ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXT.alpha.-Bungarotoxin and the competing antibody WF6 interact with different amino acids within the same cholinergic subsiteBianca M. Conti-Tronconi, Brenda M. Diethelm, Xiadong Wu, Fen Tang, Tony Bertazzon, Bernd Schroeder, Sigrid Reinhardt-Maelicke, and Alfred MaelickeCite this: Biochemistry 1991, 30, 10, 2575–2584Publication Date (Print):March 12, 1991Publication History Published online1 May 2002Published inissue 12 March 1991https://pubs.acs.org/doi/10.1021/bi00224a003https://doi.org/10.1021/bi00224a003research-articleACS PublicationsRequest reuse permissionsArticle Views47Altmetric-Citations44LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
In Myasthenia Gravis most anti-acetylcholine receptor (AChR) antibodies are against a highly conserved area of the AChR alpha-subunit called the Main Immunogenic Region (MIR). Amino acid residues critical for MIR formation have been located within the sequence alpha 67-76. In the present study, binding of anti-AChR monoclonal antibodies (mAbs) to synthetic peptide analogues of the sequence alpha 67-76 of human and Torpedo AChRs containing conservative single-residue substitutions identified the amino acid residues most important to the antigenicity of the MIR sequence, and offered clues to its tridimensional structure. Conservative substitutions of residues Asn68 and Asp71 greatly diminished mAb binding, identifying them as critical contact residues for anti-MIR mAbs. Substitutions at Asp70 and Tyr72 moderately affected binding. Cross-reactive mAbs originally raised against Electrophorus AChR bound single residue-substituted synthetic peptides in a manner consistent with the possibility that Electrophorus AChR may have a glutamic acid residue at position alpha 70 or alpha 71. Substitutions at residues Asp/Ala70 and Val/Ile70 between human and Torpedo alpha-subunits may be size-compensating, suggesting these amino acids in the native AChR may be in closer proximity than proposed in previous models of the MIR.
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In myasthenia gravis (MG) an autoimmune response against muscle acetylcholine receptor (AChR) occurs. Embryonic muscle AChR contains a gamma subunit, substituted in adult muscle by a homologous epsilon subunit. Antibodies and CD4+ cells specific for embryonic AChR have been demonstrated in MG patients. We identified sequence segments of the human gamma subunit forming epitopes recognized by four embryonic AChR-specific CD4+ T cell lines, propagated from MG patients' blood by stimulation with synthetic peptides corresponding to the human gamma subunit sequence. Each line had an individual epitope repertoire, but two 20-residue sequence regions were recognized by three lines of different HLA haplotype. Most T epitope sequences were highly diverged between the gamma and the other AChR subunits, confirming the specificity of the T cells for embryonic AChR. These T cells may have been sensitized against AChR expressed by a tissue other than innervated skeletal muscle, possibly the thymus, which expresses an embryonic muscle AChR-like protein, containing a gamma subunit. Several sequence segments forming T epitopes are similar to regions of microbial and/or mammalian proteins unrelated to the AChR. These findings are consistent with the possibility that T cell cross-reactivity between unrelated proteins ("molecular mimicry"), proposed as a cause of autoimmune responses, is not a rare event.
The sequence segment 181-200 of the Torpedo nicotinic acetylcholine receptor (nAChR) alpha subunit forms a binding site for alpha-bungarotoxin (alpha-BTX) [e.g., see Conti-Tronconi, B. M., Tang, F., Diethelm, B. M., Spencer, S. R., Reinhardt-Maelicke, S., & Maelicke, A. (1990) Biochemistry 29, 6221-6230]. Synthetic peptides corresponding to the homologous sequences of human, calf, mouse, chicken, frog, and cobra muscle nAChR alpha 1 subunits were tested for their ability to bind 125I-alpha-BTX, and differences in alpha-BTX affinity were determined by using solution (IC50S) and solid-phase (KdS) assays. Panels of overlapping peptides corresponding to the complete alpha 1 subunit of mouse and human were also tested for alpha-BTX binding, but other sequence segments forming the alpha-BTX site were not consistently detectable. The Torpedo alpha 1(181-200) and the homologous frog and chicken peptides bound alpha-BTX with higher affinity (KdS approximately 1-2 microM, IC50s approximately 1-2 microM) than the human and calf peptides (Kds approximately 3-5 microM, IC50s approximately 15 microM). The mouse peptide bound alpha-BTX weakly when attached to a solid support (Kd approximately 8 microM) but was effective in competing for 125I-alpha-BTX in solution (IC50 approximately 1 microM). The cobra nAChR alpha 1-subunit peptide did not detectably bind alpha-BTX in either assay. Amino acid substitutions were correlated with alpha-BTX binding activity peptides from different species. The role of a putative vicinal disulfide bound between Cys-192 and -193, relative to the Torpedo sequence, was determined by modifying the peptides with sulfhydryl reagents. Reduction and alkylation of the peptides decreased alpha-BTX binding, whereas oxidation of the peptides had little effect. Modifications of the cysteine/cystine residues of the cobra peptide failed to induce alpha-BTX binding activity. These results indicate that while the adjacent cysteines are likely to be involved in forming the toxin/alpha 1-subunit interface a vicinal disulfide bound was not required for alpha-BTX binding.
AbstractThe monoclonal antibody WF6 competes with acetylcholine and α-bungarotoxin (α-BGT) for binding to the Torpedo nicotinic acetylcholine receptor (nAChR) α1 subunit. Using synthetic peptides corresponding to the complete Torpedo nAChR α1 subunit, we previously mapped a continuous epitope recognized by WF6, and the prototope for α-BGT, to the sequence segment α1(181–200). Single amino acid substitution analogs have been used as an initial approach to determine the critical amino acids for WF6 and α-BGT binding. In the present study, we continue our analysis of the structural features of the WF6 epitope by comparing its cross-reactivity with synthetic peptides corresponding to the α1 subunits from the muscle nAChRs of different species, the rat brain α2, α3, α4 and α5 nAChR subtypes, and the chick brain α-BGT binding protein subunits, αBGTBP α1 and αBGTBP α2. Our results indicate that WF6 is able to cross-react with the muscle α1 subunits of different species by virtue of conservation of several critical amino acid residues between positions 190–198 of the α1 subunit. These studies further define the essential structural features of the sequence segment α1(181–200) required to form the epitope for WF6.
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