A major proportion of N-glycoproteins are transiently glucosylated in the endoplasmic reticulum.
N-linked, high-mannose-type oligosaccharides lacking glucose residues may be transiently glucosylated directly from UDP-Glc in the endoplasmic reticulum of mammalian, plant, fungal, and protozoan cells. The products formed have been identified as N-linked Glc1Man5-9GlcNAc2 and glucosidase II is apparently the enzyme responsible for the in vivo deglucosylation of the compounds. As newly glucosylated glycoproteins are immediately deglucosylated, it is unknown whether transient glucosylation involves all or nearly all N-linked glycoproteins or if, on the contrary, it only affects a minor proportion of them. In order to evaluate the molar proportion of N-linked oligosaccharides that are glucosylated, cells of the trypanosomatid protozoan Trypanosoma cruzi (a parasite transferring Man9GlcNAc2 in protein N-glycosylation) were grown in the presence of [14C]glucose and concentrations of the glucosidase II inhibitors deoxynojirimycin and castanospermine that were more than 1000-fold higher than those required to produce a 50% inhibition of the T. cruzi enzyme. About 52-53% total N-linked oligosaccharides appeared to have glucose residues. The compounds were identified as Glc1Man7-9GlcNAc2. The same percentage was obtained when cells were pulsed-chased with [14C]glucose in the presence of deoxynojirimycin for 60 min. No evidence for the presence of an endomannosidase yielding GlcMan from the glycosylated compounds was obtained. As the average number of N-linked oligosaccharides per molecule in glycoproteins is higher than one, these results indicate that more than 52-53% of total glycoproteins are glucosylated and that transient glucosylation is a major event in the normal processing of glycoproteins.
Gañán
S
S
Instituto de Investigaciones Bioquímicas, Fundación Campomar, Buenos Aires, Argentina.
Cazzulo
J J
JJ
Parodi
A J
AJ
D017485:1-Deoxynojirimycin; D000818:Animals; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005944:Glucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006596:Hexosaminidases / Q000378:metabolism; D007212:Indolizines / Q000494:pharmacology; D008361:Mannosidases / Q000494:pharmacology; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000187:drug effects / Q000201:enzymology; D000520:alpha-Glucosidases / Q000378:metabolism; D054836:Indolizidines
D017485:1-Deoxynojirimycin; D005944:Glucosamine; D005947:Glucose; D006023:Glycoproteins; D006596:Hexosaminidases; D007212:Indolizines; D008361:Mannosidases; D000520:alpha-Glucosidases; C034251:4-nitrophenyl-alpha-glucosidase; C037806:castanospermine; D054836:Indolizidines