Molecular cloning and functional expression of the alpha-scorpion toxin BotIII: pivotal role of the C-terminal region for its interaction with voltage-dependent sodium channels.
Alpha scorpion toxins bind to receptor site 3 on voltage-dependent sodium channels and inhibit their inactivation. The alpha-scorpion toxin BotIII is the most toxic protein of Buthus occitanus tunetanus. Its sequence differs only by three amino acid residues from that of AahII, the most active alpha-toxin. Due to their high affinity and selectivity for mammalian sodium channels, BotIII and AahII represent powerful tools for studying the molecular determinants of specificity for voltage-dependent sodium channels. Sequence analysis of BotIII gene has revealed two exons separated by a 381-bp intron and a signal peptide of 19 amino acids. We succeeded in expressing BotIII in significantly higher amounts than AahII the only expressed strict alpha anti-mammalian scorpion toxin reported in the literature. We have also modified specific amino acid residues of BotIII. The recombinant and the natural toxins differ by the amidation of the C-terminal residue. Toxicity and binding experiments indicated: (a) the affinity of rBotIII-OH and rAahII-OH (rBotIII-OH with the 3 mutations R10V, V51L, N64H) for the voltage-dependent sodium channels is reduced compared to the natural toxins. This data revealed the important role of the C-terminal amidation for the biological activity of BotIII and AahII; (b) the single mutation N64H is responsible for the difference of toxicity and affinity between rBotIII-OH and rAahII-OH; (c) the addition of the sequence GR to rBotIII-OH leads to the loss of biological activity. This study is in agreement with the important role attributed to the C-terminal sequence of alpha-toxins in their interaction with sodium channels receptors.
Benkhadir
Khadija
K
Laboratoire des Venins et Toxines, Institut Pasteur de Tunis, B. P 74, 1002 Tunis-Belvédère, Tunisia.
Kharrat
Riadh
R
Cestèle
Sandrine
S
Mosbah
Amor
A
Rochat
Hervé
H
El Ayeb
Mohamed
M
Karoui
Habib
H
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D015640:Ion Channel Gating / Q000187:drug effects* / Q000502:physiology; D008297:Male; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D017208:Rats, Wistar; D012604:Scorpion Venoms / Q000378:metabolism* / Q000633:toxicity; D012605:Scorpions / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015222:Sodium Channels / Q000502:physiology*
D012604:Scorpion Venoms; D015222:Sodium Channels