RECOMBINANT C-PROTEINASE AND PROCESSES, METHODS AND USES THEREOF

Abstract: The present invention is directed to the isolation and identification of the nucleic acid sequence encoding C-proteinase, the recognition of such protein's activity and applications, and tools, processes, and methods of use thereof.

Description: RECOMBINANT C-PROTEINASE AND PROCESSES, METHODS AND USES THEREOF Inventors: Darwin J. Proc op, Yoshio Hoji a, Shi-Wu Li, and Aleksander Sieron 1. STATEMENT OF RELATED CASE This application is a continuation-in-part application of provisional application, U.S. Serial No. 60/002038, filed August 8, 1995. 2. INTRODUCTION Collagen is integral to, among other things, the proper formation of connective tissue. Therefore, the over- or under-production of collagen or the production of abnormal collagen (including incorrectly processed collagen) has been linked with numerous connective tissue diseases and disorders. Consequently, control and/or modulation of collagen formation has been the focus of study. These studies include efforts to identify enzymes, including C- proteinase, critical to collagen' s proper formation and processing. The present invention is directed to the isolation and identification of the nucleic acid sequence encoding C-proteinase and the corresponding polypeptide, _he recognition of such polypeptide activity, and applications, tools, processes and methods of use thereof. 3. BACKGROUND OF THE INVENTION Collagen Structure . At present, nineteen types of collagens have been identified. These collagens, including fibrillar collagen types I, II, III, are synthesized as procollagen precursor molecules which contain amino- and carboxy-terminal peptide ext-"" -'ions . These peptide extensions, referred to - c - generally as "pro-regions, " are designated as N- and C-prope tides, respectively. Both the N-propeptide and C-propeptide are typically cleaved upon secretion of the procollagen triple helical precursor molecule from the cell to yield a mature triple helical collagen molecule. Upon cleavage, the "mature" collagen molecule is then capable of associating into highly structured collagen fibers. See e . g. , Fessler and Fessler, 1978, Annu. Rev. Biochem . 17:129-162; Bornstein and Traub, 1979, in : The Proteins (eds. Neurath, H. and Hill, R.H.), Academic Press, New York, pp. 412-632; Kivirikko et al . , 1984, in : Extracell ular Matrix Biochemistry (eds. Piez, K.A. and Reddi, A.H.), Elsevier Science Publishing Co., Inc., New York, pp. 83-118; Prockop and Kivirikko, 1984, N. Engl . J. Med . 211:376-383; Kuhn, 1987, in : Structure and Function of Collagen Types (eds. Mayne, R. and Burgeson, R.E.), Academic Press, Inc., Orlando, Florida, pp. 1-42. Diseases Associated With The Abnormal Production of Collagen . An array of critical diseases has been associated with the inappropriate or unregulated production of collagen, including pathological fibrosis or scarring, including endocardial sclerosis, idiopathic interstitial fibrosis, interstitial pulmonary fibrosis, peri uscular fibrosis, Symmers' fibrosis, pericentral fibrosis, hepatitis, derma ofibroma, biliary cirrhosis, alcoholic cirrhosis, acute pulmonary fibrosis, idiopathic pulmonary fibrosis, acute respiratory distress syndrome, kidney fibrosis/glomerulonephritis, kidney fibrosis/diabetic nephropathy, scleroderma/systemic, sclerodεrma/local, keloids, hypertrophic scars, severe joint adhesions/arthritis, myelofibrosis, corneal scarring, cystic fibrosis, muscular dystrophy (duchenne's) , cardiac fibrosis, muscular fibrosis/rεtinal separation, esophageal stricture, - j - payronles disease. Further fibrotic disorders may be induced or initiated by surgery, including scar revision/plastic surgeries, glaucoma, cataract fibrosis, corneal scarring, joint adhesions, graft vs. host disease, tendon surgery, nerve entrapment, dupuytren's contracture, OB/GYN adhesions/fibrosis, pelvic adhesions, peridural fibrosis, restenosis. One strategy for the treatment of these diseases is the inhibition of the pathological overproduction of collagen. The identification and isolation of enzymes involved in the collagen production and processing are therefore of major medical interest to provide for suitable targets for drug development. Similarly, a strategy for the treatment of diseases resulting from the pathological underproduction of collagen, where the underproduction of collagen is the consequence of improper processing of procollagen, is the administration of C-proteinase. Background Information Regarding C-Proteinase . C-proteinase is an enzyme that catalyzes the cleavage of the C-propeptide of fibrillar collagens, including type I, type II, and type III collagen. The enzyme was first observed in culture media of human and mouse fibroblasts (Goldberg et al . , 1975, Cell 4:45-50; Kessler and Goldberg, 1978, Jinal . Biochem. 86 :463- 469) , and chick tendon fibroblasts (Duskin et al . , 1978, Arch. Biochem . Biophys . lj35_:326-332; Leung et al . , 1979, J. Biol . Chem . 154:224-232) . An acidic proteinase which removes the C-terminal propeptides from type I procollagen has also been identified. Davidson et al . , 1979, Eur. J. Biochem . 100 :551. A partially purified protein having C-proteinase activity was obtained from chick calvaria in 1982. Njieha et al . , 1952, Biochemistry 23:757-764. In 1985, natural C-proteinase was isolated, purified and characterized from conditioned media of chick embryo tendons. Hojima et al . , 1985, J. Biol . Chem . 260:15996-16003. Murine C-proteinase has been subsequently purified from media of cultured mouse fibroblasts. Kessler et al . , 1986, Collagen Relat . Res . 6_:249-266; Kessler and Adar, 1989, Eur. J. Biochem . 186:115-121. Experiments conducted with these purified forms of chick and mouse C-proteinase have indicated that the enzyme is instrumental in the formation of functional collagen fibers. Fertala et al . , 1994, J. Biol . Chem. 269:11584. Generally, C-proteinase activity and the inhibition of the enzyme's activity have been determined using a wide array of assays. See e . g. , Kessler and Goldberg, 1978, Anal . Biochem. 86:463; Njieha et al . , 1982, Biochemistry 2_1=757-764. As articulated in numerous publications, the enzyme is difficult to isolate by conventional biochemical means and neither the enzyme nor the cDNA sequence encoding such enzyme was known to be available prior to the instant invention. Takahara et al . , 1994, J. Biol . Chem . £ :26280-26285, 26284 (C-proteinase's "peptide and nucleotide sequences are as yet unavailable") . Thus, despite the availability of C-proteinase related assays, large scale review and testing of potential C- proteinase inhibitors has not been performed to date. Known C-Proteinaεe Inhibitors . A number of potential C-proteinase inhibitors have been identified. For example, several metal chelators have demonstrated activity as a C-proteinase inhibitor. Likewise, chy ostatin and pepstatin A have been found to act as relatively strong inhibitors cf C-proteinase activity. α 2 -Macroglobulin, ovostatin, and fetal bovine serum appear to also, at least partially, inhibit C- proteinase activity. Similarly, dithiothreitol, SDS, concanavalin A, Zn 2* , Cu 2* , and Cd 2* possess inhibitory activity at low concentrations, and some reducing agents, several amino acids (including lysine and arginine) , phosphate, and ammonium sulfate have been found to have C-proteinase inhibitory activity at concentrations of 1-10 mM. Leung et al . , supra; Ryhanen et al . , 1982, Arch . Biochem. Biophys . 