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Purification and Characterization of a Novel Insecticidal Toxin, μ-sparatoxin-Hv2, from the Venom of the Spider Heteropoda venatoria.

ABSTRACT: The venom of the spider Heteropoda venatoria produced lethal effect to cockroaches as reported in our previous study, and could be a resource for naturally-occurring insecticides. The present study characterized a novel cockroach voltage-gated sodium channels (NaVs) antagonist, μ-sparatoxin-Hv2 (μ-SPRTX-Hv2 for short), from this venom. μ-SPRTX-Hv2 is composed of 37 amino acids and contains six conserved cysteines. We synthesized the toxin by using the chemical synthesis method. The toxin was lethal to cockroaches when intraperitoneally injected, with a LD50 value of 2.8 nmol/g of body weight. Electrophysiological data showed that the toxin potently blocked NaVs in cockroach dorsal unpaired median (DUM) neurons, with an IC50 of 833.7 ± 132.2 nM, but it hardly affected the DUM voltage-gated potassium channels (KVs) and the DUM high-voltage-activated calcium channels (HVA CaVs). The toxin also did not affect NaVs, HVA CaVs, and Kvs in rat dorsal root ganglion (DRG) neurons, as well as NaV subtypes NaV1.3⁻1.5, NaV1.7, and NaV1.8. No envenomation symptoms were observed when μ-SPRTX-Hv2 was intraperitoneally injected into mouse at the dose of 7.0 μg/g. In summary, μ-SPRTX-Hv2 is a novel insecticidal toxin from H. venatoria venom. It might exhibit its effect by blocking the insect NaVs and is a candidate for developing bioinsecticide.

SUBMITTER: Xiao Z

PROVIDER: S-EPMC6024679 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Purification and Characterization of a Novel Insecticidal Toxin, μ-sparatoxin-Hv2, from the Venom of the Spider Heteropoda venatoria.

Xiao Zhen Z Zhang Yunxiao Y Zeng Jiao J Liang Songping S Tang Cheng C Liu Zhonghua Z

Toxins 20180607 6

The venom of the spider Heteropoda venatoria produced lethal effect to cockroaches as reported in our previous study, and could be a resource for naturally-occurring insecticides. The present study characterized a novel cockroach voltage-gated sodium channels (NaVs) antagonist, μ-sparatoxin-Hv2 (μ-SPRTX-Hv2 for short), from this venom. μ-SPRTX-Hv2 is composed of 37 amino acids and contains six conserved cysteines. We synthesized the toxin by using the chemical synthesis method. ...[more]

PMID: 29880771

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Project description:BackgroundThe yellow scorpion Tityus serrulatus (Ts) is responsible for the highest number of accidents and the most severe scorpion envenoming in Brazil. Although its venom has been studied since the 1950s, it presents a number of orphan peptides that have not been studied so far. The objective of our research was to isolate and identify the components present in the fractions VIIIA and VIIIB of Ts venom, in order to search for a novel toxin. The major isolated toxins were further investigated for macrophage modulation.MethodsThe fractions VIIIA and VIIIB, obtained from Ts venom cation exchange chromatography, were rechromatographed on a C18 column (4.6 × 250 mm) followed by a reversed-phase chromatography using another C18 column (2.1 × 250 mm). The main eluted peaks were analyzed by MALDI-TOF and Edman's degradation and tested on macrophages.ResultsThe previously described toxins Ts2, Ts3-KS, Ts4, Ts8, Ts8 propeptide, Ts19 Frag-II and the novel peptide Ts19 Frag-I were isolated from the fractions VIIIA and VIIIB. Ts19 Frag-I, presenting 58 amino acid residues, a mass of 6,575 Da and a theoretical pI of 8.57, shares high sequence identity with potassium channel toxins (KTx). The toxins Ts4, Ts3-KS and the partially purified Ts19 Frag-I did not produce cytotoxic effects on macrophage murine cells line (J774.1). On the other hand, Ts19 Frag-I induced the release of nitric oxide (NO) by macrophages, while Ts4 and Ts3-KS did not affect the NO production at the tested concentration (50 μg/mL). At the same concentration, Ts19 Frag-I and Ts3-KS increased the production of interleukin-6 (IL-6). Ts19 Frag-I and Ts4 did not induce the release of IL-10, IL-1β or tumor necrosis factor-α by macrophage cells using the tested concentration (50 μg/mL).ConclusionsWe partially purified and determined the complete sequence and chemical/physical parameters of a new β-KTx, denominated Ts19 Frag-I. The toxins Ts4, Ts3-KS and Ts19 Frag-I showed no cytotoxicity toward macrophages and induced IL-6 release. Ts19 Frag-I also induced the release of NO, suggesting a pro-inflammatory activity.

Project description:The venoms of toxic animals are chemical pools composed of various proteins, peptides, and small organic molecules used for predation and defense, in which the peptidic toxins have been intensively pursued mining modulators targeting disease-related ion channels and receptors as valuable drug pioneers. In the present study, we uncovered the molecular diversity of peptide toxins in the venom of the spider Heteropoda pingtungensis (H. pingtungensis) by using a combinatory strategy of venom gland cDNA library and transcriptome sequencing (RNA-seq). An amount of 991 high-quality expressed sequence tags (ESTs) were identified from 1138 generated sequences, which fall into three categories, such as the toxin-like ESTs (531, 53.58%), the cellular component ESTs (255, 25.73%), and the no-match ESTs (205, 20.69%), as determined by gene function annotations. Of them, 190 non-redundant toxin-like peptides were identified and can be artificially grouped into 13 families based on their sequence homology and cysteine frameworks (families A-M). The predicted mature toxins contain 2-10 cysteines, which are predicted to form intramolecular disulfide bonds to stabilize their three-dimensional structures. Bioinformatics analysis showed that toxins from H. pingtungensis venom have high sequences variability and the biological targets for most toxins are unpredictable due to lack of homology to toxins with known functions in the database. Furthermore, RP-HPLC and MALDI-TOF analyses have identified a total of 110 different peptides physically existing in the H. pingtungensis venom, and many RP-HPLC fractions showed potent inhibitory activity on the heterologously expressed NaV1.7 channel. Most importantly, two novel NaV1.7 peptide antagonists, µ-Sparatoxin-Hp1 and µ-Sparatoxin-Hp2, were characterized. In conclusion, the present study has added many new members to the spider toxin superfamily and built the foundation for identifying novel modulators targeting ion channels in the H. pingtungensis venom.

Dataset Information

Purification and Characterization of a Novel Insecticidal Toxin, μ-sparatoxin-Hv2, from the Venom of the Spider Heteropoda venatoria.

Publications

Purification and Characterization of a Novel Insecticidal Toxin, μ-sparatoxin-Hv2, from the Venom of the Spider <i>Heteropoda venatoria</i>.

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