A major proportion of N-glycoproteins are transiently glucosylated in the endoplasmic reticulum

Abstract

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.

Intracellular Disposal of Incompletely Folded Human α1-Antitrypsin Involves Release from Calnexin and Post-translational Trimming of Asparagine-linked Oligosaccharides

“…Taken together, data from this study suggest that variant null(Hong Kong) is subjected to a quality control pathway identical to that recently proposed by Hammond and Helenius (11). Their model proposes that persistent association between unfolded glycoproteins and calnexin reflects a continuous cycle of binding in which assembly of the complex is facilitated by reglucosylation of asparagine-linked oligosaccharides by the ER resident enzyme UDP-glucose:glycoprotein glucosyltransferase (17)(18)(19)48). Importantly, the model predicts that permanent dissociation from the binding cycle will occur when hydrolysis of mannose residues generates an oligosaccharide unable to participate as a glucose acceptor, thereby leading to disposal of the unfolded glycoprotein.…”

Glycosylation-directed quality control of protein folding

“…This is a transient interaction, which allows the cleavage of the last glucose by glucosidase II, thereby preventing re-engagement of calnexin. Subsequently, UDP-glucose:glycoprotein glucosyltransferase (UGGT), which is a large ER protein, 'inspects' the released proteins; those still bearing unfolded domains are re-glucosylated by UGGT at the A-branch, restoring the calnexin-binding site for another round of binding and release [55][56][57][58] (FIG. 4).…”

Immunolocalization of UDP-glucose:glycoprotein glucosyltransferase indicates involvement of pre-Golgi intermediates in protein quality control

“…In the endoplasmic reticulum (ER), chaperones help proteins co-and posttranslationally to mature (14,15). Furthermore, the lectins calnexin and calreticulin bind to glycoproteins carrying monoglucosylated Glc 1 Man 9 GlcNAc 2 oligosaccharides generated by gls II (16,17) and GT (18,19), and the dissociation of such complexes can be brought about by gls II, which is located in the ER and pre-Golgi intermediates ʈ (13,20), and by endomannosidase in the pre-Golgi intermediate͞ Golgi apparatus (13). If correctly folded, glycoproteins may exit the ER.…”