Calnexin recognizes carbohydrate and protein determinants of class I major histocompatibility complex molecules.
Proper folding of nascent polypeptides is essential for their function and is monitored by intracellular "quality control" elements. The molecular chaperone calnexin participates in this process by retaining in the endoplasmic reticulum a variety of unfolded proteins, including class I major histocompatibility complex molecules. We transfected human B cell lines with genes encoding either wild-type HLA-A2 heavy chains or mutant heavy chains lacking sites for glycosylation or deficient in binding to beta 2-microglobulin (beta 2m). In CIR cells, calnexin did not associate detectably with wild-type heavy chains but bound strongly to mutant heavy chains unable to bind beta 2m. Removal of the glycosylation addition site by further mutagenesis prevented binding of mutant heavy chains to calnexin. In Daudi cells, deficient in synthesis of beta 2m, wild-type HLA-A2 heavy chains, but not a non-glycosylated mutant, bound calnexin. Castanospermine, which blocks trimming of glucose residues from asparagine-linked glycans, inhibited association of calnexin with heavy chains encoded by a second class I gene, HLA-B*0702. Although initiation of calnexin binding appears to depend on the presence of oligosaccharide on the substrate, removal of the glycan from calnexin-associated heavy chains by digestion with endoglycosidase H did not disrupt the interaction. These results suggest that calnexin first recognizes carbohydrate on substrate proteins and then binds more stably to peptide determinants, which disappear upon folding.
Zhang
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Department of Pathology, University of Pittsburgh School of Medicine, Pennsylvania 15261.
Tector
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Salter
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D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037281:Calnexin; D050260:Carbohydrate Metabolism; D002460:Cell Line; D005805:Genes, MHC Class I; D015789:HLA-A2 Antigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015793:HLA-B7 Antigen / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007212:Indolizines / Q000494:pharmacology; D009154:Mutation; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D013696:Temperature; D054836:Indolizidines
D002135:Calcium-Binding Proteins; D037281:Calnexin; D015789:HLA-A2 Antigen; D015793:HLA-B7 Antigen; D007212:Indolizines; D011134:Polysaccharides; D011506:Proteins; C037806:castanospermine; D054836:Indolizidines