Precise epitope mapping of monoclonal antibodies to the cytoplasmic side of the acetylcholine receptor α subunit

The epitopes for twelve monoclonal antibodies against the cytoplasmic side of the acetylcholine receptor (AChR) α subunit were precisely mapped using over 300 continuously overlapping synthetic peptides attached on poly(ethylene) rods. mAb cross‐reactive between Torpedo and human AChR generally bound to the homologous peptides from both species. Epitopes 4–10‐residues long were identified. One mAb could bind to either arm on both sides of a β‐turn structure. Five mAb bound to a very‐immunogenic cytoplasmic epitope on α373–380 (VICE‐α). Three of the mAb against VICE‐α were earlier found to cross‐react with non‐AChR protein(s), present in thymomas from myasthenia gravis patients but absent in thymomas from non‐myasthenics. Since VICE‐α has a potentially crucial pathogenic role, the antigenic role of each residue within it was subsequently studied by 55 analogues, most having single amino acid substitutions. All the mAb against VICE‐α bound similarly but not identically to the analogues, thus explaining their known binding heterogeneity. Lys373 proved indispensable for mAb binding. Ile376, Glu377, Gly378 and Lys380 were quite critical, while Ser 374, Ala375 and Val379 seemed rather inactive. These data should prove instructive in searches for VICE‐α‐like epitopes carrying autoantigens with potential involvement in myasthenia gravis and should further expand the applications of the anti‐(AChR) mAb in AChR studies.