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.     

N-linked glycans direct the cotranslational folding pathway of influenza hemagglutinin

For proteins that traverse the secretory pathway, folding commences cotranslationally upon translocation into the endoplasmic reticulum. In this study, we have comprehensively analyzed the earliest maturation steps of the model glycoprotein influenza hemagglutinin (HA). These steps include cleavage of the signal sequence, glycosylation, binding by the chaperones calnexin and calreticulin, and the oxidoreductase ERp57, and oxidation. Our results show that the molecular choreography of the nascent HA chain is largely directed by multiple glycans that are strategically placed to elicit the binding of lectin chaperones. These chaperones are recruited to specific nascent chain locations to regulate and facilitate glycoprotein folding, thereby suggesting that the positioning of N-linked glycans in critical regions has evolved to optimize the folding process in the cell.     

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.     

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.     

Cell surface targeting of myelin oligodendrocyte glycoprotein (MOG) in the absence of endoplasmic reticulum molecular chaperones

Myelin oligodendrocyte glycoprotein (MOG) is a type I integral membrane glycoprotein that localizes to myelin sheaths in the central nervous system. MOG has important implications in multiple sclerosis, as pathogenic anti-MOG antibodies have been detected in the sera of multiple sclerosis patients. As a membrane protein, MOG achieves its native structure in the endoplasmic reticulum where its folding is expected to be controlled by endoplasmic reticulum chaperones. Calnexin, calreticulin, and ERp57 are essential components of the endoplasmic reticulum quality control where they assist in the proper folding of newly synthesized glycoproteins. In this study, we show that expression of MOG is not affected by the absence of the endoplasmic reticulum quality control proteins calnexin, calreticulin, or ERp57. We also show that calnexin forms complexes with MOG and these interactions might be glycan-independent. Importantly, we show that cell surface targeting of MOG is not disrupted in the absence of the endoplasmic reticulum chaperones. This article is part of a special issue entitled: 11th European Symposium on Calcium.     

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.     

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.     

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.     

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.     

Quality Control in the Secretory Pathway: The Role of Calreticulin, Calnexin and BiP in the Retention of Glycoproteins with C-Terminal Truncations

Unlike properly folded and assembled proteins, most misfolded and incompletely assembled proteins are retained in the endoplasmic reticulum of mammalian cells and degraded without transport to the Golgi complex. To analyze the mechanisms underlying this unique sorting process and its fidelity, the fate of C-terminally truncated fragments of influenza hemagglutinin was determined. An assortment of different fragments was generated by adding puromycin at low concentrations to influenza virus-infected tissue culture cells. Of the fragments generated, <2% was secreted, indicating that the system for detecting defects in newly synthesized proteins is quite stringent. The majority of secreted species corresponded to folding domains within the viral spike glycoprotein. The retained fragments acquired a partially folded structure with intrachain disulfide bonds and conformation-dependent antigenic epitopes. They associated with two lectin-like endoplasmic reticulum chaperones (calnexin and calreticulin) but not BiP/GRP78. Inhibition of the association with calnexin and calreticulin by the addition of castanospermine significantly increased fragment secretion. However, it also caused association with BiP/GRP78. These results indicated that the association with calnexin and calreticulin was involved in retaining the fragments. They also suggested that BiP/GRP78 could serve as a backup for calnexin and calreticulin in retaining the fragments. In summary, the results showed that the quality control system in the secretory pathway was efficient and sensitive to folding defects, and that it involved multiple interactions with endoplasmic reticulum chaperones.     

The evolutionary history of calreticulin and calnexin genes in green plants

Calreticulin and calnexin are Ca²⁺-binding chaperones localized in the endoplasmic reticulum of eukaryotes acting in glycoprotein folding quality control and Ca²⁺ homeostasis. The evolutionary histories of calreticulin and calnexin gene families were inferred by comprehensive phylogenetic analyses using 18 completed genomes and ESTs covering the major green plants groups, from green algae to angiosperms. Calreticulin and calnexin possibly share a common origin, and both proteins are present along all green plants lineages. The calreticulin founder gene within green plants duplicated in early tracheophytes leading to two possible groups of orthologs with specialized functions, followed by lineage-specific gene duplications in spermatophytes. Calnexin founder gene in land plants was inherited from basal green algae during evolution in a very conservative copy number. A comprehensive classification in possible groups of orthologs and a catalog of calreticulin and calnexin genes from green plants are provided.     

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.     

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.     

