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

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Abstract

The nicotinic acetylcholine receptor (AChR) is a ligand-gated ion channel found in muscles and neurons. Muscle AChR, formed by five homologous subunits (α2βγδ or α2βγη), 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 α subunit has been heterologously expressed and extensively studied. Our aim was to obtain satisfactory amounts of the ECDs of the non-α 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 β (amino acids 1–221; β1–221), γ (amino acids 1–218; γ1–218), and η (amino acids 1–219; η1–219) subunits of human muscle AChR in the yeast, Pichia pastoris. β1–221 was expressed at ≈ 2 mg·L−1 culture, whereas γ1–218 and η1–219 were expressed at 0.3–0.8 mg·L−1 culture. All three recombinant polypeptides were glycosylated and soluble; β1–221 was mainly in an apparently dimeric form, whereas γ1–218 and η1–219 formed soluble oligomers. CD studies of β1–221 suggested that it has considerable β-sheet secondary structure with a proportion of α-helix. Conformation-dependent mAbs against the ECDs of the β or γ subunits specifically recognized β1–221 or γ1–218, respectively, and polyclonal rabbit antiserum raised against purified β1–221 bound to 125I-labeled α-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.

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