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Impaired Assembly Results in the Accumulation of Multiple HLA-C Heavy Chain Folding Intermediates¹

Leonardo Sibilio^*, Aline Martayan^*, Andrea Setini^2,*, Rocco Fraioli^*, Doriana Fruci, Jeffrey Shabanowitz, Donald F. Hunt and Patrizio Giacomini^3,*

^* Laboratory of Immunology, Regina Elena Cancer Institute Centro della Ricerca Sperimentale, Rome, Italy; Research Center Ospedale Bambino Gesù, Rome, Italy; and Department of Chemistry, University of Virginia, Charlottesville, VA 22904

Class I MHC H chains assemble with ₂-microglobulin (₂m) and are loaded with peptide Ags through multiple folding steps. When free of ₂m, 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, ₂m-free H chains with calnexin in ₂m-defective cells, and with calreticulin and TAP in ₂m-expressing cells. In ₂m-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 1 domain helix, reciprocally immunodepleted free HLA-C H chains, indicating the existence of a local un-/mis-folding involving the N-terminal end of the 1 domain 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.