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 is essential for efficient folding of glycoproteins sharing common structural domains

ERp57 is a member of the protein disulphide isomerase family of oxidoreductases, which are involved in native disulphide bond formation in the endoplasmic reticulum of mammalian cells. This enzyme has been shown to be associated with both calnexin and calreticulin and, therefore, has been proposed to be a glycoprotein-specific oxidoreductase. Here, we identify endogenous substrates for ERp57 by trapping mixed disulphide intermediates between enzyme and substrate. Our results demonstrate that the substrates for this enzyme are mostly heavily glycosylated, disulphide bonded proteins. In addition, we show that the substrate proteins share common structural domains, indicating that substrate specificity may involve specific structural features as well as the presence of an oligosaccharide side chain. We also show that the folding of two of the endogenous substrates for ERp57 is impaired in ERp57 knockout cells and that prevention of an interaction with calnexin or calreticulin perturbs the folding of some, but not all, substrates with multiple disulphide bonds. These results suggest a specific role for ERp57 in the isomerisation of non-native disulphide bonds in specific glycoprotein substrates.     

Separate roles and different routing of calnexin and ERp57 in endoplasmic reticulum quality control revealed by interactions with asialoglycoprotein receptor chains

The thiol oxidoreductase endoplasmic reticulum (ER)p57 interacts with newly synthesized glycoproteins through ternary complexes with the chaperones/lectins calnexin or calreticulin. On proteasomal inhibition calnexin and calreticulin concentrate in the pericentriolar endoplasmic reticulum-derived quality control compartment that we recently described. Surprisingly, ERp57 remained in an endoplasmic reticulum pattern. Using asialoglycoprotein receptor H2a and H2b as models, we determined in pulse-chase experiments that both glycoproteins initially bind to calnexin and ERp57. However, H2b, which will exit to the Golgi, dissociated from calnexin and remained bound for a longer period to ERp57, whereas the opposite was true for the endoplasmic reticulum-associated degradation substrate H2a that will go to the endoplasmic reticulum-derived quality control compartment. At 15 degrees C, ERp57 colocalized with H2b adjacent to an endoplasmic reticulum-Golgi intermediate compartment marker. Posttranslational inhibition of glucose excision prolonged association of H2a precursor to calnexin but not to ERp57. Preincubation with a low concentration (15 microg/ml) of the glucosidase inhibitor castanospermine prevented the association of H2a to ERp57 but not to calnexin. This low concentration of castanospermine accelerated the degradation of H2a, suggesting that ERp57 protects the glycoprotein from degradation and not calnexin. Our results suggest an early chaperone-mediated sorting event with calnexin being involved in the quality control retention of molecules bound for endoplasmic reticulum-associated degradation and ERp57 giving initial protection from degradation and later assisting the maturation of molecules that will exit to the Golgi.     

Folding of hepatitis C virus E1 glycoprotein in a cell-free system

The hepatitis C virus (HCV) envelope proteins, E1 and E2, form noncovalent heterodimers and are leading candidate antigens for a vaccine against HCV. Studies in mammalian cell expression systems have focused primarily on E2 and its folding, whereas knowledge of E1 folding remains fragmentary. We used a cell-free in vitro translation system to study E1 folding and asked whether the flanking proteins, Core and E2, influence this process. We translated the polyprotein precursor, in which the Core is N-terminal to E1, and E2 is C-terminal, and found that when the core protein was present, oxidation of E1 was a slow, E2-independent process. The half-time for E1 oxidation was about 5 h in the presence or absence of E2. In contrast with previous reports, analysis of three constructs of different lengths revealed that the E2 glycoprotein undergoes slow oxidation as well. Unfolded or partially folded E1 bound to the endoplasmic reticulum chaperones calnexin and (with lower efficiency) calreticulin, whereas no binding to BiP/GRP78 or GRP94 could be detected. Release from calnexin and calreticulin was used to assess formation of mature E1. When E1 was expressed in the absence of Core and E2, its oxidation was impaired. We conclude that E1 folding is a process that is affected not only by E2, as previously shown, but also by the Core. The folding of viral proteins can thus depend on complex interactions between neighboring proteins within the polyprotein precursor.     

ER quality control: towards an understanding at the molecular level.

The process of 'quality control' in the endoplasmic reticulum (ER) involves a variety of mechanisms that collectively ensure that only correctly folded, assembled and modified proteins are transported along the secretory pathway. In contrast, non-native proteins are retained and eventually targeted for degradation. Recent work provides the first structural insights into the process of glycoprotein folding in the ER involving the lectin chaperones calnexin and calreticulin. Underlying principles governing the choice of chaperone system engaged by different proteins have also been discovered.