The nicotinic acetylcholine receptor (AChR) is a member of the superfamily of ligand-gated ion channels, which also includes the glycine, c-aminobutyric acid A, and 5-HT 3 receptors [1]. Its physiological role is to mediate the fast chemical transmission of electrical signals in response to acetylcholine released from the nerve terminal to the end-plate.The muscle AChR is a transmembrane glycoprotein ( 290 kDa) located on the postsynaptic membrane of the neuromuscular junction and is composed of five The nicotinic acetylcholine receptor (AChR) is a ligand-gated ion channel found in muscles and neurons. Muscle AChR, formed by five homologous subunits (a 2 bcd or a 2 bce), is the major antigen in the autoimmune disease, myasthenia gravis (MG), in which pathogenic autoantibodies bind to, and inactivate, the AChR. The extracellular domain (ECD) of the human muscle a subunit has been heterologously expressed and extensively studied.Our aim was to obtain satisfactory amounts of the ECDs of the non-a subunits of human muscle AChR for use as starting material for the determination of the 3D structure of the receptor ECDs and for the characterization of the specificities of antibodies in sera from patients with MG. We expressed the N-terminal ECDs of the b (amino acids 1-221; b1-221), c (amino acids 1-218; c1-218), and e (amino acids 1-219; e1-219) subunits of human muscle AChR in the yeast, Pichia pastoris. b1-221 was expressed at 2 mgAEL )1 culture, whereas c1-218 and e1-219 were expressed at 0.3-0.8 mgAEL )1 culture. All three recombinant polypeptides were glycosylated and soluble; b1-221 was mainly in an apparently dimeric form, whereas c1-218 and e1-219 formed soluble oligomers. CD studies of b1-221 suggested that it has considerable b-sheet secondary structure with a proportion of a-helix. Conformation-dependent mAbs against the ECDs of the b or c subunits specifically recognized b1-221 or c1-218, respectively, and polyclonal rabbit antiserum raised against purified b1-221 bound to 125 I-labeled a-bungarotoxin-labeled human AChR. Moreover, immobilization of each ECD on Sepharose beads and incubation of the ECD-Sepharose matrices with MG sera caused a significant reduction in the concentrations of autoantibodies in the sera, showing specific binding to the recombinant ECDs. These results suggest that the expressed proteins present some near-native conformational features and are thus suitable for our purposes.Abbreviations AChR, nicotinic acetylcholine receptor; ECD, extracellular domain; MG, myasthenia gravis; b1-221, amino acids 1-221 of the human AChR b subunit; c1-218, amino acids 1-218 of the human AChR c subunit; e1-219, amino acids 1-219 of the human AChR e subunit.

Myasthenia gravis (MG) is usually caused by autoantibodies against muscle nicotinic acetylcholine receptor (AChR), which is composed of five subunits (alpha(2)betagammadelta or alpha(2)betaepsilondelta). Current treatments, including plasmapheresis, are nonspecific, causing several side effects. We aim to develop an antigen-specific alternative to plasmapheresis, since the latter removes indispensable plasma components in addition to anti-AChR antibodies. We are developing a method for the selective depletion of the anti-AChR autoantibodies from patients' plasma through the construction of "immunoadsorbent" columns carrying AChR domains. We have expressed the extracellular domains (ECDs, amino acids approximately 1-210/220) of all human muscle AChR subunits in Pichia pastoris and, in preliminary experiments, in E. coli. The ECDs were immobilized (individually or mixed) on Sepharose beads, producing Sepharose-ECD columns, which were tested for their immunoadsorbing capacity on MG sera and shown to specifically eliminate major autoantibody fractions from several MG sera. The immobilized ECDs remained stable and did not dissociate from their matrix after incubation with serum, whereas the procedure was neither toxic nor immunogenic in two experimental rabbits. Testing the intact or antibody-depleted MG sera and the affinity purified autoantibodies showed that both the intact sera and the purified autoantibodies, but not the antibody-depleted sera, could induce AChR loss in cell cultures and experimental MG in rats. This preliminary study suggests that the myasthenic potency of MG sera is entirely due to their anti-AChR antibodies and therefore their depletion should be of therapeutic value. We conclude that ECD-mediated immunoadsorption can be used as an efficient, antigen-specific therapy for MG.