JP2001505870A - Proprotein converting enzyme - Google Patents

Abstract

(57) Abstract: A cDNA clone encoding human prohormone convertase PC5 is isolated from human adrenal messenger RNA. The deduced protein sequence will encode a 915 amino acid prepro PC5 that shares a very high degree of homology with previously cloned rat and mouse analogs. PC5 mRNA is detected in many human tissues including brain, adrenal gland and thyroid, heart, placenta, lung and testis. PC5 mRNA was undetectable in liver and was present at low levels in skeletal muscle, kidney, pancreas, small intestine and stomach. Co-transfection of human PC5 with a human prorenin expression vector in cultured GH ₄ Cl cells leads to secretion of active renin. Activation of human prorenin by PC5 is based on a pair of basic amino acids at positions 42 and 43 of the prorenin prosegment and occurs only in cells containing dense core secretory granules. Human PC5 is co-localized with renin by immunohistochemistry in the globular zone of the adrenal gland, suggesting that it may participate in the activation of the local renin-angiotensin system in the human adrenal cortex. PC5 is overexpressed in atherosclerotic coronary vessels. Silencing PC5 expression with specific antisense oligonucleotides effectively suppressed proliferation of smooth muscle cells in culture. Moreover, the antisense suppressed coronary artery stenosis in a coronary vein injury model. These results suggest that PC5 silencing is applied in preventing restenosis. PC5 can be a target of choice for treating any proliferative disease, including its effects on conferred growth factors. Finally, the antisense oligonucleotide PC5 should be used to silence the activation of this enzyme towards HIV gp160, since both co-localization in CD4 ⁺ T lymphocytes and the viral glycoprotein are splittable by PC5. It is.

Description

DETAILED DESCRIPTION OF THE INVENTION                          Proprotein converting enzyme Field of the invention   The present invention relates to protein processing enzymes or prohormone convertases (PCs), especially PC5, especially human PC5. Background of the Invention   Prohormone convertases (PCs) are proteins into active proteins or enzymes. It belongs to a family of enzymes that are responsible for the maturation of clast precursors. Until now, Millie's human enzyme comprises furin, PC1, PC2, PC4 and PC7. Have been identified. Each enzyme can be restricted (for example, PC4 is Have a distribution of tissue that is restricted or ubiquitous (furin is such an example) . Even if all of these enzymes have base or dibasic residues, they are Even though they share gender, they nonetheless have different splitting characteristics. Peculiar The action of prohormone convertase is therefore dependent on the sequence of a given protein substrate. And / or substrate growth factor or hormone for a given protein Or the location of that enzyme in the corresponding tissue.   Renin is a synthesis of the vasoactive octapeptide angiotensin II (AII) Contribution to cardiovascular physiology and pathophysiology through its key role in cancer Is an aspartyl protease. The kidney is the first source of cyclically active renin While the pituitary and adrenal glands, placenta, uterus, ovaries, testes, heart, vasculature and Some additional tissues, including the brain, express the renin gene (^1-4(See inside) . The presence of supplemental components of RAS (renin-angiotensin system) in these tissues Currently, angiotensin converting enzyme (ACE) and angiotensin II receptor Including, but the actual function of various tRAs is still largely speculative In some cases, some tissues have locally active tissue renin-angiotensin system (tR AS). Renin is produced by proteolytic removal of 43 amino acids of the amino terminal prosegment. Enzymatically inactive precursor, which is converted to active renin Synthesized. The activity of RAS in any given tissue is therefore Presence of proteolytic enzymes capable of converting nin to active renin and expression of prorenin And the expression of prorenin processing enzymes (PPEs) in the same cells. I An enzyme responsive in vivo to proteolytically activate human prorenin. Has not yet been finalized. In addition, many PPEs are present in humans And may vary within the renin-producing organization. In the kidney Biochemical and microscopic studies of renin have shown that PPE candidates^FiveLys⁴² , Arg⁴³Should be selected for the resolution of human brorenin in Somatic cells⁶Would be active in the secretory granules of. Lysosomal enzyme cat Psin B is expressed in human renin / prorenin in secretory granules of JG cells and human pituitary gland. Body lactorov^7,8And high affinity for unique split sites Shows cleavage of human prorenin in vitro by gender and selectivity To⁹. Prohormone convertase PC1 is also used for transfected cells.^TenCorrection in Cleavage of human prorenin with site- and organelle specificity and adrenal gland induction Led tumor¹¹Also shows that is not a JG cell, co-locates with renin Is¹².   In an effort to identify new PPEs, we used oncogene-induced Mouse tumor (As4.1 cells¹³) Derived from established renin-expressing tissue culture cells The distribution of processing enzymes has recently been determined. One such enzyme, mouse prohormone co Invertase PC5 was found. Mouse PC5 is a specific component of human prorenin. It is possible to split.   