The influence of N-linked glycosylation on the function of platelet glycoprotein VI.
Using recombinant human glycoprotein VI (GPVI), we evaluated the effect of N-linked glycosylation at the consensus site Asparagine92-Glycine-Serine94 (N92GS94) on binding of this platelet-specific receptor to its ligands, human type I collagen, collagen-related peptide (CRP), and the snake venom C-type lectin convulxin (CVX). In COS-7 cells transiently transfected with GPVI, deglycosylation with peptide-N-glycosidase F (PNGase F; specific for complex N-linked glycans) or tunicamycin decreases the molecular weight of GPVI and reduces transfected COS-7 cell binding to both CRP and CVX. In stably transfected Dami cells, the substitutions N92A or S94A, but not L95H, resulted in a 30% to 40% decrease in adhesion to CVX, but a 90% or greater decrease in adhesion to CRP and a 65% to 70% decrease in adhesion to type I collagen. Treatment with PNGase F, but not Endoglycosidase H (Endo H) (specific for high-mannose N-linked glycans), produced an equivalent decrease in molecular weight. Neither N92A nor S94A affected the expression of GPVI, based on the direct binding of murine anti-human GPVI monoclonal antibody 204-11 to transfected Dami cells. These findings indicate that N-linked glycosylation at N92 in human GPVI is not required for surface expression, but contributes to maximal adhesion to type I collagen, CRP and, to a lesser extent, CVX.
Kunicki
Thomas J
TJ
Division of Experimental Hemostasis and Thrombosis, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, MEM-150, La Jolla, CA 92037, USA. tomk@scripps.edu
Cheli
Yann
Y
Moroi
Masaaki
M
Furihata
Kenichi
K
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D002522:Chlorocebus aethiops; D010450:Endopeptidases / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D040681:Structural Homology, Protein; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D010980:Platelet Membrane Glycoproteins; C098341:platelet membrane glycoprotein VI; D014415:Tunicamycin; D010450:Endopeptidases