High-resolution epitope mapping and fine antigenic characterization of the main immunogenic region of the acetylcholine receptor. Improving the binding activity of synthetic analogues of the region.

The main immunogenic region (MIR) of the nicotinic acetylcholine receptor (AChR) is an immunodominant area of the molecule, both in human and in experimental autoimmune myasthenia gravis. Anti-MIR monoclonal antibody (mAb) binding has been earlier localized between amino acid residues 67-76 of the AChR alpha-subunit. A thorough study of the epitope(s) for anti-MIR mAbs, by the use of a large panel of overlapping synthetic peptides and multiple peptide analogues, is now presented and offers clues for potential therapeutic applications of the obtained data. Use of all possible overlapping hexapeptides within Torpedo and human alpha 40-91 AChR and of selected peptides of various sizes, showed that the shortest peptide capable of significant antibody binding is the pentapeptide alpha 67-71. Systematic screening of peptide analogues, where each amino acid residue within alpha 67-76 and alpha 67-74 of both Torpedo and human AChRs was substituted by various amino acids, was performed. Asn68 and Asp71 were found to be indispensable for anti-MIR mAb binding, whereas Pro69 and Ala/Asp 70 were less but still significantly important. mAb binding to alpha 67-76 from various AChR species further supported the significance of these results. An additional series of selected peptide analogues was then constructed, aiming at the identification of analogues with high antigenic activity. Many analogues with either single substitutions of alpha 76 or combinations of two substitutions at alpha 73 and alpha 76 were tested. Several of these analogues (mainly His76, Arg76, Val73Ala76, His73Ala76, Val73Arg76) exhibited dramatic mAb binding enhancement. Some anti-MIR mAbs that do not bind to alpha 67-76 bound significantly to certain analogues. Such analogues could find applications in studies of therapeutic models of myasthenia gravis.