215 :230- 236. High concentrations of NaCl or Tris-HCl buffer have also been found to inhibit the C-proteinase activity. For example, it has been reported that 0.2, 0.3, and 0.5M NaCl reduces the activity of C- . . proteinase by 66, 38, and 25%, respectively, of that observed with the standard assay concentration of 0.15M. Tris-HCl buffer in a concentration of 0.2-0.5M likewise has been reported to inhibit the enzyme's activity. Hojima et al . , supra . In contrast, microbial inhibitors such as leupeptin, phosphoramidon, antipain, bestatin, elastinal, and amastatin, are considered to have weak or no effect. Background Information Regarding Bone Morphogenic Protein-1 (BMP-1 ) . A protein having the structural characteristics of C-protεinaεe was isolated in 1988 from bone tissue. Prior to the instant invention, it was believed that this protein, designated BMP-1 or "bone morphogenic protein, " was a member of the TGF-3 related protein family (Wozney et al . , 1988, Science 242:1528-1534), as BMP-1 was isolated coincidentally with BMP-2A and BMP-3. Although evidence provides that BMP-2A and BMP-3 play a key role in the stimulation of bone development and growth, the activity of BMP-l was never clearly established. Sequence comparison reveals that BMP-l contains a EGF-like domain and a region designated as "A-domain" having sequence similarity with a protease isolated from crayfish. Titany et al . , 1987, Biochemistry 26.-222. As the TGF-βl binding protein also contains EGF-like domains, it has been suggested that BMP-l could be a protease involved in the activation of TGF- jSl. Miyazono et al . , 1988, J. Biol . Chem . 263 :6407; oyznek et a 1 . , supra; Fukagawa et al . , 1994, Dev. Bio . 162:175-183. It has also been suggested that, due to homology to the Drosophila melanogaster tolloid gene product, BMP-l is involved in the overall mechanism for the dorsal-ventral patterning of the neural tube. While it has been suggested that C-proteinase ("for which [prior to this invention] peptide and nucleotide sequence are as yet unavailable") and BMP-l belong to the same structural family, BMP-l has never been associated with the formation of collagen. Takahara et al . , 1994, J. Biol . Chem. 2j69_:26280-26286. Thus, while a cDNA and polypeptide sequence of the putative bone morphogenic protein BMP-l had been identified, no correct activity or use was known for this protein until the present invention. Similarly, the structural relationship between BMP-l and C- oroteinase was not known. 4. SUMMARY OF THE INVENTION The present invention is directed to synthesized or recombinant compositions derived from the deduced amino acid and nucleic acid sequences for human C- proteinase. In one embodiment of the present invention, the composition comprises the full-length amino acid sequence for C-proteinase. In another embodiment of the present invention, the composition comprises a C-proteinase derivative having C- proteinase-like activity. In yet further embodiments of the present invention, the composition is radiolabeled or represents an analog of C-proteinase having C-proteinase-like activity. The present invention is also related to the recombinant production of C-proteinase and related compositions in a variety of recombinant expression systems. The present invention also relates to the use C- proteinase, its fragments, analogs and derivatives for use in diseases and disorders related to the abnormal production of collagen. Such polypeptides may act directly with collagen, or alternatively with other enzymes involved in the processing of collagen, i . e . , lysyl oxidase. The present invention also relates to the use of proteins, peptides and organic molecules capable of modulating the formation of collagen by affecting the interaction between C-protεinase and collagen precursor molecules, including procollagen, or alternatively, other collagen processing enzymεs and/or thε clεavage site of C-proteinase. The invention is further directed to the use of such proteins, peptidεs and/or organic molecules, either alone or in combination with other molecules, in the treatment of disorders, including disorders related to abnormal collagen formation, such as rheumatoid arthritis and scleroderma, for example. The present invention is also relatεd to the use of C-proteinase, whether labeled or unlabeled, as a tracer which could then be used to separate, by HPLC, the different C-protεinase derivatives to yield a carrier-freε tracer, in binding assays. Finally, the present invention is related to the recombinant expression and production of C-proteinase by use of the sequences of the invention. 5 . DEFINITIONS " C-proteinase " shall be construed to mean an enzyme capable of processing collagen molecules , derivatives or fragments , or their precursors by cleaving through -Alai.Asp-Asp- and/or -GlyiAsp-Glu- Thε term shall include human C-Drotieinasε and derivatives, analogs, fragments and variants thereof having C-proteinase-like activity. 6. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 (FIG. 1) generally sets forth the sequenced peptides from procollagen C-proteinase, the encoded structures of pCP-1 and pCP-2, and isolated cDNA clones. FIG. IA identifies the peptides sequenced from which pCP and described below at Table 1. FIG. IB identifies the domains encoded for pCP-1. FIG. IC identifies the domains encodεd for pCP-2. FIG. ID idεntifies the rεgions of cDNAs obtained according to the sche ε set forth below. Figure 2 (FIG. 2) sets forth a Northern blot assay of total RNA from HT-1080 cells wherεin thε filter was probed with a 32 ?-labeled clone of pCP-1 (nucleotides 837 to 2487, wherein said clone was labeled by random primer extension with 3 P to a specific activity of 4 x 10 3 cpm per μg) . The filter was exposed to X-ray film at -70° C for six hours. Lanes 1 and 2 provide two clones transfected with pCP- 1; lane 3 provides a clone transf cted with thε vector pcDNA-3 without a cDNA insert; lanes 4 and 5 set forth two clones transfected with pCP-2; and lane 6 sets forth RNA from untransfected HT-1080 host cells. Figure 3 (FIG. 3) sets forth the results of an assay conducted to determine C-proteinase activity in medium from transfected cells. Lane 1 provides R e¬ labeled type I procollagen, incubated alone; lane 2 provides "C-labeled type I procollagen, incubated with 10 units of purified chick procollagen C-proteinase; lane 3 provides 14 C-labelεd type I procollagen, incubated with proteins precipitated by PEG from 15 ml of medium from a clone transfected with pCP-1; lane 4 provides "C-labeled type I procollagen, incubated with proteins precipitated by PEG from 15 ml of medium from a clone transfected with pCP-2; lane 5 provides 14 C- labεlεd type I procollagen, incubated with proteins precipitated by PEG from 15 ml of medium from non- transfεctεd HT-1080 host cells; lane 6 provides R e¬ labeled type I procollagen, incubated with proteins partially fractionated and concεntrated by membrane filtration from 10 ml of medium from a clone transfεctεd with pCP-2. Figure 4 (FIG. 4) sets forth a chromatograph of extract from E. coli on a metal affinity column. Figure 5 (FIG. 5) sets forth the results of an assay conducted to determine C-proteinasε activity of thε rεcombinant protein expressed in Ξ . coli . Lanes 1 and 2 provide 14 C-labeled procollagen (0.2 μg; 4000 cpm) incubated in reaction buffer for fifteen (15) hours at 35°C with 0.2 μg refolded recombinant protein- activated by prior digestion for two (2) hours at 37°C with 10 μg/ml chymotrypsin. Lanes 3 and 4 provide R e¬ labeled procollagen (0.2 μg; 4000 cpm) incubated in reaction buffer for 15 hours at 35°C with 0.2 μg refolded recombinant protein activatεd by digestion with 100 μg/ml chymotrypsin. Lane 5 providεs R e¬ labeled procollagen, incubated without enzyme. Lane 5 provides R C-labelεd procollagεn, incubated with procollagen C-proteinase from chick embryos (0.2 units ; 0.006 μg) . Figure 6 (FIGs. 6A, 6B) sets forth the nucleic acid and amino acid sequence of pCP-1. Specifically, FIG. 6A sets forth the amino acid sequence and nucleic acid sεquεnce corresponding to pCP-l for positions -1 to 431 (amino acid) and -3 to 1291 (nucleic acid) . FIG. 6B sets forth the amino acid sequεnce and nucleic acid sequεncε corresponding to pCP-l for positions 431 to 731 (amino acid) and 1292 to 2431 (nucleic acid) . Figure 7 (FIG. 7) sets forth the nucleic acid and amino acid sequence of pCP-2. Specifically, FIG. 7A sets forth the amino acid sequence and nucleic acid sequencε corresponding to pCP-2 for positions -1 to 431 (amino acid) and -3 to 1291 (nucleic acid) . FIG. 7B sets forth the amino acid sequence and nucleic acid sequencε corresponding to pCP-2 for positions 431 to 911 (amino acid) and 1292 to 2731 (nucleic acid) . FIG. 7C sets forth the amino acid sequence and nucleic acid sequεnce corresponding to pCP-2 for positions 912 to 991 (amino acid) and 2732 to 3541 (nucleic acid) . 