title: Mutant forms of the extracellular domain of the human acetylcholine receptor gamma-subunit with improved solubility and enhanced antigenicity. The importance of the Cys-loop.
abstract: The muscle nicotinic acetylcholine receptor (AChR) is the prototype of the ligand-gated ion channels (or Cys-loop receptors), formed by 5 homologous subunits (alpha2betagammadelta or alpha2betagammaepsilon), and is the major autoantigen in the autoimmune disease, myasthenia gravis. Previously, we expressed the wild-type extracellular domain (ECD) of the gamma-subunit (gammaECD) of the AChR in yeast Pichia pastoris at 0.3-0.8 mg/L, in soluble but microaggregate form, to use as starting material for structural and antigenicity studies. To optimize these characteristics, we constructed and characterized four gammaECD variants: (a) mutants-1 (gammaC61S) and -2 (gammaC106S-C115S), where the non-conserved Cys of gammaECD were replaced by serines, (b) mutant-3 (gammaCysLoop), where the gamma Cys-loop region was substituted by the cognate region of the acetylcholine binding protein (AChBP) and (c) mutant-4 (gammaCysLoop-C106S-C115S), where both the C106S-C115S and Cys-loop mutations were combined. None of mutants-1 and -2 displayed any improvement, while mutant-3 and -4 were mostly in dimeric form and expressed at much higher levels (2.5 mg/L and 3.5 mg/L respectively). All four mutants and wild-type gammaECD were recognized by sera from myasthenic patients, but mutants-3 and -4 exhibited higher efficiency, compared to wild-type or mutants-1 and -2. These results suggest that the substitution of the Cys-loop region of any AChR ECD with the AChBP counterpart leads to AChR ECD of improved conformation, more suitable for structural and therapeutic studies.
authors.affiliation: Department of Biochemistry, Hellenic Pasteur Institute, GR11521 Athens, Greece.
authors.forename: Kalliopi
authors.initials: K
authors.lastname: Bitzopoulou
authors.forename: Kalliopi
authors.initials: K
authors.lastname: Kostelidou
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Poulas
authors.forename: Socrates J
authors.initials: SJ
authors.lastname: Tzartos
mesh_terms: D000595:Amino Acid Sequence; D001323:Autoantibodies / Q000097:blood; D001324:Autoantigens / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009157:Myasthenia Gravis / Q000276:immunology; D010843:Pichia / Q000235:genetics; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D012995:Solubility
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D017931:DNA Primers; D021122:Protein Subunits; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D003545:Cysteine