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Do oligosaccharide chains of the hemagglutinin-esterase (HE) protein of influenza C virus affect the formation of the intramolecular disulfide bonds?

丙型流感病毒血凝素酯酶（HE）蛋白的寡糖链是否影响分子内二硫键的形成？

基本信息

批准号：
11470074
负责人：
SUGAWARA Kanetsu
金额：
$ 9.28万
依托单位：
YAMAGATA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

项目摘要

The hem agglutinin-esterase (HE) glycoprotein of influenza C virus consists of three domains: a stem domain active in membrane fusion (F), an acetylesterase domain (E), and a receptor-binding domain R. The protein contains eight N-linked glycosylation sites, four (positions 26, 395, 552, and 603) in the F domain, three (positions 61, 131, and 144) in the E domain, and one (position 189) in the R domain. Here, we investigated the role of the individual oligosaccharide chains in antigenic properties , intracellular transport , and biological activities of the HE protein by eliminating each of the glycosylation sites. Comparison of electrophoretic mobility between the wild type and mutant proteins showed that while seven of the glycosylation sites are used, one (position 131) is not. Analysis of reactivity of the mutants with anti-HE monoclonal antibodies demonstrated that glycosylation at position 144 is essential for the formation of conformation-dependent epitopes. It was also evident that glycosylation at the two sites in the F domain (positions 26 and 603), in addition to that in the E domain (position 144) , is required for the HE molecule to be transported from the endoplasmic reticulum and that mutant Hes lacking one of these three sites failed to undergo the trimer assembly. Removal of an oligosaccharide chain at position 144 or 189 resulted in a decrease in the esterase activity. By contrast, two mutants lacking an oligosaccharide chain at position 26 or 603, which were defective not only in cell surface expression but in trimerization, possessed full enzyme activity, suggesting that the HE monomers present within the cell have acetylesterase activity. Fusion activity of cells expressing each of mutant HEs was found to be comparable with the ability of the protein to be transported to the cell surface, suggesting that there is no specific oligosaccharide involving in promoting membrane fusion.

丙型流感病毒的血凝素-酯酶（HE）糖蛋白由三个结构域组成：在膜融合中有活性的干结构域（F）、乙酰酯酶结构域（E）和受体结合结构域R。该蛋白含有8个N-连接糖基化位点，4个（位置26、395、552和603）在F结构域，3个（位置61、131和144）在E结构域，1个（位置189）在R结构域。在这里，我们通过消除每个糖基化位点来研究单个寡聚糖链在HE蛋白的抗原特性、细胞内转运和生物活性中的作用。野生型和突变体蛋白质之间的电泳迁移率的比较表明，虽然7个糖基化位点被使用，一个（位置131）不是。抗HE单克隆抗体的突变体的反应性分析表明，在位置144的糖基化是必不可少的构象依赖性表位的形成。同样明显的是，除了E结构域（位置144）中的糖基化之外，F结构域中的两个位点（位置26和603）处的糖基化是HE分子从内质网转运所需的，并且缺少这三个位点之一的突变体Hes不能进行三聚体组装。在位置144或189处去除寡糖链导致酯酶活性降低。相比之下，两个突变体缺乏寡糖链的位置26或603，这是有缺陷的，不仅在细胞表面表达，但在三聚体，具有完整的酶活性，这表明，HE单体存在于细胞内具有乙酰酯酶活性。发现表达每种突变体HE的细胞的融合活性与蛋白质被转运到细胞表面的能力相当，表明不存在参与促进膜融合的特异性寡糖。