Identification by Mutational Analysis of Amino Acid Residues Essential in the Chaperone Function of Calreticulin

Martin, Virginie; Groenendyk, Jody; Steiner, Simone S.; Guo, Lei; Dąbrowska, Monika; Parker, J. M. R.; Müller‐Esterl, Werner; Opas, Michał; Michalak, Marek

doi:10.1074/jbc.m508302200

Cited by 64 publications

(74 citation statements)

References 33 publications

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“…Transfection of crt À/À MEF with a crt-expressing plasmid restored their ability to expose ERp57 in response to MTX. However, point mutations that affect the capacity of CRT to physically interact with ERp57 (W244 and D241) 28 abolished ERp57 exposure (Figure 3f and g). In yeast, the knockout of the CRT ortholog (Dcne1) greatly reduced surface exposure of the ERP57 ortholog (pdi1 fused to GFP) following MTX treatment, underscoring the phylogenetic conservation of the obligate co-translocation of ERp57 and CRT (Figure 3h and i).…”

Section: Resultsmentioning

confidence: 99%

The co-translocation of ERp57 and calreticulin determines the immunogenicity of cell death

et al. 2008

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The exposure of calreticulin (CRT) on the plasma membrane can precede anthracycline-induced apoptosis and is required for cell death to be perceived as immunogenic. Mass spectroscopy, immunofluorescence and immunoprecipitation experiments revealed that CRT co-translocates to the surface with another endoplasmic reticulum-sessile protein, the disulfide isomerase ERp57. The knockout and knockdown of CRT or ERp57 inhibited the anthracycline-induced translocation of ERp57 or CRT, respectively. CRT point mutants that fail to interact with ERp57 were unable to restore ERp57 translocation upon transfection into crt À/À cells, underscoring that a direct interaction between CRT and ERp57 is strictly required for their co-translocation to the surface. ERp57 low tumor cells generated by retroviral introduction of an ERp57-specific shRNA exhibited a normal apoptotic response to anthracyclines in vitro, yet were resistant to anthracycline treatment in vivo. Moreover, ERp57 low cancer cells (which failed to expose CRT) treated with anthracyclines were unable to elicit an anti-tumor response in conditions in which control cells were highly immunogenic. The failure of ERp57 low cells to elicit immune responses and to respond to chemotherapy could be overcome by exogenous supply of recombinant CRT protein. These results indicate that tumors that possess an intrinsic defect in the CRT-translocating machinery become resistant to anthracycline chemotherapy due to their incapacity to elicit an anticancer immune response. The conventional treatment of cancer relies upon radiotherapy and chemotherapy, two procedures that supposedly mediate their therapeutic effects solely by intrinsic mechanisms of tumor cell death and without direct participation by the host immune system. Nevertheless, there are specific circumstances in which conventional anti-cancer treatments can induce a modality of cell death that is immunogenic, whereby dying cells elicit a tumor-specific immune response culminating in elimination of tumor cells. Although most chemotherapeutic agents kill tumor cells through a morphologically indistinguishable apoptotic pathway, they differ in their capacity to stimulate immunogenic as opposed to nonimmunogenic cell death. 1 Recently, we reported that tumor cells undergoing immunogenic cell death translocate intracellular calreticulin (endo-CRT) to their plasma membrane (PM) surface (ecto-CRT), facilitating tumor cell recognition and engulfment by dendritic cells (DC) and subsequent T-cellmediated elimination of the tumor. 2 Accordingly, anthracyclines and g-irradiation, which elicit immunogenic cell death, induce CRT exposure whereas other proapoptotic agents that induce non-immunogenic cell death fail to induce ecto-CRT. [2][3][4] Depletion of CRT abolishes the immunogenicity of cell death elicited by anthracyclines, whereas addition of exogenous CRT or enforced surface exposure of CRT by pharmacological agents that favor CRT translocation can enhance the immunogenicity of cell death. 2,4 An additional signal emitted during ...

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Section: Resultsmentioning

confidence: 99%

The co-translocation of ERp57 and calreticulin determines the immunogenicity of cell death

et al. 2008

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“…Whereas CLGN is a lectin chaperone during meiosis (21), our data suggest that CALR3 lacks broad lectin activity and no longer functions as a general chaperone for nascent N-glycoproteins. Because the essential amino acids required for oligosaccharide binding in CALR are well conserved in CALR3 (34), the lectin deficiency is likely due to differences in the divergent P domain (20,35). However, CALR3 directly associates with ADAM3 and controls its maturation.…”

Section: Discussionmentioning

confidence: 99%

Calsperin Is a Testis-specific Chaperone Required for Sperm Fertility

Ikawa

Tokuhiro

Yamaguchi

et al. 2011

Journal of Biological Chemistry

132

155

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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.

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“…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). This region was also implicated by the finding that occupancy of the lectin site with a monoglucosylated tetrasaccharide reduced the binding of several glycosy- lated and non-glycosylated proteins and that a fluorophore immobilized in the vicinity of the lectin site was quenched upon binding these proteins (22).…”

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).…”

mentioning

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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Identification by Mutational Analysis of Amino Acid Residues Essential in the Chaperone Function of Calreticulin

Cited by 64 publications

References 33 publications

The co-translocation of ERp57 and calreticulin determines the immunogenicity of cell death

The co-translocation of ERp57 and calreticulin determines the immunogenicity of cell death

Calsperin Is a Testis-specific Chaperone Required for Sperm Fertility

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

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