Miranda et al. (38) are on CD4⁺Obtained by PCR from T lymphocytes CDNA and protein sequences for the human PC6 enzyme are described. PC5 and P C6 is given a different name depending on how the same enzyme appears. I However, the sequence of Miranda et al. Has multiple locations when compared to the existing PC5 sequence. Exchange is included. In addition, the messenger RNAs encoding PC6 and PC5 The sizes are similar but not identical. Existing PC5 sequence from human adrenal gland As a result, both enzymes may be isotyped and are specifically expressed in tissues. And they may have different activities.   Prorenin and HIV gp160 are probably recognized and split by PC5 Would not be just a protein precursor to be performed. Respond to cell proliferation Many growth factors are divided by one or more PCs. They are, Platelet-derived growth factors A and B (PDGF's), epidermal growth factor (EGF), insulin Phosphorus-like growth factors I and II (IGF's), transforming growth factors α and β (T GF's). Each of these named growth factors is a typical split cytoplasm ＫK / R- (X) n-R ↓ (where n = 0, 2, 4, 6). Complete biological burial Potential is divided at these sites by one or more PC enzyme families. After cracking, they are given to these growth factors. Therefore, the manipulation of the expression of PCs is incomplete Could affect cell proliferation via significant growth factor activity.   > 450 in the United States and Canada undergoing percutaneous transluminal coronary angioplasty (PTCA) Out of 2,000 patients, 30-50% of patients have their coronary within 3-6 months The artery will restenosis. This relapse of endothelial and smooth muscle cell proliferation is very daunting It is due to his regulatory growth factor. Therefore, the enzyme can respond to this activity And the manipulation of the level of expression of this or these enzymes, It will be particularly useful for preventing restenosis. STATEMENT OF THE INVENTION:   The present invention relates to human PC5 (hPC5). We have isolated from the human adrenal gland Human prorenin with the site- and organelle-specificity predicted by hPC5 Catabolically activates and co-acts with prrenin in the spheroids of the adrenal cortex -Prove that it will be expressed. Therefore, PC5 is prorenin-processing enzyme (PPE) It is. Silencing PC5 expression is specific in suppressing renin production And applications for inhibiting renin production are described in the book It is an object of the invention. Hypertension because renin production is one of the targets of RAS Are also included. Moreover, we have found that hPC5 has been shown to treat coronary arteritis To be overexpressed. Antisense oligonucleotides are Has been shown to successfully silence hPC5 expression in smooth muscle cells in Designed from within. This antisense is effective against rabbit carotid artery injury in vivo. Carotid stenosis was suppressed in the wound model. These results indicate that the expression of PC5 was suppressed. Figure 9 shows that the mitigation method will find specific application in preventing restenosis.   PC5 is CD4 together with furin and PC7.⁺Known to be expressed in T cells It is. Three enzymes can convert HIV gp160 to its fusogenic form. Wear. Therefore, antisense structures, especially oligonucleotides that successfully suppress restenosis Reotide inhibits the expression of PC5 activity towards HIV gp160. Use will be found.   The complete amino acid and diffuse sequences of hPC5 are described below and Is another purpose. Recombination including insertion of whole or part of hPC5 Vectors and hosts are also an object of the present invention.   Oligopeptides derived from the protein sequence of hPC5 are also an object of the present invention. You.   Antibodies directed against the entire hPC5 protein or a portion thereof are also provided by the present invention. Is the purpose.   With oligonucleotide or antibody-bound P5 diffusion or protein or peptide Diagnostic methods and kits are also an object of the present invention.   The present invention is intended to illustrate the anticipated aspects of the present invention and to limit the scope of the present invention. Specific embodiments, examples and drawings that are not Will be. BRIEF DESCRIPTION OF THE FIGURES FIG. 1: Schematic representation of the isolated cDNA encoding hPC5. Subcloning The restriction enzyme sites used in are defined. Solid line is from phage library Represents an isolated clone. The hatched line indicates RT-PCR of human adrenal mRNA. Represents the portion of the cDNA separated by. Double line completes cDNA for expression Mouse PC5 cDNA (corresponding to the amino terminal group of the signal peptide) Represents the part. Figure 2: hPC5 nucleic acid and derived protein sequences (SEQ ID NO:  NOS) 1 and 2). Proposed signal peptide (solid arrow) and prosegment (open Arrow) Split site is data from mouse PC5^FifteenIs defined based on Ann The sequence with the dashed line is the signal from mouse PC5 used in the construction of the expression vector. Represents the part of the null peptide. Figure 3: Distribution for PC5 in various human tissues. Each lane is 2 μg Includes Li-A RNA. The filter was hPC5 as described in Materials and Methods. Hybridized with a radiolabeled probe for Shown on the left is a kilo salt Transfer of single chain size standard in group pairs (Kb). Absolute signals are different Because of age and hybridizing at different times Note that they cannot be compared. Figure 4: hPC5-split human protein with site and cell specificity. panel A: GH_FourCl cells are