X-Ray Structure of the Human Calreticulin Globular Domain Reveals a Peptide-Binding Area and Suggests a Multi-Molecular Mechanism

Chouquet, Anne; Païdassi, Helena; Ling, Wai Li; Frachet, Philippe; Houen, Gunnar; Arlaud, Gérard J.; Gaboriaud, Christine

doi:10.1371/journal.pone.0017886

Cited by 90 publications

(125 citation statements)

References 32 publications

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“…It turned out that anti-LRP impaired the binding of soluble PR3 to macrophages, thus suggesting (but not demonstrating) that a direct association between PR3 and LRP (or LRP-containing complexes) could occur. It also should be mentioned that CRT is also expressed at the surface of macrophages, and that homotypic CRT-CRT interactions have also been described during the phagocytosis of apoptotic cells (14,22). It thus could be foreseen that PR3-CRT association might interfere with this CRT-CRT homotypic interaction (Fig.…”

Section: Crt-pr3 Association Disables the Lrp-crt-mediated Phagocyticmentioning

confidence: 99%

“…1C). Recombinant CRT, the CRT P domain, and the CRT globular domain designed to preserve the CRT structural domain limits (15,22) were immobilized on a CM5 sensor chip. The kinetic parameters of CRT recognition were determined by recording sensorgrams at varying PR3 concentrations from 10 to 100 nM.…”

Section: Pr3 and Crt Are Colocalized On Neutrophil Membrane On Apoptosismentioning

confidence: 99%

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Proteinase 3, the Autoantigen in Granulomatosis with Polyangiitis, Associates with Calreticulin on Apoptotic Neutrophils, Impairs Macrophage Phagocytosis, and Promotes Inflammation

Gabillet

Millet

Pederzoli-Ribeil

et al. 2012

The Journal of Immunology

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Proteinase 3 (PR3) is the target of anti-neutrophil cytoplasm Abs in granulomatosis with polyangiitis, a form of systemic vasculitis. Upon neutrophil apoptosis, PR3 is coexternalized with phosphatidylserine and impaired macrophage phagocytosis. Calreticulin (CRT), a protein involved in apoptotic cell recognition, was found to be a new PR3 partner coexpressed with PR3 on the neutrophil plasma membrane during apoptosis, but not after degranulation. The association between PR3 and CRT was demonstrated in neutrophils by confocal microscopy and coimmunoprecipitation. Evidence for a direct interaction between PR3 and the globular domain of CRT, but not with its P domain, was provided by surface plasmon resonance spectroscopy. Phagocytosis of apoptotic neutrophils from healthy donors was decreased after blocking lipoprotein receptor-related protein (LRP), a CRT receptor on macrophages. In contrast, neutrophils from patients with granulomatosis with polyangiitis expressing high membrane PR3 levels showed a lower rate of phagocytosis than those from healthy controls not affected by anti-LRP, suggesting that the LRP-CRT pathway was disturbed by PR3-CRT association. Moreover, phagocytosis of apoptotic PR3-expressing cells potentiated proinflammatory cytokine in vitro by human monocyte-derived macrophages and in vivo by resident murine peritoneal macrophages, and diverted the anti-inflammatory response triggered by the phagocytosis of apoptotic cells after LPS challenge in thioglycolate-elicited murine macrophages. Therefore, membrane PR3 expressed on apoptotic neutrophils might amplify inflammation and promote autoimmunity by affecting the anti-inflammatory “reprogramming” of macrophages.

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Section: Crt-pr3 Association Disables the Lrp-crt-mediated Phagocyticmentioning

confidence: 99%

Section: Pr3 and Crt Are Colocalized On Neutrophil Membrane On Apoptosismentioning

confidence: 99%

Proteinase 3, the Autoantigen in Granulomatosis with Polyangiitis, Associates with Calreticulin on Apoptotic Neutrophils, Impairs Macrophage Phagocytosis, and Promotes Inflammation

Gabillet

Millet

Pederzoli-Ribeil

et al. 2012

The Journal of Immunology

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“…the Glycan-binding Site of Calreticulin-Previously, the crystal structure of the globular domain of human calreticulin (Protein Data Bank code 3pos) had suggested the presence of a polypeptide-binding site located on the edge of the lectin-binding site of calreticulin (6). To further examine this possibility, we asked whether occupancy of the glycan-binding site of calreticulin with the calreticulin-specific glycan Glc␣1-3Man␣1-2Man␣1-2Man G1M3 could limit interactions between calreticulin and protein substrates.…”

Section: Identification Of a Polypeptide-binding Site In Proximity Tomentioning

confidence: 99%

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

Wijeyesakere

Rizvi

Raghavan

2013

Journal of Biological Chemistry

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Background:We investigated the different modes of calreticulin-substrate binding. Results: Calreticulin binds glycosylated and nonglycosylated proteins with similar affinities but distinct kinetics and P-domain conformations. Conclusion: Successful substrate recruitment by calreticulin requires glycan and P-domain-dependent interactions. Significance: Elucidation of the distinct modes of calreticulin binding to substrate glycan and polypeptide components and their combined contributions to substrate recruitment in cells.

