The polypeptide binding conformation of calreticulin facilitates its cell-surface expression under conditions of endoplasmic reticulum stress

We define two classes of calreticulin mutants that retain glycan binding activity; those that display enhanced or reduced polypeptide-specific chaperone activity, due to conformational effects. Under normal conditions, neither set of mutants significantly impacts the ability of calreticulin to mediate assembly and trafficking of major histocompatibility complex class I molecules, which are calreticulin substrates. However, in cells treated with thapsigargin, which depletes endoplasmic reticulum calcium, major histocompatibility complex class I trafficking rates are accelerated coincident with calreticulin secretion, and detection of cell-surface calreticulin is dependent on its polypeptide binding conformations. Together, these findings identify a site on calreticulin that is an important determinant of the induction of its polypeptide binding conformation and demonstrate the relevance of the polypeptide binding conformations of calreticulin to endoplasmic reticulum stress-induced interactions.     

Structural and functional relationships between the lectin and arm domains of calreticulin

Calreticulin and calnexin are key components in maintaining the quality control of glycoprotein folding within the endoplasmic reticulum. Although their lectin function of binding monoglucosylated sugar moieties of glycoproteins is well documented, their chaperone activity in suppressing protein aggregation is less well understood. Here, we use a series of deletion mutants of calreticulin to demonstrate that its aggregation suppression function resides primarily within its lectin domain. Using hydrophobic peptides as substrate mimetics, we show that aggregation suppression is mediated through a single polypeptide binding site that exhibits a K(d) for peptides of 0.5-1 μM. This site is distinct from the oligosaccharide binding site and differs from previously identified sites of binding to thrombospondin and GABARAP (4-aminobutyrate type A receptor-associated protein). Although the arm domain of calreticulin was incapable of suppressing aggregation or binding hydrophobic peptides on its own, it did contribute to aggregation suppression in the context of the whole molecule. The high resolution x-ray crystal structure of calreticulin with a partially truncated arm domain reveals a marked difference in the relative orientations of the arm and lectin domains when compared with calnexin. Furthermore, a hydrophobic patch was detected on the arm domain that mediates crystal packing and may contribute to calreticulin chaperone function.     

Lectin-deficient calnexin is capable of binding class I histocompatibility molecules in vivo and preventing their degradation

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, we have generated six calnexin mutants defective in oligosaccharide binding 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.     

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.     

Calsperin is a testis-specific chaperone required for sperm fertility

Calnexin (CANX) and calreticulin (CALR) are homologous lectin chaperones located in the endoplasmic reticulum and cooperate to mediate nascent glycoprotein folding. In the testis, calmegin (CLGN) and calsperin (CALR3) are expressed as germ cell-specific counterparts of CANX and CALR, respectively. Here, we show that Calr3(-/-) males produced apparently normal sperm but were infertile because of defective sperm migration from the uterus into the oviduct and defective binding to the zona pellucida. Whereas CLGN was required for ADAM1A/ADAM2 dimerization and subsequent maturation of ADAM3, a sperm membrane protein required for fertilization, we show that CALR3 is a lectin-deficient chaperone directly required for ADAM3 maturation. Our results establish the client specificity of CALR3 and demonstrate that the germ cell-specific CALR-like endoplasmic reticulum chaperones have contrasting functions in the development of male fertility. The identification and understanding of the maturation mechanisms of key sperm proteins will pave the way toward novel approaches for both contraception and treatment of unexplained male infertility.     

Modes of Calreticulin Recruitment to the Major Histocompatibility Complex Class I Assembly Pathway

