Expression of the standard scorpion alpha-toxin AaH II and AaH II mutants leading to the identification of some key bioactive elements

Legros, Christian; Céard, Brigitte; Vacher, Hélène; Marchot, Pierre; Bougis, Pierre E.; Martin‐Eauclaire, Marie‐France

doi:10.1016/j.bbagen.2005.01.008

Cited by 41 publications

(33 citation statements)

References 26 publications

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“…The synaptosomal fraction (P2) from adult rat brains was prepared as previously described in the presence of protease inhibitors (Legros et al, 2005). Synaptosomes were kept frozen in liquid nitrogen until use.…”

Section: Competition Studiesmentioning

confidence: 99%

Differential effects of five ‘classical’ scorpion β-toxins on rNav1.2a and DmNav1 provide clues on species-selectivity

Bosmans

Martin‐Eauclaire²,

Tytgat

2007

Toxicology and Applied Pharmacology

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In general, scorpion β-toxins have been well examined. However, few in-depth studies have been devoted to species selectivity and affinity comparisons on the different voltage-activated Na + channels since they have become available as cloned channels that can be studied in heterologous expression systems. As a result, their classification is largely historical and dates from early in vivo experiments on mice and cockroach and fly larvae.In this study, we aimed to provide an updated overview of selectivity and affinity of scorpion β-toxins towards voltage-activated Na + channels of vertebrates or invertebrates. As pharmacological tools, we used the classic β-toxins AaHIT, Css II, Css IV, Css VI and Ts VII and tested them on the neuronal vertebrate voltage-activated Na + channel, rNa v 1.2a. For comparison, its invertebrate counterpart, DmNav1, was also tested. Both these channels were expressed in Xenopus laevis oocytes and the currents measured with the two-electrode voltage-clamp technique. We supplemented this data with several binding displacement studies on rat brain synaptosomes. The results lead us to propose a general classification and a novel nomenclature of scorpion β-toxins based on pharmacological activity.

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Section: Competition Studiesmentioning

confidence: 99%

Differential effects of five ‘classical’ scorpion β-toxins on rNav1.2a and DmNav1 provide clues on species-selectivity

Bosmans

Martin‐Eauclaire²,

Tytgat

2007

Toxicology and Applied Pharmacology

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“…However, in general these publications, even when expressing the same neurotoxin, do not present a clear description of their purification methods [3,4,8,10], and most important yet, they have not included the demonstration of biological activity in vivo of their products [3-5, 8, 10]. One of the most important issues for heterelogous expression of proteins is to find the correct folding conditions, especially for correct disulfide bridge formation that allow the recombinant molecules to be biologically active.…”

mentioning

confidence: 99%

“…Several communications have reported the production of recombinant voltagegated sodium channel toxins by heterologously expression in bacteria [3][4][5][6][7][8][9] and yeast [10].…”

mentioning

confidence: 99%

Four disulfide-bridged scorpion beta neurotoxin CssII: Heterologous expression and proper folding in vitro

Estévez

García

Schiavon

et al. 2007

Biochimica et Biophysica Acta (BBA) - General Subjects

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The gene of the four disulfide-bridged Centruroides suffusus suffusus toxin II was cloned into the expression vector pQE30 containing a 6His-tag and an FXa proteolytic cleavage region. This recombinant vector was transfected into E. coli BL21 cells and expressed under induction with isopropyl thiogalactoside (IPTG). The level of expression was 24.6 mg/L of culture medium, and the His tagged recombinant toxin (HisrCssII) was found exclusively in inclusion bodies. After solubilization the HisrCssII peptide was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisrCssII product obtained from the affinity chromatography step showed several peptide fractions having the same molecular mass of 9,392.6 Da, indicating that HisrCssII was oxidized forming several distinct disulfide bridge arrangements. The multiple forms of HisrCssII after reduction eluted from the column as a single protein component of 9,400.6 Da. Similarly, an in vitro folding of the reduced HisrCssII generated a single oxidized component of HisrCssII, which was cleaved by the proteolytic enzyme FXa to the recombinant CssII (rCssII). The molecular mass of rCssII was 7,538.6 Da as expected. Since native CssII is amidated at the C-terminal residue whereas the rCssII is heterologously expressed in the format of free carboxyl end, there is a difference of 1Da, when comparing both peptides (native versus heterologously expressed). Nevertheless, they show similar toxicity when injected intracranially into mice, and both nCssII and rCssII show the typical electrophysiological properties of beta-toxins in Na v 1.6 channels, which is for the first time demonstrated here. Binding and displacement experiments conducted with radiolabelled CssII confirms the electrophysiological results. Several problems associated with the heterologously expressed toxins containing four disulfide bridges are discussed.

