Research On The Role Of C₃₂₃ Of SR-BI In Its Mediating Cholesterol Uptake And Efflux

Purpose:To determine the change of the ability of SR-BI mediating cholesteryl esters (CEs) uptake from HDL and free cholesterol efflux from cell after C323 point mutated. And to further determine the role of C323 in SR-BI's functions.Methods:1.Western Blot is used to investigate the expression of SR-BI in cells.2.By Caveolae Prep., we determine the SR-BI localization, and double confirme it via confocal immunofluorescence images and biotinlation.3.Use iodine monochloride method to iodinate HDL and Gwynne and Mahaffee's method to trace the HDL-associated cholesteryl ester with nonhydrolyzable [1, 2(n)-3H] cholesteryl oleoyl ether. To determine the ability of SR-BI mediated cholesteryl ester uptake, selective uptake assay is carried on using double labeled HDL to trace the amount of HDL binding to cell and cholesteryl ester taken up into cells.4.SR-BI's ability of mediating free cholesterol efflux is determined by 3H cholesterol with cholesterol efflux experiment.5. Deglycosytlation is used to determine the reasons of the expression of SR-BI with molecular weight of 100KD in CHO-SR-BIC323G mutant cell line.Results:1.Western Blot indicates that CHO-SR-BIC323G cell mutant still stably express SR-BI except one difference—SR-BI in CHO-SR-BI C323G mutant express two kinds of SR-BI protein with molecular weight of 75KD and 100KD.2.Cavelae prep. indicates that SR-BI in CHO-SR-BI C323G mutant locate in the plasma membrane in caveolae, same as in positive control cell line—CHO-SR-BI , confocal images and biotin results double confirme it. 3.In cholesterol uptake assay, the results show that SR-BI of CHO-SR-BIC323G cell mutant has lost the function of binding to HDL and mediating cholesteryl ester uptake from it.4.The same phenomenon appeared in cholesterol efflux assay as in cholesterol uptake assay.5.The deglycosytlation experiment results show that the expression of SR-BI with mocecular weight of 100KD is caused by glycosylation.Conclusions:1. C323 of SR-BI is critical and required in its mediating cholesteroyl ester uptake and free cholesterol efflux.2. C323 mutating has no effect on SR-BI localization in mutant cell line.3. SR-BI of CHO-SR-BIC323G has lost the function of mediating cholesteryl ester uptake from HDL and free cholesterol efflux from cell.