Glycosylation of endothelial lipase at asparagine-116 reduces activity and the hydrolysis of native lipoproteins in vitro and in vivo.
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.
Brown
Robert J
RJ
Department of Medicine and Institute for Translational Medicine and Therapeutics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Miller
Gwen C
GC
Griffon
Nathalie
N
Long
Christopher J
CJ
Rader
Daniel J
DJ
D000818:Animals; D001216:Asparagine / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D008066:Lipolysis; D008074:Lipoproteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D011973:Receptors, LDL / Q000378:metabolism
D008074:Lipoproteins; D011973:Receptors, LDL; D001216:Asparagine; C410382:LIPG protein, human; D008049:Lipase