Calnexin is a 90-kDa integral membrane protein of the endoplasmic reticulum (ER). Calnexin binds Ca2+ and may function as a chaperone in the transition of proteins from the ER to the outer cellular membrane. Human calnexin was purified in association with the human interferon-gamma receptor and calnexin cDNA was cloned from placenta. Fragments of calnexin have been prepared as glutathione S-transferase fusion proteins and analyzed for their abilities to bind radioactively labelled Ca2+ and ruthenium red. A subdomain containing four internal repeats binds Ca2+ with the highest affinity. This sequence is highly conserved when compared to calreticulin (a luminal ER protein), an Onchocerca surface antigen, and yeast and plant calnexin homologues. Consequently, this sequence represents a conserved motif for the high-affinity binding of Ca2+, which is clearly distinct from the 'E-F hand' motif. An adjacent subdomain, also highly conserved and containing four internal repeats, fails to bind Ca2+. The carboxyl-terminal, cytosolic domain is highly charged and binds Ca2+ with moderate affinity, presumably by electrostatic interactions. The calnexin amino-terminal domain (residues 1-253) also binds Ca2+, in contrast to the amino-terminal domain of calreticulin, which is relatively less acidic. The cDNA sequences of mouse and rat calnexins have been determined. Comparison of the known mammalian calnexin sequences reveals very high conservation of sequence identity (93%-98%), suggesting that calnexin performs important cellular functions. The gene for human calnexin is located on the distal end of the long arm of human chromosome 5, at 5q35 I (Tjoelker, 1994).

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, it was 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. 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 binds 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 (Danilczyk, 2000).

Calnexin and calreticulin are molecular chaperones, which are involved in the protein folding, assembly, and retention/retrieval. Calreticulin-deficiency is lethal in utero, but the contribution of chaperone function to this phenotype is unknown. Protein folding and chaperone function of calnexin was studied in the absence of calreticulin. Protein folding is accelerated and quality control is compromised in calreticulin-deficient cells. Calnexin-substrate association is severely reduced, leading to accumulation of unfolded proteins and a triggering of the unfolded protein response (UPR). PERK and Ire1alpha and eIF2alpha are also activated in calreticulin-deficient cells. The absence of calreticulin can have devastating effects on the function of the others, compromising overall quality control of the secretory pathway and activating UPR-dependent pathways (Knee, 2003).

The endoplasmic (ER) quality control apparatus ensures that misfolded or unassembled proteins are not deployed within the cell, but are retained in the ER and degraded. A glycoprotein-specific system involving the ER lectins calnexin and calreticulin is well documented, but very little is known about mechanisms that may operate for non-glycosylated proteins. A folding mutant of a non-glycosylated membrane protein, proteolipid protein (PLP), was used to examine the quality control of this class of polypeptide. Calnexin associates with newly synthesized PLP molecules, binding stably to misfolded PLP. Calnexin also binds stably to an isolated transmembrane domain of PLP, suggesting that this chaperone is able to monitor the folding and assembly of domains within the ER membrane. Notably, this glycan-independent interaction with calnexin significantly retards the degradation of misfolded PLP. It is proposed that calnexin contributes to the quality control of non-glycosylated polytopic membrane proteins by binding to misfolded or unassembled transmembrane domains, and these findings are discussed in relation to the role of calnexin in the degradation of misfolded proteins (Swanton, 2003).

Terminally misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytoplasm and degraded by proteasomes through a mechanism known as ER-associated degradation (ERAD). EDEM, a postulated Man8B-binding protein, accelerates the degradation of misfolded proteins in the ER. Here, EDEM was shown to interact with calnexin, but not with calreticulin, through its transmembrane region. Both binding of substrates to calnexin and their release from calnexin were required for ERAD to occur. Overexpression of EDEM accelerated ERAD by promoting the release of terminally misfolded proteins from calnexin. Thus, EDEM appeared to function in the ERAD pathway by accepting substrates from calnexin (Oda, 2003).

Calreticulin and calnexin are homologous lectins that serve as molecular chaperones for glycoproteins in the endoplasmic reticulum of eukaryotic cells. 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 (Molinari, 2004).

