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Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013.

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Calnexin and Calreticulin, Molecular Chaperones of the Endoplasmic Reticulum

and .

In this chapter we present the evidence that calnexin (CNX) and calreticulin (CRT) function as molecular chaperones to assist in the folding and subunit assembly of the majority of Asn-linked glycoproteins that pass through the endoplasmic reticulum. Mechanistic insights into how this function is accomplished have been provided through diverse approaches which include interfering with the recognition of glycoproteins through CNX/CRT's lectin site, expression of CNX/CRT and model substrates in heterologous systems, gene disruption, and reconstitution of function with purified components in vitro. Furthermore, the domain organization and locations of functional sites have been revealed through mutagenesis and the recent determination of the structure of the ER luminal domain of CNX and a portion of CRT. The controversial issue of whether CNX/CRT function solely as lectins or also as “classical” chaperones that recognize the unfolded polypeptide portion of glycoproteins is presented and the evidence supporting current models is discussed in detail.

Introduction

In 1991, CNX was discovered virtually simultaneously by three groups as a protein that interacts with partially assembled class I histocompatibility molecules,1 with partial complexes of T cell receptors and membrane immunoglobulins,2 and also as a microsomal membrane protein that can be phosphorylated in vitro.3 Since then, CNX has been shown to interact transiently with a wide array of newly synthesized membrane or soluble proteins that pass though the ER.46 Given the substantial sequence identity between CNX and CRT it was not long before CRT was demonstrated to share with CNX the ability to bind transiently to diverse nascent proteins.7,8 In many cases, CNX and CRT were demonstrated to associate with folding or assembly intermediates but not with native conformers. For example, CNX binds to incompletely disulfide-bonded forms of influenza hemagglutinin (HA)9 and transferrin6,10 but dissociates at about the time these proteins become fully oxidized. In other cases it binds to individual subunits of proteins such as major histocompatibility complex (MHC) class I11 or class II12 molecules, the insulin receptor13 or integrins14 and dissociates at the time of oligomeric assembly. CRT behaves in similar fashion, binding primarily to partially oxidized HA7 or to myeloperoxidase prior to heme assembly.8 These early studies suggested that CNX and CRT are molecular chaperones, i.e., proteins that bind to non-native protein conformers by recognition of exposed hydrophobic segments and, through cycles of binding and release, prevent aggregation thereby allowing productive folding/assembly to occur more efficiently.

Another important finding was that CNX and CRT exhibit prolonged interaction with misfolded or incompletely assembled proteins and that this interaction correlates with extended residence of the non-native proteins within the ER.4,7,11,14 These prolonged interactions suggested that CNX and CRT might be components of the ER quality control system that prevents non-native proteins from being exported from the ER. Indeed both molecules have subsequently been shown directly to participate in quality control.1517 Since the topic of quality control is discussed elsewhere in this volume (see Helenius and Ellgaard entry) we will focus on the roles of CNX and CRT as molecular chaperones by examining their structures, ligand binding properties, protein binding specificities, the evidence that they assist protein folding and assembly, and the possible mechanisms whereby they effect this latter function.

Structure and Ligand Binding Properties of CNX and CRT

Mammalian CNX is a ˜570 residue type I membrane protein of the ER3,4 whereas CRT is a ˜400 amino acid soluble protein18,19 that resides primarily within the ER lumen (Fig. 6.1). They share ˜39% overall sequence identity with highest identity occurring in a central segment consisting of two tandemly repeated sequence motifs. Motif 1 [I-DP(D/E)A-KPEDWD(D/E)] is repeated four times in CNX followed by four copies of motif 2 [G-W--P-IN-P-Y]. In CRT, there are three copies of each motif. Both proteins bind Ca2+ with high affinity at a site within the tandem repeats and also have multiple sites for low affinity Ca2+ binding within the highly acidic N- and C-terminal regions of CNX20 and the C-terminal region of CRT.21 CRT also possesses two Zn2+ binding sites in its N-terminal region.22 Both CNX and CRT bind ATP although no ATPase activity has been detected as yet.2326 Furthermore, as demonstrated by chemical cross-linking27 and by direct binding experiments,28,29 CNX and CRT interact with ERp57, a thiol oxidoreductase of the ER. CRT also binds to protein disulfide isomerase under conditions of low Ca2+ concentration.30

Figure 1. Features of the primary structures of canine calnexin and rabbit calreticulin.

