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.     

Formation of native hepatitis C virus glycoprotein complexes.

The hepatitis C virus (HCV) glycoproteins (E1 and E2) interact to form a heterodimeric complex, which has been proposed as a functional subunit of the HCV virion envelope. As examined in cell culture transient-expression assays, the formation of properly folded, noncovalently associated E1E2 complexes is a slow and inefficient process. Due to lack of appropriate immunological reagents, it has been difficult to distinguish between glycoprotein molecules that undergo productive folding and assembly from those which follow a nonproductive pathway leading to misfolding and aggregation. Here we report the isolation and characterization of a conformation-sensitive E2-reactive monoclonal antibody (H2). The H2 monoclonal antibody selectively recognizes slowly maturing E1E2 heterodimers which are noncovalently linked, protease resistant, and no longer associated with the endoplasmic reticulum chaperone calnexin. This complex probably represents the native prebudding form of the HCV glycoprotein heterodimer. Besides providing a novel reagent for basic studies on HCV virion assembly and entry, this monoclonal antibody should be useful for optimizing production and isolation of native HCV glycoprotein complexes for serodiagnostic and vaccine applications.     

Antiviral effect of N-butyldeoxynojirimycin against bovine viral diarrhea virus correlates with misfolding of E2 envelope proteins and impairment of their association into E1-E2 heterodimers

The iminosugar N-butyldeoxynojirimycin (NB-DNJ), an endoplasmic reticulum alpha-glucosidase inhibitor, has an antiviral effect against bovine viral diarrhea virus (BVDV). In this report, we investigate the molecular mechanism of this inhibition by studying the folding pathway of BVDV envelope glycoproteins in the presence and absence of NB-DNJ. Our results show that, while the disulfide-dependent folding of E2 glycoprotein occurs rapidly (2.5 min), the folding of E1 occurs slowly (30 min). Both BVDV envelope glycoproteins associate rapidly with calnexin and dissociate with different kinetics. The release of E1 from the interaction with calnexin coincides with the beginning of E1 and E2 association into disulfide-linked heterodimers. In the presence of NB-DNJ, the interaction of E1 and E2 with calnexin is prevented, leading to misfolding of the envelope glycoproteins and inefficient formation of E1-E2 heterodimers. The degree of misfolding and the lack of association of E1 and E2 into disulfide-linked complexes in the presence of NB-DNJ correlate with the dose-dependent antiviral effect observed for this iminosugar.     

Assembly of a functional HCV glycoprotein heterodimer

The two HCV envelope glycoproteins E1 and E2 are released from HCV polyprotein by signal peptidase cleavages. These glycoproteins are type I transmembrane proteins with a highly glycosylated N-terminal ectodomain and a C-terminal hydrophobic anchor. After their synthesis, HCV glycoproteins E1 and E2 associate as a noncovalent heterodimer. The transmembrane domains of HCV envelope glycoproteins play a major role in E1E2 heterodimer assembly and subcellular localization. The envelope glycoprotein complex E1E2 has been proposed to be essential for HCV entry. However, for a long time, HCV entry studies have remained limited because of the lack of a robust cell culture system to amplify this virus. A few years ago, a model mimicking the entry process of HCV lifecycle has been developed by pseudotyping retroviral particles with native HCV envelope glycoproteins. This model allowed the characterization of functional E1E2 envelope glycoproteins. The data obtained can now be confirmed with the help of a newly developed cell-culture system that allows efficient amplification of HCV (HCVcc). Here, we present the recent data that have been accumulated on the assembly of the functional HCV glycoprotein heterodimer.     

Alpha-glucosidase inhibitors reduce dengue virus production by affecting the initial steps of virion morphogenesis in the endoplasmic reticulum

We report that endoplasmic reticulum alpha-glucosidase inhibitors have antiviral effects on dengue (DEN) virus. We found that glucosidase inhibition strongly affects productive folding pathways of the envelope glycoproteins prM (the intracellular glycosylated precursor of M [membrane protein]) and E (envelope protein): the proper folding of prM bearing unprocessed N-linked oligosaccharide is inefficient, and this causes delayed formation of prME heterodimer. The complexes formed between incompletely folded prM and E appear to be unstable, leading to a nonproductive pathway. Inhibition of alpha-glucosidase-mediated N-linked oligosaccharide trimming may thus prevent the assembly of DEN virus by affecting the early stages of envelope glycoprotein processing.     

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.     

Formation and intracellular localization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia and Sindbis viruses.

