ANTIGENIC PEPTIDE BINDING TO MHC CLASS-II MOLECULES AT INCREASED PEPTIDE CONCENTRATIONS

Affinity-purified major histocompatibility complex (MHC) class II mole cules are known to bind antigenic peptides in vitro. The percentage of MHC class II molecules occupied with such peptides is usually very lo w and varies significantly depending upon the sequence and size of a g iven antigenic peptide. The present study describes a method by which complete saturation of affinity-purified MHC class II with antigenic p eptide can be achieved by simply incubating purified MHC class II mole cules at neutral pH in the presence of several 100-fold molar excess o f antigenic peptide. Complexes of human HLA-DR2 and a peptide analog f rom human myelin basic protein MBP (83-102)Y-83 were selected for this study. The on-rate kinetic results showed saturation of MHC class II occupancy at 300-500-fold molar excess peptide concentrations. The spe cificity of the MBP (83-102)Y-83 peptide binding to HLA-DR2 at higher peptide concentration was demonstrated by incubating an equivalent amo unt of another epitope from myelin basic protein [MBP (1-14) peptide] as well as by competitive binding assays. The quantitation of bound pe ptide was carried out using biotinylated-MBP (83-102)Y-83 peptide whic h showed 100-125% occupancy of HLA-DR2 with a recovery of 100%. The pr esence of a single peptide entity in purified complexes was confirmed by reverse-phase narrowbore HPLC analysis of the acid extracted supern atant and by mass spectrometry analysis. Two-dimensional gel electroph oresis (IEF/SDS) of purified HLA-DR2 and DR2.MBP (83-102)Y-83 complexe s showed the absence of various endogenous polypeptides in 100% loaded complexes. These results demonstrate that higher peptide concentratio ns can be useful in generating MHC class II-peptide complexes of defin ed composition. Such complexes of MHC class II occupied with a single peptide may have significant clinical relevance for antigen-specific t herapy of various autoimmune diseases and may provide better understan ding of MHC-peptide-TCR interactions.