Abstract
Matrix metalloproteinases (MMP) are widely distributed in vertebrate tissues. One of the well-characterized gelatinases from higher vertebrates is MMP-2, named gelatinase A or 72 kDa type IV collagenase, which is active for the cleavage of denatured collagens, type IV collagen, type V collagen, and other matrix proteins. To investigate the primary structure and properties of MMP-2 from teleost as lower vertebrates, a cDNA library was prepared from mRNA of rainbow trout fibroblast and screened. Using polymerase chain reaction and degenerate oligonucleotide primers, which are specific for two highly conserved sequences found in MMP of higher vertebrates, a resultant cDNA fragment was used as a probe. A cDNA clone 3.0 kb long was isolated and found to contain an open-reading frame coding for a polypeptide of 655 amino acids. The rainbow trout polypeptide was 73% identical, at the level of amino acid sequence, to human proMMP-2 with the greatest degree of similarity occurring in the propeptide and catalytic domains and was denoted as rainbow trout proMMP-2. Then the isolated cDNA was expressed in Escherichia coli and the recombinant protein was found to degrade gelatin and human type V collagen, providing support to the hypothesis that the cDNA codes for the authentic rainbow trout proMMP-2. In contrast to human proMMP-2, rainbow trout proMMP-2 was not activated by 4-amino-phenylmercuric acetate. This is the first report of cDNA for fish proMMP to our knowledge.
