A chimeric scorpion α‐toxin displays <i>de novo</i> electrophysiological properties similar to those of α‐like toxins

Bouhaouala‐Zahar, Balkiss; Benkhalifa, Rym; Srairi, Najet; Zenouaki, I.; Ligny-Lemaire, Caroline; Drevet, Pascal; Sampiéri, Franĉois; Pelhate, M.; Ayeb, Mohamed El; Ménèz, Andre; Karoui, Hakim; Ducancel, Frédéric

doi:10.1046/j.1432-1033.2002.02918.x

Cited by 10 publications

(10 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These findings are consistent with a second mutagenesis approach on BmK-M1 in which aromatic residues (Tyr5, Tyr35, Trp38, Tyr42, Trp47) affected toxin properties, most likely by structural stabilization (Sun et al, 2003). The functional importance of residues that are located in a turn near the N terminus (8-10) was shown in a chimeric approach between Lqh␣IT and BotXIV, an ␣-like toxin (Bouhaouala-Zahar et al, 2002). All together, it is generally accepted that the so-called five-residue turn (residues 8-12 in the Lqh toxins used here) together with the C-terminal end of the toxins forms the channel interaction site.…”

Section: Discussionsupporting

confidence: 84%

Combinatorial Interaction of Scorpion Toxins Lqh-2, Lqh-3, and LqhαIT with Sodium Channel Receptor Sites-3

Leipold

Lu²,

Gordon³

et al. 2004

Mol Pharmacol

View full text Add to dashboard Cite

Scorpion ␣-toxins Lqh␣IT, Lqh-2, and Lqh-3 are representatives of three groups of ␣-toxins that differ in their preference for insects and mammals. These ␣-insect, antimammalian, and ␣-like toxins bind to voltage-gated sodium channels and slow down channel inactivation. Sodium channel mutagenesis studies using various ␣-toxins have shown that they interact with receptor site 3, which is composed mainly of a short stretch of amino-acid residues between S3 and S4 of domain 4. Variation in this region results in marked differences between various subtypes of sodium channels with respect to their sensitivity to the three Lqh toxins. We incorporated the S3-S4 linker of domain 4 from hNa V 1.2/hNa V 1.1, hNa V 1.3, hNa V 1.6, and hNa V 1.7 channels as well as individual point mutations into the rNa V 1.4 skeletal muscle sodium channel. Our data show that the affinity of Lqh-3 and Lqh␣IT to sodium channels is markedly determined by an aspartate residue (Asp1428 in rNa V 1.4); when mutated to glutamate, as is present in Na V 1.1-1.3 channels, Lqh-3-channel interactions are abolished. The interaction of Lqh-2 and Lqh␣IT, however, is strongly reduced when a lysine residue (Lys1432 in rNa V 1.4) is replaced by threonine (as in hNa V 1.7), whereas this substitution is without effect for Lqh-3. The influence of Lys1432 on Lqh-2 and Lqh␣IT strongly depends on the context of the Asp/Glu site at position 1428, giving rise to a wide variety of toxicological phenotypes by means of a combinatorial mixing and matching of only a few residues in receptor site 3.Voltage-gated sodium (Na V ) channels consist of a large (ϳ260 kDa) pore-forming ␣-subunit, composed of four homologous domains (D1-D4), each with six transmembrane segments (S1-S6) and a hairpin-like pore region between S5 and S6. Because of their structural conservation in vertebrates and invertebrates and their pivotal role in cellular excitability, Na V channels are targeted by a large variety of chemically distinct toxins, many of which do not differentiate among channel subtypes (Catterall, 1992;Gordon, 1997). However, some scorpion neurotoxins show specificity for insect or mammalian Na V channels, and others are able to differentiate between Na V subtypes in mammalian neurons . This selectivity is attributed to differences of active sites on the toxins and to variations in receptor binding sites on distinct Na V channels that, when identified, may be used for design of selective drugs.Scorpion toxins affecting Na V channels are 61-to 76-residue polypeptides that comprise two major classes, ␣-and ␤-toxins, according to their mode of action and binding properties to distinct sites (receptor sites-3 and -4, respectively) on Na V channels (Martin-Eauclaire and Couraud, 1995;Gordon et al., 1998). ␣-Toxins inhibit Na V channel inactivation in various excitable preparations, but they show vast differences in preference for insect and mammalian Na V channels. Accordingly, they are divided into classic ␣-toxins, which are highly active in mammalian brain [e.g., Lqh-2 (L...

show abstract

Section: Discussionsupporting

confidence: 84%

Combinatorial Interaction of Scorpion Toxins Lqh-2, Lqh-3, and LqhαIT with Sodium Channel Receptor Sites-3

Leipold

Lu²,

Gordon³

et al. 2004

Mol Pharmacol

View full text Add to dashboard Cite

show abstract

“…However, there are some reports (e.g. [6,7,[11][12][13]) that succeeded to express a correct and folded toxin, by reduction and refolding the product in vitro; however, such toxins have from one to four extra residues attached to their N-terminal region. The interest of our research group is to obtain the inserm-00378025, version 1 -18 Jun 2009 expression of a voltage-gated sodium channel toxin with four disulfide-bridges, capable of reproducing the same structure and function (symptoms of intoxication) in animal models, as the native peptide does.…”

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

View full text Add to dashboard Cite

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.

show abstract

“…Second, analysis of toxin sequences allowed to put forward a hypothesis regarding the differential role of the core and specificity modules , which was further substantiated by experiments on transfer of respective toxin regions to the scaffold of their counterparts from another selectivity subgroup. This was reported for Aah2, BotXIV, Lqh2, and LqhαIT . Third, it was demonstrated that the specificity modules of toxins from different selectivity subgroups are characterized by different physicochemical properties .…”

Section: Resultsmentioning

confidence: 56%

Design of sodium channel ligands with defined selectivity – a case study in scorpion alpha‐toxins

et al. 2017

View full text Add to dashboard Cite

Scorpion α-toxins are polypeptides that inhibit voltage-gated sodium channel inactivation. They are divided into mammal, insect and α-like toxins based on their relative activity toward different phyla. Several factors are currently known to influence the selectivity, which are not just particular amino acid residues but also general physical, chemical, and topological properties of toxin structural modules. The objective of this study was to change the selectivity profile of a chosen broadly active α-like toxin, BeM9 from Mesobuthus eupeus, toward mammal-selective. Based on the available information on what determines scorpion α-toxin selectivity, we designed and produced msBeM9, a BeM9 derivative, which was verified to be exclusively active toward mammalian sodium channels and, most importantly, toward the Na 1.2 isoform expressed in the brain.

show abstract

A chimeric scorpion α‐toxin displays de novo electrophysiological properties similar to those of α‐like toxins

Cited by 10 publications

References 52 publications

Combinatorial Interaction of Scorpion Toxins Lqh-2, Lqh-3, and LqhαIT with Sodium Channel Receptor Sites-3

Combinatorial Interaction of Scorpion Toxins Lqh-2, Lqh-3, and LqhαIT with Sodium Channel Receptor Sites-3

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

Design of sodium channel ligands with defined selectivity – a case study in scorpion alpha‐toxins

Contact Info

Product

Resources

About