7. DETAILED DESCRIPTION OF THE INVENTION 7.1. Isolation Of Gene Encoding C-Proteinase The C-proteinase enzyme may be isolatεd to homogeneity by application of previously described procedures, including the procedures described in Hoji a, et al . , 1985, J " . Biol . Chem. 250:1599 . In a prεfεrred embodiment, the protein is purified using thε method of Hojima, et al . and a final purification stεp in which protεin sεparation is accomplished by polyacrylamide gel elεctrophoresis in SDS. The homogenous C-proteinase enzymε may then be sequenced according to known techniques using commercially available apparatus. In one preferred method, tryptic peptides from the gel band containing the enzymε were sequenced by: (1) electroeluting the protεin band onto a filter; (2) digesting the band in si tu with trypsin; (3) separating thε tryptic peptidεs using a reverse phase C18 column (Supelco LC18DB) eluted with a gradient of 0.1% trifluoroacetic acid and 0.9% trifluoroacetic acid containing 70 % acetonitrile; (4) assaying the individual peaks from the column for homogεnεity by time-of-flight-matrix- assistεd lasεr dεsorption mass spεctromεtry (Lasεrmat; Finnigan) ; and (5) sεquencing the homogenεous fractions by Edman dεgradation. Nuclεic acid probes may be prepared using the detεrmined amino acid sequencε for C-protεinasε. Such probεs may bε synthεsizεd synthεtically and labεlεd. Prεparation techniques for such probes and others are known in the art and set forth in, for εxamplε, Sambrook, et al . , 1989, Molecular Cloning, A Laboratory Manual , 2d Edi tion, Cold Springs Harbor Laboratory Prεss, Nεw York, at Chaptεrs 10-11.-. The nucleic acid sequεncεs obtainεd using such probεs may bε sequencεd using any onε of the techniquεs gεnεrally dεscribεd in Sambrook, et al . , supra, at Chaptεr 13. Thε gεnε εncoding C-protεinasε may also bε isolatεd by pεrforming a polymεrasε chain rεaction (PCR) using onε or more degεnεratε oligonucleotide primer pools that arε designεd basεd on the deducεd nucleotidε sεquεncε of C-protεinasε, as deduced from thε amino acid sεquεncε of C-protεinase . The techniquεs usεd to idεntify thε nuclεic acid sεquence of C-proteinase using PCR arε dεscribεd in, for εxamplε, Sambrook, et al . , supra, at Chaptεr 14. Thε invεntion also rεlatεs to unknown C- protεinasε gεnεs isolated from other species and alleles of the C-proteinasε gεnε dεscribed herein, in which C-protεinasε activity εxists. Members of the C- protεinasε family arε definεd herein as those enzymεs that can procεss procollagen moleculεs at the C- terminal end of such molecule. A bacteriophage cDNA library may be screened, under conditions of reducεd stringency, using a radioactively labelεd fragmεnt of thε human C-protεinasε clonε dεscribed herein. Alternatively the human C-proteinasε sequence can bε used to design degεnεrate or fully degenerate oligonucleotide probes which can be used as PCR probεs or to scrεεn bactεriophagε cDNA librariεs . Thε PCR product may bε subclonεd and sεquεncεd to insure that the amplified sequencεs represent the C-proteinase sequencεs. The PCR fragment may be usεd to isolate a full length C-protεinase clone by radioactively labeling the amplified fragment and screεning a bactεriophagε cDNA library. Alternatively, the labelεd fragmεnt may be usεd to scrεεn a genomic library. For a reviεw of cloning stratεgiεs which may bε usεd, sεε e . g. , Sambrook, et al . , 1989, Molecular Cloning, A Laboratory Manual , 2d Edition, Cold Springs Harbor Prεss, N.Y.; and Ausubεl εt al . , 1989, Current Protocols in Molecular Biology, (Green Publishing Associates and Wilεy Intersciεncε, N.Y.) Isolation of human C-proteinase cDNA may also bε achiεvεd by construction of a cDNA library in a mammalian εxpression vector such as pcDNAl, that contains SV40 origin of replication sequences which permit high copy number expression of plasmids when transferred into COS cells. The expression of C- proteinase on the surface of transfected COS cells may be detected in a number of ways known in the art . Cells expressing the human C-proteinase may bε enriched by subjecting transfectεd cells to a FACS (fluorescent activated cell sorter) sort. In accordance with thε invεntion, C-protεinasε nuclεotidε sequences which encode C-proteinase, peptide fragments of C-proteinase, C-protemase fusion protεins or functional equivalents therεof may bε usεd to generate recombinant DNA molecules that direct the expression of the protein or a functional equivalent thereof, in appropriate host cells. Altεrnativεly, nuclεotidε sεquεnces which hybridize to portions of the C-protεinasε sequence may also be used in nucleic acid hybridization assays, Southern and Northern blot analyses, etc. Due to the inherent degεnεracy of thε genεtic code, other DNA seσuences which encode substantially the same or a functionally equivalent amino acid sequence, may be used in the practicε of thε invεntion for thε cloning and expression of the C-protεinase prσtεin. Such DNA sequencεs includε thosε which arε capablε of hybridizing to thε human C-protεinase sequencε undεr stringent conditions, and more preferably highly stringent conditions. See, e . g. , Wallace et al . , 1981, Nucleic Acid Research 9_ :879. Altered DNA sequences which may be used in accor¬ dance with thε invεntion includε dεlεtions, additions or substitutions of different nucleotidε rεsiduεs resulting in a sequence that encodes the same or a functionally equivalεnt gεnε product. Thε gεnε product itsεlf may contain dεlεtions, additions or substitutions of amino acid rεsiduεs within thε C- protεinasε sεquεncε, which rεsult in a silent change thus producing a functionally equivalεnt protein. Such amino acid substitutions may be made on thε basis of similarity in polarity, chargε, solubility, hydrophobicity, hydrophilicity, and/or thε amphipatic nature of the residuεs involvεd. For εxamplε, nεgativεly chargεd amino acids includε aspartic acid and glutamic acid; posi ivεly chargεd amino acids includε lysinε and argininε; amino acids with uncharged polar head groups having similar hydrophilicity valuεs includε thε following: leucine, isolεucinε, valine, glycine, analine, asparagine, glutamine, serine, threonine; phenylalanine, tyrosine. As usεd herein, a functionally equivalent C-proteinase refers to an enzymε which can procεss procollagen or fragments or derivatives thereof, but not necessarily with the same binding affinity of its countεrpart native C-proteinase. Thε DNA sequencεs of the invention may be engi- neerεd in ordεr to alter the εnzy ε sεquεncε for a variεty of εnds including but not limitεd to altεra- tions which modify processing and expression of