Patent application title: Peptidylarginine Deiminase 6
Jan Albert Gossen (Oss, NL)
Paul Van Den Boogaart (Oss, NL)
IPC8 Class: AC12N914FI
Class name: Chemistry: molecular biology and microbiology enzyme (e.g., ligases (6. ), etc.), proenzyme; compositions thereof; process for preparing, activating, inhibiting, separating, or purifying enzymes hydrolase (3. )
Publication date: 2009-06-25
Patent application number: 20090162915
A nucleotide acid sequence is provided encoding a peptidylarginine
deiminase 6. The gene is found to be expressed in gonads only and may be
used as target for male and female contraception. Its encoded protein can
be used to screen for small molecular weight modulators of the enzyme
12. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:3.
13. The isolated polypeptide according to claim 12, wherein the polypeptide is encoded by nucleotides 20-2077 of SEQ ID NO:4.
14. An isolated polypeptide having at least 95% sequence identity with SEQ ID NO:3.
The current invention relates to polynucleotides encoding
peptidylargininie deiminase 6, cells transfected with these
polynucleotides, proteins produced by these cells as well as to a method
to produce these proteins and its modulators.
Peptidylarginine deiminases (PADs) are a family of post-translational modification enzymes which convert peptidylarginine into citrulline in a Ca2+-dependent manner. Enzymatic deimination in vitro changes the functional properties of various proteins and alters their secondary and tertiary structures.
Sofar, five isoforms of PAD have been identified showing a broad tissue distribution. Mouse PAD1 is detected in the epidermis and uterus (Rus'd, A. A. et al. 1990, Eur. J. Biochem. 259, 660-669); murine PAD2 is widely expressed in various tissues such as brain, pituitary, spinal cord, salivary gland, pancreas, skeletal muscle, uterus, spleen, stomach and thymus (Takahara, H. et al. 1989, J. Biol. Chem. 264, 13361-13368); murine PAD3 is expressed in epidermis and hair follicles (Terakawa, H. et al. 1991, J. Biochem. (Tokyo) 110, 661-666); PAD4 (rat) is an ubiquitous enzyme being expressed in the pancreas, spleen, ovary, liver, lung, stomach, kidney, uterus, dermis, brain, heart and epidermis (Yamakoshi, A. et al. 1998, Biochim. Biophys. Acta 1386, 227-232); human PAD5 finally has been isolated as a new family member from a myeloid leukemia cell line, but its tissue distribution has not been further determined (Nakashima, K. et al. 1999, J. Biol. Chem. 274, 27786-27792).
Little is known about the physiological functions of PAD. In brain, myelin basic protein is a natural substrate and therefore, PAD plays an important part in the central nervous system. Moreover, when disregulated PAD plays a role in the aetiology of multiple sclerosis (Mastronardi, F. G. et al. 1996, Clin. Invest. 97, 349-358). PAD in the epidermis seems to be involved in the terminal processing of filaggrin, which indirectly is important for the maintenance of moisture in the upper stratum corneum (Senshu, T. et al. 1996, Biochem. Biophys. Res. Commun. 225, 712-719). Again, disregulation of this PAD may play a role in the aetiology of rheumatoid arthritis (Girbal-Neuhauser, E. et al. 1999, J. Immunol. 162, 585-594). In hair follicles finally the solubility of trichohyalin seems to be influenced by PAD; the function of this remains to be determined (Rogers, G. E. et al. 1997, J. Invest. Dermatol. 108, 700-707).
We now have found a novel PAD, which is called PAD6. The transcript has been found in mouse oocytes. Its human homologue is also described herein. The protein was found to be expressed exclusively in oocytes/ovary and testes.
Genes that are expressed specifically in male and/or female gametes may provide novel molecular targets for male and female contraception. For testis, large numbers of gene sequences expressed uniquely in germ cells have been described (Pawlak, A. et al. 1995 Genomics 26, 151-1588; Wolgemuth D. J. and Watrin F. 1991 Mamm Genome 1, 283-817). In contrast, only a few genes specifically expressed in oocytes thus far have been identified. The majority of gamete specific gene sequences identified are likely to have an essential function due to their specific expression in gametes. The latter is confirmed by studies using knockout animals indicating that gene inactivation of testis and oocyte specific genes generally results in male and/or female infertility but does not result in additional pathology in other organs and tissues (Dong, J. et al. 1996 Nature 383, 531-535; Nantel, F. et al. 1996, Nature 380, 159-162). These data provide further evidence for the specific and essential role of these genes during gametogenesis. This underlines the importance of tissue specificity as selection criteron for molecular targets for fertility regulation.
It will be clear that there is a great need for the elucidation of genes involved in fertility regulation in order to unravel the various roles these genes may play in infertility. A better knowledge of the genes involved in different stages of female and male fertility e.g. in gametogenesis and their activity and expression regulation might help to create a better insight in infertility disorders. This could eventually lead to the identification of activity modulators to be used in either in vivo or in vitro therapeutic protocols.
The present invention provides for such a gene. More specific, the present invention provides for a polynucleotide sequence encoding peptidylarginine deiminase 6 (PAD6). Preferably the polynucleotide is of mammalian origin, preferably mouse more preferably human. The RNA is expressed exclusively in reproductive organs.
The most preferred polynucleotide sequences are those encoding SEQ ID NO: 1 or SEQ ID NO:3.
The invention also includes the entire mouse mRNA sequence as indicated in SEQ ID NO:2 and more specifically the open reading frame corresponding to nucleotide sequence 6-2051 of SEQ ID NO:2. This sequence encodes a protein of 682 amino acids (SEQ ID NO:1). In addition the invention includes the entire human mRNA sequence as indicated in SEQ ID NO:4 and the open reading frame corresponding to nucleotide sequence 20-2077 of SEQ ID NO:4 This sequence encodes a protein of 686 amino acids (SEQ ID NO:3). To accommodate codon variability, the invention also includes polynucleotide sequences coding for the same amino acid sequences as the sequences disclosed herein. The sequence information as provided herein should not be so narrowly construed as to require exclusion of erroneously identified bases. The specific sequence disclosed herein can readily be used to isolate the complete genes of several other species or allelic variants. The sequence can e.g. be used to prepare probes or as a source to prepare synthetic oligonucleotides to be used as primers in DNA amplification reactions allowing the isolation and identification of the complete variant genes. In particular, polynucleotides hybridizing under stringent washing conditions with a probe prepared with PCR under standard conditions using SEQ ID NO:14 and SEQ ID NO:15 with cDNA from mammalian origin as a template, preferably human or mouse, are part of the invention. Such a probe (and its complementary sequence) is identified e.g. by the nucleotides 464-1052 of SEQ ID NO:4.
The complete genetic sequence can be used in the preparation of vector molecules for expression of the protein in suitable host cells.
Thus, in one aspect, the present invention provides for isolated polynucleotides encoding the novel PAD6 protein. Preferably the PAD6 is of human origin, but also orthologs form part of the invention.
The DNA according to the invention may be obtained from cDNA. The tissues preferably are from mammalian origin, more preferably from human origin. Preferably ribonucleic acids are isolated from oocytes or testes. Alternatively, the coding sequence might be genomic DNA, or prepared using DNA synthesis techniques. The polynucleotide may also be in the form of RNA. If the polynucleotide is DNA, it may be in single stranded or double stranded form. The single strand might be the coding strand or the non-coding (anti-sense) strand.
