The N-terminal extracellular domain (amino acids 1-210; h␣-(1-210)) of the ␣ subunit of the human muscle nicotinic acetylcholine receptor (AChR), bearing the binding sites for cholinergic ligands and the main immunogenic region, the major target for anti-AChR antibodies in patients with myasthenia gravis, was expressed in the yeast, Pichia pastoris. The recombinant protein was water-soluble and glycosylated, and fast protein liquid chromatography analysis showed it to be a monomer. h␣-(1-210) bound 125 I-␣-bungarotoxin with a high affinity (K d ‫؍‬ 5.1 ؎ 2.4 nM), and this binding was blocked by unlabeled d-tubocurarine and gallamine (K i ϳ7.5 mM). Interestingly, 125 I-␣-bungarotoxin binding was markedly impaired by in vitro deglycosylation of h␣-(1-210). Several monoclonal antibodies that show partial or strict conformation-dependent binding to the AChR were able to bind to h␣-(1-210), as did antibodies from a large proportion of myasthenic patients. These results suggest that the extracellular domain of the human AChR ␣ subunit expressed in P. pastoris has an apparently near native conformation. The correct folding of the recombinant protein, together with its relatively high expression yield, makes it suitable for structural studies on the nicotinic acetylcholine receptor and for use as an autoantigen in myasthenia gravis studies. The nicotinic acetylcholine receptor (AChR)1 at the neuromuscular junction is a member of the superfamily of ligandgated ion channels that also includes the glycine, ␥-aminobutiric acid A, and 5-HT 3 receptors (1). The AChR is a transmembrane glycoprotein (M r ϳ290000) consisting of five homologous subunits in the stoichiometry ␣ 2 ␤␥␦ (embryonic) or ␣ 2 ␤⑀␦ (adult). Each subunit consists of an N-terminal extracellular domain (ϳ210 residues) followed by three transmembrane domains, a large cytoplasmic loop, a fourth transmembrane domain, and a short, extracellular C-terminal tail (2, 3). The N-terminal extracellular domain of the ␣ chain (␣-(1-210)) contains both the binding sites for cholinergic ligands (4) and the MIR, the major target for autoantibodies in both MG and experimental models of MG (5-7). The major loop of the overlapping epitopes for several anti-MIR monoclonal antibodies (mAbs) has been localized between residues 67 and 76 of the ␣ subunit (8, 9). 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.These unique characteristics of the ␣ subunit have led to its being extensively studied in several laboratories. The expression of full-length Torpedo (15-17) or mouse (18) AChR ␣ subunits in heterologous protein expression systems has shown that the ␣ subunit, independently of other subunits, acquires a mature...

The epitopes for twelve monoclonal antibodies against the cytoplasmic side of the acetylcholine receptor (AChR) a subunit were precisely mapped using over 300 continuously overlapping synthetic peptides attached on poly(ethy1ene) rods. mAb cross-reactive between Torpedo and human AChR generally bound to the homologous peptides from both species. Epitopes 4 -10-residues long were identified. One mAb could bind to either arm on both sides of a j?-turn structure. Five mAb bound to a very-immunogenic cytoplasmic epitope on a373 -380 (VICE-a). Three of the mAb against VICE-M were earlier found to cross-react with non-AChR protein(s), present in thymomas from myasthenia gravis patients but absent in thymomas from non-myasthenics. Since VICE-a has a potentially crucial pathogenic role, the antigenic role of each residue within it was subsequently studied by 55 analogues, most having single amino acid substitutions. All the mAb against VICE-a bound similarly but not identically to the analogues, thus explaining their known binding heterogeneity. Lys373 proved indispensable for mAb binding. Ile376, Glu377, Gly378 and Lys380 were quite critical, while Ser374, Ala375 and Val379 seemed rather inactive. These data should prove instructive in searches for VICEa-like epitopes carrying autoantigens with potential involvement in myasthenia gravis and should further expand the applications of the anti-(AChR) mAb in AChR studies.

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Myasthenia gravis (MG) is an autoimmune disease, caused by autoantibodies against the muscle acetylcholine receptor (AChR), an oligomeric transmembrane glycoprotein composed of alpha(2)beta gamma delta subunits. The alpha subunit carries in its N-terminal extracellular domain the main immunogenic region (MIR), a group of conformationally dependent epitopes that seems to be a major target for the anti-AChR antibodies in MG patients. Detailed epitope studies on pathogenic anti-AChR antibodies have been hindered because the binding of most of these antibodies is conformationally dependent, which precludes the use of denatured AChR fragments. The N-terminal extracellular fragment, residues 1-207, of the human AChR alpha subunit was expressed in Escherichia coli in a denatured form, solubilized in a guanidinium hydrochloride-containing buffer, purified, and renatured using a refolding approach which employs a detergent and a cyclodextrin as 'artificial chaperones'. Compared with the non-refolded protein, the refolded molecule exhibited a dramatic improvement in terms of the binding of all anti-MIR mAb tested. Anti-MIR mAb that normally bind weakly to the denatured alpha subunit bound approximately 30-100 times better to the refolded polypeptide and other anti-MIR mAb that bind exclusively to completely conformationally dependent epitopes also bound quite efficiently. These results, in addition to providing a means for the thorough investigation of the antigenic structure of the AChR, show that the conformationally dependent MIR epitopes do not require the participation of the oligosaccharide moiety of the alpha subunit nor the contribution of neighboring subunits for antibody binding. Such AChR fragments may be used in structural studies of the AChR autoantigen, and should prove valuable in the understanding and development of therapeutic approaches for MG.

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