Recombinant C-proteinase and processes, methods and uses...

Details Recombinant C-proteinase and processes, methods and uses... Recombinant C-proteinase and processes, methods and uses...

1997-06-10

2001-07-10

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S069100, C435S252300, C435S252330, C435S320100, C536S023200, C536S023500, C530S350000

Reexamination Certificate

active

06258584

ABSTRACT:

TABLE OF CONTENTS
1. STATEMENT OF RELATED CASE
2. INTRODUCTION
3. BACKGROUND OF THE INVENTION
4. SUMMARY OF THE INVENTION
5. DEFINITIONS
6. BRIEF DESCRIPTION OF THE DRAWING
7. DETAILED DESCRIPTION OF THE INVENTION
7.1. Isolation Of Gene Encoding C-Proteinase
7.2. Uses Of The C-Proteinase Coding Sequence
7.3. Expression Of C-Proteinase
7.4. Identification Of Transfectants Or Transformants That Express C-Proteinase
7.5. Screening Of Peptide Library With C-Proteinase Or Engineered Cell Lines
7.6. Screening Of Organic Compounds With C-Proteinase Protein Or Engineered Cell Lines
8. EXAMPLES
8.1. Identification Of Partial Amino Acid Sequences Of The C-Proteinase
8.2. Preparation and Structure of cDNAs for the C-Proteinase
8.3. Expression of the cDNAs in a Mammalian Cell System
8.4. Expression In An
E. Coli
System
8.5 Synthetic Substrates For C-Proteinase
CLAIMS
ABSTRACT
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, the 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 extensions. These peptide extensions, referred to generally as “pro-regions,” are designated as N- and C-propeptides, 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.
47:129-162; 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:
Extracellular 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.
311:376-383; Kuhn, 1987, in:
Structure and Function of Collagen Types
(eds. Mayne, R. and Burgeson, R. E.), Academic Press, Inc., Orlando, Fla., 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, perimuscular fibrosis, Symmers' fibrosis, pericentral fibrosis, hepatitis, dermatofibroma, billary cirrhosis, alcoholic cirrhosis, acute pulmonary fibrosis, idiopathic pulmonary fibrosis, acute respiratory distress syndrome, kidney fibrosis/glomerulonephritis, kidney fibrosis/diabetic nephropathy, scleroderma/systemic, scleroderma/local, keloids, hypertrophic scars, severe joint adhesions/arthritis, myelofibrosis, corneal scarring, cystic fibrosis, muscular dystrophy (duchenne's), cardiac fibrosis, muscular fibrosis/retinal separation, esophageal stricture, 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,
Anal. Biochem.
86:463-469), and chick tendon fibroblasts (Duskin et al., 1978,
Arch. Biochem. Biophys.
185:326-332; Leung et al., 1979,
J. Biol. Chem.
254:224-232). 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., 1982,
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
21:757-764. 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.
269:26280-26285, 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-Proteinase 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, chymostatin and pepstatin A have been found to act as relatively strong inhibitors of C-proteinase activity.
&agr;
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; Ryhänen 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

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