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 a pericentriolar endoplasmic reticulum-derived quality control compartment. Surprisingly, ERp57 remains in an endoplasmic reticulum pattern. Using asialoglycoprotein receptors H2a and H2b as models, it was determined in pulse-chase experiments that both glycoproteins initially bind to calnexin and ERp57. However, H2b, which will exit to the Golgi, dissociates from calnexin and remains bound for a longer period to ERp57, whereas the opposite is true for the endoplasmic reticulum-associated degradation substrate H2a that will go to the endoplasmic reticulum-derived quality control compartment. At 15°C, ERp57 colocalizes with H2b adjacent to an endoplasmic reticulum-Golgi intermediate compartment marker. Posttranslational inhibition of glucose excision prolongs association of H2a precursor to calnexin but not to ERp57. Preincubation with a low concentration (15 microg/ml) of the glucosidase inhibitor castanospermine prevents the association of H2a to ERp57 but not to calnexin. This low concentration of castanospermine accelerates the degradation of H2a, suggesting that ERp57, and not calnexin, protects the glycoprotein from degradation. These 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 (Frenkel, 2004).

The UDP-glucose:glycoprotein glucosyltransferase (UGT) is a central player of glycoprotein quality control in the endoplasmic reticulum (ER). UGT reglucosylation of nonnative glycopolypeptides prevents their release from the calnexin cycle and secretion. This study compared the fate of a glycoprotein with a reversible, temperature-dependent folding defect in cells with and without UGT1. Upon persistent misfolding, tsO45 G was slowly released from calnexin and entered a second level of retention-based ER quality control by forming BiP/GRP78-associated disulfide-bonded aggregates. This correlated with loss in the ability to correct misfolding. Deletion of UGT1 did not affect the stringency of ER quality control. Rather, it accelerated release from calnexin and transfer to the second ER quality control level, but it did so after an unexpectedly long lag, showing that cycling in the calnexin chaperone system is not frenetic, as claimed by existing models, and is fully activated only upon persistent glycoprotein misfolding (Molinari, 2005).

Calnexin is a molecular chaperone that resides in the membrane of the endoplasmic reticulum. Most proteins that calnexin binds are N-glycosylated, and treatment of cells with tunicamycin or inhibitors of initial glucose trimming steps interferes with calnexin binding. To test if calnexin is a lectin that binds early oligosaccharide processing intermediates, a recombinant soluble calnexin was created. Incubation of soluble calnexin with a mixture of Glc0-3Man9GlcNAc2 oligosaccharides results in specific binding of the Glc1Man9GlcNAc2 species. Furthermore, Glc1Man5-7GlcNAc2 oligosaccharides bind relatively poorly, suggesting that, in addition to a requirement for the single terminal glucose residue, at least one of the terminal mannose residues is important for binding. To assess the involvement of oligosaccharide-protein interactions in complexes of calnexin and newly synthesized glycoproteins, alpha 1-antitrypsin or the heavy chain of the class I histocompatibility molecule were purified as complexes with calnexin and digested with endoglycosidase H. All oligosaccharides on either glycoprotein were accessible to this probe and could be removed without disrupting the association with calnexin. Furthermore, the addition of 1 M alpha-methyl glucoside or alpha-methyl mannoside had no effect on complex stability. These findings suggest that once complexes between calnexin and glycoproteins are formed, oligosaccharide binding does not contribute significantly to the overall interaction. However, it is likely that the binding of Glc1Man9GlcNAc2 oligosaccharides is a crucial event during the initial recognition of newly synthesized glycoproteins by calnexin (Ware, 1995).

