Identification of three extended antibody-binding segments in recombinant human muscle acetylcholine receptor α subunit extracellular domain 1–210

Abstract

The duplicated alpha subunits account for 40% of the total protein of the nicotinic acetylcholine receptor of muscle, and are implicated as targets for pathogenic autoantibodies in the neuromuscular disease myasthenia gravis (MG). This study reports some of the specificities of antibodies induced by a myasthenogenic recombinant protein (rH alpha 1-210) corresponding to the proposed extracellular domain of the alpha subunit of human acetylcholine receptor, residues 1-210. Antisera produced by immunizing rats, rabbits, and mice were tested with a panel of overlapping synthetic peptides (each 16 amino acids) comprising residues 1-216 of the human alpha subunit. IgG antibodies produced in all three species bound only to peptides that were clustered in three segments: segment I (residues 9-24); segment II (57-96 in rats, 57-88 in rabbits, and 57-80 in mice); and segment III (137-184 in rats, 145-184 in rabbits and mice). Monoclonal antibodies were produced by 41 independent hybridomas derived from three rats immunized with rH alpha 1-210; 12 reacted only with the recombinant or native protein, and 29 reacted additionally with peptides in segments II or III. Four mAbs bound to native human receptor; of these, three bound to peptides 57-72/65-80, 81-96, or 153-168, and one lacked peptide-binding activity. Lack of mAb reactivity with rat receptor precluded correlation of peptide reactivity with myasthenogenicity. Nevertheless, the data indicate that the human acetylcholine receptor's alpha subunit contains multiple sites in its extracellular domain that are potentially stimulatory for B cells.