Impaired assembly results in the accumulation of multiple HLA-C heavy chain folding intermediates.
Class I MHC H chains assemble with beta2-microglobulin (beta2m) and are loaded with peptide Ags through multiple folding steps. When free of beta2m, human H chains react with Abs to linear epitopes, such as L31. Immunodepletion and coimmunoprecipitation experiments, performed in this study, detected a preferential association of L31-reactive, beta2m-free H chains with calnexin in beta2m-defective cells, and with calreticulin and TAP in beta2m-expressing cells. In beta2m-defective cells, the accumulation of calnexin-bound H chains stoichiometrically exceeded their overall accumulation, a finding that supports both chaperoning preferences and distinct sorting abilities for different class I folds. No peptide species, in a mass range compatible with that of the classical class I ligands, could be detected by mass spectrometry of acidic eluates from L31-reactive HLA-Cw1 H chains. In vitro assembly experiments in TAP-defective T2 cells, and in cells expressing an intact Ag-processing machinery, demonstrated that L31 H chains are not only free of, but also unreceptive to, peptides. L31 and HC10, which bind nearly adjacent linear epitopes of the alpha1 domain alpha helix, reciprocally immunodepleted free HLA-C H chains, indicating the existence of a local un-/mis-folding involving the N-terminal end of the alpha1 domain alpha helix and peptide-anchoring residues of the class I H chain. Thus, unlike certain murine free H chains, L31-reactive H chains are not the immediate precursors of conformed class I molecules. A model inferring their precursor-product relationships with other known class I intermediates is presented.
Sibilio
Leonardo
L
Laboratory of Immunology, Regina Elena Cancer Institute Centro della Ricerca Sperimentale, Rome, Italy.
Martayan
Aline
A
Setini
Andrea
A
Fraioli
Rocco
R
Fruci
Doriana
D
Shabanowitz
Jeffrey
J
Hunt
Donald F
DF
Giacomini
Patrizio
P
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001665:Binding Sites; D037281:Calnexin / Q000378:metabolism; D037282:Calreticulin / Q000378:metabolism; D002460:Cell Line; D000939:Epitopes / Q000737:chemistry; D015236:HLA-C Antigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015395:Histocompatibility Antigens Class I / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D018448:Models, Immunological; D008958:Models, Molecular; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D014162:Transfection; D001613:beta 2-Microglobulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D000906:Antibodies; D037282:Calreticulin; D000939:Epitopes; D015236:HLA-C Antigens; D015395:Histocompatibility Antigens Class I; D001613:beta 2-Microglobulin; C477327:transporter associated with antigen processing (TAP); D037281:Calnexin