A distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin Dc1a

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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“…In contrast, 100 nM Bot IX nearly eliminates rapid inactivation of BgNa v 1 (Fig. 7B) [5][6][7]46,50].…”

Section: Electrophysiological Characteristics Of Bot IXmentioning

confidence: 99%

“…previous reports in which other scorpion toxins able to bind to both VSDII and VSDIV have been described [7,12,[46][47][48].…”

Section: Competition Binding Experiments On Rat Brain Synaptosomesmentioning

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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“…For example, the spider toxins μ-DGTX-Dc1a (Bende et al, 2014) and μ-TRTX-Ae1a (Herzig et al, 2016) are potent inhibitors of the BgNa V 1 channel from the German cockroach Blattella germanica whereas P. americana is resistant to these toxins. Thus, we next used two-electrode voltage-clamp electrophysiology to examine the effect of Ct1a on the cloned BgNa V 1 channel heterologously expressed in Xenopus oocytes.…”

Section: Resultsmentioning

confidence: 99%

“…Only three spider toxins have been previously shown to be lethal to L. cuprina , with LD 50 values of 198 pmol/g for U 1 -agatoxin-Ta1a (Ta1a) (Undheim et al, 2015), 231 pmol/g for μ-diguetoxin-Dc1a (Bende et al, 2014), and 278 pmol/g for κ-hexatoxin-Hv1c (de Araujo et al, 2013). The spider venom peptides μ-cyrtautoxin-As1a and μ-segestritoxin-Sf1a both paralyse L. cuprina , without causing lethality, with PD 50 values of 700 pmol/g (Bende et al, 2013) and 2229 pmol/g (Bende et al, 2015), respectively.…”

Section: Discussionmentioning

confidence: 99%

“…As a whole, spider venoms have been predicted to contain as many as 10 million unique peptides (King, 2011), most of which are expected to be insecticidal, but less than <0.1% of this chemical diversity has been explored to date. Nevertheless, numerous insecticidal peptides have been isolated from spider venom with a broad range of phyletic selectivity (King and Hardy, 2013; Bende et al, 2014; Herzig et al, 2016). Recently, an insecticidal spider-venom peptide was approved for control of crop pests by the United States Environmental Protection Agency (Herzig et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

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Isolation of two insecticidal toxins from venom of the Australian theraphosid spider Coremiocnemis tropix

Ikonomopoulou

Smith

Herzig

et al. 2016

Toxicon

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Sheep flystrike is caused by parasitic flies laying eggs on soiled wool or open wounds, after which the hatched maggots feed on the sheep flesh and often cause large lesions. It is a significant economic problem for the livestock industry as infestations are difficult to control due to ongoing cycles of larval development into flies followed by further egg laying. We therefore screened venom fractions from the Australian theraphosid spider Coremiocnemis tropix to identify toxins active against the sheep blowfly Lucilia cuprina, which is the primary cause of flystrike in Australia. This screen led to isolation of two insecticidal peptides, Ct1a and Ct1b, that are lethal to blowflies within 24 h of injection. The primary structure of these peptides was determined using a combination of Edman degradation and sequencing of a C. tropix venom-gland transcriptome. Ct1a and Ct1b contain 39 and 38 amino acid residues, respectively, including six cysteine residues that form three disulfide bonds. Recombinant production in bacteria (Escherichia coli) resulted in low yields of Ct1a whereas solid-phase peptide synthesis using native chemical ligation produced sufficient quantities of Ct1a for functional analyses. Synthetic Ct1a had no effect on voltage-gated sodium channels from the American cockroach Periplanata americana or the German cockroach Blattella germanica, but it was lethal to sheep blowflies with an LD50 of 1687 pmol/g.

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Toxin structures as evolutionary tools: Using conserved 3D folds to study the evolution of rapidly evolving peptides

2016

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Three-dimensional (3D) structures have been used to explore the evolution of proteins for decades, yet they have rarely been utilized to study the molecular evolution of peptides. Here, we highlight areas in which 3D structures can be particularly useful for studying the molecular evolution of peptide toxins. Although we focus our discussion on animal toxins, including one of the most widespread disulfide-rich peptide folds known, the inhibitor cystine knot, our conclusions should be widely applicable to studies of the evolution of disulfide-constrained peptides. We show that conserved 3D folds can be used to identify evolutionary links and test hypotheses regarding the evolutionary origin of peptides with extremely low sequence identity; construct accurate multiple sequence alignments; and better understand the evolutionary forces that drive the molecular evolution of peptides. Also watch the video abstract.

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A distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin Dc1a

Cited by 69 publications

References 67 publications

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

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

Isolation of two insecticidal toxins from venom of the Australian theraphosid spider Coremiocnemis tropix

Toxin structures as evolutionary tools: Using conserved 3D folds to study the evolution of rapidly evolving peptides

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