Påhlsson P, Strindhall J, Srinivas U, Lundblad A
Department of Clinical Chemistry, Faculty of Health Sciences, University Hospital, Linköping, Sweden.
Eur J Immunol. 1995 Sep;25(9):2452-9. doi: 10.1002/eji.1830250907.
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.
E选择素是一种细胞因子诱导的膜糖蛋白，能够介导白细胞与内皮细胞的黏附。它高度糖基化，含有11个N-连接糖基化位点。通过内切糖苷酶处理分析了E选择素的N-糖基化。在十二烷基硫酸钠存在下，通过聚丙烯酰胺凝胶电泳对来自人脐静脉内皮细胞（HUVEC）的免疫沉淀E选择素进行分析，结果表明E选择素对内切糖苷酶H完全耐受，但对肽N-聚糖酶F消化敏感。这表明所有N-连接寡糖链均为复合型。使用在可溶性糖基化抑制剂衣霉素或蓖麻毒蛋白存在下培养的白细胞介素-1刺激的HUVEC，研究了N-连接糖基化在E选择素表面表达和分泌中的作用。通过间接流式细胞术分析细胞表面表达。衣霉素阻断了N-糖基化，并导致E选择素的表面表达显著降低，而蓖麻毒蛋白仅略微降低E选择素的表达。还发现E选择素的去糖基化形式在体外完全能够介导HT-29细胞的黏附。总之，这些研究表明E选择素被复合型N-连接寡糖高度糖基化，并且N-糖基化对E选择素在人内皮细胞上的表达很重要。
