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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.

Type

Journal article

Journal

J Biol Chem

Publication Date

05/01/1993

Volume

268

Pages

570 - 576

Keywords

1-Deoxynojirimycin, Animals, Antiviral Agents, CHO Cells, Carbon Radioisotopes, Chromatography, Gel, Cricetinae, Glycosylation, HIV Envelope Protein gp120, HIV-1, Hexosaminidases, Mannose, Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase, Methionine, Oligosaccharides, Recombinant Proteins, Sulfur Radioisotopes, Transfection, Tritium