UDP-glucose:glycoprotein glucosyltransferase (UGGT1) promotes substrate solubility in the endoplasmic reticulum

Protein folding in the endoplasmic reticulum (ER) is error prone, and ER quality control (ERQC) processes ensure that only correctly folded proteins are exported from the ER. Glycoproteins can be retained in the ER by ERQC, and this retention contributes to multiple human diseases, termed ER storage diseases. UDP-glucose:glycoprotein glucosyltransferase (UGGT1) acts as a central component of glycoprotein ERQC, monoglucosylating deglucosylated N-glycans of incompletely folded glycoproteins and promoting subsequent reassociation with the lectin-like chaperones calreticulin and calnexin. The extent to which UGGT1 influences glycoprotein folding, however, has only been investigated for a few selected substrates. Using mouse embryonic fibroblasts lacking UGGT1 or those with UGGT1 complementation, we investigated the effect of monoglucosylation on the soluble/insoluble distribution of two misfolded α1-antitrypsin (AAT) variants responsible for AAT deficiency disease: null Hong Kong (NHK) and Z allele. Whereas substrate solubility increases directly with the number of N-linked glycosylation sites, our results indicate that additional solubility is conferred by UGGT1 enzymatic activity. Monoglucosylation-dependent solubility decreases both BiP association with NHK and unfolded protein response activation, and the solubility increase is blocked in cells deficient for calreticulin. These results suggest that UGGT1-dependent monoglucosylation of N-linked glycoproteins promotes substrate solubility in the ER.     

The ER glycoprotein quality control system

The endoplasmic reticulum (ER) is the major site for folding and sorting of newly synthesized secretory cargo proteins. One central regulator of this process is the quality control machinery, which retains and ultimately disposes of misfolded secretory proteins before they can exit the ER. The ER quality control process is highly effective and mutations in cargo molecules are linked to a variety of diseases. In mammalian cells, a large number of secretory proteins, whether membrane bound or soluble, are asparagine (N)-glycosylated. Recent attention has focused on a sugar transferase, UDP-Glucose: glycoprotein glucosyl transferase (UGGT), which is now recognized as a constituent of the ER quality control machinery. UGGT is capable of sensing the folding state of glycoproteins and attaches a single glucose residue to the Man9GlcNAc2 glycan of incompletely folded or misfolded glycoproteins. This enables misfolded glycoproteins to rebind calnexin and reenter productive folding cycles. Prolonging the time of glucose addition on misfolded glycoproteins ultimately results in either the proper folding of the glycoprotein or its presentation to an ER associated degradation machinery.     

Recognition of local glycoprotein misfolding by the ER folding sensor UDP-glucose:glycoprotein glucosyltransferase

The endoplasmic reticulum (ER) contains a stringent quality control system that ensures the correct folding of newly synthesized proteins to be exported via the secretory pathway. In this system UDP-Glc:glycoprotein glucosyltransferase (GT) serves as a glycoprotein specific folding sensor by specifically glucosylating N-linked glycans in misfolded glycoproteins thus retaining them in the calnexin/calreticulin chaperone cycle. To investigate how GT senses the folding status of glycoproteins, we generated RNase B heterodimers consisting of a folded and a misfolded domain. Only glycans linked to the misfolded domain were found to be glucosylated, indicating that the enzyme recognizes folding defects at the level of individual domains and only reglucosylates glycans directly attached to a misfolded domain. The result was confirmed with complexes of soybean agglutinin and misfolded thyroglobulin.     

Minor folding defects trigger local modification of glycoproteins by the ER folding sensor GT

UDP-glucose:glycoprotein glucosyltransferase (GT) is a key component of the glycoprotein-specific folding and quality control system in the endoplasmic reticulum. By exclusively reglucosylating incompletely folded and assembled glycoproteins, it serves as a folding sensor that prolongs the association of newly synthesized glycoproteins with the chaperone-like lectins calnexin and calreticulin. Here, we address the mechanism by which GT recognizes and labels its substrates. Using an improved inhibitor assay based on soluble conformers of pancreatic ribonuclease in its glycosylated (RNase B) and unglycosylated (RNase A) forms, we found that the protein moiety of a misfolded conformer alone is sufficient for specific recognition by GT in vitro. To investigate the relationship between recognition and glucosylation, we tested a variety of glycosylation mutants of RNase S-Protein and an RNase mutant with a local folding defect [RNase C65S, C72S], as well as a series of loop insertion mutants. The results indicated that local folding defects in an otherwise correctly folded domain could be recognized by GT. Only glycans attached to the polypeptide within the misfolded sites were glucosylated.     

