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J. Biol. Chem., Vol. 277, Issue 30, 26980-26986, July 26, 2002

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Expression of Soluble Ligand- and Antibody-binding Extracellular Domain of Human Muscle Acetylcholine Receptor  Subunit in Yeast Pichia pastoris
ROLE OF GLYCOSYLATION IN -BUNGAROTOXIN BINDING^*

Loukia Psaridi-Linardaki, Avgi Mamalaki, Michael Remoundos, and Socrates J. Tzartos^§¶

From the  Department of Biochemistry, Hellenic Pasteur Institute, 127 Vas Sofias Avenue, 11521 Athens, Greece and the ^§ Department of Pharmacy, University of Patras, 26504 Patras, Greece

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 ¹²⁵I--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~7.5 mM). Interestingly, ¹²⁵I--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.

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