Characterization And Function Analysis Of Porcine MMP-2 And TIMP-2 Genes

Matrix metalloproteinase-2 (MMP-2), also named as gelatinase A or type IV collagenase, is an important member of the MMP family, primarily, it contributes to the hydrolization of gelatine and type IV collagen for tissue remodelling. However, recent work indicates that MMP-2 also plays crucial roles in inflammation and immunity, MMP-2 affects the clearance of recruited immune cells, which is necessary to resolve inflammatory reactions. The expression of MMP-2 is up-regulated in many human as well as animal models of inflammatory and immune diseases. Tissue inhibitor of metalloproteinase-2 (TIMP-2) is the major inhibitor of MMP-2, it is also a necessary factor involved in the activation of proMMP-2 to mature MMP-2 peptide with biological activity. Based on the important roles of MMP-2 and TIMP-2 in medical and disease study, in this study, the characterization and function analysis of porcine MMP-2 and TIMP-2 were performed to provide information for further medical research using pig as animal model as well as pig genetics and breeding research.Compared with the available information of human and mouse MMP-2, the information for pig is very limited. In this study, we cloned the 5′-upstream sequence, 3′-downstream sequence as well as other missed genomic sequences of porcine MMP-2, the genomic structure and the promotor sequence were characterized and found to share high similarity with those of human MMP-2. Porcine MMP-2 was assigned to SSC6p14–p15, and closely linked to microsatellite SW1108 (53cR, LOD score 7.59) by IMpRH panel. Real-time PCR analysis revealed that the expression of porcine MMP-2 was remarkably different in diverse tissues, a high level expression was observed in the testis and uterus, relatively low expression in other tissues.15 polymorphism sites were identified within porcine MMP-2 genomic sequence. the SNP AcyI and indel MspI were selected for allele frequencies determination in different pig breeds and association study in our purebred Landrace resource population. The results showed that the SNP AcyI in exon 12 was significantly associated with white blood cell count (WBC) of neonate piglets at 0 day (P=0.0079), and classical swine fever virus antibody level (CSFV-AB) of pigs at 17 days (P=0.0461), the indel MspI in intron 4 had remarkable correlation with mean corpuscular hemoglobin (MCH) of pigs at 17 days (P<0.0001).Porcine TIMP-2 cDNA was obtained by reverse transcription polymerase chain reaction (RT-PCR), using in silico cloning strategy based on porcine ESTs database. The length of the cloned porcine TIMP-2 cDNA was 909 bp including a 663 bp entire open reading frame (ORF), which encodes a 221 amino acid protein. Multiple sequences alignment and phylogenic analysis indicated that porcine TIMP-2 protein shared high similarity with that of other species, such as Homo sapiens (97%), Mus musculus (97%) and Rattus norvegicus (97%). Protein sequence analysis showed that the deduced porcine TIMP-2 protein sequence ( pI= 7.65, MW= 24.5kD) contained 27AA leader sequence and 194 AA mature peptide, the leader sequence in pig has one more amino acid (Leu residue) than that in other 6 species. A NTR-TIMP function domain and a conserved VIRAK motif at N-terminus were predicted by bioinformatic analysis and homologous comparison, and the 12 Cys residues in the sequence were predicted to constitute 6 disulphide bonds. The expression of porcine TIMP-2 mRNA varied greatly in different tissues as well as developmental stages, it was highly expressed in testis, pituitary body, stomach, large intestine, ovary, uterus and 90 days embryonic longissimus muscle , but lowly in heart, small intestine, brain, liver and adult longissimus muscle, and a moderate expression level was detected in kidney, thymus, spleen, thyroid and 33 days embryonic longissimus muscle. The result indicated that the porcine TIMP-2 mRNA was highly expressed in organs and phases with relatively high tissue growth and/or remodelling activities, and it was in accordance with the innate biological function of TIMP-2.