Expression of soluble ligand- and antibody-binding extracellular domain of human muscle acetylcholine receptor alpha subunit in yeast Pichia pastoris. Role of glycosylation in alpha-bungarotoxin binding.
The N-terminal extracellular domain (amino acids 1-210; halpha-(1-210)) of the alpha 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. halpha-(1-210) bound (125)I-alpha-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) approximately 7.5 mm). Interestingly, (125)I-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 halpha-(1-210), as did antibodies from a large proportion of myasthenic patients. These results suggest that the extracellular domain of the human AChR alpha 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.
Psaridi-Linardaki
Loukia
L
Department of Biochemistry, Hellenic Pasteur Institute, 127 Vas Sofias Avenue, 11521 Athens, Greece.
Mamalaki
Avgi
A
Remoundos
Michael
M
Tzartos
Socrates J
SJ
D000911:Antibodies, Monoclonal / Q000378:metabolism; D001324:Autoantigens / Q000737:chemistry; D002038:Bungarotoxins / Q000494:pharmacology*; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008024:Ligands; D009132:Muscles / Q000378:metabolism*; D010843:Pichia / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011863:Radioimmunoassay; D011950:Receptors, Cholinergic / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature
D000911:Antibodies, Monoclonal; D001324:Autoantigens; D002038:Bungarotoxins; D004220:Disulfides; D008024:Ligands; D011950:Receptors, Cholinergic; D011994:Recombinant Proteins