Localization of azidophencyclidine-binding site on the nicotinic acetylcholine receptor alpha-subunit

Nicotinic acetylcholine receptors in receptor-rich membranes from Torpedo californica and from T. marmorata electric tissue were photolabeled with the non-competitive inhibitor [3H]azidophencyclidine. The receptor subunits were separated on SDS-polyacrylamide gels and the alpha-subunits recovered from the gel, were subjected to Staphylococcus aureus V8 protease cleavage. The proteolytic fragments were resolved by SDS-polyacrylamide gel electrophoresis and were identified on protein blots by 125I-labeled alpha-bungarotoxin binding and by staining with concanavalin A. The site of specific azidophencyclidine labeling has been localized to the V8-18 kDa fragment which binds toxin. Labeling of the V8-18 kDa fragment was observed in the absence and in the presence of carbamylcholine. This was found for both the species of Torpedo used here.     

Identification of a cytoplasmic region of the Torpedo nicotinic acetylcholine receptor alpha-subunit by epitope mapping.

Analysis of the binding of monoclonal antibodies (mAbs) by Torpedo nicotinic acetylcholine receptor (AChR) has demonstrated that a region of the alpha-subunit between alpha-156 and alpha-179 is exposed on the cytoplasmic surface of the nicotinic post-synaptic membrane. A panel of mAbs was produced that recognized sodium dodecyl sulfate-denatured subunits of the Torpedo AChR. Antibodies recognizing alpha-subunit were distinguished in terms of their ability to bind alpha-subunit fragments generated by Staphylococcus aureus V8 protease: an 18-kDa fragment beginning at Val-46, a 20-kDa fragment beginning at Ser-173/Ser-162, and a 10 kDa fragment beginning at Asn-339. Three mAbs, selected for binding to each of the V8-protease alpha-subunit fragments, respectively, were characterized in detail. The location of epitopes recognized by both anti-V8-18 and anti-V8-20 mAbs was determined to be within alpha-156 to alpha-179 by isolation of small immunoreactive peptides from proteolytic digests of the alpha-subunit, while the mAb reactive to V8-10 was bound to an epitope within alpha-339 to alpha-386. Quantitative evaluation of binding of the anti-V8-18 and anti-V8-20 mAbs to overlapping synthetic peptides corresponding to alpha-147 to alpha-179 localized the epitopes to distinct portions of this region. Further screening of the panel of mAbs using these synthetic peptides revealed three additional mAbs that bind in this region. The mAbs that bound the three distinct V8-protease alpha-subunit fragments were shown to bind to native AChR by indirect immunofluorescence on frozen sections of Torpedo electric organ. Binding to the native AChR was to the cytoplasmic surface of the AChR since the mAbs could bind to AChR in native vesicles, in which the AChR is oriented right-side-out, only after permeabilization of the vesicles by alkaline treatment or after scrambling of the orientation of the AChR by solubilization and reconstitution into liposomes. The location of the mAb-binding sites at the cytoplasmic surface of the AChR was visualized directly by freeze-etch immunoelectron microscopy. The identification of alpha-156 and alpha-179 as containing a cytoplasmic exposed sequence implies the existence of two non-hydrophobic transmembrane sequences between the site of N-glycosylation (Asn-141) and Cys-192, a site alkylated by the cholinergic affinity labels.     

Localization of the insulin-binding site to the cysteine-rich region of the insulin receptor alpha-subunit

Affinity-purified insulin receptor was photoaffinity labeled with a cleavable radioactive insulin photoprobe. Exhaustive digestion of the labeled alpha-subunit with endoproteinase Glu-C produced a major radioactive fragment of 23 kDa as a part of the putative insulin-binding domain. This fragment could contain either residues 205-316 or 518-633 of the alpha-subunit. Rat hepatoma cells and Chinese hamster ovary cells were transfected with cDNA encoding a human insulin receptor mutant with a deletion of the cysteine-rich region spanning amino acid residues 124-319. Insulin binding by these cells was not increased in spite of high numbers of the mutant insulin receptors being expressed. A panel of monoclonal antibodies which was specific for the receptor alpha-subunit and inhibited insulin binding immunoprecipitated the photolabeled 23-kDa receptor fragment but not the receptor mutant. A synthetic peptide containing residues 243-251 was specifically bound by agarose-insulin beads. We therefore suggest that the 23-kDa fragment contains residues 205-316, and that insulin binding occurs, in part, in the cysteine-rich region of the alpha-subunit.     