The DNA according to the invention will be very useful for in vivo or in vitro expression of the novel protein according to the invention in sufficient quantities and in substantially pure form.
The present invention further relates to polynucleotides having slight variations or having polymorphic sites. Polynucleotides having slight variations may encode variant polypeptides which retain the same biological function or activity as the natural, mature protein. Polymorphic sites are useful for diagnostic purposes.
In another aspect, the invention provides for a method to isolate a polynucleotide comprising the steps of: a) hybridizing a polynucleotide according to the present invention, or its complement, under stringent conditions against nucleic acids being (genomic) DNA RNA, or cDNA isolated preferably from tissues which highly express the polynucleotide of interest and b) isolating said nucleic acids by methods known to a skilled person in the art. The tissues preferably are from human origin. Preferably ribonucleic acids are isolated from oocytes, ovaria or testes. The hybridization conditions are preferably highly stringent.
According to the present invention the term "stringent" means washing conditions of 1×SSC, 0.1% SDS at a temperature of 65° C.; highly stringent conditions refer to a reduction in SSC towards 0.3×SSC, more preferably to 0.1×SSC. Preferably the first two washings are subsequently carried out twice each during 15-30 minutes. If there is a need to wash under highly stringent conditions an additional wash with 0.1×SSC is performed once during 15 minutes. Hybridization can be performed e.g. overnight in 0.5M phosphate buffer pH7.5/7% SDS at 65° C.
As an alternative the method to isolate the gene might comprise gene amplification methodology using primers derived from the nucleic acid according to the invention. Complete cDNAs might also be obtained by combining clones obtained by e.g. hybridization with e.g. RACE cDNA clones.
Also portions of the coding sequences coding for a functional polypeptide are part of the invention as well as allelic and species variations thereof. Sometimes, a gene is expressed in a certain tissue as a splicing variant, resulting in an altered 5' or 3' mRNA or the inclusion or exclusion of one or more exon sequences. These sequences as well as the proteins encoded by these sequences all are expected to perform the same or similar functions and form also part of the invention.
The invention also provides for peptidylarginine deiminase 6 (PAD6). Preferably the protein has a mammalian amino acid sequence, more preferably a human sequence. Most preferred are the sequences as described in SEQ ID NOs: 1 or 3. Expression can be obtained by introduction of vector molecules comprising a polynucleotide encoding PAD6 into suitable host cells. The cells can be cultured and the protein can be isolated using methods known to the person skilled in the art.
In still another aspect of the invention there are provided functional equivalents that is polypeptides encoding PAD6 activities and comprising essentially the same SEQ ID NO:1 or 3 sequence or parts thereof having variations of the sequence while still maintaining functional characteristics.
The variations that can occur in a sequence may be demonstrated by (an) amino acid difference(s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence. Amino acid substitutions that are expected not to essentially alter biological and immunological activities, have been described. Amino acid replacements between related amino acids or replacements which have occurred frequently in evolution are, inter alia Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M. D., Atlas of protein sequence and structure, Nat. Biomed. Res. Found., Washington D.C., 1978, vol. 5, suppl. 3). Based on this information Lipman and Pearson developed a method for rapid and sensitive protein comparison (Science, 1985 227, 1435-1441) and determining the functional similarity between homologous polypeptides. It will be clear that also polynucleotides coding for such variants are part of the invention.
Thus, in another aspect of the invention there are provided polypeptides comprising SEQ ID NO:1 or SEQ ID NO:3 or but also polypeptides with a similarity of 80%, preferably 90%, more preferably 95%.
As used herein the term similarity is as defined in NCBI-BLAST 2.0.10 [Aug. 26, 1999] (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25, 3389-3402). The program is used to search for sequence alignments using default settings. For amino acid alignments the BLOSUM62 matrix is used as a default and the similarity is indicated as the number of positives. No filtering of low compositional complexity is included.
Also portions of such polypeptides still capable of conferring biological effects are included. Especially portions which still are capable of converting arginine to citrulline form part of the invention. Such proteins or functional parts thereof may be functional per se, e.g. in solubilized form or they may be linked to other polypeptides (e.g. to direct it to specific subcellular compartments, to increase its stability or to facilitate its purification), either by known biotechnological ways or by chemical synthesis, to obtain chimeric proteins.
It will be clear that also polynucleotides encoding such variant polypeptides are included in the invention.
A wide variety of host cell and cloning vehicle combinations may be usefully employed in cloning the nucleic acid sequence coding for the polypeptide according to the invention.
Suitable expression vectors are for example bacterial or yeast plasmids, wide host range plasmids and vectors derived from combinations of plasmid and phage or virus DNA. Vectors derived from chromosomal DNA are also included. Furthermore an origin of replication and/or a dominant selection marker can be present in the vector according to the invention. The vectors according to the invention are suitable for transforming a host cell.
Vehicles for use in expression of the protein or parts thereof of the present invention will further comprise control sequences operably linked to the nucleic acid sequence coding for the protein. Such control sequences generally comprise a promoter sequence and sequences, which regulate and/or enhance expression levels. Of course control and other sequences can vary depending on the host cell selected.
Recombinant expression vectors comprising the DNA of the invention as well as cells transfected with said DNA or said expression vector, either transiently or stable, also form part of the present invention.
Suitable host cells according to the invention are bacterial host cells, yeast and other fungi, plant or animal host such as Chinese Hamster Ovary cells, monkey cells, or human cells. Thus, a host cell which comprises the DNA or expression vector according to the invention is also within the scope of the invention. The engineered host cells can be cultured in conventional nutrient media which can be modified e.g. for appropriate selection, amplification or induction of transcription. The culture conditions such as temperature, pH, nutrients etc. are well known to those ordinary skilled in the art.
The techniques for the preparation of the DNA or the vector according to the invention as well as the transformation or transfection of a host cell with said DNA or vector are standard and well known in the art, see for instance Sambrook et al., Molecular Cloning: A laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.
The protein according to the invention can be recovered and purified from recombinant cell cultures by common biochemical purification methods (as described in Guide to Protein purification. Edited by Murray P. Deutscher. (1990) Methods in Enzymology. Vol 182. Academic Press, inc. San Diego Calif. 92101. Harcourt Brace Jovanovich, Publishers). including ammonium sulfate precipitation, extraction, chromatography such as hydrophobic interaction chromatography, cation or anion exchange chromatography or affinity chromatography and high performance liquid chromatography. If necessary, also protein refolding steps can be included. Alternatively the protein can be expressed and purified as a fusion protein containing ("tags") which can be used for affinity purification.
Regulation of the activity of the protein according to the invention is useful in vivo for the control of follicular recruitment, but also of growth and maturation of oocytes and/or follicles. Inhibition of these processes in vivo can be used to delay (premature) menopause and/or as a contraceptive. In addition, the protein can be employed for in vitro maturation and growth of follicles e.g. from frozen ovarian tissue.
PAD gene products according to the present invention can be used for the in vivo or in vitro identification of novel substrates or analogs thereof. For this purpose e.g. peptidyl arginine deiminase assay studies can be performed with cells transformed with DNA according to the invention or an expression vector comprising DNA according to the invention, said cells expressing the PAD6 gene products according to the invention. Alternatively also the PAD6 protein itself or the substrate-binding domains thereof can be used in an assay for the identification of functional substrates or analogs.