Calnexin and calreticulin are homologous molecular chaperones of the endoplasmic reticulum. Their binding to newly synthesized glycoproteins is mediated, at least in part, by a lectin site that recognizes the early N-linked oligosaccharide processing intermediate, Glc1Man9GlcNAc2. This study compared the oligosaccharide binding specificities of calnexin and calreticulin in an effort to determine the basis for reported differences in their association with various glycoproteins. Using mono-, di-, and oligosaccharides to inhibit the binding of Glc1Man9GlcNAc2 to calreticulin and to a truncated, soluble form of calnexin, it was shown that the entire Glc alpha 1-3Man alpha 1-2Man alpha 1-2Man structure, extending from the alpha 1-3 branch point of the oligosaccharide core, is recognized by both proteins. Furthermore, analysis of the binding of monoglucosylated oligosaccharides containing progressively fewer mannose residues suggests that for both proteins the alpha 1-6 mannose branch point of the oligosaccharide core is also essential for recognition. Consistent with their essentially identical substrate specificities, calnexin and calreticulin exhibit the same relative affinities when competing for binding to the Glc1Man9GlcNAc2 oligosaccharide. Thus, differential glycoprotein binding cannot be attributed to differences in the lectin specificities or binding affinities of calnexin and calreticulin. The effects of ATP, calcium, and disulfide reduction on the lectin properties of calnexin and calreticulin was examined. Whereas oligosaccharide binding is only slightly enhanced for both proteins in the presence of high concentrations of a number of adenosine nucleotides, removal of bound calcium abrogates oligosaccharide binding, an effect that is largely reversible upon readdition of calcium. Disulfide reduction had no effect on oligosaccharide binding by calnexin, but binding by calreticulin was inhibited by 70%. Finally, deletion mutagenesis of calnexin and calreticulin identified a central proline-rich region characterized by two tandem repeat motifs as a segment capable of binding oligosaccharide. This segment bears no sequence homology to the carbohydrate recognition domains of other lectins (Vassilakos, 1998).

The three-dimensional structure of the lumenal domain of the lectin-like chaperone calnexin determined to 2.9Å resolution reveals an extended 140Å arm inserted into a beta sandwich structure characteristic of legume lectins. The arm is composed of tandem repeats of two proline-rich sequence motifs which interact with one another in a head-to-tail fashion. Identification of the ligand binding site establishes calnexin as a monovalent lectin, providing insight into the mechanism by which the calnexin family of chaperones interacts with monoglucosylated glycoproteins (Schrag, 2001).

Calnexin and calreticulin are molecular chaperones of the endoplasmic reticulum that bind to newly synthesized glycoproteins in part through a lectin site specific for monoglucosylated [Glc(1)Man(7-9)GlcNAc(2)] oligosaccharides. In addition to this lectin-oligosaccharide interaction, in vitro studies have demonstrated that calnexin and calreticulin can bind to polypeptide segments of both glycosylated and nonglycosylated proteins. However, the in vivo relevance of this latter interaction has been questioned. This study examined whether polypeptide-based interactions occur between calnexin and its substrates in vivo using the glucosidase inhibitor castanospermine or glucosidase-deficient cells to prevent the formation of monoglucosylated oligosaccharides. If care is taken to preserve weak interactions, the block in lectin-oligosaccharide binding leads to the loss of some calnexin-substrate complexes, but many others remain readily detectable. Furthermore, calnexin is capable of associating in vivo with a substrate that completely lacks Asn-linked oligosaccharides. The binding of calnexin to proteins that lack monoglucosylated oligosaccharides could not be attributed to nonspecific adsorption nor to its inclusion in protein aggregates. It is concluded that both lectin-oligosaccharide and polypeptide-based interactions occur between calnexin and diverse proteins in vivo and that the strength of the latter interaction varies substantially between protein substrates (Danilczyk, 2001).

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, the lectin, ERp57 binding, and polypeptide binding sites of calnexin and calreticulin were localized. 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. The 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 that 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 (Leach, 2002).

Calnexin is an endoplasmic reticulum chaperone that binds to substrates containing monoglucosylated oligosaccharides. Whether calnexin can also directly recognize polypeptide components of substrates is controversial. Calnexin displays significant conformational lability for a chaperone and heat treatment and calcium depletion induces the formation of calnexin dimers and higher order oligomers. These conditions enhance the chaperone activity of calnexin toward glycosylated and non-glycosylated major histocompatibility complex (MHC) class I heavy chains, and enhance calnexin binding to MHC class I heavy chains. In contrast to these observations, calnexin binding to oligosaccharide substrates has been reported to be impaired under calcium-depleting conditions. Calnexin dimers are induced in HeLa cells upon heat shock and under calcium-depleting conditions, and heat shock enhances calnexin binding to MHC class I heavy chains in HeLa cells. Virus-induced endoplasmic reticulum stress also results in the appearance of calnexin dimers. Tunicamycin treatment of HeLa cells induces a slow accumulation of calnexin dimers, the appearance of which correlates with enhanced calnexin binding to deglycosylated MHC class I heavy chains. In vitro, the presence of calnexin-specific oligosaccharides inhibits the formation of calnexin dimers and higher order structures. Together, these data indicate that polypeptide binding is favored by conditions that induce partial unfolding of calnexin monomers, whereas oligosaccharide binding is favored by conditions that enhance the structural stability (folding) of calnexin monomers. Conditions that induce the calnexin 'polypeptide-binding' conformation also induce self-association of calnexin if the concentration is sufficiently high; however, calnexin dimerization/oligomerization per se is not essential for polypeptide substrate binding (Thammavongsa, 2005).

