Cloning And Expression Of Matrix Metalloproteinases From Pacific Abalone (Haliotis Discus Hanai)

Matrix metalloproteinases(MMPs)are a family of zinc-containing endopeptidases that belong to metalloproteases.MMPs are proposed to be involved in the degradation of proteins that compose the extracellular matrix(ECM)including collagen,an important structural protein of shellfish muscle.However,few reports about the existence of MMPs genes and the interaction between MMPs and muscle proteins in shellfish was accessed.In present study,MMPs from Pacific abalone were cloned and expressed in vitro,the relative transcript level of the genes was also investigated.For the first time,the gene encoding Hdh-MMPs were cloned from Pacific abalone(Haliotis discus hanai)by RT-PCR and RACE.The full-length cDNA of Hdh-MMP-1 gene(Genbank accession number KR537291.1)was 2136 bp including an open reading frame(ORF)of 1551 bp that coding for 516 amino acid residues with an estimated molecular weight of 58.94 kDa and a theoretical pI of 5.99,while the Hdh-MMP-2(Genbank accession number KX058128.1)was 1644 bp that coded for 547 amino acids with an estimated molecular weight of 61.66 kDa and a theoretical p I of 5.19.Hdh-MMPs contained N-terminal prodomain,catalytic domain,hinge region and C-terminal hemopexin domain,which are typical in matrix metalloproteinases.Besides,three repeats of fibronectin-type II domain were found in Hdh-MMP-2,which is typical in gelatinase.Higher-order structure of Hdh-MMPs was predicted using SOPMA and SWISS-MODEL.From the multiple sequence alignment for Hdh-MMPs,similarity to Hdh-MMP-1 from other abalones as well as other MMPs were observed,moreover,Hdh-MMP-2 presented an similarity with other sequences reported in both vertebrates and invertebrates,illustrating MMPs are conserved in numerous species.Then,prokaryotic expression plasmid pET-cMMP-1 containing the catalytic domain of Hdh-MMP-1 was constructed and transformed into BL21(DE3)cells.The recombinant protein,expressed as inclusion bodies,was purified with Ni-NTA Spin column and used for preparing specific polyclonal antibody.In addition,recombinant Hdh-cMMP-1(90 k Da)was expressed in Pichia pastoris and tested active by gelatin zymography.On the other side,prokaryotic expression plasmid pET-cMMP-2 containing the catalytic domain of Hdh-MMP-2 was constructed and transformed into BL21(DE3)cells.The target protein,also expressed as inclusion bodies,was purified subsequently.But the recombinant protein was unable tohydrolyze gelatin.For purpose of acquiring active Hdh-cMMP-2,we have used the strategy to increase the yield of soluble recombinant protein by fusion of the gene encoding the target protein to the gene encoding the solubility-enhancing protein MBP(Maltose-binding protein).However,the recombinant protein still inactively after cleaving off the solubility-tag(MBP)by TEV proteinase.In order to evaluate the transcript levels for the Hdh-MMPs,tissue samples were taken from five individuals and the qPCR analysis showed that the two kind of MMPs were ubiquitously expressed in all tissues detected,including muscle,hepatopancreas,gonad,hemolymph and gill with the highest expression level in hemolymph and gill that involved in immune system,indicating the important roles MMPs played in abalone immunoregulation.After challenging by Vibrio Parahaemolyticus,the expression level of Hdh-MMPs was upregulated significantly(p<0.05),which demonstrated that MMPs play critical roles in abalone muscle under pathological conditions.Moreover,after challenging by V.Parahaemolyticus repeatedly,accompanying with upregulation expression of Hdh-MMPs,drastically decreasing of the hardness of muscle(p<0.05)was observed.The results in this study proposed a putative biological function for Hdh-MMPs involved in degradation of muscle protein,therefore affecting the texture of Pacific abalone H.discus hanai.