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

Abstract

SUMMARY: 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.

Gelatinolytic activities in muscle of Atlantic cod (Gadus morhua), spotted wolffish (Anarhichas minor) and Atlantic salmon (Salmo salar)

“…Sato et al 10 have proposed that there might exist type V collagen-specific enzymes which contribute to the softening of the muscle observed during chilled storage of rainbow trout and sardine. Recently, Saito et al 31 showed the existence of a MMP-2 in rainbow trout with enzymatic activity able to degrade type V collagen.…”

Matrix metalloproteinase 2 of grass carp Ctenopharyngodon idella (CiMMP2) is involved in the immune response against bacterial infection

“…Teleost MMP2 genes have been cloned from zebrafish [7], Fugu rubripes [8], Japanese medaka [9], Japanese flounder [10], Atlantic salmon [11] and rainbow trout [12]. Recent studies demonstrated that the MMP2 plays a pivotal role in the regeneration of zebrafish scales, fin and heart [13e15].…”

Expression profiles of matrix metalloproteinase 9 in teleost fish provide evidence for its active role in initiation and resolution of inflammation

“…Gelatin‐degrading enzymes similar to mammalian MMP‐9 have been described in fish 25–27 . cDNA of MMPs has been cloned for zebrafish, 28 rainbow trout, 29,30 medaka 31 , Japanese flounder 32 and carp 33 . The amino acid sequence of carp MMP‐9 shares 63, 61 and 58% identity with that of newt, human and chicken MMP‐9‐like enzymes, respectively 33 .…”

“…Knowledge of the biological function of piscine MMP‐9 is still limited. It has been proposed that, in fish, gelatinases participate in the disintegration of pericellular connective tissue during spawning 34 and embryonic development 35,36 as well as in post mortem degradation 25,29,30,37 . Takeuchi et al.…”