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BALB/c mice develop myasthenic symptoms after immunization with rodent acetylcholine receptor (AChR). After immunization with Torpedo AChR (TAChR), their CD4+ cells become strongly sensitized against a conserved region of the TAChR alpha subunit sequence (residues alpha 304-322), and cross-react vigorously with the homologous sequences of mouse and human AChR, which are almost identical. Therefore AChR-specific potentially autoreactive CD4+ cells exist in this strain. We immunized BALB/c mice with the synthetic TAChR sequence alpha 304-322. The CD4+ cells thus sensitized responded to TAChR, indicating that they recognize an epitope(s) produced upon TAChR processing. They recognized peptide alpha 304-322 in association with the I-Ad molecule. Anti-alpha 304-322 CD4+ cells cross-reacted well with the corresponding murine and human synthetic sequences. To identify residues involved in formation of an autoimmune epitope(s), CD4+ cells from mice immunized with peptide alpha 304-322 were challenged in vitro with single residue glycine-substituted analogues of this sequence. Substitution of residue W311, and of any residue within the sequence alpha 313-319 (RKVFIDT), consistently and, in some cases, strongly affected the CD4+ cells response. Substitution of residues in the region alpha 311-319 had variable effects in different experiments, and in general affected moderately the CD4+ response. These results suggest that anti-alpha 304-322 CD4+ cells comprise several clones, recognizing overlapping epitopes which share residues alpha 311-319. The importance of the sequence region alpha 311-319 for formation of CD4+ cell epitope(s) was verified by testing CD4+ cells sensitized to T alpha 304-322 with analogues of this sequence, carrying non-conservative substitutions at positions Q310, K314 and D318. Substitution of Q310 had minimal or no effects, while those of K314 or D318 strongly affected the CD4+ cell response.

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We propagated from myasthenia gravis (MG) patients by stimulation in vitro with synthetic sequences (alpha 48-67, alpha 304-322, gamma 75-94 and gamma 321-340) of the human muscle acetylcholine receptor (AChR), CD4+ lines against four 20-residue sequence regions of the AChR alpha and gamma subunits that are recognized by Th cells of most MG patients. Most lines secreted IL-2 and not IL-4, suggesting that they comprise Th1 cells. For three lines we verified that, as reported previously, AChR epitopes are presented by DR molecules: their response to the relevant peptide was abolished by anti-DR Abs. The DR molecules presenting AChR epitopes were identified by testing the response of the lines to the relevant peptide, using APC from donors homozygous for the different DR alleles of the line. We tested the lines with single residue-substituted analogues of the epitope sequence. The results of these experiments indicated that the lines were polyclonal and recognized overlapping epitopes. Their response was abolished by some substitutions, identifying residues common to all epitopes within a given region, whereas other substitutions reduced but did not obliterate the response, indicating residues included in some but not all epitopes recognized by the line. Comparison of the residues involved in epitope formation for different lines supported the conclusion that within the 20-residue immunodominant regions investigated here, the same sequence segment is involved in formation of epitopes, even in DR-discordant patients.

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Previous studies have identified the sequence region flanking the invariant vicinal cysteinyl residues at positions 192 and 193 of the nicotinic acetylcholine receptor alpha-subunit as containing major elements of the binding site for acetylcholine and its agonists and antagonists, including antibody WF6 (Conti-Tronconi, B. M., Diethelm, B. M., Wu, X., Tang, F., Bertazzon, T., Schröder, B., Reinhardt-Maelicke, A., and Maelicke, A. (1991) Biochemistry 30, 2575-2584). Recently we have shown that the sequence region flanking lysine alpha 125 contains elements of the binding site for physostigmine and related ligands, including antibody FK1 (Schrattenholz, A., Godovac-Zimmerman, J., Schäfer, H.-J., Albuquerque, E. X., and Maelicke, A. (1993) Eur. J. Biochem. 216, 671-677). Here we report the identification by enzyme-linked immunosorbent assay techniques, employing fragments of the Torpedo nicotinic acetylcholine receptor alpha-subunit N-terminal region and a panel of synthetic peptides matching in sequence preselected portions of this subunit, of the sequence regions alpha 118-145 and alpha 181-216 as contributing to the FK1 epitope. Of the synthetic peptides employed, alpha 118-137 displayed the highest affinity of FK1 binding. Binding of FK1 and WF6 to single residue-substituted analogs of the sequence alpha 181-200 indicated that the two antibodies have different attachment point patterns within this sequence region. These results, and those of ligand competition studies, suggest that the binding sites for FK1 and physostigmine, and those of WF6 and acetylcholine, are within the same general region of the receptor's three-dimensional structure. The sites neighbor each other, with limited overlap in the case of occupation by high molecular weight ligands.