Hepatitis C virus (HCV) encodes two putative virion glycoproteins (E1 and E2) which are released from the polyprotein by signal peptidase cleavage. In this report, we have characterized the complexes formed between E1 and E2 (called E1E2) for two different HCV strains (H and BK) and studied their intracellular localization. Vaccinia virus and Sindbis virus vectors were used to express the HCV structural proteins in three different cell lines (HepG2, BHK-21, and PK-15). The kinetics of association between E1 and E2, as studied by pulse-chase analysis and coprecipitation of E2 with an anti-E1 monoclonal antibody, indicated that formation of stable E1E2 complexes is slow. The times required for half-maximal association between E1 and E2 were 60 to 85 min for the H strain and more than 165 min for the BK strain. In the presence of nonionic detergents, two forms of E1E2 complexes were detected. The predominant form was a heterodimer of E1 and E2 stabilized by noncovalent interactions. A minor fraction consisted of heterogeneous disulfide-linked aggregates, which most likely represent misfolded complexes. Posttranslational processing and localization of the HCV glycoproteins were examined by acquisition of endoglycosidase H resistance, subcellular fractionation, immunofluorescence, cell surface immunostaining, and immunoelectron microscopy. HCV glycoproteins containing complex N-linked glycans were not observed, and the proteins were not detected at the cell surface. Rather, the proteins localized predominantly to the endoplasmic reticular network, suggesting that some mechanism exists for their retention in this compartment.     

Reconstitution of hepatitis C virus envelope glycoproteins into liposomes as a surrogate model to study virus attachment

The envelope glycoproteins, E1 and E2, of hepatitis C virus (HCV) assemble intracellularly to form a noncovalent heterodimer that is expected to be essential for viral assembly and entry. However, due to the lack of a cell culture system supporting efficient HCV replication, it is very difficult to obtain relevant information on the functions of this glycoprotein oligomer. To get better insights into its biological and biochemical properties, HCV envelope glycoprotein heterodimer expressed by a vaccinia virus recombinant was purified by immunoaffinity. Purified E1E2 heterodimer was recognized by conformation-dependent monoclonal antibodies, showing that the proteins were properly folded. In addition, it interacted with human CD81, a putative HCV receptor, as well as with human low and very low density lipoproteins, which have been shown to be associated with infectious HCV particles isolated from patients. Purified E1E2 heterodimer was also reconstituted into liposomes. E1E2-liposomes were recognized by a conformation-dependent monoclonal antibody as well as by human CD81. Together, these data indicate that E1E2-liposomes are a valuable tool to study the molecular requirements for HCV binding to target cells.     

A Retention Signal Necessary and Sufficient for Endoplasmic Reticulum Localization Maps to the Transmembrane Domain of Hepatitis C Virus Glycoprotein E2

