Abstract:
The present invention relates to the cloning of human pro-protein converting enzyme 5 (PC5) CDNA isolated from human adrenal gland messenger RNA. Additionally, this invention relates to a method for reducing restenosis occurring at an injured vascular site comprising delivering to the injured site an antisense nucleic acid to suppress the expression of human PC5.

Description:
This is a national stage application of PCT/CA97/00535 which claims the benefit of provisional application No. 60/021,008, filed Jul. 26, 1996. 
    
    
     FIELD OF THE INVENTION 
     This invention relates to protein processing enzymes or pro-hormone convertases (PCs), specifically to PC5, more specifically to the human PC5. 
     BACKGROUND OF THE INVENTION 
     Pro-hormone convertases (PCs) belong to a family of enzymes responsible for the maturation of proteic precursors into active proteins or enzymes. Up to now, many human enzymes of that family have been identified, namely furin, PC1, PC2, PC4 and PC7. Each enzyme has a tissular distribution which may be restricted (for example, PC4 is restricted to male germ cells) or ubiquitous (furin is such an example). Although all these enzymes share the properties of cleaving precursor proteins at basic or dibasic residues, they nevertheless have differing cleavage specificities. The action of a specific pro-hormone convertase is therefore governed by the cleavage sequence of a given protein substrate, and/or by the location of that enzyme in a tissue expressing or responding to a given proteic substrate growth factor or hormone. 
     Renin is an aspartyl protease which makes an important contribution to cardiovascular physiology and pathophysiology through its key role in the synthesis of the vasoactive octapeptide angiotensin II (AII). While the kidney is the primary source of circulating active renin, several additional tissues, including the pituitary and adrenal glands, placenta, uterus, ovary, testes, heart, vasculature and brain express the renin gene (reviewed in  1-4 ). The presence of additional components of the RAS (renin-angiotensin system) in these tissues, including angiotensin converting enzyme (ACE) and angiotensin II receptors, has led to the proposal that certain tissues might contain a locally active tissue renin-angiotensin system (tRAS) although the actual function of the various tRAS is still largely a matter of conjecture. 
     Renin is first synthesized as an enzymatically inactive precursor, prorenin, which is converted to active renin by the proteolytic removal of a 43 amino acid amino-terminal prosegment. The activity of the RAS within any given tissue would, therefore, be dependent on the existence of proteolytic enzymes capable of converting prorenin to active renin and on the expression of such prorenin processing enzymes (PPEs) in the same cells that express prorenin. The identity of the enzyme(s) responsible for the proteolytic activating human prorenin in vivo is still uncertain. Furthermore, it is possible that multiple PPEs exist in humans and these may differ among renin-producing tissues. Biochemical and microscopic studies of renin in the kidney suggest that candidate PPEs should be selective for cleavage of human prorenin at Lys 42 , Arg 43  of the prosegment 5  and would be active in secretory granules of the juxtaglomerular (JG) cells. 6  The lysosomal enzyme cathepsin B has been co-localized with human renin/prorenin in the secretory granules of JG cells and human pituitary lactotrophs 7,8  and has been shown to cleave human prorenin in vitro with a high affinity and selectivity for the proper cleavage site. 9  The prohormone convertase PC1 has also been shown to cleave human prorenin with the correct site-and organelle specificity in transfected cells 10  and to co-localize with renin in the adrenal medulla and derived tumors 11 , but not in JG cells. 12    
     In an effort to identify novel PPEs, we recently determined the distribution of processing enzymes in an established renin-expressing tissue culture cell line derived from an oncogene-induced mouse tumor (As4.1 cells 13 ). One such enzyme, the mouse prohormone convertase PC5, was found. Mouse PC5 is capable of partially cleaving human prorenin. 
     Miranda et al. (38) describe the cDNA and protein sequences for a human PC6 enzyme obtained by PCR from CD4 +  T lymphocytes. PC5 and PC6 are different names given to what appears to be the same enzyme. However, the sequences of Miranda et al. comprise a plurality of substitutions when compared to the present PC5 sequences. Moreover, the size of messenger RNA encoding PC6 and PC5 are similar but not identical. Since the present PC5 sequences were obtained from human adrenals, both enzymes may be isoforms, differentially expressed in tissues and they may have different activities. 
     Prorenin and HIV gp160 are most probably not the only proteic precursors to be recognized and cleaved by PC5. Many growth factors responsible for cell proliferation are cleaved by one or more PCs: they include platelet-derived-growth factors A and B (PDGF&#39;s), epidermal-growth-factor (EGF), insulin-like growth factors I and II (IGF&#39;s), transforming growth factors α and β (TGF&#39;s). Each of these named growth factors has the typical cleavage site motif K/R—(X)  n —R↓ (where n=0,2,4,6). Full biological potency is conferred to these growth factors only after cleavage at these sites, by one or more of the PC enzyme family. There is therefore a possibility that manipulating the expression of the PCs would affect cell proliferation via deficient growth factor activation. 
     Out of the &gt;450,000 patients/year in the U.S. and Canada who undergo percutaneous transluminal coronary angioplasty (PTCA), 30-50% of them will restenose their coronaries within 3-6 months. This flare-up of endothelial and smooth-muscle cells proliferation is due to the activation of numerous regulatory growth factors. Therefore, knowing which enzyme(s) is (are) responsible for this activation, and manipulating the level of expression of this or theses enzyme(s) would be particularly useful to prevent restenosis. 
     STATEMENT OF THE INVENTION 
     The present invention relates to the human PC5 (hPC5). We demonstrate that hPC5 isolated form human adrenals proteolytically activates human prorenin with the expected site- and organelle- specificity and that it is co-expressed with prorenin in the zona glomerulosa of the adrenal cortex. Therefore, PC5 is a prorenin-processing enzyme (PPE). Silencing the expression of PC5 would find a specific application in inhibiting the production of renin, and a method of inhibiting the production of renin is an object of the invention. Since the production of renin is one of targets of the RAS involved in hypertension. Furthermore, we demonstrate that hPC5 is overexpressed in atherosclerotic coronary arteries. Antisense oligonucleotides have been designed, amongst which one has been shown to successfully silence the expression of hPC5 in smooth muscle cells in culture. This antisense inhibited carotid stenosis in a in vivo rabbit carotid injury model. These results indicate that a method of silencing the expression of PC5 would find a specific application in preventing restenosis. 
     PC5 is known to be expressed in CD4 +  T cells, along with furin and PC7. The three enzymes are capable of converting HIV gp160 into its fusiogenic form. Therefore, antisense constructs, particularly the oligonucleotide that successfully inhibited restenosis, will find a use in inhibiting expression of the activity of PC5 towards HIV gp160. 
     The complete amino acid and nucleotide sequence of hPC5 is described hereinbelow and are another object of this invention. Recombinant vectors and hosts comprising as a new insert, whole or part of hPC5, are also an object of the invention. 
     Oligopeptides derived from the proteic sequence of hPC5 are also an object of the invention. 
     Antibodies directed against the whole protein hPC5 or a part thereof are also an object of the invention. 
     Diagnostic methods and kits comprising oligonucleotides or antibodies binding PC5 nucleic acids or protein or peptides are also an object of the invention. 
     This invention will be described hereinbelow by way of specific embodiments, examples and figures which purpose is to illustrate the contemplated aspects of the invention, and not to limit the scope of the invention. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG.  1 : Schematic diagram of the isolated cDNAs encoding HPC5. Restriction enzyme sites used in sub-cloning are denoted. Solid lines represent clones isolated from a phage library. Hatched lines denote the portion of the cDNA isolated by RT-PCR of human adrenal mRNA. The double line represents the portion of the mouse PC5 CDNA (corresponding to the amino terminus of the signal peptide) which was used to complete the cDNA for expression. 
     FIGS.  2 A- 2 B: Nucleotide and derived protein sequence of hPC5 (SEQ ID NOS: 1 and 2, respectively). Proposed signal peptide (solid arrow) and prosegment (open arrow) cleavage sites are denoted based on data from mouse PC5. 15  The underlined sequence represents the portion of the signal peptide from mouse PC5 which was used in the expression vector construction. 
     FIG.  3 : Distribution on PC5 RNA in various human tissues. 
     Each lane contains 2 μg poly-A RNA. Filters were hybridized with a radiolabeled probe for hPC5 as described in Materials and Methods. Shown at left is the migration of single strand size standards in kilobases (Kb). Note that the absolute is signal cannot be compared between the two filters as they were of different ages and hybridized at different times. 
     FIGS.  4 A- 4 B: hPC5 cleaves human prorenin with site and cell specificity. Panel A: GH 4 C 1  cell were co-transfected with expression vectors for the indicated proteins. Supernatants were collected 30 hrs. after transfection and assayed for % active renin [(active renin/total renin)×100]. Bars represent the mean±S.E.M of 9 independent transfections. *=P&lt;0.0001 as compared to proren+pUC, as determined by the Mann-Whitney non-parametric test. Panel B: Resulting secretion of active renin after co-transfection of CHO cells with an expression vector for prorenin and either a control plasmid (pUC) or hPC5.Bars represent the mean±S.E.M of 3 independent transfections. 
     FIG.  5 : Active renin generation in secretory granules of co-transfected GH 4 C 1  cells. Parallel wells of GH 4 C1 cells co-transfected with expression vectors for human prorenin and human PC5 were incubated for 20 min. in medium containing either 50 mmol/L NaCl (control) or 50 mmol/L KCl (a de-polarizing agent which causes the acute release of secretory granules). Percent active renin was calculated as described in the legend to FIG.  4 A. Bars represent the mean±SEM of 3 independent transfections. *=P&lt;0.005 using Student&#39;s t-test. 
     FIG.  6 : Immunodetection of hPC5 and renin/prorenin in renal cortex, human placental cotyledon and adrenal gland. Positively stained areas are denoted by solid arrows. Sections in adrenal cortex are separated by 5 mM to show co-localization in the cells of the zona glomerulosa (g) and absence of staining in the capsule (c) and zona fasciculata (f). Original magnification 25× (kidney and placenta) and 80× (adrenal gland). 
     FIG.  7 : In vivo hybridization analysis of PC5 mRNA in human coronary blood vessels in atherosclerosis. The lower panel show a vessel where a severe lesion was observed. PC5 MRNA was abundantly expressed in this vessel, in the smooth muscle cells in the neointimal formation (see arrows). In comparison, another vessel which is free of any lesion, did not express PC5 mRNA (shown in the upper panel). 
     FIG.  8 : Western blot analysis of PC5 protein in rabbit smooth muscle cells treated with either antisense, sense or mismatch PC5 oligonucleotides. The specific PC5 band is identified (see arrow) by comparison with proteins extracted from rat supraoptic nucleus (SON) and SK-N-MCIXC cells (human neuroepithelioma). In the sample extracted from antisense PC5 treatment, we observe a dramatic decrease in the level of PC5 signal (approximately 2-3 fold decrease) in comparison to the control sense or mismatch PC5 oligos. This indicates that the antisense treatment reduced significantly the protein levels of PC5 in rabbit smooth muscle cells. 
     FIG.  9 : Rabbit in vivo test of the PC5 antisense ODN as compared to the control sense and random ODNs. It shows a decreased stenosis due to the presence of a PC5 antisense when compared to the sense and random controls. 
     FIG.  10 : Proprotein convertase immunoreactivity in human atherectomy specimens. It shows the presence of the enzymes of the pro-hormone convertase family which are present in the specimens. 
     FIG.  11 : Illustrates differences between cDNA sequences of PC6 (Miranda et al. SEQ ID No. 6) and PC5 (present invention). 
     FIG.  12 : Illustrates differences between protein sequences of PC6 (Miranda et al. SEQ ID No. 7) and PC5 (present invention). 
    
