title: N-Glycosylation regulates endothelial lipase-mediated phospholipid hydrolysis in apoE- and apoA-I-containing high density lipoproteins.
abstract: Endothelial lipase (EL) is a member of the triglyceride lipase gene family with high phospholipase and low triacylglycerol lipase activities and a distinct preference for hydrolyzing phospholipids in HDL. EL has five potential N-glycosylation sites, four of which are glycosylated. The aim of this study was to determine how glycosylation affects the phospholipase activity of EL in physiologically relevant substrates. Site-directed mutants of EL were generated by replacing asparagine (N) 62, 118, 375, and 473 with alanine (A). These glycan-deficient mutants were used to investigate the kinetics of phospholipid hydrolysis in fully characterized preparations of spherical reconstituted high 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 hydrolyzed the phospholipids in (A-I)rHDL, (E2)rHDL, (E3)rHDL, and (E4)rHDL to similar extents. The phospholipase activities of EL N118A, EL N375A, and EL N473A were significantly diminished relative to that of wild-type EL, with the greatest reduction being apparent for (E3)rHDL. The phospholipase activity of EL N62A was increased up to 6-fold relative to that of wild-type EL, with the greatest enhancement of activity being observed for (E2)rHDL. These data show that individual N-linked glycans have unique and important effects on the phospholipase activity and substrate specificity of EL.
authors.affiliation: Lipid Research Group, Heart Research Institute, Camperdown, New South Wales 2050, Australia.
authors.forename: Danielle
authors.initials: D
authors.lastname: Skropeta
authors.forename: Chatri
authors.initials: C
authors.lastname: Settasatian
authors.forename: Monica R
authors.initials: MR
authors.lastname: McMahon
authors.forename: Kate
authors.initials: K
authors.lastname: Shearston
authors.forename: Daniela
authors.initials: D
authors.lastname: Caiazza
authors.forename: Kristine C
authors.initials: KC
authors.lastname: McGrath
authors.forename: Weijun
authors.initials: W
authors.lastname: Jin
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Rader
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Barter
authors.forename: Kerry-Anne
authors.initials: KA
authors.lastname: Rye
mesh_terms: D019943:Amino Acid Substitution; D016632:Apolipoprotein A-I / Q000378:metabolism*; D001057:Apolipoproteins E / Q000378:metabolism*; D002478:Cells, Cultured; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D008075:Lipoproteins, HDL / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D016632:Apolipoprotein A-I; D001057:Apolipoproteins E; D008075:Lipoproteins, HDL; D010743:Phospholipids; D011994:Recombinant Proteins; C025121:apolipoprotein E-rich HDL; C410382:LIPG protein, human; D008049:Lipase