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.     

Aminoglycoside induced nephrotoxicity: molecular modeling studies of calreticulin-gentamicin complex

Gentamicin is a member of aminoglycoside group of broad spectrum antibiotics. It impairs protein synthesis by binding to A site of the 30S subunit of bacterial ribosomes. One of the main side effects of this drug is nephrotoxicity. The drug is known to bind to calreticulin, a chaperone essential for the folding of glycosylated proteins. We provide a detailed structural insight of the calreticulin-gentamicin complex by molecular modeling and the binding of the drug in the presence of explicit solvent was analyzed by molecular dynamics simulation. The gentamicin molecule binds to the lectin site of the calreticulin and lies in the concave channel formed by the long beta sheets. It makes interactions with residues Tyr109, Asp125, Asp135, Asp317 and Trp319 which are crucial for the chaperone function of the calreticulin. The superimposing of the modeled complex with the only available crystal structure complex of calreticulin with a tetrasaccharide (Glc(1)Man(3)) shows interesting features. First, the rings of the gentamicin occupy the positions of glucose and the first two mannose sugars of the tetrasaccharide molecule. Second, the oxygen atoms of the glycosidic linkage of these two ligands have a positional deviation of 1.3 ?. The predicted binding constant of 16.9 μM is in accordance with the previous kinetic study experiments. The details therefore, strongly implicate gentamicin as a competitive inhibitor of sugar binding with calreticulin.     

The chaperone and potential mannan-binding lectin (MBL) co-receptor calreticulin interacts with MBL through the binding site for MBL-associated serine proteases

The chaperone calreticulin has been suggested to function as a C1q and collectin receptor. The interaction of calreticulin with mannan-binding lectin (MBL) was investigated by solid-phase binding assays. Calreticulin showed saturable and time-dependent binding to recombinant MBL, provided that MBL was immobilized on a solid surface or bound to mannan on a surface. The binding was non-covalent and biphasic with an initial salt-sensitive phase followed by a more stable salt-insensitive interaction. For plasma-derived MBL, known to be complexed with MBL-associated serine proteases (MASPs), no binding was observed. Interaction of calreticulin with recombinant MBL was fully inhibited by recombinant MASP-2, MASP-3 and MAp19, but not by the MASP-2 D105G and MAp19 Y59A variants characterized by defective MBL binding ability. Furthermore, MBL point mutants with impaired MASP binding showed no interaction with calreticulin. Comparative analysis of MBL with complement component C1q, its counterpart of the classical pathway, revealed that they display similar binding characteristics for calreticulin, providing further indication that calreticulin is a common co-receptor/chaperone for both proteins. In conclusion, the potential MBL co-receptor calreticulin binds to MBL at the MASP binding site and the interaction may involve a conformational change in MBL.     

Calreticulin is a thermostable protein with distinct structural responses to different divalent cation environments

Calreticulin is a soluble calcium-binding chaperone of the endoplasmic reticulum (ER) that is also detected on the cell surface and in the cytosol. Calreticulin contains a single high affinity calcium-binding site within a globular domain and multiple low affinity sites within a C-terminal acidic region. We show that the secondary structure of calreticulin is remarkably thermostable at a given calcium concentration. Rather than corresponding to complete unfolding events, heat-induced structural transitions observed for calreticulin relate to tertiary structural changes that expose hydrophobic residues and reduce protein rigidity. The thermostability and the overall secondary structure content of calreticulin are impacted by the divalent cation environment, with the ER range of calcium concentrations enhancing stability, and calcium-depleting or high calcium environments reducing stability. Furthermore, magnesium competes with calcium for binding to calreticulin and reduces thermostability. The acidic domain of calreticulin is an important mediator of calcium-dependent changes in secondary structure content and thermostability. Together, these studies indicate interactions between the globular and acidic domains of calreticulin that are impacted by divalent cations. These interactions influence the structure and stability of calreticulin, and are likely to determine the multiple functional activities of calreticulin in different subcellular environments.     