The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2). These glycoproteins interact to form a noncovalent heterodimeric complex which is retained in the endoplasmic reticulum (ER). To identify whether E1 and/or E2 contains an ER-targeting signal potentially involved in ER retention of the E1-E2 complex, these proteins were expressed alone and their intracellular localization was studied. Due to misfolding of E1 in the absence of E2, no conclusion on the localization of its native form could be drawn from the expression of E1 alone. E2 expressed in the absence of E1 was shown to be retained in the ER similarly to E1-E2 complex. Chimeric proteins in which E2 domains were exchanged with corresponding domains of a protein normally transported to the plasma membrane (CD4) were constructed to identify the sequence responsible for its ER retention. The transmembrane domain (TMD) of E2 (C-terminal 29 amino acids) was shown to be sufficient for retention of the ectodomain of CD4 in the ER compartment. Replacement of the E2 TMD by the anchor signal of CD4 or a glycosyl phosphatidylinositol (GPI) moiety led to its expression on the cell surface. In addition, replacement of the E2 TMD by the anchor signal of CD4 or a GPI moiety abolished the formation of E1-E2 complexes. Together, these results suggest that, besides having a role as a membrane anchor, the TMD of E2 is involved in both complex formation and intracellular localization.Hepatitis C virus (HCV) is an enveloped virus which belongs to the Flaviviridae family (15). Its genome encodes two membrane-associated envelope glycoproteins (E1 and E2). E1 and E2 glycoproteins interact to form a complex which has been proposed as a functional subunit of HCV virions (11, 17, 26, 41). Characterization of HCV glycoprotein complex formation expressed by using the vaccinia/T7 or Sindbis virus system indicates that a majority of HCV glycoproteins are misfolded (9, 11). Recently, we have produced a monoclonal antibody (MAb) which recognizes properly folded E2 and precipitates native HCV glycoprotein complexes but not misfolded aggregates (9). Properly folded E1 and E2 interact to form a heterodimer stabilized by noncovalent interactions, and the kinetics of association between E1 and E2 indicate that the formation of stable E1-E2 complexes is slow (half-time of association [t_1/2] ≈ 2 h). The folding of E1 and E2 has been studied and indicates that formation of intramolecular disulfide bonds is slow for E1 (t_1/2 > 1 h) whereas it is rapid for E2 (12). By using human and mouse MAbs, it has been shown that folding of a subdomain(s) of E2 correlates with acquisition of intramolecular disulfide bonds but that complete folding of E2 is slow (t_1/2 ≈ 2 h) (9, 19). In addition, E1 expressed in the absence of E2 does not fold properly, suggesting that E2 plays a chaperone-like role in the folding of E1 (32).The HCV glycoproteins are heavily modified by N-linked glycosylation and contain hydrophobic domains in their carboxy-terminal regions acting presumably as membrane anchors, giving the proteins a type I membrane topology (43). The E2 glycoprotein extends to residue 746 (position on the polyprotein), and deletions of at least 31 C-terminal amino acids lead to its secretion (47). This is in accordance with other data proposing that the hydrophobic anchor domain begins at amino acid 718 (33). However, only a shorter secreted form of E2 glycoprotein ending at amino acid 661 appears to be properly folded (32). For E1, a larger deletion (71 amino acids) seems to be necessary for its secretion, but this secreted protein is not properly folded (32).Due to the lack of an efficient cell culture replication system, HCV particle assembly and release have not been examined directly. However, the lack of complex glycans, the endoplasmic reticulum (ER) localization of expressed HCV glycoproteins (11, 41), and the absence of these proteins on the cell surface (11, 49) suggest that initial virion morphogenesis may occur by budding into intracellular vesicles. More recently, we have confirmed that the mature E1-E2 heterodimer does not leave the ER, suggesting that E1 and/or E2 contains a signal for retention of the heterodimer in this compartment (9).In this study, we show that E2 glycoprotein expressed alone is retained in the ER similarly to the E1-E2 heterodimer and that a signal for ER retention of E2 resides in its transmembrane domain (TMD) (C-terminal 29 amino acids). The evidence for this retention signal was derived from expression of chimeric proteins in which E2 domains were exchanged with corresponding domains of a protein normally transported to the plasma membrane (CD4).     

Glycopeptide specificity of the secretory protein folding sensor UDP-glucose glycoprotein:glucosyltransferase

Secretory and membrane N-linked glycoproteins undergo folding and oligomeric assembly in the endoplasmic reticulum with the aid of a folding mechanism known as the calnexin cycle. UDP–glucose glycoprotein:glucosyltransferase (UGGT) is the sensor component of the calnexin cycle, which recognizes these glycoproteins when they are incompletely folded, and transfers a glucose residue from UDP–glucose to N-linked Man9-GlcNAc2 glycans. To determine how UGGT recognizes incompletely folded glycoproteins, we used purified enzyme to glucosylate a set of Man9-GlcNAc2 glycopeptide substrates in vitro, and determined quantitatively the glucose incorporation into each glycan by mass spectrometry. A ranked order of glycopeptide specificity was found that provides the criteria for the recognition of substrates by UGGT. The preference for amino-acid residues close to N-linked glycans provides criteria for the recognition of glycopeptide substrates by UGGT.     

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.     

Role of N-linked glycans of HCV glycoprotein E1 in folding of structural proteins and formation of viral particles

Envelope proteins E1 and E2 of the hepatitis C virus (HCV) play a major role in the life cycle of a virus. These proteins are the main components of the virion and are involved in virus assembly. Envelope proteins are modified by N-linked glycosylation, which is supposed to play a role in their stability, in the assembly of the functional glycoprotein heterodimer, in protein folding, and in viral entry. The effects of N-linked glycosylation of HCV protein E1 on the assembly of structural proteins were studied using site-directed mutagenesis in a model system of Sf9 insect cells producing three viral structural proteins with the formation of virus-like particles due to the baculovirus expression system. The removal of individual N-glycosylation sites in HCV protein E1 did not affect the efficiency of its expression in insect Sf9 cells. The electrophoretic mobility of E1 increased with a decreasing number of N-glycosylation sites. The destruction of E1 glycosylation sites N1 or N5 influenced the assembly of the noncovalent E1E2 glycoprotein heterodimer, which is the prototype of the natural complex within the HCV virion. It was also shown that the lack of glycans at E1 sites N1 and N5 significantly reduced the efficiency of E1 expression in mammalian HEK293 T cells.     

