IDENTIFICATION OF A HEXAPEPTIDE THAT MIMICS A CONFORMATION-DEPENDENT BINDING-SITE OF ACETYLCHOLINE-RECEPTOR BY USE OF A PHAGE-EPITOPE LIBRARY

Monoclonal antibody (mAb) 5.5 is directed against the ligand-binding s ite of the nicotinic acetylcholine receptor. The epitope for this anti body is conformation-dependent, and the antibody does not react with s ynthetic peptides derived from the receptor sequence. We have identifi ed a ligand peptide that mimics this conformation-dependent epitope fr om a phage-epitope library composed of filamentous phage displaying ra ndom hexapeptides. Among 38 positive phage clones, individually select ed from the library, 34 positive clones carried the sequence Asp-Leu-V al-Trp-Leu-Leu (DLVWLL), 1 positive clone had the sequence Asp-Ile-Val -Trp-Leu-Leu (DIVWLL), and 3 positive clones expressed the sequence Le u-Ile-Glu-Trp-Leu-Leu (LIEWLL), none of which are significantly homolo gous with the nicotinic acetylcholine receptor alpha subunit sequence. All of these phages bind specifically to mAb 5.5. The synthetic pepti de DLVWLL inhibits binding of mAb 5.5 to the related peptide-presentin g phage and to the nicotinic acetylcholine receptor in a concentration -dependent manner; the IC50 value is of the order of 10(-4) M. Bioacti vity of the peptide ''mimotope'' DLVWLL was demonstrated in vivo in ha tched chickens by inhibition of the mAb 5.5 effect by the peptide. The neuromuscular block and myasthenia gravis-like symptoms that are indu ced in chicken by passive transfer of mAb 5.5 were specifically abolis hed by DLVWLL. This study shows the potential of a random peptide phag e-epitope library for selecting a mimotope for an antibody that recogn izes a folded form of the protein, where peptides from the linear amin o acid sequence of the protein are not applicable.