly
Thr His Cys Glu Val Asp Ile Asp Glu Cys Asp 245
250 255 Pro Asp Pro Cys His Tyr Gly Ser Cys Lys
Asp Gly Val Ala Thr Phe 260 265
270 Thr Cys Leu Cys Arg Pro Gly Tyr Thr Gly His His Cys Glu Thr
Asn 275 280 285 Ile
Asn Glu Cys Ser Ser Gln Pro Cys Arg His Gly Gly Thr Cys Gln 290
295 300 Asp Arg Asp Asn Ala Tyr
Leu Cys Phe Cys Leu Lys Gly Thr Thr Gly 305 310
315 320 Pro Asn Cys Glu Ile Asn Leu Asp Asp Cys Ala
Ser Ser Pro Cys Asp 325 330
335 Ser Gly Thr Cys Leu Asp Lys Ile Asp Gly Tyr Glu Cys Ala Cys Glu
340 345 350 Pro Gly
Tyr Thr Gly Ser Met Cys Asn Ile Asn Ile Asp Glu Cys Ala 355
360 365 Gly Asn Pro Cys His Asn Gly
Gly Thr Cys Glu Asp Gly Ile Asn Gly 370 375
380 Phe Thr Cys Arg Cys Pro Glu Gly Tyr His Asp Pro
Thr Cys Leu Ser 385 390 395
400 Glu Val Asn Glu Cys Asn Ser Asn Pro Cys Val His Gly Ala Cys Arg
405 410 415 Asp Ser Leu
Asn Gly Tyr Lys Cys Asp Cys Asp Pro Gly Trp Ser Gly 420
425 430 Thr Asn Cys Asp Ile Asn Asn Asn
Glu Cys Glu Ser Asn Pro Cys Val 435 440
445 Asn Gly Gly Thr Cys Lys Asp Met Thr Ser Gly Tyr Val
Cys Thr Cys 450 455 460
Arg Glu Gly Phe Ser Gly Pro Asn Cys Gln Thr Asn Ile Asn Glu Cys 465
470 475 480 Ala Ser Asn Pro
Cys Leu Asn Gln Gly Thr Cys Ile Asp Asp Val Ala 485
490 495 Gly Tyr Lys Cys Asn Cys Leu Leu Pro
Tyr Thr Gly Ala Thr Cys Glu 500 505
510 Val Val Leu Ala Pro Cys Ala Pro Ser Pro Cys Arg Asn Gly
Gly Glu 515 520 525
Cys Arg Gln Ser Glu Asp Tyr Glu Ser Phe Ser Cys Val Cys Pro Thr 530
535 540 Gly Trp Gln Gly Gln
Thr Cys Glu Val Asp Ile Asn Glu Cys Val Leu 545 550
555 560 Ser Pro Cys Arg His Gly Ala Ser Cys Gln
Asn Thr His Gly Gly Tyr 565 570
575 Arg Cys His Cys Gln Ala Gly Tyr Ser Gly Arg Asn Cys Glu Thr
Asp 580 585 590 Ile
Asp Asp Cys Arg Pro Asn Pro Cys His Asn Gly Gly Ser Cys Thr 595
600 605 Asp Gly Ile Asn Thr Ala
Phe Cys Asp Cys Leu Pro Gly Phe Arg Gly 610 615
620 Thr Phe Cys Glu Glu Asp Ile Asn Glu Cys Ala
Ser Asp Pro Cys Arg 625 630 635
640 Asn Gly Ala Asn Cys Thr Asp Cys Val Asp Ser Tyr Thr Cys Thr Cys
645 650 655 Pro Ala
Gly Phe Ser Gly Ile His Cys Glu Asn Asn Thr Pro Asp Cys 660
665 670 Thr Glu Ser Ser Cys Phe Asn
Gly Gly Thr Cys Val Asp Gly Ile Asn 675 680
685 Ser Phe Thr Cys Leu Cys Pro Pro Gly Phe Thr Gly
Ser Tyr Cys Gln 690 695 700
His Asp Val Asn Glu Cys Asp Ser Gln Pro Cys Leu His Gly Gly Thr 705
710 715 720 Cys Gln Asp
Gly Cys Gly Ser Tyr Arg Cys Thr Cys Pro Gln Gly Tyr 725
730 735 Thr Gly Pro Asn Cys Gln Asn Leu
Val His Trp Cys Asp Ser Ser Pro 740 745
750 Cys Lys Asn Gly Gly Lys Cys Trp Gln Thr His Thr Gln
Tyr Arg Cys 755 760 765
Glu Cys Pro Ser Gly Trp Thr Gly Leu Tyr Cys Asp Val Pro Ser Val 770
775 780 Ser Cys Glu Val
Ala Ala Gln Arg Gln Gly Val Asp Val Ala Arg Leu 785 790
795 800 Cys Gln His Gly Gly Leu Cys Val Asp
Ala Gly Asn Thr His His Cys 805 810
815 Arg Cys Gln Ala Gly Tyr Thr Gly Ser Tyr Cys Glu Asp Leu
Val Asp 820 825 830
Glu Cys Ser Pro Ser Pro Cys Gln Asn Gly Ala Thr Cys Thr Asp Tyr
835 840 845 Leu Gly Gly Tyr
Ser Cys Lys Cys Val Ala Gly Tyr His Gly Val Asn 850
855 860 Cys Ser Glu Glu Ile Asp Glu Cys
Leu Ser His Pro Cys Gln Asn Gly 865 870
875 880 Gly Thr Cys Leu Asp Leu Pro Asn Thr Tyr Lys Cys
Ser Cys Pro Arg 885 890
895 Gly Thr Gln Gly Val His Cys Glu Ile Asn Val Asp Asp Cys Asn Pro
900 905 910 Pro Val Asp
Pro Val Ser Arg Ser Pro Lys Cys Phe Asn Asn Gly Thr 915
920 925 Cys Val Asp Gln Val Gly Gly Tyr
Ser Cys Thr Cys Pro Pro Gly Phe 930 935
940 Val Gly Glu Arg Cys Glu Gly Asp Val Asn Glu Cys Leu
Ser Asn Pro 945 950 955
960 Cys Asp Ala Arg Gly Thr Gln Asn Cys Val Gln Arg Val Asn Asp Phe
965 970 975 His Cys Glu Cys
Arg Ala Gly His Thr Gly Arg Arg Cys Glu Ser Val 980
985 990 Ile Asn Gly Cys Lys Gly Lys Pro
Cys Lys Asn Gly Gly Thr Cys Ala 995 1000
1005 Val Ala Ser Asn Thr Ala Arg Gly Phe Ile Cys
Lys Cys Pro Ala 1010 1015 1020
Gly Phe Glu Gly Ala Thr Cys Glu Asn Asp Ala Arg Thr Cys Gly
1025 1030 1035 Ser Leu Arg
Cys Leu Asn Gly Gly Thr Cys Ile Ser Gly Pro Arg 1040
1045 1050 Ser Pro Thr Cys Leu Cys Leu Gly
Pro Phe Thr Gly Pro Glu Cys 1055 1060
1065 Gln Phe Pro Ala Ser Ser Pro Cys Leu Gly Gly Asn Pro
Cys Tyr 1070 1075 1080
Asn Gln Gly Thr Cys Glu Pro Thr Ser Glu Ser Pro Phe Tyr Arg 1085
1090 1095 Cys Leu Cys Pro Ala
Lys Phe Asn Gly Leu Leu Cys His Ile Leu 1100 1105
1110 Asp Tyr Ser Phe Gly Asp Leu Gly Pro Gly
Glu Pro Lys Ser Cys 1115 1120 1125
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
1130 1135 1140 Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 1145
1150 1155 Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp 1160 1165
1170 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp 1175 1180 1185
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 1190
1195 1200 Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His 1205 1210
1215 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 1220 1225 1230
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
1235 1240 1245 Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 1250
1255 1260 Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys 1265 1270
1275 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly 1280 1285 1290
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 1295
1300 1305 Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 1310 1315
1320 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu 1325 1330 1335
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
1340 1345 1350 Gly Lys
1355 601354PRTHomo sapiens 60Met Trp Gly Trp Lys Cys Leu Leu Phe Trp Ala
Val Leu Val Thr Ala 1 5 10
15 Thr Leu Cys Thr Ala Arg Cys Ala Ser Ala Ala Cys Phe His Gly Ala
20 25 30 Thr Cys
His Asp Arg Val Ala Ser Phe Tyr Cys Glu Cys Pro His Gly 35
40 45 Arg Thr Gly Leu Leu Cys His
Leu Asn Asp Ala Cys Ile Ser Asn Pro 50 55
60 Cys Asn Glu Gly Ser Asn Cys Asp Thr Asn Pro Val
Asn Gly Lys Ala 65 70 75
80 Ile Cys Thr Cys Pro Ser Gly Tyr Thr Gly Pro Ala Cys Ser Gln Asp
85 90 95 Val Asp Glu
Cys Ser Leu Gly Ala Asn Pro Cys Glu His Ala Gly Lys 100
105 110 Cys Ile Asn Thr Leu Gly Ser Phe
Glu Cys Gln Cys Leu Gln Gly Tyr 115 120
125 Thr Gly Pro Arg Cys Glu Ile Asp Val Asn Glu Cys Val
Ser Asn Pro 130 135 140
Cys Gln Asn Asp Ala Thr Cys Leu Asp Gln Ile Gly Glu Phe Gln Cys 145
150 155 160 Ile Cys Met Pro
Gly Tyr Glu Gly Val His Cys Glu Val Asn Thr Asp 165
170 175 Glu Cys Ala Ser Ser Pro Cys Leu His
Asn Gly Arg Cys Leu Asp Lys 180 185
190 Ile Asn Glu Phe Gln Cys Glu Cys Pro Thr Gly Phe Thr Gly
His Leu 195 200 205
Cys Gln Tyr Asp Val Asp Glu Cys Ala Ser Thr Pro Cys Lys Asn Gly 210
215 220 Ala Lys Cys Leu Asp
Gly Pro Asn Thr Tyr Thr Cys Val Cys Thr Glu 225 230
235 240 Gly Tyr Thr Gly Thr His Cys Glu Val Asp
Ile Asp Glu Cys Asp Pro 245 250
255 Asp Pro Cys His Tyr Gly Ser Cys Lys Asp Gly Val Ala Thr Phe
Thr 260 265 270 Cys
Leu Cys Arg Pro Gly Tyr Thr Gly His His Cys Glu Thr Asn Ile 275
280 285 Asn Glu Cys Ser Ser Gln
Pro Cys Arg His Gly Gly Thr Cys Gln Asp 290 295
300 Arg Asp Asn Ala Tyr Leu Cys Phe Cys Leu Lys
Gly Thr Thr Gly Pro 305 310 315
320 Asn Cys Glu Ile Asn Leu Asp Asp Cys Ala Ser Ser Pro Cys Asp Ser
325 330 335 Gly Thr
Cys Leu Asp Lys Ile Asp Gly Tyr Glu Cys Ala Cys Glu Pro 340
345 350 Gly Tyr Thr Gly Ser Met Cys
Asn Ile Asn Ile Asp Glu Cys Ala Gly 355 360
365 Asn Pro Cys His Asn Gly Gly Thr Cys Glu Asp Gly
Ile Asn Gly Phe 370 375 380
Thr Cys Arg Cys Pro Glu Gly Tyr His Asp Pro Thr Cys Leu Ser Glu 385
390 395 400 Val Asn Glu
Cys Asn Ser Asn Pro Cys Val His Gly Ala Cys Arg Asp 405
410 415 Ser Leu Asn Gly Tyr Lys Cys Asp
Cys Asp Pro Gly Trp Ser Gly Thr 420 425
430 Asn Cys Asp Ile Asn Asn Asn Glu Cys Glu Ser Asn Pro
Cys Val Asn 435 440 445
Gly Gly Thr Cys Lys Asp Met Thr Ser Gly Tyr Val Cys Thr Cys Arg 450
455 460 Glu Gly Phe Ser
Gly Pro Asn Cys Gln Thr Asn Ile Asn Glu Cys Ala 465 470
475 480 Ser Asn Pro Cys Leu Asn Gln Gly Thr
Cys Ile Asp Asp Val Ala Gly 485 490
495 Tyr Lys Cys Asn Cys Leu Leu Pro Tyr Thr Gly Ala Thr Cys
Glu Val 500 505 510
Val Leu Ala Pro Cys Ala Pro Ser Pro Cys Arg Asn Gly Gly Glu Cys
515 520 525 Arg Gln Ser Glu
Asp Tyr Glu Ser Phe Ser Cys Val Cys Pro Thr Gly 530
535 540 Trp Gln Gly Gln Thr Cys Glu Val
Asp Ile Asn Glu Cys Val Leu Ser 545 550
555 560 Pro Cys Arg His Gly Ala Ser Cys Gln Asn Thr His
Gly Gly Tyr Arg 565 570
575 Cys His Cys Gln Ala Gly Tyr Ser Gly Arg Asn Cys Glu Thr Asp Ile
580 585 590 Asp Asp Cys
Arg Pro Asn Pro Cys His Asn Gly Gly Ser Cys Thr Asp 595
600 605 Gly Ile Asn Thr Ala Phe Cys Asp
Cys Leu Pro Gly Phe Arg Gly Thr 610 615
620 Phe Cys Glu Glu Asp Ile Asn Glu Cys Ala Ser Asp Pro
Cys Arg Asn 625 630 635
640 Gly Ala Asn Cys Thr Asp Cys Val Asp Ser Tyr Thr Cys Thr Cys Pro
645 650 655 Ala Gly Phe Ser
Gly Ile His Cys Glu Asn Asn Thr Pro Asp Cys Thr 660
665 670 Glu Ser Ser Cys Phe Asn Gly Gly Thr
Cys Val Asp Gly Ile Asn Ser 675 680
685 Phe Thr Cys Leu Cys Pro Pro Gly Phe Thr Gly Ser Tyr Cys
Gln His 690 695 700
Asp Val Asn Glu Cys Asp Ser Gln Pro Cys Leu His Gly Gly Thr Cys 705
710 715 720 Gln Asp Gly Cys Gly
Ser Tyr Arg Cys Thr Cys Pro Gln Gly Tyr Thr 725
730 735 Gly Pro Asn Cys Gln Asn Leu Val His Trp
Cys Asp Ser Ser Pro Cys 740 745
750 Lys Asn Gly Gly Lys Cys Trp Gln Thr His Thr Gln Tyr Arg Cys
Glu 755 760 765 Cys
Pro Ser Gly Trp Thr Gly Leu Tyr Cys Asp Val Pro Ser Val Ser 770
775 780 Cys Glu Val Ala Ala Gln
Arg Gln Gly Val Asp Val Ala Arg Leu Cys 785 790
795 800 Gln His Gly Gly Leu Cys Val Asp Ala Gly Asn
Thr His His Cys Arg 805 810
815 Cys Gln Ala Gly Tyr Thr Gly Ser Tyr Cys Glu Asp Leu Val Asp Glu
820 825 830 Cys Ser
Pro Ser Pro Cys Gln Asn Gly Ala Thr Cys Thr Asp Tyr Leu 835
840 845 Gly Gly Tyr Ser Cys Lys Cys
Val Ala Gly Tyr His Gly Val Asn Cys 850 855
860 Ser Glu Glu Ile Asp Glu Cys Leu Ser His Pro Cys
Gln Asn Gly Gly 865 870 875
880 Thr Cys Leu Asp Leu Pro Asn Thr Tyr Lys Cys Ser Cys Pro Arg Gly
885 890 895 Thr Gln Gly
Val His Cys Glu Ile Asn Val Asp Asp Cys Asn Pro Pro 900
905 910 Val Asp Pro Val Ser Arg Ser Pro
Lys Cys Phe Asn Asn Gly Thr Cys 915 920
925 Val Asp Gln Val Gly Gly Tyr Ser Cys Thr Cys Pro Pro
Gly Phe Val 930 935 940
Gly Glu Arg Cys Glu Gly Asp Val Asn Glu Cys Leu Ser Asn Pro Cys 945
950 955 960 Asp Ala Arg Gly
Thr Gln Asn Cys Val Gln Arg Val Asn Asp Phe His 965
970 975 Cys Glu Cys Arg Ala Gly His Thr Gly
Arg Arg Cys Glu Ser Val Ile 980 985
990 Asn Gly Cys Lys Gly Lys Pro Cys Lys Asn Gly Gly Thr
Cys Ala Val 995 1000 1005
Ala Ser Asn Thr Ala Arg Gly Phe Ile Cys Lys Cys Pro Ala Gly
1010 1015 1020 Phe Glu Gly
Ala Thr Cys Glu Asn Asp Ala Arg Thr Cys Gly Ser 1025
1030 1035 Leu Arg Cys Leu Asn Gly Gly Thr
Cys Ile Ser Gly Pro Arg Ser 1040 1045
1050 Pro Thr Cys Leu Cys Leu Gly Pro Phe Thr Gly Pro Glu
Cys Gln 1055 1060 1065
Phe Pro Ala Ser Ser Pro Cys Leu Gly Gly Asn Pro Cys Tyr Asn 1070
1075 1080 Gln Gly Thr Cys Glu
Pro Thr Ser Glu Ser Pro Phe Tyr Arg Cys 1085 1090
1095 Leu Cys Pro Ala Lys Phe Asn Gly Leu Leu
Cys His Ile Leu Asp 1100 1105 1110
Tyr Ser Phe Gly Asp Leu Gly Pro Gly Glu Pro Lys Ser Cys Asp
1115 1120 1125 Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1130
1135 1140 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu 1145 1150
1155 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val 1160 1165 1170
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 1175
1180 1185 Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 1190 1195
1200 Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln 1205 1210 1215
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
1220 1225 1230 Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 1235
1240 1245 Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp 1250 1255
1260 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly 1265 1270 1275
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 1280
1285 1290 Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 1295 1300
1305 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg 1310 1315 1320
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
1325 1330 1335 Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 1340
1345 1350 Lys 61715PRTHomo sapiens 61Met
Pro Pro Leu Leu Ala Pro Leu Leu Cys Leu Ala Leu Leu Pro Ala 1
5 10 15 Leu Ala Ala Arg Gly Pro
Arg Cys Ala Ser Ser Pro Cys Leu His Asn 20
25 30 Gly Arg Cys Leu Asp Lys Ile Asn Glu Phe
Gln Cys Glu Cys Pro Thr 35 40
45 Gly Phe Thr Gly His Leu Cys Gln Tyr Asp Val Asp Glu Cys
Ala Ser 50 55 60
Thr Pro Cys Lys Asn Gly Ala Lys Cys Leu Asp Gly Pro Asn Thr Tyr 65
70 75 80 Thr Cys Val Cys Thr
Glu Gly Tyr Thr Gly Thr His Cys Glu Val Asp 85
90 95 Ile Asp Glu Cys Asp Pro Asp Pro Cys His
Tyr Gly Ser Cys Lys Asp 100 105
110 Gly Val Ala Thr Phe Thr Cys Leu Cys Arg Pro Gly Tyr Thr Gly
His 115 120 125 His
Cys Glu Thr Asn Ile Asn Glu Cys Ser Ser Gln Pro Cys Arg His 130
135 140 Gly Gly Thr Cys Gln Asp
Arg Asp Asn Ala Tyr Leu Cys Phe Cys Leu 145 150
155 160 Lys Gly Thr Thr Gly Pro Asn Cys Glu Ile Asn
Leu Asp Asp Cys Ala 165 170
175 Ser Ser Pro Cys Asp Ser Gly Thr Cys Leu Asp Lys Ile Asp Gly Tyr
180 185 190 Glu Cys
Ala Cys Glu Pro Gly Tyr Thr Gly Ser Met Cys Asn Ile Asn 195
200 205 Ile Asp Glu Cys Ala Gly Asn
Pro Cys His Asn Gly Gly Thr Cys Glu 210 215
220 Asp Gly Ile Asn Gly Phe Thr Cys Arg Cys Pro Glu
Gly Tyr His Asp 225 230 235
240 Pro Thr Cys Leu Ser Glu Val Asn Glu Cys Asn Ser Asn Pro Cys Val
245 250 255 His Gly Ala
Cys Arg Asp Ser Leu Asn Gly Tyr Lys Cys Asp Cys Asp 260
265 270 Pro Gly Trp Ser Gly Thr Asn Cys
Asp Ile Asn Asn Asn Glu Cys Glu 275 280
285 Ser Asn Pro Cys Val Asn Gly Gly Thr Cys Lys Asp Met
Thr Ser Gly 290 295 300
Tyr Val Cys Thr Cys Arg Glu Gly Phe Ser Gly Pro Asn Cys Gln Thr 305
310 315 320 Asn Ile Asn Glu
Cys Ala Ser Asn Pro Cys Leu Asn Gln Gly Thr Cys 325
330 335 Ile Asp Asp Val Ala Gly Tyr Lys Cys
Asn Cys Leu Leu Pro Tyr Thr 340 345
350 Gly Ala Thr Cys Glu Val Val Leu Ala Pro Cys Ala Pro Ser
Pro Cys 355 360 365
Arg Asn Gly Gly Glu Cys Arg Gln Ser Glu Asp Tyr Glu Ser Phe Ser 370
375 380 Cys Val Cys Pro Thr
Gly Trp Gln Gly Gln Thr Cys Glu Val Asp Ile 385 390
395 400 Asn Glu Cys Val Leu Ser Pro Cys Arg His
Gly Ala Ser Cys Gln Asn 405 410
415 Thr His Gly Gly Tyr Arg Cys His Cys Gln Ala Gly Tyr Ser Gly
Arg 420 425 430 Asn
Cys Glu Thr Asp Ile Asp Asp Cys Arg Pro Asn Pro Cys His Asn 435
440 445 Gly Gly Ser Cys Thr Asp
Gly Ile Asn Thr Ala Phe Cys Asp Cys Leu 450 455
460 Pro Gly Phe Arg Gly Thr Phe Cys Glu Glu Asp
Ile Asn Glu Asp Leu 465 470 475
480 Gly Pro Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
485 490 495 Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 500
505 510 Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr 515 520
525 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn 530 535 540
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 545
550 555 560 Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 565
570 575 Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser 580 585
590 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys 595 600 605
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 610
615 620 Glu Leu Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 625 630
635 640 Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu 645 650
655 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe 660 665 670
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
675 680 685 Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 690
695 700 Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 705 710 715 62714PRTHomo
sapiens 62Met Trp Gly Trp Lys Cys Leu Leu Phe Trp Ala Val Leu Val Thr Ala
1 5 10 15 Thr Leu
Cys Thr Ala Arg Cys Ala Ser Ser Pro Cys Leu His Asn Gly 20
25 30 Arg Cys Leu Asp Lys Ile Asn
Glu Phe Gln Cys Glu Cys Pro Thr Gly 35 40
45 Phe Thr Gly His Leu Cys Gln Tyr Asp Val Asp Glu
Cys Ala Ser Thr 50 55 60
Pro Cys Lys Asn Gly Ala Lys Cys Leu Asp Gly Pro Asn Thr Tyr Thr 65
70 75 80 Cys Val Cys
Thr Glu Gly Tyr Thr Gly Thr His Cys Glu Val Asp Ile 85
90 95 Asp Glu Cys Asp Pro Asp Pro Cys
His Tyr Gly Ser Cys Lys Asp Gly 100 105
110 Val Ala Thr Phe Thr Cys Leu Cys Arg Pro Gly Tyr Thr
Gly His His 115 120 125
Cys Glu Thr Asn Ile Asn Glu Cys Ser Ser Gln Pro Cys Arg His Gly 130
135 140 Gly Thr Cys Gln
Asp Arg Asp Asn Ala Tyr Leu Cys Phe Cys Leu Lys 145 150
155 160 Gly Thr Thr Gly Pro Asn Cys Glu Ile
Asn Leu Asp Asp Cys Ala Ser 165 170
175 Ser Pro Cys Asp Ser Gly Thr Cys Leu Asp Lys Ile Asp Gly
Tyr Glu 180 185 190
Cys Ala Cys Glu Pro Gly Tyr Thr Gly Ser Met Cys Asn Ile Asn Ile
195 200 205 Asp Glu Cys Ala
Gly Asn Pro Cys His Asn Gly Gly Thr Cys Glu Asp 210
215 220 Gly Ile Asn Gly Phe Thr Cys Arg
Cys Pro Glu Gly Tyr His Asp Pro 225 230
235 240 Thr Cys Leu Ser Glu Val Asn Glu Cys Asn Ser Asn
Pro Cys Val His 245 250
255 Gly Ala Cys Arg Asp Ser Leu Asn Gly Tyr Lys Cys Asp Cys Asp Pro
260 265 270 Gly Trp Ser
Gly Thr Asn Cys Asp Ile Asn Asn Asn Glu Cys Glu Ser 275
280 285 Asn Pro Cys Val Asn Gly Gly Thr
Cys Lys Asp Met Thr Ser Gly Tyr 290 295
300 Val Cys Thr Cys Arg Glu Gly Phe Ser Gly Pro Asn Cys
Gln Thr Asn 305 310 315
320 Ile Asn Glu Cys Ala Ser Asn Pro Cys Leu Asn Gln Gly Thr Cys Ile
325 330 335 Asp Asp Val Ala
Gly Tyr Lys Cys Asn Cys Leu Leu Pro Tyr Thr Gly 340
345 350 Ala Thr Cys Glu Val Val Leu Ala Pro
Cys Ala Pro Ser Pro Cys Arg 355 360
365 Asn Gly Gly Glu Cys Arg Gln Ser Glu Asp Tyr Glu Ser Phe
Ser Cys 370 375 380
Val Cys Pro Thr Gly Trp Gln Gly Gln Thr Cys Glu Val Asp Ile Asn 385
390 395 400 Glu Cys Val Leu Ser
Pro Cys Arg His Gly Ala Ser Cys Gln Asn Thr 405
410 415 His Gly Gly Tyr Arg Cys His Cys Gln Ala
Gly Tyr Ser Gly Arg Asn 420 425
430 Cys Glu Thr Asp Ile Asp Asp Cys Arg Pro Asn Pro Cys His Asn
Gly 435 440 445 Gly
Ser Cys Thr Asp Gly Ile Asn Thr Ala Phe Cys Asp Cys Leu Pro 450
455 460 Gly Phe Arg Gly Thr Phe
Cys Glu Glu Asp Ile Asn Glu Asp Leu Gly 465 470
475 480 Pro Gly Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys 485 490
495 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
500 505 510 Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 515
520 525 Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp 530 535
540 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 545 550 555
560 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
565 570 575 His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 580
585 590 Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly 595 600
605 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu 610 615 620
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 625
630 635 640 Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 645
650 655 Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe 660 665
670 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 675 680 685
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 690
695 700 Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 705 710 63745PRTHomo
sapiens 63Met Pro Pro Leu Leu Ala Pro Leu Leu Cys Leu Ala Leu Leu Pro Ala
1 5 10 15 Leu Ala
Ala Arg Gly Pro Arg Cys Ala Ser Asp Pro Cys Arg Asn Gly 20
25 30 Ala Asn Cys Thr Asp Cys Val
Asp Ser Tyr Thr Cys Thr Cys Pro Ala 35 40
45 Gly Phe Ser Gly Ile His Cys Glu Asn Asn Thr Pro
Asp Cys Thr Glu 50 55 60
Ser Ser Cys Phe Asn Gly Gly Thr Cys Val Asp Gly Ile Asn Ser Phe 65
70 75 80 Thr Cys Leu
Cys Pro Pro Gly Phe Thr Gly Ser Tyr Cys Gln His Asp 85
90 95 Val Asn Glu Cys Asp Ser Gln Pro
Cys Leu His Gly Gly Thr Cys Gln 100 105
110 Asp Gly Cys Gly Ser Tyr Arg Cys Thr Cys Pro Gln Gly
Tyr Thr Gly 115 120 125
Pro Asn Cys Gln Asn Leu Val His Trp Cys Asp Ser Ser Pro Cys Lys 130
135 140 Asn Gly Gly Lys
Cys Trp Gln Thr His Thr Gln Tyr Arg Cys Glu Cys 145 150
155 160 Pro Ser Gly Trp Thr Gly Leu Tyr Cys
Asp Val Pro Ser Val Ser Cys 165 170
175 Glu Val Ala Ala Gln Arg Gln Gly Val Asp Val Ala Arg Leu
Cys Gln 180 185 190
His Gly Gly Leu Cys Val Asp Ala Gly Asn Thr His His Cys Arg Cys
195 200 205 Gln Ala Gly Tyr
Thr Gly Ser Tyr Cys Glu Asp Leu Val Asp Glu Cys 210
215 220 Ser Pro Ser Pro Cys Gln Asn Gly
Ala Thr Cys Thr Asp Tyr Leu Gly 225 230
235 240 Gly Tyr Ser Cys Lys Cys Val Ala Gly Tyr His Gly
Val Asn Cys Ser 245 250
255 Glu Glu Ile Asp Glu Cys Leu Ser His Pro Cys Gln Asn Gly Gly Thr
260 265 270 Cys Leu Asp
Leu Pro Asn Thr Tyr Lys Cys Ser Cys Pro Arg Gly Thr 275
280 285 Gln Gly Val His Cys Glu Ile Asn
Val Asp Asp Cys Asn Pro Pro Val 290 295
300 Asp Pro Val Ser Arg Ser Pro Lys Cys Phe Asn Asn Gly
Thr Cys Val 305 310 315
320 Asp Gln Val Gly Gly Tyr Ser Cys Thr Cys Pro Pro Gly Phe Val Gly
325 330 335 Glu Arg Cys Glu
Gly Asp Val Asn Glu Cys Leu Ser Asn Pro Cys Asp 340
345 350 Ala Arg Gly Thr Gln Asn Cys Val Gln
Arg Val Asn Asp Phe His Cys 355 360
365 Glu Cys Arg Ala Gly His Thr Gly Arg Arg Cys Glu Ser Val
Ile Asn 370 375 380
Gly Cys Lys Gly Lys Pro Cys Lys Asn Gly Gly Thr Cys Ala Val Ala 385
390 395 400 Ser Asn Thr Ala Arg
Gly Phe Ile Cys Lys Cys Pro Ala Gly Phe Glu 405
410 415 Gly Ala Thr Cys Glu Asn Asp Ala Arg Thr
Cys Gly Ser Leu Arg Cys 420 425
430 Leu Asn Gly Gly Thr Cys Ile Ser Gly Pro Arg Ser Pro Thr Cys