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Autoimmune Th cells in myasthenia gravis recognize several sequence regions of the human muscle acetylcholine receptor (AChR). Most AChR Th epitopes are presented by HLA class II DR molecules (DR). Four sequence regions of the AChR alpha-subunit form Th epitopes recognized by most myasthenic patients, irrespective of their DR haplotype. In this study we first identified in five myasthenic patients the DR molecule(s) likely to be involved in presentation of T immunodominant AChR sequences. We then investigated the binding to the affinity purified DR molecules thus identified (DR2/w51, DR4/w53, and DR7/w53) and to the DR1 molecule, of a panel of overlapping synthetic peptides screening the human alpha-subunit sequence, previously used to identify AChR Th epitopes in myasthenic patients. The AChR peptides that stimulated anti-AChR autoimmune Th cells all bound the relevant DR molecules. Some AChR peptides never recognized by Th cells of myasthenic patients also bound well to one or more DR molecules. The relative ability to bind to DR molecules of different sequence regions of the AChR, i.e., an autoantigen, agrees well with the results of previous studies on the DR binding of synthetic sequences of exogenous antigens. Some peptide sequences uniquely bound one DR molecule, others bound several DR molecules, and others did not bind any of the DR molecules tested.

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T cell from H-2b mice recognize at least 12 sequence regions on the Torpedo acetylcholine receptor (TAChR) alpha, gamma and delta subunits. Immunization of C57BL/6 mice with individual synthetic TAChR sequences known to contain CD4+ epitopes resulted in most cases (10 out of 12 peptides) in anti-peptide antibody (Ab) production, indicating that short TAChR sequences contain both CD4+ and B epitopes. Immunization of C57BL/6 mice with a mixture of a CD4+ epitope peptide, from the TAChR or from an unrelated protein, plus another TAChR sequence forming a "pure" B epitope (T alpha 63-80), induced in most cases anti-peptide Ab and CD4+ cell sensitization only against the peptide containing the CD4+ epitope. However, when the T epitope peptide T alpha 360-378 was co-injected with the B epitope, Ab were also produced against the B epitope peptide. Injection of the individual peptides T alpha 360-378 and T alpha 63-80 at different and distant sites along the back of mice elicited sensitization of CD4+ cells and Ab production only against peptide T alpha 360-378. Therefore, when optimal cooperation between T and B cells occurs, spatial proximity but not covalent association of the B and the CD4+ epitope is necessary for production of Ab against the B epitope.

