Reglucosylation of glycoproteins and quality control of glycoprotein folding in the endoplasmic reticulum of yeast cells

Proteins entering the secretory pathway may be glycosylated upon transfer of an oligosaccharide (Glc₃Man₉GlcNAc₂) from a dolichol-P-P derivative to nascent polypeptide chains in the lumen of the endoplasmic reticulum (ER). Oligosaccharides are then deglucosylated by glucosidases I and II (GII). Also in the ER, glycoproteins acquire their final tertiary structures, and species that fail to fold properly are retained and eventually degraded in the proteasome. It has been proposed that in mammalian cells the monoglucosylated oligosaccharides generated either by partial deglucosylation of the transferred compound or by reglucosylation of glucose-free oligosaccharides by the UDP-Glc:glycoprotein glucosyltransferase (GT) are recognized by ER resident lectins (calnexin and/or calreticulin). GT is a sensor of glycoprotein conformation as it only glucosylates misfolded species. The lectin-monoglucosylated oligosaccharide interaction would retain glycoproteins in the ER until correctly folded, and also facilitate their acquisition of proper tertiary structures by preventing aggregation. GII would liberate glycoproteins from the calnexin/calreticulin anchor, but species not properly folded would be reglucosylated by GT, and so continue to be retained by the lectins. Only when the protein becomes properly folded would it cease to be retained by the lectins. This review presents evidence suggesting that a similar quality control mechanism of glycoprotein folding is operative in Schizosaccharomyces pombe and that the mechanism in Saccharomyces cerevisiae probably differs substantially from that occurring in mammalian and Sch. pombe cells.     

Contrasting functions of calreticulin and calnexin in glycoprotein folding and ER quality control

Calreticulin and calnexin are homologous lectins that serve as molecular chaperones for glycoproteins in the endoplasmic reticulum of eukaryotic cells. Here we show that calreticulin depletion specifically accelerates the maturation of cellular and viral glycoproteins with a modest decrease in folding efficiency. Calnexin depletion prevents proper maturation of some proteins such as influenza hemagglutinin but does not interfere appreciably with the maturation of several others. A dramatic loss of stringency in the ER quality control with transport at the cell surface of misfolded glycoprotein conformers is only observed when substrate access to both calreticulin and calnexin is prevented. Although not fully interchangeable during assistance of glycoprotein folding, calreticulin and calnexin may work, independently, as efficient and crucial factors for retention in the ER of nonnative polypeptides.     

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.     

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.     

Involvement of Endoplasmic Reticulum Chaperones in the Folding of Hepatitis C Virus Glycoproteins

The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2) which interact noncovalently to form a heterodimer (E1-E2). During the folding and assembly of HCV glycoproteins, a large portion of these proteins are trapped in aggregates, reducing the efficiency of native E1-E2 complex assembly. To better understand this phenomenon and to try to increase the efficiency of HCV glycoprotein folding, endoplasmic reticulum chaperones potentially interacting with these proteins were studied. Calnexin, calreticulin, and BiP were shown to interact with E1 and E2, whereas no interaction was detected between GRP94 and HCV glycoproteins. The association of HCV glycoproteins with calnexin and calreticulin was faster than with BiP, and the kinetics of interaction with calnexin and calreticulin were very similar. However, calreticulin and BiP interacted preferentially with aggregates whereas calnexin preferentially associated with monomeric forms of HCV glycoproteins or noncovalent complexes. Tunicamycin treatment inhibited the binding of HCV glycoproteins to calnexin and calreticulin, indicating the importance of N-linked oligosaccharides for these interactions. The effect of the co-overexpression of each chaperone on the folding of HCV glycoproteins was also analyzed. However, the levels of native E1-E2 complexes were not increased. Together, our data suggest that calnexin plays a role in the productive folding of HCV glycoproteins whereas calreticulin and BiP are probably involved in a nonproductive pathway of folding.     