In mammalian cells, the calnexin/calreticulin chaperones play a key role in glycoprotein folding and its control within the endoplasmic reticulum (ER), by interacting with folding intermediates via their monoglucosylated glycans. This lectin activity has been mapped in mammalian calnexin/calreticulin chaperones to the central region, which is a highly conserved feature of calnexin/calreticulin molecules across species. The central domain has also been implicated in Ca(2+) binding, and it has been proposed to be involved in the regulation of calcium homeostasis in the ER. This study shows that although the Schizosaccharomyces pombe calnexin is essential for viability, cells lacking its 317-amino-acid highly conserved central region are viable under normal growth conditions. However, the central region appears to be necessary for optimal growth under high ER-stress, suggesting that this region is important under extreme folding situations (such as DTT and temperature). The minimal length of calnexin required for viability spans the C-terminal 123 residues. Furthermore, cells with the central domain of the protein deleted were affected in their morphology at 37° C, probably due to a defect in cell wall synthesis, although these mutant cells exhibited the same calcium tolerance as wild-type cells at 30° C (Elagoz, 1999).

Calnexin is a molecular chaperone playing key roles in protein folding and the quality control of this process in the endoplasmic reticulum. cnx1⁺, the gene encoding the calnexin homologue in Schizosaccharomyces pombe, is essential for viability. A particular cnx1 mutant induces a novel mechanism allowing the survival of S. pombe cells in the absence of calnexin/Cnx1p. Calnexin independence is dominant in diploid cells and is inherited in a non-Mendelian manner. Remarkably, this survival pathway, bypassing the necessity for calnexin, can be transmitted by transformation of cell extracts into a wild-type naive strain, thus implicating a non-chromosomal factor. Nuclease and UV treatments of cells extracts did not obliterate transmission of calnexin independence by transformation. However, protease digestion of extracts did reduce the appearance of calnexin-independent cells, indicating that a protein element is required for calnexin-less viability. A model is discussed in which this calnexin-less survival mechanism would be activated and perpetuated by a protein component acting as a genetic element (Collin, 2004).

Calnexin, a type I integral Ca(2+)-binding protein in the endoplasmic reticulum (ER) membrane, has been implicated in various biological functions including chaperone activity, calcium homeostasis, phagocytosis, and ER stress-induced apoptosis. Caenorhabditis elegans CNX-1 is expressed in the H-shaped excretory cell, intestine, dorsal and ventral nerve cord, spermatheca, and head and tail neurons throughout development. A cnx-1 null mutant displays temperature-sensitive developmental and reproductive defects, and retarded growth under stress. Moreover, a double knockout mutant of calnexin and calreticulin exhibits additive severe defects. Interestingly, both cnx-1 transcript and protein levels are elevated under stress conditions suggesting that CNX-1 may be important for stress-induced chaperoning functions in C. elegans. Glycosidase treatment and site-directed mutagenesis confirm that CeCNX-1 is N-glycosylated at two asparagine residues of Asn(203) and Asn(571). When transgenic animals from cnx-1 mutant were generated, a glycosylation defective construct failed to rescue phenotypes of cnx-1 mutant suggesting that glycosylation is important for calnexin's functions in C. elegans (Lee, 2005).