    
     DESCRIPTION OF THE INVENTION 
     Materials and Methods 
     cDNA library construction and screening: A cDNA library derived from total human adrenal RNA was constructed by Stratagene (La Jolla, Calif.) in the phage vector Uni-Zap XR. Six hundred thousand phage plaques were screened initially using radioactive probes and standard methodologies. 14  The initial hybridization probe was a 320 base pair DNA fragment derived from reverse-transcriptase PCR of human brain RNA using information derived from an unidentified human CDNA sequence tag in Genbank (Accession # M85522) with a high degree of similarity to the previously cloned mouse PCS. 15  Fragment labeling was carried out using  32 p dCTP and a random primer labeling kit (Boehringer-Mannheim Canada, Laval, Quebec, Canada) according to manufacturer&#39;s instructions. One positive hybridizing phage (hPC5A) was identified. Its insert was sequenced in its entirety using the dideoxy-chain termination method and found to code for an 1150 base pair CDNA with a high degree of sequence similarity to mouse PCS (data not shown). A 1070 base pair fragment (excluding the poly A tail) was excised from hPC5A, labeled and used to re-screen an additional 600,000 phage from the CDNA library. A second phage clone (hPC5B) was isolated and found to contain an 1807 base pair cDNA insert overlapping hPC5A and extending toward the 5′ end of the cDNA (FIG.  1 ). 
     Reverse-transcriptase PCR: One microgram of poly A+ RNA from total human adrenal (Clontech Laboratories, Palo Alto, Calif.) was subjected to reverse transcriptase polymerase chain reaction (RT-PCR) using a published procedure 16  and the following oligonucleotides: 
     Forward oligonucleotide; derived from a region corresponding to the signal peptide of mouse PC5. 15  An artificial HindIII restriction enzyme cleavage site added to the 5′-end of the amplified fragment for the purpose of cloning is underlined: 
     5′-CCAAGCTTGGCTGCTGTGCGTGCTGGC-3′ (SEQ ID No 8) 
     Reverse oligonucleotide; derived from the 5′-end of the phage hPC5B. An internal BgIII restriction enzyme site is underlined: 
     5′-CTGCCTTCAGATCTGTAGTG-3′ (SEQ ID No 9) 
     The entire RT-PCR reaction was repeated 4 times and 4 independently derived clones of the amplified fragment were sequenced and the sequences were compared. The sequence submitted to Genbank (Accession #U49114) represents the consensus sequence, defined as any nucleotide appearing in ¾ clones. 
     Northern blot analysis: Tissue distribution of PC5 MRNA was determined by hybridizing commercially purchased nitrocellulose filters containing aliquots (2 μg) of Poly-A RNA from various human tissues (Clontech Laboratories, Palo Alto, Calif.). The probe used was a complementary RNA derived from the full length hPC5 CDNA. Probe labeling and hybridization were carried out as previously described. 17    
     Expression vector construction: A cDNA fragment from the KpnI site (FIG. 1) to just past the stop codon was excised from the phage hPC5B and combined with a KpnI to HindIII (see above) fragment derived from portions of two independent RT-PCR clones (so as to eliminate errors arising from the Taq polymerase). A region corresponding to the first 16 amino acids of the signal peptide derived from mPC5 was attached to the 5′-end by overlap-extension PCR. 18  Thus, the entire CDNA, encoding amino acids 1-16 derived from the mPC5 signal peptide and the remainder from hPC5, was subcloned into the expression vector RSV-globin 19  which places the cDNA under the control of the RSV promoter and provides a 3′ intron and polyadenylation signal from the rabbit beta globin gene. The entire subcloned fragment was subsequently verified by DNA sequencing. 
     Cell culture and transfection: GH 4 C1 cells were plated in 6-well culture dishes at a density of 5×10 5  cells per well. Twenty four hours later, medium was changed and the cells were transfected by the DEAE-dextran method using a commercial kit (CellPhect Transfection kit, Pharmacia Biotech, Baie D&#39;urfe, Quebec, Canada) according to manufacturer&#39;s instructions. Each well received 0.18 μg of either the hPC5 expression vector or a neutral plasmid vector (pUC18) and 0.18 μg of an expression vector for human prorenin (pRHR1100) or its equivalents in which amino acids 42 or 43 of the prorenin prosegment were mutated to alanine (K/A-2 and R/A-1, respectively 20 ). Supernatants were collected 30 hrs. after transfection and assayed for prorenin and renin content as previously described. 20    
     To verify that conversion of the prorenin occurred in the secretory granules, GH 4 C1 transfected with the human prorenin and hPC5 expression vectors were stimulated to release secretory granules by depolarization using a previously published technique. 21  Forty hrs. after co-transfection, the culture medium in parallel wells of transfected cells was replaced with pre-warmed medium supplemented to a final concentration of 50 mmol/L with either NaCl (control) or KCl (secretagogue). The media were collected after 20 min. and assayed for renin/prorenin. A potassium-dependent increase in the percent active renin contained in cell supernatants was taken as an indication of active renin release from the secretory granules of the transfected cells. Results shown in FIG. 5 represent the mean of three independent transfection experiments. 
     Immunolocalization of hPC5 in human tissues: Human tissue was obtained post-mortem (kidney and adrenal gland) or post-partum (placental cotyledon), fixed in Bouin&#39;s solution and embedded in paraffin. For immunolocalization, 5 μm sections were mounted on gelatin-coated slides, deparaffinized and incubated with a 1:50 dilution of a polyclonal rabbit antiserum raised against a peptide corresponding to the N-terminal 16 amino acids of rat PC5 (PC5.MAP antibody) or a 1:200 dilution of a polyclonal rabbit antiserum against recombinant human prorenin. For kidney and placental specimens, immune complexes were revealed by incubation with protein A-colloidal gold (15 nm particles) synthesized from tetra-chloroauric acid (BDH) according to the method of Ghitescu and Bendayan. 22  Gold particles were enhanced for viewing in the light microscope by incubation with silver (IntenSE ™M Silver Enhancement Kit, Amersham Life Science, Oakville, Ontario, Canada) and sections were counter-stained with hematoxylin and methyl green. Immune complexes on human adrenal sections were detected with a 1:200 dilution of biotin-labeled donkey anti-rabbit IgG and a 1:300 dilution of streptavidin-horseradish peroxidase complex (Amersham Life Science, Oakville, Ontario, Canada) and were incubated with diaminobenzidine and hydrogen peroxide (Sigma Chemicals, St. Louis, Mo.) as chromogen. All positive staining patterns were subsequently verified for specificity by omission of the first antibody. 
     RESULTS 
     The primary sequence of human PC5 is shown in FIG.  2 . We were unable to clone the extreme 5′-end of the CDNA either by the RACE protocol 16  or by using oligonucleotides based on the published sequence of mouse PC5 15,23 , possibly due to a high G/C content of the cDNA in this region. However, based on the published cDNA sequences for rat and mouse PC5 15 , we are confident that we have isolated all but the 5′-most portion of the CDNA corresponding to the first 12 amino acids of the signal peptide. By comparison with the published sequence of mouse PC5, we predict that the cDNA isolated would code for a preproPC5 of 915 amino acids, including a signal peptide and a prosegment of 32 and 84 amino acids, respectively. The deduced sequence of hPC5 is 88% identical to the previously published mouse PC5 cDNA and 96% identical to the mouse PC5 protein. 
     Northern analysis of poly A RNA from a variety of human tissues reveals a major band of approximately 6.6 Kb and a minor band at approximately 3.8 Kb (FIG.  3 ). PC5 RNA is detected in the brain, heart, placenta, lung, thyroid gland and testes and at lower levels in the skeletal muscle, kidney and pancreas, small intestine and stomach. In the adrenal gland, PC5 is particularly enriched in the cortex (FIG.  3 ). 
     Because PC5 RNA appears to be expressed in a number of tissues previously reported to contain active renin, we have tested the ability of hPC5 to cleave human prorenin in a cell co-transfection assay (FIG.  4 A). As has been previously reported 10 , when cultured rat sommatotrophic GH 4 C1 cells are co-transfected with an expression vector encoding human prorenin and a neutral plasmid vector, only unprocessed prorenin is secreted into the culture supernatant. In contrast, if the human prorenin expression vector is co-transfected with an expression vector encoding human PC5, a portion of the expressed prorenin is secreted as active renin. Co-expression of human PC5 with prorenin mutated at either of the basic residues forming the native cleavage site (Lysine 42 or Arginine 43) prevents activation. These results suggest that human PC5 activates human prorenin by proteolytic cleavage at the site previously reported for activation of renin in humans. 5  While human PC5 cleaves human prorenin in GH 4 C1 cells, there is no apparent increase in active renin secretion when co-transfections are carried out in Chinese Hamster Ovary (CHO) cells (FIG.  4 B). One obvious difference in the CHO cell line as compared to GH 4 C1 cells is their lack of secretory granules, suggesting that either human PC5 or human prorenin or both require the secretory granule environment for this proteolytic step. This conclusion is supported by the acute increase in active renin detected in the supernatants of co-transfected GH 4 C1 cells treated for 20 min. with potassium chloride (FIG.  5 ), a de-polarizing agent which causes the release of secretory granules. 21    
     Using a polyclonal antibody raised against a peptide derived from mouse PC5, we have studied the distribution of human PC5 in several human tissues (FIG.  6 ). To date, we have been unable to detect staining for PC5 in the human kidney, although our sections stain positively for renin. In the placental cotyledon, PC5 is located in the syncitiotrophoblast layer of the chorionic villi while anti-renin antibody stains primarily the chorionic mesoderm. In the adrenal gland, the antibodies against both renin and PC5 show a preferential staining of zona glomerulosa cells in the adrenal cortex (g) with very little staining of the capsule (c) and zona fasciculata (f). No staining was evident with omission of the first antibody (data not shown). Thus, our immunohistochemical studies would suggest that, of the three tissues studied, it is likely that prorenin and PC5 are only clearly co-localized in the zona glomerulosa of the human adrenal cortex. 
     DISCUSSION 
     In the present study, we describe the cloning and expression of the human prohormone convertase PC5 and its activity as a human PPE. Co-transfection assays in cultured cells demonstrates that hPC5 activates human prorenin with the expected site-specificity and that this cleavage most likely takes place in dense core secretory granules. In addition, immunohistochemistry of human tissues shows co-localization of hPC5 with renin in the zona glomerulosa of the adrenal cortex. 
