Expression of soluble ligand- and antibody-binding extracellular domain of human muscle acetylcholine receptor α subunit in yeast Pichia pastoris -: Role of glycosylation in α-bungarotoxin binding

The N-terminal extracellular domain (amino acids 1-210; halpha-(1-210)) of the a 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 pastorisr. The recombinant protein was water-soluble and glycosylated, and fast protein liquid chromatography analysis showed it to be a monomer. halpha-(1-210) bound 125 I-a-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)similar to7.5 mm). Interestingly, I-125-alpha-bungarotoxin binding was markedly impaired by in vitro deglycosylation of halpha-(1-210). Several monoclonal antibodies that show partial or strict conformation-dependent binding to the AChR were able to bind to ha-(1-210), as did antibodies from a large proportion of myasthenic patients. These results suggest that the extracellular domain of the human AChR alphasubunit 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.