Proper folding and maintenance of the native structure are central to protein function and are assisted by a family of proteins called chaperones. Calreticulin and calnexin are ER resident chaperones well conserved from worm to human. Calreticulin/calnexin knock-out mice exhibit a severe phenotype, whereas in Caenorhabditis elegans, calreticulin (crt-1^jh101)- and calnexin (cnx-1^nr2009)-null mutant worms exhibit only a mild phenotype, suggesting the possible existence of alternative chaperone machinery that can compensate for the deficiency of calreticulin and/or calnexin. In order to rapidly identify the compensatory chaperone components involved in this process, the proteome of crt-1^jh101 mutants and crt-1^jh101;cnx-1^nr2009 double mutants was examined. When grown at 20° C, it was found that five proteins were up-regulated and two proteins were down-regulated in crt-1^jh101 mutants; nine proteins were up-regulated and five proteins were down-regulated in crt-1^jh101;cnx-1^nr2009 double mutants. In addition, elevation of the cultivation temperature to 25° C, which is still permissive to growth but causes specific defects in mutants, led to the identification of several additional proteins. Interestingly, the consistent increment of heat shock protein-70 family members (hsp70) together with protein disulfide isomerase (PDI) at all the examined conditions suggests the possible compensatory function imparted by hsp70 and PDI family members in the absence of calreticulin and/or calnexin (Lee, 2006).

Calnexin is a ubiquitously expressed type I membrane protein that is exclusively localized in the endoplasmic reticulum (ER). In mammalian cells, calnexin functions as a chaperone molecule and plays a key role in glycoprotein folding and quality control within the ER by interacting with folding intermediates via their monoglucosylated glycans. In order to gain more insight into the physiological roles of calnexin, calnexin gene-deficient mice were generated. Despite its profound involvement in protein folding, calnexin is not essential for mammalian-cell viability in vivo: calnexin gene knockout mice were carried to full term, although 50% died within 48 h and the majority of the remaining mice had to be sacrificed within 4 weeks, with only a very few mice surviving to 3 months. Calnexin gene-deficient mice were smaller than their littermates and showed very obvious motor disorders, associated with a dramatic loss of large myelinated nerve fibers. Thus, the critical contribution of calnexin to mammalian physiology is tissue specific (Danzel, 2002).

To identify factors involved in the expression of ligand-gated ion channels, nicotinic acetylcholine receptors were expressed in HEK cells to characterize roles for oligosaccharide trimming, calnexin association, and targeting to the proteasome. The homologous subunits of the acetylcholine receptor traverse the membrane four times, contain at least one oligosaccharide, and are retained in the endoplasmic reticulum until completely assembled into the circular arrangement of subunits of delta-alpha-gamma-alpha-beta to enclose the ion channel. Calnexin is associated with unassembled subunits of the receptor, but appears to dissociate when subunits are assembled in various combinations. The glucosidase inhibitor castanospermine was used to block oligosaccharide processing, and thereby inhibit calnexin's interaction with the oligosaccharides in the receptor subunits. Castanospermine treatment reduces the association of calnexin with the alpha-subunit of the receptor, and diminishes the intracellular accumulation of unassembled receptor subunit protein. However, treatment with castanospermine does not appear to alter subunit folding or assembly. In contrast, co-treatment with proteasome inhibitors and castanospermine enhances the accumulation of polyubiquitin-conjugated alpha-subunits, and generally reverses the castanospermine induced loss of alpha-subunit protein. Co-transfection of cDNAs encoding the alpha- and delta-subunits, which leads to the expression of assembled alpha- and delta- subunits, also inhibits the loss of alpha-subunits expressed in the presence of castanospermine. Taken together, these observations indicate that calnexin association reduces the degradation of unassembled receptor subunits in the ubiquitin-proteasome pathway (Keller, 1998).

Calnexin is part of an ER chaperone system that monitors and promotes the proper folding and assembly of glycosylated membrane proteins. To investigate the role of calnexin in the biogenesis of the voltage-dependent Shaker K+ channel, wild-type and mutant Shaker proteins were expressed in mammalian cells. Association with calnexin was assayed by coimmunoprecipitation. Calnexin interacts transiently with wild-type Shaker protein in the ER. In contrast, calnexin failed to associate with an unglycosylated Shaker mutant that makes active, cell surface channels. Therefore, glycosylation of Shaker protein is required for association with calnexin, but calnexin is not required for the proper folding and assembly of Shaker channels. Whether calnexin is involved in the ER retention of mutant Shaker proteins defective in subunit folding, assembly, or pore formation, was investigated. Each of the mutant proteins associated transiently with calnexin during biogenesis. Calnexin dissociated from wild-type and mutant proteins with similar time courses. Thus, non-native Shaker proteins escape the folding sensor of the calnexin chaperone system. Furthermore, stable association with calnexin is not the mechanism by which these mutant proteins are retained in the ER. These results indicate that calnexin is not involved in the quality control of subunit folding, assembly, or pore formation in Shaker K+ channels (Nagaya, 1999).

