J. Lipid Res.
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Originally published In Press as doi:10.1194/jlr.M700248-JLR200 on June 3, 2007

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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 Skropeta1,*, Chatri Settasatian2,*, Monica R. McMahon*, Kate Shearston*, Daniela Caiazza*, Kristine C. McGrath*, Weijun Jin{dagger}, Daniel J. Rader{dagger}, Philip J. Barter*,§ and Kerry-Anne Rye3,*,§,**

* Lipid Research Group, Heart Research Institute, Camperdown, New South Wales 2050, Australia
{dagger} 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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N. Settasatian, P. J. Barter, and K.-A. Rye
Remodeling of apolipoprotein E-containing spherical reconstituted high density lipoproteins by phospholipid transfer protein
J. Lipid Res., January 1, 2008; 49(1): 115 - 126.
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