Kuroda K, Geyer H, Geyer R, Doerfler W, Klenk H D
Institut für Virologie, Philipps-Universität Marburg, Federal Republic of Germany.
Virology. 1990 Feb;174(2):418-29. doi: 10.1016/0042-6822(90)90095-9.
The hemagglutinin of fowl plague virus has been expressed in Spodoptera frugiperda (SF) cell cultures using a baculovirus vector. To elucidate the structure of the carbohydrate side chains, radioactively labeled oligosaccharides were liberated by treatment with endoglucosaminidase H and glycopeptidase F. Sequential degradation with exoglycosidases and chromatographic analyses revealed the presence of oligomannosidic side chains, predominantly of the structures Man5-9GlcNAc2, and the truncated oligosaccharide cores Man3GlcNAc2 and Man3[Fuc]GlcNAc2. Polyacrylamide gel electrophoresis of endoglycosidase-treated hemagglutinin showed that most side chains of the HA1 subunit are truncated, whereas the HA2 subunit has one oligomannosidic and one truncated oligosaccharide. Comparison of these results with the glycosylation pattern of hemagglutinin obtained from vertebrate cells allowed a tentative allocation of the oligosaccharides to individual glycosylation sites. The results indicate that SF cells have the capacity to trim N-glycans to trimannosyl cores and to further process these by the addition of fucose. Thus, the complex oligosaccharides found on hemagglutinin from vertebrate hosts are replaced on hemagglutinin derived from insect cells by small truncated side chains.
利用杆状病毒载体在草地贪夜蛾（SF）细胞培养物中表达了禽瘟病毒的血凝素。为了阐明碳水化合物侧链的结构，用内切葡糖胺聚糖酶H和糖肽酶F处理释放出放射性标记的寡糖。用外切糖苷酶进行顺序降解和色谱分析表明存在寡甘露糖侧链，主要结构为Man5-9GlcNAc2，以及截短的寡糖核心Man3GlcNAc2和Man3[Fuc]GlcNAc2。对经内切糖苷酶处理的血凝素进行聚丙烯酰胺凝胶电泳显示，HA1亚基的大多数侧链被截短，而HA2亚基有一个寡甘露糖侧链和一个截短的寡糖。将这些结果与从脊椎动物细胞获得的血凝素的糖基化模式进行比较，可初步将寡糖分配到各个糖基化位点。结果表明，SF细胞有能力将N-聚糖修剪为甘露糖核心，并通过添加岩藻糖进一步加工这些核心。因此，在脊椎动物宿主的血凝素上发现的复杂寡糖在昆虫细胞来源的血凝素上被小的截短侧链所取代。
