Different roles of individual N-linked oligosaccharide chains in folding, assembly, and transport of the simian virus 5 hemagglutinin-neuraminidase
Different roles of individual N-linked oligosaccharide chains in folding, assembly, and transport of the simian virus 5 hemagglutinin-neuraminidase
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DOI：
10.1128/mcb.10.5.1989-2001.1990
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发表时间：
1990-05
影响因子：
5.3
通讯作者：
D. T. Ng;Scott W. Hiebert;Robert A. Lamb
中科院分区：
文献类型：
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作者：
D. T. Ng;Scott W. Hiebert;Robert A. Lamb
利用类人猿病毒5型血凝素-神经氨酸酶（HN）蛋白研究了n-链糖基化在蛋白质成熟和转运中的作用。HN蛋白是一种模型II类完整膜糖蛋白。通过对cDNA克隆进行寡核苷酸定向诱变，去除每个潜在的n -链糖基化位点，确定了HN上的n -链糖基化位点。突变体HN蛋白在真核细胞中的表达表明，HN糖蛋白中有四个位点用于添加n -连接的低聚糖链。这些功能性糖基化位点被系统地从HN中以各种组合消除，形成一个突变体组，可以分析单个碳水化合物链和碳水化合物链组的作用。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成熟影响的严重程度取决于缺失的碳水化合物位点的数量及其在HN分子中的位置。对突变的HN蛋白的构象特异性单克隆抗体的反应模式分析表明，一个特定的碳水化合物链在促进HN的正确折叠中起主要作用。另一种碳水化合物链对HN的初始折叠并不是必需的，但它在阻止HN低聚物聚集方面发挥了作用。由于糖基化模式的改变，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形成二硫化物连接的聚集体，并与常驻内质网蛋白GRP78-BiP稳定相关，而野生型HN在其折叠过程中与GRP78-BiP形成特异性和短暂的复合物。
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.