Lectin-deficient calreticulin retains full functionality as a chaperone for class I histocompatibility molecules

Calreticulin is a molecular chaperone of the endoplasmic reticulum that uses both a lectin site specific for Glc(1)Man(5-9)GlcNAc(2) oligosaccharides and a polypeptide binding site to interact with nascent glycoproteins. The latter mode of substrate recognition is controversial. To examine the relevance of polypeptide binding to protein folding in living cells, we prepared lectin-deficient mutants of calreticulin and examined their abilities to support the assembly and quality control of mouse class I histocompatibility molecules. In cells lacking calreticulin, class I molecules exhibit inefficient loading of peptide ligands, reduced cell surface expression and aberrantly rapid export from the endoplasmic reticulum. Remarkably, expression of calreticulin mutants that are completely devoid of lectin function fully complemented all of the class I biosynthetic defects. We conclude that calreticulin can use nonlectin-based modes of substrate interaction to effect its chaperone and quality control functions on class I molecules in living cells. Furthermore, pulse-chase coimmunoisolation experiments revealed that lectin-deficient calreticulin bound to a similar spectrum of client proteins as wild-type calreticulin and dissociated with similar kinetics, suggesting that lectin-independent interactions are commonplace in cells and that they seem to be regulated during client protein maturation.     

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.     

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.     

Determination of a putative phosphate-containing peptide in calreticulin.

Calreticulin is an abundant endo/sarcoplasmic reticulum (ER/SR) protein that may carry out multiple functions inside cells. Except for calreticulin, all of the major ER/SR Ca2+-binding proteins are substrates for protein kinase CK2 in vitro, which led us to hypothesize that native calreticulin might exist in the phosphorylated form. To investigate this possibility, we purified calreticulin from cardiac microsomes and verified its identity by immunoblot analysis and sequencing of tryptic peptides. Purified calreticulin, like cardiac calsequestrin, contained endogenous phosphate as determined by a Malachite green assay for phosphate. Previous analyses of cardiac calsequestrin have localized phosphate to a single tryptic peptide containing serine phosphate on sites phosphorylated by protein kinase CK2. Using a similar procedure, we analyzed calreticulin tryptic peptides with Malachite green, localizing phosphate binding to a single calreticulin peptide 367LKEEEEDKK. As this peptide contains no phosphorylatable residues, our results suggest that calreticulin may tightly bind phosphate or a phosphate-containing molecule at this site.     

Interaction of calreticulin with amyloid beta peptide 1-42

The interaction of calreticulin with amyloid beta (Abeta) was investigated using solid phase and solution binding assays. Calreticulin bound Abeta 1-42 in a time and concentration dependent fashion. The binding was optimal at pH 5 and was stimulated by Ca2+ and inhibited by Zn2+ at pH 7. Interaction took place through the hydrophobic C-terminus of Abeta 1-42 and the polypeptide binding site of calreticulin. The results are discussed in the light of a reported role of calreticulin as a cell surface scavenger receptor.     

The anti-adhesive activity of thrombospondin is mediated by the N-terminal domain of cell surface calreticulin

