Karlsson G B, Butters T D, Dwek R A, Platt F M
Department of Biochemistry, University of Oxford, United Kingdom.
J Biol Chem. 1993 Jan 5;268(1):570-6.
The imino sugar N-butyldeoxynojirimycin (NB-DNJ) exhibits anti-HIV activity in vitro and inhibits the purified glycoprocessing enzyme alpha 1,2-glucosidase I. It has been speculated that the anti-viral activity of this compound may result from inhibition of HIV envelope glycoprotein processing. However, structural evidence that glucosidase inhibition takes place in intact cells at the anti-viral concentration (0.5 mM) is lacking. In this study, N-linked glycosylation of recombinant gp120 expressed in Chinese hamster ovary cells cultured in the presence or absence of NB-DNJ has been characterized. Immunoprecipitation, in conjunction with endoglycosidase H (endo H) digestion and SDS-polyacrylamide gel electrophoresis analysis, revealed that the glycosylation of gp120 was profoundly altered in the presence of NB-DNJ. The majority of the gp120 oligosaccharides from untreated cells were resistant to endo H. However, nearly complete endo H sensitivity was observed following treatment with 0.5 mM NB-DNJ indicating that gp120 expressed in treated cells carries immature, high mannose type oligosaccharides. In addition, using metabolic labeling with [3H]mannose, gel filtration chromatography, and digestion with highly purified glucosidases I and II, we provide the first definitive evidence that glucosidase I inhibition occurs at the anti-viral concentration of NB-DNJ. These data indicate that glucosidase inhibition is a candidate mechanism for the anti-viral activity of this compound.
亚氨基糖N-丁基脱氧野尻霉素（NB-DNJ）在体外具有抗HIV活性，并能抑制纯化的糖加工酶α1,2-葡糖苷酶I。据推测，该化合物的抗病毒活性可能源于对HIV包膜糖蛋白加工的抑制。然而，缺乏在抗病毒浓度（0.5 mM）下完整细胞中发生葡糖苷酶抑制的结构证据。在本研究中，对在中国仓鼠卵巢细胞中培养时，在有或无NB-DNJ存在的情况下表达的重组gp120的N-连接糖基化进行了表征。免疫沉淀结合内切糖苷酶H（endo H）消化和SDS-聚丙烯酰胺凝胶电泳分析表明，在NB-DNJ存在下，gp120的糖基化发生了深刻改变。未经处理细胞中的大多数gp120寡糖对内切糖苷酶H具有抗性。然而，在用0.5 mM NB-DNJ处理后，观察到几乎完全的内切糖苷酶H敏感性，这表明在处理过的细胞中表达的gp120带有未成熟的高甘露糖型寡糖。此外，使用[3H]甘露糖进行代谢标记、凝胶过滤色谱以及用高度纯化的葡糖苷酶I和II进行消化，我们提供了首个确凿证据，表明在NB-DNJ的抗病毒浓度下发生了葡糖苷酶I抑制。这些数据表明，葡糖苷酶抑制是该化合物抗病毒活性的一种可能机制。