thε genε product. For example, mutations may be intro¬ duced using techniquεs which arε well known in the art, e .g. , site-directed mutagenesis to, for example, insert new restriction sites. For example, in certain exprεssion systems such as yεast, host cells may ovεr- glycosylatε thε gene product. When using such exprεs¬ sion systεms it may bε prεferablε to alter thε C- protεinasε coding sεquεncε to εliminate any N-iinked glycosylation sitε. In anothεr εmbodi εnt of the invention, thε C- protεinasε or a modifiεd C-protεinasε sεquεncε..may bε ligatεd to a heterologous sequencε to εncodε a fusion protεin. For example, for screεning of peptide libraries it may be useful to encode a chimeric C- proteinase protεin expressing a heterologous epitopε that is rεcognized by a commercially available antibody. A fusion protεin may also bε εnginεered to contain a cleavage site located betwεεn thε C- proteinasε sεquence and thε heterologous protein sequence, so that the C-proteinase can be cleaved away from the hetεrologous moiety. In an alternate embodimεnt of thε invεntion, thε coding sequence of C-proteinase could bε synthesized in whole or in part, using chemical methods well known in the art. See, for example, Caruthers et al . , 1980, Nucleic Acids Res . Symp . Ser. 2=215-233; Crea and Horn, 1980, Nucleic Acids Res . 9.:2331; Matteucci and Caruthers, 1980, Tetrahedron Letters 21:719; and Chow and Kempε, 1981, Nucleic Acids Res . 2:2807-2817. Altεrnatively, the protein itself could be produced using chemical methods to synthesize thε C-protεinasε amino acid sεquence in whole or in part. For examplε, peptides can be synthesized by solid phase techniques, cleaved from the rεsin, and purifiεd by preparative high performance liquid chromatography. (See, e . g. , see Crεighton, 1983, Proteins Structures And Molecular Principles, W.H. Freeman and Co., N.Y. pp. 50-60) . Thε composition of the synthetic peptidεs may bε confirmεd by amino acid analysis or sequencing [ e . g. , the Edman degradation procedurε; see, Crεighton, 1983, Proteins, Structures and .-.-.lecular Principles, W.H. Frεεman and Co., N.Y., pp. 34-49) . 7.2. Uses Of The C-Proteinase Coding Sequence Thε C-protεinasε coding sεquεncε may bε usεd for diagnostic purposεs for dεtεction of C-proteinasε εxpression. Included in thε scopε of thε invεntion arε oligoribonuclεotidε sεquencεs, that includε antisεnsε RNA and DNA olεcules and ribozymes that function to inhibit translation of C-protεinasε. Antisεnsε tεchniσuεs arε known in the art and may bε appliεd herein. Ribozymes arε εnzy atic RNA molεculεs capablε of catalyzing thε specific cleavagε of RNA. Thε mεcha- nism of ribozymε action involvεs sεquεncε spεcific hybridization of thε ribozymε molεcule to complemεn- tary targεt RNA, followed by a endonuclεolytic cleav¬ age. Within the scope of thε invεntion are enginεεrεd hammerhεad motif ribozymε molεculεs that spεcifically and εfficiently catalyze endonucleolytic cleavagε of C-protεinase RNA sequences. Specific ribozyme cleavagε sitεs within any potential RNA target are initially idεntifiεd by scanning the target molecule for ribozyme cleavagε sitεs which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of betwεεn fiftεεn (15) and twεnty (20) -ibonuclεotidεs corrεsponding to thε region of the t. get c,sne containing thε clεavagε sitε may bε εvaluatεd for prεdicted structural features such as secondary structure that may render the oligonucleotide sεquεncε unsuitabl . Thε suitability of candidatε targεt may also be evaluatεd by tεsting thεir accεssibility to hybridization with complementary oligonucleotidεs, using ribonuclεase protection assays. Both antisensε RNA and DNA molecules and ribozymes of the invention may be prepared by any method known in the art for thε synthesis of RNA molecules. These include techniques for chemically synthesizing oligodεoxyribonuclεotides wεll known in thε art such as for example solid phase phosphoramidite chemical synthesis. Alternatively, RNA moleculεs may be generatεd by in vi tro and in vivo transcription of DNA sεquεnces encoding the antisense RNA moleculε. Such DNA sεσuεnces may be incorporatεd into a wide variety of vectors which incorporate suitablε RNA polymεrase promotεrs such as thε T7 or SP6 polymerase promoters. Alternatively, antisensε cDNA constructs that synthεsizε antisεnsε RNA constitutivεly or inducibly, dεpεnding on thε promotεr usεd, can bε introduced stably into cell lines. In addition, mutated forms of C-proteinasε, having a dominant negativε εffεct, may bε expressed in targeted cell populations to inhibit the activity of endogεnously εxprεssed wild-type C-proteinasε. Additionally, the DNA encoding C-protεinasε may also havε a number of uses for the diagnosis of diseasεs rεsulting from aberrant expression of the enzyme. For examplε, thε C-protεinase DNA sequεncε may bε used in hybridization assays of biopsiεs or autopsies to diagnose abnormalities of expression ( e . g. , Southern or Northern blot analysis, in situ hybridization assays) . Thε C-protεinase cDNA may bε used also as a probe to dεtεct thε εxprεssion of thε C-protεinase mRNA. In addition, the expression of C-protεinase during embryonic development may also be determined using nucleic acid encoding C-proteinasε . As dεscribed in thε literature, no deficiencies of C- proteinasε havε bεεn found in patiεnts v/ith genetic disεasεs of connectivε tissues. Thus, it has beεn gεnεrally assumεd that a gεnεtic dεficiεncy rεlatεd to C-proteinase produces death in utero . In si tu hybridizations can predict in u tero problems related to connective tissue disεasεs. Various modifications to thε DNA molεculεs may be introducεd as a means of increasing intracεllular stability and half-life. Possible modifications include but are not limited to thε addition of flanking sεquεnces of ribo- or dεoxynuclεotidεs to the 5' and/or 3' ends of thε molecule or the use of phosphorothioate or 2' O-methyl rather than phospho- diεsterasε linkagεs within thε oligodεoxyribonucleo- tide backbone . 7.3. Expression Of C-Proteinase In order to express a biologically active C- protεinasε, thε nuclεotidε sequεncε coding for thε protεin, or a functional εquivalεnt as dεscribεd in Sεction 4.1 supra , was inserted into an appropriate expression vector, i.e., a vector which contains the necessary elεmεnts for thε transcription and translation of the inserted coding sequencε. Morε spεcifically, mεthods which arε wεll known to those skilled in thε art can bε usεd to construct exprεssion vεctors containing thε C-protεinasε sequεncε and appropriatε transcriptional/translational control signals. These methods include in vi tro recombinant DNA techniques, synthetic techniquεs and in vivo recombination/genetic recombination. See e . g. , the techniquεs described in Sambrook et al . , 1989, Molecular Cloning A Laboratory Manual , Cold Spring Harbor Laboratory, N.Y. and Ausubel et al . , 1989, Current Protocols in Molecular Biology, Greenε Publishing Associatεs and Wiley Intersciεnce, N.Y. A variety of host-exprεssion vεctor systεms may bε utilized to εxprεss the C-protεinasε coding sequεncε. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the C-proteinase coding sequence; yeast transformed with recombinant yeast expression vectors containing thε C-proteinase coding sequence; insect cεll systεms infεcted with recombinant virus exprεssion vεctors ( e . g. , ■ baculovirus) containing thε C-protεinasε coding sequεncε; plant cεll systems infectεd with recombinant virus exprεssion vεctors (e . g. , cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid exprεssion vεctors ( e . g. , Ti plasmid) containing thε C-protεinase coding sequencε; or animal cεll systεms infεctεd with rεcombinant virus expression vectors ( e .g. , adenovirus, vaccinia virus, human tumor cells (including HT-1080) ) including cell lines engineerεd to contain multiplε copies of the C- proteinasε DNA either stably amplified (CHO/dhfr) or unstably amplified in double-minute chromosomεs ( e . g. , murine cell lines) . In the prεfεrrεd εmbodimεnts of the invention, HT-1080 and e. coli werε usεd as εxprεssion vεhiclεs. The expression elements of thesε systεms vary in thεir strεngth and spεcificitiεs. Depending on the host/vector systεm utilizεd, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, may be usεd in the expression vector. For example, when cloning in bacterial systems, induciblε promoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and thε likε may bε usεd; when cloning in insect cell systεms, promotεrs such as thε baculovirus polyhεdron promoter may bε usεd; whεn cloning in plant cεll systεms, promotεrs derived from thε gεnomε of plant cells ( e .g. , heat shock promoters; the promoter for thε small subunit of RU3ISC0; the promoter for the chlorophyll a/b binding protein) or from plant viruses (e.g., the 35S RNA promotεr of CaMV; thε coat protein promoter of TMV) may be used; whεn cloning in mammalian cεll systems, promoters derived from the genomε of mammalian cells (e.g., metallothionein promotεr) or from mammalian viruses (e.g., the adenovirus latε promotεr; thε vaccinia virus 7.5K promoter) may be used; when gεnεrating cell lines that contain multiple copies of the C-proteinasε DNA SV40-, BPV- and EBV-basεd vectors may be usεd with an appropriate selectable marker. In bacterial systems, a number of εxprεssion vεctors may bε advantagεously sεlεctεd dεpεnding upon thε usε intεndεd for the C-proteinasε εxprεssεd. For εxample, when large quantities of C-proteinasε arε to bε producεd to screen peptide libraries, vectors which direct the expression of high levεls of fusion protein products that are readily purified may be desirable. Such vectors include but are not limited to the E. coli exprεssion vector pUR278 (Ruther et al . , 1983, EMBO J. 2=1791) , in which thε C-protεinasε coding sεquεncε may bε ligatεd into thε vector in frame with the lac Z coding region so that a hybrid AS-lac Z protein is produced; pIN vectors (Inouye and Inouyε, 1985, Nucleic Acidε Res . 12:3101-3109; Van Heεke and Schuster, 1989, J. Biol . Chem . 251:5503-5509) , and thε likε. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with gluta¬ thione S-transferase (GST) . In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarosε beads followed by elution in the presεncε of free gluta¬ thione. The pGEX vectors arε dεsigned to include thrombin or factor Xa proteasε cleavage sites so that the cloned polypeptide of intεrεst can be released from the GST moiety. In yεast, a numbεr of vεctors containing constitutivε or induciblε promotεrs may bε used. For a reviεw, see, Current Protocols in Molecular Biology, Vol. 2, 1988, Ed. Ausubεl et al . , Grεene Publish. Assoc. & Wilεy Intεrsciεncε, Ch. 13; Grant et al . , 1987, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 1987, Acad. Press, N.Y., Vol. 153, pp. 516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., D.C, Ch. 3; 3itter, 1987, Heterologous Gene Expression in Yeast, Methods in Enzymology, Eds. Bergεr & Kimmel, Acad. Press, Ν.Y., Vol. 152, pp. 673-684; and The Molecular Biology of the Yeast Saccharomyces , 1982, Eds. Strathern et al . , Cold Spring Harbor Prεss, Vols. I and II . In casεs whεrε plant εxprεssion vectors are used, thε expression of the C-proteinasε coding sεquεncε may bε drivεn by any of a numbεr of promoters. For examplε, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al . , 1984, Nature 310 :511-514) , or the coat protein promoter of TMV (Takamatsu et al . , 1987, EMBO J. 1:307-311) may be used; altεrnativεly, plant promotεrs such as thε small subunit of RUBISCO (Coruzzi et al . , 1984, EMBO J. 2:1671-1680; 3roglie et al . , 1984, Science 224 :838- 843); or heat shock promotεrs, e . g. , soybean hspl7.5-E or hspl7.3-B (Gurley et al . , 1986, Mol . Cell . Biol . 5_:559-565) may be used. Thesε constructs can bε introducεd into plant cεlls using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transfor¬ mation, microinjection, electroporation, etc. For reviews of such techniques, see, for example, Weissbach and Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463; and Grierson and Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9. An altεrnativε expression system which could be used to exprεss C-proteinase is an insect system. In one such system, Baculovirus is usεd as a vector to exprεss foreign genes. Thε virus then grows in the insect cells. The C-proteinasε coding sequence may be cloned into non-essential regions (for examplε thε polyhedron gene) of the virus and placed under control of a Baculovirus promoter. These recombinant viruses are then used to infect insect cells in which the insertεd gεne is εxprεssεd. (E. g. , see, Smith et al . , 1983, J. Viol . £5:584; Smith, U.S. Patεnt No. 4,215,051) . In mammalian host cεlls, a numbεr of viral basεd εxprεssion systεms may bε utilizεd. In cases where an adenovirus is usεd as an expression vector, the C- proteinase coding sequence may bε ligatεd to an adεnovirus transcription/translation control co plεx, e . g. , thε latε promoter and tripartite leader sequencε. This chimeric gene may then be insεrted in the adenovirus genomε by in vitro or in vivo rεcombination. Insεrtion in a non-essential region of the viral genome ( e . g . , region El or E3) will result in a recombinant virus that is viable and capable of expressing C-protεinase i- infected hosts. See, e . g. , Logan and Shenk, 1984, Proc . Na tl . Acad . Sci . USA 21:3655-3659. Altεrnativεly, thε vaccinia 7.5K promotεr may bε usεd. See, e .g. , Mackεtt et al . , 1982, Proc . Natl . Acad. Sci . USA 29:7415-7419; Mackett et al . , 1984, J. Virol . 42:857-864; Panicali et al . , 1982, Proc . Natl . Acad . Sci . USA 79:4927-4931. In a preferrεd εmbodimεnt, thε C-protεinasε sεquεncε is εxpressed in human tumor cells, and more prefεrably HT-1080, which havε been stably transfected with calcium phosphate precipitation and a neomycin resistance gene. Specific initiation signals may also be req> .red for εfficient translation of insertεd C-Drotεinase coding sequences. These signals include the ATG initiation codon and adjacent sequεnces. In cases wherε the entire C-proteinase gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be neεdεd. Howεver, in casεs whεre only a portion of the C-proteinasε coding sεquεnce is inserted, exogenous translational control signals, including the ATG initiation codon, must bε provided. Furthermorε, the initiation codon must bε in phase with the reading frame of the-C- protεinasε coding sεquεncε to ensure translation of the εntirε insert. Thεsε exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthεtic. The efficiεncy of expression may be enhancεd by thε inclusion of appropriatε transcription enhancer elεmεnts, transcription tεrminators, etc. See, e . g. , Bitter et al . , 1987, Methodε in Enzymol . 