     Several lines of evidence suggest that the human adrenal gland contains a physiologically important local RAS: First, RNA encoding angiotensinogen and renin have been detected in preparations from the human adrenal zona glomerulosa, fasciculata and medulla 24,25 , confirming that both renin and its substrate are synthesized within the human adrenal gland. Second, ACE inhibition or blockade of angiotensin receptors inhibits aldosterone release from human adrenal tissue explants 26 , suggesting that the local RAS plays an active role in the regulation of aldosterone secretion from the adrenal gland. Third, tissue explants of human adrenal cortex and aldosterone-secreting adenomas secrete small quantities of active renin 24,26,27 , suggesting that the adrenal cortex expresses a PPE capable of activating human prorenin. Our current results suggest that PC5 could be the PPE responsible for activation of renin in the human adrenal cortex as both renin and hPC5 are immuno-detectable in the zona glomerulosa. Additional circumstantial evidence supports this conclusion: First, centrifugal fractionation of adrenal cortical cells reveals that renin is contained in the “granular” fraction, of intermediate density between vesicles and lysosomes. 28  As our current study suggests that PC5 only cleaves human prorenin in cells containing secretory granules, renin would be in the appropriate intracellular compartment to be activated by PC5 in the adrenal cortex. Second, rats transgenic for mouse Ren-2 renin [TGR(mRen-2)27] display fulminant hypertension 29  which correlates best with the expression of the mouse prorenin in the adrenal gland. 30-32  As previous studies have demonstrated that PC5 is capable of activating mouse Ren-2 prorenin, but not rat prorenin ( 23 and data not shown) it is possible that the TGR(mRen-2)27 transgenic rat is a model for activation of a tissue RAS by the fortuitous juxtaposition of prorenin with an appropriate PPE in the adrenal cortex. These results also raise the possibility that the tissue-distribution of PPEs and their apparent selectivity in activating prorenin from different species could lead to differing functions of the tissue RAS between rodents and humans. 
     The principal source of circulating active renin in humans is the JG cells of the kidney. Although low levels of hPC5 RNA can be detected by Northern blot analysis in a sample of total kidney poly-A RNA (FIG.  3 ), we were unable to localize PC5 immunostaining in kidney sections (FIG. 6) raising the possibility that PC5 is expressed at low levels in diffuse cell types in the kidney. Thus, while these results do not formally rule out PC5 as a PPE in the kidney, our inability to detect it in JG cells makes it unlikely that it plays a major role in the production of renal renin. In contrast, relatively abundant amounts of PC5 mRNA and protein were detected in the placenta although evidence suggests that placental cells in culture 33  and in vivo 34  only secrete prorenin. However, immunostaining revealed that the cells producing PC5 and prorenin in the human placenta are distinct. It is also unlikely that PC5 would activate prorenin once the two proteins are secreted due to the apparent requirement of a granular environment for the cleavage of prorenin by hPC5 in transfected cells. Thus, in contrast to the case in the adrenal gland, it is unlikely that PC5 expressed in the human placenta would activate placental prorenin. 
     In the mouse, two forms of hPC5 have been predicted based on cloned cDNAs; the first would be analogous to the hPC5 cDNA described in this study and to that cloned from rat tissues 15,23  while the second, called PC6B, is extended at its 3′-end due to a differential RNA splicing event. 35  Although the hPC5 CDNA we have cloned is only roughly 3 Kb in length, the major RNA band seen in human tissues is of approximately 6.6 Kb. The identity of the longer band hybridizing to the hPC5 probe is currently unknown. It should be noted that neither of the CDNA clones isolated from a screening of 1.2 million phage from the adrenal library was extended at its 3′-end (FIG.  1 ), although the probes used in their isolation cover the region of homology with the mouse PC6B variant. 35  In mouse tissues, expression of the PC6B variant is restricted to few tissues 35  while the abundance of the 6.6 Kb detected with the hPC5 probe is directly proportional to the abundance of the 3.8 Kb band. Hybridization of RNA blots from rodent tissues using a PC5 probe also reveals RNA bands of 3.8, 6.5 and 7.5 Kb 15,35  and use of a PC5-specific probe reveals a band at 6.5 Kb. Thus, it is possible that additional PC5 RNA species exist in mammals that are extended at their 5′-ends. Alternatively, human tissues may be particularly enriched in a homologue to PC6B which was not picked up in our screenings. Recent data suggest that the alternate C-terminal tail present on PC6B may serve to retain the enzyme in the Golgi network, while the “short” form of mouse PC5 is targeted to dense core secretory granules (N. G. Seidah, unpublished). These data and the results of our co-transfection assays (FIG. 4) would suggest that the “short” form of hPC5 described here is the form which would be active in renin processing in secretory granules. 
     The PC5 enzymes isolated from humans and mice show a remarkably high degree of conservation at the nucleotide and protein sequence levels. This degree of similarity is higher than that seen for the other mammalian PC enzymes which seem to diverge in the C-terminal half of the enzyme. 36,37  This high degree of sequence conservation may reflect an essential function of PC5 (and the C-terminus of PC5) in mammals. 
     PC5 is Linked to Smooth Muscle Proliferation 
     To investigate which PC could be a potential target of smooth muscle cell proliferation, we tested if any of the PCs were affected in the process of restenosis, wherein such proliferation is observed. Changes in PC levels in the process of restenosis is a distinct possibility since in previous studies using animal models or cell lines, we have shown that PC levels can be regulated or even be induced. We thus obtained human restenosed coronary tissues from patients. These tissues were screened for each of the PC mRNAs using in vivo hybridization histochemistry in order to obtain information within an anatomical context. Coronaries with partial or total occlusions demonstrated dramatically increased PC5 MRNA levels within smooth muscle tissues, whereas coronary tissue without occlusions were PC5 negative. These results indicate that PC5 is either strongly up-regulated or induced in the human coronary arteries during the active process of stenosis (FIG.  7 ). To our knowledge this is the first indication that a specific PC is directly linked to smooth muscle proliferation. 
     These results suggested that if PC5 enzymatic activity could somehow be inhibited or the upregulation of PC5 MRNA could be prevented, this may attenuate or stop the process of restenosis. This could occur through the inhibition of the processing function of this enzyme on the numerous growth factors that are involved in the formation of the coronary lesion. If these growth factors are not processed they will remain biologically inactive. Our approach was to test the effectiveness of PC5 antisense inhibition on smooth muscle proliferation in vitro. 
     A specific antisense oligonucleotide (ODN) was shown to drastically inhibit smooth muscle proliferation using an in vitro model of rabbit smooth muscle in culture. Incubating rabbit smooth muscle cells with a PC5 antisense 17-mer oligonucleotide shown in Table 1 caused a dose-dependent inhibition smooth muscle proliferation with a maximal inhibitory effect of 81.6%+1.6% at 10 mM (mean of three experiments done in quadruplicates). This inhibitory effect is highly significant (P=0.0001) as compared to controls which included either a sense or a mismatched oligonucleotide used at the same concentration (see Table 1). In addition we found that the expression of PC5 is decreased in the affected cells (FIG.  8 ). When compared to other targets, such as c-myc, this approach was much more effective in inhibiting smooth muscle proliferation, as the best effects of antisense c-myc resulted in 71.7+3.5% (means of three experiment done in quadruplicate) inhibition (mean of three experiment done in quadruplicates). These results are indicative of an in vivo effect since silencing PC5 would impede muscle cell proliferation and restenosis. 
     Cholesterol Conjugation of Oligonucleotides 
     Phosphorothioate antisense ODNs were synthesized on a DNA/RNA synthesizer following standard procedure (Applied Biosystems). Conjugation of oligomers with cholesterol was achieved with 3′-cholesterol-VN CPG (Clontech), a virtual nucleotide (VN) glass reagent that introduces a cholesterol label to the 3′ terminus of an oligonucleotide via solid-phase synthesis. When ODN synthesis. When ODN synthesis was completed, oligomers were removed from the column with 30% NH 4 OH (1 hour at room temperature), and then deprotected for 8 hours at 60° C. Oligos were purified and detritylated with oligonucleotide purification cartridges (Applied Biosystems), and then lyophilized with a centrifugal evaporator (Savant SpeedVac). 
     In Vivo Arterial ODN Transfection 
     New Zealand rabbits male or female (2 Kg) were intramuscularly sedated with xylazine (2 mg/Kg) and anesthetized with ketamine (100 mg/Kg) prior to surgical exposure of left carotid artery. Segments of 10 mm of carotids were transiently isolated by temporary ligatures and rinsed with 0.9% sodium chloride via a cannula until there was no more visible evidence of blood components. Carotid arteries were transfected with 80 μmol/L of antisense ODNs in a 1 cm portion either alone or conjugated to cholesterol for a period of 30 minutes. The volume infused was 100 μl, and no visible loss of volume was noted throughout the incubation period. Following transfection, the treated segments were rinsed with 0.9% sodium chloride (3×100 μl) and upon cannula removal, the arteriotomy site was repaired with microsutures, restoring normal blood flow and the neck wound closed. All experimental protocols in this project were approved by the Institutional Committee for Animal Protection of the Louis-Charles Simard Research Center. 
     Neointimal Hyperplasia Inhibition 
     A total of 36 New Zealand white rabbit carotid arteries were injured with a 2.5 mm balloon catheter serially inflated for 1 minute to 4, 6, 8 and 10 atm with gentle traction, allowing 45 seconds between inflations. Two weeks later, a second injury was imposed at the same arterial site which was then transfected in a 1 cm portion with 80 μmol/L (100 μl of volume injected) of therapeutic molecules or with 100 μL of NaCl 0.9% as control. Intimal/medial areas were evaluated by computer analysis on histological sections derived from transfected arteries two weeks following the second injury and transfection procedure. 
     The addition of a PC5 antisense 17-mer ODN shown in Table 1 at the time of a second carotid injury with a balloon catheter decreased carotid stenosis, measured as area ratio intima/media, by 40% (Area ratio intima/media sense ODN minus area ratio antisense ODN divided by area ratio sense ODN; see FIG.  9 ). This inhibitory effect is highly significant (P 0.0118 and P=0.0078) as compared to the sense and random controls, respectively. These results are the basis of a method of preventing stenosis comprising administering an effective stenosis inhibitory dose of a PC5 antisense to a subject in need for such a treatment. Other antisense oligonucleotides may be added to optimize this method of preventing restenosis, such as those silencing the expression of other convertases, namely PC2, which are also observed in atherectomy specimens (see FIG.  10 ). 
     The development of drugs based on the inhibition or the inactivation of the convertases is of great interest because the drugs can easily be delivered directly at the affected site during the intervention by the cardiologist. 
     In addition we claim that this therapeutic approach, based on the inhibition of cell growth by antisense against one of the convertases will be applicable to all proliferative diseases involving maturation of a given proteic precursor into an active protein: 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 The sequences of the oligonucleotides used are: 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Antisense 
                 GCAACTTGCCAGAGCAT 
                 SEQ ID NO: 3 
               