co-transfected with an expression vector for the indicated protein. You. The supernatant is collected 30 hours after transfer and analyzed for% active renin [(active level Nin / total renin) x 100]. Bars represent mean ± S.E.M. of 9 independent transfectants. Represents * = p <0.0001 protein + pUC as comparison with Mann-Whitney -Determined by parametric tests. Panel B: Expression for prorenin With either vector and control plasmid (pUC) or hPC5 Results of secretion of active renin after co-transfection of CHO cells. The bars are three independent importers Mean ± S.E.M. Figure 5: Co-transferred GH_FourActive renin production in secretory granules of Cl cells. Human professional GH co-transfected with expression vector for renin and human PC5_FourC1 cell Parallel wells are 50 mmol / L NaCl (control) or 50 mmol / L. KCl (a depolarizing agent causing rapid release of secretory granules) in a medium containing Incubate for 20 minutes. The percentage of active renin is as described in FIG. 4A. Is calculated as described above. Bars represent the mean ± S.E.M. of three independent transfectants. * = P <0.005 Use student'st test. Figure 6: HPC5 and renin / prorenin in renal cortex, human placental lobe and adrenal gland Immunodetection. Positive colored areas are indicated by solid arrows. Sections in the adrenal cortex Cells to show co-localization of cells in the globular band (g) and in bundles with capsules (c). band Separated at 5 mM to show absence of color in (f). Original size 25X (kidney And placenta) and 80X (adrenal gland). Figure 7: Incorporation of PC5 mRNA in human coronary vessels in atherosclerosis Hybridization analysis in vivo. Severe damage observed in lower panel Shows vessels. PC5 mRNA is abundantly expressed in smooth muscle in neointima and in this container (See arrow). In comparison, another container without any damage was PC5m Does not express RNA (shown in panel of information). Figure 8: Antisense, sense or mismatched PC5 oligonucleotides Western Blot Analysis of PC5 Protein in Rabbit Smooth Muscle Cells Analysis. A specific PC5 band was detected in rat supraoptic nucleus (SON) and SK-N-HCIXC cells (human sensory). Epithelial cells) (see arrows). Ann In samples extracted from the Chisense PC5 treatment, we used control sense Or dramatic levels of PC5 signal in comparison to mismatched PC5 oligos A decrease (almost a 2-3 fold decrease) was observed. This is because the antisense treatment 9 shows that the protein level of PC5 in smooth muscle cells is significantly reduced. Figure 9: PC5 antisense as a comparison between control sense and random ODNs. In vivo testing of rabbit ODN in rabbits. Compared to sense and random controls When compared, shows reduced stenosis due to the presence of PC5 antisense. Figure 10: Proprotein convertase immunity in human atherectomy specimens Reactivity. The presence of prohormone convertase family enzymes in the specimen Show. Figure 11: PC6 (Miranda et al. SEQ ID No. 4) and PC5 (invention) Shows differences between cDNA sequences. Figure 12: PC6 (Miranda et al. SEQ ID No. 4) and PC5 (invention) Shows differences between protein sequences. Description of the invention Materials and methods   cDNA library structure and screening: total human adrenal RNA The derived cDNA library was Strata from the phage vector Uni-Zap XR. Constructed by gene (La Jolla, CA). 600,000 phage plaques are radioactive pro And Standard Methodology¹⁴And was screened first. First hybridization The solution probe is a mouse PC5 cloned in advance.^FifteenHigh degree of similarity to Identification human cDNA sequence in Genbank (Accession # M85522) with Derived from reverse transcriptase PCR of human brain RNA using information derived from column tags It is a 320 base pair DNA fragment. Labeling of fragments³²P dC TP and random primer labeling kit (Boehringer-Mannheim Canada, Lavel, Qu ebec, Canada) according to the instructions in the instructions. One positive hybrid A pad forming phage (hPC5A) was identified. Its insertion is a dideoxy-chain end. Sequenced in its entirety using the method and highly similar to mouse PC5 Encoding a 1150 base pair cDNA having the sequence Is not shown). The 1070 base pair fragment (excludes the tail of polyA) is hPC5A Re-screen 600,000 phage added from the cDNA library Labeled and used for The second phage clone (hPC5B) Separated and 1807 base pair cDNA insertion overlapping hPC5A and cD It is found to contain an extension towards the 5 'end of NA (Figure 1).   Reverse transcriptase PCR: poly A + total human adrenal gland RNA (Clontech Laboratories, Palo Alto, CA)¹⁶And the following oligonucleotide Perform a reverse transcriptase polymerase chain reaction (RT-PCR) using: The above oligonucleotide; a region corresponding to the signal peptide of mouse PC5 Derived from^Fifteen. Artificial HindIII restriction enzyme cleavage site To the 5'-end of the amplified fragment for marking purposes: 5'-CCAAGCTTGGCTGCTGTGCGTGCTGGC-3 '   Reverse oligonucleotide; derived from the 5'-end of phage hPC5B. BgIII inside Restriction enzyme sites are underlined: 5'-CTGCCTCAGATCTGTAGTG-3 '   The entire RT-PCR reaction was repeated four times and four of the amplified fragments Independently derived clones were sequenced and their sequences compared. Jinbang The sequence (Accession # U49114) submitted to Genbank was expressed in the 3/4 clone. Represents a consensus sequence defined as any nucleotide that   Northern blot analysis: The tissue distribution of PC5 mRNA was different from that of various human tissues (Cl Aliquots (2 μl) of poly-A RNA from ontech Laboratories, Palo Alto, CA) g) for the hybridization of nitrocellulose filters purchased commercially Is determined. The probe used was derived from full-length hPC5 cDNA. Complementary RNA was used. Probe labeling and hybridization was performed as described above.