Expression of Soluble Ligand- and Antibody-binding Extracellular Domain of Human Muscle Acetylcholine Receptor α Subunit in Yeast Pichia pastoris

Abstract

The N-terminal extracellular domain (amino acids 1-210; h␣-(1-210)) of the ␣ subunit of the human muscle nicotinic acetylcholine receptor (AChR), bearing the binding sites for cholinergic ligands and the main immunogenic region, the major target for anti-AChR antibodies in patients with myasthenia gravis, was expressed in the yeast, Pichia pastoris. The recombinant protein was water-soluble and glycosylated, and fast protein liquid chromatography analysis showed it to be a monomer. h␣-(1-210) bound 125 I-␣-bungarotoxin with a high affinity (K d ‫؍‬ 5.1 ؎ 2.4 nM), and this binding was blocked by unlabeled d-tubocurarine and gallamine (K i ϳ7.5 mM). Interestingly, 125 I-␣-bungarotoxin binding was markedly impaired by in vitro deglycosylation of h␣-(1-210). Several monoclonal antibodies that show partial or strict conformation-dependent binding to the AChR were able to bind to h␣-(1-210), as did antibodies from a large proportion of myasthenic patients. These results suggest that the extracellular domain of the human AChR ␣ subunit expressed in P. pastoris has an apparently near native conformation. The correct folding of the recombinant protein, together with its relatively high expression yield, makes it suitable for structural studies on the nicotinic acetylcholine receptor and for use as an autoantigen in myasthenia gravis studies. The nicotinic acetylcholine receptor (AChR)1 at the neuromuscular junction is a member of the superfamily of ligandgated ion channels that also includes the glycine, ␥-aminobutiric acid A, and 5-HT 3 receptors (1). The AChR is a transmembrane glycoprotein (M r ϳ290000) consisting of five homologous subunits in the stoichiometry ␣ 2 ␤␥␦ (embryonic) or ␣ 2 ␤⑀␦ (adult). Each subunit consists of an N-terminal extracellular domain (ϳ210 residues) followed by three transmembrane domains, a large cytoplasmic loop, a fourth transmembrane domain, and a short, extracellular C-terminal tail (2, 3). The N-terminal extracellular domain of the ␣ chain (␣-(1-210)) contains both the binding sites for cholinergic ligands (4) and the MIR, the major target for autoantibodies in both MG and experimental models of MG (5-7). The major loop of the overlapping epitopes for several anti-MIR monoclonal antibodies (mAbs) has been localized between residues 67 and 76 of the ␣ subunit (8, 9). Previous experiments have shown that the binding sites for both acetylcholine and ␣-BTX are located close to two adjacent cysteine residues at positions 192 and 193 of the ␣ subunit (10). Moreover, other distinct regions on either the ␣ subunit (11, 12) or the adjacent ␥ or ␦ subunits (13) have been shown to contribute to ␣-BTX binding, whereas the role of glycosylation at residue ␣141 (14 -16) requires further study.These unique characteristics of the ␣ subunit have led to its being extensively studied in several laboratories. The expression of full-length Torpedo (15-17) or mouse (18) AChR ␣ subunits in heterologous protein expression systems has shown that the ␣ subunit, independently of other subunits, acquires a mature...

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.…”

Conserved High Affinity Ligand Binding and Membrane Association in the Native and Refolded Extracellular Domain of the Human Glycine Receptor α1-Subunit

“…Recently, the chicken ␣7 extracellular domain was expressed in Xenopus oocytes (10), while a soluble fusion protein of the rat ␣7 nAChR N-terminal domain and maltose binding protein has been generated in Escherichia coli (11). In addition, the extracellular domain from the human muscle AChR ␣ subunit was expressed as a soluble protein in the yeast Pichia pastoris (12), while the ␣7 nAChR extracellular domain fused to GST was expressed in E. coli and subsequently refolded (13). In an alternative approach, the x-ray structure of a molluscan ACh binding protein was solved at atomic resolution (14), and revealed structural similarity to the extracellular domain of nAChRs, as determined by electron microscopy (15).…”

Isolation of potent human Fab fragments against a novel highly immunogenic region on human muscle acetylcholine receptor which protect the receptor from myasthenic autoantibodies

“…No binding of the isolated Fab was seen either in RIA using the hybrid HaT-AChR (human a-, Torpedo b-, c-and dAChR subunits) (Fig. 5), or in ELISA with the extracellular domain of hAChR a-, b-, c-or e-subunit expressed in P. pastoris with close to native conformation ( [10], and Kostelidou et al, manuscript in preparation) (data not shown). When all the isolated Fab were tested by RIA with hAChR subunit dimers expressed in HEK293 cells, no binding to the ab or ad dimers was detected, while significant binding was observed to the ac and ae dimers for most of the Fab (Fig.…”

“…Subunit dimerization of hAChR subunit dimers isolated from transiently transfected HEK293 cells [14] was verified by the effective precipitation of 125 I-aBT-ab, ac, ae and ad dimers using mAb 73 (anti-b-subunit), 67 (anti-c-subunit), 168 (antie-subunit) or 137 (anti-d-subunit), respectively. The Nterminal extracellular domains of the hAChR a-, b-, c-, and e-subunits were expressed in Pichia pastoris ( [10], and Kostelidou et al, manuscript in preparation). The rat antiAChR mAb used in this study have been previously described [11,16,23,24].…”