MAPPING OF THE INTERLEUKIN-10 INTERLEUKIN-10 RECEPTOR COMBINING SITE/

The discontinuous interleukin-10(IL-10)/interleukin-10 receptor (n-10R ) combining site was mapped using sets of overlapping peptides derived from both binding partners bound to continuous cellulose membranes. L ow affinity binding of single regions of the discontinuous contact sit es on IL-10 and IL-10R could be identified due to (1) high peptide den sity on the membrane support, (2) incubation with high protein concent rations, (3) indirect immunodetection of the ligates after electrotran sfer onto polyvinylene difluoride membranes, and (4) use of highly ove rlapping peptide scans of different length (6-mers and 15-mers). The s ingle binding regions identified for each protein species are separate d in the protein sequences, but form continuous areas on the surface o f IL-10 (X-ray structure) and n-10R (computer model). Furthermore, fou r epitopes of neutralizing anti-IL-10 and anti-IL-10R antibodies were mapped and overlap with these binding regions. Soluble peptides (15- t o 19-mers) each spanning one of the three identified IL-10-derived rec eptor binding regions displayed no significant affinity to IL-10R as e xpected, whereas a peptide (35-mer) comprising two of these regions ha d considerably higher binding activity. The data are consistent with a previously published computer model of the IL-10/IL-10R complex. This approach should be generally applicable for the mapping of non-linear protein-protein contact sites.