title: Characterization of Amm VIII from Androctonus mauretanicus mauretanicus: a new scorpion toxin that discriminates between neuronal and skeletal sodium channels.
abstract: The venom of the scorpion Androctonus mauretanicus mauretanicus was screened by use of a specific serum directed against AaH II, the scorpion alpha-toxin of reference, with the aim of identifying new analogues. This led to the isolation of Amm VIII (7382.57 Da), which gave a highly positive response in ELISA, but was totally devoid of toxicity when injected subcutaneously into mice. In voltage-clamp experiments with rat brain type II Na+ channel rNa(v)1.2 or rat skeletal muscle Na+ channel rNa(v)1.4, expressed in Xenopus oocytes, the EC50 values of the toxin-induced slowing of inactivation were: 29+/-5 and 416+/-14 nM respectively for AmmVIII and 2.6+/-0.3 nM and 2.2+/-0.2 nM, respectively, for AaH II interactions. Accordingly, Amm VIII clearly discriminates neuronal versus muscular Na+ channel. The Amm VIII cDNA was amplified from a venom gland cDNA library and its oligonucleotide sequence determined. It shows 87% sequence homology with AaH II, but carries an unusual extension at its C-terminal end, consisting of an additional Asp due to a point mutation in the cDNA penultimate codon. We hypothesized that this extra amino acid residue could induce steric hindrance and dramatically reduce recognition of the target by Amm VIII. We constructed a model of Amm VIII based on the X-ray structure of AaH II to clarify this point. Molecular modelling showed that this C-terminal extension does not lead to an overall conformational change in Amm VIII, but drastically modifies the charge repartition and, consequently, the electrostatic dipole moment of the molecule. At last, liquid-phase radioimmunassays with poly- and monoclonal anti-(AaH II) antibodies showed the loss of conformational epitopes between AaH II and Amm VIII.
authors.affiliation: CNRS UMR 6560, Ingénierie des Protéines, Faculté de Médecine secteur Nord, Institut Jean Roche, Université de la Méditerranée, Bd Pierre Dramard, 13916, Marseille, Cedex 20, France.
authors.forename: Meriem
authors.initials: M
authors.lastname: Alami
authors.forename: Hélène
authors.initials: H
authors.lastname: Vacher
authors.forename: Frank
authors.initials: F
authors.lastname: Bosmans
authors.forename: Christiane
authors.initials: C
authors.lastname: Devaux
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Rosso
authors.forename: Pierre E
authors.initials: PE
authors.lastname: Bougis
authors.forename: Jan
authors.initials: J
authors.lastname: Tytgat
authors.forename: Hervé
authors.initials: H
authors.lastname: Darbon
authors.forename: Marie-France
authors.initials: MF
authors.lastname: Martin-Eauclaire
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004305:Dose-Response Relationship, Drug; D005260:Female; D007276:Injections, Intraventricular; D007279:Injections, Subcutaneous; D007928:Lethal Dose 50; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D012604:Scorpion Venoms / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015222:Sodium Channels / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014982:Xenopus laevis
chemical_list: C484037:Amm VIII toxin, Androctonus mauretanicus mauretanicus; D018076:DNA, Complementary; D012604:Scorpion Venoms; D015222:Sodium Channels