Leu 435 440 445 Cys
Leu Gly Pro Phe Thr Gly Pro Glu Cys Gln Phe Pro Ala Ser Ser 450
455 460 Pro Cys Leu Gly Gly Asn
Pro Cys Tyr Asn Gln Gly Thr Cys Glu Pro 465 470
475 480 Thr Ser Glu Ser Pro Phe Tyr Arg Cys Leu Cys
Pro Ala Lys Phe Asn 485 490
495 Gly Leu Leu Cys His Ile Leu Asp Tyr Ser Phe Gly Asp Leu Gly Pro
500 505 510 Gly Glu
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 515
520 525 Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys 530 535
540 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val 545 550 555
560 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
565 570 575 Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 580
585 590 Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His 595 600
605 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys 610 615 620
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 625
630 635 640 Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 645
650 655 Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro 660 665
670 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn 675 680 685
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 690
695 700 Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 705 710
715 720 Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln 725 730
735 Lys Ser Leu Ser Leu Ser Pro Gly Lys 740
745 64744PRTHomo sapiens 64Met Trp Gly Trp Lys Cys Leu Leu Phe
Trp Ala Val Leu Val Thr Ala 1 5 10
15 Thr Leu Cys Thr Ala Arg Cys Ala Ser Asp Pro Cys Arg Asn
Gly Ala 20 25 30
Asn Cys Thr Asp Cys Val Asp Ser Tyr Thr Cys Thr Cys Pro Ala Gly
35 40 45 Phe Ser Gly Ile
His Cys Glu Asn Asn Thr Pro Asp Cys Thr Glu Ser 50
55 60 Ser Cys Phe Asn Gly Gly Thr Cys
Val Asp Gly Ile Asn Ser Phe Thr 65 70
75 80 Cys Leu Cys Pro Pro Gly Phe Thr Gly Ser Tyr Cys
Gln His Asp Val 85 90
95 Asn Glu Cys Asp Ser Gln Pro Cys Leu His Gly Gly Thr Cys Gln Asp
100 105 110 Gly Cys Gly
Ser Tyr Arg Cys Thr Cys Pro Gln Gly Tyr Thr Gly Pro 115
120 125 Asn Cys Gln Asn Leu Val His Trp
Cys Asp Ser Ser Pro Cys Lys Asn 130 135
140 Gly Gly Lys Cys Trp Gln Thr His Thr Gln Tyr Arg Cys
Glu Cys Pro 145 150 155
160 Ser Gly Trp Thr Gly Leu Tyr Cys Asp Val Pro Ser Val Ser Cys Glu
165 170 175 Val Ala Ala Gln
Arg Gln Gly Val Asp Val Ala Arg Leu Cys Gln His 180
185 190 Gly Gly Leu Cys Val Asp Ala Gly Asn
Thr His His Cys Arg Cys Gln 195 200
205 Ala Gly Tyr Thr Gly Ser Tyr Cys Glu Asp Leu Val Asp Glu
Cys Ser 210 215 220
Pro Ser Pro Cys Gln Asn Gly Ala Thr Cys Thr Asp Tyr Leu Gly Gly 225
230 235 240 Tyr Ser Cys Lys Cys
Val Ala Gly Tyr His Gly Val Asn Cys Ser Glu 245
250 255 Glu Ile Asp Glu Cys Leu Ser His Pro Cys
Gln Asn Gly Gly Thr Cys 260 265
270 Leu Asp Leu Pro Asn Thr Tyr Lys Cys Ser Cys Pro Arg Gly Thr
Gln 275 280 285 Gly
Val His Cys Glu Ile Asn Val Asp Asp Cys Asn Pro Pro Val Asp 290
295 300 Pro Val Ser Arg Ser Pro
Lys Cys Phe Asn Asn Gly Thr Cys Val Asp 305 310
315 320 Gln Val Gly Gly Tyr Ser Cys Thr Cys Pro Pro
Gly Phe Val Gly Glu 325 330
335 Arg Cys Glu Gly Asp Val Asn Glu Cys Leu Ser Asn Pro Cys Asp Ala
340 345 350 Arg Gly
Thr Gln Asn Cys Val Gln Arg Val Asn Asp Phe His Cys Glu 355
360 365 Cys Arg Ala Gly His Thr Gly
Arg Arg Cys Glu Ser Val Ile Asn Gly 370 375
380 Cys Lys Gly Lys Pro Cys Lys Asn Gly Gly Thr Cys
Ala Val Ala Ser 385 390 395
400 Asn Thr Ala Arg Gly Phe Ile Cys Lys Cys Pro Ala Gly Phe Glu Gly
405 410 415 Ala Thr Cys
Glu Asn Asp Ala Arg Thr Cys Gly Ser Leu Arg Cys Leu 420
425 430 Asn Gly Gly Thr Cys Ile Ser Gly
Pro Arg Ser Pro Thr Cys Leu Cys 435 440
445 Leu Gly Pro Phe Thr Gly Pro Glu Cys Gln Phe Pro Ala
Ser Ser Pro 450 455 460
Cys Leu Gly Gly Asn Pro Cys Tyr Asn Gln Gly Thr Cys Glu Pro Thr 465
470 475 480 Ser Glu Ser Pro
Phe Tyr Arg Cys Leu Cys Pro Ala Lys Phe Asn Gly 485
490 495 Leu Leu Cys His Ile Leu Asp Tyr Ser
Phe Gly Asp Leu Gly Pro Gly 500 505
510 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala 515 520 525
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 530
535 540 Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 545 550
555 560 Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val 565 570
575 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln 580 585 590 Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 595
600 605 Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 610 615
620 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro 625 630 635
640 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
645 650 655 Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 660
665 670 Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr 675 680
685 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr 690 695 700
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 705
710 715 720 Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 725
730 735 Ser Leu Ser Leu Ser Pro Gly Lys
740 655013DNAHomo sapiens 65atgccgccgc
tcctggcgcc cctgctctgc ctggcgctgc tgcccgcgct cgccgcacga 60ggcccgcgat
gctcccagcc cggtgagacc tgcctgaatg gcgggaagtg tgaagcggcc 120aatggcacgg
aggcctgcgt ctgtggcggg gccttcgtgg gcccgcgatg ccaggacccc 180aacccgtgcc
tcagcacccc ctgcaagaac gccgggacat gccacgtggt ggaccgcaga 240ggcgtggcag
actatgcctg cagctgtgcc ctgggcttct ctgggcccct ctgcctgaca 300cccctggaca
atgcctgcct caccaacccc tgccgcaacg ggggcacctg cgacctgctc 360acgctgacgg
agtacaagtg ccgctgcccg cccggctggt cagggaaatc gtgccagcag 420gctgacccgt
gcgcctccaa cccctgcgcc aacggtggcc agtgcctgcc cttcgaggcc 480tcctacatct
gccactgccc acccagcttc catggcccca cctgccggca ggatgtcaac 540gagtgtggcc
agaagcccgg gctttgccgc cacggaggca cctgccacaa cgaggtcggc 600tcctaccgct
gcgtctgccg cgccacccac actggcccca actgcgagcg gccctacgtg 660ccctgcagcc
cctcgccctg ccagaacggg ggcacctgcc gccccacggg cgacgtcacc 720cacgagtgtg
cctgcctgcc aggcttcacc ggccagaact gtgaggaaaa tatcgacgat 780tgtccaggaa
acaactgcaa gaacgggggt gcctgtgtgg acggcgtgaa cacctacaac 840tgccgctgcc
cgccagagtg gacaggtcag tactgtaccg aggatgtgga cgagtgccag 900ctgatgccaa
atgcctgcca gaacggcggg acctgccaca acacccacgg tggctacaac 960tgcgtgtgtg
tcaacggctg gactggtgag gactgcagcg agaacattga tgactgtgcc 1020agcgccgcct
gcttccacgg cgccacctgc catgaccgtg tggcctcctt ctactgcgag 1080tgtccccatg
gccgcacagg tctgctgtgc cacctcaacg acgcatgcat cagcaacccc 1140tgtaacgagg
gctccaactg cgacaccaac cctgtcaatg gcaaggccat ctgcacctgc 1200ccctcggggt
acacgggccc ggcctgcagc caggacgtgg atgagtgctc gctgggtgcc 1260aacccctgcg
agcatgcggg caagtgcatc aacacgctgg gctccttcga gtgccagtgt 1320ctgcagggct
acacgggccc ccgatgcgag atcgacgtca acgagtgcgt ctcgaacccg 1380tgccagaacg
acgccacctg cctggaccag attggggagt tccagtgcat ctgcatgccc 1440ggctacgagg
gtgtgcactg cgaggtcaac acagacgagt gtgccagcag cccctgcctg 1500cacaatggcc
gctgcctgga caagatcaat gagttccagt gcgagtgccc cacgggcttc 1560actgggcatc
tgtgccagta cgatgtggac gagtgtgcca gcaccccctg caagaatggt 1620gccaagtgcc
tggacggacc caacacttac acctgtgtgt gcacggaagg gtacacgggg 1680acgcactgcg
aggtggacat cgatgagtgc gaccccgacc cctgccacta cggctcctgc 1740aaggacggcg
tcgccacctt cacctgcctc tgccgcccag gctacacggg ccaccactgc 1800gagaccaaca
tcaacgagtg ctccagccag ccctgccgcc acgggggcac ctgccaggac 1860cgcgacaacg
cctacctctg cttctgcctg aaggggacca caggacccaa ctgcgagatc 1920aacctggatg
actgtgccag cagcccctgc gactcgggca cctgtctgga caagatcgat 1980ggctacgagt
gtgcctgtga gccgggctac acagggagca tgtgtaacat caacatcgat 2040gagtgtgcgg
gcaacccctg ccacaacggg ggcacctgcg aggacggcat caatggcttc 2100acctgccgct
gccccgaggg ctaccacgac cccacctgcc tgtctgaggt caatgagtgc 2160aacagcaacc
cctgcgtcca cggggcctgc cgggacagcc tcaacgggta caagtgcgac 2220tgtgaccctg
ggtggagtgg gaccaactgt gacatcaaca acaatgagtg tgaatccaac 2280ccttgtgtca
acggcggcac ctgcaaagac atgaccagtg gctacgtgtg cacctgccgg 2340gagggcttca
gcggtcccaa ctgccagacc aacatcaacg agtgtgcgtc caacccatgt 2400ctgaaccagg
gcacgtgtat tgacgacgtt gccgggtaca agtgcaactg cctgctgccc 2460tacacaggtg
ccacgtgtga ggtggtgctg gccccgtgtg cccccagccc ctgcagaaac 2520ggcggggagt
gcaggcaatc cgaggactat gagagcttct cctgtgtctg ccccacgggc 2580tggcaagggc
agacctgtga ggtcgacatc aacgagtgcg ttctgagccc gtgccggcac 2640ggcgcatcct
gccagaacac ccacggcggc taccgctgcc actgccaggc cggctacagt 2700gggcgcaact
gcgagaccga catcgacgac tgccggccca acccgtgtca caacgggggc 2760tcctgcacag
acggcatcaa cacggccttc tgcgactgcc tgcccggctt ccggggcact 2820ttctgtgagg
aggacatcaa cgagtgtgcc agtgacccct gccgcaacgg ggccaactgc 2880acggactgcg
tggacagcta cacgtgcacc tgccccgcag gcttcagcgg gatccactgt 2940gagaacaaca
cgcctgactg cacagagagc tcctgcttca acggtggcac ctgcgtggac 3000ggcatcaact
cgttcacctg cctgtgtcca cccggcttca cgggcagcta ctgccagcac 3060gatgtcaatg
agtgcgactc acagccctgc ctgcatggcg gcacctgtca ggacggctgc 3120ggctcctaca
ggtgcacctg cccccagggc tacactggcc ccaactgcca gaaccttgtg 3180cactggtgtg
actcctcgcc ctgcaagaac ggcggcaaat gctggcagac ccacacccag 3240taccgctgcg
agtgccccag cggctggacc ggcctttact gcgacgtgcc cagcgtgtcc 3300tgtgaggtgg
ctgcgcagcg acaaggtgtt gacgttgccc gcctgtgcca gcatggaggg 3360ctctgtgtgg
acgcgggcaa cacgcaccac tgccgctgcc aggcgggcta cacaggcagc 3420tactgtgagg
acctggtgga cgagtgctca cccagcccct gccagaacgg ggccacctgc 3480acggactacc
tgggcggcta ctcctgcaag tgcgtggccg gctaccacgg ggtgaactgc 3540tctgaggaga
tcgacgagtg cctctcccac ccctgccaga acgggggcac ctgcctcgac 3600ctccccaaca
cctacaagtg ctcctgccca cggggcactc agggtgtgca ctgtgagatc 3660aacgtggacg
actgcaatcc ccccgttgac cccgtgtccc ggagccccaa gtgctttaac 3720aacggcacct
gcgtggacca ggtgggcggc tacagctgca cctgcccgcc gggcttcgtg 3780ggtgagcgct
gtgaggggga tgtcaacgag tgcctgtcca atccctgcga cgcccgtggc 3840acccagaact
gcgtgcagcg cgtcaatgac ttccactgcg agtgccgtgc tggtcacacc 3900gggcgccgct
gcgagtccgt catcaatggc tgcaaaggca agccctgcaa gaatgggggc 3960acctgcgccg
tggcctccaa caccgcccgc gggttcatct gcaagtgccc tgcgggcttc 4020gagggcgcca
cgtgtgagaa tgacgctcgt acctgcggca gcctgcgctg cctcaacggc 4080ggcacatgca
tctccggccc gcgcagcccc acctgcctgt gcctgggccc cttcacgggc 4140cccgaatgcc
agttcccggc cagcagcccc tgcctgggcg gcaacccctg ctacaaccag 4200gggacctgtg
agcccacatc cgagagcccc ttctaccgtt gcctgtgccc cgccaaattc 4260aacgggctct
tgtgccacat cctggactac agcttcggag atctgggccc gggcgagccc 4320aaatcttgtg
acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 4380ccgtcagtct
tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 4440gaggtcacat
gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 4500tacgtggacg
gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 4560agcacgtacc
gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 4620gagtacaagt
gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 4680aaagccaaag
ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 4740ctgaccaaga
accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 4800gccgtggagt
gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 4860ctggactccg
acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 4920cagcagggga
acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 4980cagaagagcc
tctccctgtc tccgggtaaa tga
5013662307DNAHomo sapiens 66atgccgccgc tcctggcgcc cctgctctgc ctggcgctgc
tgcccgcgct cgccgcacga 60ggcccgcgat gctcccagcc cggtgagacc tgcctgaatg
gcgggaagtg tgaagcggcc 120aatggcacgg aggcctgcgt ctgtggcggg gccttcgtgg
gcccgcgatg ccaggacccc 180aacccgtgcc tcagcacccc ctgcaagaac gccgggacat
gccacgtggt ggaccgcaga 240ggcgtggcag actatgcctg cagctgtgcc ctgggcttct
ctgggcccct ctgcctgaca 300cccctggaca atgcctgcct caccaacccc tgccgcaacg
ggggcacctg cgacctgctc 360acgctgacgg agtacaagtg ccgctgcccg cccggctggt
cagggaaatc gtgccagcag 420gctgacccgt gcgcctccaa cccctgcgcc aacggtggcc
agtgcctgcc cttcgaggcc 480tcctacatct gccactgccc acccagcttc catggcccca
cctgccggca ggatgtcaac 540gagtgtggcc agaagcccgg gctttgccgc cacggaggca
cctgccacaa cgaggtcggc 600tcctaccgct gcgtctgccg cgccacccac actggcccca
actgcgagcg gccctacgtg 660ccctgcagcc cctcgccctg ccagaacggg ggcacctgcc
gccccacggg cgacgtcacc 720cacgagtgtg cctgcctgcc aggcttcacc ggccagaact
gtgaggaaaa tatcgacgat 780tgtccaggaa acaactgcaa gaacgggggt gcctgtgtgg
acggcgtgaa cacctacaac 840tgccgctgcc cgccagagtg gacaggtcag tactgtaccg
aggatgtgga cgagtgccag 900ctgatgccaa atgcctgcca gaacggcggg acctgccaca
acacccacgg tggctacaac 960tgcgtgtgtg tcaacggctg gactggtgag gactgcagcg
agaacattga tgactgtgcc 1020agcgccgcct gcttccacgg cgccacctgc catgaccgtg
tggcctcctt ctactgcgag 1080tgtccccatg gccgcacagg tctgctgtgc cacctcaacg
acgcatgcat cagcaacccc 1140tgtaacgagg gctccaactg cgacaccaac cctgtcaatg
gcaaggccat ctgcacctgc 1200ccctcggggt acacgggccc ggcctgcagc caggacgtgg
atgagtgctc gctgggtgcc 1260aacccctgcg agcatgcggg caagtgcatc aacacgctgg
gctccttcga gtgccagtgt 1320ctgcagggct acacgggccc ccgatgcgag atcgacgtca
acgagtgcgt ctcgaacccg 1380tgccagaacg acgccacctg cctggaccag attggggagt
tccagtgcat ctgcatgccc 1440ggctacgagg gtgtgcactg cgaggtcaac acagacgagt
gtgccagcag cccctgcctg 1500cacaatggcc gctgcctgga caagatcaat gagttccagt
gcgagtgccc cacgggcttc 1560actgggcatc tgtgccagta cgatgtggac gaggatctgg
gcccgggcga gcccaaatct 1620tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg
aactcctggg gggaccgtca 1680gtcttcctct tccccccaaa acccaaggac accctcatga
tctcccggac ccctgaggtc 1740acatgcgtgg tggtggacgt gagccacgaa gaccctgagg
tcaagttcaa ctggtacgtg 1800gacggcgtgg aggtgcataa tgccaagaca aagccgcggg
aggagcagta caacagcacg 1860taccgtgtgg tcagcgtcct caccgtcctg caccaggact
ggctgaatgg caaggagtac 1920aagtgcaagg tctccaacaa agccctccca gcccccatcg
agaaaaccat ctccaaagcc 1980aaagggcagc cccgagaacc acaggtgtac accctgcccc
catcccggga tgagctgacc 2040aagaaccagg tcagcctgac ctgcctggtc aaaggcttct
atcccagcga catcgccgtg 2100gagtgggaga gcaatgggca gccggagaac aactacaaga
ccacgcctcc cgtgctggac 2160tccgacggct ccttcttcct ctacagcaag ctcaccgtgg
acaagagcag gtggcagcag 2220gggaacgtct tctcatgctc cgtgatgcat gaggctctgc
acaaccacta cacgcagaag 2280agcctctccc tgtctccggg taaatga
2307673558DNAHomo sapiens 67atgccgccgc tcctggcgcc
cctgctctgc ctggcgctgc tgcccgcgct cgccgcacga 60ggcccgcgat gctcccagcc
cggtgagacc tgcctgaatg gcgggaagtg tgaagcggcc 120aatggcacgg aggcctgcgt
ctgtggcggg gccttcgtgg gcccgcgatg ccaggacccc 180aacccgtgcc tcagcacccc
ctgcaagaac gccgggacat gccacgtggt ggaccgcaga 240ggcgtggcag actatgcctg
cagctgtgcc ctgggcttct ctgggcccct ctgcctgaca 300cccctggaca atgcctgcct
caccaacccc tgccgcaacg ggggcacctg cgacctgctc 360acgctgacgg agtacaagtg
ccgctgcccg cccggctggt cagggaaatc gtgccagcag 420gctgacccgt gcgcctccaa
cccctgcgcc aacggtggcc agtgcctgcc cttcgaggcc 480tcctacatct gccactgccc
acccagcttc catggcccca cctgccggca ggatgtcaac 540gagtgtggcc agaagcccgg
gctttgccgc cacggaggca cctgccacaa cgaggtcggc 600tcctaccgct gcgtctgccg
cgccacccac actggcccca actgcgagcg gccctacgtg 660ccctgcagcc cctcgccctg
ccagaacggg ggcacctgcc gccccacggg cgacgtcacc 720cacgagtgtg cctgcctgcc
aggcttcacc ggccagaact gtgaggaaaa tatcgacgat 780tgtccaggaa acaactgcaa
gaacgggggt gcctgtgtgg acggcgtgaa cacctacaac 840tgccgctgcc cgccagagtg
gacaggtcag tactgtaccg aggatgtgga cgagtgccag 900ctgatgccaa atgcctgcca
gaacggcggg acctgccaca acacccacgg tggctacaac 960tgcgtgtgtg tcaacggctg
gactggtgag gactgcagcg agaacattga tgactgtgcc 1020agcgccgcct gcttccacgg
cgccacctgc catgaccgtg tggcctcctt ctactgcgag 1080tgtccccatg gccgcacagg
tctgctgtgc cacctcaacg acgcatgcat cagcaacccc 1140tgtaacgagg gctccaactg
cgacaccaac cctgtcaatg gcaaggccat ctgcacctgc 1200ccctcggggt acacgggccc
ggcctgcagc caggacgtgg atgagtgctc gctgggtgcc 1260aacccctgcg agcatgcggg
caagtgcatc aacacgctgg gctccttcga gtgccagtgt 1320ctgcagggct acacgggccc
ccgatgcgag atcgacgtca acgagtgcgt ctcgaacccg 1380tgccagaacg acgccacctg
cctggaccag attggggagt tccagtgcat ctgcatgccc 1440ggctacgagg gtgtgcactg
cgaggtcaac acagacgagt gtgccagcag cccctgcctg 1500cacaatggcc gctgcctgga
caagatcaat gagttccagt gcgagtgccc cacgggcttc 1560actgggcatc tgtgccagta
cgatgtggac gagtgtgcca gcaccccctg caagaatggt 1620gccaagtgcc tggacggacc
caacacttac acctgtgtgt gcacggaagg gtacacgggg 1680acgcactgcg aggtggacat
cgatgagtgc gaccccgacc cctgccacta cggctcctgc 1740aaggacggcg tcgccacctt
cacctgcctc tgccgcccag gctacacggg ccaccactgc 1800gagaccaaca tcaacgagtg
ctccagccag ccctgccgcc acgggggcac ctgccaggac 1860cgcgacaacg cctacctctg
cttctgcctg aaggggacca caggacccaa ctgcgagatc 1920aacctggatg actgtgccag
cagcccctgc gactcgggca cctgtctgga caagatcgat 1980ggctacgagt gtgcctgtga
gccgggctac acagggagca tgtgtaacat caacatcgat 2040gagtgtgcgg gcaacccctg
ccacaacggg ggcacctgcg aggacggcat caatggcttc 2100acctgccgct gccccgaggg
ctaccacgac cccacctgcc tgtctgaggt caatgagtgc 2160aacagcaacc cctgcgtcca
cggggcctgc cgggacagcc tcaacgggta caagtgcgac 2220tgtgaccctg ggtggagtgg
gaccaactgt gacatcaaca acaatgagtg tgaatccaac 2280ccttgtgtca acggcggcac
ctgcaaagac atgaccagtg gctacgtgtg cacctgccgg 2340gagggcttca gcggtcccaa
ctgccagacc aacatcaacg agtgtgcgtc caacccatgt 2400ctgaaccagg gcacgtgtat
tgacgacgtt gccgggtaca agtgcaactg cctgctgccc 2460tacacaggtg ccacgtgtga
ggtggtgctg gccccgtgtg cccccagccc ctgcagaaac 2520ggcggggagt gcaggcaatc
cgaggactat gagagcttct cctgtgtctg ccccacgggc 2580tggcaagggc agacctgtga
ggtcgacatc aacgagtgcg ttctgagccc gtgccggcac 2640ggcgcatcct gccagaacac
ccacggcggc taccgctgcc actgccaggc cggctacagt 2700gggcgcaact gcgagaccga
catcgacgac tgccggccca acccgtgtca caacgggggc 2760tcctgcacag acggcatcaa
cacggccttc tgcgactgcc tgcccggctt ccggggcact 2820ttctgtgagg aggacatcaa
cgaggatctg ggcccgggcg agcccaaatc ttgtgacaaa 2880actcacacat gcccaccgtg
cccagcacct gaactcctgg ggggaccgtc agtcttcctc 2940ttccccccaa aacccaagga
caccctcatg atctcccgga cccctgaggt cacatgcgtg 3000gtggtggacg tgagccacga
agaccctgag gtcaagttca actggtacgt ggacggcgtg 3060gaggtgcata atgccaagac
aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 3120gtcagcgtcc tcaccgtcct
gcaccaggac tggctgaatg gcaaggagta caagtgcaag 3180gtctccaaca aagccctccc
agcccccatc gagaaaacca tctccaaagc caaagggcag 3240ccccgagaac cacaggtgta
caccctgccc ccatcccggg atgagctgac caagaaccag 3300gtcagcctga cctgcctggt
caaaggcttc tatcccagcg acatcgccgt ggagtgggag 3360agcaatgggc agccggagaa
caactacaag accacgcctc ccgtgctgga ctccgacggc 3420tccttcttcc tctacagcaa
gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 3480ttctcatgct ccgtgatgca
tgaggctctg cacaaccact acacgcagaa gagcctctcc 3540ctgtctccgg gtaaatga
3558682493DNAHomo sapiens
68atgccgccgc tcctggcgcc cctgctctgc ctggcgctgc tgcccgcgct cgccgcacga
60ggcccgcgat gtgccagcgc cgcctgcttc cacggcgcca cctgccatga ccgtgtggcc
120tccttctact gcgagtgtcc ccatggccgc acaggtctgc tgtgccacct caacgacgca
180tgcatcagca acccctgtaa cgagggctcc aactgcgaca ccaaccctgt caatggcaag
240gccatctgca cctgcccctc ggggtacacg ggcccggcct gcagccagga cgtggatgag
300tgctcgctgg gtgccaaccc ctgcgagcat gcgggcaagt gcatcaacac gctgggctcc
360ttcgagtgcc agtgtctgca gggctacacg ggcccccgat gcgagatcga cgtcaacgag
420tgcgtctcga acccgtgcca gaacgacgcc acctgcctgg accagattgg ggagttccag
480tgcatctgca tgcccggcta cgagggtgtg cactgcgagg tcaacacaga cgagtgtgcc
540agcagcccct gcctgcacaa tggccgctgc ctggacaaga tcaatgagtt ccagtgcgag
600tgccccacgg gcttcactgg gcatctgtgc cagtacgatg tggacgagtg tgccagcacc
660ccctgcaaga atggtgccaa gtgcctggac ggacccaaca cttacacctg tgtgtgcacg
720gaagggtaca cggggacgca ctgcgaggtg gacatcgatg agtgcgaccc cgacccctgc
780cactacggct cctgcaagga cggcgtcgcc accttcacct gcctctgccg cccaggctac
840acgggccacc actgcgagac caacatcaac gagtgctcca gccagccctg ccgccacggg
900ggcacctgcc aggaccgcga caacgcctac ctctgcttct gcctgaaggg gaccacagga
960cccaactgcg agatcaacct ggatgactgt gccagcagcc cctgcgactc gggcacctgt
1020ctggacaaga tcgatggcta cgagtgtgcc tgtgagccgg gctacacagg gagcatgtgt
1080aacatcaaca tcgatgagtg tgcgggcaac ccctgccaca acgggggcac ctgcgaggac
1140ggcatcaatg gcttcacctg ccgctgcccc gagggctacc acgaccccac ctgcctgtct
1200gaggtcaatg agtgcaacag caacccctgc gtccacgggg cctgccggga cagcctcaac
1260gggtacaagt gcgactgtga ccctgggtgg agtgggacca actgtgacat caacaacaat
1320gagtgtgaat ccaacccttg tgtcaacggc ggcacctgca aagacatgac cagtggctac
1380gtgtgcacct gccgggaggg cttcagcggt cccaactgcc agaccaacat caacgagtgt
1440gcgtccaacc catgtctgaa ccagggcacg tgtattgacg acgttgccgg gtacaagtgc
1500aactgcctgc tgccctacac aggtgccacg tgtgaggtgg tgctggcccc gtgtgccccc
1560agcccctgca gaaacggcgg ggagtgcagg caatccgagg actatgagag cttctcctgt
1620gtctgcccca cgggctggca agggcagacc tgtgaggtcg acatcaacga gtgcgttctg
1680agcccgtgcc ggcacggcgc atcctgccag aacacccacg gcggctaccg ctgccactgc
1740caggccggct acagtgggcg caactgcgag accgacatag atctgggccc gggcgagccc
1800aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga
1860ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct
1920gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg
1980tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac
2040agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag
2100gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc
2160aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag
2220ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc
2280gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg
2340ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg
2400cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg
2460cagaagagcc tctccctgtc tccgggtaaa tga
2493692490DNAHomo sapiens 69atgtggggct ggaagtgcct cctcttctgg gctgtgctgg
tcacagccac tctctgcact 60gccaggtgtg ccagcgccgc ctgcttccac ggcgccacct
gccatgaccg tgtggcctcc 120ttctactgcg agtgtcccca tggccgcaca ggtctgctgt
