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

doi:10.1074/jbc.M110731200

QUICK SEARCH:

	Author:		Keyword(s):
Year:		Vol:		Page:

A more recent version of this article appeared on July 19, 2002

This Article

	Full Text (Accepted Manuscript)
	All Versions of this Article: 277/30/26980 most recent M110731200v1
	Alert me when this article is cited
	Alert me if a correction is posted

Services

	Email this article to a friend
	Similar articles in this journal
	Similar articles in PubMed
	Alert me to new issues of the journal
	Download to citation manager


Citing Articles

	Citing Articles via HighWire
	Citing Articles via Google Scholar

Google Scholar

	Articles by Psaridi-Linardaki, L.
	Articles by Tzartos, S. J.
	Search for Related Content

PubMed

	PubMed Citation
	Articles by Psaridi-Linardaki, L.
	Articles by Tzartos, S. J.

Social Bookmarking

	What's this?

Papers In Press, published online ahead of print May 15, 2002
J. Biol. Chem, 10.1074/jbc.M110731200
Submitted on November 8, 2001
Revised on May 15, 2002
Accepted on May 15, 2002

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

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

Department of Biochemistry, Hellenic Pasteur Institute, Athens 115 21

Corresponding Author: tzartos{at}mail.pasteur.gr

The N-terminal extracellular domain (amino acids 1-210; h $alpha$ 1-210) of the $alpha$ subunit of the human muscle nicotinic acetylcholine receptor (hAChR), bearing the binding sites for cholinergic ligands and the main immunogenic region (MIR), 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 FPLC analysis showed it to be a monomer. h $alpha$ 1-210 bound ¹²⁵I- $alpha$ -bungarotoxin with a high affinity (Kd = 5.1 ± 2.4 nM) and this binding was efficiently blocked by unlabeled d-tubocurarine and gallamine. Interestingly, ¹²⁵I- $alpha$ -bungarotoxin binding was markedly impaired by in vitro deglycosylation of h $alpha$ 1-210. Several monoclonal antibodies that show partial or strict conformation-dependent binding to the AChR were able to bind to h $alpha$ 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 Pichia 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.

Add to CiteULike CiteULike Add to Complore Complore Add to Connotea Connotea Add to Del.icio.us Del.icio.us Add to Digg Digg Add to Reddit Reddit Add to Technorati Technorati What's this?

This article has been cited by other articles:

A. M. Person, K. L. Bills, H. Liu, S. K. Botting, J. Lindstrom, and G. B. Wells
Extracellular Domain Nicotinic Acetylcholine Receptors Formed by {alpha}4 and {beta}2 Subunits
J. Biol. Chem., December 2, 2005; 280(48): 39990 - 40002.
[Abstract] [Full Text] [PDF]

V. Avramopoulou, A. Mamalaki, and S. J. Tzartos
Soluble, Oligomeric, and Ligand-binding Extracellular Domain of the Human {alpha}7 Acetylcholine Receptor Expressed in Yeast: REPLACEMENT OF THE HYDROPHOBIC CYSTEINE LOOP BY THE HYDROPHILIC LOOP OF THE ACh-BINDING PROTEIN ENHANCES PROTEIN SOLUBILITY
J. Biol. Chem., September 10, 2004; 279(37): 38287 - 38293.
[Abstract] [Full Text] [PDF]

U. Breitinger, H.-G. Breitinger, F. Bauer, K. Fahmy, D. Glockenhammer, and C.-M. Becker
Conserved High Affinity Ligand Binding and Membrane Association in the Native and Refolded Extracellular Domain of the Human Glycine Receptor {alpha}1-Subunit
J. Biol. Chem., January 16, 2004; 279(3): 1627 - 1636.
[Abstract] [Full Text] [PDF]

All ASBMB Journals	Molecular and Cellular Proteomics
Journal of Lipid Research	ASBMB Today

				V. Avramopoulou, A. Mamalaki, and S. J. Tzartos Soluble, Oligomeric, and Ligand-binding Extracellular Domain of the Human {alpha}7 Acetylcholine Receptor Expressed in Yeast: REPLACEMENT OF THE HYDROPHOBIC CYSTEINE LOOP BY THE HYDROPHILIC LOOP OF THE ACh-BINDING PROTEIN ENHANCES PROTEIN SOLUBILITY J. Biol. Chem., September 10, 2004; 279(37): 38287 - 38293. [Abstract] [Full Text] [PDF]

				U. Breitinger, H.-G. Breitinger, F. Bauer, K. Fahmy, D. Glockenhammer, and C.-M. Becker Conserved High Affinity Ligand Binding and Membrane Association in the Native and Refolded Extracellular Domain of the Human Glycine Receptor {alpha}1-Subunit J. Biol. Chem., January 16, 2004; 279(3): 1627 - 1636. [Abstract] [Full Text] [PDF]

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

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