First chemical synthesis of a scorpion α‐toxin affecting sodium channels: The Aah I toxin of <i>Androctonus australis hector</i>

M’Barek, Sarrah Ben; Fajloun, Ziad; Cestèle, Sandrine; Devaux, Christiane; Mansuelle, Pascal; Mosbah, Ahmed; Jouirou, Besma; Mantegazza, Massimo; Rietschoten, J. Van; Ayeb, Mohamed El; Rochat, Hervé; Sabatier, Jean‐Marc; Sampiéri, Franĉois

doi:10.1002/psc.582

Cited by 12 publications

(7 citation statements)

References 68 publications

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“…The same research team obtained 1.2% yield (after denaturation and renaturation) of the recombinant toxin LqhIT2 [7]. Similarly, renaturation of toxin Aah1 performed by M'Barek et al [25] showed a recovery yield that varies from 0.3% to 2% efficiency after refolding, whereas expression and renaturation of the excitatory insectspecific toxin BmK IT-AP from Buthus martensii showed 0.5 mg/L of culture [5]. Some toxins just fold nicely, but unfortunately others do not.…”

Section: Purification In Vitro Folding and Enzymatic Cleavagementioning

confidence: 85%

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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Section: Purification In Vitro Folding and Enzymatic Cleavagementioning

confidence: 85%

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 advent of advanced recombinant (Vita et al 1995) and synthetic production techniques (M’Barek et al 2004) in combination with genetic approaches may soon yield Nav channel drug leads as well.…”

Section: Toxins Influencing Nav Channel Gating By Interacting With mentioning

confidence: 99%

Animal Toxins Influence Voltage-Gated Sodium Channel Function

Gilchrist

Olivera

Bosmans

2014

Handbook of Experimental Pharmacology

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Voltage-gated sodium (Nav) channels are essential contributors to neuronal excitability, making them the most commonly targeted ion channel family by toxins found in animal venoms. These molecules can be used to probe the functional aspects of Nav channels on a molecular level and to explore their physiological role in normal and diseased tissues. This chapter summarizes our existing knowledge of the mechanisms by which animal toxins influence Nav channels as well as their potential application in designing therapeutic drugs.

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“…However, stepwise solid-phase peptide synthesis (SPPS) of long chain α-toxins has achieved limited success, presumably due to the challenging assembly, purification and folding of these relatively large polypeptides. 11 Here, we report the efficient chemical synthesis of OD1 by combining SPPS, one-pot native chemical ligation, and in vitro protein folding. This approach produced α-toxin OD1 on a multimilligram scale, allowing detailed structural and pharmacological characterization.…”

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confidence: 99%

Chemical Engineering and Structural and Pharmacological Characterization of the α-Scorpion Toxin OD1

et al. 2013

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Scorpion α-toxins are invaluable pharmacological tools for studying voltage-gated sodium channels, but few structure-function studies have been undertaken due to their challenging synthesis. To address this deficiency, we report a chemical engineering strategy based upon native chemical ligation. The chemical synthesis of α-toxin OD1 was achieved by chemical ligation of three unprotected peptide segments. A high resolution X-ray structure (1.8 Å) of synthetic OD1 showed the typical βαββ α-toxin fold and revealed important conformational differences in the pharmacophore region when compared with other α-toxin structures. Pharmacological analysis of synthetic OD1 revealed potent α-toxin activity (inhibition of fast inactivation) at Nav1.7, as well as Nav1.4 and Nav1.6. In addition, OD1 also produced potent β-toxin activity at Nav1.4 and Nav1.6 (shift of channel activation in the hyperpolarizing direction), indicating that OD1 might interact at more than one site with Nav1.4 and Nav1.6. Investigation of nine OD1 mutants revealed that three residues in the reverse turn contributed significantly to selectivity, with the triple OD1 mutant (D9K, D10P, K11H) being 40-fold more selective for Nav1.7 over Nav1.6, while OD1 K11V was 5-fold more selective for Nav1.6 than Nav1.7. This switch in selectivity highlights the importance of the reverse turn for engineering α-toxins with altered selectivity at Nav subtypes.

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First chemical synthesis of a scorpion α‐toxin affecting sodium channels: The Aah I toxin of Androctonus australis hector

Cited by 12 publications

References 68 publications

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

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

Animal Toxins Influence Voltage-Gated Sodium Channel Function

Chemical Engineering and Structural and Pharmacological Characterization of the α-Scorpion Toxin OD1

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