gccacctcaa cgacgcatgc 180atcagcaacc cctgtaacga gggctccaac tgcgacacca
accctgtcaa tggcaaggcc 240atctgcacct gcccctcggg gtacacgggc ccggcctgca
gccaggacgt ggatgagtgc 300tcgctgggtg ccaacccctg cgagcatgcg ggcaagtgca
tcaacacgct gggctccttc 360gagtgccagt gtctgcaggg ctacacgggc ccccgatgcg
agatcgacgt caacgagtgc 420gtctcgaacc cgtgccagaa cgacgccacc tgcctggacc
agattgggga gttccagtgc 480atctgcatgc ccggctacga gggtgtgcac tgcgaggtca
acacagacga gtgtgccagc 540agcccctgcc tgcacaatgg ccgctgcctg gacaagatca
atgagttcca gtgcgagtgc 600cccacgggct tcactgggca tctgtgccag tacgatgtgg
acgagtgtgc cagcaccccc 660tgcaagaatg gtgccaagtg cctggacgga cccaacactt
acacctgtgt gtgcacggaa 720gggtacacgg ggacgcactg cgaggtggac atcgatgagt
gcgaccccga cccctgccac 780tacggctcct gcaaggacgg cgtcgccacc ttcacctgcc
tctgccgccc aggctacacg 840ggccaccact gcgagaccaa catcaacgag tgctccagcc
agccctgccg ccacgggggc 900acctgccagg accgcgacaa cgcctacctc tgcttctgcc
tgaaggggac cacaggaccc 960aactgcgaga tcaacctgga tgactgtgcc agcagcccct
gcgactcggg cacctgtctg 1020gacaagatcg atggctacga gtgtgcctgt gagccgggct
acacagggag catgtgtaac 1080atcaacatcg atgagtgtgc gggcaacccc tgccacaacg
ggggcacctg cgaggacggc 1140atcaatggct tcacctgccg ctgccccgag ggctaccacg
accccacctg cctgtctgag 1200gtcaatgagt gcaacagcaa cccctgcgtc cacggggcct
gccgggacag cctcaacggg 1260tacaagtgcg actgtgaccc tgggtggagt gggaccaact
gtgacatcaa caacaatgag 1320tgtgaatcca acccttgtgt caacggcggc acctgcaaag
acatgaccag tggctacgtg 1380tgcacctgcc gggagggctt cagcggtccc aactgccaga
ccaacatcaa cgagtgtgcg 1440tccaacccat gtctgaacca gggcacgtgt attgacgacg
ttgccgggta caagtgcaac 1500tgcctgctgc cctacacagg tgccacgtgt gaggtggtgc
tggccccgtg tgcccccagc 1560ccctgcagaa acggcgggga gtgcaggcaa tccgaggact
atgagagctt ctcctgtgtc 1620tgccccacgg gctggcaagg gcagacctgt gaggtcgaca
tcaacgagtg cgttctgagc 1680ccgtgccggc acggcgcatc ctgccagaac acccacggcg
gctaccgctg ccactgccag 1740gccggctaca gtgggcgcaa ctgcgagacc gacatagatc
tgggcccggg cgagcccaaa 1800tcttgtgaca aaactcacac atgcccaccg tgcccagcac
ctgaactcct ggggggaccg 1860tcagtcttcc tcttcccccc aaaacccaag gacaccctca
tgatctcccg gacccctgag 1920gtcacatgcg tggtggtgga cgtgagccac gaagaccctg
aggtcaagtt caactggtac 1980gtggacggcg tggaggtgca taatgccaag acaaagccgc
gggaggagca gtacaacagc 2040acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg
actggctgaa tggcaaggag 2100tacaagtgca aggtctccaa caaagccctc ccagccccca
tcgagaaaac catctccaaa 2160gccaaagggc agccccgaga accacaggtg tacaccctgc
ccccatcccg ggatgagctg 2220accaagaacc aggtcagcct gacctgcctg gtcaaaggct
tctatcccag cgacatcgcc 2280gtggagtggg agagcaatgg gcagccggag aacaactaca
agaccacgcc tcccgtgctg 2340gactccgacg gctccttctt cctctacagc aagctcaccg
tggacaagag caggtggcag 2400caggggaacg tcttctcatg ctccgtgatg catgaggctc
tgcacaacca ctacacgcag 2460aagagcctct ccctgtctcc gggtaaatga
2490704068DNAHomo sapiens 70atgccgccgc tcctggcgcc
cctgctctgc ctggcgctgc tgcccgcgct cgccgcacga 60ggcccgcgat gtgccagcgc
cgcctgcttc cacggcgcca cctgccatga ccgtgtggcc 120tccttctact gcgagtgtcc
ccatggccgc acaggtctgc tgtgccacct caacgacgca 180tgcatcagca acccctgtaa
cgagggctcc aactgcgaca ccaaccctgt caatggcaag 240gccatctgca cctgcccctc
ggggtacacg ggcccggcct gcagccagga cgtggatgag 300tgctcgctgg gtgccaaccc
ctgcgagcat gcgggcaagt gcatcaacac gctgggctcc 360ttcgagtgcc agtgtctgca
gggctacacg ggcccccgat gcgagatcga cgtcaacgag 420tgcgtctcga acccgtgcca
gaacgacgcc acctgcctgg accagattgg ggagttccag 480tgcatctgca tgcccggcta
cgagggtgtg cactgcgagg tcaacacaga cgagtgtgcc 540agcagcccct gcctgcacaa
tggccgctgc ctggacaaga tcaatgagtt ccagtgcgag 600tgccccacgg gcttcactgg
gcatctgtgc cagtacgatg tggacgagtg tgccagcacc 660ccctgcaaga atggtgccaa
gtgcctggac ggacccaaca cttacacctg tgtgtgcacg 720gaagggtaca cggggacgca
ctgcgaggtg gacatcgatg agtgcgaccc cgacccctgc 780cactacggct cctgcaagga
cggcgtcgcc accttcacct gcctctgccg cccaggctac 840acgggccacc actgcgagac
caacatcaac gagtgctcca gccagccctg ccgccacggg 900ggcacctgcc aggaccgcga
caacgcctac ctctgcttct gcctgaaggg gaccacagga 960cccaactgcg agatcaacct
ggatgactgt gccagcagcc cctgcgactc gggcacctgt 1020ctggacaaga tcgatggcta
cgagtgtgcc tgtgagccgg gctacacagg gagcatgtgt 1080aacatcaaca tcgatgagtg
tgcgggcaac ccctgccaca acgggggcac ctgcgaggac 1140ggcatcaatg gcttcacctg
ccgctgcccc gagggctacc acgaccccac ctgcctgtct 1200gaggtcaatg agtgcaacag
caacccctgc gtccacgggg cctgccggga cagcctcaac 1260gggtacaagt gcgactgtga
ccctgggtgg agtgggacca actgtgacat caacaacaat 1320gagtgtgaat ccaacccttg
tgtcaacggc ggcacctgca aagacatgac cagtggctac 1380gtgtgcacct gccgggaggg
cttcagcggt cccaactgcc agaccaacat caacgagtgt 1440gcgtccaacc catgtctgaa
ccagggcacg tgtattgacg acgttgccgg gtacaagtgc 1500aactgcctgc tgccctacac
aggtgccacg tgtgaggtgg tgctggcccc gtgtgccccc 1560agcccctgca gaaacggcgg
ggagtgcagg caatccgagg actatgagag cttctcctgt 1620gtctgcccca cgggctggca
agggcagacc tgtgaggtcg acatcaacga gtgcgttctg 1680agcccgtgcc ggcacggcgc
atcctgccag aacacccacg gcggctaccg ctgccactgc 1740caggccggct acagtgggcg
caactgcgag accgacatcg acgactgccg gcccaacccg 1800tgtcacaacg ggggctcctg
cacagacggc atcaacacgg ccttctgcga ctgcctgccc 1860ggcttccggg gcactttctg
tgaggaggac atcaacgagt gtgccagtga cccctgccgc 1920aacggggcca actgcacgga
ctgcgtggac agctacacgt gcacctgccc cgcaggcttc 1980agcgggatcc actgtgagaa
caacacgcct gactgcacag agagctcctg cttcaacggt 2040ggcacctgcg tggacggcat
caactcgttc acctgcctgt gtccacccgg cttcacgggc 2100agctactgcc agcacgatgt
caatgagtgc gactcacagc cctgcctgca tggcggcacc 2160tgtcaggacg gctgcggctc
ctacaggtgc acctgccccc agggctacac tggccccaac 2220tgccagaacc ttgtgcactg
gtgtgactcc tcgccctgca agaacggcgg caaatgctgg 2280cagacccaca cccagtaccg
ctgcgagtgc cccagcggct ggaccggcct ttactgcgac 2340gtgcccagcg tgtcctgtga
ggtggctgcg cagcgacaag gtgttgacgt tgcccgcctg 2400tgccagcatg gagggctctg
tgtggacgcg ggcaacacgc accactgccg ctgccaggcg 2460ggctacacag gcagctactg
tgaggacctg gtggacgagt gctcacccag cccctgccag 2520aacggggcca cctgcacgga
ctacctgggc ggctactcct gcaagtgcgt ggccggctac 2580cacggggtga actgctctga
ggagatcgac gagtgcctct cccacccctg ccagaacggg 2640ggcacctgcc tcgacctccc
caacacctac aagtgctcct gcccacgggg cactcagggt 2700gtgcactgtg agatcaacgt
ggacgactgc aatccccccg ttgaccccgt gtcccggagc 2760cccaagtgct ttaacaacgg
cacctgcgtg gaccaggtgg gcggctacag ctgcacctgc 2820ccgccgggct tcgtgggtga
gcgctgtgag ggggatgtca acgagtgcct gtccaatccc 2880tgcgacgccc gtggcaccca
gaactgcgtg cagcgcgtca atgacttcca ctgcgagtgc 2940cgtgctggtc acaccgggcg
ccgctgcgag tccgtcatca atggctgcaa aggcaagccc 3000tgcaagaatg ggggcacctg
cgccgtggcc tccaacaccg cccgcgggtt catctgcaag 3060tgccctgcgg gcttcgaggg
cgccacgtgt gagaatgacg ctcgtacctg cggcagcctg 3120cgctgcctca acggcggcac
atgcatctcc ggcccgcgca gccccacctg cctgtgcctg 3180ggccccttca cgggccccga
atgccagttc ccggccagca gcccctgcct gggcggcaac 3240ccctgctaca accaggggac
ctgtgagccc acatccgaga gccccttcta ccgttgcctg 3300tgccccgcca aattcaacgg
gctcttgtgc cacatcctgg actacagctt cggagatctg 3360ggcccgggcg agcccaaatc
ttgtgacaaa actcacacat gcccaccgtg cccagcacct 3420gaactcctgg ggggaccgtc
agtcttcctc ttccccccaa aacccaagga caccctcatg 3480atctcccgga cccctgaggt
cacatgcgtg gtggtggacg tgagccacga agaccctgag 3540gtcaagttca actggtacgt
ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 3600gaggagcagt acaacagcac
gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 3660tggctgaatg gcaaggagta
caagtgcaag gtctccaaca aagccctccc agcccccatc 3720gagaaaacca tctccaaagc
caaagggcag ccccgagaac cacaggtgta caccctgccc 3780ccatcccggg atgagctgac
caagaaccag gtcagcctga cctgcctggt caaaggcttc 3840tatcccagcg acatcgccgt
ggagtgggag agcaatgggc agccggagaa caactacaag 3900accacgcctc ccgtgctgga
ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 3960gacaagagca ggtggcagca
ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 4020cacaaccact acacgcagaa
gagcctctcc ctgtctccgg gtaaatga 4068714065DNAHomo sapiens
71atgtggggct ggaagtgcct cctcttctgg gctgtgctgg tcacagccac tctctgcact
60gccaggtgtg ccagcgccgc ctgcttccac ggcgccacct gccatgaccg tgtggcctcc
120ttctactgcg agtgtcccca tggccgcaca ggtctgctgt gccacctcaa cgacgcatgc
180atcagcaacc cctgtaacga gggctccaac tgcgacacca accctgtcaa tggcaaggcc
240atctgcacct gcccctcggg gtacacgggc ccggcctgca gccaggacgt ggatgagtgc
300tcgctgggtg ccaacccctg cgagcatgcg ggcaagtgca tcaacacgct gggctccttc
360gagtgccagt gtctgcaggg ctacacgggc ccccgatgcg agatcgacgt caacgagtgc
420gtctcgaacc cgtgccagaa cgacgccacc tgcctggacc agattgggga gttccagtgc
480atctgcatgc ccggctacga gggtgtgcac tgcgaggtca acacagacga gtgtgccagc
540agcccctgcc tgcacaatgg ccgctgcctg gacaagatca atgagttcca gtgcgagtgc
600cccacgggct tcactgggca tctgtgccag tacgatgtgg acgagtgtgc cagcaccccc
660tgcaagaatg gtgccaagtg cctggacgga cccaacactt acacctgtgt gtgcacggaa
720gggtacacgg ggacgcactg cgaggtggac atcgatgagt gcgaccccga cccctgccac
780tacggctcct gcaaggacgg cgtcgccacc ttcacctgcc tctgccgccc aggctacacg
840ggccaccact gcgagaccaa catcaacgag tgctccagcc agccctgccg ccacgggggc
900acctgccagg accgcgacaa cgcctacctc tgcttctgcc tgaaggggac cacaggaccc
960aactgcgaga tcaacctgga tgactgtgcc agcagcccct gcgactcggg cacctgtctg
1020gacaagatcg atggctacga gtgtgcctgt gagccgggct acacagggag catgtgtaac
1080atcaacatcg atgagtgtgc gggcaacccc tgccacaacg ggggcacctg cgaggacggc
1140atcaatggct tcacctgccg ctgccccgag ggctaccacg accccacctg cctgtctgag
1200gtcaatgagt gcaacagcaa cccctgcgtc cacggggcct gccgggacag cctcaacggg
1260tacaagtgcg actgtgaccc tgggtggagt gggaccaact gtgacatcaa caacaatgag
1320tgtgaatcca acccttgtgt caacggcggc acctgcaaag acatgaccag tggctacgtg
1380tgcacctgcc gggagggctt cagcggtccc aactgccaga ccaacatcaa cgagtgtgcg
1440tccaacccat gtctgaacca gggcacgtgt attgacgacg ttgccgggta caagtgcaac
1500tgcctgctgc cctacacagg tgccacgtgt gaggtggtgc tggccccgtg tgcccccagc
1560ccctgcagaa acggcgggga gtgcaggcaa tccgaggact atgagagctt ctcctgtgtc
1620tgccccacgg gctggcaagg gcagacctgt gaggtcgaca tcaacgagtg cgttctgagc
1680ccgtgccggc acggcgcatc ctgccagaac acccacggcg gctaccgctg ccactgccag
1740gccggctaca gtgggcgcaa ctgcgagacc gacatcgacg actgccggcc caacccgtgt
1800cacaacgggg gctcctgcac agacggcatc aacacggcct tctgcgactg cctgcccggc
1860ttccggggca ctttctgtga ggaggacatc aacgagtgtg ccagtgaccc ctgccgcaac
1920ggggccaact gcacggactg cgtggacagc tacacgtgca cctgccccgc aggcttcagc
1980gggatccact gtgagaacaa cacgcctgac tgcacagaga gctcctgctt caacggtggc
2040acctgcgtgg acggcatcaa ctcgttcacc tgcctgtgtc cacccggctt cacgggcagc
2100tactgccagc acgatgtcaa tgagtgcgac tcacagccct gcctgcatgg cggcacctgt
2160caggacggct gcggctccta caggtgcacc tgcccccagg gctacactgg ccccaactgc
2220cagaaccttg tgcactggtg tgactcctcg ccctgcaaga acggcggcaa atgctggcag
2280acccacaccc agtaccgctg cgagtgcccc agcggctgga ccggccttta ctgcgacgtg
2340cccagcgtgt cctgtgaggt ggctgcgcag cgacaaggtg ttgacgttgc ccgcctgtgc
2400cagcatggag ggctctgtgt ggacgcgggc aacacgcacc actgccgctg ccaggcgggc
2460tacacaggca gctactgtga ggacctggtg gacgagtgct cacccagccc ctgccagaac
2520ggggccacct gcacggacta cctgggcggc tactcctgca agtgcgtggc cggctaccac
2580ggggtgaact gctctgagga gatcgacgag tgcctctccc acccctgcca gaacgggggc
2640acctgcctcg acctccccaa cacctacaag tgctcctgcc cacggggcac tcagggtgtg
2700cactgtgaga tcaacgtgga cgactgcaat ccccccgttg accccgtgtc ccggagcccc
2760aagtgcttta acaacggcac ctgcgtggac caggtgggcg gctacagctg cacctgcccg
2820ccgggcttcg tgggtgagcg ctgtgagggg gatgtcaacg agtgcctgtc caatccctgc
2880gacgcccgtg gcacccagaa ctgcgtgcag cgcgtcaatg acttccactg cgagtgccgt
2940gctggtcaca ccgggcgccg ctgcgagtcc gtcatcaatg gctgcaaagg caagccctgc
3000aagaatgggg gcacctgcgc cgtggcctcc aacaccgccc gcgggttcat ctgcaagtgc
3060cctgcgggct tcgagggcgc cacgtgtgag aatgacgctc gtacctgcgg cagcctgcgc
3120tgcctcaacg gcggcacatg catctccggc ccgcgcagcc ccacctgcct gtgcctgggc
3180cccttcacgg gccccgaatg ccagttcccg gccagcagcc cctgcctggg cggcaacccc
3240tgctacaacc aggggacctg tgagcccaca tccgagagcc ccttctaccg ttgcctgtgc
3300cccgccaaat tcaacgggct cttgtgccac atcctggact acagcttcgg agatctgggc
3360ccgggcgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa
3420ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc
3480tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc
3540aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag
3600gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg
3660ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag
3720aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca
3780tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat
3840cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc
3900acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac
3960aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac
4020aaccactaca cgcagaagag cctctccctg tctccgggta aatga
4065722148DNAHomo sapiens 72atgccgccgc tcctggcgcc cctgctctgc ctggcgctgc
tgcccgcgct cgccgcacga 60ggcccgcgat gtgccagcag cccctgcctg cacaatggcc
gctgcctgga caagatcaat 120gagttccagt gcgagtgccc cacgggcttc actgggcatc
tgtgccagta cgatgtggac 180gagtgtgcca gcaccccctg caagaatggt gccaagtgcc
tggacggacc caacacttac 240acctgtgtgt gcacggaagg gtacacgggg acgcactgcg
aggtggacat cgatgagtgc 300gaccccgacc cctgccacta cggctcctgc aaggacggcg
tcgccacctt cacctgcctc 360tgccgcccag gctacacggg ccaccactgc gagaccaaca
tcaacgagtg ctccagccag 420ccctgccgcc acgggggcac ctgccaggac cgcgacaacg
cctacctctg cttctgcctg 480aaggggacca caggacccaa ctgcgagatc aacctggatg
actgtgccag cagcccctgc 540gactcgggca cctgtctgga caagatcgat ggctacgagt
gtgcctgtga gccgggctac 600acagggagca tgtgtaacat caacatcgat gagtgtgcgg
gcaacccctg ccacaacggg 660ggcacctgcg aggacggcat caatggcttc acctgccgct
gccccgaggg ctaccacgac 720cccacctgcc tgtctgaggt caatgagtgc aacagcaacc
cctgcgtcca cggggcctgc 780cgggacagcc tcaacgggta caagtgcgac tgtgaccctg
ggtggagtgg gaccaactgt 840gacatcaaca acaatgagtg tgaatccaac ccttgtgtca
acggcggcac ctgcaaagac 900atgaccagtg gctacgtgtg cacctgccgg gagggcttca
gcggtcccaa ctgccagacc 960aacatcaacg agtgtgcgtc caacccatgt ctgaaccagg
gcacgtgtat tgacgacgtt 1020gccgggtaca agtgcaactg cctgctgccc tacacaggtg
ccacgtgtga ggtggtgctg 1080gccccgtgtg cccccagccc ctgcagaaac ggcggggagt
gcaggcaatc cgaggactat 1140gagagcttct cctgtgtctg ccccacgggc tggcaagggc
agacctgtga ggtcgacatc 1200aacgagtgcg ttctgagccc gtgccggcac ggcgcatcct
gccagaacac ccacggcggc 1260taccgctgcc actgccaggc cggctacagt gggcgcaact
gcgagaccga catcgacgac 1320tgccggccca acccgtgtca caacgggggc tcctgcacag
acggcatcaa cacggccttc 1380tgcgactgcc tgcccggctt ccggggcact ttctgtgagg
aggacatcaa cgaggatctg 1440ggcccgggcg agcccaaatc ttgtgacaaa actcacacat
gcccaccgtg cccagcacct 1500gaactcctgg ggggaccgtc agtcttcctc ttccccccaa
aacccaagga caccctcatg 1560atctcccgga cccctgaggt cacatgcgtg gtggtggacg
tgagccacga agaccctgag 1620gtcaagttca actggtacgt ggacggcgtg gaggtgcata
atgccaagac aaagccgcgg 1680gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc
tcaccgtcct gcaccaggac 1740tggctgaatg gcaaggagta caagtgcaag gtctccaaca
aagccctccc agcccccatc 1800gagaaaacca tctccaaagc caaagggcag ccccgagaac
cacaggtgta caccctgccc 1860ccatcccggg atgagctgac caagaaccag gtcagcctga
cctgcctggt caaaggcttc 1920tatcccagcg acatcgccgt ggagtgggag agcaatgggc
agccggagaa caactacaag 1980accacgcctc ccgtgctgga ctccgacggc tccttcttcc
tctacagcaa gctcaccgtg 2040gacaagagca ggtggcagca ggggaacgtc ttctcatgct
ccgtgatgca tgaggctctg 2100cacaaccact acacgcagaa gagcctctcc ctgtctccgg
gtaaatga 2148732145DNAHomo sapiens 73atgtggggct ggaagtgcct
cctcttctgg gctgtgctgg tcacagccac tctctgcact 60gccaggtgtg ccagcagccc
ctgcctgcac aatggccgct gcctggacaa gatcaatgag 120ttccagtgcg agtgccccac
gggcttcact gggcatctgt gccagtacga tgtggacgag 180tgtgccagca ccccctgcaa
gaatggtgcc aagtgcctgg acggacccaa cacttacacc 240tgtgtgtgca cggaagggta
cacggggacg cactgcgagg tggacatcga tgagtgcgac 300cccgacccct gccactacgg
ctcctgcaag gacggcgtcg ccaccttcac ctgcctctgc 360cgcccaggct acacgggcca
ccactgcgag accaacatca acgagtgctc cagccagccc 420tgccgccacg ggggcacctg
ccaggaccgc gacaacgcct acctctgctt ctgcctgaag 480gggaccacag gacccaactg
cgagatcaac ctggatgact gtgccagcag cccctgcgac 540tcgggcacct gtctggacaa
gatcgatggc tacgagtgtg cctgtgagcc gggctacaca 600gggagcatgt gtaacatcaa
catcgatgag tgtgcgggca acccctgcca caacgggggc 660acctgcgagg acggcatcaa
tggcttcacc tgccgctgcc ccgagggcta ccacgacccc 720acctgcctgt ctgaggtcaa
tgagtgcaac agcaacccct gcgtccacgg ggcctgccgg 780gacagcctca acgggtacaa
gtgcgactgt gaccctgggt ggagtgggac caactgtgac 840atcaacaaca atgagtgtga
atccaaccct tgtgtcaacg gcggcacctg caaagacatg 900accagtggct acgtgtgcac
ctgccgggag ggcttcagcg gtcccaactg ccagaccaac 960atcaacgagt gtgcgtccaa
cccatgtctg aaccagggca cgtgtattga cgacgttgcc 1020gggtacaagt gcaactgcct
gctgccctac acaggtgcca cgtgtgaggt ggtgctggcc 1080ccgtgtgccc ccagcccctg
cagaaacggc ggggagtgca ggcaatccga ggactatgag 1140agcttctcct gtgtctgccc
cacgggctgg caagggcaga cctgtgaggt cgacatcaac 1200gagtgcgttc tgagcccgtg
ccggcacggc gcatcctgcc agaacaccca cggcggctac 1260cgctgccact gccaggccgg
ctacagtggg cgcaactgcg agaccgacat cgacgactgc 1320cggcccaacc cgtgtcacaa
cgggggctcc tgcacagacg gcatcaacac ggccttctgc 1380gactgcctgc ccggcttccg
gggcactttc tgtgaggagg acatcaacga ggatctgggc 1440ccgggcgagc ccaaatcttg
tgacaaaact cacacatgcc caccgtgccc agcacctgaa 1500ctcctggggg gaccgtcagt
cttcctcttc cccccaaaac ccaaggacac cctcatgatc 1560tcccggaccc ctgaggtcac
atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 1620aagttcaact ggtacgtgga
cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 1680gagcagtaca acagcacgta
ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 1740ctgaatggca aggagtacaa
gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1800aaaaccatct ccaaagccaa
agggcagccc cgagaaccac aggtgtacac cctgccccca 1860tcccgggatg agctgaccaa
gaaccaggtc agcctgacct gcctggtcaa aggcttctat 1920cccagcgaca tcgccgtgga
gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1980acgcctcccg tgctggactc
cgacggctcc ttcttcctct acagcaagct caccgtggac 2040aagagcaggt ggcagcaggg
gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 2100aaccactaca cgcagaagag
cctctccctg tctccgggta aatga 2145742238DNAHomo sapiens
74atgccgccgc tcctggcgcc cctgctctgc ctggcgctgc tgcccgcgct cgccgcacga
60ggcccgcgat gtgccagtga cccctgccgc aacggggcca actgcacgga ctgcgtggac
120agctacacgt gcacctgccc cgcaggcttc agcgggatcc actgtgagaa caacacgcct
180gactgcacag agagctcctg cttcaacggt ggcacctgcg tggacggcat caactcgttc
240acctgcctgt gtccacccgg cttcacgggc agctactgcc agcacgatgt caatgagtgc
300gactcacagc cctgcctgca tggcggcacc tgtcaggacg gctgcggctc ctacaggtgc
360acctgccccc agggctacac tggccccaac tgccagaacc ttgtgcactg gtgtgactcc
420tcgccctgca agaacggcgg caaatgctgg cagacccaca cccagtaccg ctgcgagtgc
480cccagcggct ggaccggcct ttactgcgac gtgcccagcg tgtcctgtga ggtggctgcg
540cagcgacaag gtgttgacgt tgcccgcctg tgccagcatg gagggctctg tgtggacgcg
600ggcaacacgc accactgccg ctgccaggcg ggctacacag gcagctactg tgaggacctg
660gtggacgagt gctcacccag cccctgccag aacggggcca cctgcacgga ctacctgggc
720ggctactcct gcaagtgcgt ggccggctac cacggggtga actgctctga ggagatcgac
780gagtgcctct cccacccctg ccagaacggg ggcacctgcc tcgacctccc caacacctac
840aagtgctcct gcccacgggg cactcagggt gtgcactgtg agatcaacgt ggacgactgc
900aatccccccg ttgaccccgt gtcccggagc cccaagtgct ttaacaacgg cacctgcgtg
960gaccaggtgg gcggctacag ctgcacctgc ccgccgggct tcgtgggtga gcgctgtgag
1020ggggatgtca acgagtgcct gtccaatccc tgcgacgccc gtggcaccca gaactgcgtg
1080cagcgcgtca atgacttcca ctgcgagtgc cgtgctggtc acaccgggcg ccgctgcgag
1140tccgtcatca atggctgcaa aggcaagccc tgcaagaatg ggggcacctg cgccgtggcc
1200tccaacaccg cccgcgggtt catctgcaag tgccctgcgg gcttcgaggg cgccacgtgt
1260gagaatgacg ctcgtacctg cggcagcctg cgctgcctca acggcggcac atgcatctcc
1320ggcccgcgca gccccacctg cctgtgcctg ggccccttca cgggccccga atgccagttc
1380ccggccagca gcccctgcct gggcggcaac ccctgctaca accaggggac ctgtgagccc
1440acatccgaga gccccttcta ccgttgcctg tgccccgcca aattcaacgg gctcttgtgc
1500cacatcctgg actacagctt cggagatctg ggcccgggcg agcccaaatc ttgtgacaaa
1560actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc
1620ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg
1680gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg
1740gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg
1800gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag
1860gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag
1920ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag
1980gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag
2040agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc
2100tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc
2160ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc
2220ctgtctccgg gtaaatga
2238752235DNAHomo sapiens 75atgtggggct ggaagtgcct cctcttctgg gctgtgctgg
tcacagccac tctctgcact 60gccaggtgtg ccagtgaccc ctgccgcaac ggggccaact
gcacggactg cgtggacagc 120tacacgtgca cctgccccgc aggcttcagc gggatccact
gtgagaacaa cacgcctgac 180tgcacagaga gctcctgctt caacggtggc acctgcgtgg
acggcatcaa ctcgttcacc 240tgcctgtgtc cacccggctt cacgggcagc tactgccagc
acgatgtcaa tgagtgcgac 300tcacagccct gcctgcatgg cggcacctgt caggacggct
gcggctccta caggtgcacc 360tgcccccagg gctacactgg ccccaactgc cagaaccttg
tgcactggtg tgactcctcg 420ccctgcaaga acggcggcaa atgctggcag acccacaccc
agtaccgctg cgagtgcccc 480agcggctgga ccggccttta ctgcgacgtg cccagcgtgt
cctgtgaggt ggctgcgcag 540cgacaaggtg ttgacgttgc ccgcctgtgc cagcatggag
ggctctgtgt ggacgcgggc 600aacacgcacc actgccgctg ccaggcgggc tacacaggca