The solution NMR structure of glucosylated N-glycans involved in the early stages of glycoprotein biosynthesis and folding.

Glucosylated oligomannose N-linked oligosaccharides (Glc(x)Man9GlcNAc2 where x = 1-3) are not normally found on mature glycoproteins but are involved in the early stages of glycoprotein biosynthesis and folding as (i) recognition elements during protein N-glycosylation and chaperone recognition and (ii) substrates in the initial steps of N-glycan processing. By inhibiting the first steps of glycan processing in CHO cells using the alpha-glucosidase inhibitor N-butyl-deoxynojirimycin, we have produced sufficient Glc3Man7GlcNAc2 for structural analysis by nuclear magnetic resonance (NMR) spectroscopy. Our results show the glucosyl cap to have a single, well-defined conformation independent of the rest of the saccharide. Comparison with the conformation of Man9GlcNAc2, previously determined by NMR and molecular dynamics, shows the mannose residues to be largely unaffected by the presence of the glucosyl cap. Sequential enzymatic cleavage of the glucose residues does not affect the conformation of the remaining saccharide. Modelling of the Glc3Man9GlcNAc2, Glc2Man9GlcNAc2 and Glc1Man9GlcNAc2 conformations shows the glucose residues to be fully accessible for recognition. A more detailed analysis of the conformations allows potential recognition epitopes on the glycans to be identified and can form the basis for understanding the specificity of the glucosidases and chaperones (such as calnexin) that recognize these glycans, with implications for their mechanisms of action.     

Protein quality control in the ER: The recognition of misfolded proteins

The mechanism, in molecular terms of protein quality control, specifically of how the cell recognizes and discriminates misfolded proteins, remains a challenge. In the secretory pathway the folding status of glycoproteins passing through the endoplasmic reticulum is marked by the composition of the N-glycan. The different glycoforms are recognized by specialized lectins. The folding sensor UGGT acts as an unusual molecular chaperone and covalently modifies the Man9 N-glycan of a misfolded protein by adding a glucose moiety and converts it to Glc1Man9 that rebinds the lectin calnexin. However, further links between the folding status of a glycoprotein and the composition of the N-glycan are unclear. There is little unequivocal evidence for other proteins in the ER recognizing the N-glycan and also acting as molecular chaperones. Nevertheless, based upon a few examples, we suggest that this function is carried out by individual proteins in several different complexes. Thus, calnexin binds the protein disulfide isomerase ERp57, that acts upon Glc1Man9 glycoproteins. In another example the protein disulfide isomerase ERdj5 binds specifically to EDEM (which is probably a mannosidase) and a lectin OS9, and reduces the disulfide bonds of bound glycoproteins destined for ERAD. Thus the glycan recognition is performed by a lectin and the chaperone function performed by a specific partner protein that can recognize misfolded proteins. We predict that this will be a common arrangement of proteins in the ER and that members of protein foldase families such as PDI and PPI will bind specifically to lectins in the ER. Molecular chaperones BiP and GRp94 will assist in the folding of proteins bound in these complexes as well as in the folding of non-glycoproteins.     

The Number and Location of Glycans on Influenza Hemagglutinin Determine Folding and Association with Calnexin and Calreticulin

