Main Immunogenic Region of Torpedo Electroplax and Human Muscle Acetylcholine Receptor: Localization and Microheterogeneity Revealed by the Use of Synthetic Peptides

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Abstract
Most anti-nicotinic acetylcholine receptor (AChR) antibodies in myasthenia gravis are directed against an immunodominant epitope or epitopes [main immunogenic region (MIR)] on the AChR .alpha.-subunit. Thirty-two synthetic peptides, corresponding to the complete Torpedo .alpha.-subunit sequence and to a segment of human muscle .alpha.-subunit, were used to map the epitopes for 11 monoclonal antibodies (mAbs) directed against the Torpedo and/or the human MIR and for a panel of anti-AChR mAbs directed against epitopes on the .alpha.-subunit other than the MIR. A main constitutent loop of the MIR was localized within residues .alpha.67-76. Residues 70 and 75, which are different in the Torpedo and human .alpha.-subunits, seem to be crucial in determining the binding profile for several mAbs whose binding to the peptides correlated very well with their binding pattern to native Torpedo and human AChRs. This strongly supports the identification of the peptide loop .alpha.67-76 as the actual location of the MIR on the intact AChR molecule. Residues 75 and 76 were necessary for binding of some mAbs and irrelevant for others, in agreement with earlier suggestions that the MIR comprises overlapping epitopes. Structural predictions for the sequence segment .alpha.67-76 indicate that this segment has a relatively high segmental mobility and a very strong turning potential centered around residues 68-71. The most stable structure predicted for this segment, in both the Torpedo and human .alpha.-subunits, is a hairpin loop, whose apex is a type I .beta.-turn and whose arms are .beta.-strands. This loop is highly hydrophilic, and its apex is negatively charged. All these structural properties have been proposed as characteristic of antibody binding sites. We also localized the epitopes for mAbs against non-MIR regions. Among these, the epitope for a monoclonal antibody (mAb 13) that noncompetitively inhibits channel function was localized within residues .alpha.331-351.