Figure 1

Features of the primary structures of canine calnexin and rabbit calreticulin. Regions of the two proteins that share substantial sequence identity are indicated by the white rectangles. The numbers 1 and 2 represent the two tandemly repeated sequence (more...)

Perhaps the most distinctive property of CNX and CRT is that they are both lectins with specificity for a monoglucosylated oligosaccharide present on Asn-linked glycoproteins. A lectin function was initially suggested for CNX based on the observation that treatment of human hepatoma cells with the Asn-linked glycosylation inhibitor tunicamycin prevented the association of CNX with most newly synthesized proteins.6 Subsequent experiments demonstrated that inhibitors of glucosidases I and II, ER enzymes that sequentially remove the three glucose residues from the initially attached Glc3Man9GlcNAc2 oligosaccharide (see Fig. 6.2), also inhibited the binding of CNX31 and CRT7 to most glycoproteins. This finding, coupled with the demonstration that oligosaccharides with a single terminal glucose residue are present on glycoproteins bound to CNX or CRT, led to the suggestion that the Glc1Man9GlcNAc2 oligosaccharide is the specific oligosaccharide recognized by these lectins.31 This was subsequently confirmed by direct binding experiments in vitro using purified, immobilized CNX or CRT and various radiolabeled oligosaccharides containing 0–3 glucose residues (Glc0–3Man9GlcNAc2). Only the monoglucosylated species bound to the immobilized proteins.3234 Additional binding specificity studies involving progressive removal of mannose residues revealed that the Glc1Man5–9GlcNAc2 species were capable of binding but binding of the Glc1Man4GlcNAc2 species was undetectable, indicating that both the innermost a1-6 branched mannose and the terminal glucose were important for recognition by CNX and CRT. Furthermore, binding competition experiments using monglucosylated di-, tri-, and tetrasaccharides demonstrated that the lectin sites of CNX and CRT recognize the entire glucosylated arm of the oligosaccharide, i.e., Glcα1–3Manα1–2Manα1–2Man (bold residues in Fig. 6.2).34 The presence of Ca2+ was found to be essential for the lectin functions of both CNX and CRT.34

Figure 2. Oligosaccharide binding specificity of CNX and CRT.

Figure 2

Oligosaccharide binding specificity of CNX and CRT. Shown is the Glc3Man9GlcNAc2 oligosaccharide that is initially transferred to Asn residues of nascent polypeptide chains. This is subsequently processed by the sequential action of ER glucosidases I (more...)

Recently, the structure of the ER luminal domain of canine CNX (residues 41–438, numbered as in Fig. 6.1) was solved at 2.9 Å resolution by X-ray crystallography.35 The structure consists of two distinct domains: a globular β sandwich domain (residues 41–242 and 395–438) containing two antiparallel β sheets and an elongated arm domain (residues 250–394) that extends 140 Å away from the globular domain (Fig. 6.3). The globular domain resembles both legume lectins and galectins, and, consistent with this similarity, soaking the crystal in 50 mM a-D-glucose revealed monosaccharide binding to the globular domain near the base of the arm. A bound Ca2+ ion was also present within the globular domain (Fig. 6.3) which represents a distinct site compared to previous mapping studies that localized high affinity Ca2+ binding to the repeat motifs.34 The arm consists of the repeat motifs in an extended hairpin loop with the four copies of motif 1 forming one strand of the loop and the four copies of motif 2 folding back on the motif 1 repeats to complete the hairpin. Each motif 1 interacts with a corresponding motif 2 in a head-to-tail orientation to form four distinct modules. The structure of the repeat segment, or P domain, of rat CRT was also recently solved by NMR (residues 188–288, numbered as in Fig. 6.1).36 It also exists as a hairpin with the three copies of motif 1 interacting with the three copies of motif 2 to form three modular units (Fig. 6.3). Indeed, the last copy of motif 1 and first copy of motif 2 that together form the most distal module near the loop of the hairpins in CNX and CRT are nearly superimposable in the two structures.35

Figure 3. Structures of the ER luminal segment of calnexin and of the “repeats” or arm domain of calreticulin.

