Effects of the imino sugar N-butyldeoxynojirimycin on the N-glycosylation of recombinant gp120.
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.
Karlsson
G B
GB
Department of Biochemistry, University of Oxford, United Kingdom.
Butters
T D
TD
Dwek
R A
RA
Platt
F M
FM
D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D016466:CHO Cells; D002250:Carbon Radioisotopes; D002850:Chromatography, Gel; D006224:Cricetinae; D006031:Glycosylation; D015699:HIV Envelope Protein gp120 / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D015497:HIV-1 / Q000187:drug effects / Q000378:metabolism*; D006596:Hexosaminidases; D008358:Mannose / Q000378:metabolism*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008715:Methionine / Q000378:metabolism; D009844:Oligosaccharides / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014162:Transfection; D014316:Tritium
D000998:Antiviral Agents; D002250:Carbon Radioisotopes; D015699:HIV Envelope Protein gp120; D009844:Oligosaccharides; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D014316:Tritium; D017485:1-Deoxynojirimycin; C059896:miglustat; D008715:Methionine; D006596:Hexosaminidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008358:Mannose