An unconventional mechanism for retaining improperly folded glycoproteins and facilitating acquisition of their native tertiary and quaternary structures operates in the endoplasmic reticulum. Recognition of folding glycoproteins by two resident lectins, membrane-bound calnexin and its soluble homolog, calreticulin, is mediated by protein-linked monoglucosylated oligosaccharides. These oligosaccharides contain glucose (Glc), mannose (Man), and N-acetylglucosamine (GlcNAc) in the general form Glc1Man7-9GlcNAc2. They are formed by glucosidase I- and II-catalyzed partial deglucosylation of the oligosaccharide transferred from dolichol diphosphate derivatives to Asn residues in nascent polypeptide chains (Glc3Man9GlcNAc2). Further deglucosylation of the oligosaccharides by glucosidase II liberates glycoproteins from their calnexin/calreticulin anchors. Monoglucosylated glycans are then recreated by the UDP-Glc:glycoprotein glucosyltransferase (GT), and thus recognized again by the lectins, only when linked to improperly folded protein moieties, as GT behaves as a sensor of glycoprotein conformations. The deglucosylation-reglucosylation cycle continues until proper folding is achieved. The lectin-monoglucosylated oligosaccharide interaction is one of the alternative ways by which cells retain improperly folded glycoproteins in the endoplasmic reticulum. Although it decreases the folding rate, it increases folding efficiency, prevents premature glycoprotein oligomerization and degradation, and suppresses formation of non-native disulfide bonds by hindering aggregation and thus allowing interaction of protein moieties of folding glycoproteins with classical chaperones and other proteins that assist in folding.

The biosynthesis of secretory and membrane proteins in the endoplasmic reticulum (ER) yields mostly properly folded and assembled structures with full biological activity. Such fidelity is maintained by quality control (QC) mechanisms that avoid the production of nonnative structures. QC relies on chaperone systems in the ER that monitor and assist in the folding process. When folding promotion is not sufficient, proteins are retained in the ER and eventually retranslocated to the cytosol for degradation by the ubiquitin proteasome pathway. Retention of proteins that fail QC can sometimes occur beyond the ER, and degradation can take place in lysosomes. Several diseases are associated with proteins that do not pass QC, fail to be degraded efficiently, and accumulate as aggregates. In other cases, pathology arises from the downregulation of mutated but potentially functional proteins that are retained and degraded by the QC system.

The N-glycan-dependent quality control mechanism of glycoprotein folding was proposed initially by Helenius and coworkers several years ago; with a few minor modifications, it is still valid today ( Fig. 1) (1-3).2 Glycan processing starts immediately after its transfer from a dolichol-P-P derivative to Asn residues in nascent polypeptide chains entering the lumen of the ER. 3 Removal of the outermost and following glucoses by the successive action of GI and GII exposes the Glc 1 Man 9 GlcNAc 2 epitope (Fig. 2). This structure is then recognized by two ER resident lectins (CNX and CRT) that specifically bind monoglucosylated polymannose glycans. This is followed by removal of the innermost glucose by GII, thus liberating the glycoprotein from the lectin anchor. The proteinlinked glycan is then reglucosylated by the soluble ER enzyme GT only if the protein moiety displays non-native three-dimensional structures, as this enzyme behaves as a conformational sensor. Cycles of CNX/CRT-glycoprotein binding and liberation, catalyzed by the opposing activities of GT and GII, are terminated once glycoproteins attain their native structures. Glucose-free glycoproteins then continue their transit through the secretory pathway. Alternatively, permanently misfolded glycoproteins may be then transported to the cytosol for proteasomal degradation. Lectin-glycoprotein association not only thwarts Golgi exit of folding intermediates and irreparably misfolded glycoproteins but also enhances folding efficiency by preventing aggregation and promoting proper disulfide bonding. The latter is catalyzed by an oxidoreductase of the proteindisulfide isomerase family (ERp57) that acts exclusively on glycoproteins, as it is loosely associated with CNX/CRT.GT is the only component of the quality control mechanism that senses protein conformations, as it recognizes hydrophobic amino acid patches exposed in molten globule-like conformers (4, 5). GT may also glucosylate glycoproteins in not fully assembled oligomeric complexes because it also recognizes hydrophobic surfaces exposed as a consequence of the absence of subunit components (6). The aim of this review is to give an overview of recent reports dealing with the entrance and exit of glycoproteins from CNX/CRT cycles. Getting In: GII Is Not What It Was Thought to BeThe first step in the pathway leading to the entrance of glycoproteins into CNX/CRT cycles is the removal of the outermost glucose unit from the glycan by the membrane enzyme GI. This reaction occurs almost simultaneously with glycan transfer. The rapid GI-mediated deglucosylation of the protein-linked glycan, as well as the apparent inability of the enzyme to remove in vivo (but not in vitro) the glucose from the dolichol-P-P-linked glycan, strongly suggests the existence of a supercomplex formed by the oligosaccharyltransferase, GI, and the dolichol derivative, with a very precise orientation of the components.It was assumed that the sole role of GII was that of removing glucose residues l and n (Fig. 2). Recent work has sugg...

