Characterization of cDNA clone encoding the matrix metalloproteinase 2 from rainbow trout fibroblast

Matrix metalloproteinases (MMP) are widely distributed in vertebrate tissue s. 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 t he 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 fro m mRNA of rainbow trout fibroblast and screened. Using polymerase chain rea ction and degenerate oligonucleotide primers, which are specific for two hi ghly conserved sequences found in MMP of higher vertebrates, a resultant cD NA 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 amin o acids. The rainbow trout polypeptide was 73% identical, at the level of a mino acid sequence, to human proMMP-2 with the greatest degree of similarit y occurring in the propeptide and catalytic domains and was denoted as rain bow trout proMMP-2. Then the isolated cDNA was expressed in Escherichia col i 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 tr out proMMP-2 was not activated by 4-amino-phenylmercuric acetate. This is t he first report of cDNA for fish proMMP to our knowledge.