Location of ligand-binding sites on the nicotinic acetylcholine receptor alpha-subunit

The portions of the Torpedo californica nicotinic acetylcholine receptor (AChR) alpha-subunit that contribute to the allosteric antagonist-binding site and to the agonist-binding site have been localized by affinity labeling and proteolytic mapping. [3H]Meproadifen mustard was employed as an affinity label for the allosteric antagonist-binding site and [3H]tubocurare as a photoaffinity label for the agonist-binding site. Both labels were found in a 20-kDa proteolytic fragment generated from the AChR alpha-subunit by Staphylococcus aureus V8 protease. This 20-kDa peptide also contains the 3H-labeled 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide-reactive site and binds 125I-alpha-bungarotoxin. N-terminal sequencing established that the 20-kDa fragment began at Ser-173 of the alpha-subunit. Fluorescein isothiocyanate-conjugated concanavalin A could be bound to the second of the two major V8 cleavage products, an 18-kDa peptide. This peptide was also sensitive to treatment with endo-beta-N-acetyl-glucosaminidase H, consistent with the presence of N-linked carbohydrate on this fragment. The N terminus of this peptide was found to be Val-46 of the alpha-subunit sequence. Experiments designed to map disulfide bonds within the AChR alpha-subunit indicate that no bonds exist between the 18-kDa fragment (containing Cys-128 and Cys-142) and the 20-kDa fragment (containing Cys-192, Cys-193, and Cys-222). These results establish that the 20-kDa fragment contributes to both the acetylcholine and the allosteric antagonist-binding sites, whereas there is no evidence that the 18-kDa fragment is part of either site.     

Three possible disulfides in the acetylcholine receptor alpha-subunit

The cysteinyl residues of the acetylcholine receptor alpha-subunit of Torpedo californica were analyzed. All seven cysteines could be accounted for. Three possible disulfide bridges and one unpaired cysteine were indicated. The disulfide linkages were as follows: Cys128 to Cys142; Cys192 to Cys193; Cys412 to Cys418 (Cys222 is unpaired). The identification of cysteinyl residues was accomplished by a modified protein blot procedure. Cysteinyl residues of intact nicotinic acetylcholine receptor were selectively biotinylated with 3-(N-maleimidopropionyl)biocytin and subsequently detected by the 125I-labeled avidin overlay of blotted Staphylococcus aureus V8 proteolyzed alpha-subunits. Two pairs of cysteines (Cys128/Cys142 and Cys412/Cys418) could be demonstrated only after Na(BH4) reduction of the acetylcholine receptor. Cysteine residues 192 and 193 are particularly sensitive to reduction; 0.1 mM dithiothreitol is sufficient.     

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

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.     

The main immunogenic region (MIR) of the nicotinic acetylcholine receptor and the anti-MIR antibodies

Myasthenia gravis (MG) is caused by autoantibodies against the nicotinic acetylcholine receptor (AChR) of the neuromuscular junction. The anti-AChR antibodies are heterogeneous. However, a small region on the extracellular part of the AChR alpha subunit, called the main immunogenic region (MIR), seems to be the major target of the anti-AChR antibodies, but not of the specific T-cells, in experimental animals and possibly in MG patients. The major loop of the overlapping epitopes for all testable anti-MIR monoclonal antibodies (MAbs) was localized within residues 67-76 (WNPADYGGIK for Torpedo and WNPDDYGGVK for human AChR) of the alpha subunit. The N-terminal half of alpha 67-76 is the most critical, Asn68 and Asp71 being indispensable for binding. Yet anti-MIR antibodies are functionally and structurally quite heterogeneous. Anti-MIR MAbs do not affect channel gating, but they are very potent in mediating acceleration of AChR degradation (antigenic modulation) in cell cultures and in transferring experimental MG in animals. Fab fragments of anti-MIR MAbs bound to the AChR prevent the majority of the MG patients' antibodies from binding to and causing loss of the AChR. Whether this inhibition means that most MG antibodies bind on the same small region or is a result of broad steric/allosteric effects is under current investigation.     

