Expression and characterization of soluble forms of the extracellular domains of the β, γ and ε subunits of the human muscle acetylcholine receptor

The nicotinic acetylcholine receptor (AChR) is a ligand-gated ion channel found in muscles and neurons. Muscle AChR, formed by five homologous subunits (alpha(2)beta gamma delta or alpha(2)beta gamma epsilon), 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 alpha subunit has been heterologously expressed and extensively studied. Our aim was to obtain satisfactory amounts of the ECDs of the non-alpha 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 beta (amino acids 1-221; beta 1-221), gamma (amino acids 1-218; gamma 1-218), and epsilon (amino acids 1-219; epsilon 1-219) subunits of human muscle AChR in the yeast, Pichia pastoris. beta 1-221 was expressed at approximate to 2 mg.L-1 culture, whereas gamma 1-218 and epsilon 1-219 were expressed at 0.3-0.8 mg.L-1 culture. All three recombinant polypeptides were glycosylated and soluble; beta 1-221 was mainly in an apparently dimeric form, whereas gamma 1-218 and epsilon 1-219 formed soluble oligomers. CD studies of beta 1-221 suggested that it has considerable beta-sheet secondary structure with a proportion of alpha-helix. Conformation-dependent mAbs against the ECDs of the beta or gamma subunits specifically recognized beta 1-221 or gamma 1-218, respectively, and polyclonal rabbit antiserum raised against purified beta 1-221 bound to I-125-labeled alpha-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.