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In the alpha subunit of the Torpedo nicotinic cholinergic receptor (AChR), a sequence region surrounding a pair of adjacent cysteinyl residues at positions 192 and 193 contributes to a binding site for cholinergic ligands, including the snake alpha-neurotoxins. Synthetic and biosynthetic peptides corresponding to this region bind alpha-bungarotoxin (alpha-BTX) in the absence of other structural components of the AChR and, therefore, represent a "prototope" for alpha-BTX. Using synthetic peptides corresponding to the complete AChR alpha subunits of Torpedo electroplax and mammalian muscle, we previously defined a sequence segment corresponding to a universal prototope for alpha-BTX binding between amino acid residues 181 and 200 [Conti-Tronconi, B. M., Tang, F., Diethelm, B. M., Spencer, S. R. Reinhardt-Maelicke, S., & Maelicke, A. (1990) Biochemistry 29, 6221-6230; McLane, K. E., Wu, X., & Conti-Tronconi, B. M. (1990) J. Biol. Chem. 265, 1537-1544]. To elucidate the structural requirements for alpha-BTX binding, we initially used nonconservative single amino acid substitution analogues of the parental alpha(181-200) sequence, and we found that residues at positions 188-190 (VYY), and 192-194 (CCP) and several flanking residues seemed to be involved in alpha-BTX binding [Conti-Tronconi, B. M., Diethelm, B. M., Wu, X., Tang, F., Bertazzon, A., & Maelicke, A. (1991) Biochemistry 30, 2575-2584]. In the present study, amino acid residues previously found to affect alpha-BTX binding were replaced by different conservative single amino acid substitutions, in order to determine the nature of the amino acid side-chain interactions with alpha-BTX. Whereas V188 could be replaced by Ile or Thr with minor effects on alpha-BTX binding, substitution of Phe, His, or Thr for Y189 and Y190 resulted in large to moderate decreases in alpha-BTX binding. Similarly, alpha-BTX binding activity was intolerant to substitutions of C192 or C193 with Ser, His, or Val. Structural changes of the peptide alpha(181-200) induced by substitution of P194 or P197 with two adjacent Gly residues, and insertion of a Gly between C192 and C193, were also incompatible with alpha-BTX binding. Conservative substitutions of other aliphatic and aromatic residues resulted in only minor effects on alpha-BTX binding, as did replacements of K185 and D195 that changed or maintained the charge distribution of peptide alpha (181-200). The recognition site for alpha-BTX formed by the prototope alpha(181-200), therefore, involves important interactions with Y189, Y190, C192, and C193 that are highly specific to the amino acid residue at that position.(ABSTRACT TRUNCATED AT 400 WORDS)

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We investigated the sequence regions of the human muscle acetylcholine receptor (AChR) beta subunit forming epitopes recognized by T helper cells in myasthenia gravis (MG), using overlapping synthetic peptides, 20 residues long, which screened the sequence of the AChR beta subunit. Since CD4+ lymphocytes from MG patients' blood did not respond to the peptides, we attempted propagation of beta subunit-specific T lines from six MG patients and seven healthy controls by cycles of stimulation of blood lymphocytes with the pooled peptides corresponding to the beta subunit sequence. CD4+ T lines were obtained from four patients and three controls. They secreted IL-2, not IL-4, suggesting that they comprised T helper type 1 cells. The T lines from MG patients could be propagated for several months. Three lines were tested with purified bovine muscle AChR and cross-reacted well with this antigen. All T lines were tested with the individual synthetic peptides present in the pool corresponding to the beta subunit sequence. Several beta subunit peptide sequences were recognized. Each line had an individual pattern of peptides recognition, but three sequence regions (peptides beta 181-200, beta 271-290, and the overlapping peptides beta 316-335 and beta 331-350) were recognized by most MG lines. The beta subunit-specific T lines from controls could be propagated for < 5 wk. Each line recognized several peptides, which frequently included the immunodominant regions listed above.

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The nicotinic acetylcholine receptors (AChR) are presently the best-characterized neurotransmitter receptors. They are pentamers of homologous or identical subunits, symmetrically arranged to form a transmembrane cation channel. The AChR subunits form a family of homologous proteins, derived from a common ancestor. An autoimmune response to muscle AChR causes the disease myasthenia gravis. This review summarizes recent developments in the understanding of the AChR structure and its molecular recognition by the immune system in myasthenia.