Figure 3

Structures of the ER luminal segment of calnexin and of the “repeats” or arm domain of calreticulin. The structures shown correspond to residues 41–438 of CNX and residues 188–288 of CRT (numbered as in Fig. 6.1). The CNX (more...)

The extended arm domain is an obvious candidate for a protein interaction site. In a series of mapping experiments, we prepared deletion constructs consisting of the individual globular and arm domains fused to GST. When tested for binding to radiolabeled ERp57, the globular domain failed to interact whereas the most distal three repeat modules of the CNX arm bound ERp57 as did the two most distal modules of CRT (Fig. 6.3).37 ERp57 binding to the tip of the CRT arm domain has also been reported by Ellgaard and co-workers (see Chapter 3). Since glucose is not the physiological ligand for binding to the lectin sites of CNX and CRT, we tested the single domain constructs for binding to radiolabeled Glc1Man9GlcNAc2 oligosaccharide. In this case, the globular domains of CNX and CRT retained the bulk (˜70%) of the oligosaccharide binding capability of the full length proteins whereas the arm domains consistently exhibited about 10–15% binding (Fig. 6.3).37 This clearly confirms that the globular domain contains the lectin site for the physiologically relevant oligosaccharide. Furthermore, the persistent low level binding by the arm domain, while unexplained, helps to clarify previous reports that erroneously mapped the lectin site to this segment of CNX and CRT.34,38

Differences in Binding Specificity of CNX and CRT for Newly Synthesized Glycoproteins

Two-dimensional isoelectric focusing/SDS-PAGE analysis of glycoproteins that co-immunoisolate with CNX or CRT reveals that both molecules interact with roughly 50–100 newly synthesized proteins.4,5,39 Indeed it is likely that most if not all glycoproteins bind to CNX, CRT or both at some stage in their biogenesis within the ER. However, even by one-dimensional SDS-PAGE analysis it is obvious that overlapping but distinctly different sets of glycoproteins interact with CNX versus CRT.7,17 Many individual glycoproteins have been examined for their interactions with CNX or CRT and it is clear that no specific topological category of glycoprotein is preferentially bound by either chaperone, i.e., soluble, type I or type II membrane spanning, or polytopic glycoproteins can be found associated with either CNX or CRT (reviewed in ref. 40). Some glycoproteins such as the vesicular stomatitis virus G glycoprotein7 and nicotinic acetylcholine receptor41 bind to CNX but not CRT whereas others such as influenza HA7 or the a and β subunits of the T cell receptor (TCR) interact with both.39 In some instances, simultaneous interactions of CNX and CRT with an individual glycoprotein molecule have been reported.42,43 There are also examples of temporal differences in chaperone interaction, as exemplified by the human MHC class I molecule. The free class I heavy chain (H chain) initially binds exclusively to CNX but, upon H chain assembly with the β2-microglobulin subunit, CNX dissociates and is rapidly replaced by CRT. CRT then remains bound during assembly of a muti-component complex that facilitates loading of peptide ligands onto the class I molecule for subsequent display at the cell surface to cytotoxic T cells.44,45

Some studies suggest that it is the distinct topological relationship between CNX, CRT and the oligosaccharide chains of the various glycoproteins they bind that influences substrate selection. When CRT was expressed as a membrane-anchored protein in human hepatoma cells, the pattern of interacting glycoproteins resembled that of CNX.46 Similar results were obtained in a separate study in which CRT was expressed as a membrane-anchored protein in mouse L cells and a CNX-like pattern of interacting proteins was obtained. Conversely, when CNX was expressed as a soluble protein in L cells, its substrate specificity switched to resemble that of CRT.17 In a comprehensive study examining the effect of altering oligosaccharide location on a substrate glycoprotein, influenza HA, it was observed that CRT interacted preferentially with the rapidly folding top/hinge domain of HA which is presumably more accessible to the ER lumen. However, CNX was less discriminating in its interactions, binding to both the top/hinge domain and the membrane-proximal stem domain.43 Collectively, these findings are consistent with the view that the distinct membrane versus soluble topologies of the lectin sites of CNX and CRT play a role in substrate selection. Interestingly, the substrate preferences of CNX and CRT can be overcome under some circumstances. For example, although free MHC class I H chains normally bind exclusively to CNX in mouse cells, when co-expressed in Drosophila cells with mammalian CRT but not CNX, CRT can substitute for the chaperone and quality control functions of CNX.17