It was found, in cell-free assays, that the Man8GlcNAc2 and Man7GlcNAc2 isomers having the mannose unit to which the glucose is added were glucosylated by the rat liver glucosyltransferase at 50 and 15%, respectively, of the rate of Man9GlcNAc2 glucosylation. This indicates that processing by endoplasmic reticulum mannosidases decreases the extent of glycoprotein glucosylation. All five different glycoproteins tested (bovine and porcine thyroglobulins, phytohemagglutinin, soybean agglutinin, and bovine pancreas ribonuclease B) were found to be poorly glucosylated or not glucosylated unless they were subjected to treatments that modified their native conformations. The effect of denaturation was not to expose the oligosaccharides but to make protein determinants, required for enzymatic activity, accessible to the glucosyltransferase because (a) cleavage of denatured glycoproteins by unspecific (Pronase) or specific (trypsin) proteases abolished their glucose acceptor capacities almost completely except when the tryptic peptides were held together by disulfide bonds and (b) high mannose oligosaccharides in native glycoproteins, although poorly glucosylated or not glucosylated, were accessible to macromolecular probes as concanavalin A-Sepharose, endo-beta-N-acetylglucosaminidase H, and jack bean alpha-mannosidase. In addition, denatured, endo-beta-N-acetylglucosaminidase H deglycosylated glycoproteins were found to be potent inhibitors of the glucosylation of denatured glycoproteins. It is suggested that in vivo only unfolded, partially folded, and malfolded glycoproteins are glucosylated and that glucosylation stops upon adoption of the correct conformation, a process that hides the protein determinants (possibly hydrophobic amino acids) from the glucosyltransferase.

The UDP‐Glc:glycoprotein glucosyltransferase is a soluble enzyme of the endoplasmic reticulum that glucosylates protein‐linked Man7–9GlcNAc2 to form the monoglucosylated derivatives. In vivo the reaction products are immediately deglucosylated by glucosidase II. The glucosyltransferase has a unique property: it glucosylates misfolded, but not native, glycoproteins. It has been proposed that the glucosyltransferase participates, together with calnexin, in the control mechanism by which only properly folded glycoproteins can exit from the endoplasmic reticulum. In this paper it is demonstrated that the glucosyltransferase recognizes two elements in the acceptor substrates: the innermost N‐acetylglucosamine unit of the oligosaccharide and protein domains exposed in denatured, but not in native, conformations. Both determinants have to be covalently linked. In many cases the first element is not accessible to macromolecular probes in native conformations. Concerning the protein domains, it is demonstrated here that the glucosyltransferase interacts with hydrophobic amino acids exposed in denatured conformations. More disordered conformations, i.e. those exposing more hydrophobic amino acids, were found to be those having higher glucose acceptor capacity. It is suggested that both accessibility of the innermost N‐acetylglucosamine unit and binding to hydrophobic patches determine the exclusive glucosylation of misfolded conformations by the glucosyltransferase.

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The N-glycan-dependent quality control of glycoprotein folding prevents endoplasmic to Golgi exit of folding intermediates, irreparably misfolded glycoproteins and incompletely assembled multimeric complexes. It also enhances folding efficiency by preventing aggregation and facilitating formation of proper disulfide bonds. The control mechanism essentially involves four components, resident lectin-chaperones that recognize monoglucosylated polymannose glycans, a lectin-associated oxidoreductase acting on monoglucosylated glycoproteins, a glucosyltransferase that creates monoglucosytlated epitopes in protein-linked glycans and a glucosidase that removes the glucose units added by the glucosyltransferase. This last enzyme is the only mechanism component sensing glycoprotein conformations as it creates monoglucosylated glycans exclusively in not properly folded species or in not completely assembled complexes. The glucosidase is a dimeric heterodimer composed of a catalytic subunit and an additional one that is partially responsible for the ER localization of the enzyme and for the enhancement of the deglucosylation rate as its mannose 6-phosphate receptor homologous domain presents the substrate to the catalytic site. This review deals with our present knowledge on the glucosyltransferase and the glucosidase.

We have investigated the structure of the glycosylphosphatidylinositol (GPI) anchor and the O-linked glycan chains of the 40/45-kDa glycoprotein from the cell surface of the protozoan parasite Trypanosoma cruzi. This glycoconjugate is the major acceptor for sialic acid transferred by trans-sialidase of T. cruzi Y-strain, epimastigote form. The GPI anchor was liberated by treatment with hot alkali, and the phosphoinositol-oligosaccharide moiety was characterized and shown to have the following structure. [formula: see text] Unusually the glucosamine was 6-O-substituted with 2-aminoethylphosphonate, and 2-aminoethylphosphonate was also present on the third mannose residue distal to glucosamine, partially replacing the ethanolamine phosphate. The beta-eliminated reduced oligosaccharide chains showed that two novel classes of O-linked N-acetylglucosamine oligosaccharide were present. The first series had the structures Galp beta 1-3GlcNAc-ol; Galp beta 1-6(Galp beta 1-3)GlcNAc-ol; and Galp beta 1-2Galp beta 1-6(Galp beta 1-3)GlcNAc-ol, whereas the other series had a 1-4 linkage to N-acetylglucosaminitol and had structures Galp beta 1-4GlcNAc-ol, Galp beta 1-6(Galp beta 1-4)GlcNAc-ol, and Galp beta 1-2Galp beta 1-6(Galp beta 1-4)GlcNAc-ol. We have also investigated the kinetics of in vitro sialylation of these O-linked oligosaccharides by the T. cruzi transsialidase and have shown that incorporation of one molecule of sialic acid hinders entry of a second molecule when two potential acceptor sites are present.