Two-dimensional 1H-NMR study of synthetic peptides containing the main immunogenic region of the Torpedo acetylcholine receptor

A comparative 1H-NMR spectral study of a synthetic decapeptide containing the main immunogenic region of the Torpedo acetylcholine receptor (AChR; WNPADYGGIK, representing the alpha 67-76 fragment of Torpedo AChR) with four analogous peptides (WNP3-D5YGGIK, WNPAA5YGGIK, WNPADYGGA9K, and WNPD4DYGGV9K) has been carried out in dimethyl sulfoxide. One- and two-dimensional nmr experiments [correlated spectroscopy (COSY), relayed COSY, and phase-sensitive nuclear Overhauser enhancement spectroscopy (NOESY)] were performed to obtain complete assignments of the proton resonances. The presence of strong and multiple short- and long-range NOEs, and especially a strong long-range NOE between the two Asn2-C alpha H and Gly7-C alpha H protons, argues in favor of a rigid folded structure in all five cases. Temperature dependence measurements indicate the existence of three intramolecular interactions involving the Asp3, Gly8, and Lys10 amide protons.     

The main immunogenic region of the nicotinic acetylcholine receptor: interaction of monoclonal antibodies with synthetic peptides

Monoclonal antibodies to the main immunogenic region of the nicotinic acetylcholine receptor have been studied with regard to their binding to synthetic peptides. It was found that monoclonal antibody 210 to the main immunogenic region binds to the synthetic fragment spanning residues 66 to 76 of the alpha subunits of the acetylcholine receptor from human muscle, but not to the homologous sequence from Xenopus. This parallels the reactivities of antibodies to the main immunogenic region with intact receptors from two species, and confirms the biological significance of the weak interactions observed between antibodies to this region and synthetic peptides. It also suggests that N alpha 68 and D alpha 71 are critical contact residues.     

Rabies virus binding to an acetylcholine receptor alpha-subunit peptide

The binding of 125I-labeled rabies virus to a synthetic peptide comprising residues 173-204 of the alpha 1-subunit of the nicotinic acetylcholine receptor was investigated. Binding of rabies virus to the receptor peptide was dependent on pH, could be competed with by unlabeled homologous virus particles, and was saturable. Synthetic peptides of snake venom, curaremimetic neurotoxins and of the structurally similar segment of the rabies virus glycoprotein, were effective in competing with labeled virus binding to the receptor peptide at micromolar concentrations. Similarly, synthetic peptides of the binding domain on the acetylcholine receptor competed for binding. These findings suggest that both rabies virus and neurotoxins bind to residues 173-204 of the alpha 1-subunit of the acetylcholine receptor. Competition studies with shorter alpha-subunit peptides within this region indicate that the highest affinity virus binding determinants are located within residues 179-192. A rat nerve alpha 3-subunit peptide, that does not bind alpha-bungarotoxin, inhibited binding of virus to the alpha 1 peptide, suggesting that rabies binds to neuronal nicotinic acetylcholine receptors. These studies indicate that synthetic peptides of the glycoprotein binding domain and of the receptor binding domain may represent useful antiviral agents by targeting the recognition event between the viral attachment protein and the host cell receptor, and inhibiting attachment of virus to the receptor.     

Antibodies against an alpha-bungarotoxin-binding peptide of the alpha-subunit of the acetylcholine receptor

Polyclonal and monoclonal antibodies were raised against a peptide comprising residues 173-204 of the alpha-subunit of the acetylcholine receptor. The polyclonal and pooled monoclonal antibodies inhibited up to 50% of 125I-alpha-bungarotoxin binding to peptide 173-204. Some of the antibodies recognized native receptor but did not significantly affect alpha-bungarotoxin binding. Epitope mapping revealed that the antibodies are directed against residues 183-194 indicating this region is a major determinant of toxin binding. This region is most likely conformationally constrained in the native receptor.     

