The influence of N-linked glycosylation on the function of platelet glycoprotein VI

• Published in: Blood 2005-10, Vol.106 (8), p.2744-2749
• Main Authors: Kunicki, Thomas J., Cheli, Yann, Moroi, Masaaki, Furihata, Kenichi
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Blood coagulation. Blood cells; Blood Platelets - drug effects; Blood Platelets - metabolism; Cell Adhesion - drug effects; Cell Line; Chlorocebus aethiops; Endopeptidases - metabolism; Fundamental and applied biological sciences. Psychology; Glycosylation - drug effects; Hemostasis, Thrombosis, and Vascular Biology; Humans; Models, Molecular; Molecular and cellular biology; Mutation - genetics; Platelet; Platelet Membrane Glycoproteins - chemistry; Platelet Membrane Glycoproteins - genetics; Platelet Membrane Glycoproteins - metabolism; Protein Structure, Tertiary; Structural Homology, Protein; Transfection; Tunicamycin - pharmacology
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2005-04-1454

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.