Amino Acid Residues Within the Sequence Region α55–74 of Torpedo Nicotinic Acetylcholine Receptor Interacting with Antibodies to the Main Immunogenic Region and with Snake α-Neurotoxins

Abstract

The sequence region 55–74 of the α-subunit of the acetylcholine receptor (AChR) from Torpedo californica electroplax comprises the amino-terminal end of a sequence segment—residues α67–76—forming the main immunogenic region (MIR), which is most frequently recognized by anti-AChR autoantibodies in myasthenia gravis. The synthetic sequence α55–74 of Torpedo AChR binds α-bungarotoxin (αBTX), suggesting that amino acid residues within this sequence region may contribute to formation of an αBTX binding site.

Using single-residue substituted synthetic analogues of the sequence α55–74 of Torpedo AChR, in which each residue was sequentially substituted by either glycine or alanine, we sought identification of the amino acids involved in interaction with α-neurotoxins and with three different anti-MIR monoclonal antibodies (mAbs 6, 22, and 198). Substitution of Arg₅₅, Arg₅₇, Trp₆₀, Arg₆₄, Leu₆₅, Arg₆₆, Trp₆₇, or Asn₆₈ strongly inhibited α-toxin binding, whereas substitutions of Ile₆₁, Val₆₃, Pro₆₉, Ala₇₀, Asp₇₁, or Tyr₇₂ had marginal effects. Substitutions within the region α68–72 significantly diminished binding of anti-MIR mAbs, although residue preferences differed among mAbs. Further, substituting Trp₆₀ substantially reduced binding of mAb 198, and moderately affected binding of mAb 6, and substitution of Asp₆₂ slightly but consistently affected binding of mAbs 6 and 22.