¹⁷ .   Expression vector structure: cDNA sequence from KpnI site (Fig. 1) to immediately after stop codon The fragment was excised from phage hPC5B, containing KpnI and two independent RT-Ps. The HindIII (see above) fragment derived from a portion of the CR clone was ligated ( (To eliminate errors arising from Tag polymerase). derived from mPC5 The region corresponding to the first 16 amino acids of the signal peptide is the overlap extension Attached to 5'-end by PCR¹⁸. Thus, the entire cDNA, mPC5 Coded amino acids 1-16 derived from gnal peptide and residue from hPC5 One places the cDNA under the control of the RSV promoter and Provides a 3 'intron and polyadenylation signal from the human globulin gene Expression vector RSV-globulin¹⁹Subcloned into Subcloned The entire fragment is subsequently verified by DNA sequencing.   Cell culture and transfer: GH_FourCl cells are 5x10 per well^Five6-cells in cell concentration Plated in L culture dishes. After 24 hours, the medium is changed and the cells are Kit (CellPect Transfection kit, Pharmacia Biotech, Bair D'urfe, Quebec , Canada) according to the instructions in the manual and transferred by the DEAE-dextran method. It is. Each well contains an hPC5 expression vector or a neutral plasmid vector (pUC18) 0.18 μg of the expression vector or prorenin processor for human prorenin. The equivalents of amino acids 42 and 43 mutated to alanine (respectively , K / A-2 and R / A-1). 30 hours after transfer Captured and analyzed for prorenin and renin as described above²⁰.   To demonstrate the conversion of prorenin generated in secretory granules, GH transfected with hPC5 expression vector_FourCl is a known technique^{twenty one}Using It is stimulated by releasing secretory granules by depolarization. Co-populated 40 After a time, the medium in the parallel wells of the transferred cells is NaCl (control) or K Replace with preheated medium supplemented to a final concentration of either 50 mM of Cl (secretagogue) did. The medium was collected after 20 minutes and analyzed for renin / prorenin. Cell supernatant Potassium that increases the percentage of active renin contained in Treated as an indicator of active renin released from secretory granules. The results shown in FIG. , Represents the average of three independent transfer experiments.   Immunolocalization of hPC5 in human tissues: Human tissues are fused in Bouin's solution. Post-mortem (kidney and adrenal) or paraffin-embedded Obtained Post-partum (placental lobe). 5 μm for immunolocalization Sections are mounted on gelatin-coated slides, deparaffinized, and rat PC Polyclonal strain raised against a peptide corresponding to the N-terminal 16 amino acids of 5 1:50 dilution of Saki antiserum (PC5.MAP antibody) or poly against recombinant human prorenin Incubated with a 1: 200 dilution of clonal rabbit antiserum. Kidney and placenta For specimens, the immune complex was obtained by Gitescu and Bendayan's method.^{twenty two}According to tetraclo Incubation with protein A-colloidal gold (15 nm particles) synthesized from chloroauric acid (BDH) It was revealed by the solution. Gold particles are silver (IntenSE^TMM silver Enhancement Kit, Amersham Life Science, Oakville, Ontario, Canada) Increases the visibility under the microscope light, and the sections are hematoxylin and methylgrease. Was counter-colored. Immune complexes on human adrenal gland sections were labeled with biotin-label. Intellectualized Lobalite-Rabbit IgG 1: 200 dilution and Streptavidin-Horsra Dish peroxidase complex (Amersham Life Science, Oakville, Ontari o, Canada) and diaminobenzen as chromogen. Incubated with digidine and hydrogen peroxide. All positive coloring patterns are Specificity was substantially demonstrated by omitting the first antibody. result   The initial sequence of human PC5 is shown in FIG. We are the RACE protocol¹⁶Ma Or mouse PC5^15,23Using oligonucleotides based on the known sequence of Can not clone the 5'-end of the top of the cDNA, This may be due to the high G / C content of the known sequence of the cDNA in this region. I However, rat and mouse PC5^FifteenBased on known cDNA sequences for Each is the 5′-end of the cDNA corresponding to the first 12 amino acids of the signal peptide. I was convinced that the minutes were separated, but not all. Ratio to the known sequence of mouse PC5 By comparison, we found that the isolated cDNA was signal peptide and 32 and 8 respectively. Encodes a 915 amino acid prepro PC5, including a 4 amino acid prosegment Predict that this will happen. The deduced sequence of hPC5 is 88% homologous to mouse PC5 cDNA and 96% homologous to mouse PC5 protein. Is homologous to   Northern analysis of polyA RNA from various human tissues showed a major 6.6 Kb The band required and a minor band of about 3.8 Kb are shown (FIG. 3). PC5 RNA is in the brain, Skeletal muscle, kidney and pancreas in the heart, placenta, lung, thyroid and testis, and at low levels; Detected in small intestine and stomach. In the adrenal gland, PC5 is particularly abundant in the cortex (FIG. 3).   