Calnexin and calreticulin are homologous molecular chaperones that promote proper folding, oligomeric assembly, and quality control of newly synthesized glycoproteins in the endoplasmic reticulum (ER). Both are lectins that bind to substrate glycoproteins that have monoglucosylated N-linked oligosaccharides. Their binding to newly translated influenza virus hemagglutinin (HA), and various mutants thereof, was analyzed in microsomes after in vitro translation and expression in live CHO cells. A large fraction of the HA molecules was found to occur in ternary HA– calnexin–calreticulin complexes. In contrast to calnexin, calreticulin was found to bind primarily to early folding intermediates. Analysis of HA mutants with different numbers and locations of N-linked glycans showed that although the two chaperones share the same carbohydrate specificity, they display distinct binding properties; calreticulin binding depends on the oligosaccharides in the more rapidly folding top/hinge domain of HA whereas calnexin is less discriminating. Calnexin's binding was reduced if the HA was expressed as a soluble anchor-free protein rather than membrane bound. When the co- and posttranslational folding and trimerization of glycosylation mutants was analyzed, it was observed that removal of stem domain glycans caused accelerated folding whereas removal of the top domain glycans (especially the oligosaccharide attached to Asn81) inhibited folding. In summary, the data established that individual N-linked glycans in HA have distinct roles in calnexin/calreticulin binding and in co- and posttranslational folding.     

Endoplasmic reticulum glucosidase II is inhibited by its end products

The calnexin/calreticulin cycle is a quality control system responsible for promoting the folding of newly synthesized glycoproteins entering the endoplasmic reticulum (ER). The association of calnexin and calreticulin with the glycoproteins is regulated by ER glucosidase II, which hydrolyzes Glc 2Man X GlcNAc 2 glycans to Glc 1Man X GlcNAc 2 and further to Glc 0Man X GlcNAc 2 ( X represents any number between 5 and 9). To gain new insights into the reaction mechanism of glucosidase II, we developed a kinetic model that describes the interactions between glucosidase II, calnexin/calreticulin, and the glycans. Our model accurately reconstructed the hydrolysis of glycans with nine mannose residues and glycans with seven mannose residues, as measured by Totani et al. [Totani, K., Ihara, Y., Matsuo, I., and Ito, Y. (2006) J. Biol. Chem. 281, 31502-31508]. Intriguingly, our model predicted that glucosidase II was inhibited by its nonglucosylated end products, where the inhibitory effect of Glc 0Man 7GlcNAc 2 was much stronger than that of Glc 0Man 9GlcNAc 2. These predictions were confirmed experimentally. Moreover, our model suggested that glycans with a different number of mannose residues can be equivalent substrates of glucosidase II, in contrast to what had been previously thought. We discuss the possibility that nonglucosylated glycans, existing in the ER, might regulate the entry of newly synthesized glycoproteins into the calnexin/calreticulin cycle. Our model also shows that glucosidase II does not interact with monoglucosylated glycans while they are bound to calnexin or calreticulin.     

Conformational requirements for glycoprotein reglucosylation in the endoplasmic reticulum

Newly synthesized glycoproteins interact during folding and quality control in the ER with calnexin and calreticulin, two lectins specific for monoglucosylated oligosaccharides. Binding and release are regulated by two enzymes, glucosidase II and UDP-Glc:glycoprotein:glycosyltransferase (GT), which cyclically remove and reattach the essential glucose residues on the N-linked oligosaccharides. GT acts as a folding sensor in the cycle, selectively reglucosylating incompletely folded glycoproteins and promoting binding of its substrates to the lectins. To investigate how nonnative protein conformations are recognized and directed to this unique chaperone system, we analyzed the interaction of GT with a series of model substrates with well defined conformations derived from RNaseB. We found that conformations with slight perturbations were not reglucosylated by GT. In contrast, a partially structured nonnative form was efficiently recognized by the enzyme. When this form was converted back to a nativelike state, concomitant loss of recognition by GT occurred, reproducing the reglucosylation conditions observed in vivo with isolated components. Moreover, fully unfolded conformers were poorly recognized. The results indicated that GT is able to distinguish between different nonnative conformations with a distinct preference for partially structured conformers. The findings suggest that discrete populations of nonnative conformations are selectively reglucosylated to participate in the calnexin/calreticulin chaperone pathway.     