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We tested the response of CD4+ cells and/or total lymphocytes from the blood of 22 myasthenic patients and 10 healthy controls to overlapping synthetic peptides, 20 residues long, to screen the sequence of the gamma and delta subunits of human muscle acetylcholine receptor (AChR). The gamma subunit is part of the AChR expressed in embryonic muscle and is substituted in the AChRs of most adult muscles by an epsilon subunit. The delta subunit is present in both embryonic and adult AChRs. Adult extrinsic ocular muscles, which are preferentially and sometimes uniquely affected by myasthenic symptoms, and thymus, which has a still obscure but important role in the pathogenesis of myasthenia gravis, express the embryonic gamma subunit. Anti-AChR CD4+ responses were more easily detected after CD8+ depletion. All responders recognized epitopes on both the gamma and delta subunits and had severe symptoms. In four patients the CD4+ cell response was tested twice, when the symptoms were severe and during a period of remission. Consistently, the response was only detectable, or larger, when the patients were severely affected.

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Affinity labeling techniques have been used to investigate the location of high-affinity binding sites for cholinergic agonists on the Torpedo acetylcholine receptor and the extent of overlap of these sites with those for long alpha-neurotoxins. Following reduction of the receptor by dithiothreitol, reaction with [3H]bromoacetylcholine leads to covalent incorporation into each of the two alpha subunits. At high concentrations of [3H]bromoacetylcholine (240 microM) and with prolonged incubation times (1-2 h), this labeling was not inhibited by either alpha-bungarotoxin or alpha-najatoxin. Following maximum labeling by [3H]bromoacetylcholine, no residual high binding sites for [125I]-alpha-bungarotoxin could be detected in the membrane-bound receptor, but 50% of the original sites were recovered by receptor solubilization. Since it has previously been reported that one of the two sites for alpha-bungarotoxin in the membrane-bound receptor is readily reversible but is converted to a high-affinity state by solubilization [Conti-Tronconi, B. M., Tang, F., Walgrave, S., & Gallagher, W. (1990) Biochemistry 29, 1046-1054], these results demonstrate that the covalently bound agonist inhibits the binding of alpha-bungarotoxin only to its higher affinity site in the membrane. When [3H]bromoacetylcholine labeling was carried out after reduction of the receptor by sodium borohydride rather than dithiothreitol, both alpha and gamma subunits of the receptor were labeled. Labeling of both subunits was completely inhibited if the receptor was first reduced with dithiothreitol and the alpha subunit sites were previously covalently labeled by unlabeled bromoacetylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)

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Experimental autoimmune myasthenia gravis is induced in C57BL/6 mice by injection of Torpedo nicotinic acetylcholine receptor (TAChR). We investigated here the presence of cryptic CD4+ epitopes on the TAChR molecule, and their relationship with potentially autoreactive CD4+ cells, which survived clonal deletion. CD4+ cells from C57BL/6 mice immunized with native or denatured TAChR were challenged in vitro with overlapping synthetic peptides, 20-residue long, screening the sequences of TAChR alpha, gamma, and delta subunits. Only three epitopes on the alpha subunit were recognized consistently. Mice immunized with large doses (nanomoles) of TAChR clearly recognized only the immunodominant sequence T alpha 150-169. Anti-TAChR CD4+ cells did not cross-react with murine alpha subunit sequences, or with any synthetic sequence of human gamma and delta subunits, which are very similar to the corresponding murine subunits. To facilitate recognition of cryptic epitopes, we injected mice with pools of synthetic peptides corresponding to the sequences of TAChR alpha, gamma, and delta subunits. In addition to the three immunodominant alpha subunit epitopes, other epitopes were recognized by CD4+ cells within the sequences T alpha 304-322, T gamma 105-124, T gamma 120-139, T gamma 401-420, T gamma 357-376, T delta 16-35, T delta 61-80, T delta 121-140, and T delta 301-320. CD4+ cells thus sensitized cross-reacted with the mammalian sequences alpha 304-322, gamma 105-124, gamma 120-139, and delta 301-320. Mice were immunized with large doses (approximately 40 nmol) of individual TAChR synthetic cryptic epitopes. CD4+ cells sensitized to five cryptic epitopes (the ones listed above plus delta 121-140) cross-reacted with autologous sequences. We determined the dose dependence of the sensitization of CD4+ cells in vivo to the strongly immunodominant epitope peptide T alpha 150-169 and to the cryptic epitope peptides T gamma 120-139 and T delta 301-320 by immunizing mice with increasing doses of peptide (approximately 1.2 to approximately 20 nmol), and testing the in vitro anti-peptide response of the CD4+ cells. No difference was found for the epitopes tested. Doses of 3 to 10 micrograms induced a strong CD4+ sensitization, and the dose dependence of the in vitro response of the sensitized cells to the relevant peptide was comparable. Production of cryptic epitopes upon in vitro TAChR processing was investigated by testing peptide-sensitized CD4+ cells with native TAChR: only two cryptic epitopes were produced.

