Abstract
Molecular polymorphism influences the strong association of HLA-B27 with ankylosing spondylitis through an unknown mechanism. Natural subtypes and site-directed mutants were used to analyze the effect of altering the peptide-binding site of this molecule on its stability, interaction with tapasin, folding, and export. The disease-associated subtypes B*2705, B*2702, and B*2704 showed higher thermostability at 50 °C than all other subtypes and mutants, except some mimicking B*2702 polymorphism. The lowest values were found among pocket B mutants, most of which interacted strongly with tapasin, but otherwise there was no correlation between thermostability and tapasin interaction. Mutants resulting in increased hydrophobicity frequently acquired their maximal thermostability faster than those with increased polarity, suggesting that this process is largely driven by the thermodynamics of peptide binding. Folding, export, and tendency to misfold were influenced by polymorphism all along the peptide-binding site and were not specifically dependent on any particular region or structural feature. Frequent uncoupling of thermostability, folding/misfolding, and export can be explained by the distinct effect of mutations on the acquisition of a folded conformation, the optimization rate of B27-peptide complexes, and their quality control in the endoplasmic reticulum, all of which largely depend on the ways in which mutations alter peptide binding, without excluding additional effects on interactions with tapasin or other proteins involved in folding and export. The similarity of the generally disease-associated B*2707 to nondisease-associated subtypes in all the features analyzed suggests that molecular properties other than antigen presentation may not currently explain the relationship between HLA-B27 polymorphism and ankylosing spondylitis.
Highlights
Hmy2.C1R; mAb, monoclonal antibody; Endoglycosidase H (Endo H), endoglycosidase H; H/M ratio, ratio between the heavy chain (HC) precipitated with HC10 and with ME1
The dissociation of pulse-labeled HLA-B27-peptide complexes was assessed by immunoprecipitation with ME1 after incubation of the lysates obtained at 0, 2, and 4-h chase times, at temperatures ranging from 4 to 50 °C
The thermostability was measured as the percentage of the HC immunoprecipitated with ME1 at a given temperature, relative to the amount immunoprecipitated at 4 °C (Figs. 1 and supplemental Figs. 2S–S6)
Summary
Cell Lines, Antibodies, and Western Blot—HMy2.C1R (C1R) is an HLA-A-negative human B cell line expressing low levels of HLA-B35 [24]. The protein expression level of HLA-B27 variants in C1R transfectant cells, relative to B*2705, is shown in supplemental Fig. S1 For some of these variants, these data were reported previously [17] and are included here for comparison. They were pulselabeled with 500 –1000 Ci/ml [35S]Met/Cys (GE Healthcare) and chased at various times with RPMI 1640 medium supplemented with 1 mM cold L-Met and L-Cys at the same temperature.
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