Methods to determine peptidyl arginine deiminase activity of expressed gene products in in vitro and in vivo assays to determine biological activity of gene products are well known. See e.g. Lamensa, F. E. W. and Moscarello, M. A. 1993 J. Neurochem. 61, 987-996. In this assay arginine in α-Nbenzoyl-Larginine ethyl esther (BAEE) is converted in citrulline which can easily be measured after precipitation with perchloric acid.
Another example of determining the enzymatic activity of PAD6 makes use of the inactivation of a protein e.g. Soybean Trypsin Inhibitor (STI) (Takahara, H. et al. 1985, J. Biol. Chem. 260, 8378-8383. When an essential arginine in STI is converted into citrulline it is no longer able to inhibit the proteolytic activity of trypsin. This can be used as the basis for a two-step assay for the determination of PAD activity. The assay consists of two steps. In the first reaction PAD converts the arginine position 63) in STI into a citrulline inactivating the STI. In the second reaction trypsin and a fluorescent substrate are added and trypsin activity is measured.
Alternatively modulation of the PAD6 activity can also be obtained by downregulation of the expression level of the protein e.g. by using anti-sense nucleic acids through triple-helix formation (Cooney et al., 1988, Science 241, 456-459) or by binding to the mRNA, or by influencing in RNA stability or protein interactions by small molecules. This in itself could also lead to regulation of fertility i.e. contraception or treatment of infertility.
Thus, the present invention provides for a method for identifying compounds that affect the enzymatic function of the protein according to the invention. The method comprises the steps of
a) contacting the PAD6 protein with an arginine containing substrateb) contacting said mixture with a test compoundc) measuring the arginine to citrulline conversion andd) comparing said conversion with peptidylarginine deiminase activity in the absence of a test compound.
The arginine to citrulline conversion can easily be measured e.g. by analytical methods like HPLC, altered proteolytic sensitivity of the peptide, change in activity properties of the peptide or specific antibody recognition. As a substrate peptides or proteins comprising arginine can be used, but also synthetic compounds such as α-N-benzoyl-L-arginine ethyl ester can be used. However, the amino and carboxyl groups have to be substituted or have to be in a peptide bonded form.
Alternatively, the present invention provides for a method to identify compounds that modulate the PAD6 mRNA stability or the PAD6 expression levels.
The present invention thus provides for a quick and economic method to screen for therapeutic agents for fertility control related to the activity of PAD6. The method according to the invention furthermore provides for the selection of selective therapeutic agents discriminating between different peptidylarginine deiminases thus leading to a more effective therapeutic agent and/or diminishing of side effects. The method is especially suited to be used for the high throughput screening of numerous potential target compounds.
Compounds which modulate the peptidylarginine deiminase 6 function may be employed in therapeutic treatments by modulating the PAD of the present invention.
The invention also provides for a method for the formulation of a pharmaceutical composition comprising mixing the modulator compounds identified with a pharmaceutically acceptable carrier.
Pharmaceutical acceptable carriers are well known to those skilled in the art and include, for example, sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil, olive oil, sesame oil and water.
Furthermore the pharmaceutical composition may comprise one or more stabilizers such as, for example, carbohydrates including sorbitol, mannitol, starch, sucrosedextrin and glucose, proteins such as albumin or casein, and buffers like alkaline phosphates. Methods for making preparations and intravenous admixtures are disclosed in Remingtons's Pharmaceutical Sciences, pp. 1463-1497 (16th ed. 1980, Mack Publ. Co of Easton, Pa., USA).
Thus, the modulator compounds identified by using the peptidylarginine deiminase according to the invention are useful in the preparation of a pharmaceutical. The pharmaceutical is to be used for control of fertility disorders.
The following examples are illustrative for the invention and should in no way be interpreted as limiting the scope of the invention.
LEGENDS TO THE FIGURES
FIG. 1 RT-PCR analysis (30 cycles) of mouse PAD6 expression in various mouse tissues (upper panel). In the lower panel GAPDH controls in the absence and presence of RT are shown.
FIG. 2 ISH (In Situ Hybridization) analysis using clone 1B11 as\a probe on ovaries from young (7 days) and adult mice. S=secondary follicle A=antral follicle
FIG. 3 RT-PCR analysis (30 cycles) of human PAD6 expression in various human tissues (upper panel). In the lower panel GAPDH controls in the absence and presence of RT are shown.
FIG. 4 Human multiple tissue northern blots (Clontech) hybridised with hPAD6 probe.
FIG. 5 Fluorescence measurement to determine PAD activity. STI (0.17 μg) was pre-incubated in the absence (filled square) or presence of 0.5 μg GST-PAD6 (filled triangle), 1.0 μg GST-PAD6 (open triangle), or 0.1 μg rabbit muscle PAD (open square; Sigma cat. No P1584) respectively. Subsequently, Nα-benzoyl-L-Arginine-7-amido-4-methylcoumarin (100 μM; Sigma cat. No. B7260) and trypsin (0.25 μg) were added, and fluorescence determined.
Preparation of Mouse cDNA Clones
Generation of Oocyte cDNA Library.
Total RNA was isolated from 2172 denuded mouse oocytes, treated in vitro for 15 h with 50 μM FF-MAS, according to the RNAzol B® RNA isolation protocol (Campro scientific). RNAzol B was added directly to the frozen cell pellets containing approximately 100 oocytes each. Homogenates were pooled and extracted with 0.1 volume of chloroform, shaken for 15 seconds and incubated on ice for 10 minutes. After centrifugation for 15 minutes at 14000 rpm at 4° C. the aqueous phase was collected. Total RNA was precipitated by adding an equal volume of isopropanol followed by o/n incubation at 4° C. RNA was centrifuged for 45 minutes at 14000 rpm at 4° C., the pellet was washed once with 700 μl of 70% ethanol followed by centrifugation at 14000 rpm at 4° C. for 30 minutes. The air-dried pellet was finally resuspended in 7.5 μl Rnase free water (Ambion). The total amount of RNA isolated using this procedure was determined using the Ribogreen® RNA quantitation kit (Molecular Probes).
For cDNA synthesis, the SMART® PCR library construction kit (Clontech) was used. The following modifications were introduced. An oligodT(18) primer with EcoRI restriction site (Pharmacia) was annealed to the 3' end of the mRNA and the SMART® oligo extended with an EcoRI restriction site was annealed to the 5' end of the mRNA. The first strand cDNA synthesis reaction was in a reaction buffer containing 50 mM Tris (pH 8.3), 75 mM KCl, 6 mM MgCl2, 2 mM DTT, 1 mM dNTP mix and 200 units Superscript II RNase H Reverse transcriptase (Gibco BRL) for 1 hour at 42° C. Subsequently first strand cDNA was amplified by PCR using a Perkin Elmer thermocycler (9600). The PCR was performed in a total volume of 100 μl reaction buffer containing 1× Klen Taq PCR buffer (Clontech), 0.2 mM dNTP mix (Clontech), 0.2 mM 5' EcoRI-SMART primer, 0.2 mM NotI-EcoRI-dT(18) primer (Pharmacia) and 1× Advantage Klen Taq Polymerase Mix (Clontech) starting with 1 minute denaturation at 95° C. followed by 28 cycles of 15 seconds at 95° C. and 5 minutes at 68° C.
