Study On Matrix Metalloproteinase 1 And Tissue Inhibitor Of Metalloproteinase In Haliotis Discus Hannai

Autolysis refers to the process when the body was stimulated by physical,chemical or biological factors,and endogenous proteases were induced to destroy tissue structure,leading to muscle softening eventually.During this process,extracellular matrix（ECM）was greatly degradated.The main component of ECM is collagen,and type I collagen accounts for 80-90%of the total collagen.Since matrix metalloproteinases（MMPs）could specifically degradate collagen,and the activity of MMPs was precisely regulated by the tissue inhibitors of metalloproteinases（TIMPs）,indicating that MMPs and TIMPs play a crucial role in autolysis process.This study was conducted in Haliotis discus hannai.Recombinant MMP-1 and TIMP protein were generated by gene engineering technique.The object of this research is to study their properties and interaction relationship,explore their function in type I collagen degradation and reveal the mechanism of abalone autolysis.At the beginning of this study,the results indicated that collagenases have participated in the post-mortem autolysis of Haliotis discus hannai by analysing the degradation of type I collage which was co-incubation with abalone muscle homogenate.rMMP1c protein was expressed by using the Escherichia coli prokaryotic expression system.rMMP1c with high purity and enzyme activity was renatured from inclusion body.The optimum temperature and p H of rMMP1c were 37℃ and p H 7.0,showing reliable thermal stability and p H stability.The T_mvalue of rMMP1c was 67.0±0.9℃.At low concentrations,EDTA and 1,10-phenanthroline could inhibit the activity rMMP1c completely.Ba²⁺,Ca²⁺,Mg²⁺significantly elevated the activity of rMMP1c.rTIMP protein was expressed by HEK 293F human embryonic kidney cell expression system,and results showed that it had good inhibitory activity to rMMP1c.rTIMP showed good thermal stability and p H stability,and its T_mvalue was 73.1±0.6°C.PAS staining showed that rTIMP was a glycoprotein.Inhibition kinetics experiment showed that rTIMP was a competitive/non-competitive inhibitor of rMMP1c.Biolayer Interferometry Technology analysis protein-protein interaction showed that rTIMP could effectively bind and dissociate with rMMP1c,and their affinity constant K_Dvalue was 0.2628μM.After co-incubating rMMP1c and type I collagen,rMMP1c could completely degradate theγ-β-αchains of type I collagen in turn,which indicated that rMMP1c could significantly increase type I collagen degradation,fitting to collagenases characteristics.The molecular weight distribution of type I collagen degradation products was analyzed by reverse phase high performance liquid chromatography,showing that the large molecular weight parts of type I collagen were degraded into small molecular protein and polypeptide by rMMP1c.Besides,rTIMP could significantly inhibit the degradation activity of rMMP1c to type I collagen.Our study not only provides theoretical basis for understanding the autolysis process of abalone,but also offers certain directive significance for abalone storage and processing.