Characterization of Amm VIII from Androctonus mauretanicus mauretanicus: a new scorpion toxin that discriminates between neuronal and skeletal sodium channels.
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.
Alami
Meriem
M
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.
Vacher
Hélène
H
Bosmans
Frank
F
Devaux
Christiane
C
Rosso
Jean-Pierre
JP
Bougis
Pierre E
PE
Tytgat
Jan
J
Darbon
Hervé
H
Martin-Eauclaire
Marie-France
MF
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
C484037:Amm VIII toxin, Androctonus mauretanicus mauretanicus; D018076:DNA, Complementary; D012604:Scorpion Venoms; D015222:Sodium Channels