Calnexin discriminates between protein conformational states and functions as a molecular chaperone in vitro.

Although calnexin is thought to function as a molecular chaperone for glycoproteins, a prevalent view is that it cannot distinguish between protein conformational states, binding solely through its lectin site to monoglucosylated oligosaccharides. Using purified components in vitro, calnexin effectively prevented the aggregation not only of glycoproteins bearing monoglucosylated oligosaccharides but also proteins lacking N-glycans, an effect enhanced by ATP. It also suppressed the thermal denaturation of nonglycosylated proteins and enhanced their refolding in conjunction with other cellular components. Calnexin formed stable complexes with unfolded conformers of these proteins but not with the native molecules. Therefore, in addition to being a lectin, calnexin functions as a bona fide molecular chaperone capable of interacting with polypeptide segments of folding 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.     

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.     

Functional relationship between calreticulin, calnexin, and the endoplasmic reticulum luminal domain of calnexin

Calnexin is a membrane protein of the endoplasmic reticulum (ER) that functions as a molecular chaperone and as a component of the ER quality control machinery. Calreticulin, a soluble analog of calnexin, is thought to possess similar functions, but these have not been directly demonstrated in vivo. Both proteins contain a lectin site that directs their association with newly synthesized glycoproteins. Although many glycoproteins bind to both calnexin and calreticulin, there are differences in the spectrum of glycoproteins that each binds. Using a Drosophila expression system and the mouse class I histocompatibility molecule as a model glycoprotein, we found that calreticulin does possess apparent chaperone and quality control functions, enhancing class I folding and subunit assembly, stabilizing subunits, and impeding export of assembly intermediates from the ER. Indeed, the functions of calnexin and calreticulin were largely interchangeable. We also determined that a soluble form of calnexin (residues 1-387) can functionally replace its membrane-bound counterpart. However, when calnexin was expressed as a soluble protein in L cells, the pattern of associated glycoproteins changed to resemble that of calreticulin. Conversely, membrane-anchored calreticulin bound to a similar set of glycoproteins as calnexin. Therefore, the different topological environments of calnexin and calreticulin are important in determining their distinct substrate specificities.     

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.     

ERp57 Functions as a Subunit of Specific Complexes Formed with the ER Lectins Calreticulin and Calnexin

ERp57 is a lumenal protein of the endoplasmic reticulum (ER) and a member of the protein disulfide isomerase (PDI) family. In contrast to archetypal PDI, ERp57 interacts specifically with newly synthesized glycoproteins. In this study we demonstrate that ERp57 forms discrete complexes with the ER lectins, calnexin and calreticulin. Specific ERp57/calreticulin complexes exist in canine pancreatic microsomes, as demonstrated by SDS-PAGE after cross-linking, and by native electrophoresis in the absence of cross-linking. After in vitro translation and import into microsomes, radiolabeled ERp57 can be cross-linked to endogenous calreticulin and calnexin while radiolabeled PDI cannot. Likewise, radiolabeled calreticulin is cross-linked to endogenous ERp57 but not PDI. Similar results were obtained in Lec23 cells, which lack the glucosidase I necessary to produce glycoprotein substrates capable of binding to calnexin and calreticulin. This observation indicates that ERp57 interacts with both of the ER lectins in the absence of their glycoprotein substrate. This result was confirmed by a specific interaction between in vitro synthesized calreticulin and ERp57 prepared in solution in the absence of other ER components. We conclude that ERp57 forms complexes with both calnexin and calreticulin and propose that it is these complexes that can specifically modulate glycoprotein folding within the ER lumen.     