               
                   
                   
               
               
                   
                 Sense 
                 ATGCTCTGGCAAGTTGC 
                 SEQ ID NO. 4 
               
               
                   
                   
               
               
                   
                 Random 
                 AATCCGTGAGACCAGTC 
                 SEQ ID NO. 5. 
               
               
                   
                   
               
             
          
         
       
     
     PC5 is Involved in the Cleavage of HIV gp160 into gp120 and gp41 
     As mentioned above, PC5, PC7 and furin are known to be present in CD4 +  T lymphocytes. All three enzymes cleave HIV gp160 to gp120 and gp41 as well as a synthetic peptide covering the junction wherein cleavage occurs in gp160. Since the 17-mer antisense ODN defined in Seq ID. No. 3 successfully silenced the expression of PC5 and prevented restenosis, the same oligonucleotide as well as any other oligonucleotide or construct having an equivalent silencing function will find use in inhibiting the action of PC5 on HIV gp160 in CD4 +  T lymphocytes. To optimize the inhibition of conversion of gp160 into its fusiogenic form, a cocktail comprising antisense molecules to PC5, PC7 and furin is contemplated as part of the present invention. Appropriate vehicles such as liposomes may be used to deliver these antisense molecules to the target tissues. 
     This invention has been described hereinbelow. It may be apparent to the skilled reader that modifications can be made thereto without departing from the above teachings. These modifications are considered as part of the scope of the present invention, as defined in the appended claims. 
     REFERENCES 
     1. Frohlich E D, Iwata T, Sasaki 0. Clinical and physiologic significance of local tissue renin-angiotensin systems.  Am J Med . 1990;87 (suppl 6B):6B-19S-6B-23S 
     2. Johnston C I, Burrell L M, Perich R, Jandeleit K, Jackson B. The tissue renin-angiotensin system and its functional role.  Clin Exper Pharmacol Physiol . 1992;19:1-5 
     3. Gardner D G, Gertz B J, Deschepper C F, Kim D Y. Gene for the rat atrial natriuretic peptide is regulated by glucocorticoids in vitro.  J Clin Invest . 1988;82:1275-1281 
     4. Lee M A, Bohm M, Paul M, Ganten D. Tissue renin-angiotensin systems. Their role in cardiovascular disease.  Circulation . 1993;87:IV7-I13 
     5. Do Y S, Shinagawa T, Tam H, Inagami T, Hsueh W A. Characterization of pure human renal renin.  J Biol Chem . 1987;262:1037-1043 
     6. Taugner R, Kim S J, Murakami K, Waldherr R. The fate of prorenin during granulopoiesis in epithelioid cells. Immunocytochemical experiments with antisera against renin and different portions of the renin prosegment.  Histochemistry . 1987;86:249-253 
     7. Taugner R, Hackenthal E. On the character of the secretory granules in juxtaglomerular epithelioid cells.  Int Rev Cytol . 1988;110:93-131 
     8. Saint-Andre J P, Rohmer V, Pinet F, Rousselet M C, Bigorgne J C, Corvol P. Renin and cathepsin B in human pituitary lactotroph cells. An ultrastructural study.  Histochemistry . 1989;91:291-297 
     9. Wang P H, Do Y S, Macaulay L, Shinagawa T, Anderson P W, Baxter J D, Hsueh W A. Identification of renal cathepsin B as a human prorenin-processing enzyme.  J Biol Chem . 1991;266:12633-12638 
     10. Benjannet S, Reudelhuber T, Mercure C, Rondeau N, Chretien M, Seidah N G. Proprotein conversion is determined by a multiplicity of factors including convertase processing, substrate specificity, and intracellular environment. Cell type-specific processing of human prorenin by the convertase PC1 . J Biol Chem . 1992;267:11417-11423 
     11. Konoshita T, Gasc J M, Villard E, Takeda R, Seidah N G, Corvol P, Pinet F. Expression of PC2 and PC1/PC3 in human pheochromocytomas.  Mol Cell Endocrinol . 1994;99:307-314 
     12. Reudelhuber T L, Ramla D, Chiu L, Mercure C, Seidah N G. Proteolytic processing of human prorenin in renal and non-renal tissues.  Kidney Int . 1994;46:1522-1524 
     13. Sigmund C D, Okuyama K, Ingelfinger J, Jones C A, Mullins J J, Kane C, Kim U, Wu C Z, Kenny L, Rustum Y, Dzau V J, Gross K W. Isolation and characterization of renin-expressing cell lines from transgenic mice containing a renin-promoter viral oncogene fusion construct.  J Biol Chem . 1990;265:19916-19922 
     14. Sambrook J, Fritsch E F, Maniatis T.  Molecular cloning: A laboratory manual . Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 1989; 
     15. Lusson J, Vieau D, Hamelin J, Day R, Chretien M, Seidah N G. cDNA structure of the mouse and rat subtilisin/kexin-like PC5: a candidate proprotein convertase expressed in endocrine and nonendocrine cells.  Proc Natl Acad Sci USA . 1993;90:6691-6695 
     16 . Current Protocols in Molecular Biology . New York: John Wiley and Sons, Inc. 1996; 
     17. Day R, Schafer M K, Watson S J, Chretien M, Seidah N G. Distribution and regulation of the prohormone convertases PC1 and PC2 in the rat pituitary.  Molec Endocrinol . 1992;6:485-497 
     18. Horton R M, Hunt H D, Ho S N, Pullen J K, Pease L R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension.  Gene . 1989;77:61-68 
     19. Chu W N, Baxter J D, Reudelhuber T L. A targeting sequence for dense core secretory granules resides in the protein moiety of human prorenin.  Molec Endocrinol . 1990;4:1905-1913 
     20. Chu W N, Mercure C, Baxter J D, Reudelhuber T L. Molecular determinants of human prorenin processing.  Hypertension . 1992;20:782-787 
     21. Scammell J G, Burrage T G, Dannies P S. Hormonal induction of secretory granules in a pituitary tumor cell line.  Endocrinology . 1986;119:1543-1548 
     22. Ghitescu L, Bendayan M. Immunolabeling efficiency of protein A-gold complexes.  J Histochem Cytochem . 1990;38:1523-1530 
     23. Nakagawa T, Hosaka M, Torii S, Watanabe T, Murakami K, Nakayama K. Identification and functional expression of a new member of the mammalian Kex2-like processing endoprotease family: Its striking structural similarity to PACE-4 . J Biochem . 1993;113:132-135 
     24. Racz K, Pinet F, Gasc J M, Guyene T T, Corvol P. Coexpression of renin, angiotensinogen, and their messenger ribonucleic acids in adrenal tissues.  J Clin Endocrinol Metab . 1992;75:730-737 
     25. Paul M, Wagner J, Dzau V J. Gene expression of the renin-angiotensin system in human tissues. Quantitative analysis by the polymerase chain reaction.  J Clin Invest . 1993;91:2058-2064 
     26. Fallo F, Pistorello M, Pedini F, D&#39;Agostino D, Mantero F, Boscaro M. in vitro evidence for local generation of renin and angiotensin II/III immunoreactivity by the human adrenal gland.  Acta Endocrinologica . 1991;125:319-330 
     27. Sarzani R, Fallo F, Dessi-Fulgheri P, Pistorello M, Lanari A, Paci VM, Mantero F, Rappelli A. Local renin-angiotensin system in human adrenals and aldosteronomas.  Hypertension . 1992;19:702-707 
     28. Mizuno K, Hoffman L H, McKenzie J C, Inagami T. Presence of renin secretory granules in rat adrenal gland and stimulation of renin secretion by angiotensin II but not by adrenocorticotropin.  J Clin Invest . 1988;82:1007-1016 
     29. Mullins J J, Peters J, Ganten D. Fulminant hypertension in transgenic rats harbouring the mouse Ren-2 gene.  Nature . 1990;344:541-544 
     30. Yamaguchi T, Tokita Y, Franco-Saenz R, Mulrow P J, Peters J, Ganten D. Zonal distribution and regulation of adrenal renin in a transgenic model of hypertension in the rat.  Endocrinology . 1992;131:1955-1962 
     31. Rocco S, Rebuffat P, Cimolato M, Opocher G, Peters J, Mazzocchi G, Ganten D, Mantero F, Nussdorfer G G. Zona glomerulosa of the adrenal gland in a transgenic strain of rat: a morphologic and functional study.  Cell  &amp;  Tiss Res . 1994;278:21-28 
     32. Tokita Y, Franco-Saenz R, Mulrow P J, Ganten D. Effects of nephrectomy and adrenalectomy on the renin-angiotensin system of transgenic rats TGR(mRen2)27 . Endocrinology . 1994;134:253-257 
     33. Duncan K G, Haidar M A, Baxter J D, Reudelhuber T L. Regulation of human renin expression in chorion cell primary cultures.  Proc Natl Acad Sci USA . 1990;87:7588-7592 
     34. Lenz T, James G D, Laragh J H, Sealey J E. Prorenin secretion from human placenta perfused in vitro.  Am J Physiol . 1991;260:E876-E882 
     35. Nakagawa T, Murakami K, Nakayama K. Identification of an isoform with an extremely large Cys-rich region of PC6, a Kex2-like processing endoprotease.  FEBS Lett . 1993;327:165-171 
     36. Seidah N G, Hamelin J, Gaspar A M, Day R, Chretien M. The cDNA sequence of the human pro-hormone and pro-protein convertase PC1 . DNA  &amp;  Cell Biology . 1992;11:283-289 
     37. Seidah N G, Day R, Marcinkiewicz M, Chretien M. Mammalian paired basic amino acid convertases of prohormones and proproteins.  Ann NY Acad Sci . 1993;680:135-146 
     38. Miranda L, Wolf J, Pichuantes S, Duke R, Franzusoff A. Isolation of the human PC6 gene encoding the putative host proteas for HIV-1 gp160 processing in CD4 +  T lymphocytes.  Proc. Natl. Acad. Sci. USA  1996;93:7695-7700. 
     
