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

C型流感病毒的HE糖蛋白由三个结构域组成：一个在膜融合中活跃的茎结构域(F)、一个乙酰酯酶结构域(E)和一个受体结合结构域R。该蛋白含有8个n连接的糖基化位点，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，研究了单个寡糖链在HE蛋白的抗原性、细胞内运输和生物活性中的作用。野生型和突变型蛋白之间的电泳迁移率比较表明，虽然使用了7个糖基化位点，但有一个（位置131）没有使用。突变体与抗he单克隆抗体的反应性分析表明，144位的糖基化对于构象依赖性表位的形成至关重要。同样明显的是，除了E结构域（位置144）外，F结构域的两个位点（位置26和603）的糖基化是HE分子从内质网运输所必需的，缺乏这三个位点之一的突变HE无法进行三聚体组装。去除144或189位的低聚糖链导致酯酶活性降低。相比之下，在26号或603号位置缺乏寡糖链的两个突变体，不仅在细胞表面表达上有缺陷，而且在三聚体化上也有缺陷，但具有充分的酶活性，这表明细胞内存在的HE单体具有乙酰酯酶活性。研究发现，表达每种突变HEs的细胞的融合活性与蛋白质转运到细胞表面的能力相当，这表明没有特异性的低聚糖参与促进膜融合。

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.