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“…However, either these mutants were destabilizing (45), or they did not impair aggregation suppression function under physiological conditions of the ER (28). More recently, interest has focused around the lectin binding site because a crystal structure of the Crt globular domain detected an N-terminal affinity tag interacting with the edge of the lectin site (31). Indeed, a mutation at Trp-319 within the lectin site has been reported to attenuate aggregation suppression in vitro (32).…”

Section: Discussionmentioning

confidence: 99%

“…Several protein binding sites have been identified on Crt such as those specific for GABARAP (29) and thrombospondin (30), but interacting peptides derived from these proteins fail to compete in aggregation suppression assays, indicative of distinct binding sites (24). Additional binding sites have been proposed in proximity to the lectin site (22,31,32), but these either do not exhibit the expected specificity for hydrophobic substrates (22) or would be expected to be blocked upon addition of monoglucosylated oligosaccharide (31), a treatment that does not affect the binding of hydrophobic peptides to Crt (24).…”

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confidence: 99%

Contributions of the Lectin and Polypeptide Binding Sites of Calreticulin to Its Chaperone Functions in Vitro and in Cells

Lum

Ahmad

Hong

et al. 2016

Journal of Biological Chemistry

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Calreticulin is a lectin chaperone of the endoplasmic reticulum that interacts with newly synthesized glycoproteins by binding to Glc 1 Man 9 GlcNAc 2 oligosaccharides as well as to the polypeptide chain. In vitro, the latter interaction potently suppresses the aggregation of various non-glycosylated proteins. Although the lectin-oligosaccharide association is well understood, the polypeptide-based interaction is more controversial because the binding site on calreticulin has not been identified, and its significance in the biogenesis of glycoproteins in cells remains unknown. In this study, we identified the polypeptide binding site responsible for the in vitro aggregation suppression function by mutating four candidate hydrophobic surface patches. Mutations in only one patch, P19K/I21E and Y22K/ F84E, impaired the ability of calreticulin to suppress the thermally induced aggregation of non-glycosylated firefly luciferase. These mutants also failed to bind several hydrophobic peptides that act as substrate mimetics and compete in the luciferase aggregation suppression assay. To assess the relative contributions of the glycan-dependent and -independent interactions in living cells, we expressed lectin-deficient, polypeptide bindingdeficient, and doubly deficient calreticulin constructs in calreticulin-negative cells and monitored the effects on the biogenesis of MHC class I molecules, the solubility of mutant forms of ␣ 1 -antitrypsin, and interactions with newly synthesized glycoproteins. In all cases, we observed a profound impairment in calreticulin function when its lectin site was inactivated. Remarkably, inactivation of the polypeptide binding site had little impact. These findings indicate that the lectin-based mode of client interaction is the predominant contributor to the chaperone functions of calreticulin within the endoplasmic reticulum. Membrane-bound calnexin (Cnx)3 and its soluble paralog calreticulin (Crt) are glycoprotein-specific chaperones of the endoplasmic reticulum (ER). As components of the ER quality control system, they retain glycoprotein folding intermediates within this organelle and assist folding by preventing aggregation and by recruiting folding catalysts such as the thiol oxidoreductase ERp57 and peptidyl-prolyl isomerase cyclophilin B (for reviews, see Refs. 1 and 2). Both chaperones consist of a globular lectin domain and an extended arm or P domain (3-5) with the tip of the arm domain comprising the binding site for ERp57 (6) and cyclophilin B (7). The specificity for glycoproteins resides within their lectin domains, which recognize a monoglucosylated oligosaccharide-processing intermediate of composition Glc 1 Man 9 GlcNAc 2 (8 -10). Cycles of chaperone binding and release are regulated by the availability of the terminal glucose residue on this oligosaccharide with glucose removal catalyzed by glucosidase II and its readdition by UDPglucose:glycoprotein glucosyltransferase I (11). UDP-glucose: glycoprotein glucosyltransferase I acts as the folding sensor in the cycle, only...

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X-Ray Structure of the Human Calreticulin Globular Domain Reveals a Peptide-Binding Area and Suggests a Multi-Molecular Mechanism

Cited by 90 publications

References 32 publications

Proteinase 3, the Autoantigen in Granulomatosis with Polyangiitis, Associates with Calreticulin on Apoptotic Neutrophils, Impairs Macrophage Phagocytosis, and Promotes Inflammation

Proteinase 3, the Autoantigen in Granulomatosis with Polyangiitis, Associates with Calreticulin on Apoptotic Neutrophils, Impairs Macrophage Phagocytosis, and Promotes Inflammation

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

Contributions of the Lectin and Polypeptide Binding Sites of Calreticulin to Its Chaperone Functions in Vitro and in Cells

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