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The symptoms of myasthenia gravis are primarily or exclusively due to an autoimmune response against the muscle nicotinic acetylcholine receptor (AChR) and this has been the object of intensive investigations for almost 20 years. A detailed picture at the molecular level of the interaction of this autoantigen with the key elements involved in the autoimmune response, such as anti-AChR antibodies, the T-cell receptor and restricting major histocompatibility complex molecules, is now emerging for both human myasthenia gravis and its experimental model, experimental autoimmune myasthenia gravis. Here, Maria Pia Protti and colleagues focus on the molecular interactions occurring in human myasthenia gravis and summarize recent information on pathogenic mechanisms of the autoimmune response, and the structure of epitopes recognized by B cells and CD4+ T cells of myasthenic patients on the AChR molecule.

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kappa-Flavotoxin (kappa-FTX), a snake neurotoxin that is a selective antagonist of certain neuronal nicotinic acetylcholine receptors (AChRs), has recently been isolated and characterized [Grant, G. A., Frazier, M. W., & Chiappinelli, V. A. (1988) Biochemistry 27, 1532-1537]. Like the related snake toxin kappa-bungarotoxin (kappa-BTX), kappa-FTX binds with high affinity to alpha 3 subtypes of neuronal AChRs, even though there are distinct sequence differences between the two toxins. To further characterize the sequence regions of the neuronal AChR alpha 3 subunit involved in formation of the binding site for this family of kappa-neurotoxins, we investigated kappa-FTX binding to overlapping synthetic peptides screening the alpha 3 subunit sequence. A sequence region forming a "prototope" for kappa-FTX was identified within residues alpha 3 (51-70), confirming the suggestions of previous studies on the binding of kappa-BTX to the alpha 3 subunit [McLane, K. E., Tang, F., & Conti-Tronconi, B. M. (1990) J. Biol. Chem. 265, 1537-1544] and alpha-bungarotoxin to the Torpedo AChR alpha subunit [Conti-Tronconi, B. M., Tang, F., Diethelm, B. M., Spencer, S. R., Reinhardt-Maelicke, S., & Maelicke, A. (1990) Biochemistry 29, 6221-6230] that this sequence region is involved in formation of a cholinergic site. Single residue substituted analogues, where each residue of the sequence alpha 3 (51-70) was sequentially replaced by a glycine, were used to identify the amino acid side chains involved in the interaction of this prototope with kappa-FTX.(ABSTRACT TRUNCATED AT 250 WORDS)

