Effects of the imino sugar N-butyldeoxynojirimycin on the N-glycosylation of recombinant gp120.
Effects of the imino sugar N-butyldeoxynojirimycin on the N-glycosylation of recombinant gp120.
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DOI：
10.1016/s0021-9258(18)54189-2
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发表时间：
1993-01
期刊：
影响因子：
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通讯作者：
G. Karlsson;T. Butters;R. Dwek;F. Platt
中科院分区：
文献类型：
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作者：
G. Karlsson;T. Butters;R. Dwek;F. Platt
亚氨基糖n -丁基脱氧诺吉霉素（NB-DNJ）在体外表现出抗HIV活性，并抑制纯化的糖苷加工酶α 1,2-葡萄糖苷酶i。据推测，该化合物的抗病毒活性可能与抑制HIV包膜糖蛋白加工有关。然而，缺乏结构证据表明葡萄糖苷酶在抗病毒浓度（0.5 mM）的完整细胞中发生抑制。在本研究中，研究了在存在或不存在NB-DNJ的情况下，中国仓鼠卵巢细胞中表达的重组gp120的n -链糖基化。免疫沉淀、内糖苷酶H （endo H）消化和sds -聚丙烯酰胺凝胶电泳分析显示，NB-DNJ存在时gp120的糖基化发生了深刻的改变。然而，在0.5 mM NB-DNJ处理后，观察到几乎完全的endo H敏感性，这表明在处理细胞中表达的gp120携带未成熟的高甘露糖型低聚糖。此外，通过使用[3H]甘露糖代谢标记、凝胶过滤层析和高纯度葡萄糖苷酶I和II的消化，我们首次提供了葡萄糖苷酶I在NB-DNJ抗病毒浓度下发生抑制的明确证据。这些数据表明葡萄糖苷酶抑制是该化合物抗病毒活性的候选机制。
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.