The main immunogenic region of the nicotinic acetylcholine receptor. Identification of amino acid residues interacting with different antibodies

In myasthenia gravis a highly conserved area of the nicotinic receptor (AcChR) dominates the autoantibody response (main immunogenic region, MIR), and it is formed by residues within the sequence segment 67-76 of the AcChR alpha-subunit. We have studied the binding of eight anti-MIR mAb to synthetic peptides containing the sequence segment 67-76 of the human alpha-subunit, and peptide analogues containing single residue substitutions of this sequence. We used also a peptide where both Asp70 and Asp71 were substituted by glycine residues. The binding of six anti-MIR mAb was strongly influenced by several substitutions. All these mAb required residues Asn68, and Pro69 for binding. Five of them required also Asp71 and Tyr72. Substitution of Asp70, which is an Ala residue in Torpedo AcChR, was irrelevant for the binding of an anti-Torpedo and an anti-Electrophorus mAb, and moderately reduced the binding of an anti-human mAb (no. 203). Substitution of Trp67 moderately reduced the binding of some of these mAbs. A mAb of this group (the antihuman mAb no. 198) bound in a manner only slightly influenced by ionic strength, whereas the binding of the other five mAb of this group was very sensitive to the ionic strength. Two anti-Electrophorus MIR mAb bound similarly to all peptide analogues in low ionic strength. At high ionic strength only the peptide analogue where Asp 70 was changed to a Gly residue bound significantly. This may indicate that the Electrophorus MIR has an uncharged residue at this position, as does Torpedo AcChR. Residues at position 73, 74, 75, and 76 were of little or no importance for the binding of all anti-MIR mAb. A free amino terminus was essential for the binding of most mAb. The results of competition experiments between different peptides and native AcChR for mAb binding were consistent with those obtained in direct binding experiments.     

The crystal structure of the Fab fragment of a rat monoclonal antibody against the main immunogenic region of the human muscle acetylcholine receptor.

The crystal structure of the Fab fragment of a rat monoclonal antibody, number 192, with a very high affinity (Kd = 0.05 nM) for the main immunogenic region of the human muscle acetylcholine receptor (AChR), has been determined and refined to 2.4 A resolution by X-ray crystallographic methods. The overall structure is similar to a Fab (NC6.8) from a murine antibody, used as a search model in molecular replacement. Structural comparisons with known antibody structures showed that the conformations of the hypervariable regions H1, H2, L1, L2, L3 of Fab192 adopt the canonical structures 1, 1, 2, 1, and 1, respectively. The surface of the antigen-binding site is relatively planar, as expected for an antibody against a large protein antigen, with an accessible area of 2865 A2. Analysis of the electrostatic surface potential of the antigen-binding site shows that the bottom of the cleft formed in the center of the site appears to be negatively charged. The structure will be useful in the rational design of very high affinity humanized mutants of Fab192, appropriate for therapeutic approaches of the model autoimmune disease myasthenia gravis.     

The regions of alpha-neurotoxin binding on the extracellular part of the alpha-subunit of human acetylcholine receptor

A set of 18 synthetic uniform overlapping peptides spanning the entire extracellular part (residues 1–210) of the -subunit of human acetylcholine receptor were studied for their binding activity of¹²⁵I-labeled -bungarotoxin and cobratoxin. A major toxin-binding region was found to reside within peptide 122–138. In addition, low-binding activities were obtained with peptides 34–49 and 194–210. It is concluded that the region within residues 122–138 constitutes a universal major toxin-binding region for acetylcholine receptor of various species.     

Human nicotinic acetylcholine receptor alpha-subunit isoforms: origins and expression.

A majority of the autoantibodies in the disease myasthenia gravis (MG) are directed against the alpha-subunit of the muscle nicotinic acetylcholine receptor (AChR). Unlike AChR alpha-subunits previously characterised from other species, the human alpha-subunit exists as two isoforms. The isoforms are generated by alternate splicing of an additional exon located between exons P3 and P4, termed P3A. The 25 amino acids encoded by the P3A exon are incorporated into the extracellular region of the alpha-subunit, and so may be relevant to the pathogenesis of MG. Genomic sequences from rhesus monkey, and from dog and cat, which are susceptible to MG, were characterised between AChR alpha-subunit exons P3 and P4. Although regions homologous to the P3A exon were identified for each of these species, analysis by RT-PCR showed that they are not expressed. At variance with a previous report, constitutive expression of mRNA encoding the human P3A+ alpha-subunit isoform was not detected in heart, kidney, liver, lung or brain. Differential expression of the two alpha-subunit isoforms was not seen during fetal muscle development or in muscle from MG patients. In all cases where mRNAs encoding the two alpha-subunit isoforms have been detected, they are present at an approximate 1:1 ratio.     