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The synthesis of AChR antibodies requires intervention of AChR-specific Th cells. Because of the paucity of anti-AChR Th cells in the blood of myasthenia gravis (MG) patients, direct studies of these autoimmune cells in the blood are seldom possible. Propagation in vitro of anti-AChR T cells from MG patients by cycles of stimulation with AChR antigens selectively enriches and expands the autoimmune T-cell clones, allowing investigation of their function and epitope specificity. Torpedo electroplax AChR was initially used for propagation of anti-AChR T-cell lines. Those studies demonstrated the feasibility of in vitro propagation of AChR-specific T cells. These are bona fide CD4+ Th cells, which stimulate production in vitro of anti-AChR antibodies by B cells of myasthenic patients and recognize equally well denatured and native AChR, suggesting the usefulness of synthetic human AChR sequences as antigens for propagation of the autoimmune Th cells. We used pools of overlapping synthetic peptides, corresponding to the complete sequences of the human AChR alpha-, beta-, gamma-, and delta-subunits, to propagate AChR-specific Th cells from the blood of MG patients. The AChR sequence regions forming epitopes recognized by the autoimmune T cells were determined by challenging the lines with individual synthetic peptides, 20 residues long, screening the AChR subunit sequences. Although each line had an individual pattern of epitope recognition--as expected from their different HLA-DR haplotype--some peptides were recognized by most of all the CD4+ T-cell lines, irrespective of their DR haplotype. The existence of immunodominant regions of the AChR sequence was verified by investigating the response of unselected CD4+ cells from the blood of a relatively large number of MG patients to the individual peptides screening the human alpha-, gamma-, and delta-subunit sequences. Those studies confirmed that each patient has an individual pattern of peptide recognition. The studies also identified a large number of T epitopes of the human AChR and verified the existence of sequence regions immunodominant for T-helper sensitization, because a limited number of sequence regions, including all those immunodominant for the T-helper lines, were recognized by most patients. Anti-AChR CD4+ T lines could be propagated from some healthy controls only for a brief period of time. They recognized AChR sequences poorly, suggesting a low affinity of their T-cell receptors for the corresponding AChR epitopes.(ABSTRACT TRUNCATED AT 400 WORDS)

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Binding of positively charged radiolabeled synthetic peptides to human major histocompatibility complex class II DR molecules, purified by affinity chromatography from lymphoblastoid B cell lines of different haplotypes, is rapidly, quantitatively, and specifically assayed by selective adsorption of the complexes between peptide and DR molecules onto DEAE-cellulose paper disks. This assay can be used as a revealing system of the ability of unlabeled test peptides to competitively inhibit the binding between the radiolabeled peptide and the DR molecules, thus measuring the binding of the competitor peptides, irrespective of their charge properties, to different DR molecules.

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We have PCR-amplified cDNA sequence of the "embryonic" gamma subunit of muscle acetylcholine receptor (AChR) from adult bovine extraocular muscle (EOM). We cloned and sequenced this product and used it to probe Northern blots. We detected the gamma subunit in EOM mRNA, but not in control skeletal muscle. The presence of gamma subunit in EOMs may explain their preferential involvement in myasthenia gravis, where an autoimmune response to the gamma subunit is generally present.

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Monoclonal antibodies (mAbs) were derived from mice immunized with synthetic peptide sequence regions of the alpha subunit of the nicotinic acetylcholine receptor from Torpedo electric tissue (TAChR). Sequence-specific mAbs were obtained against the following peptides: alpha 1-20, alpha 291-308, alpha 304-322, alpha 332-350, alpha 346-364, alpha 360-378, alpha 376-393, alpha 390-409, and alpha 420-437. The ability of mAbs to recognize native TAChR was quantitated by immunoprecipitation of TAChR solubilized in the nondenaturing detergent Triton X-100. mAbs against peptide alpha 304-322, alpha 332-350, and alpha 360-378 cross-reacted with most or all Triton-solubilized TAChR molecules and, in immunoelectron microscopy experiments, bound to the cytoplasmic surface of AChR-rich postsynaptic membrane fragments. Two mAbs specific for the sequence alpha 376-393, proposed to form an amphypathic alpha helix possibly involved in formation of the ion channel, recognized only approximately 35% of Triton-solubilized TAChR molecules and did not react with membrane-bound TAChR. All of these sequence-specific antibodies recognized SDS-denatured TAChR alpha subunit in Western blots. MAbs specific for the amino-terminal sequence region of the alpha subunit, alpha 1-20, and for the sequences alpha 291-308, alpha 346-364, and alpha 390-409 did not recognize native TAChR. A mAb directed against the carboxyl-terminal region, alpha 420-437, recognized with low apparent titer Triton-solubilized TAChR, not membrane-bound TAChR. In conclusion, a complex membrane protein, TAChR, contains several continuous sequence segments exposed on the TAChR surface, because different mAbs raised against certain synthetic sequences recognized most or all native TAChR molecules. By analogy, it should be possible for most proteins of known sequence to raise anti-peptide antibodies fully cross-reactive with the native cognate protein.

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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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