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Journal of Lipid Research, Vol. 48, 2047-2057, September 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

N-Glycosylation regulates endothelial lipase-mediated phospholipid hydrolysis in apoE- and apoA-I-containing high density lipoproteins

Danielle Skropeta^1,*, Chatri Settasatian^2,*, Monica R. McMahon^*, Kate Shearston^*, Daniela Caiazza^*, Kristine C. McGrath^*, Weijun Jin, Daniel J. Rader, Philip J. Barter^*, and Kerry-Anne Rye^3,*,,**

^* Lipid Research Group, Heart Research Institute, Camperdown, New South Wales 2050, Australia
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104
Department of Medicine, University of Sydney, New South Wales 2006, Australia
^** Department of Medicine, University of Melbourne, Victoria 3010, Australia

Published, JLR Papers in Press, June 3, 2007.

¹ Present address of D. Skropeta: Department of Chemistry, University of Wollongong, New South Wales 2522, Australia.

² Present address of C. Settasatian: Department of Pathology, Khon Kaen University, Khon Kaen, 40002, Thailand.

³ To whom correspondence should be addressed. e-mail: ryek{at}hri.org.au

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.

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

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