After purification on a Qiaquick spin column (QIAGEN) the cDNA was digested with EcoRI (Pharmacia) at 37° C. followed by heat inactivation at 70° C. for 10 minutes. cDNA was purified twice using two subsequent Qiaquick spin columns and finally resuspended in 50 μl 10 mM Tris-CL (pH 8.5). DNA concentration was determined by measuring absorbance at 260 nm using a Genequant spectrophotometer.
Size Fractionation of cDNA
cDNA was size fractionated using agarose gel electrophoresis and extracted from the gel matrix using the QiaexII Agarose Gel Extraction Kit (Qiagen). DNA was eluted in 20 μl H2O, purified on a Qiaquick spin column (Qiagen) and eluted in 50 μl H2O. The samples were precipitated by adding 0.1 volume of 3M Sodium Acetate, 10 μg of glycogen and 2.5 volumes of ethanol (96% V/v) followed by 1 h incubation at -20° C. The size fractionated cDNA was collected by centrifugation at 14.000 rpm for 20 minutes at 4° C. The DNA pellet was washed with 70% ethanol and air dried before it was dissolved in MQ. DNA concentration was determined using the PicoGreen® dsDNA Quantitation Kit (Molecular Probes).
After EcoRI digestion, 200 ng oocyte cDNA was ligated into 500 ng of predigested and dephosphorylated λGT11 phage arms in a buffer containing 50 mM Tris-Cl pH 7.8, 10 mM MgCl2, 10 mM dithiotreitol, 1 mM ATP, and 750 units/ml T4 ligase (Pharmacia). The reactions were incubated o/n at 16° C. The complete ligation reaction was finally packaged into a Max Plax® packaging extract (Epicentre) as described in the product information sheet.
Isolation and Characterization of Mouse PAD6
PCR Amplification of Phage Clones
Single plaques were incubated for at least one hour in 100 μl λ phage buffer (10 mM Tris-HCL pH 8.3, 100 mM NaCl2 and 10 mM MgCl2). From each eluted plaque 2.5 μl was PCR-amplified using λGT11 primers (SEQ ID NO:5 and SEQ ID NO:6). PCR reactions were performed on the PE9700 (9600 mode, Perkin Elmer), one cycle of 5 min at 94° C., 30 cycles of 30 sec at 94° C., 30 sec at 55° C. and 3 min at 72° C., followed by one cycle of 5 min at 72° C. PCR products were analyzed by agarose gel electrophoresis and selected on size, purity and concentration. Only single bands of 500 bp or more were selected for sequencing.
DNA Sequence Analysis
750 clones from the mouse oocyte cDNA library were analyzed by DNA sequencing after insert amplification by PCR. Sequence analysis was performed using the Big Dye DNA sequencing ready reaction protocol (Perkin Elmer) and samples were analyzed on the ABI377 automatic DNA sequencer (Perkin Elmer). Sequences were blasted against several databases a.o.: gb111rod, genpept, EMrodESTs59 and EMhumanESTs59 databases using BLASTN or TBLASTN in an automated procedure and annotated on basis of homology to gene(s) with known functions.
Identification and Characterization of PAD6.
One of the sequences obtained shows strong homology with peptidyl arginine deiminase III. Based on homology searches it has been established that this clone, 1B11, encodes a novel peptidyl argine deiminase that has been termed PAD6.
The 5'-end of mouse PAD6 cDNA could be amplified from a mouse ovary cDNA library. The cDNA of this library had been cloned directionally into NotI-SalI sites (5'-3') of the pSPORT vector (Life Technologies). This vector contains the M13 forward and SP6 promotor sequences 5' from the NotI site which have been used in the 5' RACE PCR in combination with two PAD6 specific reverse primers. The first PCR was performed with the M13F primer (SEQ ID NO:7) and the gene specific reverse primer (SEQ ID NO:8). This PCR product was diluted fifty times and one microliter of this dilution was used as template in the nested PCR with the SP6 primer (SEQ ID NO:9) and the nested gene specific reverse primer (SEQ ID NO:10). Both PCR reactions were performed in a total volume of 50 μl reaction buffer containing 1× Klen Taq PCR buffer (Clontech), 0.2 mM dNTP mix (Clontech) and 1× Advantage Klen Taq Polymerase Mix (Clontech) starting with 5 minutes of denaturation at 94° C. followed by 30 cycles of 30 seconds at 94° C., 30 seconds at 56° C., 3 minutes at 72° C. with an final extension of 5 minutes at 72° C.
Bands in the nested PCR products were cloned in the TA Topo PCR2.1 vector (Invitrogen) following the product information sheet and sequenced. It was found that a 1800 bp 5' RACE fragment completed the mouse PAD6 clone. The sequence of the full-length mouse cDNA is given in SEQ ID NO:2.
Based on DNA sequence information obtained, gene specific PCR primer sets were designed and used in RT-PCR experiments to confirm the tissue-specific expression profile. The data obtained (FIG. 1) confirm the oocyte/ovary- (and testis-) specific expression for mouse PAD6. (SEQ ID NO:8 and SEQ ID NO:13 were used as primers).
In Situ Hybridization (ISH)
To further study the expression of PAD6 in the gonads, in situ hybridization (ISH) was performed on sections of mouse ovary and testis.
Ovaries of day 7 and adult mice were fixed in 4% buffered formalin for 24 hours at room temperature. The tissues were embedded in paraffin. Paraffin sections (5 μm) were cut, mounted on Superfrost plus microscope slides, and allowed to dry overnight at 37° C. The slides were baked at 60° C. for two hours.
Tissue sections were dewaxed in xylene en rehydrated in descending concentrations of ethanol. Slides were washed for 20 min shaking in 0.2M HCl, followed by two washes in DEPC (di-ethylpyrocarbonate) treated Milli Q. The sections were treated with proteinase K (1 μg/ml) in digest buffer (100 mM Tris, 50 mM EDTA pH 8) for 30 min at 37° C. Digestion was stopped in prechilled 0.2% glycine in PBS for 10 min shaking at room temperature (RT). The slides were acetylated for 5 min with 0.25% acetic anhydride in 0.1 M triethanolamine buffer, followed by two washes in DEPC treated Milli Q. Sections were prehybridised at hybridisation temperature in a humid chamber with prehybridisation mix, containing 52% formamide, 21 mM Tris, 1 mM EDTA, 0.33 M NaCl, 10% dextran sulphate, 1×Denhardt's solution, 100 μg/ml salmon sperm DNA, 100 μg/ml tRNA and 250 μg/ml yeast total RNA. The slides were covered with a glass coverslip. After two hours coverslips were replaced by coverslips holding 100 μl probe hybridization mix, containing prehybridization mix with the following additions: 0.1 mM DTT, 0.1% sodium thiosulphate, 0.1% SDS and 200 ng/ml DIG-labeled probe.
DIG-labeled probes were generated by in vitro transcription from a linear DNA template, using DIG-dUTP and DNA-dependent RNA polymerases (SP6 and T7). The promoter site of each RNA polymerase was attached to gene specific sequences allowing the generation of a PCR fragment containing the SP6 promoter site at the 5' and the T7 promoter at the 3' site. In general, probes from about 250-500 nucleotides were made located at the 5' end of SEQ ID NO:2. After in vitro transcription a small amount of the probe was analyzed on a 1.5% agarose gel to confirm successful in vitro transcription. Probe concentrations were estimated by spotting serial dilutions (including control DIG-RNA (100 ng/μl)) on a Hybond N.sup.+ membrane followed by anti-DIG alkaline phosphatase Fab' fragments (anti-DIG-AP) and NBT/BCIP colour substrate incubation.
