title: Contributions of the Lectin and Polypeptide Binding Sites of Calreticulin to Its Chaperone Functions in Vitro and in Cells.
abstract: Calreticulin is a lectin chaperone of the endoplasmic reticulum that interacts with newly synthesized glycoproteins by binding to Glc1Man9GlcNAc2 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 binding-deficient, 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 α1-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.
authors.affiliation: From the Departments of Biochemistry and Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada and.
authors.forename: Ronnie
authors.initials: R
authors.lastname: Lum
authors.affiliation: From the Departments of Biochemistry and Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada and.
authors.forename: Samar
authors.initials: S
authors.lastname: Ahmad
authors.affiliation: From the Departments of Biochemistry and Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada and.
authors.forename: Seo Jung
authors.initials: SJ
authors.lastname: Hong
authors.affiliation: From the Departments of Biochemistry and Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada and.
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Chapman
authors.affiliation: the Groupe de Recherche Axé sur la Structure des Proteines, Department of Biochemistry, McGill University, Montréal, Québec H3G 0B1, Canada.
authors.forename: Guennadi
authors.initials: G
authors.lastname: Kozlov
authors.affiliation: From the Departments of Biochemistry and Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada and david.williams@utoronto.ca.
authors.forename: David B
authors.initials: DB
authors.lastname: Williams
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D037282:Calreticulin / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D005347:Fibroblasts / Q000378:metabolism*; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D000515:alpha 1-Antitrypsin / Q000096:biosynthesis / Q000235:genetics
chemical_list: D037282:Calreticulin; D015395:Histocompatibility Antigens Class I; D018832:Molecular Chaperones; D000515:alpha 1-Antitrypsin
keywords: endoplasmic reticulum (ER); lectin; molecular chaperone; protein aggregation; protein folding