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HEM凝集素-酯酶(HE)糖蛋白由3个结构域组成：膜融合活性干区(F)、乙酰酯酶区(E)和受体结合区R。该蛋白含有8个N-连接的糖基化位点，F区有4个(26、395、552和603位)，E区有3个(61、131和144位)，R区有1个(189位)。在这里，我们通过消除每个糖基化位点来研究单个寡糖链在HE蛋白的抗原性、细胞内转运和生物活性中的作用。比较野生型和突变型蛋白的凝胶迁移率表明，虽然使用了7个糖基化位点，但有1个(131位)没有使用。对突变体与抗HE单抗的反应性分析表明，144位的糖基化对于构象依赖表位的形成是必不可少的。同样明显的是，除了E结构域(144位)外，F结构域(26和603位)的两个位点的糖基化也是HE分子从内质网运输所必需的，缺少这三个位点之一的突变体Hes无法进行三聚体组装。去掉144或189位的寡糖链会导致酯酶活性下降。而在26位和603位缺失寡糖链的两个突变体，不仅在细胞表面表达存在缺陷，而且在三聚化方面也存在缺陷，它们都具有完全的酶活性，这表明细胞内存在的HE单体具有乙酰酯酶活性。表达每个突变型HES的细胞的融合活性与蛋白被运输到细胞表面的能力相当，这表明没有特定的寡糖参与促进膜融合。

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.