In VitroFolding and Functional Analysis of an Anti-insect Selective Scorpion Depressant Neurotoxin Produced inEscherichia coli

Turkov, Michael; Rashi, Sharon; Zilberberg, Noam; Gordon, Dalia; Khalifa, Rym Ben; Stankiewicz, Maria; Pelhate, M.; Gurevitz, Michael

doi:10.1006/prep.1997.0724

Cited by 59 publications

(34 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Similarly, storage of highly disulfidebridged and compact peptide toxins seems to be problematic. For example, in vitro expression of scorpion toxins almost invariably results in minute quantities of active peptide (14). Therefore, keeping a prevenom ready would undoubtedly be an important aspect of conserving by reducing the unnecessary use of venom.…”

Section: Discussionmentioning

confidence: 99%

“…Other than the antimicrobial peptides, almost all neurotoxins in the venom are reported to be highly folded, disulfide-bridged molecules (13). Low yields of expression of these peptides in recombinant systems also hint at the unique and difficult folding and storage requirements (14). However, used sparingly, venom is an excellent tool for both offense and defense.…”

mentioning

confidence: 99%

See 1 more Smart Citation

One scorpion, two venoms: Prevenom ofParabuthus transvaalicusacts as an alternative type of venom with distinct mechanism of action

Inceoglu

Langó

Jie

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

154

105

View full text Add to dashboard Cite

Scorpion venom is a complex mixture of salts, small molecules, peptides, and proteins. Scorpions employ this valuable tool in several sophisticated ways for subduing prey, deterring predators, and possibly during mating. Here, a subtle but clever strategy of venom utilization by scorpions is reported. Scorpions secrete a small quantity of transparent venom when initially stimulated that we propose to name prevenom. If secretion continues, a cloudy and dense venom that is white in color is subsequently released. The prevenom contains a combination of high K ؉ salt and several peptides including some that block rectifying K ؉ channels and elicit significant pain and toxicity because of a massive local depolarization. The presence of high extracellular K ؉ in the prevenom can depolarize cells and also decrease the local electrochemical gradient making it more difficult to reestablish the resting potential. When this positive change to the K ؉ equilibrium potential is combined with the blockage of rectifying K ؉ channels, this further delays the recovery of the resting potential, causing a prolonged effect. We propose that the prevenom of scorpions is used as a highly efficacious predator deterrent and for immobilizing small prey while conserving metabolically expensive venom until a certain level of stimuli is reached, after which the venom is secreted.

show abstract

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

One scorpion, two venoms: Prevenom ofParabuthus transvaalicusacts as an alternative type of venom with distinct mechanism of action

Inceoglu

Langó

Jie

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

154

105

View full text Add to dashboard Cite

show abstract

“…DNA Manipulations-A synthetic Css4 gene was constructed using eight overlapping oligonucleotide primers designed with a codon usage based on the nucleotide sequences of long-chain scorpion toxin genes that have been successfully expressed in E. coli (Table I) (9,17,18). A mixture of 1 M of each of primers 1-8 (Table I) heated to 95°C was cooled stepwise to 4°C for annealing.…”

Section: Methodsmentioning

confidence: 99%

“…An NdeI-BamHI fragment was cleaved out and subcloned into the corresponding sites of pET-14b (Novagen). Expression in E. coli BL21 cells and in vitro folding of the recombinant His-Css4 (fusion of His tag and a thrombin cleavage sequence to the N-terminal side of Css4) was performed following established protocols (18). The average yield of His-Css4 was 2 mg from 1 liter of E. coli culture.…”

Section: Methodsmentioning

confidence: 99%

“…Css4 -Protocols that were previously shown to be useful for the production and extensive mutagenesis of a number of long-chain scorpion toxins using the expression vector pET-11c (9,10,17,18,26) were unsuccessful in attempts to overexpress Css4. However, large quantities of recombinant toxin, His-Css4, were obtained using the expression vector pET-14b (see "Materials and Methods").…”

