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Journal of Lipid Research, Vol. 48, 1132-1139, May 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Glycosylation of endothelial lipase at asparagine-116 reduces activity and the hydrolysis of native lipoproteins in vitro and in vivo

Robert J. Brown, Gwen C. Miller, Nathalie Griffon, Christopher J. Long and Daniel J. Rader¹

Department of Medicine and Institute for Translational Medicine and Therapeutics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

Published, JLR Papers in Press, February 24, 2007.

¹ To whom correspondence should be addressed. e-mail: rader{at}mail.med.upenn.edu

We previously identified that four of five putative N-linked glycosylation sites of human endothelial lipase (EL) are utilized and suggested that the substitution of asparagine-116 (Asn-116) with alanine (Ala) (N116A) increased the hydrolytic activity of EL. The current study demonstrates that mutagenesis of either Asn-116 to threonine (Thr) or Thr-118 to Ala also disrupted the glycosylation of EL and enhanced catalytic activity toward synthetic substrates by 3-fold versus wild-type EL. Furthermore, we assessed the hydrolysis of native lipoprotein lipids by EL-N116A. EL-N116A exhibited a 5-fold increase in LDL hydrolysis and a 1.8-fold increase in HDL₂ hydrolysis. Consistent with these observations, adenovirus-mediated expression of EL-N116A in mice significantly reduced the levels of both LDL and HDL cholesterol beyond the reductions observed by the expression of wild-type EL alone. Finally, we introduced Asn-116 of EL into the analogous positions within LPL and HL, resulting in N-linked glycosylation at this site. Glycosylation at this site suppressed the LPL hydrolysis of synthetic substrates, LDL, HDL₂, and HDL₃ but had little effect on HL activity. These data suggest that N-linked glycosylation at Asn-116 reduces the ability of EL to hydrolyze lipids in LDL and HDL₂.

Supplementary key words lipase • lipoprotein hydrolysis • adenovirus • glycosylation • site-directed mutagenesis • heparin • heparan sulfate proteoglycan

Abbreviations: A/A, antibiotic/antimycotic; DPPC, dipalmitoylphosphatidyl choline; EL, endothelial lipase; FPLC, fast-performance liquid chromatography; HDL-C, high density lipoprotein cholesterol; LDLR, low density lipoprotein receptor; PL, phospholipid; TC, total cholesterol; TG, triglyceride

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