Glycosylation of Asn-76 in mouse GPIHBP1 is critical for its appearance on the cell surface and the binding of chylomicrons and lipoprotein lipase.
GPIHBP1 is a glycosylphosphatidylinositol-anchored protein in the lymphocyte antigen 6 (Ly-6) family that recently was identified as a platform for the lipolytic processing of triglyceride-rich lipoproteins. GPIHBP1 binds both LPL and chylomicrons and is expressed on the luminal face of microvascular endothelial cells. Here, we show that mouse GPIHBP1 is N-glycosylated at Asn-76 within the Ly-6 domain. Human GPIHBP1 is also glycosylated. The N-linked glycan could be released from mouse GPIHBP1 with N-glycosidase F, endoglycosidase H, or endoglycosidase F1. The glycan was marginally sensitive to endoglycosidase F2 digestion but resistant to endoglycosidase F3 digestion, suggesting that the glycan on GPIHBP1 is of the oligomannose type. Mutating the N-glycosylation site in mouse GPIHBP1 results in an accumulation of GPIHBP1 in the endoplasmic reticulum and a markedly reduced amount of the protein on the cell surface. Consistent with this finding, cells expressing a nonglycosylated GPIHBP1 lack the ability to bind LPL or chylomicrons. Eliminating the N-glycosylation site in a truncated soluble version of GPIHBP1 causes a modest reduction in the secretion of the protein. These studies demonstrate that N-glycosylation of GPIHBP1 is important for the trafficking of GPIHBP1 to the cell surface.
Beigneux
Anne P
AP
Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA. abeigneux@mednet.ucla.edu
Gin
Peter
P
Davies
Brandon S J
BS
Weinstein
Michael M
MM
Bensadoun
André
A
Ryan
Robert O
RO
Fong
Loren G
LG
Young
Stephen G
SG
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002914:Chylomicrons / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers; D006031:Glycosylation; D006801:Humans; D008071:Lipoprotein Lipase / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D021381:Protein Transport; D018110:Receptors, Lipoprotein / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D002914:Chylomicrons; D017931:DNA Primers; C472779:GPI-HBP1 protein, mouse; D018110:Receptors, Lipoprotein; D001216:Asparagine; D008071:Lipoprotein Lipase