The hybridization was carried out overnight (16 hours) in a humid chamber at 42° C. or 50° C. Slides were then washed in 2×SSC, shaking for 15 min, followed by washes in 2×SSC, 1×SSC and 0.1×SSC for 15 min shaking at hybridization temperature. Sections were digested by Ribonuclease A (20 μg/ml) in RNase buffer (0.6 M NaCl, 20 mM Tris, 10 mM EDTA) for 1 hour at 37° C. After two washes (5 min shaking RT) in prechilled PBS and one wash in buffer 1 (100 mM maleic acid, 150 mM NaCl), the sections were incubated for 30 min with blocking solution (1 g/ml blocking reagent in buffer 1). Then the sections were incubated with anti-DIG-AP, diluted 1:500 in blocking solution, for 1 hour at RT. After two washes in buffer 1 (15 min shaking RT), the slides were carefully wiped dry around the tissue and the sections were encircled with a DAKO-pen. The sections were covered with NBT/BCIP colour development reagent and incubated in a humid chamber at RT. After two hours the sections were examined under a microscope. If no or only weak staining was observed the incubation was continued overnight at 4° C. and the next day at RT. Finally, the slides were rinsed in water and optionally counterstained with Mayer's hematoxyline 1:5 for three seconds. Slides were mounted in Kaisers glycerol gelatin.
As shown in FIG. 2, PAD6 is expressed in the ovary exclusively in oocytes.
PAD6 mRNA has high expression levels in oocytes of primary, secondary and antral follicles, but is also expressed in oocytes from primordial follicles. Based on the data obtained sofar the expression level of PAD6 miRNA decreases in oocytes of antral follicles suggesting that the function of PAD6 is most likely required during early stages of oogenesis. Although RT-PCR data revealed testis expression of PAD6, no expression above background level of PAD6 mRNA was detected using ISH analysis suggesting low levels of expression of PAD6 in the testis.
Isolation and Characterization of Human PAD6
A BLAST search using the full-length mouse PAD6 cDNA as a query against the EM63hsGeno(new) databases identified the human homologue of PAD6. This search only identified the C-terminal region of the coding sequence of human PAD6. To extend the sequence in the 5' direction primers were designed and a 5' RACE PCR was performed on human ovary Marathon Ready cDNA (Clontech) using the Marathon Ready® cDNA user manual. The first PCR was performed under the following conditions: a denaturation of 30 seconds at 94° C., 5 cycles of 5 seconds at 94° C. and 3 minutes at 72° C., 5 cycles of 5 seconds at 94° C. and 3 minutes at 70° C. and 25 cycles of 5 seconds at 94° C. and 3 minutes at 68° C. A 50-fold dilution of this first PCR product served as template in the second, nested PCR reaction using the same PCR conditions. An expected band of ≈650 bp was cloned in the TA Topo PCR2.1 vector (Invitrogen) and sequenced. This clone contained (by homology) the first 5' 500 base pairs of the coding sequence of human PAD6, thus completing the coding sequence of human PAD6.
PCR primers were selected to amplify the fall length human PAD6 cDNA human ovary RNA. For isolation of human PAD6 cDNA the primers SEQ ID NO:11 and SEQ ID NO:12 were used on Marathon Ready ovary cDNA (Clontech). PCR conditions were: denaturation for 5 minutes at 94° C. followed by 5 cycles of 30 seconds at 94° C. and 3 minutes at 68° C., 28 cycles of 30 seconds at 94° C., 30 seconds at 62° C. and 3 minutes at 72° C. with a final extension of 7 minutes at 72° C.
The full length amplificates of three independent PCR reactions were cloned into the PCR2.1 Topo vector (Invitrogen) and sequenced to determine the consensus nucleotide sequences. Its sequence is shown in SEQ ID NO:4.
Gene specific PCR primer sets were designed (SEQ ID NO:14 and SEQ ID NO:15) and used in RT-PCR experiments to determine the expression profile of human PAD6. RT-PCR on RNA from human testis, uterus, kidney, thymus, liver, brain, heart, lung and spleen, revealed PAD6 expression only in testis (FIG. 3).
Multiple Tissue Northern Blots (Clontech) of human tissues were hybridised with the PCR fragment of human PAD6 (approximately 590 bp; PCR product of primers SEQ ID No:14 and SEQ ID NO:15 extending from nucleotides 464-1052 in SEQ ID NO:4). Probes were labelled with [32P]dCTP and Ready to Go Labellings beads (AP Biotech) according to the manufacturer's instruction using an incubation time of 60 minutes at 37° C. The n-on-incorporated dNTP's were removed on a spin column of Sephadex G50 in a 1 ml syringe.
The blots were prehybridised in Express hybmix (Clontech) for at least one hour at 65° C. For hybridisation 4-8×107 cpm of the denaturated probes were added to the prehybridisation mixture. The blots were hybridised at 65° C. overnight and washed once with 2×SSC, 0.1% SDS at room temperature, twice with 1×SSC, 0.1% SDS at 65° C. and once with 0.1×SSC, 0.1% SDS at 65° C. The hybridised blots were analysed with the STORM 840 Phosphor imager (Molecular Dynamics), scanned on 200 micron and printed with a range of 0-50 after exposure of three days to Kodak storage phosphor screens GP (Molecular dynamics).
In FIG. 4 a single band in ovary with an estimated length of about 3 kB can be seen showing up only in ovary. No signal could be detected in testis, most likely because the level of PAD6 expression in testis is too low to be detected on Northern blots. In situ hybridisation analysis corroborates these results: PAD6 expression could be detected in all types of follicles of human and monkey ovaries and are in this respect similar to the in situ data in mouse. By in situ hybridisation no expression was detected in testis (data not shown).
Expression of Human PAD6 and Determination of PAD6 Activity
Full-length human PAD6 was cloned into the bacterial expression vector pGEX4T1 (AP Biotech) using the Rapid DNA Ligation kit (Boehringer). The recombinant construct (pGEXhPAD6) was characterised by restriction enzyme digestion.
E. coli BL-21 cells transformed with pGEXhPAD6 were grown in 2×YT medium at 25° C. to a cell density of 1.0 at 650 nm. After addition of 0.1 mM isopropyl-β-D-thiogalactopyranoside the culture was grown for an additional 5 hours at 25° C. The cells were centrifuged and resuspended in 0.1 vol of the original culture volume of 20 mM Tris-HCl, pH 7.6, 1 mM EDTA and lysed by sonication on ice. The sonicate was centrifuged at 15000×g for 30 minutes at 4° C. (Sorvall, SS34 rotor) and to the supernatant 1M NaCl, 0.1% of Triton X-100 and 50% glutathion-Sepharose 4B beads in PBS (Pharmacia Biotech, 1 ml to an equivalent of 250 ml initial culture) was added, followed by incubation at 4° C. for 60 minutes with gentle agitation. The beads were then washed three times with 10 bed volumes of a buffer containing 20 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.1% Triton X-100 and 0.1 M NaCl at RT for 5 minutes with gentle agitation. The recombinant hPAD6-gst fusion protein was eluted from the beads in several steps with 10 to 100 mM reduced glutathione in 50 mM Tris-Cl pH 8.0, 0.1 M NaCl and 0.1% Triton X-100 at 4° C. for 30 minutes with gentle agitation. The eluates were stored with 10% glycerol at -20° C. for determination of enzymatic activity. The purity of the protein was estimated to be 90% based on SDS PAGE analysis.