       
         
           
             9 
           
           
             1 
             2819 
             DNA 
             Homo sapiens 
             
               CDS 
               (1)..(2745) 
             
           
            1
atg gac tgg gac tgg ggg aac cgc tgc agc cgc ccg gga cgg cgg gat       48
Met Asp Trp Asp Trp Gly Asn Arg Cys Ser Arg Pro Gly Arg Arg Asp
  1               5                  10                  15
ctg ctg tgc gtg ctg gcg ctg ctc ggg ggc tgc ctg ctc ccc gtg tgt       96
Leu Leu Cys Val Leu Ala Leu Leu Gly Gly Cys Leu Leu Pro Val Cys
             20                  25                  30
cgg acg cgc gtc tac acc aac cac tgg gca gtc aaa atc gcc ggg ggc      144
Arg Thr Arg Val Tyr Thr Asn His Trp Ala Val Lys Ile Ala Gly Gly
         35                  40                  45
ttc ccg gag gcc aac cgt atc gcc agc aag tac gga ttc atc aac ata      192
Phe Pro Glu Ala Asn Arg Ile Ala Ser Lys Tyr Gly Phe Ile Asn Ile
     50                  55                  60
gga cag ata ggg gcc ctg aag gac tac tac cac ttc tac cat agc agg      240
Gly Gln Ile Gly Ala Leu Lys Asp Tyr Tyr His Phe Tyr His Ser Arg
 65                  70                  75                  80
acg att aaa agg tca gtt atc tcg agc aga ggg acc cac agt ttc att      288
Thr Ile Lys Arg Ser Val Ile Ser Ser Arg Gly Thr His Ser Phe Ile
                 85                  90                  95
tca atg gaa cca aag gtg gaa tgg atc caa cag caa gtg gta aaa aag      336
Ser Met Glu Pro Lys Val Glu Trp Ile Gln Gln Gln Val Val Lys Lys
            100                 105                 110
cgg aca aag agg gat tat gac ttc agt cgt gcc cag tct acc tat ttc      384
Arg Thr Lys Arg Asp Tyr Asp Phe Ser Arg Ala Gln Ser Thr Tyr Phe
        115                 120                 125
aat gat ccc aag tgg ccc agc atg tgg tat atg cac tgc agt gac aat      432
Asn Asp Pro Lys Trp Pro Ser Met Trp Tyr Met His Cys Ser Asp Asn
    130                 135                 140
aca cat ccc tgc cag tct gac atg aat atc gaa gga gcc tgg aag aga      480
Thr His Pro Cys Gln Ser Asp Met Asn Ile Glu Gly Ala Trp Lys Arg
145                 150                 155                 160
ggc tac acg gga aag aac att gtg gtc act atc ctg gat gac gga att      528
Gly Tyr Thr Gly Lys Asn Ile Val Val Thr Ile Leu Asp Asp Gly Ile
                165                 170                 175
gag aga acc cat cca gat ctg atg caa aac tac gat gct ctg gca agt      576
Glu Arg Thr His Pro Asp Leu Met Gln Asn Tyr Asp Ala Leu Ala Ser
            180                 185                 190
tgc gac gtg aat ggg aat gac ttg gac cca atg cct cgt tat gat gca      624
Cys Asp Val Asn Gly Asn Asp Leu Asp Pro Met Pro Arg Tyr Asp Ala
        195                 200                 205
agc aac gag aac aag cat ggg act cgc tgt gct gga gaa gtg gca gcc      672
Ser Asn Glu Asn Lys His Gly Thr Arg Cys Ala Gly Glu Val Ala Ala
    210                 215                 220
gct gca aac aat tcg cac tgc aca gtc gga att gct ttc aac gcc aag      720
Ala Ala Asn Asn Ser His Cys Thr Val Gly Ile Ala Phe Asn Ala Lys
225                 230                 235                 240
atc gga gga gtg cga atg ctg gac gga gat gtc acg gac atg gtt gaa      768
Ile Gly Gly Val Arg Met Leu Asp Gly Asp Val Thr Asp Met Val Glu
                245                 250                 255
gca aaa tca gtt agc ttc aac ccc cag cac gtg cac att tac agc gcc      816
Ala Lys Ser Val Ser Phe Asn Pro Gln His Val His Ile Tyr Ser Ala
            260                 265                 270
agc tgg ggc ccg gat gat gat ggc aag act gtg gac gga cca gcc ccc      864
Ser Trp Gly Pro Asp Asp Asp Gly Lys Thr Val Asp Gly Pro Ala Pro
        275                 280                 285
ctc acc cgg caa gcc ttt gaa aac ggc gtt aga atg ggg cgg aga ggc      912
Leu Thr Arg Gln Ala Phe Glu Asn Gly Val Arg Met Gly Arg Arg Gly
    290                 295                 300
ctc ggc tct gtg ttt gtt tgg gca tct gga aat ggt gga agg agc aaa      960
Leu Gly Ser Val Phe Val Trp Ala Ser Gly Asn Gly Gly Arg Ser Lys
305                 310                 315                 320
gac cac tgc tcc tgt gat ggc tac acc aac agc atc tac acc atc tcc     1008
Asp His Cys Ser Cys Asp Gly Tyr Thr Asn Ser Ile Tyr Thr Ile Ser
                325                 330                 335
atc agc agc act gca gaa agc gga aag aaa cct tgg tac ctg gaa gag     1056
Ile Ser Ser Thr Ala Glu Ser Gly Lys Lys Pro Trp Tyr Leu Glu Glu
            340                 345                 350
tgt tca tcc acg ctg gcc aca acc tac agc agc ggg gag tcc tac gat     1104
Cys Ser Ser Thr Leu Ala Thr Thr Tyr Ser Ser Gly Glu Ser Tyr Asp
        355                 360                 365
aag aaa atc atc act aca gat ctg agg cag cgt tgc acg gac aac cac     1152
Lys Lys Ile Ile Thr Thr Asp Leu Arg Gln Arg Cys Thr Asp Asn His
    370                 375                 380
act ggg acg tca gcc tca gcc ccc atg gct gca ggc atc att gcg ctg     1200
Thr Gly Thr Ser Ala Ser Ala Pro Met Ala Ala Gly Ile Ile Ala Leu
385                 390                 395                 400
gcc ctg gaa gcc aat ccg ttt ctg acc tgg aga gac gta cag cat gtt     1248
Ala Leu Glu Ala Asn Pro Phe Leu Thr Trp Arg Asp Val Gln His Val
                405                 410                 415
att gtc agg act tcc cgt gcg gga cat ttg aac gct aat gac tgg aaa     1296
Ile Val Arg Thr Ser Arg Ala Gly His Leu Asn Ala Asn Asp Trp Lys
            420                 425                 430
acc aat gct gct ggt ttt aag gtg agc cat ctt tat gga ttt gga ctg     1344
Thr Asn Ala Ala Gly Phe Lys Val Ser His Leu Tyr Gly Phe Gly Leu
        435                 440                 445
atg gac gca gaa gcc atg gtg atg gag gca gag aag tgg acc acc gtt     1392
Met Asp Ala Glu Ala Met Val Met Glu Ala Glu Lys Trp Thr Thr Val
    450                 455                 460
ccc cgg cag cac gtg tgt gtg gag agc aca gac cga caa atc aag aca     1440
Pro Arg Gln His Val Cys Val Glu Ser Thr Asp Arg Gln Ile Lys Thr
465                 470                 475                 480
atc cgc cct aac agt gca gtg cgc tcc atc tac aaa gct tca ggc tgc     1488
Ile Arg Pro Asn Ser Ala Val Arg Ser Ile Tyr Lys Ala Ser Gly Cys
                485                 490                 495
tcg gat aac ccc aac cgc cat gtc aac tac ctg gag cac gtc gtt gtg     1536
Ser Asp Asn Pro Asn Arg His Val Asn Tyr Leu Glu His Val Val Val
            500                 505                 510
cgc atc acc atc acc cac ccc agg aga gga gac ctg gcc atc tac ctg     1584
Arg Ile Thr Ile Thr His Pro Arg Arg Gly Asp Leu Ala Ile Tyr Leu
        515                 520                 525
acc tcg ccc tct gga act agg tct cag ctt ttg gcc aac agg cta ttt     1632
Thr Ser Pro Ser Gly Thr Arg Ser Gln Leu Leu Ala Asn Arg Leu Phe
    530                 535                 540
gat cac tcc atg gaa gga ttc aaa aac tgg gag ttc atg acc att cat     1680
Asp His Ser Met Glu Gly Phe Lys Asn Trp Glu Phe Met Thr Ile His
545                 550                 555                 560
tgc tgg gga gaa aga gct gct ggt gac tgg gtc ctt gaa gtt tat gat     1728
Cys Trp Gly Glu Arg Ala Ala Gly Asp Trp Val Leu Glu Val Tyr Asp
                565                 570                 575
act ccc tct cag cta agg aac ttt aag act cca ggt aaa ttg aaa gaa     1776
Thr Pro Ser Gln Leu Arg Asn Phe Lys Thr Pro Gly Lys Leu Lys Glu
            580                 585                 590
tgg tct ttg gtc ctc tac ggc acc tcc gtg cag cca tat tca cca acc     1824
Trp Ser Leu Val Leu Tyr Gly Thr Ser Val Gln Pro Tyr Ser Pro Thr
        595                 600                 605
aat gaa ttt ccg aaa gtg gaa cgg ttc cgc tat agc cga gtt gaa gac     1872
Asn Glu Phe Pro Lys Val Glu Arg Phe Arg Tyr Ser Arg Val Glu Asp
    610                 615                 620
ccc aca gac gac tat ggc aca gag gat tat gca ggt ccc tgc gac cct     1920
Pro Thr Asp Asp Tyr Gly Thr Glu Asp Tyr Ala Gly Pro Cys Asp Pro
625                 630                 635                 640
gag tgc agt gag gtt ggc tgt gac ggg cca gga cca gac cac tgc aat     1968
Glu Cys Ser Glu Val Gly Cys Asp Gly Pro Gly Pro Asp His Cys Asn
                645                 650                 655
gac tgt ttg cac tac tac tac aag ctg aaa aac aat acc agg atc tgt     2016
Asp Cys Leu His Tyr Tyr Tyr Lys Leu Lys Asn Asn Thr Arg Ile Cys
            660                 665                 670
gtc tcc agc tgc ccc cct ggc cac tac cac gcc gac aag aag cgc tgc     2064
Val Ser Ser Cys Pro Pro Gly His Tyr His Ala Asp Lys Lys Arg Cys
        675                 680                 685
agg aag tgt gcc ccc aac tgt gag tcc tgc ttt ggg agc cat ggt gac     2112
Arg Lys Cys Ala Pro Asn Cys Glu Ser Cys Phe Gly Ser His Gly Asp
    690                 695                 700
caa tgc atg tcc tgc aaa tat gga tac ttt ctg aat gaa gaa acc aac     2160
Gln Cys Met Ser Cys Lys Tyr Gly Tyr Phe Leu Asn Glu Glu Thr Asn
705                 710                 715                 720
agc tgt gtt act cac tgc cct gat ggg tca tat cag gat acc aag aaa     2208
Ser Cys Val Thr His Cys Pro Asp Gly Ser Tyr Gln Asp Thr Lys Lys
                725                 730                 735
aat ctt tgc cgg aaa tgc agt gaa aac tgc aag aca tgt act gaa ttc     2256
Asn Leu Cys Arg Lys Cys Ser Glu Asn Cys Lys Thr Cys Thr Glu Phe
            740                 745                 750
cat aac tgt aca gaa tgt agg gat ggg tta agc ctg cag gga tcc cgg     2304
His Asn Cys Thr Glu Cys Arg Asp Gly Leu Ser Leu Gln Gly Ser Arg
        755                 760                 765
tgc tct gtc tcc tgt gaa gat gga cgg tat ttc aac ggc cag gac tgc     2352
Cys Ser Val Ser Cys Glu Asp Gly Arg Tyr Phe Asn Gly Gln Asp Cys
    770                 775                 780
cag ccc tgc cac cgc ttc tgc gcc act tgt gct ggg gca gga gct gat     2400
Gln Pro Cys His Arg Phe Cys Ala Thr Cys Ala Gly Ala Gly Ala Asp
785                 790                 795                 800
ggg tgc att aac tgc aca gag ggc tac ttc atg gag gat ggg aga tgc     2448
Gly Cys Ile Asn Cys Thr Glu Gly Tyr Phe Met Glu Asp Gly Arg Cys
                805                 810                 815
gtg cag agc tgt agt atc agc tat tac ttt gac cac tct tca gag aat     2496
Val Gln Ser Cys Ser Ile Ser Tyr Tyr Phe Asp His Ser Ser Glu Asn
            820                 825                 830
gga tac aaa tcc tgc aaa aaa tgt gat atc agt tgt ttg acg tgc aat     2544
Gly Tyr Lys Ser Cys Lys Lys Cys Asp Ile Ser Cys Leu Thr Cys Asn
        835                 840                 845
ggc cca gga ttc aag aac tgt aca agc tgc cct agt ggg tat ctc tta     2592
Gly Pro Gly Phe Lys Asn Cys Thr Ser Cys Pro Ser Gly Tyr Leu Leu
    850                 855                 860
gac tta gga atg tgt caa atg gga gcc att tgc aag gat gca acg gaa     2640
Asp Leu Gly Met Cys Gln Met Gly Ala Ile Cys Lys Asp Ala Thr Glu
865                 870                 875                 880
gag tcc tgg gcg gaa gga ggc ttc tgt atg ctt gtg aaa aag aac aat     2688
Glu Ser Trp Ala Glu Gly Gly Phe Cys Met Leu Val Lys Lys Asn Asn
                885                 890                 895
ctg tgc caa cgg aag gtt ctt caa caa ctt tgc tgc aaa aca tgt aca     2736
Leu Cys Gln Arg Lys Val Leu Gln Gln Leu Cys Cys Lys Thr Cys Thr
            900                 905                 910
ttt caa ggc tgagcagcca tcttagattt ctttgttcct gtagacttat             2785
Phe Gln Gly
        915
agattattcc atattattaa aaagaaaaaa aaaa                               2819
 