An unglycosylated form of the Shaker potassium channel protein is retained in the endoplasmic reticulum (ER) and degraded by proteasomes in mammalian cells despite apparently normal folding and assembly. These results suggest that channel proteins with a native structure can be substrates for ER-associated degradation. This hypothesis was tested using the wild-type Shaker protein. Wild-type Shaker is degraded by cytoplasmic proteasomes when it is trapped in the ER and prevented from interacting with calnexin. Neither condition alone is sufficient to destabilize the protein. Proteasomal degradation of the wild-type protein is abolished when ER mannosidase I trimming of the core glycan is inhibited. These results indicate that transient interaction with calnexin provides long-term protection from ER-associated degradation (Khanna, 2004).

Long QT syndrome type 2 is caused by mutations in the human ether-a-go-go-related gene (hERG). The N470D mutation is retained in the endoplasmic reticulum (ER) but can be rescued to the plasma membrane by hERG channel blocker E-4031. The mechanisms of ER retention and how E-4031 rescues the N470D mutant are poorly understood. In this study, the interaction of hERG channels with the ER chaperone protein calnexin was investigated. Using coimmunoprecipitation, it was shown that the immature forms of both wild type hERG and N470D associate with calnexin. The association requires N-linked glycosylation of hERG channels. Pulse-chase analysis revealed that N470D has a prolonged association with calnexin compared with wild type hERG and that E-4031 shortens the time course of calnexin association with N470D. To test whether the prolonged association of N470D with calnexin is due to defective folding of mutant channels, hERG channel folding was studied using the trypsin digestion method. It was found that N470D and the immature form of wild type hERG are more sensitive to trypsin digestion than the mature form of wild type hERG. In the presence of E-4031, N470D becomes more resistant to trypsin even when its ER-to-Golgi transport is blocked by brefeldin A. These results suggest that defective folding of N470D contributes to its prolonged association with calnexin and ER retention and that E-4031 may restore proper folding of the N470D channel leading to its cell surface expression (Gong, 2005).

A mammalian semipermeabilized cell system has been establishes that faithfully reconstitutes the proteasome-mediated degradation of major histocompatibility complex Class I heavy chain. Degradation was shown to require unfolding of the protein and was shown to be cytosol- and ATP-dependent and dislocation and degradation was shown to require proteasome activity. When the interaction of heavy chain with calnexin is prevented, the rate of degradation is accelerated, suggesting that an interaction with calnexin stabilizes the heavy chain. Stabilization of the heavy chain to degradation was also achieved either by preventing mannose trimming or by removal of the N-linked glycosylation site. This demonstrates that glycosylation and mannose trimming are required to ensure heavy chain degradation. When degradation or mannose trimming is inhibited, the heavy chain forms a prolonged interaction with immunoglobulin heavy chain binding protein, ERp57, and protein disulfide isomerase. Taken together, these results indicate that calnexin association and mannose trimming provide a mechanism to regulate the folding, assembly, and degradation of glycoproteins entering the secretory pathway (Wilson, 2000).

Members of the CD1 family of membrane glycoproteins can present antigenic lipids to T lymphocytes. Like major histocompatibility complex class I molecules, they form a heterodimeric complex of a heavy chain and beta(2)-microglobulin [beta(2)m] in the endoplasmic reticulum (ER). Binding of lipid antigens, however, takes place in endosomal compartments, similar to class II molecules, and on the plasma membrane. Unlike major histocompatibility complex class I or CD1b molecules, which need beta(2)m to exit the ER, CD1d can be expressed on the cell surface as either a free heavy chain or associated with beta(2)m. These differences led to an investigation of early events of CD1d biosynthesis and maturation and the role of ER chaperones in its assembly. CD1d associates in the ER with both calnexin and calreticulin and with the thiol oxidoreductase ERp57 in a manner dependent on glucose trimming of its N-linked glycans. Complete disulfide bond formation in the CD1d heavy chain was substantially impaired if the chaperone interactions were blocked by the glucosidase inhibitors castanospermine or N-butyldeoxynojirimycin. The formation of at least one of the disulfide bonds in the CD1d heavy chain is coupled to its glucose trimming-dependent association with ERp57, calnexin, and calreticulin (Kang, 2002).

