An unstable beta 2-microglobulin: major histocompatibility complex class I heavy chain intermediate dissociates from calnexin and then is stabilized by binding peptide.
Proper assembly of the class I heavy chain (HC), beta 2-microglobulin (beta 2m), and peptide must occur in the endoplasmic reticulum (ER) in order for MHC class I molecules to be expressed on the cell surface. Newly synthesized class I HC bind calnexin, an ER resident chaperone. These calnexin-associated class I HC appeared to lack the stable association with beta 2m in peptide transporter-deficient T2 cells since beta 2m-unassociated class I HC-specific HC10 antibody, but not beta 2m-associated class I HC-specific W6/32 antibody, coimmunoprecipitated calnexin. To determine the precursor-product relationship of the pool of HC that bind peptide, class I-restricted peptides were added to lysates of T2 cells in vitro. These peptides stabilized preexisting beta 2m-associated HC complexes (beta 2m+:HC:pep-), but had no significant effect on the preexisting pool of calnexin-associated HC that lack beta 2m. Release of HC from calnexin appeared to be controlled by the binding of beta 2m, since beta 2m-deficient FO-1 cells showed a prolonged association of class I HC with calnexin, while beta 2m-transfected FO-1 cells displayed a more rapid dissociation of class I HC from calnexin. Consistent with this result, the dissociation of class I HC from calnexin did not appear to be dependent on peptide binding since the dissociation rates were similar in peptide transporter-deficient T2 cells and in wild-type T1 cells. From these observations, we speculate that in the stepwise assembly of class I molecules, calnexin may mediate dimerization of class I HC with beta 2m, and that the unstable beta 2m+:HC:pep- complexes, after dissociation from calnexin, subsequently bind peptide to complete the assembly.
Department of Rheumatology and Immunology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115.
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D000818:Animals; D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D037281:Calnexin; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D015796:HLA-B27 Antigen / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D011485:Protein Binding; D013601:T-Lymphocytes; D014162:Transfection; D001613:beta 2-Microglobulin / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D015796:HLA-B27 Antigen; D015395:Histocompatibility Antigens Class I; D046911:Macromolecular Substances; D008565:Membrane Proteins; D001613:beta 2-Microglobulin; D037281:Calnexin; D008715:Methionine