Study On Matrix Metalloproteinase-2 From Muscle Of Silver Carp(Hypophthalmichthys Molitrix) And Its Endogenous Inhibitor

Endogenous proteases and their inhibitors are closely related to muscle metabolism and postmortem storage,so it is of great significance and application value to study proteases and their inhibitors.In this study,matrix metalloproteinase 2(MMP2)and its endogenous inhibitor which are involved in the degradation of collagen in fish were studied.Silver carp was used as raw material.The physicochemical properties of the enzyme and its inhibitor and their interaction mechanism were analyzed.The enzyme activity of silver carp MMP2 is high in fish muscle,but it is difficult to purify and obtain natural enzyme for its trivial content in muscle.Therefore,in order to isolate the endogenous inhibitor of MMP2 in silver carp,the recombinant catalytic domain of MMP2(rHm-MMP2c)was expressed and purified.The protease with high activity and high yield provided a basis for purifying its endogenous inhibitors.In this thesis,to study the decomposition and autolysis of muscle in aquatic animals,the molecular cloning and expression of rHm-MMP2c were studied and the basic properties were illuminated.The sequence of the rHm-MMP2c was cloned and the c DNA was 996 bp in length,encoding 332 amino acids.The theoretical molecular weight of rHm-MMP2c was 37 k Da and the isoelectric point was 4.38 based on the analysis of DNAMAN.The expression level of MMP2 was highest in blood,the expression level in brain and muscle gill was equivalent by real time quantitative PCR.rHm-MMP2c was expressed in E.coli.The enzyme was expressed in supernatant and showed high degradation on I type collagen and gelatin.rHm-MMP2c was purified by Ni column.rHm-MMP2c showed high activity at p H 9.0 and 37?.EDTA,EGTA and 1,10-phenanthroline had strong inhibitory effect on this enzyme indicating rHm-MMP2c was a typical metalloproteinase.CD spectra showed that the secondary structure of rHm-MMP2c was dominated by random coiling,followed by?-sheet and the denaturation temperature of rHm-MMP2c was 50.1±0.1?.rHm-MMP2c can degrade type I collagen effectively.The kinetic parameters of rHm-MMP2c showed the Michaelis constant(K_m)constant was 26 nmol/L and the catalytic number(k_cat)was 54.7/s.Endogenous inhibitor was screened and isolated from skeleton muscle of silver carp.The inhibitor was isolated and purified by 60?heating,50-90%ammonium sulfate fractionation and continuous column chromatography including DEAE-Sepharose,Phenyl-Sepharose and Superdex 200/300.SDS-PAGE showed that the molecular mass of the inhibitor was 30 k Da.The inhibitor had high inhibitory activity against rHm-MMP2c and the inhibitory activity(IC₅₀)was3.33?mol/L.It was identified as apolipoprotein A-?(Apo A-?)by mass spectrometry.Apo A-? was stable at 20-70? and p H 6.0-11.0.The secondary structure of apolipoprotein was dominated by?-helical(48.6%)and the denaturation temperature of rHm-MMP2c was 75.3±0.3°C The inhibition kinetics showed that Apo A-? exhibited a competitive inhibition on rHm-MMP2c,and the inhibition constant K_iwas 1.31?mol/L.Western blot analysis of enzyme and inhibitor showed that Apo A-? could inhibit the degradation of gelatin by rHM-MMP2c.In order to further elucidate the inhibition mechanism of Apo A-? on rHm-MMP2c,molecular docking and molecular dynamics(MD)simulations of rHm-MMP2c with its substrate and Apo A-? were performed.The docking results showed that Apo A-? inhibited the activity of rHm-MMP2c by a reversible competitive inhibition which was consistent with the result of inhibitory kinetics.The inhibition may be due to the steric hindrance formed by the inhibitor,which prevented the substrate from binding to the enzyme.MD simulation showed that Apo A-? was more tightly bound to rHm-MMP2c during 100 ns simulation.Hydrogen bonding force plays leading role in the binding process,which affected the binding of the substrate to rHm-MMP2c.In this thesis,the catalytic domain of silver carp MMP2 was expressed efficiently with high activity,and a new endogenous inhibitor of silver carp MMP2 was screened with the enzyme.The interaction between rHm-MMP2c and Apo A-? was studied,which provided a basis for the separation of endogenous inhibitors in the future.