PH dependent binding of high and low affinity myelin basic protein peptides to purified HLA-DR2

Abstract

Major histocompatibility complex (MHC) class II molecules are cell surface glycoproteins and are known to display processed antigens on the surface of antigen presenting cells (APC). Within the APC, the loading of processed antigenic peptides to MHC class II molecules is known to take place in the endosomal compartment at acidic pH environment. The present study describes the in vitro effect of pH on binding of four biotinylated myelin basic protein (MBP) peptides to affinity purified HLA-DR2 containing a mixture of DRB1 ∗1501 and DRB5 ∗0101 beta chain. The binding affinity of the selected peptides are in the order of MBP(83–102)Y 83 > MBP(124–143) > MBP(143–168) > MBP(1–14). Most of these peptides in association with HLA-DR2 are considered as immunodominant epitopes for human multiple sclerosis autoimmune disorder. One epitope, MBP(1–14), had almost no affinity to purified HLA-DR2 and was used as a control peptide in all binding assays. The quantitation of the bound peptide at various pH was carried out by antibody capture of complexes followed by avidin-alkaline phosphatase detection system. Among four peptides tested, only the highest affinity MBP(83–102)Y 83 peptide showed maximum binding to purified HLA-DR2 at acidic pH. Two other epitopes, MBP(124–143) and MBP(143–168), showed maximum binding at basic and neutral pH values, respectively. The binding of only high affinity peptides, MBP(83–102)Y 83 and MBP(124–143), was significantly affected by changing the pH of the binding buffer. Such alteration in pH of the binding buffer resulted in 100% occupancy of DR2 with both high affinity MBP peptides. In contrast, no significant increase in binding of the low affinity MBP(143–168) peptide was observed at altered pH values. The specificity of the increased binding of high affinity peptides to HLA-DR2 at optimum pH was demonstrated by competitive binding assays using non-biotinylated peptides. Finally, the stability of various MBP peptide bound complexes was tested at 4°, 25° and 37°C which correlates well with their affinity to HLA-DR2. These results suggest that pH plays an important role in in vitro binding of antigenic peptides and such manipulation of binding conditions can be utilized in generating 100% loaded MHC class II with high affinity antigenic peptides. Since high affinity peptides are generally considered as major immunodominant epitopes, the in vitro pH dependent binding can be utilized in screening immunodominant epitopes of various autoantigens and generating complexes of defined composition.