PC5 RNA is expressed in many tissues previously reported to contain active renin We found that in the cell co-transfection assay, we The ability of hPC5 to cleave prorenin is tested (FIG. 4A). Conventional report As advertised^TenCultivated rat somatotrophic GH_FourCl cells Co-transfected with toprorenin encoding expression vector and neutral plasmid vector In this case, only unprocessed prorenin is secreted into the culture supernatant. In contrast If the human prorenin expression vector co-exists with the expression vector encoding human PC5, -If transferred, some of the expressed prorenin is secreted as active renin . Any of the unique cleavage site (lysine 42 or arginine 43) forming base residues Co-expression of prorenin mutated with and hPC5 prevents activation. these The results indicate that human PC5 was previously reported for renin activation in humans.^Five Protein at site This suggests that the proteolytic cleavage activates human prorenin. Human PC5 Is GH_FourWhile dividing human prorenin in Cl cells, the co-transfectant Increased secretion of active renin when transported into Chinese hamster ovary (CHO) cells Is not found (FIG. 4B). GH_FourCHO cell line for comparison with Cl cells The apparent difference in the above is that either human PC5 or human prorenin or both Its lack of secretory granules suggests a requirement for a distributed granule environment during the proteolytic process It is a fall. The conclusion is that potassium chloride (FIG. 5), a deagglomeration that causes the release of secretory granules Extreme agent^{twenty one}And transferred GH for 20 minutes_FourActivity detected in Cl cell supernatant This is supported by a sharp increase in renin (FIG. 5).   Polyclonal antibodies raised against peptides derived from mouse PC5 We have been experimenting with the distribution of human PC5 in several human tissues. 6). To date, we believe that even if sections were stained positive by renin, No coloration by PC5 could be detected in the kidneys of the mice. Placental lobe PC5 is localized in the syncytiotrophoblast of the chorionic villi, and anti-renin antibody is The hair mesoderm is first colored. In the adrenal gland, anti-bodies against both renin and PC5 The body consists of spheres in the adrenal cortex (g) with very little coloration of the capsule (c) and the fascicle A preferential coloring of the band was indicated. No staining is evident from the deletion of the first antibody (Data not shown). Thus, our immunohistochemical study is based on three experiments That prorenin and PC5 are clearly co-localized in the globular zone of the human adrenal cortex Would suggest that this is only the case. Commentary   In this study, we cloned human prohormone convertase PC5 and The expression and its activation as human PPE are described. In cultured cells The co-transfer assay shows that hPC5 has the expected site-specificity of human prorenin And that this splitting is most likely in dense core secretory granules. Demonstrate. In addition, the immunohistochemistry of human tissues indicates that renin in the globular zone of the adrenal cortex Shows co-localization of hPC5. Several lines of evidence suggest that human adrenal glands contain physiologically important local RAS Suggests: First, the RNA encoding angiotensinogen and renin is Detected in preparations from human adrenal globular gland, fasciculus and medulla^24,25, Reni This is the same idea that protein and its substrate are synthesized inside the human adrenal gland. Second ACE suppression or blockade of angiotensin receptor is dependent on human adrenal tissue translocation Suppresses aldosterone released from plant²⁶, Local RAS is aldo from adrenal gland Suggests playing an active role in regulating sterone secretion. Third, human Adrenal cortical tissue grafts and aldosterone-secreting adenomas are low in active renin Secretes^24,26,27, Adrenal cortex expresses PPE capable of activating human prorenin Suggest that. Our current results show that human side effects such as both renin and PC5 PC5 can be PPE responsive for renin activation in renal cortex Suggest that is immunologically detectable in the globular zone. Additional circumstantial evidence supports this conclusion: First, the centrifugal fractionation of adrenocortical cells Shows that renin is in the "granule" fraction of intermediate density between vesicles and lysosomes To be included in²⁸. In our current study, PC5 contains secretory granules. Suggests that it cleaves only human prorenin in cells, and that renin is It will be in the appropriate intracellular compartment to be activated by PC5. Second, Rat mutations caused by mouse REN-2 renin [TGR (mRen-2) 27] Hypertension is best correlated with prorenin expression in rats²⁹ Represents^30-32. As a previous study, PC5 activates mouse Ren-2 prorenin Yes, but not rat prorenin (^{twenty three}And data are not shown). TGR (mRen-2) 27 transgenic mice In the tissue R by unexpected close juxtaposition of appropriate PPE and prorenin This seems to be a model for activation of AS. These results also indicate that PP Es tissue-dispersion and their manifestation in activated prorenin from different species Selectivity may lead to differential function of RAS tissue between rodents and humans Also occur.   