Molecular cloning of cDNA coding for a matrix metalloproteinase from Atlantic salmon
2002
Nagashima, K. (Hokkaido. Food Processing Research Center, Ebetsu (Japan)) | Shimizu, T. | Yasokawa, D. | Nakagawa, R. | Okumura, Y.
To isolate cDNA coding for matrix metalloproteinases (MMPs) from fish, we first investigated Ca2+ -dependent gelatinolytic activities in conditioned media of eight fish cell lines using gelatin-zymography with and without Ca2+. As a result, we found the major activity at positions of 60, 66, 70 and 80 kDa, respectively in some cell lines. RT-PCR was performed using total RNA from ASE cells, which were derived from Atlantic salmon and expressed the gelatinolytic enzyme of 66 kDa, and using degenerate primers that were designed on the basis of the conserved amino acid sequences corresponding to cysteine switch and catalytic domain of MMP. Amplified DNA was 329 bp long and its deduced amino acid sequence was highly homologous to known MMPs. Based on this nucleotide sequence, we performed 5' and 3' RACE and finally obtained 1714 bp long cDNA coding for a protein of 462 amino acid residues. The amino acid sequence showed higher homologies (54-560/0 identities) to MMP13s (collagenase 3) from several species than other MMPs, suggesting that the cloned cDNA coded for a salmon counterpart of MMP13.
Показать больше [+] Меньше [-]