Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-07-26
2003-05-20
Patterson, Jr., Charles L. (Department: 1632)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S023000, C435S069100, C435S252300, C435S320100, C514S002600, C536S023200
Reexamination Certificate
active
06566116
ABSTRACT:
TECHNICAL FIELD
This application is based on PCT/JP97/01433, having an International filing date of Apr. 24, 1997, which claims the priority date of Apr. 24, 1996 of Japanese application number 8-104902.
BACKGROUND ART
The extracellular matrix, which is a cell-supporting tissue composed mainly of collagens and proteoglycans, is profoundly involved in such events as cell development, inflammation, and tissue repair. The enzymes known to be associated with the degradation of extracellular matrix are (1) cathepsin D, etc. which belongs to the aspartic proteaseas, (2) cathepsin B, H, L, etc. which belong to the cysteine proteases, (3) plasmin, kallikrein, neutrophil elastase, tryptase, chymase, cathepsin G, etc. which belong to the serine proteases, and (4) metalloproteases are known. Also called matrix metalloproteases, these metalloproteases are known to be playing central roles in the degradation of extracellular matrix.
So far, in humans, 13 kinds of matrix metalloproteases such as collagenases, gelatinases stromelysins, and membrane-type matrix metalloproteases have been cloned and their nucleotide sequences and amino acid sequences have been reported (T. Takino et al., Journal of Biological Chemistry, 270, 23013, 1995; J. M. P. Freije et al., Journal of Biological Chemistry, 269, 16766, 1994; H. Wills et al., European Journal of Biochemistry, 231, 602, 1995). All of these enzymes are zinc-dependent metalloproteases, in which the amino acid sequence of the zinc-binding domain: His-Glu-X-Gly-His-Ser-Leu-Gly-Leu-X-His-Ser is well conserved, and their activities are inhibited by o-phenanthroline. Each of these enzymes is secreted in the latent form which is inactive with a propeptide at the N-terminus of the active enzyme. A conserved domain consisting in the amino acid sequence of Met-Arg-Lys-Pro-Arg-Cys-Gly-Val-Pro-Asp is located near the C-terminal region of the propeptide. This domain is called “cysteine switch”, and it controls a protease activity by coordinating the zinc atom at active center with cysteine in the domain. While the latent enzymes are activated upon cleavage of the propeptide, three kinds of inhibitor proteins, named TIMP, have been reported and known to performing strict control of activity. It is also known that, in vitro, the latent enzymes are activated by treatment with trypsin or aminophenyl-mercuric acetate.
Matrix metalloproteases are not only involved in the degradation of the extracellular matrix such as collagens, gelatins which are denatured collagens, proteoglycans, fibronectins, laminins, elastins, etc. but also are in charge of activation of other matrix metalloproteases and inactivation of protease inhibitors such as al-protease inhibitor. Furthermore, it is known that these metalloproteases are associated with solubilization of membrane proteins and cell surface proteins such as TNF, Fas ligand, IL-6 receptor, TNF-receptor, etc. and, as a result, modulate the death, differentiation, proliferation inhibition, proliferation and gene expression of cells.
It is known that physiologically matrix metallo protease activities are elevated in ovulation, development and differentiation, osteogenesis, atretic uterus, vascularization, and other events. In morbid states, those metalloprotease activities are elevated in rheumatoid arthritis, osteoarthritis, cancer (metastasis and invasion), peridontitis, corneal ulcer, gastric ulcer, myocardiopathy, aneurysm, otosclerosis, epidermolysis bullosa, premature labor, and atherosclerosis, among other conditions. Conversely, it is known that the enzyme activities are suppressed in fibroid lung, hepatolienal fibrosis, hepatocirrhosis, osteopetrosis, etc. Recently the fourth membrane-type matrix metalloprotease has been cloned (X. S. Puente et al., Cancer Research, 56, 944, 1996), suggesting the likelihood that there exist still other novel matrix metalloproteases.
Any novel matrix metalloproteases of human origin make it possible to develop new drugs which inhibit or stimulate the activity of the metalloprotease and are useful for the prevention and treatment of various matrix metalloprotease-associated morbidities, such as rheumatoid arthritis, and osteoarthritis. Therefore, in the technological area to which the present invention pertains, there has been a standing need for isolating novel human matrix metalloproteases and developing a method for high production of the proteins.
The inventors of the present invention have made extensive research for solving the above problems and succeeded in cloning cDNAs each having a novel nucleotide sequence from human liver-derived and rat liver-derived cDNA libraries. They have found that the proteins encoded by these cDNAs are matrix metalloproteases. The present inventors have made further investigations based on these findings, and accomplished the present invention.
DISCLOSURE OF INVENTION
The present invention provides:
(1) A protein comprising an amino acid sequence represented by SEQ ID NO:1 or a substantially equivalent thereto, or a salt thereof,
(2) The protein according to claim
1
, which comprises an amino acid sequence represented by SEQ ID NO:2,
(3) The protein according to (1), which is a metalloprotease,
(4) A partial peptide of the protein according to (1), or a salt thereof, which shows the activity of the protein according to (1),
(5) An isolated DNA which contains a DNA comprising a nucleotide sequence coding for a protein according to (1),
(6) The DNA according to (5), which comprises a nucleotide sequence represented by SEQ ID NO:4,
(7) The DNA according to (5), which comprises a nucleotide sequence represented by SEQ ID NO:8,
(8) A recombinant vector comprising the DNA according to (5),
(9) A transformant carrying the recombinant vector according to (8),
(10) A process for producing a protein or a salt thereof according to (1), which comprises culturing a transformant according to (9) under conditions suitable to express the protein,
(11) A pharmaceutical composition which comprises the protein according to (1) or the partial peptide according to (4),
(12) The pharmaceutical composition according to (11) which is a therapeutic or prophylactic composition for diabetic nephropathy, glomerulonephritis, pulmonary fibrosis, hepatolienal fibrosis, hepatocirrhosis, osteopetrosis or herniated disk,
(13) A pharmaceutical composition which comprises the DNA according to (5),
(14) The pharmaceutical composition according to (13) which is a therapeutic or prophylactic composition for diabetic nephropathy, glomerulonephritis, pulmonary fibrosis, hepatolienal fibrosis, hepatocirrhosis, osteopetrosis or herniated disk,
(15) An antibody against the protein according to (1) or the partial peptide according to (4),
(16) A method for screening for a compound which activates or inhibits a proteolytic activity of the protein according to (1) or the partial peptide according to (4), which comprises measuring and comparing a proteolytic activity of the protein according to (1) or the partial peptide according to (4), in case of (i) a substrate is contacted with the protein according to (1) or the partial peptide according to (4) and (ii) a substrate and a test compound are contacted with the protein according to (1) or the partial peptide according to (4),
(17) A kit for screening for a compound which activates or inhibits a proteolytic activity of the protein according to (1) or the partial peptide according to (4), which comprises the protein according to (1) or the partial peptide according to (4),
(18) A compound which activates or inhibits a proteolytic activity of the protein according to (1) or the partial peptide according to (4), which is identified by the screening method according to (16) or the kit according to (17),
(19) A pharmaceutical composition which comprises the compound which inhibits a proteolytic activity of the protein according to (1) or the partial peptide according to (4), which is identified by the screening method according to (16) or the kit according to (17),
(20) A method for treating or preventing diabetic nephropathy, glomerulonephritis
Atsushi Nishimura
Koji Yoshimura
Yuichi Hikichi
Conlin David G.
Edwards & Angell LLP
Intellectual Property Practice Group
Moore William W.
Patterson Jr. Charles L.
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