The major source of cyclic active renin in humans is kidney JG cells. for example, Low levels of hPC5 RNA are present in total kidney poly-A RNA samples Even if it could be detected by Northern blot analysis in Off PC5 immunostaining in pieces could not be localized (FIG. 6) and in the nucleic acid cell types in the kidney At low levels PC5 raises the possibility of expression. Thus, these results are PC5 could not be formally excluded as a PPE in JG cells, Detecting whether or not it is likely to play a major role in renin production That is impossible for us. In contrast, the phase of PC5 mRNA and prorenin Conversely abundant amounts, even if the evidence is that placental cells in culture³³And in vivo³⁴But professional It is detected in the placenta, even if it suggests that it secretes only renin. But However, the cells producing PC5 and prorenin in the human placenta are clear This was shown by immunostaining. It also produces prorenin in two proteins at once PC5, which would be activated, was converted to prorenin by hPC5 in the transfected cells. It also does not appear to be secreted due to the obvious demands of the granule environment for splitting. Scratch In contrast, PC5 expressed in the human placenta was treated with placental prole It is unlikely that it will activate nin.   In mice, two forms of hPC5 are based on cloned cDNAs. The first is the hPC5 cDNA described in this study. And cloned from rat tissue.^15,23And PC The second, termed 6B, is characterized by its 3'-end due to the differential RNA splicing phenomenon. Extended by³⁵. Even if the hPC5 cDNA that we clone The major R, found in human tissues, even if only about 3 Kb The NA band is approximately 6.6 Kb. Hybridizes to hPC5 probe The homology of the longer band is currently unknown. It's an adrenal library Of cDNA clones isolated from a screen of 1.2 million phages from In any case, even if the probe used to separate it was homologous to the mouse PC6B mutant Even if it covers a typical area³⁵Note that it is not extended at the 3'-end It should be noted (Fig. 1). In mouse tissues, the expression of the PC6B mutant Organization³⁵While many 6.6K detected by the hPC5 probe b is directly proportional to a number of 3.8 Kb bands. Using a PC5 probe Hybridization of RNA blots from dental animal tissues also showed 3.8, 6.5. And also the 7.5 Kb RNA band^15,35Use of PC5-specific probes Use for 6. Represents the band at 5 Kb. Thus, an additional PC5 RNA species has its 5'-end May be present in mammals prolonged. Alternatively, human tissues are In screening for homology to PC6B not picked up Can be particularly enriched. Recent data indicate the presence of alternating C-terminal tails on PC6B. While presence can help maintain enzymes in the Golgi network, mouse PC The "short" form of 5 suggests that it is targeted for dense core secretory granules (N .G.Seidah, unpublished). These data and our co-transfer assay (FIG. 4) The "short" form of hPC5 described here activates renin processing in secretory granules Would suggest a form that would be.   The PC5 enzyme isolated from humans and mice has nucleotide and protein sequence levels. Shows a significantly higher degree of conversion in the A similar measure is the C-terminus of the enzyme. Than found in other mammalian PC enzymes that appear to diverge in half of the ,taller than^36,37. A high degree of sequence conversion indicates that PC5 (and PC5 (C-terminal). PC5 is linked to smooth muscle proliferation PC is a potential target for smooth muscle cell proliferation To investigate that we can determine if any of the PCs is a process of restenosis, just If such growth was observed, it was tested for any effect. Restenotic Changes in the PC level in the process are prior to using animal models or cell lines. Since in testing, there is a distinct possibility that we may adjust or even elicit PC levels. Shows that it can be derived. Thus, we used restenotic human coronary vein from a patient. Got tissue. These tissues are used in vivo to obtain information in anatomical technical content. Screen for each PC mRNA using hybridization histochemistry in vivo Was. Some coronary arteries or total occlusions occur when the coronary artery tissue is PC5-negative. PC5 mRNA levels in smooth muscle tissue show a dramatic increase, whereas no occlusion Was. These results indicate that during the active process of PC5 stenosis, PC5 (See FIG. 7). About our findings Thus, this is the first indication that specific PCs are directly linked to smooth muscle proliferation. It is.   These results indicate that if enzymatically active PC5 is somehow suppressed, If possible, or if improved regulation of PC5 mRNA can be prevented, Suggests that the process can be weakened or stopped. This is the shape of the coronary lesion Inhibition of the processing function of this enzyme for many growth factors involved in growth Can be done. If these growth factors are not processed, they Will remain inactive. Our approach is smooth in vitro To test the efficacy of PC5 antisense suppression in muscle growth.   