Investigation On Repair Function Of HMsrA Upon Oxidition Damaged Low Density Lipoprotein

Object: Atherosclerosis(As) is the leading cause of death in most developed countries. The mechanism of it is very complicated and involved in many pathophysiological process. Low density lipoprotein(LDL) critically contributes to the risk of human As. Oxidative modification of LDL increases its atherogenic potential. Oxidative stress occurs when oxidative substances surpass the eliminative ability of antioxidants. As is correlated closely with oxidative stress. Methionine sulfoxide reductase(MsrA) exists in many organisms and is one of the most important antioxidants in vivo. We try to investigate whether human methionine sulfoxide reductase(hMsrA) can protect oxidative damage to human LDL in vitro and the oxidation mechanism of LDL, which may contribute to further research on antioxidation function of hMsrA in vivo.Methods: To isolate LDL by using polyanions precipitation and short-time density gradient ultracentrifugation. The recombinant plasmid harboring hMsrA gene was transformed into E. Coli. M15(pREP4) bacterial. After induction, hMsrA protein was expressed and extracted by breakdown of bacterials. The protein was purified through high-efficient Ni-NTA agarose column and its activity was determined. To oxidate LDL by AAPH and evaluate the protective action of hMsrA by using SDS-PAGE TBA method and lipoprotein agarose-gel electrophoresis.Results: (l)hMsrA can be expressed in prokaryotic E.Coli. and successfully purified by Ni-NTA agarose column with native activity; (2)hMsrA can prevent apoB-100 from breaking down; (3)hMsrA can downregulate the amount of MDA, reflecting its inhibitive ability on lipid peroxidaion; (4)hMsrA can decrease REM of ox-LDL, which denotes reduction of oxidative extent on LDL.Conclusion: hMsrA can protect LDL from oxidative damage, which may function through reducing methionine sulfoxide(Met(O)) of apolipoproteinB100 to methionine(Met).