gctactgtga ggacctggtg 660gacgagtgct cacccagccc ctgccagaac ggggccacct
gcacggacta cctgggcggc 720tactcctgca agtgcgtggc cggctaccac ggggtgaact
gctctgagga gatcgacgag 780tgcctctccc acccctgcca gaacgggggc acctgcctcg
acctccccaa cacctacaag 840tgctcctgcc cacggggcac tcagggtgtg cactgtgaga
tcaacgtgga cgactgcaat 900ccccccgttg accccgtgtc ccggagcccc aagtgcttta
acaacggcac ctgcgtggac 960caggtgggcg gctacagctg cacctgcccg ccgggcttcg
tgggtgagcg ctgtgagggg 1020gatgtcaacg agtgcctgtc caatccctgc gacgcccgtg
gcacccagaa ctgcgtgcag 1080cgcgtcaatg acttccactg cgagtgccgt gctggtcaca
ccgggcgccg ctgcgagtcc 1140gtcatcaatg gctgcaaagg caagccctgc aagaatgggg
gcacctgcgc cgtggcctcc 1200aacaccgccc gcgggttcat ctgcaagtgc cctgcgggct
tcgagggcgc cacgtgtgag 1260aatgacgctc gtacctgcgg cagcctgcgc tgcctcaacg
gcggcacatg catctccggc 1320ccgcgcagcc ccacctgcct gtgcctgggc cccttcacgg
gccccgaatg ccagttcccg 1380gccagcagcc cctgcctggg cggcaacccc tgctacaacc
aggggacctg tgagcccaca 1440tccgagagcc ccttctaccg ttgcctgtgc cccgccaaat
tcaacgggct cttgtgccac 1500atcctggact acagcttcgg agatctgggc ccgggcgagc
ccaaatcttg tgacaaaact 1560cacacatgcc caccgtgccc agcacctgaa ctcctggggg
gaccgtcagt cttcctcttc 1620cccccaaaac ccaaggacac cctcatgatc tcccggaccc
ctgaggtcac atgcgtggtg 1680gtggacgtga gccacgaaga ccctgaggtc aagttcaact
ggtacgtgga cggcgtggag 1740gtgcataatg ccaagacaaa gccgcgggag gagcagtaca
acagcacgta ccgtgtggtc 1800agcgtcctca ccgtcctgca ccaggactgg ctgaatggca
aggagtacaa gtgcaaggtc 1860tccaacaaag ccctcccagc ccccatcgag aaaaccatct
ccaaagccaa agggcagccc 1920cgagaaccac aggtgtacac cctgccccca tcccgggatg
agctgaccaa gaaccaggtc 1980agcctgacct gcctggtcaa aggcttctat cccagcgaca
tcgccgtgga gtgggagagc 2040aatgggcagc cggagaacaa ctacaagacc acgcctcccg
tgctggactc cgacggctcc 2100ttcttcctct acagcaagct caccgtggac aagagcaggt
ggcagcaggg gaacgtcttc 2160tcatgctccg tgatgcatga ggctctgcac aaccactaca
cgcagaagag cctctccctg 2220tctccgggta aatga
2235762003PRTHomo sapiens 76Met Gln Pro Pro Ser Leu
Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu 1 5
10 15 Cys Val Ser Val Val Arg Pro Arg Gly Leu Leu
Cys Gly Ser Phe Pro 20 25
30 Glu Pro Cys Ala Asn Gly Gly Thr Cys Leu Ser Leu Ser Leu Gly
Gln 35 40 45 Gly
Thr Cys Gln Cys Ala Pro Gly Phe Leu Gly Glu Thr Cys Gln Phe 50
55 60 Pro Asp Pro Cys Gln Asn
Ala Gln Leu Cys Gln Asn Gly Gly Ser Cys 65 70
75 80 Gln Ala Leu Leu Pro Ala Pro Leu Gly Leu Pro
Ser Ser Pro Ser Pro 85 90
95 Leu Thr Pro Ser Phe Leu Cys Thr Cys Leu Pro Gly Phe Thr Gly Glu
100 105 110 Arg Cys
Gln Ala Lys Leu Glu Asp Pro Cys Pro Pro Ser Phe Cys Ser 115
120 125 Lys Arg Gly Arg Cys His Ile
Gln Ala Ser Gly Arg Pro Gln Cys Ser 130 135
140 Cys Met Pro Gly Trp Thr Gly Glu Gln Cys Gln Leu
Arg Asp Phe Cys 145 150 155
160 Ser Ala Asn Pro Cys Val Asn Gly Gly Val Cys Leu Ala Thr Tyr Pro
165 170 175 Gln Ile Gln
Cys His Cys Pro Pro Gly Phe Glu Gly His Ala Cys Glu 180
185 190 Arg Asp Val Asn Glu Cys Phe Gln
Asp Pro Gly Pro Cys Pro Lys Gly 195 200
205 Thr Ser Cys His Asn Thr Leu Gly Ser Phe Gln Cys Leu
Cys Pro Val 210 215 220
Gly Gln Glu Gly Pro Arg Cys Glu Leu Arg Ala Gly Pro Cys Pro Pro 225
230 235 240 Arg Gly Cys Ser
Asn Gly Gly Thr Cys Gln Leu Met Pro Glu Lys Asp 245
250 255 Ser Thr Phe His Leu Cys Leu Cys Pro
Pro Gly Phe Ile Gly Pro Asp 260 265
270 Cys Glu Val Asn Pro Asp Asn Cys Val Ser His Gln Cys Gln
Asn Gly 275 280 285
Gly Thr Cys Gln Asp Gly Leu Asp Thr Tyr Thr Cys Leu Cys Pro Glu 290
295 300 Thr Trp Thr Gly Trp
Asp Cys Ser Glu Asp Val Asp Glu Cys Glu Thr 305 310
315 320 Gln Gly Pro Pro His Cys Arg Asn Gly Gly
Thr Cys Gln Asn Ser Ala 325 330
335 Gly Ser Phe His Cys Val Cys Val Ser Gly Trp Gly Gly Thr Ser
Cys 340 345 350 Glu
Glu Asn Leu Asp Asp Cys Ile Ala Ala Thr Cys Ala Pro Gly Ser 355
360 365 Thr Cys Ile Asp Arg Val
Gly Ser Phe Ser Cys Leu Cys Pro Pro Gly 370 375
380 Arg Thr Gly Leu Leu Cys His Leu Glu Asp Met
Cys Leu Ser Gln Pro 385 390 395
400 Cys His Gly Asp Ala Gln Cys Ser Thr Asn Pro Leu Thr Gly Ser Thr
405 410 415 Leu Cys
Leu Cys Gln Pro Gly Tyr Ser Gly Pro Thr Cys His Gln Asp 420
425 430 Leu Asp Glu Cys Leu Met Ala
Gln Gln Gly Pro Ser Pro Cys Glu His 435 440
445 Gly Gly Ser Cys Leu Asn Thr Pro Gly Ser Phe Asn
Cys Leu Cys Pro 450 455 460
Pro Gly Tyr Thr Gly Ser Arg Cys Glu Ala Asp His Asn Glu Cys Leu 465
470 475 480 Ser Gln Pro
Cys His Pro Gly Ser Thr Cys Leu Asp Leu Leu Ala Thr 485
490 495 Phe His Cys Leu Cys Pro Pro Gly
Leu Glu Gly Gln Leu Cys Glu Val 500 505
510 Glu Thr Asn Glu Cys Ala Ser Ala Pro Cys Leu Asn His
Ala Asp Cys 515 520 525
His Asp Leu Leu Asn Gly Phe Gln Cys Ile Cys Leu Pro Gly Phe Ser 530
535 540 Gly Thr Arg Cys
Glu Glu Asp Ile Asp Glu Cys Arg Ser Ser Pro Cys 545 550
555 560 Ala Asn Gly Gly Gln Cys Gln Asp Gln
Pro Gly Ala Phe His Cys Lys 565 570
575 Cys Leu Pro Gly Phe Glu Gly Pro Arg Cys Gln Thr Glu Val
Asp Glu 580 585 590
Cys Leu Ser Asp Pro Cys Pro Val Gly Ala Ser Cys Leu Asp Leu Pro
595 600 605 Gly Ala Phe Phe
Cys Leu Cys Pro Ser Gly Phe Thr Gly Gln Leu Cys 610
615 620 Glu Val Pro Leu Cys Ala Pro Asn
Leu Cys Gln Pro Lys Gln Ile Cys 625 630
635 640 Lys Asp Gln Lys Asp Lys Ala Asn Cys Leu Cys Pro
Asp Gly Ser Pro 645 650
655 Gly Cys Ala Pro Pro Glu Asp Asn Cys Thr Cys His His Gly His Cys
660 665 670 Gln Arg Ser
Ser Cys Val Cys Asp Val Gly Trp Thr Gly Pro Glu Cys 675
680 685 Glu Ala Glu Leu Gly Gly Cys Ile
Ser Ala Pro Cys Ala His Gly Gly 690 695
700 Thr Cys Tyr Pro Gln Pro Ser Gly Tyr Asn Cys Thr Cys
Pro Thr Gly 705 710 715
720 Tyr Thr Gly Pro Thr Cys Ser Glu Glu Met Thr Ala Cys His Ser Gly
725 730 735 Pro Cys Leu Asn
Gly Gly Ser Cys Asn Pro Ser Pro Gly Gly Tyr Tyr 740
745 750 Cys Thr Cys Pro Pro Ser His Thr Gly
Pro Gln Cys Gln Thr Ser Thr 755 760
765 Asp Tyr Cys Val Ser Ala Pro Cys Phe Asn Gly Gly Thr Cys
Val Asn 770 775 780
Arg Pro Gly Thr Phe Ser Cys Leu Cys Ala Met Gly Phe Gln Gly Pro 785
790 795 800 Arg Cys Glu Gly Lys
Leu Arg Pro Ser Cys Ala Asp Ser Pro Cys Arg 805
810 815 Asn Arg Ala Thr Cys Gln Asp Ser Pro Gln
Gly Pro Arg Cys Leu Cys 820 825
830 Pro Thr Gly Tyr Thr Gly Gly Ser Cys Gln Thr Leu Met Asp Leu
Cys 835 840 845 Ala
Gln Lys Pro Cys Pro Arg Asn Ser His Cys Leu Gln Thr Gly Pro 850
855 860 Ser Phe His Cys Leu Cys
Leu Gln Gly Trp Thr Gly Pro Leu Cys Asn 865 870
875 880 Leu Pro Leu Ser Ser Cys Gln Lys Ala Ala Leu
Ser Gln Gly Ile Asp 885 890
895 Val Ser Ser Leu Cys His Asn Gly Gly Leu Cys Val Asp Ser Gly Pro
900 905 910 Ser Tyr
Phe Cys His Cys Pro Pro Gly Phe Gln Gly Ser Leu Cys Gln 915
920 925 Asp His Val Asn Pro Cys Glu
Ser Arg Pro Cys Gln Asn Gly Ala Thr 930 935
940 Cys Met Ala Gln Pro Ser Gly Tyr Leu Cys Gln Cys
Ala Pro Gly Tyr 945 950 955
960 Asp Gly Gln Asn Cys Ser Lys Glu Leu Asp Ala Cys Gln Ser Gln Pro
965 970 975 Cys His Asn
His Gly Thr Cys Thr Pro Lys Pro Gly Gly Phe His Cys 980
985 990 Ala Cys Pro Pro Gly Phe Val Gly
Leu Arg Cys Glu Gly Asp Val Asp 995 1000
1005 Glu Cys Leu Asp Gln Pro Cys His Pro Thr Gly
Thr Ala Ala Cys 1010 1015 1020
His Ser Leu Ala Asn Ala Phe Tyr Cys Gln Cys Leu Pro Gly His
1025 1030 1035 Thr Gly Gln
Trp Cys Glu Val Glu Ile Asp Pro Cys His Ser Gln 1040
1045 1050 Pro Cys Phe His Gly Gly Thr Cys
Glu Ala Thr Ala Gly Ser Pro 1055 1060
1065 Leu Gly Phe Ile Cys His Cys Pro Lys Gly Phe Glu Gly
Pro Thr 1070 1075 1080
Cys Ser His Arg Ala Pro Ser Cys Gly Phe His His Cys His His 1085
1090 1095 Gly Gly Leu Cys Leu
Pro Ser Pro Lys Pro Gly Phe Pro Pro Arg 1100 1105
1110 Cys Ala Cys Leu Ser Gly Tyr Gly Gly Pro
Asp Cys Leu Thr Pro 1115 1120 1125
Pro Ala Pro Lys Gly Cys Gly Pro Pro Ser Pro Cys Leu Tyr Asn
1130 1135 1140 Gly Ser
Cys Ser Glu Thr Thr Gly Leu Gly Gly Pro Gly Phe Arg 1145
1150 1155 Cys Ser Cys Pro His Ser Ser
Pro Gly Pro Arg Cys Gln Lys Pro 1160 1165
1170 Gly Ala Lys Gly Cys Glu Gly Arg Ser Gly Asp Gly
Ala Cys Asp 1175 1180 1185
Ala Gly Cys Ser Gly Pro Gly Gly Asn Trp Asp Gly Gly Asp Cys 1190
1195 1200 Ser Leu Gly Val Pro
Asp Pro Trp Lys Gly Cys Pro Ser His Ser 1205 1210
1215 Arg Cys Trp Leu Leu Phe Arg Asp Gly Gln
Cys His Pro Gln Cys 1220 1225 1230
Asp Ser Glu Glu Cys Leu Phe Asp Gly Tyr Asp Cys Glu Thr Pro
1235 1240 1245 Pro Ala
Cys Thr Pro Ala Tyr Asp Gln Tyr Cys His Asp His Phe 1250
1255 1260 His Asn Gly His Cys Glu Lys
Gly Cys Asn Thr Ala Glu Cys Gly 1265 1270
1275 Trp Asp Gly Gly Asp Cys Arg Pro Glu Asp Gly Asp
Pro Glu Trp 1280 1285 1290
Gly Pro Ser Leu Ala Leu Leu Val Val Leu Ser Pro Pro Ala Leu 1295
1300 1305 Asp Gln Gln Leu Phe
Ala Leu Ala Arg Val Leu Ser Leu Thr Leu 1310 1315
1320 Arg Val Gly Leu Trp Val Arg Lys Asp Arg
Asp Gly Arg Asp Met 1325 1330 1335
Val Tyr Pro Tyr Pro Gly Ala Arg Ala Glu Glu Lys Leu Gly Gly
1340 1345 1350 Thr Arg
Asp Pro Thr Tyr Gln Glu Arg Ala Ala Pro Gln Thr Gln 1355
1360 1365 Pro Leu Gly Lys Glu Thr Asp
Ser Leu Ser Ala Gly Phe Val Val 1370 1375
1380 Val Met Gly Val Asp Leu Ser Arg Cys Gly Pro Asp
His Pro Ala 1385 1390 1395
Ser Arg Cys Pro Trp Asp Pro Gly Leu Leu Leu Arg Phe Leu Ala 1400
1405 1410 Ala Met Ala Ala Val
Gly Ala Leu Glu Pro Leu Leu Pro Gly Pro 1415 1420
1425 Leu Leu Ala Val His Pro His Ala Gly Thr
Ala Pro Pro Ala Asn 1430 1435 1440
Gln Leu Pro Trp Pro Val Leu Cys Ser Pro Val Ala Gly Val Ile
1445 1450 1455 Leu Leu
Ala Leu Gly Ala Leu Leu Val Leu Gln Leu Ile Arg Arg 1460
1465 1470 Arg Arg Arg Glu His Gly Ala
Leu Trp Leu Pro Pro Gly Phe Thr 1475 1480
1485 Arg Arg Pro Arg Thr Gln Ser Ala Pro His Arg Arg
Arg Pro Pro 1490 1495 1500
Leu Gly Glu Asp Ser Ile Gly Leu Lys Ala Leu Lys Pro Lys Ala 1505
1510 1515 Glu Val Asp Glu Asp
Gly Val Val Met Cys Ser Gly Pro Glu Glu 1520 1525
1530 Gly Glu Glu Val Gly Gln Ala Glu Glu Thr
Gly Pro Pro Ser Thr 1535 1540 1545
Cys Gln Leu Trp Ser Leu Ser Gly Gly Cys Gly Ala Leu Pro Gln
1550 1555 1560 Ala Ala
Met Leu Thr Pro Pro Gln Glu Ser Glu Met Glu Ala Pro 1565
1570 1575 Asp Leu Asp Thr Arg Gly Pro
Asp Gly Val Thr Pro Leu Met Ser 1580 1585
1590 Ala Val Cys Cys Gly Glu Val Gln Ser Gly Thr Phe
Gln Gly Ala 1595 1600 1605
Trp Leu Gly Cys Pro Glu Pro Trp Glu Pro Leu Leu Asp Gly Gly 1610
1615 1620 Ala Cys Pro Gln Ala
His Thr Val Gly Thr Gly Glu Thr Pro Leu 1625 1630
1635 His Leu Ala Ala Arg Phe Ser Arg Pro Thr
Ala Ala Arg Arg Leu 1640 1645 1650
Leu Glu Ala Gly Ala Asn Pro Asn Gln Pro Asp Arg Ala Gly Arg
1655 1660 1665 Thr Pro
Leu His Ala Ala Val Ala Ala Asp Ala Arg Glu Val Cys 1670
1675 1680 Gln Leu Leu Leu Arg Ser Arg
Gln Thr Ala Val Asp Ala Arg Thr 1685 1690
1695 Glu Asp Gly Thr Thr Pro Leu Met Leu Ala Ala Arg
Leu Ala Val 1700 1705 1710
Glu Asp Leu Val Glu Glu Leu Ile Ala Ala Gln Ala Asp Val Gly 1715
1720 1725 Ala Arg Asp Lys Trp
Gly Lys Thr Ala Leu His Trp Ala Ala Ala 1730 1735
1740 Val Asn Asn Ala Arg Ala Ala Arg Ser Leu
Leu Gln Ala Gly Ala 1745 1750 1755
Asp Lys Asp Ala Gln Asp Asn Arg Glu Gln Thr Pro Leu Phe Leu
1760 1765 1770 Ala Ala
Arg Glu Gly Ala Val Glu Val Ala Gln Leu Leu Leu Gly 1775
1780 1785 Leu Gly Ala Ala Arg Glu Leu
Arg Asp Gln Ala Gly Leu Ala Pro 1790 1795
1800 Ala Asp Val Ala His Gln Arg Asn His Trp Asp Leu
Leu Thr Leu 1805 1810 1815
Leu Glu Gly Ala Gly Pro Pro Glu Ala Arg His Lys Ala Thr Pro 1820
1825 1830 Gly Arg Glu Ala Gly
Pro Phe Pro Arg Ala Arg Thr Val Ser Val 1835 1840
1845 Ser Val Pro Pro His Gly Gly Gly Ala Leu
Pro Arg Cys Arg Thr 1850 1855 1860
Leu Ser Ala Gly Ala Gly Pro Arg Gly Gly Gly Ala Cys Leu Gln
1865 1870 1875 Ala Arg
Thr Trp Ser Val Asp Leu Ala Ala Arg Gly Gly Gly Ala 1880
1885 1890 Tyr Ser His Cys Arg Ser Leu
Ser Gly Val Gly Ala Gly Gly Gly 1895 1900
1905 Pro Thr Pro Arg Gly Arg Arg Phe Ser Ala Gly Met
Arg Gly Pro 1910 1915 1920
Arg Pro Asn Pro Ala Ile Met Arg Gly Arg Tyr Gly Val Ala Ala 1925
1930 1935 Gly Arg Gly Gly Arg
Val Ser Thr Asp Asp Trp Pro Cys Asp Trp 1940 1945
1950 Val Ala Leu Gly Ala Cys Gly Ser Ala Ser
Asn Ile Pro Ile Pro 1955 1960 1965
Pro Pro Cys Leu Thr Pro Ser Pro Glu Arg Gly Ser Pro Gln Leu
1970 1975 1980 Asp Cys
Gly Pro Pro Ala Leu Gln Glu Met Pro Ile Asn Gln Gly 1985
1990 1995 Gly Glu Gly Lys Lys 2000
77237PRTHomo sapiens 77Asp Leu Gly Pro Gly Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys 1 5 10
15 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu 20 25 30 Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 35
40 45 Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys 50 55
60 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys 65 70 75
80 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
85 90 95 Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 100
105 110 Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys 115 120
125 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser 130 135 140
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 145
150 155 160 Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 165
170 175 Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly 180 185
190 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln 195 200 205
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 210
215 220 His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 225 230
235 781410PRTHomo sapiens 78Gln Pro Pro Ser Leu Leu Leu Leu Leu
Leu Leu Leu Leu Leu Leu Cys 1 5 10
15 Val Ser Val Val Arg Pro Arg Gly Leu Leu Cys Gly Ser Phe
Pro Glu 20 25 30
Pro Cys Ala Asn Gly Gly Thr Cys Leu Ser Leu Ser Leu Gly Gln Gly
35 40 45 Thr Cys Gln Cys
Ala Pro Gly Phe Leu Gly Glu Thr Cys Gln Phe Pro 50
55 60 Asp Pro Cys Gln Asn Ala Gln Leu
Cys Gln Asn Gly Gly Ser Cys Gln 65 70
75 80 Ala Leu Leu Pro Ala Pro Leu Gly Leu Pro Ser Ser
Pro Ser Pro Leu 85 90
95 Thr Pro Ser Phe Leu Cys Thr Cys Leu Pro Gly Phe Thr Gly Glu Arg
100 105 110 Cys Gln Ala
Lys Leu Glu Asp Pro Cys Pro Pro Ser Phe Cys Ser Lys 115
120 125 Arg Gly Arg Cys His Ile Gln Ala
Ser Gly Arg Pro Gln Cys Ser Cys 130 135
140 Met Pro Gly Trp Thr Gly Glu Gln Cys Gln Leu Arg Asp
Phe Cys Ser 145 150 155
160 Ala Asn Pro Cys Val Asn Gly Gly Val Cys Leu Ala Thr Tyr Pro Gln
165 170 175 Ile Gln Cys His
Cys Pro Pro Gly Phe Glu Gly His Ala Cys Glu Arg 180
185 190 Asp Val Asn Glu Cys Phe Gln Asp Pro
Gly Pro Cys Pro Lys Gly Thr 195 200
205 Ser Cys His Asn Thr Leu Gly Ser Phe Gln Cys Leu Cys Pro
Val Gly 210 215 220
Gln Glu Gly Pro Arg Cys Glu Leu Arg Ala Gly Pro Cys Pro Pro Arg 225
230 235 240 Gly Cys Ser Asn Gly
Gly Thr Cys Gln Leu Met Pro Glu Lys Asp Ser 245
250 255 Thr Phe His Leu Cys Leu Cys Pro Pro Gly
Phe Ile Gly Pro Asp Cys 260 265
270 Glu Val Asn Pro Asp Asn Cys Val Ser His Gln Cys Gln Asn Gly
Gly 275 280 285 Thr
Cys Gln Asp Gly Leu Asp Thr Tyr Thr Cys Leu Cys Pro Glu Thr 290
295 300 Trp Thr Gly Trp Asp Cys
Ser Glu Asp Val Asp Glu Cys Glu Thr Gln 305 310
315 320 Gly Pro Pro His Cys Arg Asn Gly Gly Thr Cys
Gln Asn Ser Ala Gly 325 330
335 Ser Phe His Cys Val Cys Val Ser Gly Trp Gly Gly Thr Ser Cys Glu
340 345 350 Glu Asn
Leu Asp Asp Cys Ile Ala Ala Thr Cys Ala Pro Gly Ser Thr 355
360 365 Cys Ile Asp Arg Val Gly Ser
Phe Ser Cys Leu Cys Pro Pro Gly Arg 370 375
380 Thr Gly Leu Leu Cys His Leu Glu Asp Met Cys Leu
Ser Gln Pro Cys 385 390 395
400 His Gly Asp Ala Gln Cys Ser Thr Asn Pro Leu Thr Gly Ser Thr Leu
405 410 415 Cys Leu Cys
Gln Pro Gly Tyr Ser Gly Pro Thr Cys His Gln Asp Leu 420
425 430 Asp Glu Cys Leu Met Ala Gln Gln
Gly Pro Ser Pro Cys Glu His Gly 435 440
445 Gly Ser Cys Leu Asn Thr Pro Gly Ser Phe Asn Cys Leu
Cys Pro Pro 450 455 460
Gly Tyr Thr Gly Ser Arg Cys Glu Ala Asp His Asn Glu Cys Leu Ser 465
470 475 480 Gln Pro Cys His
Pro Gly Ser Thr Cys Leu Asp Leu Leu Ala Thr Phe 485
490 495 His Cys Leu Cys Pro Pro Gly Leu Glu
Gly Gln Leu Cys Glu Val Glu 500 505
510 Thr Asn Glu Cys Ala Ser Ala Pro Cys Leu Asn His Ala Asp
Cys His 515 520 525
Asp Leu Leu Asn Gly Phe Gln Cys Ile Cys Leu Pro Gly Phe Ser Gly 530
535 540 Thr Arg Cys Glu Glu
Asp Ile Asp Glu Cys Arg Ser Ser Pro Cys Ala 545 550
555 560 Asn Gly Gly Gln Cys Gln Asp Gln Pro Gly
Ala Phe His Cys Lys Cys 565 570
575 Leu Pro Gly Phe Glu Gly Pro Arg Cys Gln Thr Glu Val Asp Glu
Cys 580 585 590 Leu
Ser Asp Pro Cys Pro Val Gly Ala Ser Cys Leu Asp Leu Pro Gly 595
600 605 Ala Phe Phe Cys Leu Cys
Pro Ser Gly Phe Thr Gly Gln Leu Cys Glu 610 615
620 Val Pro Leu Cys Ala Pro Asn Leu Cys Gln Pro
Lys Gln Ile Cys Lys 625 630 635
640 Asp Gln Lys Asp Lys Ala Asn Cys Leu Cys Pro Asp Gly Ser Pro Gly
645 650 655 Cys Ala
Pro Pro Glu Asp Asn Cys Thr Cys His His Gly His Cys Gln 660
665 670 Arg Ser Ser Cys Val Cys Asp
Val Gly Trp Thr Gly Pro Glu Cys Glu 675 680
685 Ala Glu Leu Gly Gly Cys Ile Ser Ala Pro Cys Ala
His Gly Gly Thr 690 695 700
Cys Tyr Pro Gln Pro Ser Gly Tyr Asn Cys Thr Cys Pro Thr Gly Tyr 705
710 715 720 Thr Gly Pro
Thr Cys Ser Glu Glu Met Thr Ala Cys His Ser Gly Pro 725
730 735 Cys Leu Asn Gly Gly Ser Cys Asn
Pro Ser Pro Gly Gly Tyr Tyr Cys 740 745
750 Thr Cys Pro Pro Ser His Thr Gly Pro Gln Cys Gln Thr
Ser Thr Asp 755 760 765
Tyr Cys Val Ser Ala Pro Cys Phe Asn Gly Gly Thr Cys Val Asn Arg 770
775 780 Pro Gly Thr Phe
Ser Cys Leu Cys Ala Met Gly Phe Gln Gly Pro Arg 785 790
795 800 Cys Glu Gly Lys Leu Arg Pro Ser Cys
Ala Asp Ser Pro Cys Arg Asn 805 810
815 Arg Ala Thr Cys Gln Asp Ser Pro Gln Gly Pro Arg Cys Leu
Cys Pro 820 825 830
Thr Gly Tyr Thr Gly Gly Ser Cys Gln Thr Leu Met Asp Leu Cys Ala
835 840 845 Gln Lys Pro Cys
Pro Arg Asn Ser His Cys Leu Gln Thr Gly Pro Ser 850
855 860 Phe His Cys Leu Cys Leu Gln Gly
Trp Thr Gly Pro Leu Cys Asn Leu 865 870
875 880 Pro Leu Ser Ser Cys Gln Lys Ala Ala Leu Ser Gln
Gly Ile Asp Val 885 890
895 Ser Ser Leu Cys His Asn Gly Gly Leu Cys Val Asp Ser Gly Pro Ser
900 905 910 Tyr Phe Cys
His Cys Pro Pro Gly Phe Gln Gly Ser Leu Cys Gln Asp 915
920 925 His Val Asn Pro Cys Glu Ser Arg
Pro Cys Gln Asn Gly Ala Thr Cys 930 935
940 Met Ala Gln Pro Ser Gly Tyr Leu Cys Gln Cys Ala Pro
Gly Tyr Asp 945 950 955
960 Gly Gln Asn Cys Ser Lys Glu Leu Asp Ala Cys Gln Ser Gln Pro Cys
965 970 975 His Asn His Gly
Thr Cys Thr Pro Lys Pro Gly Gly Phe His Cys Ala 980
985 990 Cys Pro Pro Gly Phe Val Gly Leu
Arg Cys Glu Gly Asp Val Asp Glu 995 1000
1005 Cys Leu Asp Gln Pro Cys His Pro Thr Gly Thr
Ala Ala Cys His 1010 1015 1020
Ser Leu Ala Asn Ala Phe Tyr Cys Gln Cys Leu Pro Gly His Thr
1025 1030 1035 Gly Gln Trp
Cys Glu Val Glu Ile Asp Pro Cys His Ser Gln Pro 1040
1045 1050 Cys Phe His Gly Gly Thr Cys Glu
Ala Thr Ala Gly Ser Pro Leu 1055 1060
1065 Gly Phe Ile Cys His Cys Pro Lys Gly Phe Glu Gly Pro
Thr Cys 1070 1075 1080
Ser His Arg Ala Pro Ser Cys Gly Phe His His Cys His His Gly 1085
1090 1095 Gly Leu Cys Leu Pro
Ser Pro Lys Pro Gly Phe Pro Pro Arg Cys 1100 1105
1110 Ala Cys Leu Ser Gly Tyr Gly Gly Pro Asp
Cys Leu Thr Pro Pro 1115 1120 1125
Ala Pro Lys Gly Cys Gly Pro Pro Ser Pro Cys Leu Tyr Asn Gly
1130 1135 1140 Ser Cys
Ser Glu Thr Thr Gly Leu Gly Gly Pro Gly Phe Arg Cys 1145
1150 1155 Ser Cys Pro His Ser Ser Pro
Gly Pro Arg Cys Gln Lys Pro Gly 1160 1165
1170 Asp Leu Gly Pro Gly Glu Pro Lys Ser Cys Asp Lys
Thr His Thr 1175 1180 1185
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 1190
1195 1200 Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 1205 1210
1215 Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp 1220 1225 1230
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
1235 1240 1245 Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 1250
1255 1260 Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn 1265 1270
1275 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala 1280 1285 1290
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 1295
1300 1305 Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 1310 1315
1320 Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser 1325 1330 1335
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
1340 1345 1350 Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 1355
1360 1365 Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly 1370 1375
1380 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His 1385 1390 1395
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 1400
1405 1410 79791PRTHomo sapiens 79Met Gln Pro Pro Ser
Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu 1 5
10 15 Cys Val Ser Val Val Arg Pro Arg Gly Leu
Leu Cys Gly Ser Phe Pro 20 25
30 Glu Pro Cys Ala Asn Gly Gly Thr Cys Leu Ser Leu Ser Leu Gly
Gln 35 40 45 Gly
Thr Cys Gln Cys Ala Pro Gly Phe Leu Gly Glu Thr Cys Gln Phe 50
55 60 Pro Asp Pro Cys Gln Asn
Ala Gln Leu Cys Gln Asn Gly Gly Ser Cys 65 70
75 80 Gln Ala Leu Leu Pro Ala Pro Leu Gly Leu Pro
Ser Ser Pro Ser Pro 85 90
95 Leu Thr Pro Ser Phe Leu Cys Thr Cys Leu Pro Gly Phe Thr Gly Glu
100 105 110 Arg Cys
Gln Ala Lys Leu Glu Asp Pro Cys Pro Pro Ser Phe Cys Ser 115
120 125 Lys Arg Gly Arg Cys His Ile
Gln Ala Ser Gly Arg Pro Gln Cys Ser 130 135
140 Cys Met Pro Gly Trp Thr Gly Glu Gln Cys Gln Leu
Arg Asp Phe Cys 145 150 155
160 Ser Ala Asn Pro Cys Val Asn Gly Gly Val Cys Leu Ala Thr Tyr Pro
165 170 175 Gln Ile Gln
Cys His Cys Pro Pro Gly Phe Glu Gly His Ala Cys Glu 180
185 190 Arg Asp Val Asn Glu Cys Phe Gln
Asp Pro Gly Pro Cys Pro Lys Gly 195 200
205 Thr Ser Cys His Asn Thr Leu Gly Ser Phe Gln Cys Leu
Cys Pro Val 210 215 220
