Role of N‐linked glycosylation in expression of E‐selectin on human endothelial cells

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Abstract
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.