Reconstitution of conformationally dependent epitopes on the N-terminal extracellular domain of the human muscle acetylcholine receptor α subunit expressed in Escherichia coli: implications for myasthenia gravis therapeutic approaches

• Published in: International immunology 2000-09, Vol.12 (9), p.1255-1265
• Main Authors: Tsouloufis, Theodoros, Mamalaki, Avgi, Remoundos, Michael, Tzartos, Socrates J.
• Format: Article
• Language: English
• Subjects: acetylcholine receptor; acetylcholine receptors; AChR acetylcholine receptor; Antibodies, Monoclonal - immunology; artificial chaperones; Cloning, Molecular; conformationally dependent epitopes; CTAB cetyltrimethyl-ammonium bromide; Cyclodextrins; Detergents; Enzyme-Linked Immunosorbent Assay; Epitopes - immunology; Escherichia coli; GSH reduced glutathione; GSSG oxidized glutathione; GuHCl guanidine hydrochloride; Humans; Hα1–207 human AChR α subunit amino acid residues 1–207; main immunogenic region; methyl-β-CD methyl-β-cyclodextrin; MG myasthenia gravis; MIR main immunogenic region; Muscle, Skeletal - immunology; Muscle, Skeletal - metabolism; myasthenia gravis; Myasthenia Gravis - immunology; N-dimethyl-3-ammonio-1-propanesulfonate; NTA nitrilo-tri-acetic acid; Peptides - immunology; Protein Conformation; Protein Denaturation - drug effects; Receptors, Cholinergic - biosynthesis; Receptors, Cholinergic - genetics; Receptors, Cholinergic - immunology; Recombinant Proteins - immunology; renaturation; TTAB tetradecyltrimethylammonium bromide; Z 3–14 N-tetradecyl-N; α-Bgt α-bungarotoxin; α-CD α-cyclodextrin
• ISSN: 0953-8178; 1460-2377; 1460-2377
• DOI: 10.1093/intimm/12.9.1255

Myasthenia gravis (MG) is an autoimmune disease, caused by autoantibodies against the muscle acetylcholine receptor (AChR), an oligomeric transmembrane glycoprotein composed of α2βγδ subunits. The α 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 α 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 α 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 α 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.