AMINO-ACID-RESIDUES WITHIN THE SEQUENCE REGION-ALPHA-55-74 OF TORPEDONICOTINIC ACETYLCHOLINE-RECEPTOR INTERACTING WITH ANTIBODIES TO THE MAIN IMMUNOGENIC REGION AND WITH SNAKE ALPHA-NEUROTOXINS

The sequence region 55-74 of the alpha-subunit of the acetylcholine re ceptor (AChR) from Torpedo californica electroplax comprises the amino -terminal end of a sequence segment-residues alpha67-76-forming the ma in immunogenic region (MIR), which is most frequently recognized by an ti-AChR autoantibodies in myasthenia gravis. The synthetic sequence al pha55-74 of Torpedo AChR binds alpha-bungarotoxin (alphaBTX), suggesti ng that amino acid residues within this sequence region may contribute to formation of an alphaBTX binding site. Using single-residue substi tuted synthetic analogues of the sequence alpha55-74 of Torpedo AChR, in which each residue was sequentially substituted by either glycine o r alanine, we sought identification of the amino acids involved in int eraction with alpha-neurotoxins and with three different anti-MIR mono clonal antibodies (mAbs 6, 22, and 198). Substitution of Arg55, Arg57, Trp60, Arg64, Leu65, Arg66, Trp67, or Asn68 strongly inhibited alpha- toxin binding, whereas substitutions of Ile61, Val63, Pro69, Ala70, As p71, or Tyr72 had marginal effects. Substitutions within the region al pha68-72 significantly diminished binding of anti-MIR mAbs, although r esidue preferences differed among mAbs. Further, substituting Trp60 su bstantially reduced binding of mAb 198, and moderately affected bindin g of mAb 6, and substitution of Asp62 slightly but consistently affect ed binding of mAbs 6 and 22.