Gly Gln Glu Gly Pro Arg Cys Glu Leu Arg Ala Gly Pro Cys Pro Pro 225
230 235 240 Arg Gly Cys Ser
Asn Gly Gly Thr Cys Gln Leu Met Pro Glu Lys Asp 245
250 255 Ser Thr Phe His Leu Cys Leu Cys Pro
Pro Gly Phe Ile Gly Pro Asp 260 265
270 Cys Glu Val Asn Pro Asp Asn Cys Val Ser His Gln Cys Gln
Asn Gly 275 280 285
Gly Thr Cys Gln Asp Gly Leu Asp Thr Tyr Thr Cys Leu Cys Pro Glu 290
295 300 Thr Trp Thr Gly Trp
Asp Cys Ser Glu Asp Val Asp Glu Cys Glu Thr 305 310
315 320 Gln Gly Pro Pro His Cys Arg Asn Gly Gly
Thr Cys Gln Asn Ser Ala 325 330
335 Gly Ser Phe His Cys Val Cys Val Ser Gly Trp Gly Gly Thr Ser
Cys 340 345 350 Glu
Glu Asn Leu Asp Asp Cys Ile Ala Ala Thr Cys Ala Pro Gly Ser 355
360 365 Thr Cys Ile Asp Arg Val
Gly Ser Phe Ser Cys Leu Cys Pro Pro Gly 370 375
380 Arg Thr Gly Leu Leu Cys His Leu Glu Asp Met
Cys Leu Ser Gln Pro 385 390 395
400 Cys His Gly Asp Ala Gln Cys Ser Thr Asn Pro Leu Thr Gly Ser Thr
405 410 415 Leu Cys
Leu Cys Gln Pro Gly Tyr Ser Gly Pro Thr Cys His Gln Asp 420
425 430 Leu Asp Glu Cys Leu Met Ala
Gln Gln Gly Pro Ser Pro Cys Glu His 435 440
445 Gly Gly Ser Cys Leu Asn Thr Pro Gly Ser Phe Asn
Cys Leu Cys Pro 450 455 460
Pro Gly Tyr Thr Gly Ser Arg Cys Glu Ala Asp His Asn Glu Cys Leu 465
470 475 480 Ser Gln Pro
Cys His Pro Gly Ser Thr Cys Leu Asp Leu Leu Ala Thr 485
490 495 Phe His Cys Leu Cys Pro Pro Gly
Leu Glu Gly Gln Leu Cys Glu Val 500 505
510 Glu Thr Asn Glu Cys Ala Ser Ala Pro Cys Leu Asn His
Ala Asp Cys 515 520 525
His Asp Leu Leu Asn Gly Phe Gln Cys Ile Cys Leu Pro Gly Phe Ser 530
535 540 Gly Thr Arg Cys
Glu Glu Asp Ile Asp Glu Asp Leu Gly Pro Gly Glu 545 550
555 560 Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro 565 570
575 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 580 585 590
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
595 600 605 Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 610
615 620 Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr 625 630
635 640 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp 645 650
655 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
660 665 670 Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 675
680 685 Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys 690 695
700 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp 705 710 715
720 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
725 730 735 Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 740
745 750 Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser 755 760
765 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser 770 775 780
Leu Ser Leu Ser Pro Gly Lys 785 790 801170PRTHomo
sapiens 80Met Gln Pro Pro Ser Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu
1 5 10 15 Cys Val
Ser Val Val Arg Pro Arg Gly Leu Leu Cys Gly Ser Phe Pro 20
25 30 Glu Pro Cys Ala Asn Gly Gly
Thr Cys Leu Ser Leu Ser Leu Gly Gln 35 40
45 Gly Thr Cys Gln Cys Ala Pro Gly Phe Leu Gly Glu
Thr Cys Gln Phe 50 55 60
Pro Asp Pro Cys Gln Asn Ala Gln Leu Cys Gln Asn Gly Gly Ser Cys 65
70 75 80 Gln Ala Leu
Leu Pro Ala Pro Leu Gly Leu Pro Ser Ser Pro Ser Pro 85
90 95 Leu Thr Pro Ser Phe Leu Cys Thr
Cys Leu Pro Gly Phe Thr Gly Glu 100 105
110 Arg Cys Gln Ala Lys Leu Glu Asp Pro Cys Pro Pro Ser
Phe Cys Ser 115 120 125
Lys Arg Gly Arg Cys His Ile Gln Ala Ser Gly Arg Pro Gln Cys Ser 130
135 140 Cys Met Pro Gly
Trp Thr Gly Glu Gln Cys Gln Leu Arg Asp Phe Cys 145 150
155 160 Ser Ala Asn Pro Cys Val Asn Gly Gly
Val Cys Leu Ala Thr Tyr Pro 165 170
175 Gln Ile Gln Cys His Cys Pro Pro Gly Phe Glu Gly His Ala
Cys Glu 180 185 190
Arg Asp Val Asn Glu Cys Phe Gln Asp Pro Gly Pro Cys Pro Lys Gly
195 200 205 Thr Ser Cys His
Asn Thr Leu Gly Ser Phe Gln Cys Leu Cys Pro Val 210
215 220 Gly Gln Glu Gly Pro Arg Cys Glu
Leu Arg Ala Gly Pro Cys Pro Pro 225 230
235 240 Arg Gly Cys Ser Asn Gly Gly Thr Cys Gln Leu Met
Pro Glu Lys Asp 245 250
255 Ser Thr Phe His Leu Cys Leu Cys Pro Pro Gly Phe Ile Gly Pro Asp
260 265 270 Cys Glu Val
Asn Pro Asp Asn Cys Val Ser His Gln Cys Gln Asn Gly 275
280 285 Gly Thr Cys Gln Asp Gly Leu Asp
Thr Tyr Thr Cys Leu Cys Pro Glu 290 295
300 Thr Trp Thr Gly Trp Asp Cys Ser Glu Asp Val Asp Glu
Cys Glu Thr 305 310 315
320 Gln Gly Pro Pro His Cys Arg Asn Gly Gly Thr Cys Gln Asn Ser Ala
325 330 335 Gly Ser Phe His
Cys Val Cys Val Ser Gly Trp Gly Gly Thr Ser Cys 340
345 350 Glu Glu Asn Leu Asp Asp Cys Ile Ala
Ala Thr Cys Ala Pro Gly Ser 355 360
365 Thr Cys Ile Asp Arg Val Gly Ser Phe Ser Cys Leu Cys Pro
Pro Gly 370 375 380
Arg Thr Gly Leu Leu Cys His Leu Glu Asp Met Cys Leu Ser Gln Pro 385
390 395 400 Cys His Gly Asp Ala
Gln Cys Ser Thr Asn Pro Leu Thr Gly Ser Thr 405
410 415 Leu Cys Leu Cys Gln Pro Gly Tyr Ser Gly
Pro Thr Cys His Gln Asp 420 425
430 Leu Asp Glu Cys Leu Met Ala Gln Gln Gly Pro Ser Pro Cys Glu
His 435 440 445 Gly
Gly Ser Cys Leu Asn Thr Pro Gly Ser Phe Asn Cys Leu Cys Pro 450
455 460 Pro Gly Tyr Thr Gly Ser
Arg Cys Glu Ala Asp His Asn Glu Cys Leu 465 470
475 480 Ser Gln Pro Cys His Pro Gly Ser Thr Cys Leu
Asp Leu Leu Ala Thr 485 490
495 Phe His Cys Leu Cys Pro Pro Gly Leu Glu Gly Gln Leu Cys Glu Val
500 505 510 Glu Thr
Asn Glu Cys Ala Ser Ala Pro Cys Leu Asn His Ala Asp Cys 515
520 525 His Asp Leu Leu Asn Gly Phe
Gln Cys Ile Cys Leu Pro Gly Phe Ser 530 535
540 Gly Thr Arg Cys Glu Glu Asp Ile Asp Glu Cys Arg
Ser Ser Pro Cys 545 550 555
560 Ala Asn Gly Gly Gln Cys Gln Asp Gln Pro Gly Ala Phe His Cys Lys
565 570 575 Cys Leu Pro
Gly Phe Glu Gly Pro Arg Cys Gln Thr Glu Val Asp Glu 580
585 590 Cys Leu Ser Asp Pro Cys Pro Val
Gly Ala Ser Cys Leu Asp Leu Pro 595 600
605 Gly Ala Phe Phe Cys Leu Cys Pro Ser Gly Phe Thr Gly
Gln Leu Cys 610 615 620
Glu Val Pro Leu Cys Ala Pro Asn Leu Cys Gln Pro Lys Gln Ile Cys 625
630 635 640 Lys Asp Gln Lys
Asp Lys Ala Asn Cys Leu Cys Pro Asp Gly Ser Pro 645
650 655 Gly Cys Ala Pro Pro Glu Asp Asn Cys
Thr Cys His His Gly His Cys 660 665
670 Gln Arg Ser Ser Cys Val Cys Asp Val Gly Trp Thr Gly Pro
Glu Cys 675 680 685
Glu Ala Glu Leu Gly Gly Cys Ile Ser Ala Pro Cys Ala His Gly Gly 690
695 700 Thr Cys Tyr Pro Gln
Pro Ser Gly Tyr Asn Cys Thr Cys Pro Thr Gly 705 710
715 720 Tyr Thr Gly Pro Thr Cys Ser Glu Glu Met
Thr Ala Cys His Ser Gly 725 730
735 Pro Cys Leu Asn Gly Gly Ser Cys Asn Pro Ser Pro Gly Gly Tyr
Tyr 740 745 750 Cys
Thr Cys Pro Pro Ser His Thr Gly Pro Gln Cys Gln Thr Ser Thr 755
760 765 Asp Tyr Cys Val Ser Ala
Pro Cys Phe Asn Gly Gly Thr Cys Val Asn 770 775
780 Arg Pro Gly Thr Phe Ser Cys Leu Cys Ala Met
Gly Phe Gln Gly Pro 785 790 795
800 Arg Cys Glu Gly Lys Leu Arg Pro Ser Cys Ala Asp Ser Pro Cys Arg
805 810 815 Asn Arg
Ala Thr Cys Gln Asp Ser Pro Gln Gly Pro Arg Cys Leu Cys 820
825 830 Pro Thr Gly Tyr Thr Gly Gly
Ser Cys Gln Thr Leu Met Asp Leu Cys 835 840
845 Ala Gln Lys Pro Cys Pro Arg Asn Ser His Cys Leu
Gln Thr Gly Pro 850 855 860
Ser Phe His Cys Leu Cys Leu Gln Gly Trp Thr Gly Pro Leu Cys Asn 865
870 875 880 Leu Pro Leu
Ser Ser Cys Gln Lys Ala Ala Leu Ser Gln Gly Ile Asp 885
890 895 Val Ser Ser Leu Cys His Asn Gly
Gly Leu Cys Val Asp Ser Gly Pro 900 905
910 Ser Tyr Phe Cys His Cys Pro Pro Gly Phe Gln Gly Ser
Leu Cys Gln 915 920 925
Asp His Val Asn Pro Asp Leu Gly Pro Gly Glu Pro Lys Ser Cys Asp 930
935 940 Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 945 950
955 960 Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile 965 970
975 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu 980 985 990
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
995 1000 1005 Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 1010
1015 1020 Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn 1025 1030
1035 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala 1040 1045 1050
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 1055
1060 1065 Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 1070 1075
1080 Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser 1085 1090 1095
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
1100 1105 1110 Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 1115
1120 1125 Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly 1130 1135
1140 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His 1145 1150 1155
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 1160
1165 1170 81839PRTHomo sapiens 81Met Gln Pro Pro Ser
Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu 1 5
10 15 Cys Val Ser Val Val Arg Pro Arg Gly Leu
Leu Cys Ile Ala Ala Thr 20 25
30 Cys Ala Pro Gly Ser Thr Cys Ile Asp Arg Val Gly Ser Phe Ser
Cys 35 40 45 Leu
Cys Pro Pro Gly Arg Thr Gly Leu Leu Cys His Leu Glu Asp Met 50
55 60 Cys Leu Ser Gln Pro Cys
His Gly Asp Ala Gln Cys Ser Thr Asn Pro 65 70
75 80 Leu Thr Gly Ser Thr Leu Cys Leu Cys Gln Pro
Gly Tyr Ser Gly Pro 85 90
95 Thr Cys His Gln Asp Leu Asp Glu Cys Leu Met Ala Gln Gln Gly Pro
100 105 110 Ser Pro
Cys Glu His Gly Gly Ser Cys Leu Asn Thr Pro Gly Ser Phe 115
120 125 Asn Cys Leu Cys Pro Pro Gly
Tyr Thr Gly Ser Arg Cys Glu Ala Asp 130 135
140 His Asn Glu Cys Leu Ser Gln Pro Cys His Pro Gly
Ser Thr Cys Leu 145 150 155
160 Asp Leu Leu Ala Thr Phe His Cys Leu Cys Pro Pro Gly Leu Glu Gly
165 170 175 Gln Leu Cys
Glu Val Glu Thr Asn Glu Cys Ala Ser Ala Pro Cys Leu 180
185 190 Asn His Ala Asp Cys His Asp Leu
Leu Asn Gly Phe Gln Cys Ile Cys 195 200
205 Leu Pro Gly Phe Ser Gly Thr Arg Cys Glu Glu Asp Ile
Asp Glu Cys 210 215 220
Arg Ser Ser Pro Cys Ala Asn Gly Gly Gln Cys Gln Asp Gln Pro Gly 225
230 235 240 Ala Phe His Cys
Lys Cys Leu Pro Gly Phe Glu Gly Pro Arg Cys Gln 245
250 255 Thr Glu Val Asp Glu Cys Leu Ser Asp
Pro Cys Pro Val Gly Ala Ser 260 265
270 Cys Leu Asp Leu Pro Gly Ala Phe Phe Cys Leu Cys Pro Ser
Gly Phe 275 280 285
Thr Gly Gln Leu Cys Glu Val Pro Leu Cys Ala Pro Asn Leu Cys Gln 290
295 300 Pro Lys Gln Ile Cys
Lys Asp Gln Lys Asp Lys Ala Asn Cys Leu Cys 305 310
315 320 Pro Asp Gly Ser Pro Gly Cys Ala Pro Pro
Glu Asp Asn Cys Thr Cys 325 330
335 His His Gly His Cys Gln Arg Ser Ser Cys Val Cys Asp Val Gly
Trp 340 345 350 Thr
Gly Pro Glu Cys Glu Ala Glu Leu Gly Gly Cys Ile Ser Ala Pro 355
360 365 Cys Ala His Gly Gly Thr
Cys Tyr Pro Gln Pro Ser Gly Tyr Asn Cys 370 375
380 Thr Cys Pro Thr Gly Tyr Thr Gly Pro Thr Cys
Ser Glu Glu Met Thr 385 390 395
400 Ala Cys His Ser Gly Pro Cys Leu Asn Gly Gly Ser Cys Asn Pro Ser
405 410 415 Pro Gly
Gly Tyr Tyr Cys Thr Cys Pro Pro Ser His Thr Gly Pro Gln 420
425 430 Cys Gln Thr Ser Thr Asp Tyr
Cys Val Ser Ala Pro Cys Phe Asn Gly 435 440
445 Gly Thr Cys Val Asn Arg Pro Gly Thr Phe Ser Cys
Leu Cys Ala Met 450 455 460
Gly Phe Gln Gly Pro Arg Cys Glu Gly Lys Leu Arg Pro Ser Cys Ala 465
470 475 480 Asp Ser Pro
Cys Arg Asn Arg Ala Thr Cys Gln Asp Ser Pro Gln Gly 485
490 495 Pro Arg Cys Leu Cys Pro Thr Gly
Tyr Thr Gly Gly Ser Cys Gln Thr 500 505
510 Leu Met Asp Leu Cys Ala Gln Lys Pro Cys Pro Arg Asn
Ser His Cys 515 520 525
Leu Gln Thr Gly Pro Ser Phe His Cys Leu Cys Leu Gln Gly Trp Thr 530
535 540 Gly Pro Leu Cys
Asn Leu Pro Leu Ser Ser Cys Gln Lys Ala Ala Leu 545 550
555 560 Ser Gln Gly Ile Asp Val Ser Ser Leu
Cys His Asn Gly Gly Leu Cys 565 570
575 Val Asp Ser Gly Pro Ser Tyr Phe Cys His Cys Pro Pro Gly
Phe Gln 580 585 590
Gly Ser Leu Cys Gln Asp His Val Asn Pro Asp Leu Gly Pro Gly Glu
595 600 605 Pro Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 610
615 620 Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys 625 630
635 640 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val 645 650
655 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
660 665 670 Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 675
680 685 Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp 690 695
700 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu 705 710 715
720 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
725 730 735 Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 740
745 750 Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp 755 760
765 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys 770 775 780
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 785
790 795 800 Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 805
810 815 Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser 820 825
830 Leu Ser Leu Ser Pro Gly Lys 835
82834PRTHomo sapiens 82Met Trp Gly Trp Lys Cys Leu Leu Phe Trp Ala Val
Leu Val Thr Ala 1 5 10
15 Thr Leu Cys Thr Ala Arg Cys Ile Ala Ala Thr Cys Ala Pro Gly Ser
20 25 30 Thr Cys Ile
Asp Arg Val Gly Ser Phe Ser Cys Leu Cys Pro Pro Gly 35
40 45 Arg Thr Gly Leu Leu Cys His Leu
Glu Asp Met Cys Leu Ser Gln Pro 50 55
60 Cys His Gly Asp Ala Gln Cys Ser Thr Asn Pro Leu Thr
Gly Ser Thr 65 70 75
80 Leu Cys Leu Cys Gln Pro Gly Tyr Ser Gly Pro Thr Cys His Gln Asp
85 90 95 Leu Asp Glu Cys
Leu Met Ala Gln Gln Gly Pro Ser Pro Cys Glu His 100
105 110 Gly Gly Ser Cys Leu Asn Thr Pro Gly
Ser Phe Asn Cys Leu Cys Pro 115 120
125 Pro Gly Tyr Thr Gly Ser Arg Cys Glu Ala Asp His Asn Glu
Cys Leu 130 135 140
Ser Gln Pro Cys His Pro Gly Ser Thr Cys Leu Asp Leu Leu Ala Thr 145
150 155 160 Phe His Cys Leu Cys
Pro Pro Gly Leu Glu Gly Gln Leu Cys Glu Val 165
170 175 Glu Thr Asn Glu Cys Ala Ser Ala Pro Cys
Leu Asn His Ala Asp Cys 180 185
190 His Asp Leu Leu Asn Gly Phe Gln Cys Ile Cys Leu Pro Gly Phe
Ser 195 200 205 Gly
Thr Arg Cys Glu Glu Asp Ile Asp Glu Cys Arg Ser Ser Pro Cys 210
215 220 Ala Asn Gly Gly Gln Cys
Gln Asp Gln Pro Gly Ala Phe His Cys Lys 225 230
235 240 Cys Leu Pro Gly Phe Glu Gly Pro Arg Cys Gln
Thr Glu Val Asp Glu 245 250
255 Cys Leu Ser Asp Pro Cys Pro Val Gly Ala Ser Cys Leu Asp Leu Pro
260 265 270 Gly Ala
Phe Phe Cys Leu Cys Pro Ser Gly Phe Thr Gly Gln Leu Cys 275
280 285 Glu Val Pro Leu Cys Ala Pro
Asn Leu Cys Gln Pro Lys Gln Ile Cys 290 295
300 Lys Asp Gln Lys Asp Lys Ala Asn Cys Leu Cys Pro
Asp Gly Ser Pro 305 310 315
320 Gly Cys Ala Pro Pro Glu Asp Asn Cys Thr Cys His His Gly His Cys
325 330 335 Gln Arg Ser
Ser Cys Val Cys Asp Val Gly Trp Thr Gly Pro Glu Cys 340
345 350 Glu Ala Glu Leu Gly Gly Cys Ile
Ser Ala Pro Cys Ala His Gly Gly 355 360
365 Thr Cys Tyr Pro Gln Pro Ser Gly Tyr Asn Cys Thr Cys
Pro Thr Gly 370 375 380
Tyr Thr Gly Pro Thr Cys Ser Glu Glu Met Thr Ala Cys His Ser Gly 385
390 395 400 Pro Cys Leu Asn
Gly Gly Ser Cys Asn Pro Ser Pro Gly Gly Tyr Tyr 405
410 415 Cys Thr Cys Pro Pro Ser His Thr Gly
Pro Gln Cys Gln Thr Ser Thr 420 425
430 Asp Tyr Cys Val Ser Ala Pro Cys Phe Asn Gly Gly Thr Cys
Val Asn 435 440 445
Arg Pro Gly Thr Phe Ser Cys Leu Cys Ala Met Gly Phe Gln Gly Pro 450
455 460 Arg Cys Glu Gly Lys
Leu Arg Pro Ser Cys Ala Asp Ser Pro Cys Arg 465 470
475 480 Asn Arg Ala Thr Cys Gln Asp Ser Pro Gln
Gly Pro Arg Cys Leu Cys 485 490
495 Pro Thr Gly Tyr Thr Gly Gly Ser Cys Gln Thr Leu Met Asp Leu
Cys 500 505 510 Ala
Gln Lys Pro Cys Pro Arg Asn Ser His Cys Leu Gln Thr Gly Pro 515
520 525 Ser Phe His Cys Leu Cys
Leu Gln Gly Trp Thr Gly Pro Leu Cys Asn 530 535
540 Leu Pro Leu Ser Ser Cys Gln Lys Ala Ala Leu
Ser Gln Gly Ile Asp 545 550 555
560 Val Ser Ser Leu Cys His Asn Gly Gly Leu Cys Val Asp Ser Gly Pro
565 570 575 Ser Tyr
Phe Cys His Cys Pro Pro Gly Phe Gln Gly Ser Leu Cys Gln 580
585 590 Asp His Val Asn Pro Asp Leu
Gly Pro Gly Glu Pro Lys Ser Cys Asp 595 600
605 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly 610 615 620
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 625
630 635 640 Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 645
650 655 Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His 660 665
670 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg 675 680 685
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 690
695 700 Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 705 710
715 720 Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr 725 730
735 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu 740 745 750
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
755 760 765 Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 770
775 780 Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp 785 790
795 800 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His 805 810
815 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
820 825 830 Gly Lys
831080PRTHomo sapiens 83Met Gln Pro Pro Ser Leu Leu Leu Leu Leu Leu Leu
Leu Leu Leu Leu 1 5 10
15 Cys Val Ser Val Val Arg Pro Arg Gly Leu Leu Cys Ile Ala Ala Thr
20 25 30 Cys Ala Pro
Gly Ser Thr Cys Ile Asp Arg Val Gly Ser Phe Ser Cys 35
40 45 Leu Cys Pro Pro Gly Arg Thr Gly
Leu Leu Cys His Leu Glu Asp Met 50 55
60 Cys Leu Ser Gln Pro Cys His Gly Asp Ala Gln Cys Ser
Thr Asn Pro 65 70 75
80 Leu Thr Gly Ser Thr Leu Cys Leu Cys Gln Pro Gly Tyr Ser Gly Pro
85 90 95 Thr Cys His Gln
Asp Leu Asp Glu Cys Leu Met Ala Gln Gln Gly Pro 100
105 110 Ser Pro Cys Glu His Gly Gly Ser Cys
Leu Asn Thr Pro Gly Ser Phe 115 120
125 Asn Cys Leu Cys Pro Pro Gly Tyr Thr Gly Ser Arg Cys Glu
Ala Asp 130 135 140
His Asn Glu Cys Leu Ser Gln Pro Cys His Pro Gly Ser Thr Cys Leu 145
150 155 160 Asp Leu Leu Ala Thr
Phe His Cys Leu Cys Pro Pro Gly Leu Glu Gly 165
170 175 Gln Leu Cys Glu Val Glu Thr Asn Glu Cys
Ala Ser Ala Pro Cys Leu 180 185
190 Asn His Ala Asp Cys His Asp Leu Leu Asn Gly Phe Gln Cys Ile
Cys 195 200 205 Leu
Pro Gly Phe Ser Gly Thr Arg Cys Glu Glu Asp Ile Asp Glu Cys 210
215 220 Arg Ser Ser Pro Cys Ala
Asn Gly Gly Gln Cys Gln Asp Gln Pro Gly 225 230
235 240 Ala Phe His Cys Lys Cys Leu Pro Gly Phe Glu
Gly Pro Arg Cys Gln 245 250
255 Thr Glu Val Asp Glu Cys Leu Ser Asp Pro Cys Pro Val Gly Ala Ser
260 265 270 Cys Leu
Asp Leu Pro Gly Ala Phe Phe Cys Leu Cys Pro Ser Gly Phe 275
280 285 Thr Gly Gln Leu Cys Glu Val
Pro Leu Cys Ala Pro Asn Leu Cys Gln 290 295
300 Pro Lys Gln Ile Cys Lys Asp Gln Lys Asp Lys Ala
Asn Cys Leu Cys 305 310 315
320 Pro Asp Gly Ser Pro Gly Cys Ala Pro Pro Glu Asp Asn Cys Thr Cys
325 330 335 His His Gly
His Cys Gln Arg Ser Ser Cys Val Cys Asp Val Gly Trp 340
345 350 Thr Gly Pro Glu Cys Glu Ala Glu
Leu Gly Gly Cys Ile Ser Ala Pro 355 360
365 Cys Ala His Gly Gly Thr Cys Tyr Pro Gln Pro Ser Gly
Tyr Asn Cys 370 375 380
Thr Cys Pro Thr Gly Tyr Thr Gly Pro Thr Cys Ser Glu Glu Met Thr 385
390 395 400 Ala Cys His Ser
Gly Pro Cys Leu Asn Gly Gly Ser Cys Asn Pro Ser 405
410 415 Pro Gly Gly Tyr Tyr Cys Thr Cys Pro
Pro Ser His Thr Gly Pro Gln 420 425
430 Cys Gln Thr Ser Thr Asp Tyr Cys Val Ser Ala Pro Cys Phe
Asn Gly 435 440 445
Gly Thr Cys Val Asn Arg Pro Gly Thr Phe Ser Cys Leu Cys Ala Met 450
455 460 Gly Phe Gln Gly Pro
Arg Cys Glu Gly Lys Leu Arg Pro Ser Cys Ala 465 470
475 480 Asp Ser Pro Cys Arg Asn Arg Ala Thr Cys
Gln Asp Ser Pro Gln Gly 485 490
495 Pro Arg Cys Leu Cys Pro Thr Gly Tyr Thr Gly Gly Ser Cys Gln
Thr 500 505 510 Leu
Met Asp Leu Cys Ala Gln Lys Pro Cys Pro Arg Asn Ser His Cys 515
520 525 Leu Gln Thr Gly Pro Ser
Phe His Cys Leu Cys Leu Gln Gly Trp Thr 530 535
540 Gly Pro Leu Cys Asn Leu Pro Leu Ser Ser Cys
Gln Lys Ala Ala Leu 545 550 555
560 Ser Gln Gly Ile Asp Val Ser Ser Leu Cys His Asn Gly Gly Leu Cys
565 570 575 Val Asp
Ser Gly Pro Ser Tyr Phe Cys His Cys Pro Pro Gly Phe Gln 580
585 590 Gly Ser Leu Cys Gln Asp His
Val Asn Pro Cys Glu Ser Arg Pro Cys 595 600
605 Gln Asn Gly Ala Thr Cys Met Ala Gln Pro Ser Gly
Tyr Leu Cys Gln 610 615 620
Cys Ala Pro Gly Tyr Asp Gly Gln Asn Cys Ser Lys Glu Leu Asp Ala 625
630 635 640 Cys Gln Ser
Gln Pro Cys His Asn His Gly Thr Cys Thr Pro Lys Pro 645
650 655 Gly Gly Phe His Cys Ala Cys Pro
Pro Gly Phe Val Gly Leu Arg Cys 660 665
670 Glu Gly Asp Val Asp Glu Cys Leu Asp Gln Pro Cys His
Pro Thr Gly 675 680 685
Thr Ala Ala Cys His Ser Leu Ala Asn Ala Phe Tyr Cys Gln Cys Leu 690
695 700 Pro Gly His Thr
Gly Gln Trp Cys Glu Val Glu Ile Asp Pro Cys His 705 710
715 720 Ser Gln Pro Cys Phe His Gly Gly Thr
Cys Glu Ala Thr Ala Gly Ser 725 730
735 Pro Leu Gly Phe Ile Cys His Cys Pro Lys Gly Phe Glu Gly
Pro Thr 740 745 750
Cys Ser His Arg Ala Pro Ser Cys Gly Phe His His Cys His His Gly
755 760 765 Gly Leu Cys Leu
Pro Ser Pro Lys Pro Gly Phe Pro Pro Arg Cys Ala 770
775 780 Cys Leu Ser Gly Tyr Gly Gly Pro
Asp Cys Leu Thr Pro Pro Ala Pro 785 790
795 800 Lys Gly Cys Gly Pro Pro Ser Pro Cys Leu Tyr Asn
Gly Ser Cys Ser 805 810
815 Glu Thr Thr Gly Leu Gly Gly Pro Gly Phe Arg Cys Ser Cys Pro His
820 825 830 Ser Ser Pro
Gly Pro Arg Cys Gln Lys Pro Gly Asp Leu Gly Pro Gly 835
840 845 Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala 850 855
860 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro 865 870 875
880 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
885 890 895 Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 900
905 910 Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln 915 920
925 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln 930 935 940
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 945
950 955 960 Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 965