153 :516-544. In addition, a host cεll strain may bε chosen which modulates the expression of the inserted sequεnces, or modifiεs and processes thε gεnε product in thε specific fashion desirεd. Such modifications ( e . g . , glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of thε protein. Different host cells have charac¬ teristic and specific mechanisms for the post-transla¬ tional processing and modification of proteins. Appropriate cells lines or host systems can be chosen to ensurε thε corrεct modification and procεssing of thε forεign protein εxprεssεd. To this εnd, εukaryotic host cεlls which possεss thε cellular achinεry for propεr procεssing of thε primary transcript, glycosylation, and phosphorylation of the gεnε product may bε used. Such mammalian host cεlls include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, WI38, HT-1080 etc. For long-term, high-yield production of recombi¬ nant proteins, stablε εxprεssion is prεfεrrεd. For example, cell lines which 'stably exprεss C-protεinase may be engineerεd. Rathεr than using εxprεssion vectors which contain viral origins of replication, host cells can be transformed with C-proteinasε DNA controllεd by appropriatε εxpression control elemεnts ( e . g. , promotεr, enhancer, sequεncεs, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of foreign DNA, enginεεrεd cεlls may bε allowεd to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the rεcombinant plasmid confεrs rεsistancε to thε selection and allows cells to stably integratε thε plasmid into their chromosomεs and grow to form foci which in turn can bε clonεd and expanded into cell lines. A number of selεction systems may bε used, including but not limited to the herpes simplex virus thymidine kinase (Wiglεr et al . , 1977, Cell 11:223) , hypoxanthine-guanine phosphoribosyltransferasε (Szybalska and Szybalski, 1962, Proc . Natl . Acad . Sci . USA 48_:2026) , and adεnine phosphoribosyltransferasε (Lowy et al. , 1980, Cell 22. -. B11) genεs can be employed in tk", hgprt" or aprt" cells, respectivεly. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler et al . , 1980, Proc . Natl . Acad. Sci . USA 27:3567; O'Hare et al . , 1981, Proc . Natl . Acad. Sci . USA 26 1527); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc . Natl . Acad. Sci . USA 28:2072) ; neo, which confers resistance to the aminoglycoside G-418 (Colberre- Garapin et al . , 1981, J. Mol . Biol . 150-.1) ; and hygro, which confers rεsistancε to hygromycin (Santεrre et al . , 1984, Gene 30:147) genεs. Recently, additional sεlεctable gεnεs have beεn dεscribεd, na εly trpB, which allows cεlls to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman and Mulligan, 1988, Proc . Natl . Acad. Sci . USA 85. : 8041 ) , and ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2- (difluoro εthyl) -DL-ornithine, DFMO (McConloguε, 1987, In: Current Communicationε in Molecular Biology, Cold Spring Harbor Laboratory εd.) . 7.4. Identification Of Transfectants Or Transformants That Express C-Proteinaεe The host cells which contain the coding sequεncε and which express the biologically active gene product may be identified by at least four general approaches: (a) DNA-DNA or DNA-RNA hybridization; (b) the presεncε or absεncε of "markεr" gene functions; (c) assεssing thε lεvεl of transcription as mεasured by the expression of C- proteinase mRNA transcripts in thε host cεll; and (d) dεtεction of the gεnε product as mεasurεd by an assay or by its biological activity. In the first approach, the prεsεncε of thε C- protεinasε coding sεσuεncε insεrtεd in thε εxprεssion vεctor can bε dεtectεd by DNA-DNA or DNA-RNA hybridization using probes comprising nucleotide sεquεnces that are homologous to the C-proteinase coding sequence, respectively, or portions or derivatives thεrεof. In the second approach, the recombinant εxpres¬ sion vector/host system can be identified and selected based upon the presεnce or absence of certain "marker" gene functions ( e . g. , resistance to antibiotics, rεsistancε to mεthotrεxatε, transformation phεnotypε, occlusion body formation in baculovirus, εtc. ) . For example, in a prεfεrrεd εmbodiment, the C-oroteinasε coding sεquence is inserted within a neomycin- rεsistancε markεr gene sequence of the vεctor, and rεcombinants containing the C-proteinasε coding sequencε can bε idεntified by the absence of thε markεr gene function. Alternatively, a marker gene can be placed in tandem with thε C-protεinase sequence under thε control of the same or different promoter used to control the exprεssion of the C-proteinase coding sεquεncε. Exprεssion of thε markεr in rεsponsε to induction or selection indicates εxprεssion of thε C-protεinase coding sequence. In the third approach, transcriptional activity for the C-proteinasε coding rεgion can be assessεd by hybridization assays. For εxamplε, RNA can bε isolatεd and analyzεd by Northern blot using a probe homologous to the C-proteinasε coding sεquεnce or particular portions therεof. Alternatively, total nucleic acids of the host cell may be extractεd and assayεd for hybridization to such probεs. Thε fourth approach involvεs thε detection of the biologically active C-proteinasε gene product. A number of assays can be used to detεct C-protεinasε activity, including, but not limited to, thosε assays dεscribεd in Kεsslεr and Goldbεrg, 1978, Anal . Biochem . 26:463; and Njiεha et al . , 1982, Biochemiεtry 21:757-764. 7.5. Screening Of Peptide Library With C- Proteinase Or Engineered Cell Lines Random peptidε librariεs consisting of all possiblε combinations of amino acids attached to a solid phase support may be used to identify pεptidεs that are able to modulate and/or inhibit C-protεinase activity by binding to C-proteinase. The screening of peptidε librariεs may havε thεrapεutic valuε in thε discovery of pharmaceutical agents that act to inhibit the biological activity of the protein. Idεntification of olεcules that are able to bind to C-proteinase may be accomplished by screening a peptidε library with rεcombinant soluble C-proteinasε. Methods for εxprεssion and purification of thε εnzymε arε dεscribed above and may bε usεd to εxpress recombinant full length C-proteinasε or fragments, analogs, or dεrivativεs thereof depending on the functional domains of interεst . To identify and isolate the peptidε/solid phasε support that intεracts and forms a complex with C- proteinasε it is necεssary to labεl or " tag" thε C- proteinase moleculε. Thε C-protεinasε protεin may bε labεlεd according to wεll-known tεchniquεs, including iodination labεlling with 15 I . Additionally, thε C- proteinase protein also may bε conjugated to enzymes such as alkaline phosphatase or horseradish peroxidasε or to other rεagεnts such as fluorescent labels which may include fluorescεin isothyiocynatε (FITC) , phycoεrythrin (PE) or rhodaminε. Conjugation of any givεn label, to C-proteinasε, may bε pεrformεd using techniques that are routine in the art . Altεrnativεly, C-protεinasε εxprεssion vεctors may be enginεεrεd to εxprεss a chimεric protεin containing an εpiropε for which a commεrcially availablε antibody εxis . Thε εpitopε spεcific antibody may bε tagged using mεthods wεll known in thε art including labeling with enzymes, fluorescent dyes or colored or magnetic beads . The " tagged" C-proteinasε is incubatεd with thε random peptide library for 30 minutes to one hour at 22°C to allow complex formation between C-proteinasε and peptide species within thε library. Thε library is then washed to remove any unbound protεin. If C- proteinase has been conjugatεd to alkalinε phosphatasε or horseradish peroxidase the wholε library is pourεd into a petri dish containing a substrates for εither alkalinε phosphatasε or pεroxidasε, for εxamplε, 5- bromo-4-chloro-3-indoyl phosphatε (BCIP) or 3,3' ,4,4"-diamnobεnzidine (DAB), respectively. After incubating for several minutes, the peptide/solid phase-C-protεinasε complex changes color, and can be easily identified and isolated physically under a dissecting microscope with a micromanipulator. If a fluorescent tagged C-protεinasε molεculε has bεen used, complεxεs may bε isolated by fluorescent activated sorting. If a chimeric C-proteinasε expressing a heterologous epitopε has been used, detection of the peptide/C-proteinase complex may be accomplished by using a labeled epitopε spεcific antibody. Oncε isolatεd, thε idεntity of the peptidε attached to the solid phase support may be determinεd by oeptide sεσuεncing. 