Abstract
Major histocompatibility complex class II (MHC II) expressed on the surface of antigen-presenting cells (APCs) displays peptides to CD4⁺ T cells. Depletion of membrane cholesterol from APCs by methyl β-cyclodextrin treatment compromises peptide-MHC II complex formation coupled with impaired binding of conformational antibody, which binds close to the peptide binding groove of MHC II. Interestingly, the total cell surface of MHC II remains unaltered. These defects can be corrected by restoring membrane cholesterol. In silico docking studies with a three-dimensional model showed the presence of a cholesterol binding site in the transmembrane domain of MHC II (TM-MHC-II). From the binding studies it was clear that cholesterol, indeed, interacts with the TM-MHC-II and alters its conformation. Mutation of cholesterol binding residues (F240, L243, and F246) in the TM-MHC-II decreased the affinity for cholesterol. Furthermore, transfection of CHO cells with full-length mutant MHC II, but not wild-type MHC II, failed to activate antigen-specific T cells coupled with decreased binding of conformation-specific antibodies. Thus, cholesterol-induced conformational change of TM-MHC-II may allosterically modulate the peptide binding groove of MHC II leading to T cell activation.
Highlights
Major histocompatibility complex class II (MHC II) expressed on the surface of antigen-presenting cells (APCs) displays peptides to CD4+ T cells
Our study showed that treatment of M⌽s with methyl -cyclodextrin (m-CD) (m-M⌽) reduces membrane cholesterol by ف50% and treatment of m-M⌽s with liposomal cholesterol (mM⌽-CL) restored membrane cholesterol by ف95%
normal macrophage (N-M⌽), m-M⌽s, m-M⌽-CL, and m-M⌽-CL-AN were used as APCs to stimulate MHC II-restricted T cell hybridoma
Summary
FBS, penicillin-streptomycin, sodium bicarbonate, HEPES, -ME, cholesterol, Tris, EDTA, EGTA, PMSF, protease inhibitor cocktail, m-CD, Giemsa, RPMI-1640, and 22-NBD-cholesterol were purchased from Sigma. The IL-2 assay kit was purchased from BD. The Amplex Red reagent kit was purchased from Invitrogen. All amino acids and trifluoroethanol (TFE) were purchased from Merck
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