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 m olecular 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 hu man B cell Lines with genes encoding either wild-type HLA-A2 heavy cha ins or mutant heavy chains lacking sites for glycosylation or deficien t in binding to beta(2)-microglobulin (beta(2)m) In CIR cells, calnexi n did not associate detectably with wild-type heavy chains but bound s trongly to mutant heavy chains unable to bind beta(2)m Removal of the glycosylation addition site by further mutagenesis prevented binding o f mutant heavy chains to calnexin, In Daudi cells, deficient in synthe sis of beta(2)m, wild-type HLA-A2 heavy chains, but not a nonglycosyla ted 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 th e presence of oligosaccharide on the substrate, removal of the glycan from calnexin-associated heavy chains by digestion with endoglycosidas e H did not disrupt the interaction, These results suggest that calnex in first recognizes carbohydrate on substrate proteins and then binds more stably to peptide determinants, which disappear upon folding.