Major histocompatibility complex (MHC) class I molecules are ligands for T-cell receptors of CD8⁺ T cells and inhibitory receptors of natural killer cells. Assembly of the heavy chain, light chain, and peptide components of MHC class I molecules occurs in the endoplasmic reticulum (ER). Specific assembly factors and generic ER chaperones, collectively called the MHC class I peptide loading complex (PLC), are required for MHC class I assembly. Calreticulin has an important role within the PLC and induces MHC class I cell surface expression, but the interactions and mechanisms involved are incompletely understood. We show that interactions with the thiol oxidoreductase ERp57 and substrate glycans are important for the recruitment of calreticulin into the PLC and for its functional activities in MHC class I assembly. The glycan and ERp57 binding sites of calreticulin contribute directly or indirectly to complexes between calreticulin and the MHC class I assembly factor tapasin and are important for maintaining steady-state levels of both tapasin and MHC class I heavy chains. A number of destabilizing conditions and mutations induce generic polypeptide binding sites on calreticulin and contribute to calreticulin-mediated suppression of misfolded protein aggregation in vitro. We show that generic polypeptide binding sites per se are insufficient for stable recruitment of calreticulin to PLC substrates in cells. However, such binding sites could contribute to substrate stabilization in a step that follows the glycan and ERp57-dependent recruitment of calreticulin to the PLC.     

Delineation of the lectin site of the molecular chaperone calreticulin

Calreticulin (CRT) is a soluble molecular chaperone of the endoplasmic reticulum that functions to promote protein folding as well as to retain misfolded proteins. Similar to its membrane-bound paralog calnexin (CNX), CRT is a lectin that preferentially interacts with glycoproteins bearing Glc1Man5-9GlcNAc2 oligosaccharides. Although the lectin site of CNX has been delineated through X-ray crystallographic and mutagenic studies, the corresponding site for CRT has not been as well characterized. To address this issue, we attempted to construct lectin-deficient CRT mutants, using the structure of CNX as a guide to identify potential oligosaccharide-binding residues. Mutation of 4 such CRT residues (Y109, K111, Y128, D317) completely abrogated oligosaccharide binding. In contrast, mutation of CRT residues M131 and D160, which correspond to important residues in the lectin site of CNX, had no effect on oligosaccharide binding. These findings suggest that the organization of the lectin site in CRT largely resembles that of CNX but is not identical. The deficiency in oligosaccharide binding by the mutants was not due to misfolding because they exhibited wild-type protease digestion patterns, were capable of binding the thiol oxidoreductase ERp57, and functioned just as efficiently as wild-type CRT in suppressing the aggregation of the nonglycosylated substrate citrate synthase. However, they were impaired in their ability to suppress the aggregation of the glycosylated substrate jack bean alpha-mannosidase. This provides the first direct demonstration of the importance of CRT's lectin site in suppressing the aggregation of nonnative glycoproteins.     

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

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

Major histocompatibility complex class I molecules expressed with monoglucosylated N-linked glycans bind calreticulin independently of their assembly status

The assembly of major histocompatibility complex (MHC) class I molecules with peptides in the endoplasmic reticulum (ER) is a critical step in the presentation of viral antigens to CD8+ T cells. This process is subject to quality control restrictions that prevent free class I heavy chains (HCs) and peptide-free HC-beta(2)-microglobulin (beta(2)m) dimers from exiting the ER. The lectin-like chaperone calreticulin associates with HC-beta(2)m heterodimers prior to peptide binding, but its precise role in regulating the subsequent events of peptide association and ER to Golgi transport remains undefined. In vitro analysis of the assembly process has been limited by the specificity of calreticulin for monoglucosylated N-linked glycans, which are transient biosynthetic intermediates. To address this problem, we developed a novel expression system using Saccharomyces cerevisiae glycosylation mutants to produce class I HC bearing N-linked oligosaccharides with the specific structure Glc(1)Man(9)GlcNAc(2). The monoglucosylated glycan proved to be both necessary and sufficient for in vitro binding of calreticulin to MHC class I molecules. Calreticulin bound as efficiently to peptide-loaded MHC class I complexes as it did to folding intermediates created in vitro, namely free class I HC and empty HC-beta(2)m heterodimers. Thus, calreticulin is unable to discriminate between native and non-native MHC class I conformations and therefore unlikely to play a role in the recognition and release of peptide-loaded complexes from the ER. Furthermore, the recombinant expression system developed in this study can be used to produce a broad range of calreticulin substrates to elucidate its general mechanism of activity in vitro.     