7.6. Screening Of Organic Compounds With C- Proteinase Protein Or Engineered Cell Lines Cell lines that express C-proteinase may be used to screen for moleculεs that modulate C- proteinase activity or collagen formation. Such moleculεs may includε small organic or inorganic compounds, or other molecules that modulate C- proteinasε activity or that promote or prevent the formation of collagen. Synthetic compounds, natural products, and other sources of potentially biologically active materials can be screenεd in a numbεr of ways. The ability of a test molecule to interfere with C-proteinase-procollagen binding and/or C-proteinase- processing enzyme binding may be mεasurεd using standard biochemical techniσuεs. Othεr responses, such as activation or suppression of catalytic activity may also be monitorεd. These assays may be performed using conventional techniσuεs dεvεlopεd for these purposεs in thε coursε of screening. Various εmbodimεnts arε dεscribed below for screening, identification and evaluation of compounds that interact with C-proteinasε or its targets, which compounds may affect various cellular processes including the formation and production of collagen. The following preparations and examples are givεn to εnablε thosε skilled in the art to more clearly understand and to practice the presεnt invention. The presεnt invention is not to be limitεd in scopε by the exemplified embodimεnts which are intendεd as illustrations of singlε aspects of the invention, and methods which are functionally equivalεnt are within the scopε of the invention. Indeed, various modifications of thε invention in addition to those dεscribεd herein will become apparent to thosε skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. 8. EXAMPLES 8.1. Identification Of Partial Amino Acid Sequences Of The C-Proteinase Typε I procollagεn C-protεinasε was purifiεd to homogeneity from organ cultures of chick embryo according to the mεthods set forth in Hojima et al . , 1985, J. Biol . Chem. 260.:15996-16003. In the final step, the protein was separated by polyacrylamide gel electrophoresis in SDS. The protein band was electroeluted onto a filter and digested in si tu with trypsin. The tryptic peptides werε sεparated on a rεvεrsε phasε C18 column (Supelco LC18DB) , and εlutεd with a gradiεnt of 0.1 % trifluoroacetic acid and 0.9% trifluoroacetic acid containing 70% acεtonitril . Individual pεaks from thε column wεrε assayεd for homogeneity by time-of-flight-matrix-assisted laser desorption mass spectrometry (Lasεrmat; Finnigan) . Homogenous fractions were sequencεd by Edman dεgradation with an automated instrument . Nine sequεncεs of diffεrεnt peptides werε obtained, as set forth below in TABLE 1 and in FIG. IA. TABLE 1 Amino Acid Position Observed tolloid-like Peptidε Sεquεncε' 3 ?-l/pC: P-l BMP-l/pC?-2 1 MEPQΞVESLGΞTYDFDSIMH 252-274 252-274 YAR 2 NTFSR 275-279 275-279 3 GIFLDT 230-285 280-285 4 EVN KP 290-302 290-302 YFEAGVRSPIGQR 5 T 401-411 401-411 LPEPIVSSDSR 6 AYDYLEVR 489-496 489-496 7 LWLK 525-528 525-528 8 R C R 547-562 547-562 EVDECSRPNNGGXEOK 9 N CD K 731-751 SGFVLHDMKHDCKEAGSSHR Observed sequences are from tryptic peptides from chick pC?. Sequences above the continuous sequences are different are different amino acids encoded by human cDNAs. With minor conservative substitutions attributable to the species differεncε, eight of the peptidεs contained sequencεs found in thε protεin initially identified as human BMP-l, as set forth in Wozney e al . , Science 242:1528-1534. The ninth peptide had a sequence of 20 amino acids found in thε C-tεrminal domain of onε of thε longer forms of BMP-l, as identified in mouse (Fukagawa et al . , 1994, Develop. Biol . 163 =175-183) and human (Takahara et al . , 1994, J. Biol . Chem . 269:32572-32578) tissues. 8.2. Preparation and Structure of cDNAS for the C-Proteinase To isolate cDNAs for procollagen C- proteinase, total RNA was extracted from normal human skin fibroblasts (RNAεasy; Qiagεn) and reverse transcribed with random primers (First Strand cDNA Synthesis Kit; Pharmacia) . The cDNA was amplified by PCR with a pair of primers designed on the basis of the amino acid sequεnce of two of thε pεptidε fragmεnts (pεptidεs 1 and 6 in TABLE 1) from thε chick C-protεinasεs, as dεcibεd abovε. Spεcifically, thε primεrs used wεre designated B-3 (ATGACTTCGACAGCATCA TGC) and B-4 (CTCCAGATAGTCGTAGGCACA) . The PCR producr was 3 P-labeled with random primεrs (Primε-It; Stratagene) and used as a probε to screen a cDNA library prepared from human skin fibroblasts (CRL 1252 (patient with osteogenesis impεrfecta) ; ATCC) insertεd into a lambda phagε (ZAP II; Stratagεnε) . Fivε positivε clonεs ranging in sizε from 0.16 to 2.9 kb were obtained (FIG. ID). Thε most 3'-cDNA sεσuεncε extended to 3,560 base pairs. Analysis of overlapping sequεnces of thε clonεs indicatεd that the cDNAs coded for two proteins of different length, onε of 730 amino acids (pCP-l, sεε FIG. 6) and a sεcond of 986 amino acids (pCP-2, see FIG. 7). The domains εncoded by the two proteins, pCP-l and pCP-2, are set forth at FIG. IB and FIG. IC, respectivεly. As set forth in FIGs. IA and IB, thε first 702 codons for thε two proteins werε identical. Beginning with the codon for amino acid 703, however, pCP-2 had a new sequence that coded for a second EGF-like domain and a fourth and fifth CU3 domain. Thε sεquεncεs of pCP-l were identical to the previously published sequencεs of BMP-l sεt forth in Wozney et al . , supra, with thε εxcεption of thε codon at amino acid position 24. Contrary to thε BMP-l sequencε reportεd in Woznε, et al . , supra, setting forth a -GAC- codon (encoding aspartate at amino acid position 24), thε sεquence obtained according to the above method included a - AAC- codon (encoding asparagine at amino acid position 24) . 8.3. Expression of the cDNAs in a Mammalian Cell System To express the cDNAs for procollagen C- protεinasε, ovεrlapping clones of the cDNAs were cleaved and ligated to generatε full-length cDNAs. The cDNAs were inserted into the expression vector pcDNA3 (InVitrogen) and used to prεparε stable transfectants of a HT-1080 human tumor cεll linε by calcium phosphatε prεcipitation with a commercial kit (Promεga) . Cεlls were initially cultured for twenty-four (24) hours in a high glucose DMEM medium containing 10% fetal bovine serum (Cellgrow; MediaTech) on 80 cm 2 culture dishes with 10 s cells per dish. The cells werε transfεctεd by incubation for eighteen hours with a calcium phosphate precipitatε containing 10 μg of thε linεarizεd plasmid. Thε culturεs werε incubated in fresh medium for an additional twenty-four (24) hours, passed after 1"10 dilution in 80 cm 2 dishes, and then grown under selεction with 400 μg/ml of G 18 (GIBCO/BRL) for twelve (12) days. Neomycin-resistant clones were transferred to 12-well microtiter plates, grown to confluency, and cultured in two 24-well plates for an additional twenty-four (24) hours. Total RNA was extracted from the cell layer in one well (RNAeasy; Qiagen) and used for Northern blot assay after separation by elεctrophoresis on a 1% agarose gel and transfer to nitrocellulosε filters. The filtεrs wεrε probed using a 32 P-labεlεd cDNA for thε shortεst cDNA for procollagεn C-protεinase (pCP- 1) . Two clones, each transfεctεd with pCP-l or pCP-2, had high levels of corresponding mRNAs, as set forth in FIG. 2. Medium from neomycin-resistant clonεs expressing either pCP-l or pCP-2 contained εnzymatic activity that specifically cleaved type I procollagen into the products predicted by the introduction of C- protεinasε. Spεcifically, mεdium protεins from positivε clonεs were fractionated either by PEG precipitation or by membrane filtration. For PEG precipitation, 2 x IO 7 cells in 175 cm 2 flasks were incubated for 24 hours in serum-frεε DMEM and thε medium (15 ml) was precipitated, as described above . The precipitatεd protεins wεrε partially solubilizεd in 20 μl; of reaction buffer. For fractionation by membrane filtration, 30 ml of the medium were passed through a filter with a high molecular weight cut-off (XM300; Amicon) . A second filter (50kDa Ultrafree 15; Millipore) was used to concentrate the flowthrough about 500-fold and to transfer the sample to reaction buffer. 