文章基本信息

标题：N-Glycosylation regulates endothelial lipase-mediated phospholipid hydrolysis in apoE- and apoA-I-containing high density lipoproteins
本地全文：下载
作者：Skropeta, Danielle ; Settasatian, Chatri ; McMahon, Monica R. 等
期刊名称：JLR Papers In Press
印刷版ISSN：0022-2275
电子版ISSN：1539-7262
出版年度：2007
卷号：48
期号：9
页码：2047-2057
DOI：10.1194/jlr.M700248-JLR200
出版社：American Society for Biochemistry and Molecular Biology
摘要：Endothelial lipase (EL) is a member of the triglyceride lipasegene family with high phospholipase and low triacylglycerollipase activities and a distinct preference for hydrolyzingphospholipids in HDL. EL has five potential N-glycosylationsites, four of which are glycosylated. The aim of this studywas to determine how glycosylation affects the phospholipaseactivity of EL in physiologically relevant substrates. Site-directedmutants of EL were generated by replacing asparagine (N) 62,118, 375, and 473 with alanine (A). These glycan-deficient mutantswere used to investigate the kinetics of phospholipid hydrolysisin fully characterized preparations of spherical reconstitutedhigh density lipoprotein (rHDL) containing apolipoprotein E2(apoE2) [(E2)rHDL], apoE3 [(E3)rHDL], apoE4 [(E4)rHDL], or apoA-I[(A-I)rHDL] as the sole apolipoprotein. Wild-type EL hydrolyzedthe phospholipids in (A-I)rHDL, (E2)rHDL, (E3)rHDL, and (E4)rHDLto similar extents. The phospholipase activities of EL N118A,EL N375A, and EL N473A were significantly diminished relativeto that of wild-type EL, with the greatest reduction being apparentfor (E3)rHDL. The phospholipase activity of EL N62A was increasedup to 6-fold relative to that of wild-type EL, with the greatestenhancement of activity being observed for (E2)rHDL. These datashow that individual N-linked glycans have unique and importanteffects on the phospholipase activity and substrate specificityof EL. Supplementary key words N-linked glycans • site-directed mutagenesis • apolipoprotein E • apolipoprotein A-I • phospholipase kinetics • Michaelis-Menten constant • maximum velocity • binding affinity Abbreviations: apoE, apolipoprotein E; CE, cholesteryl ester; EL, endothelial lipase; rHDL, reconstituted high density lipoprotein; UC, unesterified cholesterol