Acidification and disulfide reduction can be sufficient to allow intact proteins to bind class II MHC.
The hypothesis that reduction of disulfide bonds and exposure to low pH may be sufficient to allow intact proteins to bind class II MHC was addressed in this study. Functional assays were used to determine minimal conditions sufficient to bypass the requirement for Ag processing. Fixed APC, pulsed with HEL at pH 5 in the presence of a reducing agent, were observed to stimulate I-Ed-restricted T cell hybridomas. Activity required both low pH and reducing agent. Fixed APC also stimulated cytochrome c-specific T cells after exposure to Ag at pH 5 but not pH 7. No reducing agent was required. Peptide was considerably more potent in these assays than intact cytochrome c. By contrast, highly purified cytochrome c was equal in potency with a peptide in competition binding assays using purified I-Ek. The protein did not bind I-Ad or I-Ak. This suggested that cytochrome c can efficiently bind I-Ek at low pH without the requirement for proteolytic cleavage. The capacity of HEL to bind purified I-Ed was strictly dependent on the presence of reducing agent. The reducing agent alone had no effect. A small panel of proteins was screened for class II binding at low pH in competition assays. Horse myoglobin and human hemoglobin were particularly potent in their capacity to inhibit binding of biotin-labeled peptide to purified class II. Unlabeled peptide inhibited binding of labeled peptide to I-Ad at pH 5 and pH 7. By contrast, intact horse myoglobin was active only at pH 5. This result suggested that low pH may enhance the binding of horse myoglobin to I-Ad through effects on the structure of both proteins. Our observations support the hypothesis that low pH and disulfide reduction can be sufficient to allow partially unfolded or structurally destabilized proteins to bind class II MHC.
Department of Pathology, Emory University School of Medicine, Atlanta, GA 30322.
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Jensen
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003574:Cytochrome c Group / Q000378:metabolism; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D000949:Histocompatibility Antigens Class II / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009113:Muramidase / Q000378:metabolism; D009211:Myoglobin / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D013601:T-Lymphocytes / Q000276:immunology*
D003574:Cytochrome c Group; D004220:Disulfides; D000949:Histocompatibility Antigens Class II; C024138:I-E-antigen; D009211:Myoglobin; D011506:Proteins; D009113:Muramidase; D004229:Dithiothreitol