Molecular Chaperone Functions of CNX and CRT

Several approaches have been used to study the involvement of CNX and CRT in glycoprotein folding and subunit assembly. The most common is to use inhibitors of ER glucosidases I and II, such as castanospermine (CAS) or deoxynojirimycin (DNJ), to prevent the formation of monoglucosylated oligosaccharides. This approach does not permit an examination of the individual functions of CNX or CRT. It is also limited in that the oligosaccharides of all cellular glycoproteins are affected and hence the possibility exists that any observed alteration in glycoprotein folding may not be a direct consequence of impaired CNX or CRT binding. Nevertheless, the accumulated data are consistent with a role for these molecules in enhancing correct folding of many glycoproteins. For example, treatment of dog pancreas microsomes with CAS doubled the rate of disulfide oxidation and oligomerization of influenza HA, but decreased overall folding efficiency by increasing aggregate formation and enhancing degradation.47 In the case of MHC class I molecules, CST treatment increased aggregate formation and reduced assembly efficiency in murine cells48 and slowed disulfide formation in human cells.49 CST or DNJ treatment also abolished expression of tyrosinase activity in Cos 750 or B16 melanoma cells,51 caused premature dimerization and misfolding of the insulin receptor,13 inhibited folding of the VSV G glycoprotein52 and HIV gp120 glycoprotein,53 and decreased folding, assembly and surface expression of the nicotinic acetylcholine receptor.54

An independent approach that does not utilize glucosidase inhibitors involves heterologous expression of mouse MHC class I subunits in Drosophila melanogaster cells in the absence or presence of mammalian CNX or CRT. It was found that co-expression of CNX increased folding efficiency of the H chain subunit, stabilized it against rapid degradation, and enhanced its assembly with the β2-microglobulin subunit by as much as five-fold.48 In a subsequent study, using the same approach, CRT was shown to exert similar effects as those observed for CNX.17

Finally, the functions of CNX and CRT have been examined by disrupting expression of the corresponding genes in a variety of cell lines and organisms. Surprisingly, in a CNX-deficient human leukemia cell line, there was no observable phenotype in MHC class I assembly, intracellular transport, or antigen presentation function.55,56 However, this might be explained by compensatory action of CRT since, as shown in the Drosophila experiments above, the functions of CNX and CRT were largely interchangeable at least during early stages of class I folding and assembly. In contrast, in fibroblasts derived from CRT-deficient mice, newly synthesized class I molecules were prematurely released from the ER and were profoundly deficient in assembling with their peptide ligands57 (also see entry by T. Elliott in this volume). This suggests that CNX may be less flexible than CRT in assuming a solo role in enhancing class I assembly and participating in quality control.

CRT-deficient mice have been produced which exhibit an embryonic lethal phenotype. Severe defects in heart development were observed which may be more related to CRT's role in Ca2+ homeostasis than to a molecular chaperone function.58 Recently, both the CNX and CRT genes were disrupted in the amoeba Dictyostelium discoideum.59 The double mutants were viable, exhibiting a moderately reduced growth rate, and were capable of chemotactic responses to cAMP. The most notable defect was a severe impairment in phagocytosis. However, since phagocytosis is strongly dependent on cytosolic Ca2+ concentration it is unclear if the defect is due to a lack of CNX/CRT's chaperone functions or a loss of their Ca2+ storage capacity.59 In yeast cells, only the CNX gene is present and gene disruption experiments have demonstrated that CNX is essential for viability in Schizosaccharomyces pombe60,61 whereas growth is normal in CNX-deficient Saccharomyces cerevisiae cells.62 The basis for the lethal phenotype in S. pombe is unclear. It appears not to be due to a lack of lectin-mediated interactions of CNX with monoglucosylated glycoproteins since various mutations that prevent the formation of monoglucosylated oligosaccharides in this organism do not show a discernable phenotype under normal growth conditions.63,64 There is some evidence that CNX deficiency in S. cerevisiae affects chaperone/quality control function since the cell-surface expression of the normally ER-retained ste2–3p allele of the α-pheromone receptor is increased as is the secretion of heterologously expressed mammalian α1-antitrypsin.62