Promiscuous activity of ER glucosidase II discovered through donor specificity analysis of UGGT

In glycoprotein quality control system in the endoplasmic reticulum (ER), UGGT (UDP-glucose:glycoprotein glucosyltransferase) and glucosidase II (G-II) play key roles. UGGT serves as a glycoprotein folding sensor by virtue of its unique specificity to glucosylate glycoproteins at incompletely folded stage. By using various UDP-Glc analogues, we first analyzed donor specificity of UGGT, which was proven to be rather narrow. However, marginal activity was observed with UDP-galactose and UDP-glucuronic acid as well as with 3-, 4- and 6-deoxy glucose analogues to give corresponding transfer products. Intriguingly, G-II smoothly converted all of them back to Man(9)GlcNAc(2), providing an indication that G-II has a promiscuous activity as a broad specificity hexosidase.     

The ER protein folding sensor UDP-glucose glycoprotein-glucosyltransferase modifies substrates distant to local changes in glycoprotein conformation

We present in vitro data that explain the recognition mechanism of misfolded glycoproteins by UDP-glucose glycoprotein-glucosyltransferase (UGGT). The glycoprotein exo-(1,3)-beta-glucanase (beta-Glc) bearing two glycans unfolds in a pH-dependent manner to become a misfolded substrate for UGGT. In the crystal structure of this glycoprotein, the local hydrophobicity surrounding each glycosylation site coincides with the differential recognition of N-linked glycans by UGGT. We introduced a single F280S point mutation, producing a beta-Glc protein with full enzymatic activity that was both recognized as misfolded and monoglucosylated by UGGT. Contrary to current views, these data show that UGGT can modify N-linked glycans positioned at least 40 A from localized regions of disorder and sense subtle conformational changes within structurally compact, enzymatically active glycoprotein substrates.     

Cloning and characterization of mammalian UDP-glucose glycoprotein: glucosyltransferase and the development of a specific substrate for this enzyme

The endoplasmic reticulum enzyme UDP-glucose glycoprotein:glucosyltransferase (UGGT) has the unique property of recognizing incompletely folded glycoproteins and, if they carry an N -linked Man(9)GlcNAc(2)oligosaccharide, of catalyzing the addition of a glucose residue from UDP-glucose. Using peptide sequence information, we have isolated the complete cDNA of rat liver UGGT and expressed it in insect cells. The cDNA specifies an open reading frame which codes for a protein of 1527 residues including an 18 amino acid signal peptide. The protein has a C-terminal tetrapeptide (HEEL) characteristic of endoplasmic reticulum luminal proteins. The purified recombinant enzyme shows the same preference for unfolded polypeptides with N -linked Man(9)GlcNAc(2)glycans as the enzyme purified from rat liver. A genetically engineered Saccharomyces cerevisiae strain capable of producing glyco-proteins with Man(9)GlcNAc(2)core oligosaccharides was constructed and secreted acid phosphatase (G0-AcP) was purified. G0-AcP was used as an acceptor glycoprotein for UGGT and found to be a better substrate than the previously used soybean agglutinin and thyroglobulin. Recombinant rat UGGT has a K (m) of 44 microM for UDP-glucose. A proteolytic fragment of UGGT was found to retain enzymatic activity thus localizing the catalytic site of the enzyme to the C-terminal 37 kDa of the protein. Using site-directed mutagenesis and photoaffinity labeling, we have identified residues D1334, D1336, Q1429, and N1433 to be necessary for the catalytic activity of the enzyme.     

Glucosidase II and N-glycan mannose content regulate the half-lives of monoglucosylated species in vivo

Glucosidase II (GII) sequentially removes the two innermost glucose residues from the glycan (Glc(3)Man(9)GlcNAc(2)) transferred to proteins. GII also participates in cycles involving the lectin/chaperones calnexin (CNX) and calreticulin (CRT) as it removes the single glucose unit added to folding intermediates and misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase (UGGT). GII is a heterodimer in which the α subunit (GIIα) bears the active site, and the β subunit (GIIβ) modulates GIIα activity through its C-terminal mannose 6-phosphate receptor homologous (MRH) domain. Here we report that, as already described in cell-free assays, in live Schizosaccharomyces pombe cells a decrease in the number of mannoses in the glycan results in decreased GII activity. Contrary to previously reported cell-free experiments, however, no such effect was observed in vivo for UGGT. We propose that endoplasmic reticulum α-mannosidase-mediated N-glycan demannosylation of misfolded/slow-folding glycoproteins may favor their interaction with the lectin/chaperone CNX present in S. pombe by prolonging the half-lives of the monoglucosylated glycans (S. pombe lacks CRT). Moreover, we show that even N-glycans bearing five mannoses may interact in vivo with the GIIβ MRH domain and that the N-terminal GIIβ G2B domain is involved in the GIIα-GIIβ interaction. Finally, we report that protists that transfer glycans with low mannose content to proteins have nevertheless conserved the possibility of displaying relatively long-lived monoglucosylated glycans by expressing GIIβ MRH domains with a higher specificity for glycans with high mannose content.     