A polypeptide binding conformation of calreticulin is induced by heat shock, calcium depletion, or by deletion of the C-terminal acidic region

It is widely believed that the chaperone activity of calreticulin is mediated by its ability to bind glycoproteins containing monoglucosylated oligosaccharides. However, calreticulin is also a polypeptide binding protein. Here we show that heat shock, calcium depletion, or deletion of the C-terminal acidic domain enhance binding of purified calreticulin to polypeptide substrates and enhance calreticulin's chaperone activity. These conditions also enhance calreticulin oligomerization, but oligomerization per se is not required for enhanced polypeptide binding. In cells, calreticulin oligomerization intermediates accumulate in response to conditions that induce protein misfolding (heat shock and tunicamycin treatments), and upon calcium depletion. Additionally, in cells, calreticulin binds to deglycosylated major histocompatibility complex class I heavy chains when significant levels of calreticulin oligomerization intermediates are induced. Thus, cell stress conditions that generate nonnative substrates of calreticulin also affect the conformational properties of calreticulin itself, and enhance its binding to substrates, independent of substrate glucosylation.     

Transient, lectin-like association of calreticulin with folding intermediates of cellular and viral glycoproteins.

The soluble, calcium-binding protein calreticulin shares high sequence homology with calnexin, a transmembrane chaperone of glycoprotein folding. Our experiments demonstrated that calreticulin, like calnexin, associated transiently with numerous newly synthesized proteins in the endoplasmic reticulum. The population of proteins that bound to calreticulin was partially overlapping with those that bound to calnexin. Hemagglutinin (HA) of influenza virus was shown to associate with both calreticulin and calnexin. Using HA as a model substrate, it was found that both calreticulin- and calnexin-bound HA corresponded primarily to incompletely disulfide-bonded folding intermediates and conformationally trapped forms. Binding of all substrates was oligosaccharide-dependent and required the trimming of glucose residues from asparagine-linked core glycans by glucosidases I and II. In vitro, alpha-mannosidase digestion of calreticulin-bound HA indicated that calreticulin was specific for monoglucosylated glycans. Thus, calreticulin appeared to be a lectin with similar oligosaccharide specificity as its membrane-bound homologue, calnexin. Both are therefore likely to play an important role in glycoprotein maturation and quality control in the endoplasmic reticulum.     

The use of calnexin and calreticulin by cellular and viral glycoproteins

Calnexin and calreticulin are homologous lectin chaperones that assist maturation of cellular and viral glycoproteins in the mammalian endoplasmic reticulum. Calnexin and calreticulin share the same specificity for monoglucosylated protein-bound N-glycans but associate with a distinct set of newly synthesized polypeptides. We report here that most calnexin substrates do not associate with calreticulin even upon selective calnexin inactivation, while BiP associates more abundantly with nascent polypeptides under these conditions. Calreticulin associated more abundantly with orphan calnexin substrates only in infected cells and preferentially with polypeptides of viral origin, showing stronger dependence of model viral glycoproteins on endoplasmic reticulum lectins. This may explain why inactivation of the calnexin cycle affects viral replication and infectivity but not viability of mammalian cells.     

Potent lectin-independent chaperone function of calnexin under conditions prevalent within the lumen of the endoplasmic reticulum

Calnexin is a membrane-bound chaperone of the endoplasmic reticulum (ER) that participates in the folding and quality control of newly synthesized glycoproteins. Binding to glycoproteins occurs through a lectin site with specificity for Glc1Man9GlcNAc2 oligosaccharides as well as through a polypeptide binding site that recognizes non-native protein conformations. The latter interaction is somewhat controversial because it is based on observations that calnexin can suppress the aggregation of non-glycosylated substrates at elevated temperature or at low calcium concentrations, conditions that may affect the structural integrity of calnexin. Here, we examine the ability of calnexin to interact with a non-glycosylated substrate under physiological conditions of the ER lumen. We show that the soluble ER luminal domain of calnexin can indeed suppress the aggregation of non-glycosylated firefly luciferase at 37 degrees C and at the normal resting ER calcium concentration of 0.4 mM. However, gradual reduction of calcium below the resting level was accompanied by a progressive loss of native calnexin structure as assessed by thermal stability, protease sensitivity, intrinsic fluorescence, and bis-ANS binding. These assays permitted the characterization of a single calcium binding site on calnexin with a Kd = 0.15 +/- 0.05 mM. We also show that the suppression of firefly luciferase aggregation by calnexin is strongly enhanced in the presence of millimolar concentrations of ATP and that the Kd for ATP binding to calnexin in the presence of 0.4 mM calcium is 0.7 mM. ATP did not alter the overall stability of calnexin but instead triggered the localized exposure of a hydrophobic site on the chaperone. These findings demonstrate that calnexin is a potent molecular chaperone that is capable of suppressing the aggregation of substrates through polypeptide-based interactions under conditions that exist within the ER lumen.     