Mechanisms of Chaperone Action—The “Lectin Only” versus “Dual Binding” Controversy

There is a debate concerning how CNX and CRT interact with folding glycoproteins which centers on whether the association is solely lectin-oligosaccharide based or if there is an additional protein-protein interaction. The two models are depicted in Figure 6.4. In the “lectin-only” model originally proposed by Helenius and co-workers,31,65 cycles of CNX/CRT binding and release are controlled by the availability of the terminal glucose residue on monoglucosylated Asn-linked oligosaccharides. Initial binding occurs following the trimming of the precursor Glc3Man9GlcNAc2 oligosaccharide to the monoglucosylated form by the sequential action of glucosidases I and II. Dissociation then occurs through the further action of glucosidase II (probably during transient glycoprotein release controlled by the low affinity of oligosaccharide binding [Kd ˜ 1–2 μM])66 and, if folding does not occur rapidly, re-binding can occur through reglucosylation of the glycoprotein by UDP-glucose:glycoprotein glucosyltransferase (UGGT). UGGT is the folding sensor in the cycle since it will only reglucosylate non-native glycoproteins.67,68 In this model, CNX and CRT do not function as molecular chaperones in that they lack the ability to suppress aggregation through binding to exposed hydrophobic segments of the unfolded glycoprotein. Rather they are thought to recruit other ER chaperones and folding enzymes such as ERp57 to the unfolded subtrate which in turn are responsible for promoting more efficient folding. Indeed the interaction of ERp57 with CNX or CRT has been shown in vitro to enhance dramatically the formation of disulfide bonds within monoglucosylated RNase B that is bound to the lectin site of CNX or CRT.28 The lectin-oligosaccharide based binding also effects retention of non-native glycoproteins in the ER and thus provides the basis for the functions of CNX/CRT in quality control.

Figure 4. Mechanisms of calnexin and calreticulin action as described by the “lectin-only” and “dual-binding” models.

Figure 4

Mechanisms of calnexin and calreticulin action as described by the “lectin-only” and “dual-binding” models. Details of the models are described in the text. ERp57 catalyzes disulfide bond formation and isomerization within (more...)

The “dual binding” model proposed by Williams and co-workers,25,32 incorporates the central aspects of the lectin-only model but, in addition, proposes the existence of a second substrate binding site on CNX/CRT that recognizes exposed hydrophobic segments of the unfolded polypeptide chain. Substrate dissociation involves not only the action of glucosidase II but a change in affinity of the polypeptide binding site, possibly regulated by a shift from an ATP-bound to an ADP-bound or unbound state.25,26 Again, if folding does not occur rapidly, the glycoprotein is reglucosylated by UGGT and can then re-bind in dual fashion to the ATP form of CNX/CRT. In this model, both UGGT and CNX/CRT act as folding sensors. The central difference between the models is that CNX and CRT function as classical molecular chaperones that suppress aggregation in addition to being capable of recruiting folding factors such as ERp57.