N-linked oligosaccharides are necessary and sufficient for association of glycosylated forms of bovine RNase with calnexin and calreticulin.

Calnexin and calreticulin are lectin-like molecular chaperones that promote folding and assembly of newly synthesized glycoproteins in the endoplasmic reticulum. While it is well established that they interact with substrate monoglucosylated N-linked oligosaccharides, it has been proposed that they also interact with polypeptide moieties. To test this notion, glycosylated forms of bovine pancreatic ribonuclease (RNase) were translated in the presence of microsomes and their folding and association with calnexin and calreticulin were monitored. When expressed with two N-linked glycans in the presence of micromolar concentrations of deoxynojirimycin, this small soluble protein was found to bind firmly to both calnexin and calreticulin. The oligosaccharides were necessary for association, but it made no difference whether the RNase was folded or not. This indicated that unlike other chaperones, calnexin and calreticulin do not select their substrates on the basis of folding status. Moreover, enzymatic removal of the oligosaccharide chains using peptide N-glycosidase F or removal of the glucoses by ER glucosidase II resulted in dissociation of the complexes. This indicated that the lectin-like interaction, and not a protein-protein interaction, played the central role in stabilizing RNase-calnexin/calreticulin complexes.     

Substrate-specific requirements for UGT1-dependent release from calnexin

Newly synthesized glycoproteins displaying monoglucosylated N-glycans bind to the endoplasmic reticulum (ER) chaperone calnexin, and their maturation is catalyzed by the calnexin-associated oxidoreductase ERp57. Folding substrates are eventually released from calnexin, and terminal glucoses are removed from N-glycans. The UDP-glucose:glycoprotein glucosyltransferase (UGT1, UGGT, GT) monitors the folding state of polypeptides released from calnexin and adds back a glucose residue on N-glycans of nonnative polypeptides, thereby prolonging retention in the calnexin chaperone system for additional folding attempts. Here we show that for certain newly synthesized glycoproteins UGT1 deletion has no effect on binding to calnexin. These proteins must normally complete their folding program in one binding event. Other proteins normally undergo multiple binding events, and UGT1 deletion results in their premature release from calnexin. For other proteins, UGT1 deletion substantially delays release from calnexin, unexpectedly showing that UGT1 activity might be required for a structural maturation needed for substrate dissociation from calnexin and export from the ER.     

More than one glycan is needed for ER glucosidase II to allow entry of glycoproteins into the calnexin/calreticulin cycle

Nascent and newly synthesized glycoproteins enter the calnexin (Cnx)/calreticulin (Crt) cycle when two out of three glucoses in the core N-linked glycans have been trimmed sequentially by endoplasmic reticulum (ER) glucosidases I (GI) and II (GII). By analyzing arrested glycopeptides in microsomes, we found that GI removed the outermost glucose immediately after glycan addition. However, although GII associated with singly glycosylated nascent chains, trimming of the second glucose only occurred efficiently when a second glycan was present in the chain. Consistent with a requirement for multiple glycans to activate GII, pancreatic RNase in live cells needed more than one glycan to enter the Cnx/Crt cycle. Thus, whereas GI trimming occurs as an automatic extension of glycosylation, trimming by GII is a regulated process. By adjusting the number and location of glycans, glycoproteins can instruct the cell to engage them in an individually determined folding and quality control pathway.     

