Mapping of a cholinergic binding site by means of synthetic peptides, monoclonal antibodies, and .alpha.-bungarotoxin

Abstract

Previous studies by several laboratories have identified a narrow sequence region of the nicotinic acetylcholine receptor (AChR) alpha subunit, flanking the cysteinyl residues at positions 192 and 193, as containing major elements of, if not all, the binding site for cholinergic ligands. In the present study, we used a panel of synthetic peptides as representative structural elements of the AChR to investigate whether additional segments of the AChR sequences are able to bind alpha-bungarotoxin (alpha-BTX) and several alpha-BTX-competitive monoclonal antibodies (mAbs). The mAbs used (WF6, WF5, and W2) were raised against native Torpedo AChR, specifically recognize the alpha subunit, and bind to AChR is inhibited by all cholinergic ligands. WF6 competes with agonists, but not with low mol. wt. antagonists, for AChR binding. The synthetic peptides used in this study were approximately 20 residue long, overlapped each other by 4-6 residues, and corresponded to the complete sequence of Torpedo AChR alpha subunit. Also, overlapping peptides, corresponding to the sequence segments of each Torpedo AChR subunit homologous to alpha 166-203, were synthesized. alpha-BTX bound to a peptide containing the sequence alpha 181-200 and also, albeit to a lesser extent, to a peptide containing the sequence alpha 55-74. WF6 bound to alpha 181-200 and to a lesser extent to alpha 55-74 and alpha 134-153. The two other mAbs predominantly bound to alpha 55-74, and to a lesser extent to alpha 181-200. Peptides alpha 181-200 and alpha 55-74 both inhibited binding of 125I-alpha-BTX to native Torpedo AChR. None of the peptides corresponding to sequence segments from other subunits bound alpha-BTX or WF6, or interfered with their binding. Therefore, the cholinergic binding site is not a single narrow sequence region, but rather two or more discontinuous sequence segments within the N-terminal extracellular region of the AChR alpha subunit, folded together in the native structure of the receptor, contribute to form a cholinergic binding region. Such a structural arrangement is similar to the "discontinuous epitopes" observed by X-ray diffraction studies of antibody-antigen complexes [reviewed in Davies et al. (1988)].

Expression of Soluble Ligand- and Antibody-binding Extracellular Domain of Human Muscle Acetylcholine Receptor α Subunit in Yeast Pichia pastoris

“…(iii) ␣-BTX bound to h␣-(1-210) with a high affinity (K d ϭ 5.1 Ϯ 2.4 nM). High affinity binding of ␣-BTX to the AChR ␣ subunit is routinely used to indicate correct folding of the recombinant molecule as the formation of the binding site requires the coordination of several post-translational events that draw together different regions of the ␣ subunit (11,12). (iv) The small competitive antagonists, dtubocurarine and gallamine, inhibited ␣-BTX binding to h␣- .…”

“…Previous experiments have shown that the binding sites for both acetylcholine and ␣-BTX are located close to two adjacent cysteine residues at positions 192 and 193 of the ␣ subunit (10). Moreover, other distinct regions on either the ␣ subunit (11,12) or the adjacent ␥ or ␦ subunits (13) have been shown to contribute to ␣-BTX binding, whereas the role of glycosylation at residue ␣141 (14 -16) requires further study.…”

A second pathway of activation of the Torpedo acetylcholine receptor channel

“…Further supported by our binding studies, we established in this way a new class of channel-activating site(s) at the Torpedo nAChR that is clearly distinct in location and pharmacology from the cholinergic sites. Epitope mapping studies employing synthetic peptides [25,36] and affinity labeling studies are presently under way in order to locate conclusively in the primary structure of the nAChR the site from which eserine exerts its ion-flux-stimulating activity. In addition to these sites, separate sites exist (probably the local anaesthetic binding sites previously identified [37,381) from which eserine, at high concentrations, acts as a direct blocker of the acetylcholine-activated channel [12].…”

“…None of our antibodies raised against Torpedo nAChR [22,25, 291 displayed such properties, but two others were found that did. Antibody FK1 (Fels, G., Kuhlmann, J. and Maelicke, A., unpublished) was raised against hindleg muscle cell membranes from newborn rats [30]; antibody PK1 was raised against a synthetic peptide conjugated to bovine serum albumin [31].…”

Anti-CTLA-4 Antibody Treatment Triggers Determinant Spreading and Enhances Murine Myasthenia Gravis

“…In the present study significant levels of diversified Abs against extracellular regions on the ␣ subunit could be detected only in anti-CTLA-4 Ab-treated mice. T cell recognition of ␣ 182-198 , the cholinergic binding site on AChR, for instance, may lead to production of pathogenic Abs, which directly impair AChR function (42). Previous reports have demonstrated that two ␣ 146 -162 specific T cell lines caused Torpedo AChRprimed B cells to differentiate and secrete fully cross-reactive Abs against the murine epitope ␣ 122-138 that dominate the Ab response in vitro (32).…”