Section: Expression and Characterization Of Recombinantmentioning

confidence: 99%

See 1 more Smart Citation

Common Features in the Functional Surface of Scorpion β-Toxins and Elements That Confer Specificity for Insect and Mammalian Voltage-gated Sodium Channels

Cohen

Karbat

Gilles

et al. 2005

Journal of Biological Chemistry

Self Cite

125

View full text Add to dashboard Cite

Scorpion ␤-toxins that affect the activation of mammalian voltage-gated sodium channels (Na v s) have been studied extensively, but little is known about their functional surface and mode of interaction with the channel receptor. To enable a molecular approach to this question, we have established a successful expression system for the anti-mammalian scorpion ␤-toxin, Css4, whose effects on rat brain Na v s have been well characterized. A recombinant toxin, His-Css4, was obtained when fused to a His tag and a thrombin cleavage site and had similar binding affinity for and effect on Na currents of rat brain sodium channels as those of the native toxin isolated from the scorpion venom. Molecular dissection of His-Css4 elucidated a functional surface of 1245 Å 2 composed of the following: 1) a cluster of residues associated with the ␣-helix, which includes a putative "hot spot" (this cluster is conserved among scorpion ␤-toxins and contains their "pharmacophore"); 2) a hydrophobic cluster associated mainly with the ␤2 and ␤3 strands, which is likely to confer the specificity for mammalian Na v s; 3) a single bioactive residue (Trp-58) in the C-tail; and 4) a negatively charged residue (Glu-15) involved in voltage sensor trapping as inferred from our ability to uncouple toxin binding from activity upon its substitution. This study expands our understanding about the mode of action of scorpion ␤-toxins and illuminates differences in the functional surfaces that may dictate their specificities for mammalian versus insect sodium channels.

show abstract

Scorpion neurotoxins: structure/function relationships and application in agriculture

et al. 2000

Self Cite

View full text Add to dashboard Cite

Continued use of non‐specific chemical insecticides poses potential risks to the environment and to human health resulting from non‐target toxicity and increased insect resistance to these agents. Scorpions produce anti‐insect selective polypeptide toxins that bind to and modulate voltage‐sensitive ion channels in excitable tissues, thus offering alternative, environmentally safer means for insect pest control. Despite this potential, little is known about their structural elements dictating anti‐insect preference, which may be useful for the design of selective insecticides. We used a bacterial system for expression and genetic dissection of two pharmacologically distinct scorpion toxins: alpha and excitatory. By exploiting a multi‐disciplinary approach consisting of mutagenesis, protein chemistry, electrophysiology, binding and toxicity assays, and structural studies, we elucidated the bioactive surface of two anti‐insect toxins, LqhαIT and Bj‐xtrIT. In both polypeptides the bioactive surface is composed of residues surrounding the C‐terminal region. In addition, a direct, immediate approach in using the toxin genes was demonstrated by engineering baculoviruses with cDNAs encoding LqhIT2 (depressant toxin), and LqhIT1 (excitatory toxin) resulting in viral vectors with significantly improved insecticidal efficacy. © 2000 Society of Chemical Industry

show abstract

In VitroFolding and Functional Analysis of an Anti-insect Selective Scorpion Depressant Neurotoxin Produced inEscherichia coli

Cited by 59 publications

References 39 publications

One scorpion, two venoms: Prevenom ofParabuthus transvaalicusacts as an alternative type of venom with distinct mechanism of action

One scorpion, two venoms: Prevenom ofParabuthus transvaalicusacts as an alternative type of venom with distinct mechanism of action

Common Features in the Functional Surface of Scorpion β-Toxins and Elements That Confer Specificity for Insect and Mammalian Voltage-gated Sodium Channels

Scorpion neurotoxins: structure/function relationships and application in agriculture

Contact Info

Product

Resources

About