Ng D T, Hiebert S W, Lamb R A
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500.
Mol Cell Biol. 1990 May;10(5):1989-2001. doi: 10.1128/mcb.10.5.1989-2001.1990.
The role of N-linked glycosylation in protein maturation and transport has been studied by using the simian virus 5 hemagglutinin-neuraminidase (HN) protein, a model class II integral membrane glycoprotein. The sites of N-linked glycosylation on HN were identified by eliminating each of the potential sites for N-linked glycosylation by oligonucleotide-directed mutagenesis on a cDNA clone. Expression of the mutant HN proteins in eucaryotic cells indicated that four sites are used in the HN glycoprotein for the addition of N-linked oligosaccharide chains. These functional glycosylation sites were systematically eliminated in various combinations from HN to form a panel of mutants in which the roles of individual carbohydrate chains and groups of carbohydrate chains could be analyzed. Alterations in the normal glycosylation pattern resulted in the impairment of HN protein folding and assembly which, in turn, affected the intracellular transport of HN. The severity of the consequences on HN maturation depended on both the number of deleted carbohydrate sites and their position in the HN molecule. Analysis of the reactivity pattern of HN conformation-specific monoclonal antibodies with the mutant HN proteins indicated that one specific carbohydrate chain plays a major role in promoting the correct folding of HN. Another carbohydrate chain, which is not essential for the initial folding of HN was found to play a role in preventing the aggregation of HN oligomers. The HN molecules which were misfolded, owing to their altered glycosylation pattern, were retained in the endoplasmic reticulum. Double-label immunofluorescence experiments indicate that misfolded HN and folded HN are segregated in the same cell. Misfolded HN forms disulfide-linked aggregates and is stably associated with the resident endoplasmic reticulum protein, GRP78-BiP, whereas wild-type HN forms a specific and transient complex with GRP78-BiP during its folding process.
通过使用猴病毒5血凝素-神经氨酸酶（HN）蛋白（一种典型的II类整合膜糖蛋白），研究了N-连接糖基化在蛋白质成熟和运输中的作用。通过对cDNA克隆进行寡核苷酸定向诱变，消除每个潜在的N-连接糖基化位点，从而确定了HN上N-连接糖基化的位点。在真核细胞中表达突变型HN蛋白表明，HN糖蛋白中有四个位点用于添加N-连接寡糖链。从HN中系统地以各种组合消除这些功能性糖基化位点，以形成一组突变体，从而可以分析单个糖链和糖链组的作用。正常糖基化模式的改变导致HN蛋白折叠和组装受损，进而影响HN的细胞内运输。对HN成熟产生的后果的严重程度取决于缺失的糖基化位点的数量及其在HN分子中的位置。用突变型HN蛋白分析HN构象特异性单克隆抗体的反应模式表明，一条特定的糖链在促进HN的正确折叠中起主要作用。发现另一条对HN的初始折叠不是必需的糖链在防止HN寡聚体聚集方面发挥作用。由于糖基化模式改变而错误折叠的HN分子保留在内质网中。双标记免疫荧光实验表明，错误折叠的HN和折叠的HN在同一细胞中分离。错误折叠的HN形成二硫键连接的聚集体，并与内质网驻留蛋白GRP78-BiP稳定结合，而野生型HN在其折叠过程中与GRP78-BiP形成特定的瞬时复合物。
