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

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 lipo- protein 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 tuni- camycin demonstrated that EL is a glycosylated protein. Each putative glycosylation site was examined by site-directed mu- tagenesis of the asparagine (Asn). Mutation of Asn-60 mark- edly reduced secretion and slightly increased specific activ- ity. 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 . Muta- tion of Asn-373 did not influence secretion but significantly reduced specific activity, as a result of a decrease in appar- ent V max . Mutation of Asn-471 resulted in no reduction in se- cretion or specific activity. Mutation of Asn-449 resulted in no change in secretion, activity, or molecular mass, indicat- ing that the site is not utilized. The ability of mutants se- creted at normal levels to mediate bridging between LDL and cell surfaces was examined. The Asn-373 mutant dem- onstrated a 3-fold decrease in bridging compared with wild-type EL, whereas Asn-116 and Asn-471 were similar to wild-type EL. —Miller, G. C., C. J. Long, E. D. Bojilova, D. Marchadier, K. O. Badellino, N. Blanchard, I. V. Fuki, J. M. Glick, and D. J. Rader. Role of N-linked glycosylation in the secretion and activity of endothelial lipase. J. Lipid Res. 2004. 45: 2080-2087.