Specific antisense oligonucleotides (ODNs) are Was demonstrated to rapidly suppress smooth muscle proliferation. PC5 Incubation with antisense 17-mer oligonucleotide The heron smooth muscle cells were 81.6% ± 1.6% at 10 mM (3 out of 4 Table 1 showing the dosage-dependent inhibition of smooth muscle proliferation with the maximum inhibitory effect of It was shown to. Inhibitory effects can be achieved at the same concentration with sense or mismatch oligonucleotides. Significantly higher (P = 0.0001) compared to controls containing either (See Table 1). In addition, we have found that PC5 expression reduces affected cells. (FIG. 8). When compared to other targets such as c-myc, this approach 71.7 + 3.5% (average of 3 experiments out of 4) suppression in chisense c-myc Smooth muscle gain compared to the best efficacy resulting from a control (mean of three of the four tested) It is significantly more effective in controlling breeding. These results indicate that sedated PC5 is smooth Demonstrates in vivo efficacy as it would prevent muscle cell proliferation and restenosis. Cholesterol binding of oligonucleotides   Phosphorothioate antisense ODNs are prepared using the following standard procedure for DNA / R It is synthesized by an NA synthesizer (provided by Biosystems). Cholesterol and olives The binding of gomato is performed via 3'-cholesterol-VN CPG (Clontech) and solid-phase synthesis. Virtual nucleosides that induce cholesterol labeling at the 3 'end of gonucleotides Achieved by the tide (VN) glass reagent. At the time of ODN synthesis. ODN synthesis succeeded If the oligomer is 30% NH_FourRemoved from the column by OH (1 hour at room temperature) And then deprotected at 60 ° C. for 8 hours. Oligos are purified and oligonutrient Oh Detritylated with a tide purification cartridge (provided by Biosystem) and centrifuged Lyophilized by concentration (Savant SpeedVac). In vivo arterial ODN transfer   Male or female New Zealand rabbits (2 Kg) were treated with xylazine (2 mg / Kg) in muscle. They were quiescent and anesthetized with ketamine (100 mg / Kg) prior to surgical exposure of the left carotid artery. Ten mm carotid segments are temporarily tied and have no visible evidence of blood components Rinsed with 0.9% sodium chloride through a cannula until clean. Carotid 1cm Either of the moieties alone was transferred with 80 μmol / L antisense ODNs Or bind to cholesterol during any 30 minute deadline. Leached capacity Is 100 μl and an invisible loss of volume Recorded through. After transfer, the treated segment was 0.9% sodium chloride For rinsing (3 × 100 μl) and cannula removal, the arteriotomy site is micro-suture And restored normal blood flow and closed the wound. In this project All experimental protocols are based on the Institutional Committee for Animal Protectio Approved by n of Louis-Charles Simard Reseach Center. Suppress neointimal hyperplasia   A total of 36 New Zealand white rabbit carotid arteries with gentle traction Continuous inflation at 4, 6, 8, and 10 atmospheres for 1 minute and 45 seconds between inflation. It was injured by a 2.5 mm balloon catheter. Two weeks later, second loss The scarring is 80 μmol / L (100 μl of injected volume) of therapeutic molecule or control. Same neck movement transferred within 1 cm with 100 μL of 0.9% NaCl Added to the pulse site. Intima / media area is second damaged and transferred Two weeks after the procedure, the control in the histological section derived from the transferred carotid artery Evaluated by pewter analysis.   At the time when the carotid artery was damaged for the second time by the balloon catheter, it is shown in Table 1. The addition of PC5 antisense 17-mer-ODN reduces the area ratio of intima / inside to 40%. (Divide by area ratio intima / inner sense ODN minus area ratio sense ODN Was Area ratio antisense ODN; see FIG. 9) Reduces measured carotid artery stenosis. this The inhibitory effect is as compared with each of the sense and random control, High significance (P = 0.0118 and P = 0.0078). These results indicate that such treatments To administer a dosage effective for controlling stenosis of PC5 antisense to necessary patients This is the basis of the method for preventing stenosis. Other antisense oligonucleotides Is another converter called PC2, also observed in the atherectomy sample. Make it most effective in preventing restenosis, such as silencing the expression of lysase This method can be added (see FIG. 10).   The drug is readily and directly accessible to affected sites during interventions by cardiologists. The development of drugs based on the inhibition or inactivation of convertases is Always interesting.   In addition, we anticipate that cell growth is achieved by antisense to one of the convertases. This therapeutic approach, which is based on inhibition, allows the active Ensuring that it would be applicable to all proliferative disorders, including protein maturation Request.                                  Table 1   The sequence of the oligonucleotide used was: PC5 is involved in the division of HIV gp160 into gp120 and gp41.   As described above, PC5, PC7 and furin are CD4⁺Present in T lymphocytes It is known that All three enzymes have the same H IV gp160 is split into gp120 and gp41, where the split is gp160 Happens in. The 17-mer antisense ODN represented in SEQ ID NO: 3 is PC5 Successfully silenced the onset of expression and prevented restenosis. With a tide Also any other oligonucleotide or structure with equivalent silencing function is CD4⁺In suppressing the activity of PC5 on HIV gp160 in T lymphocytes Use will be found. suppression of the conversion of gp160 to its fusogenic form Includes antisense molecules to PC5, PC7 and Furin for optimization Cocktails are contemplated as part of the present invention. A suitable vehicle such as a liposome Can be used to transport these antisense molecules to target tissues.   The invention will now be described below. Without departing from the above teachings. It will be clear to the skilled reader that modifications can be made to it . These modifications are intended to form part of the scope of the present invention as set forth in the appended claims. To be considered.