There is a large body of evidence that argues both for and against the two models. Support for the lectin-only model comes from the finding that cells lacking glucosidases I and II or treatment of cells with glycosylation or glucosidase inhibitors usually results in a dramatic reduction in the amounts of glycoproteins co-immunoisolating with CNX or CRT.68,31,42,48,50,69,70 In addition, treatment of cells with glucosidase inhibitors after complexes are formed impairs complex dissociation supporting the view that glucosidase II is important for complex dissociation.10,65,71 Furthermore, cycles of deglucosylation and reglucosylation have been clearly demonstrated in microsomal and cellular systems and have been demonstrated to be important for efficient glycoprotein folding.10,65,71,72 However, what is frequently overlooked in reviewing these studies is that there is quite a spectrum of effects observed. For example, under conditions where glycosylation or glucosidase activity are inhibited, complexes are not detected between CNX or CRT and the α and β subunits of the T cell receptor,5 influenza HA,31 VSV G glycoprotein,52 RNase B,73 myeloperoxidase,8 cruzipain,74 and tyrosinase.50 However, complexes can readily be detected at normal or reduced levels with the ϵ and δ subunits of the T cell receptor,16,75 P glycoprotein,76 erythrocyte AE1,77 acid phosphatase,78 MHC class II α and β chains,79 MHC class II invariant chain,80 MHC class I H chain,81 and HIV gp160.42 Interestingly, CAS treatment almost completely prevented the formation of complexes between CNX and coagulation factors V and VIII but only partially inhibited the formation of complexes with CRT.82 Furthermore, CAS prevented the formation of complexes between CNX and the a subunit of the acetylcholine receptor in one study but had little apparent effect on complex formation in another study in which a different detergent was employed for cell lysis and recovery of CNX-α-subunit complexes.41,54 When the entire spectrum of CNX or CRT associated proteins were examined variable results have also been apparent. For example, Kearse et al. observed strong association of many proteins with CNX following CAS treatment or in the glucosidase II-deficient PhaR2.7 cell line, even though associations with TCRα and TCRβ were virtually eliminated.5 In contrast, Helenius and co-workers observed an almost complete elimination of CNX- or CRT-associated proteins in PhaR2.7 cells or in CAS-treated cells.7,70 Therefore, it appears that there are significant differences in the extent to which individual glycoproteins may bind to CNX or CRT via lectin-oligosaccharide independent interactions and that varying results can arise depending on the specific cell lysis and immune isolation conditions employed.

There have been two reports in which the interactions of CRT and/or CNX were studied with different conformational forms of monoglucosylated RNase B.73,83 These studies, conducted either with purified components in vitro or in a microsomal system with in vitro translated RNase B, demonstrated that binding to CNX and CRT was absolutely dependent on the presence of monoglucosylated oligosaccharide whereas the conformational status of the polypeptide chain did not affect the interaction. These studies have been highly cited in support of the lectin-only model but they suffer from one major drawback. A hydropathy plot of RNase reveals that this protein lacks hydrophobic segments considered essential for the binding of molecular chaperones that recognize substrates via protein-protein interactions.84 Consistent with this lack of hydrophobic character, RNase fails to aggregate even upon heating to 100°C.

There are a number of lines of evidence to support the concept that CNX and CRT are capable of recognizing glycoproteins via protein-protein interactions, i.e., the dual-binding model. First, pre-formed complexes between CNX and either membrane-bound (MHC class I and II molecules) or soluble glycoproteins (α1-antitrypsin) could not be dissociated by enzymatic removal of oligosaccharides.32,79,85 However, it has been speculated that the observed lack of dissociation may be due to the trapping of the two species within the same detergent micelle.73,83,86 Such an argument cannot be applied to the interaction with α1-antitrypsin but rather it has been suggested that this substrate, being non-native, might become insoluble upon dissociation and thereby associate with CNX non-specifically.73,83,86 Second, there are many examples of CNX or CRT interacting at normal or reduced levels with proteins that either completely lack Asn-linked oligosaccharides or, as described above, with glycoproteins lacking monoglucosylated oligosaccharides through glucosidase deficiency or inhibition.16,41,75,76,78,80,82,87,88 These studies, particularly those with non-glycosylated proteins, have been criticized on the basis that the substrate may aggregate and trap CNX or CRT non-specifically.73,83,86 Indeed CNX has been detected in association with aggregates of non-glycosylated VSV G protein.89 Third, both CNX and CRT have been shown to bind specifically to non-glycosylated peptides both in vitro and in vivo.9093 In one study, the binding of 39 different peptides to CRT was examined and a marked preference for hydrophobic peptides lacking acidic residues was noted. There also appeared to be a minimum length requirement of ˜ 10 residues.91 Fourth, and perhaps most compelling, is that the purified ER luminal domain of CNX (S-CNX) and CRT were capable of functioning as molecular chaperones in vitro to suppress thermally-induced aggregation not only of glycoproteins bearing monoglucosylated oligosaccharides but also of non-glycosylated proteins such as citrate synthase (CS) and malate dehydrogenase (MDH).25,26 As expected for molecular chaperones, S-CNX and CRT discriminated between native and non-native conformers of CS and MDH, forming stable complexes with unfolded forms but not the enzymatically active species. Aggregation suppression of both glycosylated and non-glycosylated proteins was enhanced in the presence of ATP but not ADP, consistent with a role for ATP in the dual binding model (Fig. 6.4). S-CNX and CRT were also shown to participate in the refolding of denatured CS by maintaining the non-native protein in a refolding-competent conformation. These experiments demonstrated that S-CNX and CRT do indeed utilize a polypeptide-based mode of substrate interaction to function as bona fide molecular chaperones in vitro.