Thrombospondin (TSP) induces reorganization of the actin cytoskeleton and restructuring of focal adhesions through binding of amino acids (aa) 17-35 (hep I peptide) of thrombospondin to a cell surface form of calreticulin (CRT). In this report we provide further evidence for the involvement of calreticulin in thrombospondin signaling and characterize thrombospondin-calreticulin interactions. Wild type but not crt(-/-) cells respond to hep I/TSP. Responsiveness can be restored by incubation of cells with exogenous calreticulin or by transfection with calreticulin. Thrombospondin forms complexes with the CRT-N-domain that are enhanced by physiologic levels of calcium and zinc. Consistent with thrombospondin/CRT-N-domain binding, only the CRT-N-domain blocks hep I- and thrombospondin-stimulated focal adhesion disassembly. A series of glutathione S-transferase-N-domain mutants were used to map the sequence within the N-domain that interacts with TSP/hep I. A construct containing aa 1-43 but not a construct of aa 1-31 supported thrombospondin binding and focal adhesion disassembly. A series of overlapping peptides were used to further map the thrombospondin-binding site. Peptides spanning aa 19-36 (RWIESKHKSDFGKFVLSS) blocked hep I-stimulated focal adhesion disassembly, indicating that the TSP/hep I-binding site is located to this sequence in calreticulin. A mutant fusion protein lacking aa 19-36 (glutathione S-transferase-CRTDeltahep I) failed to restore responsiveness to hep I in crt(-/-) cells, bind thrombospondin, or competitively block focal adhesion disassembly, providing evidence for the role of this calreticulin sequence in mediating thrombospondin signaling.     

The primary substrate binding site in the b' domain of ERp57 is adapted for endoplasmic reticulum lectin association

ERp57 is a member of the protein disulfide isomerase (PDI) family that is located in the endoplasmic reticulum (ER) and characterized by its specificity for glycoproteins. Substrate selection by ERp57 is dependent upon its formation of discrete complexes with two ER resident lectins, soluble calreticulin and membrane-bound calnexin. It is these two lectins that directly associate with glycoproteins bearing correctly trimmed oligosaccharide side chains. Thus, ERp57 is presented with a preselected set of substrates upon which it can act, and the specific binding of calreticulin and calnexin to ERp57 is pivotal to the functions of the resulting complexes. To gain further insights into the formation of these ERp57-ER lectin complexes, we have investigated the regions of ERp57 that are specifically required for its binding to calreticulin. Using a quantitative pull-down assay to investigate the binding of ERp57/PDI chimeras to calreticulin, we define the b and b' domains of ERp57 as the minimal elements that are sufficient for complex formation. This analysis further identifies a novel role for the distinctive C-terminal extension of ERp57 in reconstituting complex formation to wild type levels. Using our understanding of substrate binding to the b' domain of PDI as a paradigm, we show that alterations to specific residues in the b' domain of ERp57 dramatically reduce or completely abolish its binding to calreticulin. On the basis of these data, we propose a model where the region of ERp57 equivalent to the primary substrate binding site of archetypal PDI is occupied by calreticulin and suggest that the ER lectins act as adaptor molecules that define the substrate specificity of ERp57.     

Glycan-dependent and -independent Interactions Contribute to Cellular Substrate Recruitment by Calreticulin

Calreticulin is an endoplasmic reticulum chaperone with specificity for monoglucosylated glycoproteins. Calreticulin also inhibits precipitation of nonglycosylated proteins and thus contains generic protein-binding sites, but their location and contributions to substrate folding are unknown. We show that calreticulin binds glycosylated and nonglycosylated proteins with similar affinities but distinct interaction kinetics. Although both interactions involve the glycan-binding site or its vicinity, the arm-like proline-rich (P-) domain of calreticulin contributes to binding non/deglycosylated proteins. Correspondingly, ensemble FRET spectroscopy measurements indicate that glycosylated and nonglycosylated proteins induce “open” and “closed” P-domain conformations, respectively. The co-chaperone ERp57 influences substrate-binding kinetics and induces a closed P-domain conformation. Together with analysis of the interactions of calreticulin with cellular proteins, these findings indicate that the recruitment of monoglucosylated proteins to calreticulin is kinetically driven, whereas the P-domain and co-chaperone contribute to stable substrate binding. Substrate sequestration in the cleft between the glycan-binding site and P-domain is a likely mechanism for calreticulin-assisted protein folding.     

Expression and purification of recombinant and native calreticulin.

