Molecular Cloning, Expression And Characterization Of Matrix Metalloproteinase-2from Common Carp (Cyprinus Carpio)

Matrix metalloproteinases (MMPs) are metal ion-dependent proteinases that are largelyresponsible for degrading extracellular matrix (ECM) proteins. MMPs involved in physiologicaland pathological processes, and can be found in animals, plants and microorganisms. MMPshave been proposed to participate in the post-mortem degradation of fish muscle, collagenaseand gelatinase played key roles in these processes. Matrix metalloproteinases-2(MMP-2) played critical roles in the metabolism of collagens while seldom reports wereavailable for their existence in aquatic animals. In the present study, MMP-2was cloned andexpressed in E.coli.In our study, the gene encoding MMP-2was cloned from common carp (Cyprinus carpio)by RT-PCR and RACE. The full-length cDNA of MMP-2was2789bp and the complete openreading frame was1971bp, encoding a protein composed of657amino acid residues. Thededuced MMP-2protein contains four conserved domains, including a29amino acids signalpeptide located at the N-terminus, a propeptide domain, three repeats of fibronectin-typeⅡdomain inserted in the catalytic domain and a C-terminal hemopexin-like domain. The3Dstructure of MMP-2showed that it is a monomer contains catalytic domain, fibronectin-typeⅡdomain and hemopexin-like domain.Due to the structural feature of MMP-2, the catalytic domain of MMP-2containing351amino acid residues was expressed in E. coli. SDS-PAGE showed that the recombinant MMP-2with molecular mass of approximately38kDa was in the form of inclusion body. Therecombinant MMP-2was further purified by immobilized metal ion affinity chromatography.After renaturation, similar to native MMP-2, the recombinant MMP-2exhibited highhydrolyzing activity toward gelatin as appeared on gelatin zymography.Furthermore, the activity of recombinant MMP-2at different temperatures and pH wasdetected by gelatin zymography. Clear proteolytic bands could be observed from30°C to45°C,and the clearest band was at40°C which was thus regarded as its optimal temperature. Highproteolytic activity in the pH range from7.0to9.0, and the highest activity was pH8.0. Theactivity of recombinant MMP-2was completely suppressed by metalloproteinase inhibitors,including EDTA, EGTA and1,10-phenanthroline whereas other proteinase inhibitors did notshow any inhibitory effect. Divalent metal ion Ca2+was essential for the gelatinolytic activity,suggesting that it is a calcium-dependent metalloproteinase. Moreover, the recombinant MMP-2 hydrolyzed native typeⅠ collagen effectively at37°C and even at4°C, indicating its involvementin the texture softening of fish muscle during post-mortem stage. However, degradation ofmyofibrillar proteins by recombinant MMP-2was not observed, implying MMP-2is specificallyactive on collagen and gelatin.In order to identify the role of fibronectin-typeⅡdomain of MMP-2, we tried to expressrecombinant proteins recombinant Fnt and recombinant△Fnt. The result showed that comparedwith recombinant MMP-2, both recombinant Fnt and recombinant△Fnt did not show anygelatinolytic activity, suggesting that the fibronectin-typeⅡdomain plays a crucial role forcatalytic activity of MMP-2. This study is helpful for understanding the biochemical role in fishmuscle and during the post-mortem tenderization of fish muscle, and screening the inhibitors ofMMP-2.