Recognition of native hepatitis C virus E1E2 heterodimers by a human monoclonal antibody

The majority of hepatitis C virus (HCV)-infected individuals progress from acute to chronic disease, despite the presence of a strong humoral immune response to the envelope glycoproteins E1 and E2. When expressed in mammalian cells, E1 and E2 form both noncovalently linked E1E2 heterodimers, believed to be properly folded, and disulfide-linked, high-molecular-weight aggregates that are misfolded. Previously, we identified 10 human monoclonal antibodies (HMAbs) that bind E2 glycoproteins from different genotypes. Here we demonstrate that one of these HMAbs, CBH-2, is unique in its ability to distinguish between properly folded and misfolded envelope proteins. This HMAb recognizes HCV-E2 only when complexed with E1. The E1E2 complexes recognized by CBH-2 are noncovalently linked heterodimers and not misfolded disulfide-linked, high-molecular-weight aggregates. The E1E2 heterodimers seen by CBH-2 no longer associate with the endoplasmic reticulum chaperone calnexin and are likely to represent the prebudding form of the HCV virion.     

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.     

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.     

UDP-GlC:glycoprotein glucosyltransferase-glucosidase II,the ying-yang of the ER quality control

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.     

Palmitoylated calnexin is a key component of the ribosome-translocon complex

A third of the human genome encodes N-glycosylated proteins. These are co-translationally translocated into the lumen/membrane of the endoplasmic reticulum (ER) where they fold and assemble before they are transported to their final destination. Here, we show that calnexin, a major ER chaperone involved in glycoprotein folding is palmitoylated and that this modification is mediated by the ER palmitoyltransferase DHHC6. This modification leads to the preferential localization of calnexin to the perinuclear rough ER, at the expense of ER tubules. Moreover, palmitoylation mediates the association of calnexin with the ribosome-translocon complex (RTC) leading to the formation of a supercomplex that recruits the actin cytoskeleton, leading to further stabilization of the assembly. When formation of the calnexin-RTC supercomplex was affected by DHHC6 silencing, mutation of calnexin palmitoylation sites or actin depolymerization, folding of glycoproteins was impaired. Our findings thus show that calnexin is a stable component of the RTC in a manner that is exquisitely dependent on its palmitoylation status. This association is essential for the chaperone to capture its client proteins as they emerge from the translocon, acquire their N-linked glycans and initiate folding.     

How sugars convey information on protein conformation in the endoplasmic reticulum

The N-glycan-dependent quality control of glycoprotein folding prevents endoplasmic reticulum to Golgi exit of folding intermediates, irreparably misfolded glycoproteins and not completely 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 and a glucosidase that creates monoglucosylated epitopes in glycans transferred in protein N-glycosylation or 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 purpose of the review is to describe the most significant recent findings on the mechanism of glycoprotein folding and assembly quality control and to discuss the main still unanswered questions.     

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.     

Calnexin Improves the Folding Efficiency of Mutant Rhodopsin in the Presence of Pharmacological Chaperone 11-cis-Retinal

The lectin chaperone calnexin (Cnx) is important for quality control of glycoproteins, and the chances of correct folding of a protein increase the longer the protein interacts with Cnx. Mutations in glycoproteins increase their association with Cnx, and these mutant proteins are retained in the endoplasmic reticulum. However, until now, the increased interaction with Cnx was not known to increase the folding of mutant glycoproteins. Because many human diseases result from glycoprotein misfolding, a Cnx-assisted folding of mutant glycoproteins could be beneficial. Mutations of rhodopsin, the glycoprotein pigment of rod photoreceptors, cause misfolding resulting in retinitis pigmentosa. Despite the critical role of Cnx in glycoprotein folding, surprisingly little is known about its interaction with rhodopsin or whether this interaction could be modulated to increase the folding of mutant rhodopsin. Here, we demonstrate that Cnx preferentially associates with misfolded mutant opsins associated with retinitis pigmentosa. Furthermore, the overexpression of Cnx leads to an increased accumulation of misfolded P23H opsin but not the correctly folded protein. Finally, we demonstrate that increased levels of Cnx in the presence of the pharmacological chaperone 11-cis-retinal increase the folding efficiency and result in an increase in correct folding of mutant rhodopsin. These results demonstrate that misfolded rather than correctly folded rhodopsin is a substrate for Cnx and that the interaction between Cnx and mutant, misfolded rhodopsin, can be targeted to increase the yield of folded mutant protein.