Proteins, their production and use

#The present invention relates to a novel matrix metalloprotease and a DNA coding for the metalloprotease.

#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.

#FIG. 1 shows an amino acid sequence of a human liver-derived metalloprotease of the present invention.

#FIG. 2 shows an amino acid sequence of a partial peptide of the human liver-derived metalloprotease of the present invention, wherein 97 amino acid residues are deleted from the N-terminus of the amino acid sequence shown in FIG. 1.

#FIG. 3 shows an amino acid sequence of a partial peptide of a human liver-derived metalloprotease of the present invention, wherein 92 amino acid residues are deleted from the N-terminus of the amino acid sequence shown in FIG. 1.

#The following examples are intended to illustrate the present invention in further detail and should by no means be construed as defining the scope of the invention. Incidentally, the gene manipulations using Escherichia coli were made according to the protocol described in Molecular Cloning.

#Cloning of a Gene Coding for Human Liver-Derived Metalloprotease

#The cloning of the cDNA was carried out using Gene Trapper Positive Selection System (GIBCO/BRL).