The influence of N-linked glycosylation on the function of platelet glycoprotein VI
The influence of N-linked glycosylation on the function of platelet glycoprotein VI
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DOI：
10.1182/blood-2005-04-1454
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发表时间：
2005-10-15
期刊：
影响因子：
20.3
通讯作者：
Furihata, K
中科院分区：
文献类型：
--
作者：
Kunicki, TJ;Cheli, Y;Furihata, K
使用重组人糖蛋白 VI (GPVI)，我们评估了共有位点天冬酰胺 (92)-甘氨酸-丝氨酸 (94) (N(92)GS(94)) 处的 N 连接糖基化对该血小板特异性受体与其配体、人 I 型胶原、胶原相关肽 (CRIP) 和蛇毒 C 型凝集素惊厥素 (CVX) 结合的影响。在瞬时转染 GPVI 的 COS-7 细胞中，用肽-N-糖苷酶 F（PNGase F；对复杂的 N 连接聚糖具有特异性）或衣霉素去糖基化可降低 GPVI 的分子量，并减少转染的 COS-7 细胞与 CRP 和 CVX 的结合。在稳定转染的 Dami 细胞中，N92A 或 S94A（而非 L95H）的取代导致对 CVX 的粘附力降低 30% 至 40%，但对 CRP 的粘附力降低 90% 或更多，对 I 型胶原蛋白的粘附力降低 65% 至 70%。使用 PNGase F 而不是内切糖苷酶 H (Endo H)（特异于高甘露糖 N 连接聚糖）处理，会产生同等的分子量下降。根据鼠抗人 GPVI 单克隆抗体 204-11 与转染的 Dami 细胞的直接结合，N92A 和 S94A 都不影响 GPVI 的表达。这些发现表明，人 GPVI 中 N92 处的 N 联糖基化不是表面表达所必需的，但有助于最大程度地粘附 I 型胶原、CRP 以及较小程度的 CVX。
Using recombinant human glycoprotein VI (GPVI), we evaluated the effect of N-linked glycosylation at the consensus site Asparagine(92)-Glycine-Serine(94) (N(92)GS(94)) on binding of this platelet-specific receptor to its ligands, human type I collagen, collagen-related peptide (CRIP), 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 antihuman 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.