Subsequent studies compared the relative potencies of the ER Hsp70 chaperone, BiP, and S-CNX to suppress aggregation and promote folding of monoglucosylated glycoproteins and non-glycoproteins.94 S-CNX was just as potent as BiP at suppressing the aggregation of non-glycosylated CS but was much more effective than BiP when presented with monoglucosylated jack bean α-mannosidase or chicken IgY. Upon deglycosylation of the substrates, S-CNX lost its advantage but still could suppress aggregation, consistent with a dual mode of interaction with the monoglucosylated glycoproteins. This latter study indicates that S-CNX (and presumably CRT) are more potent molecular chaperones for monoglucosylated glycoproteins than is an Hsp70 chaperone that is restricted solely to polypeptide-based interactions. 94 Presumably a dual mode of substrate binding increases overall binding avidity relative to other ER chaperones such as BiP or Grp94.

Proponents of the lectin-only model have questioned the in vitro chaperone experiments in terms of their relevance to the in vivo situation.86,95 To address this issue, Danilczyk et al. developed an extremely mild immunoisolation procedure in an effort to detect polypeptide-based CNX-substrate interactions in lysates of radiolabeled cells. It was reasoned that if a dual mode of CNX-substrate interaction exists in living cells and one interferes with the lectin-oligosaccharide component (e.g., by CAS treatment), then the remaining protein-protein interaction might be relatively weak and lost using more typical isolation conditions.81 It was demonstrated that in glucosidase I or II-deficient cells or in CAS-treated wild type cells the interaction of CNX with many newly synthesized proteins was preserved whereas binding to other proteins was either reduced or eliminated. Analysis of complexes with specific glycoproteins revealed that CAS-treatment did not eliminate CNX binding to a human MHC class I molecule or to the MHC class II invariant chain. Furthermore, removal of all glycosylation sites from a mouse MHC class I molecule failed to ablate CNX binding. In each of these cases, sedimentation studies revealed that the specific substrate was neither insoluble nor present in aggregated form.81 Consequently, there appears to be sufficient evidence to support a dual mode of CNX and CRT binding to at least certain glycoproteins both in vitro and in living cells.

Concluding Remarks

A decade has passed since the discovery of CNX and intensive study on the functions of this protein and those of CRT have clearly established their roles as molecular chaperones that assist glycoprotein folding and participate in ER quality control. The extent to which the cell relies on the functions of CNX and CRT relative to other ER chaperones has been difficult to assess. Certainly the lectin-oligosaccharide component of the interaction is dispensable for viability since glucosidase I and glucosidase II deficient mammalian and yeast cells grow normally. Mixed results have been obtained when the CRT and CNX genes have been disrupted either singly or in combination, with phenotypes ranging from subtle to essential. Much of the complexity can be attributed to the redundant nature of ER chaperones wherein the synthesis of BiP or GRp94 is upregulated as a compensatory response to impairments in the CNX/CRT system.69,96 Also, the role of CNX and CRT in ER Ca2+ homeostasis in addition to their chaperone functions complicates interpretation of results. However, the most contentious issue is still the relative roles of lectin-oligosaccharide versus protein-protein modes of substrate interaction in vivo. With portions of the lectin site well-defined and ongoing progress in delineating ERp57, peptide and ATP binding sites, there will be much interest in examining the in vitro and in vivo functions of CNX and CRT mutants that are selectively deficient in the binding of each ligand.

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