title: Expression of the standard scorpion alpha-toxin AaH II and AaH II mutants leading to the identification of some key bioactive elements.
abstract: The AaH II toxin from the scorpion Androctonus australis Hector is considered to be the standard alpha-toxin because it selectively binds with the highest known affinity to site 3 of mammalian voltage-activated Na+ channels (Na(v)) on rat brain synaptosomes but does not bind to insect synaptosomes. We generated two different constructs in pMALp allowing us to produce AaH II fused with the maltose-binding protein (MBP) in E. coli. We obtained reasonable amounts of recombinant AaH II after cleavage by enterokinase at the site DDDDK. We show that the introduction of a net negative charge at the C-terminus by the suppression of H64 amidation and the addition of an extra residue to the C-terminus (G65) led to fully active AaH II mutants, exhibiting exactly the same affinity as the native toxin for its target on rat brain synaptosomes. In contrast, the mutation of residue K58 into V, I or E residues drastically reduced toxin activity.
authors.affiliation: Ingénierie des Protéines CNRS FRE 2738, Institut Fédératif de Recherche Jean Roche, Faculté de Médecine Secteur Nord, Université de la Méditerranée, Bd Pierre Dramard, 13916, Marseille, cedex 20, France.
authors.forename: Christian
authors.initials: C
authors.lastname: Legros
authors.forename: Brigitte
authors.initials: B
authors.lastname: Céard
authors.forename: Hélène
authors.initials: H
authors.lastname: Vacher
authors.forename: Pascale
authors.initials: P
authors.lastname: Marchot
authors.forename: Pierre E
authors.initials: PE
authors.lastname: Bougis
authors.forename: Marie-France
authors.initials: MF
authors.lastname: Martin-Eauclaire
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009498:Neurotoxins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D030162:Reptilian Proteins; D012604:Scorpion Venoms / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D015222:Sodium Channels / Q000187:drug effects; D013329:Structure-Activity Relationship; D013574:Synaptosomes / Q000378:metabolism
chemical_list: D009498:Neurotoxins; D011993:Recombinant Fusion Proteins; D030162:Reptilian Proteins; D012604:Scorpion Venoms; D015222:Sodium Channels; C053969:scorpion toxin II, Androctonus