           
             2 
             915 
             PRT 
             Homo sapiens 
           
            2
Met Asp Trp Asp Trp Gly Asn Arg Cys Ser Arg Pro Gly Arg Arg Asp
  1               5                  10                  15
Leu Leu Cys Val Leu Ala Leu Leu Gly Gly Cys Leu Leu Pro Val Cys
             20                  25                  30
Arg Thr Arg Val Tyr Thr Asn His Trp Ala Val Lys Ile Ala Gly Gly
         35                  40                  45
Phe Pro Glu Ala Asn Arg Ile Ala Ser Lys Tyr Gly Phe Ile Asn Ile
     50                  55                  60
Gly Gln Ile Gly Ala Leu Lys Asp Tyr Tyr His Phe Tyr His Ser Arg
 65                  70                  75                  80
Thr Ile Lys Arg Ser Val Ile Ser Ser Arg Gly Thr His Ser Phe Ile
                 85                  90                  95
Ser Met Glu Pro Lys Val Glu Trp Ile Gln Gln Gln Val Val Lys Lys
            100                 105                 110
Arg Thr Lys Arg Asp Tyr Asp Phe Ser Arg Ala Gln Ser Thr Tyr Phe
        115                 120                 125
Asn Asp Pro Lys Trp Pro Ser Met Trp Tyr Met His Cys Ser Asp Asn
    130                 135                 140
Thr His Pro Cys Gln Ser Asp Met Asn Ile Glu Gly Ala Trp Lys Arg
145                 150                 155                 160
Gly Tyr Thr Gly Lys Asn Ile Val Val Thr Ile Leu Asp Asp Gly Ile
                165                 170                 175
Glu Arg Thr His Pro Asp Leu Met Gln Asn Tyr Asp Ala Leu Ala Ser
            180                 185                 190
Cys Asp Val Asn Gly Asn Asp Leu Asp Pro Met Pro Arg Tyr Asp Ala
        195                 200                 205
Ser Asn Glu Asn Lys His Gly Thr Arg Cys Ala Gly Glu Val Ala Ala
    210                 215                 220
Ala Ala Asn Asn Ser His Cys Thr Val Gly Ile Ala Phe Asn Ala Lys
225                 230                 235                 240
Ile Gly Gly Val Arg Met Leu Asp Gly Asp Val Thr Asp Met Val Glu
                245                 250                 255
Ala Lys Ser Val Ser Phe Asn Pro Gln His Val His Ile Tyr Ser Ala
            260                 265                 270
Ser Trp Gly Pro Asp Asp Asp Gly Lys Thr Val Asp Gly Pro Ala Pro
        275                 280                 285
Leu Thr Arg Gln Ala Phe Glu Asn Gly Val Arg Met Gly Arg Arg Gly
    290                 295                 300
Leu Gly Ser Val Phe Val Trp Ala Ser Gly Asn Gly Gly Arg Ser Lys
305                 310                 315                 320
Asp His Cys Ser Cys Asp Gly Tyr Thr Asn Ser Ile Tyr Thr Ile Ser
                325                 330                 335
Ile Ser Ser Thr Ala Glu Ser Gly Lys Lys Pro Trp Tyr Leu Glu Glu
            340                 345                 350
Cys Ser Ser Thr Leu Ala Thr Thr Tyr Ser Ser Gly Glu Ser Tyr Asp
        355                 360                 365
Lys Lys Ile Ile Thr Thr Asp Leu Arg Gln Arg Cys Thr Asp Asn His
    370                 375                 380
Thr Gly Thr Ser Ala Ser Ala Pro Met Ala Ala Gly Ile Ile Ala Leu
385                 390                 395                 400
Ala Leu Glu Ala Asn Pro Phe Leu Thr Trp Arg Asp Val Gln His Val
                405                 410                 415
Ile Val Arg Thr Ser Arg Ala Gly His Leu Asn Ala Asn Asp Trp Lys
            420                 425                 430
Thr Asn Ala Ala Gly Phe Lys Val Ser His Leu Tyr Gly Phe Gly Leu
        435                 440                 445
Met Asp Ala Glu Ala Met Val Met Glu Ala Glu Lys Trp Thr Thr Val
    450                 455                 460
Pro Arg Gln His Val Cys Val Glu Ser Thr Asp Arg Gln Ile Lys Thr
465                 470                 475                 480
Ile Arg Pro Asn Ser Ala Val Arg Ser Ile Tyr Lys Ala Ser Gly Cys
                485                 490                 495
Ser Asp Asn Pro Asn Arg His Val Asn Tyr Leu Glu His Val Val Val
            500                 505                 510
Arg Ile Thr Ile Thr His Pro Arg Arg Gly Asp Leu Ala Ile Tyr Leu
        515                 520                 525
Thr Ser Pro Ser Gly Thr Arg Ser Gln Leu Leu Ala Asn Arg Leu Phe
    530                 535                 540
Asp His Ser Met Glu Gly Phe Lys Asn Trp Glu Phe Met Thr Ile His
545                 550                 555                 560
Cys Trp Gly Glu Arg Ala Ala Gly Asp Trp Val Leu Glu Val Tyr Asp
                565                 570                 575
Thr Pro Ser Gln Leu Arg Asn Phe Lys Thr Pro Gly Lys Leu Lys Glu
            580                 585                 590
Trp Ser Leu Val Leu Tyr Gly Thr Ser Val Gln Pro Tyr Ser Pro Thr
        595                 600                 605
Asn Glu Phe Pro Lys Val Glu Arg Phe Arg Tyr Ser Arg Val Glu Asp
    610                 615                 620
Pro Thr Asp Asp Tyr Gly Thr Glu Asp Tyr Ala Gly Pro Cys Asp Pro
625                 630                 635                 640
Glu Cys Ser Glu Val Gly Cys Asp Gly Pro Gly Pro Asp His Cys Asn
                645                 650                 655
Asp Cys Leu His Tyr Tyr Tyr Lys Leu Lys Asn Asn Thr Arg Ile Cys
            660                 665                 670
Val Ser Ser Cys Pro Pro Gly His Tyr His Ala Asp Lys Lys Arg Cys
        675                 680                 685
Arg Lys Cys Ala Pro Asn Cys Glu Ser Cys Phe Gly Ser His Gly Asp
    690                 695                 700
Gln Cys Met Ser Cys Lys Tyr Gly Tyr Phe Leu Asn Glu Glu Thr Asn
705                 710                 715                 720
Ser Cys Val Thr His Cys Pro Asp Gly Ser Tyr Gln Asp Thr Lys Lys
                725                 730                 735
Asn Leu Cys Arg Lys Cys Ser Glu Asn Cys Lys Thr Cys Thr Glu Phe
            740                 745                 750
His Asn Cys Thr Glu Cys Arg Asp Gly Leu Ser Leu Gln Gly Ser Arg
        755                 760                 765
Cys Ser Val Ser Cys Glu Asp Gly Arg Tyr Phe Asn Gly Gln Asp Cys
    770                 775                 780
Gln Pro Cys His Arg Phe Cys Ala Thr Cys Ala Gly Ala Gly Ala Asp
785                 790                 795                 800
Gly Cys Ile Asn Cys Thr Glu Gly Tyr Phe Met Glu Asp Gly Arg Cys
                805                 810                 815
Val Gln Ser Cys Ser Ile Ser Tyr Tyr Phe Asp His Ser Ser Glu Asn
            820                 825                 830
Gly Tyr Lys Ser Cys Lys Lys Cys Asp Ile Ser Cys Leu Thr Cys Asn
        835                 840                 845
Gly Pro Gly Phe Lys Asn Cys Thr Ser Cys Pro Ser Gly Tyr Leu Leu
    850                 855                 860
Asp Leu Gly Met Cys Gln Met Gly Ala Ile Cys Lys Asp Ala Thr Glu
865                 870                 875                 880
Glu Ser Trp Ala Glu Gly Gly Phe Cys Met Leu Val Lys Lys Asn Asn
                885                 890                 895
Leu Cys Gln Arg Lys Val Leu Gln Gln Leu Cys Cys Lys Thr Cys Thr
            900                 905                 910
Phe Gln Gly
        915
 
           
             3 
             17 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequencesynthetic 
             
           
            3
gcaacttgcc agagcat                                                    17
 
           
             4 
             17 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequencesynthetic 
             
           
            4
atgctctggc aagttgc                                                    17
 
           
             5 
             17 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequencesynthetic 
             
           
            5
aatccgtgag accagtc                                                    17
 
           
             6 
             2766 
             DNA 
             Homo sapiens 
             
               CDS 
               (13)..(2757) 
             
