Antigenic role of single residues within the main immunogenic region of the nicotinic acetylcholine receptor

Abstract

The target of most of the autoantibodies against the acetylcholine receptor (AChR) in myasthenic sera is the main immunogenic region (MIR) on the extracellular side of the AChR alpha-subunit. Binding of anti-MIR monoclonal antibodies (mAbs) has been recently localized between residues alpha 67 and alpha 76 of Torpedo californica electric organ (WNPADYGGIK) and human muscle (WNPDDYGGVK) AChR. In order to evaluate the contribution of each residue to the antigenicity of the MIR, we synthesized peptides corresponding to residues alpha 67-76 from Torpedo and human AChRs, together with 13 peptide analogues. Nine of these analogues had one residue of the Torpedo decapeptide replaced by L-alanine, three had a structure which was intermediate between those of the Torpedo and human alpha 67-76 decapeptides, and one had D-alanine in position 73. Binding studies employing six anti-MIR mAbs and all 15 peptides revealed that some residues (Asn68 and Asp71) are indispensable for binding by all mAbs tested, whereas others are important only for binding by some mAbs. Antibody binding was mainly restricted to residues alpha 68-74, the most critical sequence being alpha 68-71. Fish electric organ and human MIR form two distinct groups of strongly overlapping epitopes. Some peptide analogues enhanced mAb binding compared with Torpedo and human peptides, suggesting that the construction of a very antigenic MIR is feasible.

High‐resolution epitope mapping and fine antigenic characterization of the main immunogenic region of the acetylcholine receptor

“…Although anti-MIR antibodies bind with high affinity to the intact AChR, they bind with low afinity to the synthetic MIR peptides. However, it was noticed both in earlier studies (Papadouli et al, 1990;Bellone et al, 1989) and in the present work that some analogues, especially of a73 and a76, often enhanced binding of the tested anti-MIR mAbs. Subsequent study of selected single and double substitutions on a73 and a76 showed that some of these substitutions had a dramatically enhanced mAb binding activity (Fig.…”

“…However, the binding efficiency of anti-MIR mAbs to a67-76 peptides, as well as to any denatured AChR fragment, is very low in comparison to that of the intact AChR. A~~~~ the above-studied ~1~ substitutions, those on a73 and a76 appeared to enhance anti-MIR mAb binding (Papadouli et al, 1990). Further study ofthe enhancemerit effect towards the design of an MIR peptide with antigenicity to that of the MIR on the intact AChR, would have potential clinical applications.…”

“…The above results on the enhancement of mAb binding induced by substitutions on a73, as well as earlier observations on enhancement of mAb binding when substituting Lys76 by Ala on the decapeptide a67-76 (Papadouli et al, 1990), prompted us to search systematically for analogues with improved binding activity.…”

“…Limited a67-76 analogue studies (having Ala or Gly substitutions) suggested that certain amino acid residues such as Am68 and Asp71 may be indispensable for the antigenicity of the MIR (Bellone et al, 1989;Papadouli et al, 1990). Similarly, mutant AChRs resulting from site-directed mutagenesis at positions a68 and/or a71 did not exhibit any binding activity with anti-MIR mAbs (Saedi et al, 1990).…”

Bacterial expression of a single‐chain Fv fragment which efficiently protects the acetylcholine receptor against antigenic modulation caused by myasthenic antibodies

“…Binding of several anti-MIR monoclonal antibodies (mAb) has been localised within residues 67-76 of the a-subunit [7]. The antigenic role of each residue within a67-76 has been examined and it has been shown that residues a68-71 are [I 115231 the most critical [8,91. The conformation of the a67-76 peptide, either free or bound on an anti-MIR mAb, has been elucidated by two-dimensional nuclear magnetic resonance (NMR) studies [lo, 111.…”