20 μl of each sample was incubated at 35°C for three (3) hours with 5 μl of reaction buffer containing 1 μg of "C-labεled typε I procollagεn purified from chick embryo fibroblasts . The rεaction products wεrε sεparatεd on a 7.5% SDS-polyacrylamidε gel without reduction and an image generated on a phosphor storage imager. As sεt forth in FIG. 3, thεrε was no detectable C-proteinase activity in medium from untransfected cells. Separation of the samples by polyacrylamide gel electrophoresis under rεducing conditions demonstrated that the Cl subunit from the proαl(I) chain and thε C2 subunit from the proαl(I) chain resemblεd thε εxpectεd sizε and wεrε obtained in the expected ratio of 2:1. Completε clεavage of typε I procollagεn to pNc-l(I) chains, pNc.2(I) chains and thε C-propεptidε were obtained with medium from a clone transfectεd with pC?-2 and partially fractionatεd and concεntrated by membrane filtration (as set forth in lane 6 of FIG. 3) . 8.4. Expression In An E. coli System To express the protein in the E. coli system, a CDNA containing the complete coding sequεnces for . PCP-2 was insertεd into thε εxprεssion vector that introduced as "tag" coding for 6 histidine residues at the 5'-end of thε coding sεquences (pQΞ-32 Vector; Qiagen) . The vector was then used to transfect E. coli cells that werε grown with and without induction with 1 Mm IPTG for onε to four hours at 30°C. A protεin band with an apparεnt molecular weight of about 100 kDa appearεd in thε cεlls induced with the IPTG. The cell pellet was lysed in buffer containing lysozyme, sonicated, reincubated in lysis buffer containing Triton X-100 and then extractεd with 6 M guanidinε hydrochloridε and 200 Mm NaCl in 10 Mm Tris- Hcl buffεr (Ph 7.6) . The solubilized protein was chromatographed on a metal affinity column (Talon; Clontεch) and εlutεd with 100 Mm imidazolε. Thε gεl fractions wεrε assayεd by SDS PAGE and silvεr staining. As indicatεd in FIG. 4, thε rεcombinant protεin was rεcovεrεd in apparεntly homogenous form. To refold thε protein, a method used to refold recombinant interstitial collagenase from E. coli inclusion bodies was used. Specifically, the protein from the metal affinity column was diluted to an intermediate strength of denaturant (2 M guanidinε hydrochloridε) so as to prevent precipitation but to allow formation of critical intermεdiatεs in the refolding process ( see, e . g . , Brems, 1988, Biochemistry 22:4541-4546; Ptitsyn, 1994, Protein Eng. 2:593-596) . Thε protein was then dialyzεd against a nεutral isotonic buffer. No C-proteinasε activity was observed when the recombinant protein was incubated in a standard reaction system without prior activation of the protein by digestion with chymotrypsin. Specific C- proteinase activity was observed (Lanes 1 to 4 in FIG. 5) after limitεd digestion with chymotrypsin. The specific activity of the re-foldεd recombinant protein was about l/30-th of the specific activity of C- protεinase isolatεd from chick εmbryo tεndons (Lane 6 in FIG 5) . The yield of recombinant protein was about 2 mg/l . 8.5. Synthetic Substrates For C-Proteinase Synthetic substrates for C-proteinase werε obtainεd by tεsting a sεries of synthetic peptides with sequεncεs found in and around the C-proteinase cleavagε sitεs of -Ala-Asp- and -Gly-Asp- in thε four proor chains of typεs 1, 11 and 111 procollagen (see, 11) . Although it has been reportεd that at concεntrations of 1 to 5 Mm thε peptides compεtitivεly rεducεd cleavage of a "C-labelεd procollagεn substratε, pεptidε cleavage was not originally detected using this reportεd concentration. When the enzyme concentration was increased 40- to 100-fold, thε rεaction ti ε was concurrently incrεasεd from two (2) hours to εight (8) to twεnty-four (24) hours, and the reaction products were analyzed by HPLC, followed by laser desorption mass spectrometry, four of the eight propeptides were specifically and completely cleaved by thε enzyme . All references cited within thε body of the instant specification are herεby incorporated by reference in their entirety.

Claims: CLAIMS What Is Claimed: 1. A nuclεic acid sequence encoding C- protεinas . 2. The nucleic acid sequence of Claim 1 wherein said sequεncε is set forth at Figure 6. 3. The nucleic acid sequence of Claim 1 wherεin said sεquence is set forth at Figure 7. . A nucleic acid sequence encoding C- proteinase comprising: (a) the nucleic acid sequence of Figure 6 or Figure 8 ; (b) the nucleic acid sεquεncε which can hybridizε to thε nucleic acid sequence of Figure 5 or Figure 8 under highly stringent hybridization conditions; or (c) the nucleic acid sequεncε which is a degenerate sequencε of thε sεquεnces of step (a) or (b) . 5 A polypεptidε encoded by thε sequence of Claim 1. 6. A polypeptide of Claim 5 encoded by the amino acid sequεncε of Figurε 6. 7. A polypeptidε of Claim 5 encoded by thε amino acid sεquεnce of Figure 7. 8. An antifibrotic compound having thε ability to bind to the protein of Claim 5 for the purpose of interrupting said polypeptid 's interaction with collagen. 9. A mεthod for trεating disorders or diseases related to fibrosis comprising administration of a pharmaceutically effectivε amount of thε compound of Claim 8 to a patient in neεd. 10. A mεthod for trεating disordεrs or disεases • •elated to fibrosis comprising administration of a armaceutically effεctivε amount of C-proteinase. 11. A mεthod for using thε DNA sεquεnce of Claim 1 to identify compounds useful in modulating the activity of C-proteinasε . 12. A procεss for producing rεco binant C- protεinasε comprising: (a) culturing a cεll in a nutrient medium under conditions suitable for the expression of human C-proteinasε, whεrein said cell is transformed with an εxprεssion vεctor comprisεd of a promotεr sεquεncε opεrably linked to a nucleotidε sεquεncε encoding human C-protεinase; and (b) isolating the human C-proteinasε. 13. A procεss for producing rεcombinant C- protεinasε comprising: (a) culturing a cεll in a nutrient medium under conditions suitablε for the expression of human C-proteinasε, whεrεin said cεll is transformεd with an exprεssion vεctor comprisεd of a promotεr sεquεncε operably linked to the nucleotidε sequencε of Claim 1; and (b) isolating thε human C-protεinasε. 14. A process for producing rεcombinant C- protεinasε comprising: (a) culturing a cεll in a nutriεnt medium under conditions suitable for the expression of human C-proteinasε, wherein said cell is transformed with an expression vector comprised of a promoter sequencε operably linked to thε nuclεotidε sεquεnce of Claim ; and (b) isolating thε human C-protεinasε.

Inventors: HOJIMA YOSHIO, LI SHI-WU, PROCKOP DARWIN J, SIERON ALEKSANDER, HOJIMA, YOSHIO, LI, SHI-WU, PROCKOP, DARWIN, J., SIERON, ALEKSANDER
Applicants: UNIV JEFFERSON, THOMAS JEFFERSON UNIVERSITY
Application year: 1996
Main classification: C12N5
IPC codes: A61K31, A61K38, A61K45, A61P1, A61P9, A61P11, A61P17, A61P19, A61P21, A61P27, A61P43, C07C259, C07C311, C07C323, C07D207, C07F9, C07K5, C07K14, C12N5, C12N9, C12N15, C12R1