Calreticulin is a 60-kDa Ca(2+)-binding protein of the endo(sarco)plasmic reticulum membranes of a variety of cellular systems. The protein binds approximately 25 mol of Ca2+ with low affinity and approximately 1 mol of Ca2+ with high affinity and is believed to be a site for Ca2+ binding/storage in the lumen of the endo(sarco)plasmic reticulum. In the present study, we describe purification procedures for the isolation of recombinant and native calreticulin. Recombinant calreticulin was expressed in Escherichia coli, using the glutathione S-transferase fusion protein system, and was purified to homogeneity on glutathione-Sepharose followed by Mono Q FPLC chromatography. A selective ammonium sulfate precipitation method was developed for the purification of native calreticulin. The protein was purified from ammonium sulfate precipitates by diethylaminoethyl-Sephadex and hydroxylapatite chromatography procedures, which eliminates the need to prepare membrane fractions. The purification procedures reported here for recombinant and native calreticulin yield homogeneous preparations of the proteins, as judged by the HPLC reverse-phase chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified native and recombinant calreticulin were identified by their NH2-terminal amino acid sequences, by their Ca2+ binding properties, and by their reactivity with anticalreticulin antibodies.     

Structure of a pancreatic alpha-amylase bound to a substrate analogue at 2.03 A resolution.

The structure of pig pancreatic alpha-amylase in complex with carbohydrate inhibitor and proteinaceous inhibitors is known but the successive events occurring at the catalytic center still remain to be elucidated. The X-ray structure analysis of a crystal of pig pancreatic alpha-amylase (PPA, EC 3.2.1.1.) soaked with an enzyme-resistant substrate analogue, methyl 4,4'-dithio-alpha-maltotrioside, showed electron density corresponding to the binding of substrate analogue molecules at the active site and at the "second binding site." The electron density observed at the active site was interpreted in terms of overlapping networks of oligosaccharides, which show binding of substrate analogue molecules at subsites prior to and subsequent to the cleavage site. A weaker patch of density observed at subsite -1 (using a nomenclature where the site of hydrolysis is taken to be between subsites -1 and +1) was modeled with water molecules. Conformational changes take place upon substrate analogue binding and the "flexible loop" that constitutes the surface edge of the active site is observed in a specific conformation. This confirms that this loop plays an important role in the recognition and binding of the ligand. The crystal structure was refined at 2.03 A resolution, to an R-factor of 16.0 (Rfree, 18.5).     

Calreticulin, a component of the endoplasmic reticulum and of cytotoxic lymphocyte granules, regulates perforin-mediated lysis in the hemolytic model system.

Cytotoxic lymphocytes kill virally infected cells with specialized cytotoxic granules containing perforin, a protein that forms toxic pores in the target cell membrane. These specialized cytotoxic granules also contain calreticulin, an endoplasmic reticulum chaperone protein. The calcium-independent association of perforin and calreticulin prompted our evaluation of calreticulin's potential to function as a regulatory molecule that protects cytotoxic lymphocytes from their own perforin. We report here that 10(-7) M calreticulin blocked perforin-mediated lysis in the hemolytic model system using erythrocytes as targets. Previously, we found that millimolar levels of calcium in the hemolytic assays dissociate high-affinity perforin-calreticulin complexes, which makes it unlikely that perforin associates with calreticulin in solution when hemolysis is blocked. Calreticulin may affect perforin at the erythrocyte membrane. We observed calcium-dependent binding of calreticulin to erythrocyte membranes with a Kd of 2.7 x 10(-7) M and a saturation average of 10(5) molecules calreticulin per erythrocyte. At concentrations that blocked hemolysis, calreticulin occupied many of the calreticulin membrane-binding sites and was in molar excess of perforin. These observations open the possibilities that membrane-bound calreticulin prevents hydrophobic entry of perforin into membranes and (or) prevents perforin from assembling into polyperforin pores.     

