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Journal of Lipid Research, Vol. 45, 2080-2087, November 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology

Role of N-linked glycosylation in the secretion and activity of endothelial lipase

Gwen C. Miller^1,*, Christopher J. Long^1,*, Ekaterina D. Bojilova^*, Dawn Marchadier^*, Karen O. Badellino^*, Nadine Blanchard^*, Ilia V. Fuki^*, Jane M. Glick and Daniel J. Rader^2,*

^* Department of Medicine and Center for Experimental Therapeutics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104
Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

² To whom correspondence should be addressed. e-mail: rader{at}mail.med.upenn.edu

Human endothelial lipase (EL), a member of the triglyceride lipase gene family, has five potential N-linked glycosylation sites, two of which are conserved in both lipoprotein lipase and hepatic lipase. Reduction in molecular mass of EL after treatment with glycosidases and after treatment of EL-expressing cells with the glycosylation inhibitor tunicamycin demonstrated that EL is a glycosylated protein. Each putative glycosylation site was examined by site-directed mutagenesis of the asparagine (Asn). Mutation of Asn-60 markedly reduced secretion and slightly increased specific activity. Mutation of Asn-116 did not influence secretion but increased specific activity. In both cases, this resulted from decreased apparent K_m and increased apparent V_max. Mutation of Asn-373 did not influence secretion but significantly reduced specific activity, as a result of a decrease in apparent V_max. Mutation of Asn-471 resulted in no reduction in secretion or specific activity. Mutation of Asn-449 resulted in no change in secretion, activity, or molecular mass, indicating that the site is not utilized. The ability of mutants secreted at normal levels to mediate bridging between LDL and cell surfaces was examined.

The Asn-373 mutant demonstrated a 3-fold decrease in bridging compared with wild-type EL, whereas Asn-116 and Asn-471 were similar to wild-type EL.

Abbreviations: A/A, antibiotic/antimycotic; EL, endothelial lipase; Endo F, endoglycosidase F; HSPG, heparan sulfate proteoglycan; PBST, PBS plus 0.02% Tween 20

Supplementary key words lipase kinetics • maximum velocity • Michaelis-Menten constant • glycosidase • bridging • secretion • site-directed mutagenesis

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