[Procedure of Amendment] Article 184-8, Paragraph 1 of the Patent Act [Submission date] October 29, 1998 (1998.10.29) [Correction contents]                                The scope of the claims 1. Use of a compound capable of suppressing PC5 expression or activation, PC5 was used in the manufacture of a medical device to prevent stenosis, Proprotein convertase having an amino acid sequence contained in 1 Use that is. 2. The compound is capable of hybridizing the PC5 gene and silencing its expression. A functional antisense nucleic acid, wherein the PC5 gene has the nucleic acid sequence of SEQ ID NO: 1. The use according to claim 1, comprising or a variant thereof. 3. 3. The method according to claim 2, wherein the antisense nucleic acid has the nucleic acid sequence of SEQ ID NO: 3. Use. 4. Effective amount of compound capable of suppressing PC5 expression or activation and pharmaceutically An anti-restenosis composition comprising an acceptable carrier, wherein PC5 has SEQ ID NO: 1. A proprotein convertase having an amino acid sequence contained therein or a variant thereof Some compositions. 5. The compound is capable of hybridizing the PC5 gene and silencing its expression. A possible antisense nucleic acid, wherein the PC5 gene has the nucleic acid sequence of SEQ ID NO: 1. 5. The composition according to claim 4, comprising a sequence or a variant thereof. 6. 6. The method according to claim 5, wherein the antisense nucleic acid has the nucleic acid sequence of SEQ ID NO: 3. Composition. 7. A method comprising an effective amount of a compound capable of suppressing PC5 expression or activation. This is a medical device for preventing stenosis, wherein PC5 has the amino acid sequence contained in SEQ ID NO: 1. A medical device which is a proprotein converting enzyme or a mutant thereof. 8. The compound is capable of hybridizing the PC5 gene and silencing its expression. A possible antisense nucleic acid, wherein the PC5 gene has the nucleic acid sequence of SEQ ID NO: 1. 5. The medical device according to claim 4, comprising a row or a variant thereof. 9. 6. The method according to claim 5, wherein the antisense nucleic acid has the nucleic acid sequence of SEQ ID NO: 3. On-board medical device.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 15/09 ZNA C12N 15/00 ZNAA (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE GH, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ , PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (71) Applicant Schletian, Michel Canada Quebec H2V 1Z8 Montreal Coto Sainte-Catherine 1 Apartment 1404 (71) Applicant Rudruber, Tim Canada Quebec H9X 2P4 Beddurfe Warwick Drive 671 (71) Applicant Le Clark, Guy Quebec J7A 4K1 Rose Mail Lorraine 327 (72) Inventor Dei-Robert Quebec H2W 3S6 Saint-Drotet-Lu-Piet, Canada Le 833 (72) Inventor Sadar, Neville G. Québec H3E 1S6 Ile de Seur de Gaspe 200 apartment in Canada 1412 (72) Inventor Martel, Remi in Quebec H1V 1R7 Montreal La Fontaine 4865 (72) Inventor Shrethian, Michelle Quebec H2V 1Z8 Montreal Coto Sainte-Catherine 1 Apartment 1404 (72) Inventor Rudruber, Tim Canada Quebec H9X 2P4 Bedurfe Warwick Drive 671 (72) Inventor Le Clark, Guy Quebec, Canada J7A 4K1 Rosemail Lorraine 327

Claims (1)

[Claims] 1. A purified human proprotein converting enzyme designated PC5. 2. A purified human proprotein converting enzyme designated PC5 isolated from the adrenal gland. 3. The proprotein converting enzyme according to claim 2, which has the amino acid sequence of SEQ ID NO: 2. 4. An isolated nucleic acid encoding the proprotein converting enzyme according to claim 1, 2 or 3. 5. The isolated nucleic acid according to claim 4, which has the nucleic acid sequence of SEQ ID NO: 1. 6. An oligonucleotide having at least 15 nucleic acids that is a part of the nucleic acid according to claim 4 or 5 or a complementary sequence thereof. 7. An oligonucleotide having the nucleic acid sequence set forth in SEQ ID NO: 3. 8. Contacting the proprotein converting enzyme according to claim 1, 2, or 3 with a protein precursor under conditions that maintain the activity of the enzyme, and recovering the protein; A method of convertible to a mature protein by PC5. 9. A method for inhibiting muscle cell proliferation, comprising contacting a muscle cell with the oligonucleotide of claim 7 or any equivalent silencing antisense molecule. 10. 10. The method of claim 9, which results in prevention of restenosis. 11. A method for silencing PC5 expression in a cell, comprising the step of contacting the cell with a complementary sequence of the nucleic acid according to claim 4 or 5, or a portion thereof having at least 15 nucleic acids. 12. 9. The method of claim 8, wherein said precursor is HIV gp160. 13. The method according to claim 11, wherein the cells are CD4 ⁺ T lymphocytes. 14． 14. The method of claim 13, wherein said cells are HIV infected T lymphocytes. 15. 15. The method of claim 14, wherein PC5 results in suppression of HIV gp160 splitting. 16. 16. The method of any one of claims 11 and 13 to 15, wherein said portion of said complementary sequence comprises the oligonucleotide set forth in SEQ ID NO: 3. 17． 16. The method of claim 15, wherein said portion of said complementary sequence comprises the oligonucleotide set forth in SEQ ID NO: 3. 18. 18. The method of claim 17, further comprising an antisense silencing molecule for PC7 and furin.