TY - JOUR
T1 - Impaired assembly results in the accumulation of multiple HLA-C heavy chain folding intermediates
AU - Sibilio, Leonardo
AU - Martayan, Aline
AU - Setini, Andrea
AU - Fraioli, Rocco
AU - Fruci, Doriana
AU - Shabanowitz, Jeffrey
AU - Hunt, Donald F.
AU - Giacomini, Patrizio
PY - 2005/11/15
Y1 - 2005/11/15
N2 - Class I MHC H chains assemble with β2-microglobulin (β2m) and are loaded with peptide Ags through multiple foiding steps. When free of β2m, human H chains react with Abs to linear epitopes, such as L31. Immunodepletion and coimmunoprecipitation experiments, performed in tins study, detected a preferential association of L31-reactive, β2m-free H chains with calnexin in β2m-defective cells, and with calreticulin and TAP in β2m-expressing cells. In β2m-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 Iigands, could be detected by mass spectrometry of acidic ehiates 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 α1 domain α helix, reciprocally immunodepleted free HLA-C H chains, indicating the existence of a local un-/mis-folding involving the N-terminal end of the α domain α 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.
AB - Class I MHC H chains assemble with β2-microglobulin (β2m) and are loaded with peptide Ags through multiple foiding steps. When free of β2m, human H chains react with Abs to linear epitopes, such as L31. Immunodepletion and coimmunoprecipitation experiments, performed in tins study, detected a preferential association of L31-reactive, β2m-free H chains with calnexin in β2m-defective cells, and with calreticulin and TAP in β2m-expressing cells. In β2m-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 Iigands, could be detected by mass spectrometry of acidic ehiates 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 α1 domain α helix, reciprocally immunodepleted free HLA-C H chains, indicating the existence of a local un-/mis-folding involving the N-terminal end of the α domain α 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.
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M3 - Article
C2 - 16272320
AN - SCOPUS:27744522695
VL - 175
SP - 6651
EP - 6658
JO - Journal of Immunology
JF - Journal of Immunology
SN - 0022-1767
IS - 10
ER -