           
            6
agcgtcggga cc atg gat tgg gat tgg ggg aac cgc tgc agc cgc ccg gga     51
              Met Asp Trp Asp Trp Gly Asn Arg Cys Ser Arg Pro Gly
                1               5                  10
cgg cgg gac ctg ctg tgc gtg ctg gca ctg ctc gcc ggc tgt ctg ctc       99
Arg Arg Asp Leu Leu Cys Val Leu Ala Leu Leu Ala Gly Cys Leu Leu
     15                  20                  25
ccg gta tgc cgg acg cgc gtc tac acc aac cac tgg gca gtg aag atc      147
Pro Val Cys Arg Thr Arg Val Tyr Thr Asn His Trp Ala Val Lys Ile
 30                  35                  40                  45
gcc ggc ggc ttc gcg gag gca gat cgc ata gcc agc aag tac gga ttc      195
Ala Gly Gly Phe Ala Glu Ala Asp Arg Ile Ala Ser Lys Tyr Gly Phe
                 50                  55                  60
atc aac gta gga cag atc ggt gca ctg aag gac tac tat cac ttc tac      243
Ile Asn Val Gly Gln Ile Gly Ala Leu Lys Asp Tyr Tyr His Phe Tyr
             65                  70                  75
cat agt agg acc att aaa agg tct gtt ctc tcg agc aga gga acc cac      291
His Ser Arg Thr Ile Lys Arg Ser Val Leu Ser Ser Arg Gly Thr His
         80                  85                  90
agt ttc att tca atg gaa cca aag gtg gag tgg atc caa cag caa gtg      339
Ser Phe Ile Ser Met Glu Pro Lys Val Glu Trp Ile Gln Gln Gln Val
     95                 100                 105
gtg aaa aaa aga acc aag agg gat tat gac ctc agc cat gcc cag tca      387
Val Lys Lys Arg Thr Lys Arg Asp Tyr Asp Leu Ser His Ala Gln Ser
110                 115                 120                 125
acc tac ttc aat gat ccc aag tgg cca agt atg tgg tac atg cac tgt      435
Thr Tyr Phe Asn Asp Pro Lys Trp Pro Ser Met Trp Tyr Met His Cys
                130                 135                 140
agc gac aat aca cat ccc tgc cag tct gac atg aat atc gaa gga gcc      483
Ser Asp Asn Thr His Pro Cys Gln Ser Asp Met Asn Ile Glu Gly Ala
            145                 150                 155
tgg aag aga ggc tac acg gga aag aac att gtg gtc act atc ctg gat      531
Trp Lys Arg Gly Tyr Thr Gly Lys Asn Ile Val Val Thr Ile Leu Asp
        160                 165                 170
gac gga att gag aga acc cat cca gat ctg atg caa aac tac gat gct      579
Asp Gly Ile Glu Arg Thr His Pro Asp Leu Met Gln Asn Tyr Asp Ala
    175                 180                 185
ctg gca agt tgc gac gtg aat ggg aat gac ttg gac cca atg cct cgt      627
Leu Ala Ser Cys Asp Val Asn Gly Asn Asp Leu Asp Pro Met Pro Arg
190                 195                 200                 205
tat gat gca agc aac gag aac aag cat ggg act cgc tgt gct gga gaa      675
Tyr Asp Ala Ser Asn Glu Asn Lys His Gly Thr Arg Cys Ala Gly Glu
                210                 215                 220
gtg gca gcc gct gca aac aat tcg cac tgc aca gtc gga att gct ttc      723
Val Ala Ala Ala Ala Asn Asn Ser His Cys Thr Val Gly Ile Ala Phe
            225                 230                 235
aac gcc aag atc gga gga gtg cga atg ctg gac gga gat gtc acg gac      771
Asn Ala Lys Ile Gly Gly Val Arg Met Leu Asp Gly Asp Val Thr Asp
        240                 245                 250
atg gtt gaa gca aaa tca gtt agc ttc aac ccc cag cac gtg cac att      819
Met Val Glu Ala Lys Ser Val Ser Phe Asn Pro Gln His Val His Ile
    255                 260                 265
tac agc gcc agc tgg ggc ccg gat gat gat ggc aag act gtg gac gga      867
Tyr Ser Ala Ser Trp Gly Pro Asp Asp Asp Gly Lys Thr Val Asp Gly
270                 275                 280                 285
cca gcc ccc ctc acc cgg caa gcc ttt gaa aac ggc gtt aga atg ggg      915
Pro Ala Pro Leu Thr Arg Gln Ala Phe Glu Asn Gly Val Arg Met Gly
                290                 295                 300
cgg aga ggc ctc ggc tct gtg ttt gtt tgg gca tct gga aat ggt gga      963
Arg Arg Gly Leu Gly Ser Val Phe Val Trp Ala Ser Gly Asn Gly Gly
            305                 310                 315
agg agc aaa gac cac tgc tcc tgt gat ggc tac acc aac agc atc tac     1011
Arg Ser Lys Asp His Cys Ser Cys Asp Gly Tyr Thr Asn Ser Ile Tyr
        320                 325                 330
acc atc tcc atc agc agc act gca gaa agc gga aag aaa cct tgg tac     1059
Thr Ile Ser Ile Ser Ser Thr Ala Glu Ser Gly Lys Lys Pro Trp Tyr
    335                 340                 345
ctg gaa gag tgt tca tcc acg ctg gcc aca acc tac agc agc ggg gag     1107
Leu Glu Glu Cys Ser Ser Thr Leu Ala Thr Thr Tyr Ser Ser Gly Glu
350                 355                 360                 365
tcc tac gat aag aaa atc atc act aca gat ctg agg cag cgt tgc acg     1155
Ser Tyr Asp Lys Lys Ile Ile Thr Thr Asp Leu Arg Gln Arg Cys Thr
                370                 375                 380
gac aac cac act ggg acg tca gcc tca gcc ccc atg gct gca ggc atc     1203
Asp Asn His Thr Gly Thr Ser Ala Ser Ala Pro Met Ala Ala Gly Ile
            385                 390                 395
att gcg ctg gcc ctg gaa gcc aat ccg ttt ctg acc tgg aga gac gta     1251
Ile Ala Leu Ala Leu Glu Ala Asn Pro Phe Leu Thr Trp Arg Asp Val
        400                 405                 410
cag cat gtt att gtc agg act tcc cgt gcg gga cat ttg aac gct aat     1299
Gln His Val Ile Val Arg Thr Ser Arg Ala Gly His Leu Asn Ala Asn
    415                 420                 425
gac tgg aaa acc aat gct gct ggt ttt aag gtg agc cat ctt tat gga     1347
Asp Trp Lys Thr Asn Ala Ala Gly Phe Lys Val Ser His Leu Tyr Gly
430                 435                 440                 445
ttt gga ctg atg gac gca gaa gcc atg gtg atg gag gca gag aag tgg     1395
Phe Gly Leu Met Asp Ala Glu Ala Met Val Met Glu Ala Glu Lys Trp
                450                 455                 460
acc acc gtt ccc cgg cag cac gtg tgt gtg gag agc aca gac cga caa     1443
Thr Thr Val Pro Arg Gln His Val Cys Val Glu Ser Thr Asp Arg Gln
            465                 470                 475
atc aag aca atc cgc cct aac agt gca gtg cgc tcc atc tac aaa gct     1491
Ile Lys Thr Ile Arg Pro Asn Ser Ala Val Arg Ser Ile Tyr Lys Ala
        480                 485                 490
tca ggc tgc tcg gat aac ccc aac cgc cat gtc aac tac ctg gag cac     1539
Ser Gly Cys Ser Asp Asn Pro Asn Arg His Val Asn Tyr Leu Glu His
    495                 500                 505
gtc gtt gtg cgc atc acc atc acc cac ccc agg aga gga gac ctg gcc     1587
Val Val Val Arg Ile Thr Ile Thr His Pro Arg Arg Gly Asp Leu Ala
510                 515                 520                 525
atc tac ctg acc tcg ccc tct gga act agg tct cag ctt ttg gcc aac     1635
Ile Tyr Leu Thr Ser Pro Ser Gly Thr Arg Ser Gln Leu Leu Ala Asn
                530                 535                 540
agg cta ttt gat cac tcc atg gaa gga ttc aaa aac tgg gag ttc atg     1683
Arg Leu Phe Asp His Ser Met Glu Gly Phe Lys Asn Trp Glu Phe Met
            545                 550                 555
acc att cat tgc tgg gga gaa aga gct gct ggt gac tgg gtc ctt gaa     1731
Thr Ile His Cys Trp Gly Glu Arg Ala Ala Gly Asp Trp Val Leu Glu
        560                 565                 570
gtt tat gat act ccc tct cag cta agg aac ttt aag act cca ggt aaa     1779
Val Tyr Asp Thr Pro Ser Gln Leu Arg Asn Phe Lys Thr Pro Gly Lys
    575                 580                 585
ttg aaa gaa tgg tct ttg gtc ctc tac ggc acc tcc gtg cgg cca tat     1827
Leu Lys Glu Trp Ser Leu Val Leu Tyr Gly Thr Ser Val Arg Pro Tyr
590                 595                 600                 605
tca cca acc aat gaa ttt ccg aaa gtg gaa cgg ttc cgc tat agc cga     1875
Ser Pro Thr Asn Glu Phe Pro Lys Val Glu Arg Phe Arg Tyr Ser Arg
                610                 615                 620
gtt gaa gac ccc aca gac gac tat ggc aca gag gat tat gca ggt ccc     1923
Val Glu Asp Pro Thr Asp Asp Tyr Gly Thr Glu Asp Tyr Ala Gly Pro
            625                 630                 635
tgc gac cct gag tgc agt gag gtt ggc tgt gac ggg cca gga cca gac     1971
Cys Asp Pro Glu Cys Ser Glu Val Gly Cys Asp Gly Pro Gly Pro Asp
        640                 645                 650
cac tgc aat gac tgt ttg cac tac tac tac aag ctg aaa aac aat acc     2019
His Cys Asn Asp Cys Leu His Tyr Tyr Tyr Lys Leu Lys Asn Asn Thr
    655                 660                 665
agg atc tgt gtc tcc agc tgc ccc cct ggc cac tac cac gcc gac aag     2067
Arg Ile Cys Val Ser Ser Cys Pro Pro Gly His Tyr His Ala Asp Lys
670                 675                 680                 685
aag cgc tgc agg aag tgt gcc ccc aac tgt gag tcc tgc ttt ggg agc     2115
Lys Arg Cys Arg Lys Cys Ala Pro Asn Cys Glu Ser Cys Phe Gly Ser
                690                 695                 700
cat ggt gac caa tgc atg tcc tgc aaa tat gga tac ttt ctg aat gaa     2163
His Gly Asp Gln Cys Met Ser Cys Lys Tyr Gly Tyr Phe Leu Asn Glu
            705                 710                 715
gaa acc aac agc tgt gtt act cac tgc cct gat ggg tca tat cag gat     2211
Glu Thr Asn Ser Cys Val Thr His Cys Pro Asp Gly Ser Tyr Gln Asp
        720                 725                 730
acc aag aaa aat ctt tgc cgg aaa tgc agt gaa aac tgc aag aca tgt     2259
Thr Lys Lys Asn Leu Cys Arg Lys Cys Ser Glu Asn Cys Lys Thr Cys
    735                 740                 745
act gaa ttc cat aac tgt aca gaa tgt agg gat ggg tta agc ctg cag     2307
Thr Glu Phe His Asn Cys Thr Glu Cys Arg Asp Gly Leu Ser Leu Gln
750                 755                 760                 765
gga tcc cgg tgc tct gtc tcc tgt gaa gat gga cgg tat ttc aac ggc     2355
Gly Ser Arg Cys Ser Val Ser Cys Glu Asp Gly Arg Tyr Phe Asn Gly
                770                 775                 780
cag gac tgc cag ccc tgc cac cgc ttc tgc gcc act tgt gct ggg gca     2403
Gln Asp Cys Gln Pro Cys His Arg Phe Cys Ala Thr Cys Ala Gly Ala
            785                 790                 795
gga gct gat ggg tgc att aac tgc aca gag ggc tac ttc atg gag gat     2451
Gly Ala Asp Gly Cys Ile Asn Cys Thr Glu Gly Tyr Phe Met Glu Asp
        800                 805                 810
ggg aga tgc gtg cag agc tgt agt atc agc tat tac ttt gac cac tct     2499
Gly Arg Cys Val Gln Ser Cys Ser Ile Ser Tyr Tyr Phe Asp His Ser
    815                 820                 825
tca gag aat gga tac aaa tcc tgc aaa aaa tgt gat atc agt tgt ttg     2547
Ser Glu Asn Gly Tyr Lys Ser Cys Lys Lys Cys Asp Ile Ser Cys Leu
830                 835                 840                 845
acg tgc aat ggc cca gga ttc aag aac tgt aca agc tgc cct agt ggg     2595
Thr Cys Asn Gly Pro Gly Phe Lys Asn Cys Thr Ser Cys Pro Ser Gly
                850                 855                 860
tat ctc tta gac tta gga atg tgt caa atg gga gcc att tgc aag gat     2643
Tyr Leu Leu Asp Leu Gly Met Cys Gln Met Gly Ala Ile Cys Lys Asp
            865                 870                 875
gca acg gaa gag tcc tgg gcg gaa gga ggc ttc tgt atg ctt gtg aaa     2691
Ala Thr Glu Glu Ser Trp Ala Glu Gly Gly Phe Cys Met Leu Val Lys
        880                 885                 890
aag aac aat ctg tgc caa cgg aag gtt ctt caa caa ctt tgc tgc aaa     2739
Lys Asn Asn Leu Cys Gln Arg Lys Val Leu Gln Gln Leu Cys Cys Lys
    895                 900                 905
aca tgt aca ttc caa ggc tgagcagcc                                   2766
Thr Cys Thr Phe Gln Gly
910                 915
 
