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.
Leach
Michael R
MR
Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Williams
David B
DB
D000818:Animals; D001665:Binding Sites; D037281:Calnexin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004330:Drosophila; D004926:Escherichia coli; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018832:Molecular Chaperones / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation
D015395:Histocompatibility Antigens Class I; D018832:Molecular Chaperones; D037281:Calnexin