Localisation of the main immunogenic region of the nicotinic acetylcholine receptor

Barkas, T.; Gabriel, Jean-Marc; Juillerat, Marcel A.; Kokla, Anna; Tzartos, Socrates J.

doi:10.1016/0014-5793(86)80254-x

Cited by 25 publications

(10 citation statements)

References 18 publications

(8 reference statements)

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“…a-Bungarotoxin bound on blots to all mouse fusion proteins containing residues 160-216, and not to fusion proteins lacking this sequence (Table 1). This is in agreement with experiments based on the use of affinity labeling, which show binding of ligand to Cys 192,193 (15) and immunoblots of protease digests ofTorpedo nAChR (12,16). aBTX binding to fusion protein X4Ql was also tested in solution with the DEAEcellulose filter assay (17).…”

supporting

confidence: 85%

“…The direct proof presented here corroborates our previous work in which, using protease digestion and immunoblots of the bacteriallysates. Samples 78 separated fragments, we localized the MIR of Torpedo (electric ray) nAChR as NH2-terminal to residues 151-169 (12). Recently, Ramam et al (13), using similar methods, have provided further indirect evidence that the MIR of Torpedo nAChR is located between residues 46 and 127.…”

mentioning

confidence: 95%

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Mapping the Main Immunogenic Region and Toxin-Binding Site of the Nicotinic Acetylcholine Receptor

Barkas

Mauron

Roth

et al. 1987

Science

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160

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The alpha-chain of the nicotinic acetylcholine receptor carries the binding sites both for cholinergic ligands and for most experimentally induced or naturally occurring antibodies to the native receptor. By means of expression cloning in Escherichia coli, fusion proteins were derived from specific fragments of a complementary DNA encoding the mouse alpha-chain, allowing the mapping of the toxin-binding site to residues 160-216 and the main immunogenic region to residues 6-85. This approach permits the independent study of different functional domains of a complex receptor molecule and should be generally applicable to other proteins for which complementary DNA clones are available.

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supporting

confidence: 85%

mentioning

confidence: 95%

Mapping the Main Immunogenic Region and Toxin-Binding Site of the Nicotinic Acetylcholine Receptor

Barkas

Mauron

Roth

et al. 1987

Science

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160

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“…2). Of these antibodies, evidence of binding (Table 4) to mammalian AChR was obtained for B3, D6, G3, G10, 7B2 and G. The monoclonal antibody D6 competes (A. Vincent, unpublished data) with a monoclonal (M35, Lindstrom et al 1981) known to be directed against the 'main immunogenic region' (MIR) of the receptor (Tzartos & Lindstrom, 1980;Barkas, Gabriel, Juillerat, Kokla & Tzartos, 1986), suggesting that D6 binds to the MIR. Furthermore, D6 cross-reacts with AChR from many species (Whiting et al 1986).…”

Section: Region Of Antibodymentioning

confidence: 99%

“…Furthermore, the monoclonal antibodies against human receptor compete amongst themselves (Whiting et al 1986) for sites near to or at (e.g. D6) the MIR (see above), which is located on the a-subunit (Lindstrom, 1985;Barkas et al 1986). However, our antibody preparations raised against Torpedo subunits each cross-react with all the other subunits of the AChR , presumably because of sequence homology.…”

Section: Region Of Antibodymentioning

confidence: 99%

Action of antibodies directed against the acetylcholine receptor on channel function at mouse and rat motor end‐plates.

Dolly

Gwilt

Lacey

et al. 1988

The Journal of Physiology

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1. The acute effects of antibodies (both polyclonal and monoclonal) raised against the acetylcholine receptor were studied at mouse and rat end-plates. Isolated muscles were incubated in solutions containing antibody for 2 1/4 to 3 1/2 h. Intracellular microelectrode techniques were then used to record miniature end-plate potentials (MEPPs) and voltage noise. 2. Most antibody preparations investigated did not reduce MEPP amplitudes as compared with controls. One monoclonal (C7) and one polyclonal (J) preparation irreversibly reduced MEPP amplitudes. Both preparations caused reductions in acetylcholine-induced depolarization and associated channel opening frequency (from voltage noise analysis). Single-channel depolarization was not altered by these antibodies. 3. On the basis of these and previous results, four antibody binding regions on the receptor surface were distinguished according to whether channel function and/or alpha-bungarotoxin binding were affected. Although most antibody preparations did not affect channel function, monoclonal antibody C7 appeared to alter function by acting on the channel itself so as to prevent channel opening.

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“…Anti-MIR mAb binding has been gradually localized between amino acid residues 1-151 (Barkas et al, 1986), 46-120 (Ratnam et al, 1986), 37-85 (Barkas et al, 1987), 61-76 (Barkas et al, 1988) 67-78 (Wood et al, 1989 and finally 67-76 of the AChR ol-subunit . The conformation of the MIR decapeptide, either free (in dimethyl sulfoxide) or bound on the corresponding anti-MIR mAb, has been elucidated by the use of two-dimensional NMR experiments (Cung et al, 1989(Cung et al, , 1992.…”

mentioning

confidence: 99%

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

Papadouli

Sakarellos

Tzartos

1993

European Journal of Biochemistry

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The main immunogenic region (MIR) of the nicotinic acetylcholine receptor (AChR) is an immunodominant area of the molecule, both in human and in experimental autoimmune myasthenia gravis. Anti-MII$ monoclonal antibody (mAb) binding has been earlier localized between amino acid residues 67-76 of the AChR a-subunit. A thorough study of the epitope(s) for anti-MIR mAbs, by the use of a large panel of overlapping synthetic peptides and multiple peptide analogues, is now presented and offers clues for potential therapeutic applications of the obtained data.Use of all possible overlapping hexapeptides within Torpedo and human 1x40-91 AChR and of selected peptides of various sizes, showed that the shortest peptide capable of significant antibody binding is the pentapeptide a67 -71. Systematic screening of peptide analogues, where each amino acid residue within a67-76 and a67-74 of both Torpedo and human AChRs was substituted by various amino acids, was performed. Asn68 and Asp71 were found to be indispensable for anti-MIR mAb binding, whereas Pro69 and Ala/Asp70 were less but still significantly important. mAb binding to a67-76 from various AChR species further supported the significance of these results. An additional series of selected peptide analogues was then constructed, aiming at the identification of analogues with high antigenic activity. Many analogues with either single substitutions of a76 or combinations of two substitutions at a73 and a76 were tested. Several of these analogues (mainly His76, Arg76, Va173Ala76, His73Ala76, Va173Arg76) exhibited dramatic mAb binding enhancement. Some anti-MIR mAbs that do not bind to a67 -76 bound significantly to certain analogues. Such analogues could find applications in studies of therapeutic models of myasthenia gravis.

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Localisation of the main immunogenic region of the nicotinic acetylcholine receptor

Cited by 25 publications

References 18 publications

Mapping the Main Immunogenic Region and Toxin-Binding Site of the Nicotinic Acetylcholine Receptor

Mapping the Main Immunogenic Region and Toxin-Binding Site of the Nicotinic Acetylcholine Receptor

Action of antibodies directed against the acetylcholine receptor on channel function at mouse and rat motor end‐plates.

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

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