A cell-based reglucosylation assay demonstrates the role of GT1 in the quality control of a maturing glycoprotein

The endoplasmic reticulum (ER) protein GT1 (UDP-glucose: glycoprotein glucosyltransferase) is the central enzyme that modifies N-linked carbohydrates based upon the properties of the polypeptide backbone of the maturing substrate. GT1 adds glucose residues to nonglucosylated proteins that fail the quality control test, supporting ER retention through persistent binding to the lectin chaperones calnexin and calreticulin. How GT1 functions in its native environment on a maturing substrate is poorly understood. We analyzed the reglucosylation of a maturing model glycoprotein, influenza hemagglutinin (HA), in the intact mammalian ER. GT1 reglucosylated N-linked glycans in the slow-folding stem domain of HA once the nascent chain was released from the ribosome. Maturation mutants that disrupted the oxidation or oligomerization of HA also supported region-specific reglucosylation by GT1. Therefore, GT1 acts as an ER quality control sensor by posttranslationally reglucosylating glycans on slow-folding or nonnative domains to recruit chaperones specifically to critical aberrant regions.     

Localization of the lectin,ERp57 binding,and polypeptide binding sites of calnexin and calreticulin

Calnexin and calreticulin are membrane-bound and soluble chaperones, respectively, of the endoplasmic reticulum (ER) which interact transiently with a broad spectrum of newly synthesized glycoproteins. In addition to sharing substantial sequence identity, both calnexin and calreticulin bind to monoglucosylated oligosaccharides of the form Glc(1)Man(5-9)GlcNAc(2), interact with the thiol oxidoreductase, ERp57, and are capable of acting as chaperones in vitro to suppress the aggregation of non-native proteins. To understand how these diverse functions are coordinated, we have localized the lectin, ERp57 binding, and polypeptide binding sites of calnexin and calreticulin. Recent structural studies suggest that both proteins consist of a globular domain and an extended arm domain comprised of two sequence motifs repeated in tandem. Our results indicate that the primary lectin site of calnexin and calreticulin resides within the globular domain, but the results also point to a much weaker secondary site within the arm domain which lacks specificity for monoglucosylated oligosaccharides. For both proteins, a site of interaction with ERp57 is centered on the arm domain, which retains approximately 50% of binding compared with full-length controls. This site is in addition to a Zn(2+)-dependent site located within the globular domain of both proteins. Finally, calnexin and calreticulin suppress the aggregation of unfolded proteins via a polypeptide binding site located within their globular domains but require the arm domain for full chaperone function. These findings are integrated into a model that describes the interaction of glycoprotein folding intermediates with calnexin and calreticulin.     

Reglucosylation by UDP-glucose:glycoprotein glucosyltransferase 1 delays glycoprotein secretion but not degradation

UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1) is a central quality control gatekeeper in the mammalian endoplasmic reticulum (ER). The reglucosylation of glycoproteins supports their rebinding to the carbohydrate-binding ER molecular chaperones calnexin and calreticulin. A cell-based reglucosylation assay was used to investigate the role of UGT1 in ER protein surveillance or the quality control process. UGT1 was found to modify wild-type proteins or proteins that are expected to eventually traffic out of the ER through the secretory pathway. Trapping of reglucosylated wild-type substrates in their monoglucosylated state delayed their secretion. Whereas terminally misfolded substrates or off-pathway proteins were most efficiently reglucosylated by UGT1, the trapping of these mutant substrates in their reglucosylated or monoglucosylated state did not delay their degradation by the ER-associated degradation pathway. This indicated that monoglucosylated mutant proteins were actively extracted from the calnexin/calreticulin binding-reglucosylation cycle for degradation. Therefore trapping proteins in their monoglucosylated state was sufficient to delay their exit to the Golgi but had no effect on their rate of degradation, suggesting that the degradation selection process progressed in a dominant manner that was independent of reglucosylation and the glucose-containing A-branch on the substrate glycans.     

Transient Association of Calnexin and Calreticulin with Newly Synthesized G1 and G2 Glycoproteins of Uukuniemi Virus (Family Bunyaviridae)

The membrane glycoproteins G1 and G2 of Uukuniemi virus, a member of the Bunyaviridae family, are cotranslationally cleaved from a common precursor in the endoplasmic reticulum (ER). Here, we show that newly made G1 and G2 associate transiently with calnexin and calreticulin, two lectins involved in glycoprotein folding in the ER. Stable complexes between G1-G2 and calnexin or calreticulin could be immunoprecipitated after solubilization of virus-infected BHK21 cells with the detergents digitonin or Triton X-100. In addition, G1-G2-calnexin complexes could be recovered after solubilization with CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate), while G1-G2-calreticulin complexes were not readily detected by using this detergent. Only endoglycosidase H-sensitive forms of G1 were found complexed with calnexin. Pulse-chase experiments showed that G1 and G2 associated with both chaperones transiently for up to 120 min. Sequential immunoprecipitations with anticalreticulin and anticalnexin antisera indicated that about 50% of newly synthesized G1 and G2 was associated with either calnexin or calreticulin. Our previous results have shown that newly synthesized G1 and G2 transiently interact also with the ER chaperone BiP and with protein disulfide isomerase (R. Persson and R. F. Pettersson, J. Cell Biol. 112:257-266, 1991). Taking all of this into consideration, we conclude that the folding of G1 and G2 in the ER is catalyzed by at least four different folding factors.     

Association of the thyrotropin receptor with calnexin, calreticulin and BiP. Efects on the maturation of the receptor.

The thyrotropin receptor (TSHR) is a member of the G protein-coupled receptor superfamily. It has by now been clearly established that the maturation of the glycoproteins synthesized in the endoplasmic reticulum involves interactions with molecular chaperones, which promote the folding and assembly of the glycoproteins. In this study, we investigated whether calnexin (CNX), calreticulin (CRT) and BiP, three of the main molecular chaperones present in the endoplasmic reticulum, interact with the TSHR and what effects these interactions might have on the folding of the receptor. In the first set of experiments, we observed that in a K562 cell line expressing TSHR, about 50% of the receptor synthesized was degraded by the proteasome after ubiquitination. In order to determine whether TSHR interact with CNX, CRT and BiP, coimmunoprecipitation experiments were performed. TSHR was found to be associated with all three molecular chaperones. To study the role of the interactions between CNX and CRT and the TSHR, we used castanospermine, a glucosidase I and II inhibitor that blocks the interactions between these chaperones and glycoproteins. In K562 cells expressing the TSHR, these drugs led to a faster degradation of the receptor, which indicates that these interactions contribute to stabilizing the receptor after its synthesis. The overexpression of calnexin and calreticulin in these cells stabilizes the receptor during the first hour after its synthesis, whereas the degradation of TSHR increased in a cell line overexpressing BiP and the quantity of TSHR able to acquire complex type oligosaccharides decreased. These results show that calnexin, calreticulin and BiP all interact with TSHR and that the choice made between these two chaperone systems is crucial because each of them has distinct effects on the folding and stability of this receptor at the endoplasmic reticulum level.