Determination of amino acids critical to the main immunogenic region of intact acetylcholine receptors by in vitro mutagenesis

Abstract

The main immunogenic region (MIR) of the acetylcholine receptor (AChR) is the target for the majority of high-affinity autoantibodies produced in myasthenia gravis patients. Some monoclonal antibodies (mAbs) to the MIR bind specifically, but with low affinity, to synthetic AChR α subunit peptides with the sequence α67–76. Studies of synthetic peptides suggest that amino acids α68 and α71 may be especially important to the antigenic structure of the MIR. We have studied the contribution of amino acids α68 and α71 to the antigenicity of the MIR on intact AChR by replacing α68 (N) and α71 (D) of Torpedo AChR α with α68 (D) and α71 (K) by site-directed mutagenesis, expressing the mutated transcripts in Xenopus oocytes along with wild-type Torpedo β,γ andδ subunits, and analyzing the expressed AChR for the binding of mAbs to the MIR. These mutations of the MIR greatly diminished binding of mAbs to the MIR. Thus, both α68 and α71 are critical to the antigenicity of the MIR in intact AChRs.