Calreticulin‐dependent recycling in the early secretory pathway mediates optimal peptide loading of MHC class I molecules

Calreticulin is a lectin chaperone of the endoplasmic reticulum (ER). In calreticulin‐deficient cells, major histocompatibility complex (MHC) class I molecules travel to the cell surface in association with a sub‐optimal peptide load. Here, we show that calreticulin exits the ER to accumulate in the ER–Golgi intermediate compartment (ERGIC) and the cis‐Golgi, together with sub‐optimally loaded class I molecules. Calreticulin that lacks its C‐terminal KDEL retrieval sequence assembles with the peptide‐loading complex but neither retrieves sub‐optimally loaded class I molecules from the cis‐Golgi to the ER, nor supports optimal peptide loading. Our study, to the best of our knowledge, demonstrates for the first time a functional role of intracellular transport in the optimal loading of MHC class I molecules with antigenic peptide.     

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

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

X-ray structure of the human calreticulin globular domain reveals a peptide-binding area and suggests a multi-molecular mechanism

In the endoplasmic reticulum, calreticulin acts as a chaperone and a Ca(2+)-signalling protein. At the cell surface, it mediates numerous important biological effects. The crystal structure of the human calreticulin globular domain was solved at 1.55 ? resolution. Interactions of the flexible N-terminal extension with the edge of the lectin site are consistently observed, revealing a hitherto unidentified peptide-binding site. A calreticulin molecular zipper, observed in all crystal lattices, could further extend this site by creating a binding cavity lined by hydrophobic residues. These data thus provide a first structural insight into the lectin-independent binding properties of calreticulin and suggest new working hypotheses, including that of a multi-molecular mechanism.     

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

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

Gentamicin binds to the lectin site of calreticulin and inhibits its chaperone activity

Recently, it became clear that aminoglycoside antibiotics affect protein-protein interactions involving protein disulfide isomerase as well as protein synthesis in the endoplasmic reticulum. In this study, we used affinity column chromatography to screen gentamicin-binding proteins in microsomes derived from bovine kidney in order to learn about the possible mechanisms of gentamicin-associated nephrotoxicity. One of the gentamicin-binding proteins was identified as calreticulin (CRT) by N-terminal amino acid sequence analysis. Interestingly, gentamicin inhibited the chaperone and oxidative refolding activities of CRT when N-glycosylated substrates such as alpha1-antitrypsin and alpha-mannosidase were used as substrates, but it did not inhibit the chaperone activity of CRT when unglycosylated citrate synthase was used. Moreover, CRT suppressed the aggregation of deglycosylated and denatured alpha-mannosidase, but gentamicin did not inhibit its chaperone activity. Experiments with domain mutants suggest that the lectin site of CRT is the main target for gentamicin binding and that binding of gentamicin to this site inhibits the chaperone activity of CRT.     

Polypeptide substrate recognition by calnexin requires specific conformations of the calnexin protein

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. We found that calnexin displayed significant conformational lability for a chaperone and that heat treatment and calcium depletion induced the formation of calnexin dimers and higher order oligomers. These conditions enhanced the chaperone activity of calnexin toward glycosylated and non-glycosylated major histocompatibility complex (MHC) class I heavy chains, and enhanced 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 were induced in HeLa cells upon heat shock and under calcium-depleting conditions, and heat shock enhanced calnexin binding to MHC class I heavy chains in HeLa cells. Virus-induced endoplasmic reticulum stress also resulted in the appearance of calnexin dimers. Tunicamycin treatment of HeLa cells induced a slow accumulation of calnexin dimers, the appearance of which correlated with enhanced calnexin binding to deglycosylated MHC class I heavy chains. In vitro, the presence of calnexin-specific oligosaccharides inhibited 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.     

Tapasin enhances assembly of transporters associated with antigen processing-dependent and -independent peptides with HLA-A2 and HLA-B27 expressed in insect cells.