Calreticulin promotes folding of functional human leukocyte antigen class I molecules in vitro

The assembly of MHC class I molecules with beta(2)-microglobulin and peptides is assisted by the housekeeping chaperones calnexin, calreticulin, and Erp57 and the dedicated accessory protein, tapasin. Tapasin and calreticulin are essential for efficient MHC class I assembly, but their precise action during class I assembly remains to be elucidated. Previous in vitro studies have demonstrated that the lectin calreticulin interacts with monoglucosylated MHC class I heavy chains, whatever their state of assembly with light chains and peptide, and inhibits their aggregation above physiological temperature. We used a soluble single chain HLA-A2/beta(2)-microglobulin molecule, A2SC, to study the effect of calreticulin on the peptide binding capacity of HLA class I molecules. Calreticulin inhibited the formation of A2SC aggregates both when co-expressed in insect cells and during incubations at elevated temperature. Calreticulin dramatically enhanced acquisition of peptide binding capacity when added to denatured A2SC molecules during refolding at 4 degrees C. However, it had no effect on the rapid loss of A2SC peptide binding capacity at physiological temperature. We conclude that calreticulin promotes the folding of HLA class I molecules to a state in which, at low temperature, they spontaneously acquire peptide binding capacity. However, it does not induce or maintain a peptide-receptive state of the class I-binding site, which is likely to be promoted by one or several other components of the class I loading complexes. By being amenable to complementation with additional proteins, the described system should be useful for identification of these components.     

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.     

The interactions of calreticulin with immunoglobulin G and immunoglobulin Y

Calreticulin is a chaperone of the endoplasmic reticulum (ER) assisting proteins in achieving the correctly folded structure. Details of the binding specificity of calreticulin are still a matter of debate. Calreticulin has been described as an oligosaccharide-binding chaperone but data are also accumulating in support of calreticulin as a polypeptide binding chaperone. In contrast to mammalian immunoglobulin G (IgG), which has complex type N-glycans, chicken immunoglobulin Y (IgY) possesses a monoglucosylated high mannose N-linked glycan, which is a ligand for calreticulin. Here, we have used solid and solution-phase assays to analyze the in vitro binding of calreticulin, purified from human placenta, to human IgG and chicken IgY in order to compare the interactions. In addition, peptides from the respective immunoglobulins were included to further probe the binding specificity of calreticulin. The experiments demonstrate the ability of calreticulin to bind to denatured forms of both IgG and IgY regardless of the glycosylation state of the proteins. Furthermore, calreticulin exhibits binding to peptides (glycosylated and non-glycosylated) derived from trypsin digestion of both immunoglobulins. Additionally, calreticulin peptide binding was examined with synthetic peptides covering the IgG Cγ2 domain demonstrating interaction with approximately half the peptides. Our results show that the dominant binding activity of calreticulin in vitro is toward the polypeptide moieties of IgG and IgY even in the presence of the monoglucosylated high mannose N-linked oligosaccharide on IgY.     

Autocrine regulation of T cell motility by calreticulin-thrombospondin-1 interaction

The mechanisms regulating T lymphocyte migration within the extracellular matrix are not understood. We show in this study that the thrombospondin-1 binding site of calreticulin, spanning aa 19-32, is a major triggering factor for T cell motility and migration within a three-dimensional collagen type 1 matrix, and that exogenous motogenic factors such as chemokines can stimulate migration via a calreticulin-thrombospondin-1 pathway. Endogenous calreticulin binding to the N-terminal domain of endogenous thrombospondin-1 elicited a motogenic signal to the T cells through the C-terminal domain of thrombospondin-1 and its cell surface receptor integrin-associated protein (CD47). Our data further revealed that thrombospondin-1 was expressed on the cell surface with a high turnover, and that PI3K and the Janus family of tyrosine kinases were required for T cell motility mediated through calreticulin, thrombospondin-1, and CD47. These results unveil an autocrine mechanism of calreticulin-thrombospondin-1-CD47 interaction for the control of T cell motility and migration within three-dimensional extracellular matrix substrata.