970 975 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr 980 985
990 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser 995 1000 1005
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 1010
1015 1020 Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 1025 1030
1035 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly 1040 1045 1050
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
1055 1060 1065 Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 1070 1075
1080 841075PRTHomo sapiens 84Met Trp Gly Trp Lys Cys Leu
Leu Phe Trp Ala Val Leu Val Thr Ala 1 5
10 15 Thr Leu Cys Thr Ala Arg Cys Ile Ala Ala Thr
Cys Ala Pro Gly Ser 20 25
30 Thr Cys Ile Asp Arg Val Gly Ser Phe Ser Cys Leu Cys Pro Pro
Gly 35 40 45 Arg
Thr Gly Leu Leu Cys His Leu Glu Asp Met Cys Leu Ser Gln Pro 50
55 60 Cys His Gly Asp Ala Gln
Cys Ser Thr Asn Pro Leu Thr Gly Ser Thr 65 70
75 80 Leu Cys Leu Cys Gln Pro Gly Tyr Ser Gly Pro
Thr Cys His Gln Asp 85 90
95 Leu Asp Glu Cys Leu Met Ala Gln Gln Gly Pro Ser Pro Cys Glu His
100 105 110 Gly Gly
Ser Cys Leu Asn Thr Pro Gly Ser Phe Asn Cys Leu Cys Pro 115
120 125 Pro Gly Tyr Thr Gly Ser Arg
Cys Glu Ala Asp His Asn Glu Cys Leu 130 135
140 Ser Gln Pro Cys His Pro Gly Ser Thr Cys Leu Asp
Leu Leu Ala Thr 145 150 155
160 Phe His Cys Leu Cys Pro Pro Gly Leu Glu Gly Gln Leu Cys Glu Val
165 170 175 Glu Thr Asn
Glu Cys Ala Ser Ala Pro Cys Leu Asn His Ala Asp Cys 180
185 190 His Asp Leu Leu Asn Gly Phe Gln
Cys Ile Cys Leu Pro Gly Phe Ser 195 200
205 Gly Thr Arg Cys Glu Glu Asp Ile Asp Glu Cys Arg Ser
Ser Pro Cys 210 215 220
Ala Asn Gly Gly Gln Cys Gln Asp Gln Pro Gly Ala Phe His Cys Lys 225
230 235 240 Cys Leu Pro Gly
Phe Glu Gly Pro Arg Cys Gln Thr Glu Val Asp Glu 245
250 255 Cys Leu Ser Asp Pro Cys Pro Val Gly
Ala Ser Cys Leu Asp Leu Pro 260 265
270 Gly Ala Phe Phe Cys Leu Cys Pro Ser Gly Phe Thr Gly Gln
Leu Cys 275 280 285
Glu Val Pro Leu Cys Ala Pro Asn Leu Cys Gln Pro Lys Gln Ile Cys 290
295 300 Lys Asp Gln Lys Asp
Lys Ala Asn Cys Leu Cys Pro Asp Gly Ser Pro 305 310
315 320 Gly Cys Ala Pro Pro Glu Asp Asn Cys Thr
Cys His His Gly His Cys 325 330
335 Gln Arg Ser Ser Cys Val Cys Asp Val Gly Trp Thr Gly Pro Glu
Cys 340 345 350 Glu
Ala Glu Leu Gly Gly Cys Ile Ser Ala Pro Cys Ala His Gly Gly 355
360 365 Thr Cys Tyr Pro Gln Pro
Ser Gly Tyr Asn Cys Thr Cys Pro Thr Gly 370 375
380 Tyr Thr Gly Pro Thr Cys Ser Glu Glu Met Thr
Ala Cys His Ser Gly 385 390 395
400 Pro Cys Leu Asn Gly Gly Ser Cys Asn Pro Ser Pro Gly Gly Tyr Tyr
405 410 415 Cys Thr
Cys Pro Pro Ser His Thr Gly Pro Gln Cys Gln Thr Ser Thr 420
425 430 Asp Tyr Cys Val Ser Ala Pro
Cys Phe Asn Gly Gly Thr Cys Val Asn 435 440
445 Arg Pro Gly Thr Phe Ser Cys Leu Cys Ala Met Gly
Phe Gln Gly Pro 450 455 460
Arg Cys Glu Gly Lys Leu Arg Pro Ser Cys Ala Asp Ser Pro Cys Arg 465
470 475 480 Asn Arg Ala
Thr Cys Gln Asp Ser Pro Gln Gly Pro Arg Cys Leu Cys 485
490 495 Pro Thr Gly Tyr Thr Gly Gly Ser
Cys Gln Thr Leu Met Asp Leu Cys 500 505
510 Ala Gln Lys Pro Cys Pro Arg Asn Ser His Cys Leu Gln
Thr Gly Pro 515 520 525
Ser Phe His Cys Leu Cys Leu Gln Gly Trp Thr Gly Pro Leu Cys Asn 530
535 540 Leu Pro Leu Ser
Ser Cys Gln Lys Ala Ala Leu Ser Gln Gly Ile Asp 545 550
555 560 Val Ser Ser Leu Cys His Asn Gly Gly
Leu Cys Val Asp Ser Gly Pro 565 570
575 Ser Tyr Phe Cys His Cys Pro Pro Gly Phe Gln Gly Ser Leu
Cys Gln 580 585 590
Asp His Val Asn Pro Cys Glu Ser Arg Pro Cys Gln Asn Gly Ala Thr
595 600 605 Cys Met Ala Gln
Pro Ser Gly Tyr Leu Cys Gln Cys Ala Pro Gly Tyr 610
615 620 Asp Gly Gln Asn Cys Ser Lys Glu
Leu Asp Ala Cys Gln Ser Gln Pro 625 630
635 640 Cys His Asn His Gly Thr Cys Thr Pro Lys Pro Gly
Gly Phe His Cys 645 650
655 Ala Cys Pro Pro Gly Phe Val Gly Leu Arg Cys Glu Gly Asp Val Asp
660 665 670 Glu Cys Leu
Asp Gln Pro Cys His Pro Thr Gly Thr Ala Ala Cys His 675
680 685 Ser Leu Ala Asn Ala Phe Tyr Cys
Gln Cys Leu Pro Gly His Thr Gly 690 695
700 Gln Trp Cys Glu Val Glu Ile Asp Pro Cys His Ser Gln
Pro Cys Phe 705 710 715
720 His Gly Gly Thr Cys Glu Ala Thr Ala Gly Ser Pro Leu Gly Phe Ile
725 730 735 Cys His Cys Pro
Lys Gly Phe Glu Gly Pro Thr Cys Ser His Arg Ala 740
745 750 Pro Ser Cys Gly Phe His His Cys His
His Gly Gly Leu Cys Leu Pro 755 760
765 Ser Pro Lys Pro Gly Phe Pro Pro Arg Cys Ala Cys Leu Ser
Gly Tyr 770 775 780
Gly Gly Pro Asp Cys Leu Thr Pro Pro Ala Pro Lys Gly Cys Gly Pro 785
790 795 800 Pro Ser Pro Cys Leu
Tyr Asn Gly Ser Cys Ser Glu Thr Thr Gly Leu 805
810 815 Gly Gly Pro Gly Phe Arg Cys Ser Cys Pro
His Ser Ser Pro Gly Pro 820 825
830 Arg Cys Gln Lys Pro Gly Asp Leu Gly Pro Gly Glu Pro Lys Ser
Cys 835 840 845 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 850
855 860 Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 865 870
875 880 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His 885 890
895 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
900 905 910 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 915
920 925 Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly 930 935
940 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 945 950 955
960 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
965 970 975 Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 980
985 990 Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu 995 1000
1005 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro 1010 1015 1020
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
1025 1030 1035 Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 1040
1045 1050 Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser 1055 1060
1065 Leu Ser Leu Ser Pro Gly Lys 1070
1075 85681PRTHomo sapiens 85Met Gln Pro Pro Ser Leu Leu Leu Leu Leu Leu
Leu Leu Leu Leu Leu 1 5 10
15 Cys Val Ser Val Val Arg Pro Arg Gly Leu Leu Cys Ala Ser Ala Pro
20 25 30 Cys Leu
Asn His Ala Asp Cys His Asp Leu Leu Asn Gly Phe Gln Cys 35
40 45 Ile Cys Leu Pro Gly Phe Ser
Gly Thr Arg Cys Glu Glu Asp Ile Asp 50 55
60 Glu Cys Arg Ser Ser Pro Cys Ala Asn Gly Gly Gln
Cys Gln Asp Gln 65 70 75
80 Pro Gly Ala Phe His Cys Lys Cys Leu Pro Gly Phe Glu Gly Pro Arg
85 90 95 Cys Gln Thr
Glu Val Asp Glu Cys Leu Ser Asp Pro Cys Pro Val Gly 100
105 110 Ala Ser Cys Leu Asp Leu Pro Gly
Ala Phe Phe Cys Leu Cys Pro Ser 115 120
125 Gly Phe Thr Gly Gln Leu Cys Glu Val Pro Leu Cys Ala
Pro Asn Leu 130 135 140
Cys Gln Pro Lys Gln Ile Cys Lys Asp Gln Lys Asp Lys Ala Asn Cys 145
150 155 160 Leu Cys Pro Asp
Gly Ser Pro Gly Cys Ala Pro Pro Glu Asp Asn Cys 165
170 175 Thr Cys His His Gly His Cys Gln Arg
Ser Ser Cys Val Cys Asp Val 180 185
190 Gly Trp Thr Gly Pro Glu Cys Glu Ala Glu Leu Gly Gly Cys
Ile Ser 195 200 205
Ala Pro Cys Ala His Gly Gly Thr Cys Tyr Pro Gln Pro Ser Gly Tyr 210
215 220 Asn Cys Thr Cys Pro
Thr Gly Tyr Thr Gly Pro Thr Cys Ser Glu Glu 225 230
235 240 Met Thr Ala Cys His Ser Gly Pro Cys Leu
Asn Gly Gly Ser Cys Asn 245 250
255 Pro Ser Pro Gly Gly Tyr Tyr Cys Thr Cys Pro Pro Ser His Thr
Gly 260 265 270 Pro
Gln Cys Gln Thr Ser Thr Asp Tyr Cys Val Ser Ala Pro Cys Phe 275
280 285 Asn Gly Gly Thr Cys Val
Asn Arg Pro Gly Thr Phe Ser Cys Leu Cys 290 295
300 Ala Met Gly Phe Gln Gly Pro Arg Cys Glu Gly
Lys Leu Arg Pro Ser 305 310 315
320 Cys Ala Asp Ser Pro Cys Arg Asn Arg Ala Thr Cys Gln Asp Ser Pro
325 330 335 Gln Gly
Pro Arg Cys Leu Cys Pro Thr Gly Tyr Thr Gly Gly Ser Cys 340
345 350 Gln Thr Leu Met Asp Leu Cys
Ala Gln Lys Pro Cys Pro Arg Asn Ser 355 360
365 His Cys Leu Gln Thr Gly Pro Ser Phe His Cys Leu
Cys Leu Gln Gly 370 375 380
Trp Thr Gly Pro Leu Cys Asn Leu Pro Leu Ser Ser Cys Gln Lys Ala 385
390 395 400 Ala Leu Ser
Gln Gly Ile Asp Val Ser Ser Leu Cys His Asn Gly Gly 405
410 415 Leu Cys Val Asp Ser Gly Pro Ser
Tyr Phe Cys His Cys Pro Pro Gly 420 425
430 Phe Gln Gly Ser Leu Cys Gln Asp His Val Asn Pro Asp
Leu Gly Pro 435 440 445
Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 450
455 460 Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 465 470
475 480 Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val 485 490
495 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr 500 505 510
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
515 520 525 Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 530
535 540 Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys 545 550
555 560 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln 565 570
575 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
580 585 590 Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 595
600 605 Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn 610 615
620 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu 625 630 635
640 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
645 650 655 Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 660
665 670 Lys Ser Leu Ser Leu Ser Pro Gly Lys
675 680 86676PRTHomo sapiens 86Met Trp Gly
Trp Lys Cys Leu Leu Phe Trp Ala Val Leu Val Thr Ala 1 5
10 15 Thr Leu Cys Thr Ala Arg Cys Ala
Ser Ala Pro Cys Leu Asn His Ala 20 25
30 Asp Cys His Asp Leu Leu Asn Gly Phe Gln Cys Ile Cys
Leu Pro Gly 35 40 45
Phe Ser Gly Thr Arg Cys Glu Glu Asp Ile Asp Glu Cys Arg Ser Ser 50
55 60 Pro Cys Ala Asn
Gly Gly Gln Cys Gln Asp Gln Pro Gly Ala Phe His 65 70
75 80 Cys Lys Cys Leu Pro Gly Phe Glu Gly
Pro Arg Cys Gln Thr Glu Val 85 90
95 Asp Glu Cys Leu Ser Asp Pro Cys Pro Val Gly Ala Ser Cys
Leu Asp 100 105 110
Leu Pro Gly Ala Phe Phe Cys Leu Cys Pro Ser Gly Phe Thr Gly Gln
115 120 125 Leu Cys Glu Val
Pro Leu Cys Ala Pro Asn Leu Cys Gln Pro Lys Gln 130
135 140 Ile Cys Lys Asp Gln Lys Asp Lys
Ala Asn Cys Leu Cys Pro Asp Gly 145 150
155 160 Ser Pro Gly Cys Ala Pro Pro Glu Asp Asn Cys Thr
Cys His His Gly 165 170
175 His Cys Gln Arg Ser Ser Cys Val Cys Asp Val Gly Trp Thr Gly Pro
180 185 190 Glu Cys Glu
Ala Glu Leu Gly Gly Cys Ile Ser Ala Pro Cys Ala His 195
200 205 Gly Gly Thr Cys Tyr Pro Gln Pro
Ser Gly Tyr Asn Cys Thr Cys Pro 210 215
220 Thr Gly Tyr Thr Gly Pro Thr Cys Ser Glu Glu Met Thr
Ala Cys His 225 230 235
240 Ser Gly Pro Cys Leu Asn Gly Gly Ser Cys Asn Pro Ser Pro Gly Gly
245 250 255 Tyr Tyr Cys Thr
Cys Pro Pro Ser His Thr Gly Pro Gln Cys Gln Thr 260
265 270 Ser Thr Asp Tyr Cys Val Ser Ala Pro
Cys Phe Asn Gly Gly Thr Cys 275 280
285 Val Asn Arg Pro Gly Thr Phe Ser Cys Leu Cys Ala Met Gly
Phe Gln 290 295 300
Gly Pro Arg Cys Glu Gly Lys Leu Arg Pro Ser Cys Ala Asp Ser Pro 305
310 315 320 Cys Arg Asn Arg Ala
Thr Cys Gln Asp Ser Pro Gln Gly Pro Arg Cys 325
330 335 Leu Cys Pro Thr Gly Tyr Thr Gly Gly Ser
Cys Gln Thr Leu Met Asp 340 345
350 Leu Cys Ala Gln Lys Pro Cys Pro Arg Asn Ser His Cys Leu Gln
Thr 355 360 365 Gly
Pro Ser Phe His Cys Leu Cys Leu Gln Gly Trp Thr Gly Pro Leu 370
375 380 Cys Asn Leu Pro Leu Ser
Ser Cys Gln Lys Ala Ala Leu Ser Gln Gly 385 390
395 400 Ile Asp Val Ser Ser Leu Cys His Asn Gly Gly
Leu Cys Val Asp Ser 405 410
415 Gly Pro Ser Tyr Phe Cys His Cys Pro Pro Gly Phe Gln Gly Ser Leu
420 425 430 Cys Gln
Asp His Val Asn Pro Asp Leu Gly Pro Gly Glu Pro Lys Ser 435
440 445 Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu 450 455
460 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu 465 470 475
480 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
485 490 495 His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 500
505 510 Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr 515 520
525 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn 530 535 540
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 545
550 555 560 Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 565
570 575 Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val 580 585
590 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val 595 600 605
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 610
615 620 Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 625 630
635 640 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val 645 650
655 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu 660 665 670 Ser
Pro Gly Lys 675 87629PRTHomo sapiens 87Met Gln Pro Pro Ser
Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu 1 5
10 15 Cys Val Ser Val Val Arg Pro Arg Gly Leu
Leu Cys Ala Asp Ser Pro 20 25
30 Cys Arg Asn Arg Ala Thr Cys Gln Asp Ser Pro Gln Gly Pro Arg
Cys 35 40 45 Leu
Cys Pro Thr Gly Tyr Thr Gly Gly Ser Cys Gln Thr Leu Met Asp 50
55 60 Leu Cys Ala Gln Lys Pro
Cys Pro Arg Asn Ser His Cys Leu Gln Thr 65 70
75 80 Gly Pro Ser Phe His Cys Leu Cys Leu Gln Gly
Trp Thr Gly Pro Leu 85 90
95 Cys Asn Leu Pro Leu Ser Ser Cys Gln Lys Ala Ala Leu Ser Gln Gly
100 105 110 Ile Asp
Val Ser Ser Leu Cys His Asn Gly Gly Leu Cys Val Asp Ser 115
120 125 Gly Pro Ser Tyr Phe Cys His
Cys Pro Pro Gly Phe Gln Gly Ser Leu 130 135
140 Cys Gln Asp His Val Asn Pro Cys Glu Ser Arg Pro
Cys Gln Asn Gly 145 150 155
160 Ala Thr Cys Met Ala Gln Pro Ser Gly Tyr Leu Cys Gln Cys Ala Pro
165 170 175 Gly Tyr Asp
Gly Gln Asn Cys Ser Lys Glu Leu Asp Ala Cys Gln Ser 180
185 190 Gln Pro Cys His Asn His Gly Thr
Cys Thr Pro Lys Pro Gly Gly Phe 195 200
205 His Cys Ala Cys Pro Pro Gly Phe Val Gly Leu Arg Cys
Glu Gly Asp 210 215 220
Val Asp Glu Cys Leu Asp Gln Pro Cys His Pro Thr Gly Thr Ala Ala 225
230 235 240 Cys His Ser Leu
Ala Asn Ala Phe Tyr Cys Gln Cys Leu Pro Gly His 245
250 255 Thr Gly Gln Trp Cys Glu Val Glu Ile
Asp Pro Cys His Ser Gln Pro 260 265
270 Cys Phe His Gly Gly Thr Cys Glu Ala Thr Ala Gly Ser Pro
Leu Gly 275 280 285
Phe Ile Cys His Cys Pro Lys Gly Phe Glu Gly Pro Thr Cys Ser His 290
295 300 Arg Ala Pro Ser Cys
Gly Phe His His Cys His His Gly Gly Leu Cys 305 310
315 320 Leu Pro Ser Pro Lys Pro Gly Phe Pro Pro
Arg Cys Ala Cys Leu Ser 325 330
335 Gly Tyr Gly Gly Pro Asp Cys Leu Thr Pro Pro Ala Pro Lys Gly
Cys 340 345 350 Gly
Pro Pro Ser Pro Cys Leu Tyr Asn Gly Ser Cys Ser Glu Thr Thr 355
360 365 Gly Leu Gly Gly Pro Gly
Phe Arg Cys Ser Cys Pro His Ser Ser Pro 370 375
380 Gly Pro Arg Cys Gln Lys Pro Gly Asp Leu Gly
Pro Gly Glu Pro Lys 385 390 395
400 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
405 410 415 Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 420
425 430 Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val 435 440
445 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val 450 455 460
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 465
470 475 480 Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 485
490 495 Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala 500 505
510 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro 515 520 525
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 530
535 540 Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 545 550
555 560 Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr 565 570
575 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu 580 585 590
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
595 600 605 Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 610
615 620 Leu Ser Pro Gly Lys 625
88624PRTHomo sapiens 88Met Trp Gly Trp Lys Cys Leu Leu Phe Trp Ala
Val Leu Val Thr Ala 1 5 10
15 Thr Leu Cys Thr Ala Arg Cys Ala Asp Ser Pro Cys Arg Asn Arg Ala
20 25 30 Thr Cys
Gln Asp Ser Pro Gln Gly Pro Arg Cys Leu Cys Pro Thr Gly 35
40 45 Tyr Thr Gly Gly Ser Cys Gln
Thr Leu Met Asp Leu Cys Ala Gln Lys 50 55
60 Pro Cys Pro Arg Asn Ser His Cys Leu Gln Thr Gly
Pro Ser Phe His 65 70 75
80 Cys Leu Cys Leu Gln Gly Trp Thr Gly Pro Leu Cys Asn Leu Pro Leu
85 90 95 Ser Ser Cys
Gln Lys Ala Ala Leu Ser Gln Gly Ile Asp Val Ser Ser 100
105 110 Leu Cys His Asn Gly Gly Leu Cys
Val Asp Ser Gly Pro Ser Tyr Phe 115 120
125 Cys His Cys Pro Pro Gly Phe Gln Gly Ser Leu Cys Gln
Asp His Val 130 135 140
Asn Pro Cys Glu Ser Arg Pro Cys Gln Asn Gly Ala Thr Cys Met Ala 145
150 155 160 Gln Pro Ser Gly
Tyr Leu Cys Gln Cys Ala Pro Gly Tyr Asp Gly Gln 165
170 175 Asn Cys Ser Lys Glu Leu Asp Ala Cys
Gln Ser Gln Pro Cys His Asn 180 185
190 His Gly Thr Cys Thr Pro Lys Pro Gly Gly Phe His Cys Ala
Cys Pro 195 200 205
Pro Gly Phe Val Gly Leu Arg Cys Glu Gly Asp Val Asp Glu Cys Leu 210
215 220 Asp Gln Pro Cys His
Pro Thr Gly Thr Ala Ala Cys His Ser Leu Ala 225 230
235 240 Asn Ala Phe Tyr Cys Gln Cys Leu Pro Gly
His Thr Gly Gln Trp Cys 245 250
255 Glu Val Glu Ile Asp Pro Cys His Ser Gln Pro Cys Phe His Gly
Gly 260 265 270 Thr
Cys Glu Ala Thr Ala Gly Ser Pro Leu Gly Phe Ile Cys His Cys 275
280 285 Pro Lys Gly Phe Glu Gly
Pro Thr Cys Ser His Arg Ala Pro Ser Cys 290 295
300 Gly Phe His His Cys His His Gly Gly Leu Cys
Leu Pro Ser Pro Lys 305 310 315
320 Pro Gly Phe Pro Pro Arg Cys Ala Cys Leu Ser Gly Tyr Gly Gly Pro
325 330 335 Asp Cys
Leu Thr Pro Pro Ala Pro Lys Gly Cys Gly Pro Pro Ser Pro 340
345 350 Cys Leu Tyr Asn Gly Ser Cys
Ser Glu Thr Thr Gly Leu Gly Gly Pro 355 360
365 Gly Phe Arg Cys Ser Cys Pro His Ser Ser Pro Gly
Pro Arg Cys Gln 370 375 380
Lys Pro Gly Asp Leu Gly Pro Gly Glu Pro Lys Ser Cys Asp Lys Thr 385
390 395 400 His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 405
410 415 Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg 420 425
430 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro 435 440 445
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 450
455 460 Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 465 470
475 480 Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr 485 490
495 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr 500 505 510
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
515 520 525 Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 530
535 540 Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser 545 550
555 560 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp 565 570
575 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
580 585 590 Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 595
600 605 Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 610 615
620 896012DNAHomo sapiens 89atgcagcccc cttcactgct
gctgctgctg ctgctgctgc tgctgctatg tgtctcagtg 60gtcagaccca gagggctgct
gtgtgggagt ttcccagaac cctgtgccaa tggaggcacc 120tgcctgagcc tgtctctggg
acaagggacc tgccagtgtg cccctggctt cctgggtgag 180acgtgccagt ttcctgaccc
ctgccagaac gcccagctct gccaaaatgg aggcagctgc 240caagccctgc ttcccgctcc
cctagggctc cccagctctc cctctccatt gacacccagc 300ttcttgtgca cttgcctccc
tggcttcact ggtgagagat gccaggccaa gcttgaagac 360ccttgtcctc cctccttctg
ttccaaaagg ggccgctgcc acatccaggc ctcgggccgc 420ccacagtgct cctgcatgcc
tggatggaca ggtgagcagt gccagcttcg ggacttctgt 480tcagccaacc catgtgttaa
tggaggggtg tgtctggcca cataccccca gatccagtgc 540cactgcccac cgggcttcga
gggccatgcc tgtgaacgtg atgtcaacga gtgcttccag 600gacccaggac cctgccccaa
aggcacctcc tgccataaca ccctgggctc cttccagtgc 660ctctgccctg tggggcagga
gggtccacgt tgtgagctgc gggcaggacc ctgccctcct 720aggggctgtt cgaatggggg
cacctgccag ctgatgccag agaaagactc cacctttcac 780ctctgcctct gtcccccagg
tttcataggc ccagactgtg aggtgaatcc agacaactgt 840gtcagccacc agtgtcagaa
tgggggcact tgccaggatg ggctggacac ctacacctgc 900ctctgcccag aaacctggac
aggctgggac tgctccgaag atgtggatga gtgtgagacc 960cagggtcccc ctcactgcag
aaacgggggc acctgccaga actctgctgg tagctttcac 1020tgcgtgtgtg tgagtggctg
gggcggcaca agctgtgagg agaacctgga tgactgtatt 1080gctgccacct gtgccccggg
atccacctgc attgaccggg tgggctcttt ctcctgcctc 1140tgcccacctg gacgcacagg
actcctgtgc cacttggaag acatgtgtct gagccagccg 1200tgccatgggg atgcccaatg
cagcaccaac cccctcacag gctccacact ctgcctgtgt 1260cagcctggct attcggggcc
cacctgccac caggacctgg acgagtgtct gatggcccag 1320caaggcccaa gtccctgtga
acatggcggt tcctgcctca acactcctgg ctccttcaac 1380tgcctctgtc cacctggcta
cacaggctcc cgttgtgagg ctgatcacaa tgagtgcctc 1440tcccagccct gccacccagg
aagcacctgt ctggacctac ttgccacctt ccactgcctc 1500tgcccgccag gcttagaagg
gcagctctgt gaggtggaga ccaacgagtg tgcctcagct 1560ccctgcctga accacgcgga
ttgccatgac ctgctcaacg gcttccagtg catctgcctg 1620cctggattct ccggcacccg
atgtgaggag gatatcgatg agtgcagaag ctctccctgt 1680gccaatggtg ggcagtgcca
ggaccagcct ggagccttcc actgcaagtg tctcccaggc 1740tttgaagggc cacgctgtca
aacagaggtg gatgagtgcc tgagtgaccc atgtcccgtt 1800ggagccagct gccttgatct
tccaggagcc ttcttttgcc tctgcccctc tggtttcaca 1860ggccagctct gtgaggttcc
cctgtgtgct cccaacctgt gccagcccaa gcagatatgt 1920aaggaccaga aagacaaggc
caactgcctc tgtcctgatg gaagccctgg ctgtgcccca 1980cctgaggaca actgcacctg
ccaccacggg cactgccaga gatcctcatg tgtgtgtgac 2040gtgggttgga cggggccaga
gtgtgaggca gagctagggg gctgcatctc tgcaccctgt 2100gcccatgggg ggacctgcta
cccccagccc tctggctaca actgcacctg ccctacaggc 2160tacacaggac ccacctgtag
tgaggagatg acagcttgtc actcagggcc atgtctcaat 2220ggcggctcct gcaaccctag
ccctggaggc tactactgca cctgccctcc aagccacaca 2280gggccccagt gccaaaccag
cactgactac tgtgtgtctg ccccgtgctt caatgggggt 2340acctgtgtga acaggcctgg
caccttctcc tgcctctgtg ccatgggctt ccagggcccg 2400cgctgtgagg gaaagctccg
ccccagctgt gcagacagcc cctgtaggaa tagggcaacc 2460tgccaggaca gccctcaggg
tccccgctgc ctctgcccca ctggctacac cggaggcagc 2520tgccagactc tgatggactt