Assembly of HLA class I-peptide complexes is assisted by multiple proteins that associate with HLA molecules in loading complexes. These include the housekeeping chaperones calnexin and calreticulin and two essential proteins, the transporters associated with antigen processing (TAP) for peptide supply, and the protein tapasin which is thought to act as a specialized chaperone. We dissected functional effects of processing cofactors by co-expressing in insect cells various combinations of the human proteins HLA-A2, HLA-B27, beta(2)-microglobulin, TAP, calnexin, calreticulin, and tapasin. Stability at 37 degrees C and surface expression of class I dimers correlated closely in baculovirus-infected Sf9 cells, suggesting that these cells retain empty dimers in the endoplasmic reticulum. Both HLA molecules form substantial quantities of stable complexes with insect cell-produced peptide pools. These pools are TAP-selected cytosolic peptides for HLA-B27 but endoplasmic reticulum-derived, i.e. TAP-independent peptides for HLA-A2. This discrepancy may be due to peptide selection by human TAP which is much better adapted to the HLA-B27 than to the HLA-A2 ligand preferences. HLA class I assembly with peptides from TAP-dependent and -independent pools was enhanced strongly by tapasin. Thus, tapasin acts as a chaperone and/or peptide editor that facilitates assembly of peptides with HLA class I molecules independently of mediating their interaction with TAP and/or retention in the endoplasmic reticulum.     

Calreticulin displays in vivo peptide-binding activity and can elicit CTL responses against bound peptides.

Calreticulin is an endoplasmic reticulum (ER) chaperone that displays lectin activity and contributes to the folding pathways for nascent glycoproteins. Calreticulin also participates in the reactions yielding assembly of peptides onto nascent MHC class I molecules. By chemical and immunological criteria, we identify calreticulin as a peptide-binding protein and provide data indicating that calreticulin can elicit CTL responses to components of its bound peptide pool. In an adoptive immunotherapy protocol, dendritic cells pulsed with calreticulin isolated from B16/F10.9 murine melanoma, E.G7-OVA, or EL4 thymoma tumors elicited a CTL response to as yet unknown tumor-derived Ags or the known OVA Ag. To evaluate the relative efficacy of calreticulin in eliciting CTL responses, the ER chaperones GRP94/gp96, BiP, ERp72, and protein disulfide isomerase were purified in parallel from B16/F10.9, EL4, and E.G7-OVA tumors, and the capacity of the proteins to elicit CTL responses was compared. In both the B16/F10.9 and E.G7-OVA models, calreticulin was as effective as or more effective than GRP94/gp96 in eliciting CTL responses. Little to no activity was observed for BiP, ERp72, and protein disulfide isomerase. The observed antigenic activity of calreticulin was recapitulated in in vitro experiments, in which it was observed that pulsing of bone marrow dendritic cells with E.G7-OVA-derived calreticulin elicited sensitivity to lysis by OVA-specific CD8+ T cells. These data identify calreticulin as a peptide-binding protein and indicate that calreticulin-bound peptides can be re-presented on dendritic cell class I molecules for recognition by CD8+ T cells.     

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

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

Identification by mutational analysis of amino acid residues essential in the chaperone function of calreticulin

Calreticulin is a Ca2+ -binding chaperone that resides in the lumen of the endoplasmic reticulum and is involved in the regulation of intracellular Ca2+ homeostasis and in the folding of newly synthesized glycoproteins. In this study, we have used site-specific mutagenesis to map amino acid residues that are critical in calreticulin function. We have focused on two cysteine residues (Cys(88) and Cys(120)), which form a disulfide bridge in the N-terminal domain of calreticulin, on a tryptophan residue located in the carbohydrate binding site (Trp(302)), and on certain residues located at the tip of the "hairpin-like" P-domain of the protein (Glu(238), Glu(239), Asp(241), Glu(243), and Trp(244)). Calreticulin mutants were expressed in crt(-/-) fibroblasts, and bradykinin-dependent Ca2+ release was measured as a marker of calreticulin function. Bradykinin-dependent Ca2+ release from the endoplasmic reticulum was rescued by wild-type calreticulin and by the Glu(238), Glu(239), Asp(241), and Glu(243) mutants. The Cys(88) and Cys(120) mutants rescued the calreticulin-deficient phenotype only partially ( approximately 40%), and the Trp(244) and Trp(302) mutants did not rescue it at all. We identified four amino acid residues (Glu(239), Asp(241), Glu(243), and Trp(244)) at the hairpin tip of the P-domain that are critical in the formation of a complex between ERp57 and calreticulin. Although the Glu(239), Asp(241), and Glu(243) mutants did not bind ERp57 efficiently, they fully restored bradykinin-dependent Ca2+ release in crt(-/-) cells. This indicates that binding of ERp57 to calreticulin may not be critical for the chaperone function of calreticulin with respect to the bradykinin receptor.