THE INFLUENCE OF EXOGENOUS PEPTIDE ON BETA(2)-MICROGLOBULIN EXCHANGE IN THE HLA COMPLEX - ANALYSIS IN REAL-TIME

We used an optical biosensor to determine the relative binding affinit y of peptides to purified HLA class I molecules. In this assay we moni tor beta(2)-microglobulin (beta(2)m) exchange within the HLA-A2 molecu le, whereby native beta(2)m in the complex is replaced by plm immobili zed at the surface of the biosensor. Quantitative kinetic measurements permit us to obtain association rate (k(ass)), dissociation rate (k(d iss)) and affinity constants (K-A) for the beta(2)m exchange reaction, alone, (control) and in the presence of exogenous peptide. Wa tested a panel of six peptides which had been designed and synthesized with a n HLA-A2 binding motif, and had also been tested by the T2-cell bindin g assay, along with control peptides. The biosensor results demonstrat e that exogenous peptide influences the dynamics of beta(2)m exchange in a sequence-specific manner. Five of six peptides increased the asso ciation rate. decreased the dissociation rate, and significantly incre ased the affinity (K-A = 1.55-1.88 x 10(9) M-1) of HLA-A2 for immobili zed beta(2)m compared with the control (K-A = 1.14 +/- 0.04 x 10(9) M- 1), demonstrating stabilization of the complex. One peptide was unable to stabilize the complex, as also shown in the T2 binding assay. Howe ver. analysis of peptide sequences demonstrated that the HLA-A2 second ary motif as well as primary motif residues are required for HLA-A2 st abilization. Further experiments demonstrated that beta(2)m exchange a lone cannot stabilize the HLA class I complex at the cell surface unti l a peptide of sufficient binding affinity is bound. Hence kinetics eq ual to or below the control values in our biosensor assay probably rep resent an unstable complex in vivo. Unlike other methods described for the analysis of peptide stabilization, this approach is significantly faster, provides full kinetic analysis, and is simpler, since it requ ires no labeling of peptides. Furthermore, this may have important imp lications in the assessment of peptide vaccines.