Site-directed removal of N-glycosylation sites in the bovine cation-dependent mannose 6-phosphate receptor: effects on ligand binding, intracellular targetting and association with binding immunoglobulin protein.
Site-directed removal of N-glycosylation sites in the bovine cation-dependent mannose 6-phosphate receptor: effects on ligand binding, intracellular targetting and association with binding immunoglobulin protein.
复制标题
牛阳离子依赖性甘露糖 6-磷酸受体中 N-糖基化位点的定点去除：对配体结合、细胞内靶向以及与结合免疫球蛋白的关联的影响。
DOI：
10.1042/bj2950841
复制
发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Dahms,NM
中科院分区：
文献类型：
--
作者：
Zhang,Y;Dahms,NM
牛阳离子依赖的甘露糖6-磷酸受体(CD-MPR)含有5个潜在的N-连接糖基化位点，其中4个已被利用。为了评价这些寡糖的功能，我们利用定点突变技术产生了缺乏各种潜在糖基化位点的糖基化缺陷CD-MPR突变体。突变体被构建在受体的全长和可溶截短形式(STOP155构建)中，并在转染的COS-1细胞中检测它们的性质。结果表明，与未糖化的受体相比，CD-MPR上的单链寡糖链，特别是第87位，显著增强了其与甘露糖6-磷酸(Man-6-P)的结合能力。此外，在第87位和在第31或81位有一个单一的寡糖链的存在促进了STOP155 CD-MPR的分泌。脉冲标记转基因的COS-1细胞，然后用结合免疫球蛋白(Bip)特异性抗体和CD-MPR特异性抗体进行免疫沉淀，表明Bip与非糖基化形式的受体结合，而不与野生型CD-MPR结合。此外，CD-MPR的各种糖基化缺陷形式与BiP的关联与它们结合Man-6-P的能力呈负相关。根据这些结果，我们得出结论，牛CD-MPR的N-糖基化有助于将新生的多肽链折叠成有利于细胞内运输和配体结合的构象。
The bovine cation-dependent mannose 6-phosphate receptor (CD-MPR) contains five potential N-linked glycosylation sites, four of which are utilized. To evaluate the function of these oligosaccharides, site-directed mutagenesis was used to generate glycosylation-deficient CD-MPR mutants lacking various potential glycosylation sites. The mutants were constructed in both a full-length and a soluble truncated (STOP155 construct) form of the receptor and their properties were examined in transfected COS-1 cells. The results showed that the presence of a single oligosaccharide chain, particularly at position 87, on the CD-MPR significantly enhanced its mannose 6-phosphate (Man-6-P)-binding ability when compared with non-glycosylated receptors. In addition, the presence of a single oligosaccharide chain at position 87, and to a lesser degree at position 31 or 81, promoted the secretion of the STOP155 CD-MPR. Pulse-labelling of transfected COS-1 cells followed by immunoprecipitation with binding immunoglobulin protein (BiP)-specific and CD-MPR-specific antibodies indicated that BiP associated with the non-glycosylated forms of the receptor but not with the wild-type CD-MPR. Furthermore, the association of the various glycosylation-deficient forms of the CD-MPR with BiP correlated inversely with their ability to bind Man-6-P. From these results we conclude that N-glycosylation of the bovine CD-MPR facilities the folding of the nascent polypeptide chain into a conformation that is conductive for intracellular transport and ligand binding.