International Immunology, Vol. 12, No. 9, 1255-1265,
September 2000
© 2000 Japanese Society for Immunology
Reconstitution of conformationally dependent epitopes on the N-terminal extracellular domain of the human muscle acetylcholine receptor 
subunit expressed in Escherichia coli: implications for myasthenia gravis therapeutic approaches
Department of Biochemistry, Hellenic Pasteur Institute, 127 Vas. Sofias Avenue, 11521 Athens, Greece
Correspondence to: S. J. Tzartos
Myasthenia gravis (MG) is an autoimmune disease, caused by autoantibodies against the muscle acetylcholine receptor (AChR), an oligomeric transmembrane glycoprotein composed of 
2ß
subunits. The 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 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 subunit bound ~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 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.
Keywords: acetylcholine receptor, artificial chaperones, conformationally dependent epitopes, main immunogenic region, myasthenia gravis, renaturation
Transmitting editor: J.-F. Bach
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