atgtgcccag aagccctgcc cacgcaattc ccactgcctc 2580cagactgggc cctccttcca
ctgcttgtgc ctccagggat ggaccgggcc tctctgcaac 2640cttccactgt cctcctgcca
gaaggctgca ctgagccaag gcatagacgt ctcttccctt 2700tgccacaatg gaggcctctg
tgtcgacagc ggcccctcct atttctgcca ctgcccccct 2760ggattccaag gcagcctgtg
ccaggatcac gtgaacccat gtgagtccag gccttgccag 2820aacggggcca cctgcatggc
ccagcccagt gggtatctct gccagtgtgc cccaggctac 2880gatggacaga actgctcaaa
ggaactcgat gcttgtcagt cccaaccctg tcacaaccat 2940ggaacctgta ctcccaaacc
tggaggattc cactgtgcct gccctccagg ctttgtgggg 3000ctacgctgtg agggagacgt
ggacgagtgt ctggaccagc cctgccaccc cacaggcact 3060gcagcctgcc actctctggc
caatgccttc tactgccagt gtctgcctgg acacacaggc 3120cagtggtgtg aggtggagat
agacccctgc cacagccaac cctgctttca tggagggacc 3180tgtgaggcca cagcaggatc
acccctgggt ttcatctgcc actgccccaa gggttttgaa 3240ggccccacct gcagccacag
ggccccttcc tgcggcttcc atcactgcca ccacggaggc 3300ctgtgtctgc cctcccctaa
gccaggcttc ccaccacgct gtgcctgcct cagtggctat 3360gggggtcctg actgcctgac
cccaccagct cctaaaggct gtggccctcc ctccccatgc 3420ctatacaatg gcagctgctc
agagaccacg ggcttggggg gcccaggctt tcgatgctcc 3480tgccctcaca gctctccagg
gccccggtgt cagaaacccg gagccaaggg gtgtgagggc 3540agaagtggag atggggcctg
cgatgctggc tgcagtggcc cgggaggaaa ctgggatgga 3600ggggactgct ctctgggagt
cccagacccc tggaagggct gcccctccca ctctcggtgc 3660tggcttctct tccgggacgg
gcagtgccac ccacagtgtg actctgaaga gtgtctgttt 3720gatggctacg actgtgagac
ccctccagcc tgcactccag cctatgacca gtactgccat 3780gatcacttcc acaacgggca
ctgtgagaaa ggctgcaaca ctgcagagtg tggctgggat 3840ggaggtgact gcaggcctga
agatggggac ccagagtggg ggccctccct ggccctgctg 3900gtggtactga gccccccagc
cctagaccag cagctgtttg ccctggcccg ggtgctgtcc 3960ctgactctga gggtaggact
ctgggtaagg aaggatcgtg atggcaggga catggtgtac 4020ccctatcctg gggcccgggc
tgaagaaaag ctaggaggaa ctcgggaccc cacctatcag 4080gagagagcag cccctcaaac
gcagcccctg ggcaaggaga ccgactccct cagtgctggg 4140tttgtggtgg tcatgggtgt
ggatttgtcc cgctgtggcc ctgaccaccc ggcatcccgc 4200tgtccctggg accctgggct
tctactccgc ttccttgctg cgatggctgc agtgggagcc 4260ctggagcccc tgctgcctgg
accactgctg gctgtccacc ctcatgcagg gaccgcaccc 4320cctgccaacc agcttccctg
gcctgtgctg tgctccccag tggccggggt gattctcctg 4380gccctagggg ctcttctcgt
cctccagctc atccggcgtc gacgccgaga gcatggagct 4440ctctggctgc cccctggttt
cactcgacgg cctcggactc agtcagctcc ccaccgacgc 4500cggcccccac taggcgagga
cagcattggt ctcaaggcac tgaagccaaa ggcagaagtt 4560gatgaggatg gagttgtgat
gtgctcaggc cctgaggagg gagaggaggt gggccaggct 4620gaagaaacag gcccaccctc
cacgtgccag ctctggtctc tgagtggtgg ctgtggggcg 4680ctccctcagg cagccatgct
aactcctccc caggaatctg agatggaagc ccctgacctg 4740gacacccgtg gacctgatgg
ggtgacaccc ctgatgtcag cagtttgctg tggggaagta 4800cagtccggga ccttccaagg
ggcatggttg ggatgtcctg agccctggga acctctgctg 4860gatggagggg cctgtcccca
ggctcacacc gtgggcactg gggagacccc cctgcacctg 4920gctgcccgat tctcccggcc
aaccgctgcc cgccgcctcc ttgaggctgg agccaacccc 4980aaccagccag accgggcagg
gcgcacaccc cttcatgctg ctgtggctgc tgatgctcgg 5040gaggtctgcc agcttctgct
ccgtagcaga caaactgcag tggacgctcg cacagaggac 5100gggaccacac ccttgatgct
ggctgccagg ctggcggtgg aagacctggt tgaagaactg 5160attgcagccc aagcagacgt
gggggccaga gataaatggg ggaaaactgc gctgcactgg 5220gctgctgccg tgaacaacgc
ccgagccgcc cgctcgcttc tccaggccgg agccgataaa 5280gatgcccagg acaacaggga
gcagacgccg ctattcctgg cggcgcggga aggagcggtg 5340gaagtagccc agctactgct
ggggctgggg gcagcccgag agctgcggga ccaggctggg 5400ctagcgccgg cggacgtcgc
tcaccaacgt aaccactggg atctgctgac gctgctggaa 5460ggggctgggc caccagaggc
ccgtcacaaa gccacgccgg gccgcgaggc tgggcccttc 5520ccgcgcgcac ggacggtgtc
agtaagcgtg cccccgcatg ggggcggggc tctgccgcgc 5580tgccggacgc tgtcagccgg
agcaggccct cgtgggggcg gagcttgtct gcaggctcgg 5640acttggtccg tagacttggc
tgcgcggggg ggcggggcct attctcattg ccggagcctc 5700tcgggagtag gagcaggagg
aggcccgacc cctcgcggcc gtaggttttc tgcaggcatg 5760cgcgggcctc ggcccaaccc
tgcgataatg cgaggaagat acggagtggc tgccgggcgc 5820ggaggcaggg tctcaacgga
tgactggccc tgtgattggg tggccctggg agcttgcggt 5880tctgcctcca acattccgat
cccgcctcct tgccttactc cgtccccgga gcggggatca 5940cctcaacttg actgtggtcc
cccagccctc caagaaatgc ccataaacca aggaggagag 6000ggtaaaaaat ag
60129081DNAHomo sapiens
90atgcagcccc cttcactgct gctgctgctg ctgctgctgc tgctgctatg tgtctcagtg
60gtcagaccca gagggctgct g
819166DNAHomo sapiens 91atgtggggct ggaagtgcct cctcttctgg gctgtgctgg
tcacagccac tctctgcact 60gccagg
6692714DNAHomo sapiens 92gatctgggcc cgggcgagcc
caaatcttgt gacaaaactc acacatgccc accgtgccca 60gcacctgaac tcctgggggg
accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 120ctcatgatct cccggacccc
tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 180cctgaggtca agttcaactg
gtacgtggac ggcgtggagg tgcataatgc caagacaaag 240ccgcgggagg agcagtacaa
cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 300caggactggc tgaatggcaa
ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 360cccatcgaga aaaccatctc
caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 420ctgcccccat cccgggatga
gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 480ggcttctatc ccagcgacat
cgccgtggag tgggagagca atgggcagcc ggagaacaac 540tacaagacca cgcctcccgt
gctggactcc gacggctcct tcttcctcta cagcaagctc 600accgtggaca agagcaggtg
gcagcagggg aacgtcttct catgctccgt gatgcatgag 660gctctgcaca accactacac
gcagaagagc ctctccctgt ctccgggtaa atga 714934236DNAHomo sapiens
93atgcagcccc cttcactgct gctgctgctg ctgctgctgc tgctgctatg tgtctcagtg
60gtcagaccca gagggctgct gtgtgggagt ttcccagaac cctgtgccaa tggaggcacc
120tgcctgagcc tgtctctggg acaagggacc tgccagtgtg cccctggctt cctgggtgag
180acgtgccagt ttcctgaccc ctgccagaac gcccagctct gccaaaatgg aggcagctgc
240caagccctgc ttcccgctcc cctagggctc cccagctctc cctctccatt gacacccagc
300ttcttgtgca cttgcctccc tggcttcact ggtgagagat gccaggccaa gcttgaagac
360ccttgtcctc cctccttctg ttccaaaagg ggccgctgcc acatccaggc ctcgggccgc
420ccacagtgct cctgcatgcc tggatggaca ggtgagcagt gccagcttcg ggacttctgt
480tcagccaacc catgtgttaa tggaggggtg tgtctggcca cataccccca gatccagtgc
540cactgcccac cgggcttcga gggccatgcc tgtgaacgtg atgtcaacga gtgcttccag
600gacccaggac cctgccccaa aggcacctcc tgccataaca ccctgggctc cttccagtgc
660ctctgccctg tggggcagga gggtccacgt tgtgagctgc gggcaggacc ctgccctcct
720aggggctgtt cgaatggggg cacctgccag ctgatgccag agaaagactc cacctttcac
780ctctgcctct gtcccccagg tttcataggc ccagactgtg aggtgaatcc agacaactgt
840gtcagccacc agtgtcagaa tgggggcact tgccaggatg ggctggacac ctacacctgc
900ctctgcccag aaacctggac aggctgggac tgctccgaag atgtggatga gtgtgagacc
960cagggtcccc ctcactgcag aaacgggggc acctgccaga actctgctgg tagctttcac
1020tgcgtgtgtg tgagtggctg gggcggcaca agctgtgagg agaacctgga tgactgtatt
1080gctgccacct gtgccccggg atccacctgc attgaccggg tgggctcttt ctcctgcctc
1140tgcccacctg gacgcacagg actcctgtgc cacttggaag acatgtgtct gagccagccg
1200tgccatgggg atgcccaatg cagcaccaac cccctcacag gctccacact ctgcctgtgt
1260cagcctggct attcggggcc cacctgccac caggacctgg acgagtgtct gatggcccag
1320caaggcccaa gtccctgtga acatggcggt tcctgcctca acactcctgg ctccttcaac
1380tgcctctgtc cacctggcta cacaggctcc cgttgtgagg ctgatcacaa tgagtgcctc
1440tcccagccct gccacccagg aagcacctgt ctggacctac ttgccacctt ccactgcctc
1500tgcccgccag gcttagaagg gcagctctgt gaggtggaga ccaacgagtg tgcctcagct
1560ccctgcctga accacgcgga ttgccatgac ctgctcaacg gcttccagtg catctgcctg
1620cctggattct ccggcacccg atgtgaggag gatatcgatg agtgcagaag ctctccctgt
1680gccaatggtg ggcagtgcca ggaccagcct ggagccttcc actgcaagtg tctcccaggc
1740tttgaagggc cacgctgtca aacagaggtg gatgagtgcc tgagtgaccc atgtcccgtt
1800ggagccagct gccttgatct tccaggagcc ttcttttgcc tctgcccctc tggtttcaca
1860ggccagctct gtgaggttcc cctgtgtgct cccaacctgt gccagcccaa gcagatatgt
1920aaggaccaga aagacaaggc caactgcctc tgtcctgatg gaagccctgg ctgtgcccca
1980cctgaggaca actgcacctg ccaccacggg cactgccaga gatcctcatg tgtgtgtgac
2040gtgggttgga cggggccaga gtgtgaggca gagctagggg gctgcatctc tgcaccctgt
2100gcccatgggg ggacctgcta cccccagccc tctggctaca actgcacctg ccctacaggc
2160tacacaggac ccacctgtag tgaggagatg acagcttgtc actcagggcc atgtctcaat
2220ggcggctcct gcaaccctag ccctggaggc tactactgca cctgccctcc aagccacaca
2280gggccccagt gccaaaccag cactgactac tgtgtgtctg ccccgtgctt caatgggggt
2340acctgtgtga acaggcctgg caccttctcc tgcctctgtg ccatgggctt ccagggcccg
2400cgctgtgagg gaaagctccg ccccagctgt gcagacagcc cctgtaggaa tagggcaacc
2460tgccaggaca gccctcaggg tccccgctgc ctctgcccca ctggctacac cggaggcagc
2520tgccagactc tgatggactt atgtgcccag aagccctgcc cacgcaattc ccactgcctc
2580cagactgggc cctccttcca ctgcttgtgc ctccagggat ggaccgggcc tctctgcaac
2640cttccactgt cctcctgcca gaaggctgca ctgagccaag gcatagacgt ctcttccctt
2700tgccacaatg gaggcctctg tgtcgacagc ggcccctcct atttctgcca ctgcccccct
2760ggattccaag gcagcctgtg ccaggatcac gtgaacccat gtgagtccag gccttgccag
2820aacggggcca cctgcatggc ccagcccagt gggtatctct gccagtgtgc cccaggctac
2880gatggacaga actgctcaaa ggaactcgat gcttgtcagt cccaaccctg tcacaaccat
2940ggaacctgta ctcccaaacc tggaggattc cactgtgcct gccctccagg ctttgtgggg
3000ctacgctgtg agggagacgt ggacgagtgt ctggaccagc cctgccaccc cacaggcact
3060gcagcctgcc actctctggc caatgccttc tactgccagt gtctgcctgg acacacaggc
3120cagtggtgtg aggtggagat agacccctgc cacagccaac cctgctttca tggagggacc
3180tgtgaggcca cagcaggatc acccctgggt ttcatctgcc actgccccaa gggttttgaa
3240ggccccacct gcagccacag ggccccttcc tgcggcttcc atcactgcca ccacggaggc
3300ctgtgtctgc cctcccctaa gccaggcttc ccaccacgct gtgcctgcct cagtggctat
3360gggggtcctg actgcctgac cccaccagct cctaaaggct gtggccctcc ctccccatgc
3420ctatacaatg gcagctgctc agagaccacg ggcttggggg gcccaggctt tcgatgctcc
3480tgccctcaca gctctccagg gccccggtgt cagaaacccg gagatctggg cccgggcgag
3540cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg
3600ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc
3660cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac
3720tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac
3780aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc
3840aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc
3900tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat
3960gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac
4020atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc
4080gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg
4140tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac
4200acgcagaaga gcctctccct gtctccgggt aaatga
4236942376DNAHomo sapiens 94atgcagcccc cttcactgct gctgctgctg ctgctgctgc
tgctgctatg tgtctcagtg 60gtcagaccca gagggctgct gtgtgggagt ttcccagaac
cctgtgccaa tggaggcacc 120tgcctgagcc tgtctctggg acaagggacc tgccagtgtg
cccctggctt cctgggtgag 180acgtgccagt ttcctgaccc ctgccagaac gcccagctct
gccaaaatgg aggcagctgc 240caagccctgc ttcccgctcc cctagggctc cccagctctc
cctctccatt gacacccagc 300ttcttgtgca cttgcctccc tggcttcact ggtgagagat
gccaggccaa gcttgaagac 360ccttgtcctc cctccttctg ttccaaaagg ggccgctgcc
acatccaggc ctcgggccgc 420ccacagtgct cctgcatgcc tggatggaca ggtgagcagt
gccagcttcg ggacttctgt 480tcagccaacc catgtgttaa tggaggggtg tgtctggcca
cataccccca gatccagtgc 540cactgcccac cgggcttcga gggccatgcc tgtgaacgtg
atgtcaacga gtgcttccag 600gacccaggac cctgccccaa aggcacctcc tgccataaca
ccctgggctc cttccagtgc 660ctctgccctg tggggcagga gggtccacgt tgtgagctgc
gggcaggacc ctgccctcct 720aggggctgtt cgaatggggg cacctgccag ctgatgccag
agaaagactc cacctttcac 780ctctgcctct gtcccccagg tttcataggc ccagactgtg
aggtgaatcc agacaactgt 840gtcagccacc agtgtcagaa tgggggcact tgccaggatg
ggctggacac ctacacctgc 900ctctgcccag aaacctggac aggctgggac tgctccgaag
atgtggatga gtgtgagacc 960cagggtcccc ctcactgcag aaacgggggc acctgccaga
actctgctgg tagctttcac 1020tgcgtgtgtg tgagtggctg gggcggcaca agctgtgagg
agaacctgga tgactgtatt 1080gctgccacct gtgccccggg atccacctgc attgaccggg
tgggctcttt ctcctgcctc 1140tgcccacctg gacgcacagg actcctgtgc cacttggaag
acatgtgtct gagccagccg 1200tgccatgggg atgcccaatg cagcaccaac cccctcacag
gctccacact ctgcctgtgt 1260cagcctggct attcggggcc cacctgccac caggacctgg
acgagtgtct gatggcccag 1320caaggcccaa gtccctgtga acatggcggt tcctgcctca
acactcctgg ctccttcaac 1380tgcctctgtc cacctggcta cacaggctcc cgttgtgagg
ctgatcacaa tgagtgcctc 1440tcccagccct gccacccagg aagcacctgt ctggacctac
ttgccacctt ccactgcctc 1500tgcccgccag gcttagaagg gcagctctgt gaggtggaga
ccaacgagtg tgcctcagct 1560ccctgcctga accacgcgga ttgccatgac ctgctcaacg
gcttccagtg catctgcctg 1620cctggattct ccggcacccg atgtgaggag gatatcgatg
aggatctggg cccgggcgag 1680cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc
cagcacctga actcctgggg 1740ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca
ccctcatgat ctcccggacc 1800cctgaggtca catgcgtggt ggtggacgtg agccacgaag
accctgaggt caagttcaac 1860tggtacgtgg acggcgtgga ggtgcataat gccaagacaa
agccgcggga ggagcagtac 1920aacagcacgt accgtgtggt cagcgtcctc accgtcctgc
accaggactg gctgaatggc 1980aaggagtaca agtgcaaggt ctccaacaaa gccctcccag
cccccatcga gaaaaccatc 2040tccaaagcca aagggcagcc ccgagaacca caggtgtaca
ccctgccccc atcccgggat 2100gagctgacca agaaccaggt cagcctgacc tgcctggtca
aaggcttcta tcccagcgac 2160atcgccgtgg agtgggagag caatgggcag ccggagaaca
actacaagac cacgcctccc 2220gtgctggact ccgacggctc cttcttcctc tacagcaagc
tcaccgtgga caagagcagg 2280tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg
aggctctgca caaccactac 2340acgcagaaga gcctctccct gtctccgggt aaatga
2376953513DNAHomo sapiens 95atgcagcccc cttcactgct
gctgctgctg ctgctgctgc tgctgctatg tgtctcagtg 60gtcagaccca gagggctgct
gtgtgggagt ttcccagaac cctgtgccaa tggaggcacc 120tgcctgagcc tgtctctggg
acaagggacc tgccagtgtg cccctggctt cctgggtgag 180acgtgccagt ttcctgaccc
ctgccagaac gcccagctct gccaaaatgg aggcagctgc 240caagccctgc ttcccgctcc
cctagggctc cccagctctc cctctccatt gacacccagc 300ttcttgtgca cttgcctccc
tggcttcact ggtgagagat gccaggccaa gcttgaagac 360ccttgtcctc cctccttctg
ttccaaaagg ggccgctgcc acatccaggc ctcgggccgc 420ccacagtgct cctgcatgcc
tggatggaca ggtgagcagt gccagcttcg ggacttctgt 480tcagccaacc catgtgttaa
tggaggggtg tgtctggcca cataccccca gatccagtgc 540cactgcccac cgggcttcga
gggccatgcc tgtgaacgtg atgtcaacga gtgcttccag 600gacccaggac cctgccccaa
aggcacctcc tgccataaca ccctgggctc cttccagtgc 660ctctgccctg tggggcagga
gggtccacgt tgtgagctgc gggcaggacc ctgccctcct 720aggggctgtt cgaatggggg
cacctgccag ctgatgccag agaaagactc cacctttcac 780ctctgcctct gtcccccagg
tttcataggc ccagactgtg aggtgaatcc agacaactgt 840gtcagccacc agtgtcagaa
tgggggcact tgccaggatg ggctggacac ctacacctgc 900ctctgcccag aaacctggac
aggctgggac tgctccgaag atgtggatga gtgtgagacc 960cagggtcccc ctcactgcag
aaacgggggc acctgccaga actctgctgg tagctttcac 1020tgcgtgtgtg tgagtggctg
gggcggcaca agctgtgagg agaacctgga tgactgtatt 1080gctgccacct gtgccccggg
atccacctgc attgaccggg tgggctcttt ctcctgcctc 1140tgcccacctg gacgcacagg
actcctgtgc cacttggaag acatgtgtct gagccagccg 1200tgccatgggg atgcccaatg
cagcaccaac cccctcacag gctccacact ctgcctgtgt 1260cagcctggct attcggggcc
cacctgccac caggacctgg acgagtgtct gatggcccag 1320caaggcccaa gtccctgtga
acatggcggt tcctgcctca acactcctgg ctccttcaac 1380tgcctctgtc cacctggcta
cacaggctcc cgttgtgagg ctgatcacaa tgagtgcctc 1440tcccagccct gccacccagg
aagcacctgt ctggacctac ttgccacctt ccactgcctc 1500tgcccgccag gcttagaagg
gcagctctgt gaggtggaga ccaacgagtg tgcctcagct 1560ccctgcctga accacgcgga
ttgccatgac ctgctcaacg gcttccagtg catctgcctg 1620cctggattct ccggcacccg
atgtgaggag gatatcgatg agtgcagaag ctctccctgt 1680gccaatggtg ggcagtgcca
ggaccagcct ggagccttcc actgcaagtg tctcccaggc 1740tttgaagggc cacgctgtca
aacagaggtg gatgagtgcc tgagtgaccc atgtcccgtt 1800ggagccagct gccttgatct
tccaggagcc ttcttttgcc tctgcccctc tggtttcaca 1860ggccagctct gtgaggttcc
cctgtgtgct cccaacctgt gccagcccaa gcagatatgt 1920aaggaccaga aagacaaggc
caactgcctc tgtcctgatg gaagccctgg ctgtgcccca 1980cctgaggaca actgcacctg
ccaccacggg cactgccaga gatcctcatg tgtgtgtgac 2040gtgggttgga cggggccaga
gtgtgaggca gagctagggg gctgcatctc tgcaccctgt 2100gcccatgggg ggacctgcta
cccccagccc tctggctaca actgcacctg ccctacaggc 2160tacacaggac ccacctgtag
tgaggagatg acagcttgtc actcagggcc atgtctcaat 2220ggcggctcct gcaaccctag
ccctggaggc tactactgca cctgccctcc aagccacaca 2280gggccccagt gccaaaccag
cactgactac tgtgtgtctg ccccgtgctt caatgggggt 2340acctgtgtga acaggcctgg
caccttctcc tgcctctgtg ccatgggctt ccagggcccg 2400cgctgtgagg gaaagctccg
ccccagctgt gcagacagcc cctgtaggaa tagggcaacc 2460tgccaggaca gccctcaggg
tccccgctgc ctctgcccca ctggctacac cggaggcagc 2520tgccagactc tgatggactt
atgtgcccag aagccctgcc cacgcaattc ccactgcctc 2580cagactgggc cctccttcca
ctgcttgtgc ctccagggat ggaccgggcc tctctgcaac 2640cttccactgt cctcctgcca
gaaggctgca ctgagccaag gcatagacgt ctcttccctt 2700tgccacaatg gaggcctctg
tgtcgacagc ggcccctcct atttctgcca ctgcccccct 2760ggattccaag gcagcctgtg
ccaggatcac gtgaacccag atctgggccc gggcgagccc 2820aaatcttgtg acaaaactca
cacatgccca ccgtgcccag cacctgaact cctgggggga 2880ccgtcagtct tcctcttccc
cccaaaaccc aaggacaccc tcatgatctc ccggacccct 2940gaggtcacat gcgtggtggt
ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 3000tacgtggacg gcgtggaggt
gcataatgcc aagacaaagc cgcgggagga gcagtacaac 3060agcacgtacc gtgtggtcag
cgtcctcacc gtcctgcacc aggactggct gaatggcaag 3120gagtacaagt gcaaggtctc
caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 3180aaagccaaag ggcagccccg
agaaccacag gtgtacaccc tgcccccatc ccgggatgag 3240ctgaccaaga accaggtcag
cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 3300gccgtggagt gggagagcaa
tgggcagccg gagaacaact acaagaccac gcctcccgtg 3360ctggactccg acggctcctt
cttcctctac agcaagctca ccgtggacaa gagcaggtgg 3420cagcagggga acgtcttctc
atgctccgtg atgcatgagg ctctgcacaa ccactacacg 3480cagaagagcc tctccctgtc
tccgggtaaa tga 3513963243DNAHomo sapiens
96atgcagcccc cttcactgct gctgctgctg ctgctgctgc tgctgctatg tgtctcagtg
60gtcagaccca gagggctgct gtgtattgct gccacctgtg ccccgggatc cacctgcatt
120gaccgggtgg gctctttctc ctgcctctgc ccacctggac gcacaggact cctgtgccac
180ttggaagaca tgtgtctgag ccagccgtgc catggggatg cccaatgcag caccaacccc
240ctcacaggct ccacactctg cctgtgtcag cctggctatt cggggcccac ctgccaccag
300gacctggacg agtgtctgat ggcccagcaa ggcccaagtc cctgtgaaca tggcggttcc
360tgcctcaaca ctcctggctc cttcaactgc ctctgtccac ctggctacac aggctcccgt
420tgtgaggctg atcacaatga gtgcctctcc cagccctgcc acccaggaag cacctgtctg
480gacctacttg ccaccttcca ctgcctctgc ccgccaggct tagaagggca gctctgtgag
540gtggagacca acgagtgtgc ctcagctccc tgcctgaacc acgcggattg ccatgacctg
600ctcaacggct tccagtgcat ctgcctgcct ggattctccg gcacccgatg tgaggaggat
660atcgatgagt gcagaagctc tccctgtgcc aatggtgggc agtgccagga ccagcctgga
720gccttccact gcaagtgtct cccaggcttt gaagggccac gctgtcaaac agaggtggat
780gagtgcctga gtgacccatg tcccgttgga gccagctgcc ttgatcttcc aggagccttc
840ttttgcctct gcccctctgg tttcacaggc cagctctgtg aggttcccct gtgtgctccc
900aacctgtgcc agcccaagca gatatgtaag gaccagaaag acaaggccaa ctgcctctgt
960cctgatggaa gccctggctg tgccccacct gaggacaact gcacctgcca ccacgggcac
1020tgccagagat cctcatgtgt gtgtgacgtg ggttggacgg ggccagagtg tgaggcagag
1080ctagggggct gcatctctgc accctgtgcc catgggggga cctgctaccc ccagccctct
1140ggctacaact gcacctgccc tacaggctac acaggaccca cctgtagtga ggagatgaca
1200gcttgtcact cagggccatg tctcaatggc ggctcctgca accctagccc tggaggctac
1260tactgcacct gccctccaag ccacacaggg ccccagtgcc aaaccagcac tgactactgt
1320gtgtctgccc cgtgcttcaa tgggggtacc tgtgtgaaca ggcctggcac cttctcctgc
1380ctctgtgcca tgggcttcca gggcccgcgc tgtgagggaa agctccgccc cagctgtgca
1440gacagcccct gtaggaatag ggcaacctgc caggacagcc ctcagggtcc ccgctgcctc
1500tgccccactg gctacaccgg aggcagctgc cagactctga tggacttatg tgcccagaag
1560ccctgcccac gcaattccca ctgcctccag actgggccct ccttccactg cttgtgcctc
1620cagggatgga ccgggcctct ctgcaacctt ccactgtcct cctgccagaa ggctgcactg
1680agccaaggca tagacgtctc ttccctttgc cacaatggag gcctctgtgt cgacagcggc
1740ccctcctatt tctgccactg cccccctgga ttccaaggca gcctgtgcca ggatcacgtg
1800aacccatgtg agtccaggcc ttgccagaac ggggccacct gcatggccca gcccagtggg
1860tatctctgcc agtgtgcccc aggctacgat ggacagaact gctcaaagga actcgatgct
1920tgtcagtccc aaccctgtca caaccatgga acctgtactc ccaaacctgg aggattccac
1980tgtgcctgcc ctccaggctt tgtggggcta cgctgtgagg gagacgtgga cgagtgtctg
2040gaccagccct gccaccccac aggcactgca gcctgccact ctctggccaa tgccttctac
2100tgccagtgtc tgcctggaca cacaggccag tggtgtgagg tggagataga cccctgccac
2160agccaaccct gctttcatgg agggacctgt gaggccacag caggatcacc cctgggtttc
2220atctgccact gccccaaggg ttttgaaggc cccacctgca gccacagggc cccttcctgc
2280ggcttccatc actgccacca cggaggcctg tgtctgccct cccctaagcc aggcttccca
2340ccacgctgtg cctgcctcag tggctatggg ggtcctgact gcctgacccc accagctcct
2400aaaggctgtg gccctccctc cccatgccta tacaatggca gctgctcaga gaccacgggc
2460ttggggggcc caggctttcg atgctcctgc cctcacagct ctccagggcc ccggtgtcag
2520aaacccggag atctgggccc gggcgagccc aaatcttgtg acaaaactca cacatgccca
2580ccgtgcccag cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc
2640aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc
2700cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc
2760aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc
2820gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc
2880ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag
2940gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc
3000ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg
3060gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac
3120agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg
3180ttgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa
3240tga
3243973228DNAHomo sapiens 97atgtggggct ggaagtgcct cctcttctgg gctgtgctgg
tcacagccac tctctgcact 60gccaggtgta ttgctgccac ctgtgccccg ggatccacct
gcattgaccg ggtgggctct 120ttctcctgcc tctgcccacc tggacgcaca ggactcctgt
gccacttgga agacatgtgt 180ctgagccagc cgtgccatgg ggatgcccaa tgcagcacca
accccctcac aggctccaca 240ctctgcctgt gtcagcctgg ctattcgggg cccacctgcc
accaggacct ggacgagtgt 300ctgatggccc agcaaggccc aagtccctgt gaacatggcg
gttcctgcct caacactcct 360ggctccttca actgcctctg tccacctggc tacacaggct
cccgttgtga ggctgatcac 420aatgagtgcc tctcccagcc ctgccaccca ggaagcacct
gtctggacct acttgccacc 480ttccactgcc tctgcccgcc aggcttagaa gggcagctct
gtgaggtgga gaccaacgag 540tgtgcctcag ctccctgcct gaaccacgcg gattgccatg
acctgctcaa cggcttccag 600tgcatctgcc tgcctggatt ctccggcacc cgatgtgagg
aggatatcga tgagtgcaga 660agctctccct gtgccaatgg tgggcagtgc caggaccagc
ctggagcctt ccactgcaag 720tgtctcccag gctttgaagg gccacgctgt caaacagagg
tggatgagtg cctgagtgac 780ccatgtcccg ttggagccag ctgccttgat cttccaggag
ccttcttttg cctctgcccc 840tctggtttca caggccagct ctgtgaggtt cccctgtgtg
ctcccaacct gtgccagccc 900aagcagatat gtaaggacca gaaagacaag gccaactgcc
tctgtcctga tggaagccct 960ggctgtgccc cacctgagga caactgcacc tgccaccacg
ggcactgcca gagatcctca 1020tgtgtgtgtg acgtgggttg gacggggcca gagtgtgagg
cagagctagg gggctgcatc 1080tctgcaccct gtgcccatgg ggggacctgc tacccccagc
cctctggcta caactgcacc 1140tgccctacag gctacacagg acccacctgt agtgaggaga
tgacagcttg tcactcaggg 1200ccatgtctca atggcggctc ctgcaaccct agccctggag
gctactactg cacctgccct 1260ccaagccaca cagggcccca gtgccaaacc agcactgact
actgtgtgtc tgccccgtgc 1320ttcaatgggg gtacctgtgt gaacaggcct ggcaccttct
cctgcctctg tgccatgggc 1380ttccagggcc cgcgctgtga gggaaagctc cgccccagct
gtgcagacag cccctgtagg 1440aatagggcaa cctgccagga cagccctcag ggtccccgct
gcctctgccc cactggctac 1500accggaggca gctgccagac tctgatggac ttatgtgccc
agaagccctg cccacgcaat 1560tcccactgcc tccagactgg gccctccttc cactgcttgt
gcctccaggg atggaccggg 1620cctctctgca accttccact gtcctcctgc cagaaggctg
cactgagcca aggcatagac 1680gtctcttccc tttgccacaa tggaggcctc tgtgtcgaca
gcggcccctc ctatttctgc 1740cactgccccc ctggattcca aggcagcctg tgccaggatc
acgtgaaccc atgtgagtcc 1800aggccttgcc agaacggggc cacctgcatg gcccagccca
gtgggtatct ctgccagtgt 1860gccccaggct acgatggaca gaactgctca aaggaactcg
atgcttgtca gtcccaaccc 1920tgtcacaacc atggaacctg tactcccaaa cctggaggat
tccactgtgc ctgccctcca 1980ggctttgtgg ggctacgctg tgagggagac gtggacgagt
gtctggacca gccctgccac 2040cccacaggca ctgcagcctg ccactctctg gccaatgcct
tctactgcca gtgtctgcct 2100ggacacacag gccagtggtg tgaggtggag atagacccct
gccacagcca accctgcttt 2160catggaggga cctgtgaggc cacagcagga tcacccctgg
gtttcatctg ccactgcccc 2220aagggttttg aaggccccac ctgcagccac agggcccctt
cctgcggctt ccatcactgc 2280caccacggag gcctgtgtct gccctcccct aagccaggct
tcccaccacg ctgtgcctgc 2340ctcagtggct atgggggtcc tgactgcctg accccaccag
ctcctaaagg ctgtggccct 2400ccctccccat gcctatacaa tggcagctgc tcagagacca
cgggcttggg gggcccaggc 2460tttcgatgct cctgccctca cagctctcca gggccccggt
gtcagaaacc cggagatctg 2520ggcccgggcg agcccaaatc ttgtgacaaa actcacacat
gcccaccgtg cccagcacct 2580gaactcctgg ggggaccgtc agtcttcctc ttccccccaa
aacccaagga caccctcatg 2640atctcccgga cccctgaggt cacatgcgtg gtggtggacg
tgagccacga agaccctgag 2700gtcaagttca actggtacgt ggacggcgtg gaggtgcata
atgccaagac aaagccgcgg 2760gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc
tcaccgtcct gcaccaggac 2820tggctgaatg gcaaggagta caagtgcaag gtctccaaca
aagccctccc agcccccatc 2880gagaaaacca tctccaaagc caaagggcag ccccgagaac
cacaggtgta caccctgccc 2940ccatcccggg atgagctgac caagaaccag gtcagcctga
cctgcctggt caaaggcttc 3000tatcccagcg acatcgccgt ggagtgggag agcaatgggc
agccggagaa caactacaag 3060accacgcctc ccgtgctgga ctccgacggc tccttcttcc
tctacagcaa gctcaccgtg 3120gacaagagca ggtggcagca ggggaacgtc ttctcatgct
ccgtgatgca tgaggctctg 3180cacaaccact acacgcagaa gagcctctcc ctgtctccgg
gtaaatga 3228982520DNAHomo sapiens 98atgcagcccc cttcactgct
gctgctgctg ctgctgctgc tgctgctatg tgtctcagtg 60gtcagaccca gagggctgct
gtgtattgct gccacctgtg ccccgggatc cacctgcatt 120gaccgggtgg gctctttctc
ctgcctctgc ccacctggac gcacaggact cctgtgccac 180ttggaagaca tgtgtctgag
ccagccgtgc catggggatg cccaatgcag caccaacccc 240ctcacaggct ccacactctg
cctgtgtcag cctggctatt cggggcccac ctgccaccag 300gacctggacg agtgtctgat
ggcccagcaa ggcccaagtc cctgtgaaca tggcggttcc 360tgcctcaaca ctcctggctc
cttcaactgc ctctgtccac ctggctacac aggctcccgt 420tgtgaggctg atcacaatga
gtgcctctcc cagccctgcc acccaggaag cacctgtctg 480gacctacttg ccaccttcca
ctgcctctgc ccgccaggct tagaagggca gctctgtgag 540gtggagacca acgagtgtgc
ctcagctccc tgcctgaacc acgcggattg ccatgacctg 600ctcaacggct tccagtgcat
ctgcctgcct ggattctccg gcacccgatg tgaggaggat 660atcgatgagt gcagaagctc
tccctgtgcc aatggtgggc agtgccagga ccagcctgga 720gccttccact gcaagtgtct
cccaggcttt gaagggccac gctgtcaaac agaggtggat 780gagtgcctga gtgacccatg
tcccgttgga gccagctgcc ttgatcttcc aggagccttc 840ttttgcctct gcccctctgg
tttcacaggc cagctctgtg aggttcccct gtgtgctccc 900aacctgtgcc agcccaagca
gatatgtaag gaccagaaag acaaggccaa ctgcctctgt 960cctgatggaa gccctggctg
tgccccacct gaggacaact gcacctgcca ccacgggcac 1020tgccagagat cctcatgtgt
gtgtgacgtg ggttggacgg ggccagagtg tgaggcagag 1080ctagggggct gcatctctgc
accctgtgcc catgggggga cctgctaccc ccagccctct 1140ggctacaact gcacctgccc
tacaggctac acaggaccca cctgtagtga ggagatgaca 1200gcttgtcact cagggccatg
tctcaatggc ggctcctgca accctagccc tggaggctac 1260tactgcacct gccctccaag
ccacacaggg ccccagtgcc aaaccagcac tgactactgt 1320gtgtctgccc cgtgcttcaa
tgggggtacc tgtgtgaaca ggcctggcac cttctcctgc 1380ctctgtgcca tgggcttcca
gggcccgcgc tgtgagggaa agctccgccc cagctgtgca 1440gacagcccct gtaggaatag
ggcaacctgc caggacagcc ctcagggtcc ccgctgcctc 1500tgccccactg gctacaccgg
aggcagctgc cagactctga tggacttatg tgcccagaag 1560ccctgcccac gcaattccca
ctgcctccag actgggccct ccttccactg cttgtgcctc 1620cagggatgga ccgggcctct
ctgcaacctt ccactgtcct cctgccagaa ggctgcactg 1680agccaaggca tagacgtctc
ttccctttgc cacaatggag gcctctgtgt cgacagcggc 1740ccctcctatt tctgccactg
cccccctgga ttccaaggca gcctgtgcca ggatcacgtg 1800aacccagatc tgggcccggg
cgagcccaaa tcttgtgaca aaactcacac atgcccaccg 1860tgcccagcac ctgaactcct
ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 1920gacaccctca tgatctcccg
gacccctgag gtcacatgcg tggtggtgga cgtgagccac 1980gaagaccctg aggtcaagtt
caactggtac gtggacggcg tggaggtgca taatgccaag 2040acaaagccgc gggaggagca
gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 2100ctgcaccagg actggctgaa
tggcaaggag tacaagtgca aggtctccaa caaagccctc 2160ccagccccca tcgagaaaac
catctccaaa gccaaagggc agccccgaga accacaggtg 2220tacaccctgc ccccatcccg
ggatgagctg accaagaacc aggtcagcct gacctgcctg 2280gtcaaaggct tctatcccag
cgacatcgcc gtggagtggg agagcaatgg gcagccggag 2340aacaactaca agaccacgcc
tcccgtgctg gactccgacg gctccttctt cctctacagc 2400aagctcaccg tggacaagag
caggtggcag caggggaacg tcttctcatg ctccgtgatg 2460catgaggctc tgcacaacca
ctacacgcag aagagcctct ccctgtctcc gggtaaatga 2520992505DNAHomo sapiens
99atgtggggct ggaagtgcct cctcttctgg gctgtgctgg tcacagccac tctctgcact
60gccaggtgta ttgctgccac ctgtgccccg ggatccacct gcattgaccg ggtgggctct
120ttctcctgcc tctgcccacc tggacgcaca ggactcctgt gccacttgga agacatgtgt
180ctgagccagc cgtgccatgg ggatgcccaa tgcagcacca accccctcac aggctccaca
240ctctgcctgt gtcagcctgg ctattcgggg cccacctgcc accaggacct ggacgagtgt
300ctgatggccc agcaaggccc aagtccctgt gaacatggcg gttcctgcct caacactcct
360ggctccttca actgcctctg tccacctggc tacacaggct cccgttgtga ggctgatcac
420aatgagtgcc tctcccagcc ctgccaccca ggaagcacct gtctggacct acttgccacc
480ttccactgcc tctgcccgcc aggcttagaa gggcagctct gtgaggtgga gaccaacgag
540tgtgcctcag ctccctgcct gaaccacgcg gattgccatg acctgctcaa cggcttccag
600tgcatctgcc tgcctggatt ctccggcacc cgatgtgagg aggatatcga tgagtgcaga
660agctctccct gtgccaatgg tgggcagtgc caggaccagc ctggagcctt ccactgcaag
720tgtctcccag gctttgaagg gccacgctgt caaacagagg tggatgagtg cctgagtgac
780ccatgtcccg ttggagccag ctgccttgat cttccaggag ccttcttttg cctctgcccc
840tctggtttca caggccagct ctgtgaggtt cccctgtgtg ctcccaacct gtgccagccc
900aagcagatat gtaaggacca gaaagacaag gccaactgcc tctgtcctga tggaagccct
960ggctgtgccc cacctgagga caactgcacc tgccaccacg ggcactgcca gagatcctca
1020tgtgtgtgtg acgtgggttg gacggggcca gagtgtgagg cagagctagg gggctgcatc
1080tctgcaccct gtgcccatgg ggggacctgc tacccccagc cctctggcta caactgcacc
1140tgccctacag gctacacagg acccacctgt agtgaggaga tgacagcttg tcactcaggg
1200ccatgtctca atggcggctc ctgcaaccct agccctggag gctactactg cacctgccct
1260ccaagccaca cagggcccca gtgccaaacc agcactgact actgtgtgtc tgccccgtgc
1320ttcaatgggg gtacctgtgt gaacaggcct ggcaccttct cctgcctctg tgccatgggc
1380ttccagggcc cgcgctgtga gggaaagctc cgccccagct gtgcagacag cccctgtagg
1440aatagggcaa cctgccagga cagccctcag ggtccccgct gcctctgccc cactggctac
1500accggaggca gctgccagac tctgatggac ttatgtgccc agaagccctg cccacgcaat
1560tcccactgcc tccagactgg gccctccttc cactgcttgt gcctccaggg atggaccggg
1620cctctctgca accttccact gtcctcctgc cagaaggctg cactgagcca aggcatagac
1680gtctcttccc tttgccacaa tggaggcctc tgtgtcgaca gcggcccctc ctatttctgc
1740cactgccccc ctggattcca aggcagcctg tgccaggatc acgtgaaccc agatctgggc
1800ccgggcgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa
1860ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc
1920tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc
1980aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag
2040gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg
2100ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag
2160aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca
2220tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat
2280cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc
2340acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac
2400aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac
2460aaccactaca cgcagaagag cctctccctg tctccgggta aatga
25051002046DNAHomo sapiens 100atgcagcccc cttcactgct gctgctgctg ctgctgctgc
tgctgctatg tgtctcagtg 60gtcagaccca gagggctgct gtgtgcctca gctccctgcc
tgaaccacgc ggattgccat 120gacctgctca acggcttcca gtgcatctgc ctgcctggat
tctccggcac ccgatgtgag 180gaggatatcg atgagtgcag aagctctccc tgtgccaatg
gtgggcagtg ccaggaccag 240cctggagcct tccactgcaa gtgtctccca ggctttgaag
ggccacgctg tcaaacagag 300gtggatgagt gcctgagtga cccatgtccc gttggagcca
gctgccttga tcttccagga 360gccttctttt gcctctgccc ctctggtttc acaggccagc
tctgtgaggt tcccctgtgt 420gctcccaacc tgtgccagcc caagcagata tgtaaggacc
agaaagacaa ggccaactgc 480ctctgtcctg atggaagccc tggctgtgcc ccacctgagg
acaactgcac ctgccaccac 540gggcactgcc agagatcctc atgtgtgtgt gacgtgggtt
ggacggggcc agagtgtgag 600gcagagctag ggggctgcat ctctgcaccc tgtgcccatg
gggggacctg ctacccccag 660ccctctggct acaactgcac ctgccctaca ggctacacag
gacccacctg tagtgaggag 720atgacagctt gtcactcagg gccatgtctc aatggcggct
cctgcaaccc tagccctgga 780ggctactact gcacctgccc tccaagccac acagggcccc
agtgccaaac cagcactgac 840tactgtgtgt ctgccccgtg cttcaatggg ggtacctgtg
tgaacaggcc tggcaccttc 900tcctgcctct gtgccatggg cttccagggc ccgcgctgtg
agggaaagct ccgccccagc 960tgtgcagaca gcccctgtag gaatagggca acctgccagg
acagccctca gggtccccgc 1020tgcctctgcc ccactggcta caccggaggc agctgccaga
ctctgatgga cttatgtgcc 1080cagaagccct gcccacgcaa ttcccactgc ctccagactg
ggccctcctt ccactgcttg 1140tgcctccagg gatggaccgg gcctctctgc aaccttccac
tgtcctcctg ccagaaggct 1200gcactgagcc aaggcataga cgtctcttcc ctttgccaca
atggaggcct ctgtgtcgac 1260agcggcccct cctatttctg ccactgcccc cctggattcc
aaggcagcct gtgccaggat 1320cacgtgaacc cagatctggg cccgggcgag cccaaatctt
gtgacaaaac tcacacatgc 1380ccaccgtgcc cagcacctga actcctgggg ggaccgtcag
tcttcctctt ccccccaaaa 1440cccaaggaca ccctcatgat ctcccggacc cctgaggtca
catgcgtggt ggtggacgtg 1500agccacgaag accctgaggt caagttcaac tggtacgtgg
acggcgtgga ggtgcataat 1560gccaagacaa agccgcggga ggagcagtac aacagcacgt
accgtgtggt cagcgtcctc 1620accgtcctgc accaggactg gctgaatggc aaggagtaca
agtgcaaggt ctccaacaaa 1680gccctcccag cccccatcga gaaaaccatc tccaaagcca
aagggcagcc ccgagaacca 1740caggtgtaca ccctgccccc atcccgggat gagctgacca
agaaccaggt cagcctgacc 1800tgcctggtca aaggcttcta tcccagcgac atcgccgtgg
agtgggagag caatgggcag 1860ccggagaaca actacaagac cacgcctccc gtgctggact
ccgacggctc cttcttcctc 1920tacagcaagc tcaccgtgga caagagcagg tggcagcagg
ggaacgtctt ctcatgctcc 1980gtgatgcatg aggctctgca caaccactac acgcagaaga
gcctctccct gtctccgggt 2040aaatga
20461012031DNAHomo sapiens 101atgtggggct ggaagtgcct
cctcttctgg gctgtgctgg tcacagccac tctctgcact 60gccaggtgtg cctcagctcc
ctgcctgaac cacgcggatt gccatgacct gctcaacggc 120ttccagtgca tctgcctgcc
tggattctcc ggcacccgat gtgaggagga tatcgatgag 180tgcagaagct ctccctgtgc
caatggtggg cagtgccagg accagcctgg agccttccac 240tgcaagtgtc tcccaggctt
tgaagggcca cgctgtcaaa cagaggtgga tgagtgcctg 300agtgacccat gtcccgttgg
agccagctgc cttgatcttc caggagcctt cttttgcctc 360tgcccctctg gtttcacagg
ccagctctgt gaggttcccc tgtgtgctcc caacctgtgc 420cagcccaagc agatatgtaa
ggaccagaaa gacaaggcca actgcctctg tcctgatgga 480agccctggct gtgccccacc
tgaggacaac tgcacctgcc accacgggca ctgccagaga 540tcctcatgtg tgtgtgacgt
gggttggacg gggccagagt gtgaggcaga gctagggggc 600tgcatctctg caccctgtgc
ccatgggggg acctgctacc cccagccctc tggctacaac 660tgcacctgcc ctacaggcta
cacaggaccc acctgtagtg aggagatgac agcttgtcac 720tcagggccat gtctcaatgg
cggctcctgc aaccctagcc ctggaggcta ctactgcacc 780tgccctccaa gccacacagg
gccccagtgc caaaccagca ctgactactg tgtgtctgcc 840ccgtgcttca atgggggtac
ctgtgtgaac aggcctggca ccttctcctg cctctgtgcc 900atgggcttcc agggcccgcg
ctgtgaggga aagctccgcc ccagctgtgc agacagcccc 960tgtaggaata gggcaacctg
ccaggacagc cctcagggtc cccgctgcct ctgccccact 1020ggctacaccg gaggcagctg
ccagactctg atggacttat gtgcccagaa gccctgccca 1080cgcaattccc actgcctcca
gactgggccc tccttccact gcttgtgcct ccagggatgg 1140accgggcctc tctgcaacct
tccactgtcc tcctgccaga aggctgcact gagccaaggc 1200atagacgtct cttccctttg
ccacaatgga ggcctctgtg tcgacagcgg cccctcctat 1260ttctgccact gcccccctgg
attccaaggc agcctgtgcc aggatcacgt gaacccagat 1320ctgggcccgg gcgagcccaa
atcttgtgac aaaactcaca catgcccacc gtgcccagca 1380cctgaactcc tggggggacc
gtcagtcttc ctcttccccc caaaacccaa ggacaccctc 1440atgatctccc ggacccctga
ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct 1500gaggtcaagt tcaactggta
cgtggacggc gtggaggtgc ataatgccaa gacaaagccg 1560cgggaggagc agtacaacag
cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag 1620gactggctga atggcaagga
gtacaagtgc aaggtctcca acaaagccct cccagccccc 1680atcgagaaaa ccatctccaa
agccaaaggg cagccccgag aaccacaggt gtacaccctg 1740cccccatccc gggatgagct
gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc 1800ttctatccca gcgacatcgc
cgtggagtgg gagagcaatg ggcagccgga gaacaactac 1860aagaccacgc ctcccgtgct
ggactccgac ggctccttct tcctctacag caagctcacc 1920gtggacaaga gcaggtggca
gcaggggaac gtcttctcat gctccgtgat gcatgaggct 1980ctgcacaacc actacacgca
gaagagcctc tccctgtctc cgggtaaatg a 20311021890DNAHomo sapiens
102atgcagcccc cttcactgct gctgctgctg ctgctgctgc tgctgctatg tgtctcagtg
60gtcagaccca gagggctgct gtgtgcagac agcccctgta ggaatagggc aacctgccag
120gacagccctc agggtccccg ctgcctctgc cccactggct acaccggagg cagctgccag
180actctgatgg acttatgtgc ccagaagccc tgcccacgca attcccactg cctccagact
240gggccctcct tccactgctt gtgcctccag ggatggaccg ggcctctctg caaccttcca
300ctgtcctcct gccagaaggc tgcactgagc caaggcatag acgtctcttc cctttgccac
360aatggaggcc tctgtgtcga cagcggcccc tcctatttct gccactgccc ccctggattc
420caaggcagcc tgtgccagga tcacgtgaac ccatgtgagt ccaggccttg ccagaacggg
480gccacctgca tggcccagcc cagtgggtat ctctgccagt gtgccccagg ctacgatgga
540cagaactgct caaaggaact cgatgcttgt cagtcccaac cctgtcacaa ccatggaacc
600tgtactccca aacctggagg attccactgt gcctgccctc caggctttgt ggggctacgc
660tgtgagggag acgtggacga gtgtctggac cagccctgcc accccacagg cactgcagcc
720tgccactctc tggccaatgc cttctactgc cagtgtctgc ctggacacac aggccagtgg
780tgtgaggtgg agatagaccc ctgccacagc caaccctgct ttcatggagg gacctgtgag
840gccacagcag gatcacccct gggtttcatc tgccactgcc ccaagggttt tgaaggcccc
900acctgcagcc acagggcccc ttcctgcggc ttccatcact gccaccacgg aggcctgtgt
960ctgccctccc ctaagccagg cttcccacca cgctgtgcct gcctcagtgg ctatgggggt
1020cctgactgcc tgaccccacc agctcctaaa ggctgtggcc ctccctcccc atgcctatac
1080aatggcagct gctcagagac cacgggcttg gggggcccag gctttcgatg ctcctgccct
1140cacagctctc cagggccccg gtgtcagaaa cccggagatc tgggcccggg cgagcccaaa
1200tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg
1260tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag
1320gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac
1380gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc
1440acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag
1500tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa
1560gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggatgagctg
1620accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc
1680gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg
1740gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag
1800caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag
1860aagagcctct ccctgtctcc gggtaaatga
18901031875DNAHomo sapiens 103atgtggggct ggaagtgcct cctcttctgg gctgtgctgg
tcacagccac tctctgcact 60gccaggtgtg cagacagccc ctgtaggaat agggcaacct
gccaggacag ccctcagggt 120ccccgctgcc tctgccccac tggctacacc ggaggcagct
gccagactct gatggactta 180tgtgcccaga agccctgccc acgcaattcc cactgcctcc
agactgggcc ctccttccac 240tgcttgtgcc tccagggatg gaccgggcct ctctgcaacc
ttccactgtc ctcctgccag 300aaggctgcac tgagccaagg catagacgtc tcttcccttt
gccacaatgg aggcctctgt 360gtcgacagcg gcccctccta tttctgccac tgcccccctg
gattccaagg cagcctgtgc 420caggatcacg tgaacccatg tgagtccagg ccttgccaga
acggggccac ctgcatggcc 480cagcccagtg ggtatctctg ccagtgtgcc ccaggctacg
atggacagaa ctgctcaaag 540gaactcgatg cttgtcagtc ccaaccctgt cacaaccatg
gaacctgtac tcccaaacct 600ggaggattcc actgtgcctg ccctccaggc tttgtggggc
tacgctgtga gggagacgtg 660gacgagtgtc tggaccagcc ctgccacccc acaggcactg
cagcctgcca ctctctggcc 720aatgccttct actgccagtg tctgcctgga cacacaggcc
agtggtgtga ggtggagata 780gacccctgcc acagccaacc ctgctttcat ggagggacct
gtgaggccac agcaggatca 840cccctgggtt tcatctgcca ctgccccaag ggttttgaag
gccccacctg cagccacagg 900gccccttcct gcggcttcca tcactgccac cacggaggcc
tgtgtctgcc ctcccctaag 960ccaggcttcc caccacgctg tgcctgcctc agtggctatg
ggggtcctga ctgcctgacc 1020ccaccagctc ctaaaggctg tggccctccc tccccatgcc
tatacaatgg cagctgctca 1080gagaccacgg gcttgggggg cccaggcttt cgatgctcct
gccctcacag ctctccaggg 1140ccccggtgtc agaaacccgg agatctgggc ccgggcgagc
ccaaatcttg tgacaaaact 1200cacacatgcc caccgtgccc agcacctgaa ctcctggggg
gaccgtcagt cttcctcttc 1260cccccaaaac ccaaggacac cctcatgatc tcccggaccc
ctgaggtcac atgcgtggtg 1320gtggacgtga gccacgaaga ccctgaggtc aagttcaact
ggtacgtgga cggcgtggag 1380gtgcataatg ccaagacaaa gccgcgggag gagcagtaca
acagcacgta ccgtgtggtc 1440agcgtcctca ccgtcctgca ccaggactgg ctgaatggca
aggagtacaa gtgcaaggtc 1500tccaacaaag ccctcccagc ccccatcgag aaaaccatct
ccaaagccaa agggcagccc 1560cgagaaccac aggtgtacac cctgccccca tcccgggatg
agctgaccaa gaaccaggtc 1620agcctgacct gcctggtcaa aggcttctat cccagcgaca
tcgccgtgga gtgggagagc 1680aatgggcagc cggagaacaa ctacaagacc acgcctcccg
tgctggactc cgacggctcc 1740ttcttcctct acagcaagct caccgtggac aagagcaggt
ggcagcaggg gaacgtcttc 1800tcatgctccg tgatgcatga ggctctgcac aaccactaca
cgcagaagag cctctccctg 1860tctccgggta aatga
187510424PRTHomo sapiens 104Met Gln Pro Pro Ser Leu
Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu 1 5
10 15 Cys Val Ser Val Val Arg Pro Arg
20 10569DNAHomo sapiens 105atgcagcccc cttcactgct
gctgctgctg ctgctgctgc tgctgctatg tgtctcagtg 60gtcagaccc
6910612DNAHomo sapiens
106cagaaacccg ga
1210712DNAHomo sapiens 107tccgggtttc tg
121084PRTHomo sapiens 108Gln Lys Pro Gly 1
10917DNAHomo sapiens 109cagaaacccg gagatct
1711017DNAHomo sapiens 110agatctccgg gtttctg
1711124DNAHomo sapiens
111cagaaacccg gagatctggg cccg
2411224DNAHomo sapiens 112cgggcccaga tctccgggtt tctg
241138PRTHomo sapiens 113Gln Lys Pro Gly Asp Leu
Gly Pro 1 5 11412DNAHomo sapiens
114gatatcgatg ag
1211512DNAHomo sapiens 115ctcatcgata tc
121164PRTHomo sapiens 116Asp Ile Asp Glu 1
11717DNAHomo sapiens 117gatatcgatg aggatcc
1711817DNAHomo sapiens 118ggatcctcat cgatatc
1711924DNAHomo sapiens
119gatatcgatg aggatctggg cccg
2412024DNAHomo sapiens 120cgggcccaga tcctcatcga tatc
241218PRTHomo sapiens 121Asp Ile Asp Glu Asp Leu
Gly Pro 1 5 12212DNAHomo sapiens
122cacgtgaacc ca
1212312DNAHomo sapiens 123tgggttcacg tg
121244PRTHomo sapiens 124His Val Asn Pro 1
12517DNAHomo sapiens 125cacgtgaacc cagatct
1712617DNAHomo sapiens 126agatctgggt tcacgtg
1712724DNAHomo sapiens
127cacgtgaacc cagatctggg cccg
2412824DNAHomo sapiens 128cgggcccaga tctgggttca cgtg
241298PRTHomo sapiens 129His Val Asn Pro Asp Leu
Gly Pro 1 5 13023PRTHomo sapiens 130Met Pro
Pro Leu Leu Ala Pro Leu Leu Cys Leu Ala Leu Leu Pro Ala 1 5
10 15 Leu Ala Ala Arg Gly Pro Arg
20 13127PRTHomo sapiens 131Met Gln Pro Pro Ser
Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu 1 5
10 15 Cys Val Ser Val Val Arg Pro Arg Gly Leu
Leu 20 25
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