Structural characterization of the main immunogenic region of the Torpedo acetylcholine receptor

Abstract

To develop antigen-specific immunotherapies for autoimmune diseases, knowledge of the molecular structure of targeted immunological hotspots will guide the production of reagents to inhibit and halt production of antigen specific attack agents. To this end we have identified three noncontiguous segments of the Torpedo nicotinic acetylcholine receptor (AChR) α-subunit that contribute to the conformationally sensitive immunological hotspot on the AChR termed the main immunogenic region (MIR): α(1-12), α(65-79), and α(110-115). This region is the target of greater than 50% of the anti-AChR Abs in serum from patients with myasthenia gravis (MG) and animals with experimental autoimmune myasthenia gravis (EAMG). Many monoclonal antibodies (mAbs) raised in one species against an electric organ AChR cross react with the neuromuscular AChR MIR in several species. Probing the Torpedo AChR α-subunit with mAb 132A, a disease inducing anti-MIR mAb raised against the Torpedo AChR, we have determined that two of the three MIR segments, α(1-12) and α(65-79), form a complex providing the signature components recognized by mAb 132A. These two segments straddle a third, α(110-115), that seems not to contribute specific side chains for 132A recognition, but is necessary for optimum antibody binding. This third segment appears to form a foundation upon which the three-dimensional 132A epitope is anchored.

Keywords: 3,3′diaminobenzidine; 4-hydroxyazobenzene-2-carboxylic acid; AChR; AChR main immunogenic region (MIR); Anti-MIR monoclonal antibody; Btx; Conformational epitope structure; DAB; E-AChR; EAMG; Electrophorus electric organ AChR; Fusion peptide screening; HABA; IPTG; MCS; MG; MIR; N-hydroxysuccinimide; NHS; PVDF; Pathogenic Abs; Site-specific mutagenesis; T-AChR; TAE; Torpedo electric organ AChR; Tris, acetic acid, EDTA buffer, pH 8.5; Western blots; aa; acetylcholine receptor; amino acid; experimental autoimmune myasthenia gravis; isopropylthiogalactopyranoside; main immunogenic region; multiple cloning site; myasthenia gravis; polyvinylidene difluoride; α-bungarotoxin.