Recombinant human acetylcholine receptor alpha-subunit induces chronic experimental autoimmune myasthenia gravis

A synthetic gene encoding the 210 N-terminal residues of the alpha-subunit of the nicotinic acetylcholine receptor (AChR) of human skeletal muscle was cloned into an inducible expression plasmid to produce a fusion protein in high yield in Escherichia coli. Like native human AChR, the recombinant human alpha 1-210 protein induced AChR-binding, AChR-modulating, and AChR-blocking autoantibodies in rats when injected once intradermally as an emulsion in CFA, with Bordetella pertussis vaccine as supplementary adjuvant. The minimum dose of recombinant protein required to induce biochemical signs of experimental autoimmune myasthenia gravis (EAMG) with 100% incidence was 2.2 micrograms. With 6.6 to 22 micrograms, serum levels of autoantibodies were persistent, and clinically apparent EAMG lasted more than a month. Clinical, electrophysiological, and biochemical indices of EAMG induced by doses of 66 micrograms or more were more uniformly severe and persistent, with 33% fatality. Rats receiving a control extract of E. coli containing plasmid without the alpha 1-210 codon insert, with adjuvants, did not develop autoantibodies or signs of EAMG. This highly reproducible new model of EAMG induced by a recombinant human autoantigen should be valuable for testing Ag-specific immunotherapeutic strategies that might be applicable to treating acquired myasthenia gravis in humans.     

Localization of the functional sites on the alpha chain of acetylcholine receptor

A comprehensive synthetic approach, previously developed in this laboratory, has been applied to systematically screen the entire extracellular part (residues 1-210) of the alpha chain of the Torpedo californica acetylcholine receptor (AChR) for the profiles of the continuous regions that are recognized by antibodies against free, or membrane-sequestered, AChR; the regions recognized by AChR-primed T cells; the regions that bind alpha-bungarotoxin and cobratoxin; and an acetylcholine-binding region. Eight continuous antigenic sites were localized in this part of the alpha chain by all of the antisera tested. The sites were independent of the host species from which the antisera were obtained and were also similar to antisera against the isolated pentameric AChR or against the membrane-sequestered AChR. Six regions were found to stimulate AChR-primed T cells (T sites). Three of the T sites coincided with regions recognized by antibodies. At least two T sites had no detectable antibody responses directed to them. Five toxin-binding regions were localized, and may constitute distinct sites or, alternatively, different faces in one (or more) sites. Some of these regions coincided with regions recognized by anti-AChR antibodies. One of the toxin-binding regions bound acetylcholine, and immunization with this peptide induced experimental autoimmune myasthenia gravis.     

Design, synthesis, and conformational study of biologically active photolabeled analogues of the main immunogenic region of the acetylcholine receptor

Photoaffinity labeling is a powerful tool for the characterization of the molecular basis of ligand binding to acceptor molecules, which provides important insights for mapping the bimolecular interfaces. The autoimmune disease myasthenia gravis is caused by autoantibodies against the acetylcholine receptor (AChR). The majority of the anti-AChR antibodies bind to the "main immunogenic region" (MIR) of the AChR. To identify the contact points between the complementarity determining regions of the anti-MIR antibodies that recognize the MIR contact sites of the AChR, we present here three photoreactive dodecapeptide MIR analogues containing the photolabel p-benzoyl-L-phenylalanine (Bpa) moiety, either in position 1 or 11. The structure of the produced 12-mers was analyzed using two-dimensional (1)H-NMR spectroscopy, whereas their binding to anti-MIR monoclonal antibodies (mAbs) was determined by immunochemical assays. In all cases the modifications resulted in conservation of the beta-turn conformation of the N-terminus, which has been proved essential for antibody recognition and increased anti-MIR binding relative to the MIR decapeptide.