Role of N-linked glycosylation in expression of E-selectin on human endothelial cells.
E-selectin is a cytokine-inducible membrane glycoprotein capable of mediating adhesion of leukocytes to endothelial cells. It is highly glycosylated, containing 11 sites for N-linked glycosylation. N-Glycosylation of E-selectin was analyzed by endoglycosidase treatment. Analysis of immunoprecipitated E-selectin from human umbilical vein endothelial cells (HUVEC) by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate showed that E-selectin was completely resistant to endoglycosidase H, but sensitive to peptide N-glycanase F digestion. This suggested that all N-linked oligosaccharide chains were of the complex type. The role of N-linked glycosylation in surface expression and secretion of E-selectin was studied using interleukin-1-stimulated HUVEC, cultured in the presence of the soluble glycosylation inhibitors tunicamycin or castanospermine. Cell surface expression was analyzed by indirect flow cytometry. N-Glycosylation was blocked by tunicamycin, and resulted in a significantly reduced surface expression of E-selectin, whereas castanospermine only marginally reduced E-selectin expression. The deglycosylated forms of E-selectin were also found to be fully capable of mediating adhesion of HT-29 cells in vitro. In conclusion, these studies show that E-selectin is heavily glycosylated with complex type N-linked oligosaccharides and that N-glycosylation is important for expression of E-selectin on human endothelial cells.
Påhlsson
P
P
Department of Clinical Chemistry, Faculty of Health Sciences, University Hospital, Linköping, Sweden.
Strindhall
J
J
Srinivas
U
U
Lundblad
A
A
D002448:Cell Adhesion; D019040:E-Selectin / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D006026:Glycoside Hydrolases; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D007212:Indolizines / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014415:Tunicamycin / Q000494:pharmacology; D054836:Indolizidines
D019040:E-Selectin; D004791:Enzyme Inhibitors; D006026:Glycoside Hydrolases; D007212:Indolizines; D007375:Interleukin-1; D014415:Tunicamycin; C037806:castanospermine; D054836:Indolizidines