Basak S, Compans R W
Virology. 1983 Jul 15;128(1):77-91. doi: 10.1016/0042-6822(83)90320-3.
The biological and antigenic roles of glycosylation were investigated in the influenza hemagglutinin (HA) glycoprotein using the glycosylation inhibitor tunicamycin (TM). Under conditions where only the nonglycosylated form of HA was detected by immunoprecipitation and gel electrophoresis, the migration of glycoproteins to the cell surface was observed by immunofluorescence using either monospecific or monoclonal antibody to the HA polypeptide. Analysis of the surface fluorescence in TM-treated infected cells by a fluorescence-activated cell sorter (FACS) showed that all cells exhibited fluorescence in the complete absence of glycosylation. The relative amount of HA antigen on cell surfaces was found to be reduced by only 30-40% in TM-treated cells, and this reflected a similar reduction in intracellular synthesis. Electron microscopic studies using ferritin labeling also demonstrated that the nonglycosylated HA glycoprotein was present in significant amounts on surfaces of infected cells. Virions with nonglycosylated glycoproteins were purified, and were found to have an approximate 30-fold decrease in both hemagglutinin and neuraminidase specific activities. The possible role of oligosaccharides in antigenic variation among various H1N1 strains was investigated. Immunoprecipitation reactions involving five different monoclonal antibodies and five antigenic variants of A/USSR/90/77 revealed no major antigenic differences between the glycosylated and nonglycosylated forms of HA.
使用糖基化抑制剂衣霉素（TM）研究了流感血凝素（HA）糖蛋白中糖基化的生物学和抗原作用。在通过免疫沉淀和凝胶电泳仅检测到非糖基化形式的HA的条件下，使用针对HA多肽的单特异性或单克隆抗体通过免疫荧光观察糖蛋白向细胞表面的迁移。用荧光激活细胞分选仪（FACS）分析TM处理的感染细胞中的表面荧光，结果表明在完全没有糖基化的情况下所有细胞均表现出荧光。发现在TM处理的细胞中，细胞表面HA抗原的相对量仅减少了30%-40%，这反映了细胞内合成的类似减少。使用铁蛋白标记的电子显微镜研究也表明，非糖基化的HA糖蛋白大量存在于感染细胞的表面。纯化了具有非糖基化糖蛋白的病毒粒子，发现其血凝素和神经氨酸酶的比活性均下降了约30倍。研究了寡糖在各种H1N1毒株抗原变异中的可能作用。涉及五种不同单克隆抗体和A/USSR/90/77的五种抗原变体的免疫沉淀反应显示，HA的糖基化形式和非糖基化形式之间没有主要的抗原差异。