Calnexin is a membrane-bound lectin of the endoplasmic reticulum (ER) that binds transiently to newly synthesized glycoproteins. By interacting with oligosaccharides of the form Glc(1)Man(9)GlcNAc(2), calnexin enhances the folding of glycoprotein substrates, retains misfolded variants in the ER, and in some cases participates in their degradation. Calnexin has also been shown to bind polypeptides in vivo that do not possess a glycan of this form and to function in vitro as a molecular chaperone for nonglycosylated proteins. To test the relative importance of the lectin site compared with the polypeptide-binding site, six calnexin mutants defective in oligosaccharide binding were generated using site-directed mutagenesis. Expressed as glutathione S-transferase fusions, these mutants were still capable of binding ERp57, a thiol oxidoreductase, and preventing the aggregation of a nonglycosylated substrate, citrate synthase. They were, however, unable to bind Glc(1) Man(9)GlcNAc(2) oligosaccharide and were compromised in preventing the aggregation of the monoglucosylated substrate jack bean alpha-mannosidase. Two of these mutants were then engineered into full-length calnexin for heterologous expression in Drosophila cells along with the murine class I histocompatibility molecules K(b) and D(b) as model glycoproteins. In this system, lectin site-defective calnexin was able to replace wild type calnexin in forming a complex with K(b) and D(b) heavy chains and preventing their degradation. Thus, at least for class I molecules, the lectin site of calnexin is dispensable for some of its chaperone functions (Leach, 2004).

This study investigated how asparagine (N)-linked glycosylation affects assembly of acetylcholine receptors (AChRs) in the endoplasmic reticulum (ER). Block of N-linked glycosylation inhibits AChR assembly whereas block of glucose trimming partially blocks assembly at the late stages. Removal of each of seven glycans has a distinct effect on AChR assembly, ranging from no effect to total loss of assembly. Because the chaperone calnexin associates with N-linked glycans, calnexin interactions with AChR subunits was examined. Calnexin rapidly associates with 50% or more of newly synthesized AChR subunits, but not with subunits after maturation. Block of N-linked glycosylation or trimming does not alter calnexin-AChR subunit associations nor do subunit mutations prevent N-linked glycosylation. Additionally, calnexin associations with subunits lacking N-linked glycans occurs without subunit aggregation or misfolding. These data indicate that calnexin associates with AChR subunits without N-linked glycan interactions. Furthermore, calnexin-subunit associations only occur early in AChR assembly and have no role in events later that require N-linked glycosylation (Wanamaker, 2005).

Apolipoprotein(a) [apo(a)] is a component of atherogenic lipoprotein(a) [Lp(a)]. Differences in the extent of endoplasmic reticulum (ER) associated degradation (ERAD) of apo(a) allelic variants contribute to the >1000-fold variation in plasma Lp(a) levels. Using human apo(a) transgenic mouse hepatocytes, the role of the ER chaperones calnexin (CNX) and calreticulin (CRT), and ER mannosidase I in apo(a) intracellular targeting, was analyzed. Co-immunoprecipitation and pulse-chase analyses revealed similar kinetics of apo(a) interaction with CNX and CRT, peaking 15-30 min after apo(a) synthesis. Trapping of apo(a) N-linked glycans in their monoglucosylated form, by posttranslational inhibition of ER glucosidase activity with castanospermine (CST), enhances apo(a)-CNX/CRT interaction and prevents both apo(a) secretion and ERAD. Delay of CST addition until 20 or 30 min after apo(a) synthesis [when no apo(a) had yet undergone degradation or Golgi-specific carbohydrate modification] allows a portion of apo(a) to be secreted or degraded. These results are consistent with a transient apo(a)-CNX/CRT association and suggest that events downstream of CNX/CRT interaction determine apo(a) intracellular targeting. Inhibition of ER mannosidase I with deoxymannojirimycin or kifunensine has no effect on apo(a) secretion, but inhibits proteasome-mediated apo(a) ERAD even under conditions where apo(a)-CNX/CRT interaction is prevented. These results suggest a role for an additional, mannose-specific, ER lectin in targeting secretory proteins to the proteasome for destruction (Wang, 2000).

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. 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 associates 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 (Pieren, 2005).