Determination of PAD6 Enzyme Activity.
The activity of the PAD was determined by the formation of citrulline in Soybean Trypsin Inhibitor (STI) as a substrate. In contrast to the original STI, citrullinated STI is unable to inhibit trypsin activity. Therefore, an increased activity of trypsin, as detected with a fluorescent trypsin substrate, indicates PAD activity.
For PAD activity, the reaction mixture consisted of 100 mM HEPES (pH 7.5), 5 mM CaCl2, 2 mM DTT, 0.17 μg STI and an aliquot of the purified enzyme solution [either GST-PAD6 or the commercial available PAD (Sigma), derived form rabbit muscle] in a final volume of 20 μl. After incubation of the assay mixture for 30 minutes at 37° C., 10 μl of the fluorescent substrate Nα-benzoyl-L-Arginine-7-amido-4-methylcoumarin [400 μM in 100 mM HEPES (pH 7.5), 50 mM EDTA] and 101 of trypsin solution [0.25 μg in 100 mM HEPES (pH 7.5)] were added subsequently. Fluorescence measurements (excitation 360 nm, emission 460 nm) were started directly in a Victor V at room temperature, and were continued for one hour.
PAD6 activity could be detected as can be seen in FIG. 5.
151682PRTMus musculus 1Met Ser Phe Gln Asn Ser Leu Ser Leu Ser Leu Val Asn Pro Thr His 1 5 10 15Ala Leu Cys Met Val Gly Met Glu Ile Thr Leu Asp Ile Ser Lys Cys 20 25 30Ala Pro Asp Lys Cys Lys Ser Phe Thr Ile Arg Gly Ser Pro Arg Ile 35 40 45Leu Ile His Ile Ser Ser Ser Val Ile Ala Gly Lys Glu Asp Thr Val 50 55 60Val Trp Arg Ser Met Asn His Pro Thr Val Ala Leu Val Arg Met Val 65 70 75 80Ala Pro Ser Pro Thr Val Asp Glu Asp Lys Val Leu Val Ser Tyr Phe 85 90 95Cys Pro Asp Gln Glu Val Pro Thr Ala Thr Ala Val Leu Phe Leu Thr 100 105 110Gly Ile Glu Ile Ser Leu Glu Ala Asp Ile Tyr Arg Asp Gly Gln Leu 115 120 125Asp Met Pro Ser Asp Lys Gln Ala Lys Lys Lys Trp Met Trp Gly Met 130 135 140Asn Gly Trp Gly Ala Ile Leu Leu Val Asn Cys Ser Pro Asn Ala Val145 150 155 160Gly Gln Pro Asp Glu Gln Ser Phe Gln Glu Gly Pro Arg Glu Ile Gln 165 170 175Asn Leu Ser Gln Met Asn Val Thr Val Glu Gly Pro Thr Ser Ile Leu 180 185 190Gln Asn Tyr Gln Leu Ile Leu His Thr Ser Glu Glu Glu Ala Lys Lys 195 200 205Thr Arg Val Tyr Trp Ser Gln Arg Gly Ser Ser Ala Tyr Glu Leu Val 210 215 220Val Gly Pro Asn Lys Pro Val Tyr Leu Leu Pro Thr Phe Glu Asn Arg225 230 235 240Arg Lys Glu Ala Phe Tyr Val Glu Ala Thr Glu Phe Pro Ser Pro Ser 245 250 255Phe Ser Gly Leu Ile Ser Leu Ser Leu Ser Leu Val Glu Lys Ala His 260 265 270Asp Glu Cys Ile Pro Glu Ile Pro Leu Tyr Lys Asp Thr Val Met Phe 275 280 285Arg Val Ala Pro Tyr Ile Phe Met Pro Ser Thr Gln Met Pro Leu Glu 290 295 300Val Tyr Leu Cys Arg Glu Leu Gln Leu Gln Gly Phe Val Asp Ser Val305 310 315 320Thr Lys Leu Ser Glu Lys Ser Lys Val Gln Val Val Lys Val Tyr Glu 325 330 335Asp Pro Asn Arg Gln Ser Lys Trp Leu Gln Asp Glu Met Ala Phe Cys 340 345 350Tyr Thr Gln Ala Pro His Lys Thr Val Ser Leu Ile Leu Asp Thr Pro 355 360 365Arg Val Ser Lys Leu Glu Asp Phe Pro Met Lys Tyr Thr Leu Thr Pro 370 375 380Gly Ser Gly Tyr Leu Ile Arg Gln Thr Glu Asp His Arg Val Ala Ser385 390 395 400Leu Asp Ser Ile Gly Asn Leu Met Val Ser Pro Pro Val Lys Ala Gln 405 410 415Gly Lys Asp Tyr Pro Leu Gly Arg Val Leu Ile Gly Gly Ser Phe Tyr 420 425 430Pro Ser Ser Glu Gly Arg Asp Met Asn Lys Gly Leu Arg Glu Phe Val 435 440 445Tyr Ala Gln Gln Val Gln Ala Pro Val Glu Leu Phe Ser Asp Trp Leu 450 455 460Met Thr Gly His Met Asp Gln Phe Met Cys Phe Val Pro Thr Asn Asp465 470 475 480Lys Asn Asn Asp Gln Lys Asp Phe Arg Leu Leu Leu Ala Ser Pro Ser 485 490 495Ala Cys Phe Glu Leu Phe Glu Gln Lys Gln Lys Glu Gly Tyr Gly Asn 500 505 510Val Thr Leu Phe Glu Asp Ile Gly Ala Glu Gln Leu Leu Ser Asn Gly 515 520 525Arg Glu Ser Lys Thr Ile Ser Gln Ile Leu Ala Asp Lys Ser Phe Arg 530 535 540Glu Gln Asn Thr Tyr Val Glu Lys Cys Ile Ser Leu Asn Arg Thr Leu545 550 555 560Leu Lys Thr Glu Leu Gly Leu Glu Asp Lys Asp Ile Ile Leu Ile Pro 565 570 575Gln Leu Phe Cys Leu Glu Gln Leu Thr Asn Val Pro Ser Asn Gln Gln 580 585 590Ser Thr Lys Leu Phe Ala Arg Pro Tyr Phe Pro Asp Met Leu Gln Ile 595 600 605Ile Val Leu Gly Lys Asn Leu Gly Ile Pro Lys Pro Phe Gly Pro Lys 610 615 620Ile Asn Gly Thr Cys Cys Leu Glu Glu Lys Val Cys Gly Leu Leu Glu625 630 635 640Pro Leu Gly Leu Lys Cys Thr Phe Ile Asp Asp Phe Asp Cys Tyr Leu 645 650 655Ala Asn Ile Gly Asp Val Cys Ala Ser Ala Ile Ile Asn Arg Val Pro 660 665 670Phe Ala Phe Lys Trp Trp Lys Met Thr Pro 675 68022055DNAMus musculus 2cagccatgtc ttttcagaac tcactcagcc tgtctctggt caatcccacc catgccctct 60gcatggtagg catggaaatc accttggaca tcagcaagtg tgcaccagac aagtgcaagt 120ctttcaccat ccgtggttcc cccaggatct tgatccacat ctctagctcc gtcatcgctg 180gcaaagagga