EDEM1 regulates ER-associated degradation by accelerating de-mannosylation of folding-defective polypeptides and by inhibiting their covalent aggregation

Proteins expressed in the endoplasmic reticulum (ER) are covalently modified by co-translational addition of pre-assembled core glycans (glucose(3)-mannose(9)-N-acetylglucosamine(2)) to asparagines in Asn-X-Ser/Thr motifs. N-Glycan processing is essential for protein quality control in the ER. Cleavages and re-additions of the innermost glucose residue prolong folding attempts in the calnexin cycle. Progressive loss of mannoses is a symptom of long retention in the ER and elicits preparation of terminally misfolded polypeptides for dislocation into the cytosol and proteasome-mediated degradation. The ER stress-induced protein EDEM1 regulates disposal of folding-defective glycoproteins and has been described as a mannose-binding lectin. Here we show that elevation of the intralumenal concentration of EDEM1 accelerates ER-associated degradation (ERAD) by accelerating de-mannosylation of terminally misfolded glycoproteins and by inhibiting formation of covalent aggregates upon release of terminally misfolded ERAD candidates from calnexin. Acceleration of Man(9) or Man(5)N-glycans dismantling upon overexpression was fully blocked by substitution in EDEM1 of one catalytic residue conserved amongst alpha1,2-mannosidases, thus suggesting that EDEM1 is an active mannosidase. This mutation did not affect the chaperone function of EDEM1.     

Hepatitis C virus glycoprotein folding: disulfide bond formation and association with calnexin.

The hepatitis C virus (HCV) glycoproteins (E1 and E2) are released from the polyprotein by signal peptidase-mediated cleavage and interact to form a heterodimer. Since properly folded subunits are usually required for specific recognition and stable oligomer formation, the rate of stable E1E2 complex formation, which is low, may be limited by the rate of HCV E1 and/or E2 folding. In this study, the folding of the HCV E1 and E2 glycoproteins was monitored by observing the kinetics of intramolecular disulfide bond formation. The association/dissociation of E1 and E2 with calnexin was also examined, since this molecular chaperone appears to play a major role in quality control via retention of incompletely folded or misfolded proteins in the endoplasmic reticulum. Our results indicate that the disulfide-dependent folding of E2 occurs rapidly and appears to be complete upon cleavage of the precursor E2-NS2. In contrast, folding of E1 is slow (> 1 h), suggesting that this step may be rate limiting for E1E2 oligomerization. Both HCV glycoproteins associated rapidly with calnexin, but dissociation was slow, consistent with the slow folding and assembly of E1E2 glycoprotein complexes. These results suggest a role for prolonged association with calnexin in the folding and assembly of HCV glycoprotein heterodimer complexes.     

Htm1p, a mannosidase-like protein, is involved in glycoprotein degradation in yeast

Misfolded proteins are recognized in the endoplasmic reticulum (ER), transported back to the cytoplasm and degraded by the proteasome. Processing intermediates of N-linked oligosaccharides on incompletely folded glycoproteins have an important role in their folding/refolding, and also in their targeting to proteolytic degradation. In Saccharomyces cerevisiae, we have identified a gene coding for a non-essential protein that is homologous to mannosidase I (HTM1) and that is required for degradation of glycoproteins. Deletion of the HTM1 gene does not affect oligosaccharide trimming. However, deletion of HTM1 does reduce the rate of degradation of the mutant glycoproteins such as carboxypeptidase Y, ABC-transporter Pdr5-26p and oligosaccharyltransferase subunit Stt3-7p, but not of mutant Sec61-2p, a non-glycoprotein. Our results indicate that although Htm1p is not involved in processing of N-linked oligosaccharides, it is required for their proteolytic degradation. We propose that this mannosidase homolog is a lectin that recognizes Man8GlcNAc2 oligosaccharides that serve as signals in the degradation pathway.     

Folding of the human immunodeficiency virus type 1 envelope glycoprotein in the endoplasmic reticulum

The lumen of the endoplasmic reticulum (ER) provides a unique folding environment that is distinct from other organelles supporting protein folding. The relatively oxidizing milieu allows the formation of disulfide bonds. N-linked oligosaccharides that are attached during synthesis play multiple roles in the folding process of glycoproteins. They stabilize folded domains and increase protein solubility, which prevents aggregation of folding intermediates. Glycans mediate the interaction of newly synthesized glycoproteins with some resident ER folding factors, such as calnexin and calreticulin. Here we present an overview of the present knowledge on the folding process of the heavily glycosylated human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein in the ER.