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“…For example, replacing the conserved 58 Lys residue in AaH II by a hydrophobic (Val and Ile) or acidic (Glu) amino acid leads to an inactive analogue [49]. This crucial Lys is also conserved in Bot IX and may play a similar functional role.…”

Section: Discussionmentioning

confidence: 99%

“…Using a liquid-phase radioimmunoassay set up with experimental conditions similar to those used with other scorpion toxins [33,49], a weak competition with 125 I-Bot I bound to its specific antibodies is observed when using increasing concentrations of Bot IX. The IC 0.5 concentrations are 0.6 AE 0.2 nM for Bot I and 0.1 AE 0.3 lM for Bot IX (Fig.…”

Section: Antigenic Profiling Of Bot IXmentioning

confidence: 99%

The scorpion toxin Bot IX is a potent member of the α‐like family and has a unique N‐terminal sequence extension

et al. 2016

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We report the detailed chemical, immunological and pharmacological characterization of the a-toxin Bot IX from the Moroccan scorpion Buthus occitanus tunetanus venom. Bot IX, which consists of 70 amino acids, is a highly atypical toxin. It carries a unique N-terminal sequence extension and is highly lethal in mice. Voltage clamp recordings on oocytes expressing rat Nav1.2 or insect BgNav1 reveal that, similar to other a-like toxins, Bot IX inhibits fast inactivation of both variants. Moreover, Bot IX belongs to the same structural/immunological group as the a-like toxin Bot I. Remarkably, radioiodinated Bot IX competes efficiently with the classical a-toxin AaH II from Androctonus australis, and displays one of the highest affinities for Nav channels.Keywords: Nav channel; scorpion toxin; a-like toxin Eukaryotic voltage-gated Na + (Na v ) channels are targeted by animal venom toxins that bind to diverse regions on the extracellular side within the voltagesensing domain (VSD) or pore-region [1]. As such, they are fitting tools to study the functional and structural properties of mammalian and insect Na v channels, and may inspire the rational design of novel channel modulators [2]. Typically, scorpion toxins targeting Na v channels are small peptides (60-70 amino acid residues) that are cross-linked by four conserved disulphide bonds [1,3] and are divided into a-and btype toxins according to their binding properties in competition assays on rat brain synaptosomes [4,5]. However, accumulating reports on the characterization of more than one hundred scorpion toxins able to modulate Na v channel function led to a revision of their classification, one that primarily takes into account their pharmacological response in electrophysiological experiments with voltage-gated ion channels. Specifically, a-toxins interact with the voltage-sensor domain (VSD) in domain IV of the channel [6-12] to induce a prolongation of the action potential by blocking channel fast inactivation, with toxin binding being dependent on the membrane potential [1]. In contrast, b-toxins bind primarily to the VSD in domain II and act on channel activation by shifting its voltage-dependency toward more negative potentials [3,5]. Scorpion a-toxins are further divided based on their preference for insect versus mammalian Na v channels [13,14]. The classical a-toxins are highly lethal in mammals and bind with nanomolar affinities to rat brain synaptosomes but are inactive when injected into insects. This subfamily of toxins is mainly found in Old World Buthidae venoms and, in Abbreviation AaH I to III, toxin I to III of Androctonus australis Hector; Amm V, toxin V of Androctonus mauretanicus mauretanicus; Bom, Buthus occitanus mardochei; Bot I to XI, toxin I to XI of Buthus occitanus tunetanus; CD, circular dichroism; CpA, carboxypeptidase A; DMAA, dimethylalylamine; HPLC, high performance liquid chromatography; Lqq V, toxin V of Leiurus quinquestriatus quinquestriatus; PTH-aa, phenylthiohydantoinaminoacid; RCM-toxin, reduced and S-carboxymethylated t...

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Expression of the standard scorpion alpha-toxin AaH II and AaH II mutants leading to the identification of some key bioactive elements

Cited by 41 publications

References 26 publications

Differential effects of five ‘classical’ scorpion β-toxins on rNav1.2a and DmNav1 provide clues on species-selectivity

Differential effects of five ‘classical’ scorpion β-toxins on rNav1.2a and DmNav1 provide clues on species-selectivity

Four disulfide-bridged scorpion beta neurotoxin CssII: Heterologous expression and proper folding in vitro

The scorpion toxin Bot IX is a potent member of the α‐like family and has a unique N‐terminal sequence extension

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