           
             7 
             915 
             PRT 
             Homo sapiens 
           
            7
Met Asp Trp Asp Trp Gly Asn Arg Cys Ser Arg Pro Gly Arg Arg Asp
  1               5                  10                  15
Leu Leu Cys Val Leu Ala Leu Leu Ala Gly Cys Leu Leu Pro Val Cys
             20                  25                  30
Arg Thr Arg Val Tyr Thr Asn His Trp Ala Val Lys Ile Ala Gly Gly
         35                  40                  45
Phe Ala Glu Ala Asp Arg Ile Ala Ser Lys Tyr Gly Phe Ile Asn Val
     50                  55                  60
Gly Gln Ile Gly Ala Leu Lys Asp Tyr Tyr His Phe Tyr His Ser Arg
 65                  70                  75                  80
Thr Ile Lys Arg Ser Val Leu Ser Ser Arg Gly Thr His Ser Phe Ile
                 85                  90                  95
Ser Met Glu Pro Lys Val Glu Trp Ile Gln Gln Gln Val Val Lys Lys
            100                 105                 110
Arg Thr Lys Arg Asp Tyr Asp Leu Ser His Ala Gln Ser Thr Tyr Phe
        115                 120                 125
Asn Asp Pro Lys Trp Pro Ser Met Trp Tyr Met His Cys Ser Asp Asn
    130                 135                 140
Thr His Pro Cys Gln Ser Asp Met Asn Ile Glu Gly Ala Trp Lys Arg
145                 150                 155                 160
Gly Tyr Thr Gly Lys Asn Ile Val Val Thr Ile Leu Asp Asp Gly Ile
                165                 170                 175
Glu Arg Thr His Pro Asp Leu Met Gln Asn Tyr Asp Ala Leu Ala Ser
            180                 185                 190
Cys Asp Val Asn Gly Asn Asp Leu Asp Pro Met Pro Arg Tyr Asp Ala
        195                 200                 205
Ser Asn Glu Asn Lys His Gly Thr Arg Cys Ala Gly Glu Val Ala Ala
    210                 215                 220
Ala Ala Asn Asn Ser His Cys Thr Val Gly Ile Ala Phe Asn Ala Lys
225                 230                 235                 240
Ile Gly Gly Val Arg Met Leu Asp Gly Asp Val Thr Asp Met Val Glu
                245                 250                 255
Ala Lys Ser Val Ser Phe Asn Pro Gln His Val His Ile Tyr Ser Ala
            260                 265                 270
Ser Trp Gly Pro Asp Asp Asp Gly Lys Thr Val Asp Gly Pro Ala Pro
        275                 280                 285
Leu Thr Arg Gln Ala Phe Glu Asn Gly Val Arg Met Gly Arg Arg Gly
    290                 295                 300
Leu Gly Ser Val Phe Val Trp Ala Ser Gly Asn Gly Gly Arg Ser Lys
305                 310                 315                 320
Asp His Cys Ser Cys Asp Gly Tyr Thr Asn Ser Ile Tyr Thr Ile Ser
                325                 330                 335
Ile Ser Ser Thr Ala Glu Ser Gly Lys Lys Pro Trp Tyr Leu Glu Glu
            340                 345                 350
Cys Ser Ser Thr Leu Ala Thr Thr Tyr Ser Ser Gly Glu Ser Tyr Asp
        355                 360                 365
Lys Lys Ile Ile Thr Thr Asp Leu Arg Gln Arg Cys Thr Asp Asn His
    370                 375                 380
Thr Gly Thr Ser Ala Ser Ala Pro Met Ala Ala Gly Ile Ile Ala Leu
385                 390                 395                 400
Ala Leu Glu Ala Asn Pro Phe Leu Thr Trp Arg Asp Val Gln His Val
                405                 410                 415
Ile Val Arg Thr Ser Arg Ala Gly His Leu Asn Ala Asn Asp Trp Lys
            420                 425                 430
Thr Asn Ala Ala Gly Phe Lys Val Ser His Leu Tyr Gly Phe Gly Leu
        435                 440                 445
Met Asp Ala Glu Ala Met Val Met Glu Ala Glu Lys Trp Thr Thr Val
    450                 455                 460
Pro Arg Gln His Val Cys Val Glu Ser Thr Asp Arg Gln Ile Lys Thr
465                 470                 475                 480
Ile Arg Pro Asn Ser Ala Val Arg Ser Ile Tyr Lys Ala Ser Gly Cys
                485                 490                 495
Ser Asp Asn Pro Asn Arg His Val Asn Tyr Leu Glu His Val Val Val
            500                 505                 510
Arg Ile Thr Ile Thr His Pro Arg Arg Gly Asp Leu Ala Ile Tyr Leu
        515                 520                 525
Thr Ser Pro Ser Gly Thr Arg Ser Gln Leu Leu Ala Asn Arg Leu Phe
    530                 535                 540
Asp His Ser Met Glu Gly Phe Lys Asn Trp Glu Phe Met Thr Ile His
545                 550                 555                 560
Cys Trp Gly Glu Arg Ala Ala Gly Asp Trp Val Leu Glu Val Tyr Asp
                565                 570                 575
Thr Pro Ser Gln Leu Arg Asn Phe Lys Thr Pro Gly Lys Leu Lys Glu
            580                 585                 590
Trp Ser Leu Val Leu Tyr Gly Thr Ser Val Arg Pro Tyr Ser Pro Thr
        595                 600                 605
Asn Glu Phe Pro Lys Val Glu Arg Phe Arg Tyr Ser Arg Val Glu Asp
    610                 615                 620
Pro Thr Asp Asp Tyr Gly Thr Glu Asp Tyr Ala Gly Pro Cys Asp Pro
625                 630                 635                 640
Glu Cys Ser Glu Val Gly Cys Asp Gly Pro Gly Pro Asp His Cys Asn
                645                 650                 655
Asp Cys Leu His Tyr Tyr Tyr Lys Leu Lys Asn Asn Thr Arg Ile Cys
            660                 665                 670
Val Ser Ser Cys Pro Pro Gly His Tyr His Ala Asp Lys Lys Arg Cys
        675                 680                 685
Arg Lys Cys Ala Pro Asn Cys Glu Ser Cys Phe Gly Ser His Gly Asp
    690                 695                 700
Gln Cys Met Ser Cys Lys Tyr Gly Tyr Phe Leu Asn Glu Glu Thr Asn
705                 710                 715                 720
Ser Cys Val Thr His Cys Pro Asp Gly Ser Tyr Gln Asp Thr Lys Lys
                725                 730                 735
Asn Leu Cys Arg Lys Cys Ser Glu Asn Cys Lys Thr Cys Thr Glu Phe
            740                 745                 750
His Asn Cys Thr Glu Cys Arg Asp Gly Leu Ser Leu Gln Gly Ser Arg
        755                 760                 765
Cys Ser Val Ser Cys Glu Asp Gly Arg Tyr Phe Asn Gly Gln Asp Cys
    770                 775                 780
Gln Pro Cys His Arg Phe Cys Ala Thr Cys Ala Gly Ala Gly Ala Asp
785                 790                 795                 800
Gly Cys Ile Asn Cys Thr Glu Gly Tyr Phe Met Glu Asp Gly Arg Cys
                805                 810                 815
Val Gln Ser Cys Ser Ile Ser Tyr Tyr Phe Asp His Ser Ser Glu Asn
            820                 825                 830
Gly Tyr Lys Ser Cys Lys Lys Cys Asp Ile Ser Cys Leu Thr Cys Asn
        835                 840                 845
Gly Pro Gly Phe Lys Asn Cys Thr Ser Cys Pro Ser Gly Tyr Leu Leu
    850                 855                 860
Asp Leu Gly Met Cys Gln Met Gly Ala Ile Cys Lys Asp Ala Thr Glu
865                 870                 875                 880
Glu Ser Trp Ala Glu Gly Gly Phe Cys Met Leu Val Lys Lys Asn Asn
                885                 890                 895
Leu Cys Gln Arg Lys Val Leu Gln Gln Leu Cys Cys Lys Thr Cys Thr
            900                 905                 910
Phe Gln Gly
        915
 
           
             8 
             27 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            8
ccaagcttgg ctgctgtgcg tgctggc                                         27
 
           
             9 
             19 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            9
ctgcctcaga tctgtagtg                                                  19