cactgtggtc tggaggtcaa tgaaccatcc cacagtggca ttggtgagga 240tggtggcgcc cagccccact gtggatgaag acaaggtgct ggtctcctac ttctgtcctg 300accaagaagt ccccacggcc acagctgtgc tgtttctcac cggcatcgag atctccctgg 360aggcagacat ctatcgagat ggacaactgg acatgccaag tgataagcaa gctaagaaaa 420aatggatgtg gggtatgaac ggctggggag ccatcctgct tgtgaattgt agccctaatg 480ctgtgggcca gcctgatgaa cagtcctttc aggagggccc cagagaaata cagaacctgt 540ctcagatgaa tgtaactgtg gagggcccca ccagcatcct acagaattac cagttgatcc 600tacatacctc cgaagaagag gcgaagaaga caagagtcta ctggtctcag agaggctcct 660ctgcgtatga actggtggtg ggacccaaca agcctgtcta tctcctgcct acctttgaga 720accgtaggaa agaggctttc tacgtagaag ccacggaatt cccatctccc agcttctcgg 780gcctgatctc cttgtcactc tccctagtag aaaaggctca cgacgagtgc atcccagaga 840ttccgctcta taaggataca gtgatgttcc gggtggcacc ttatatcttc atgcccagca 900cccagatgcc tctagaggtt tacctgtgca gggagctaca gctgcaaggc tttgtggact 960cagtgaccaa gctgagcgag aagagcaaag tgcaggtggt aaaggtctat gaggacccca 1020accgccagag caagtggctc caggacgaga tggctttctg ctatactcag gctcctcaca 1080agacggtgtc attgatcctt gacaccccaa gggtttccaa gctggaagac ttccccatga 1140aatacacact gacccctggc tctggctacc tgatccgaca aactgaggac caccgggtgg 1200ctagcctgga ttccatcggg aacctgatgg tatctccgcc tgtcaaggct cagggcaaag 1260actaccctct agggagggtc ctcattggtg gcagctttta ccccagctct gagggccggg 1320acatgaacaa gggcctgcga gaattcgtgt atgcccagca ggtgcaggcc cctgtggaac 1380tcttctcgga ctggctgatg accggtcaca tggatcaatt catgtgcttt gtccctacca 1440atgataaaaa caacgaccag aaggacttcc gcctgctgct ggccagcccc agtgcctgct 1500ttgagctgtt cgaacagaag cagaaggaag gctatgggaa cgtgaccctg tttgaagaca 1560ttggagcaga acagctcctt tctaatggga gggagagcaa aactatttcc caaatcctgg 1620ctgacaagag ttttcgagag cagaacacct atgttgagaa gtgtatcagc ctgaaccgca 1680ccctcctgaa gacagaactg ggattggagg acaaggacat catcctgatc ccgcagctct 1740tctgcctgga gcagctgacg aatgtcccct ccaaccagca gagcaccaaa ctcttcgcga 1800ggccgtactt ccccgacatg ctgcagataa tcgtgttggg caagaacctt ggaatcccca 1860agccctttgg gcccaaaatc aatggcacct gctgcctaga agagaaagtg tgtggattac 1920tggagcccct gggtctcaag tgcaccttca ttgatgattt tgactgctac ctggccaaca 1980taggggacgt ctgtgccagt gccatcataa acagggtgcc atttgcattc aagtggtgga 2040agatgacccc ataaa 20553686PRTHomo sapiens 3Met Ser Phe Gln Ser Ile Ile His Leu Ser Leu Asp Ser Pro Val His 1 5 10 15Ala Val Cys Val Leu Gly Thr Glu Ile Cys Leu Asp Leu Ser Gly Cys 20 25 30Ala Pro Gln Lys Cys Gln Cys Phe Thr Ile His Gly Ser Gly Arg Val 35 40 45Leu Ile Asp Val Ala Asn Thr Val Ile Ser Glu Lys Glu Asp Ala Thr 50 55 60Ile Trp Trp Pro Leu Ser Asp Pro Thr Tyr Ala Thr Val Lys Met Thr 65 70 75 80Ser Pro Ser Pro Ser Val Asp Ala Asp Lys Val Ser Val Thr Tyr Tyr 85 90 95Gly Pro Asn Glu Asp Ala Pro Val Gly Thr Ala Val Leu Tyr Leu Thr 100 105 110Gly Ile Glu Val Ser Leu Glu Val Asp Ile Tyr Arg Asn Gly Gln Val 115 120 125Glu Met Ser Ser Asp Lys Gln Ala Lys Lys Lys Trp Ile Trp Gly Pro 130 135 140Ser Gly Trp Gly Ala Ile Leu Leu Val Asn Cys Asn Pro Ala Asp Val145 150 155 160Gly Gln Gln Leu Glu Asp Lys Lys Thr Lys Lys Val Ile Phe Ser Glu 165 170 175Glu Ile Thr Asn Leu Ser Gln Met Thr Leu Asn Val Gln Gly Pro Ser 180 185 190Cys Ile Leu Lys Lys Tyr Arg Leu Val Leu His Thr Ser Lys Glu Glu 195 200 205Ser Lys Lys Ala Arg Val Tyr Trp Pro Gln Lys Asp Asn Ser Ser Thr 210 215 220Phe Glu Leu Val Leu Gly Pro Asp Gln His Ala Tyr Thr Leu Ala Leu225 230 235 240Leu Gly Asn His Leu Lys Glu Thr Phe Tyr Val Glu Ala Ile Ala Phe 245 250 255Pro Ser Ala Glu Phe Ser Gly Leu Ile Ser Tyr Ser Val Ser Leu Val 260 265 270Glu Glu Ser Gln Asp Pro Ser Ile Pro Glu Thr Val Leu Tyr Lys Asp 275 280 285Thr Val Val Phe Arg Val Ala Pro Cys Val Phe Ile Pro Cys Thr Gln 290 295 300Val Pro Leu Glu Val Tyr Leu Cys Arg Glu Leu Gln Leu Gln Gly Phe305 310 315 320Val Asp Thr Val Thr Lys Leu Ser Glu Lys Ser Asn Ser Gln Val Ala 325 330 335Ser Val Tyr Glu Asp Pro Asn Arg Leu Gly Arg Trp Leu Gln Asp Glu 340 345 350Met Ala Phe Cys Tyr Thr Gln Ala Pro His Lys Thr Thr Ser Leu Ile 355 360 365Leu Asp Thr Pro Gln Ala Ala Asp Leu Asp Glu Phe Pro Met Lys Tyr 370 375 380Ser Leu Ser Pro Gly Ile Gly Tyr Met Ile Gln Asp Thr Glu Asp His385 390 395 400Lys Val Ala Ser Met Asp Ser Ile Gly Asn Leu Met Val Ser Pro Pro 405 410 415Val Lys Val Gln Gly Lys Glu Tyr Pro Leu Gly Arg Val Leu Ile Gly 420 425 430Ser Ser Phe Tyr Pro Ser Ala Glu Gly Arg Ala Met Ser Lys Thr Leu 435 440 445Arg Asp Phe Leu Tyr Ala Gln Gln Val Gln Ala Pro Val Glu Leu Tyr 450 455 460Ser Asp Trp Leu Met Thr Gly His Val Asp Glu Phe Met Cys Phe Ile465 470 475 480Pro Thr Asp Asp Lys Asn Glu Gly Lys Lys Gly Phe Leu Leu Leu Leu 485 490 495Ala Ser Pro Ser Ala Cys Tyr Lys Leu Phe Arg Glu Lys Gln Lys Glu 500 505 510Gly Tyr Gly Asp