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

Abstract

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.

Genomic Differentiation Between Temperate and Tropical Australian Populations ofDrosophila melanogaster

“…Structural homology modeling suggests that this E/D polymorphism occurs in the main immunogenic region (MIR) of the protein (Figure 8b). This region constitutes a loop sandwiched between b2 and b3 that binds autoimmune antibodies in myasthenia gravis patients in the homologous human muscle acetylcholine receptor (Tsouloufis et al 2000;Dellisanti et al 2007). The fact that the I/V polymorphism is found in close proximity to this region suggests the possibility that differentiation at NtR could affect interactions with other molecules, possibly those relating to the immune system.…”

Isolation and characterization of human anti-acetylcholine receptor monoclonal antibodies from transgenic mice expressing human immunoglobulin loci

“…The recombinant polypeptides used as immunogens were expressed and purified as previously described [27,28]. Briefly, the human AChR a-ECD (1-207) cloned into PET-15b was expressed in E. coli as inclusion bodies.…”

Rapid Purification of Recombinant Anthrax-Protective Antigen under Nondenaturing Conditions

“…Studies with other proteins have shown that reconstitution of conformation-dependent epitopes of a denatured protein is a very complex problem. For example, researchers in pursuit of producing correctly folded, renatured N-terminal domain of human acetylcholine receptor (21), tried several expression vectors and renaturation conditions including immobilization of protein on Ni-NTA column to remove denaturant, its gradual removal by dialysis and rapid dilution of denaturant. Correct conformation (as a Cells plated in 96-well tissue culture plates were treated with media containing EF (1 g/ml) and indicated concentration of PA from either of the sources.…”