Ala Leu Leu Phe Asp Glu Leu Arg Ala Asp Gln Leu 515 520 525Leu Ser Asn Gly Arg Glu Ala Lys Thr Ile Asp Gln Leu Leu Ala Asp 530 535 540Glu Ser Leu Lys Lys Gln Asn Glu Tyr Val Glu Lys Cys Ile His Leu545 550 555 560Asn Arg Asp Ile Leu Lys Thr Glu Leu Gly Leu Val Glu Gln Asp Ile 565 570 575Ile Glu Ile Pro Gln Leu Phe Cys Leu Glu Lys Leu Thr Asn Ile Pro 580 585 590Ser Asp Gln Gln Pro Lys Arg Ser Phe Ala Arg Pro Tyr Phe Pro Asp 595 600 605Leu Leu Arg Met Ile Val Met Gly Lys Asn Leu Gly Ile Pro Lys Pro 610 615 620Phe Gly Pro Gln Ile Lys Gly Thr Cys Cys Leu Glu Glu Lys Ile Cys625 630 635 640Cys Leu Leu Glu Pro Leu Gly Phe Lys Cys Thr Phe Ile Asn Asp Phe 645 650 655Asp Cys Tyr Leu Thr Glu Val Gly Asp Ile Cys Ala Cys Ala Asn Ile 660 665 670Arg Arg Val Pro Phe Ala Phe Lys Trp Trp Lys Met Val Pro 675 680 68542092DNAHomo sapiens 4tagcgtggag ggccgagcca tgtccttcca gagtatcatc cacctgtccc tggacagccc 60tgtccatgcc gtttgtgtgt tgggcacaga aatctgcttg gatctcagcg ggtgtgcccc 120ccagaagtgc cagtgcttca ccatccatgg ctctgggagg gtcttgatcg atgtggccaa 180cacggtgatt tctgagaagg aggacgccac catctggtgg cccctgtctg atcccacgta 240cgccacagtg aagatgacat cgcccagccc ttccgtggat gcggataagg tctcggtcac 300atactatggg cccaacgagg atgcccccgt gggcacagct gtgctgtacc tcactggcat 360tgaggtctct ctagaggtag acatctaccg caatgggcaa gttgagatgt caagtgacaa 420acaggctaag aaaaaatgga tctggggtcc cagcggttgg ggtgccatcc tgcttgtgaa 480ttgcaaccct gctgatgtgg gccagcaact tgaggacaag aaaaccaaga aagtgatctt 540ttcagaggaa ataacgaatc tgtcccagat gactctgaat gtccaaggcc ccagctgtat 600cttaaagaaa tatcggctag tcctccatac ctccaaggaa gagtcgaaga aggcgagagt 660ctactggccc caaaaagaca actccagtac ctttgagttg gtgctggggc ccgaccagca 720cgcctatacc ttggccctcc tcgggaacca cttgaaggag actttctacg ttgaagctat 780agcattccca tctgccgaat tctcaggcct catctcctac tctgtgtccc tggtggagga 840gtctcaagac ccgtcaattc cagagactgt gctgtacaaa gacacggtgg tgttccgggt 900ggctccctgt gtcttcattc cctgtaccca ggtgcctctg gaggtttacc tgtgcaggga 960gctgcagctg cagggttttg tggacacagt gacgaagctg agtgagaaga gcaacagcca 1020ggtggcatct gtctatgagg accccaaccg cctgggcagg tggctccagg atgagatggc 1080cttctgctac acccaggctc cccacaagac aacgtccttg atcctcgaca cacctcaggc 1140cgccgatctc gatgagttcc ccatgaagta ctcactgagc cctggtattg gctacatgat 1200ccaggacact gaggaccata aagtggccag catggattcc attgggaacc tgatggtgtc 1260cccacctgtc aaggtccaag ggaaagagta cccgctgggc agagtcctca ttggcagcag 1320cttttacccc agcgcagagg gccgggccat gagtaagacc ctccgagact tcctctatgc 1380ccagcaggtc caagcgccgg tggagctcta ctcagattgg ctaatgactg gccacgtgga 1440tgagttcatg tgcttcatcc ccacagatga caagaatgag ggcaaaaagg gcttcctgct 1500gctcctggcc agccccagtg cctgctataa actgttccga gagaaacaga aggaaggcta 1560tggcgacgct cttctgtttg atgagcttag agcagatcag ctcctgtcta atggaaggga 1620agccaaaacc atcgaccaac ttctggctga tgaaagcctg aagaagcaga atgaatacgt 1680ggagaagtgc attcacctga accgtgacat cctgaagacg gagctgggcc tggtggaaca 1740ggacatcatc gagattcccc agctgttctg cttggagaag ctgactaaca tcccctctga 1800ccagcagccc aagaggtcct ttgcgaggcc atacttccct gacctgttgc ggatgattgt 1860gatgggcaag aacctgggga tccccaagcc ttttgggccc caaatcaagg ggacctgctg 1920cctggaagaa aagatttgct gcttgctgga gcccctgggc ttcaagtgca ccttcatcaa 1980tgactttgac tgttacctga cagaggtcgg agacatctgt gcctgtgcca acatccgccg 2040ggtgcccttt gccttcaaat ggtggaagat ggtaccttag acccaggccc ta 2092524DNAArtificial SequenceDescription of Artificial Sequence Synthetic Primer 5ttgacaccag accaactggt aatg 24624DNAArtificial SequenceDescription of Artificial Sequence Synthetic Primer 6ggtggcgacg actcctggag cccg 24717DNAArtificial SequenceDescription of Artificial Sequence Synthetic Primer 7gttttcccag tcacgac 17820DNAArtificial SequenceDescription of Artificial Sequence Synthetic Primer 8cgtcagctgc tccagcagaa 20919DNAArtificial SequenceDescription of Artificial Sequence Synthetic Primer 9gatttaggtg acactatag 191020DNAArtificial SequenceDescription of Artificial Sequence Synthetic Primer 10gtgcggttca ggctgataca 201121DNAArtificial SequenceDescription of Artificial Sequence Synthetic Primer 11agcgtggagg gccgagccat g 211224DNAArtificial SequenceDescription of Artificial Sequence Synthetic Primer 12agggcctggg tctaaggtac catc 241320DNAArtificial SequenceDescription of Artificial Sequence Synthetic Primer 13ctgatgaccg gcacatggat 201420DNAArtificial SequenceDescription of Artificial Sequence Synthetic Primer 14ggcggttggg gtcctcatag 201520DNAArtificial SequenceDescription of Artificial Sequence Synthetic Primer 15gccatcctgc ttgtgaattg 20
Patent applications by Jan Albert Gossen, Oss NL
Patent applications by Paul Van Den Boogaart, Oss NL
Patent applications in class Hydrolase (3. )
Patent applications in all subclasses Hydrolase (3. )