Isolation and pharmacological characterisation of delta-atracotoxin-Hv1b, a vertebrate-selective sodium channel toxin

delta-Atracotoxins (delta-ACTXs) are peptide toxins isolated from the venom of Australian funnel-web spiders that slow sodium current inactivation in a similar manner to scorpion alpha-toxins. We have isolated and determined the amino acid sequence of a novel delta-ACTX, designated delta-ACTX-Hv1b, from the venom of the funnel-web spider Hadronyche versuta. This 42 residue toxin shows 67% sequence identity with delta-ACTX-Hv1a previously isolated from the same spider. Under whole-cell voltage-clamp conditions, the toxin had no effect on tetrodotoxin (TTX)-resistant sodium currents in rat dorsal root ganglion neurones but exerted a concentration-dependent reduction in peak TTX-sensitive sodium current amplitude accompanied by a slowing of sodium current inactivation similar to other delta-ACTXs. However, delta-ACTX-Hv1b is approximately 15-30-fold less potent than other delta-ACTXs and is remarkable for its complete lack of insecticidal activity. Thus, the sequence differences between delta-ACTX-Hv1a and -Hv1b provide key insights into the residues that are critical for targeting of these toxins to vertebrate and invertebrate sodium channels.     

Isolation of delta-missulenatoxin-Mb1a, the major vertebrate-active spider delta-toxin from the venom of Missulena bradleyi (Actinopodidae)

The present study describes the isolation and pharmacological characterisation of the neurotoxin delta-missulenatoxin-Mb1a (delta-MSTX-Mb1a) from the venom of the male Australian eastern mouse spider, Missulena bradleyi. This toxin was isolated using reverse-phase high-performance liquid chromatography and was subsequently shown to cause an increase in resting tension, muscle fasciculation and a decrease in indirect twitch tension in a chick biventer cervicis nerve-muscle bioassay. Interestingly, these effects were neutralised by antivenom raised against the venom of the Sydney funnel-web spider Atrax robustus. Subsequent whole-cell patch-clamp electrophysiology on rat dorsal root ganglion neurones revealed that delta-MSTX-Mb1a caused a reduction in peak tetrodotoxin (TTX)-sensitive sodium current, a slowing of sodium current inactivation and a hyperpolarising shift in the voltage at half-maximal activation. In addition, delta-MSTX-Mb1a failed to affect TTX-resistant sodium currents. Subsequent Edman degradation revealed a 42-residue peptide with unusual N- and C-terminal cysteines and a cysteine triplet (Cys(14-16)). This toxin was highly homologous to a family of delta-atracotoxins (delta-ACTX) from Australian funnel-web spiders including conservation of all eight cysteine residues. In addition to actions on sodium channel gating and kinetics to delta-ACTX, delta-MSTX-Mb1a caused significant insect toxicity at doses up to 2000 pmol/g. Delta-MSTX-Mb1a therefore provides evidence of a highly conserved spider delta-toxin from a phylogenetically distinct spider family that has not undergone significant modification.     

Isolation and pharmacological characterisation of δ-atracotoxin-Hv1b, a vertebrate-selective sodium channel toxin

δ-Atracotoxins (δ-ACTXs) are peptide toxins isolated from the venom of Australian funnel-web spiders that slow sodium current inactivation in a similar manner to scorpion α-toxins. We have isolated and determined the amino acid sequence of a novel δ-ACTX, designated δ-ACTX-Hv1b, from the venom of the funnel-web spider Hadronyche versuta. This 42 residue toxin shows 67% sequence identity with δ-ACTX-Hv1a previously isolated from the same spider. Under whole-cell voltage-clamp conditions, the toxin had no effect on tetrodotoxin (TTX)-resistant sodium currents in rat dorsal root ganglion neurones but exerted a concentration-dependent reduction in peak TTX-sensitive sodium current amplitude accompanied by a slowing of sodium current inactivation similar to other δ-ACTXs. However, δ-ACTX-Hv1b is approximately 15–30-fold less potent than other δ-ACTXs and is remarkable for its complete lack of insecticidal activity. Thus, the sequence differences between δ-ACTX-Hv1a and -Hv1b provide key insights into the residues that are critical for targeting of these toxins to vertebrate and invertebrate sodium channels.     

Discovery of an MIT-like atracotoxin family: spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins

This project identified a novel family of six 66–68 residue peptides from the venom of two Australian funnel-web spiders, Hadronyche sp. 20 and H. infensa: Orchid Beach (Hexathelidae: Atracinae), that appear to undergo N- and/or C-terminal post-translational modifications and conform to an ancestral protein fold. These peptides all show significant amino acid sequence homology to atracotoxin-Hvf17 (ACTX–Hvf17), a non-toxic peptide isolated from the venom of H. versuta, and a variety of AVIT family proteins including mamba intestinal toxin 1 (MIT1) and its mammalian and piscine orthologs prokineticin 1 (PK1) and prokineticin 2 (PK2). These AVIT family proteins target prokineticin receptors involved in the sensitization of nociceptors and gastrointestinal smooth muscle activation. Given their sequence homology to MIT1, we have named these spider venom peptides the MIT-like atracotoxin (ACTX) family. Using isolated rat stomach fundus or guinea-pig ileum organ bath preparations we have shown that the prototypical ACTX–Hvf17, at concentrations up to 1 μM, did not stimulate smooth muscle contractility, nor did it inhibit contractions induced by human PK1 (hPK1). The peptide also lacked activity on other isolated smooth muscle preparations including rat aorta. Furthermore, a FLIPR Ca²⁺ flux assay using HEK293 cells expressing prokineticin receptors showed that ACTX–Hvf17 fails to activate or block hPK1 or hPK2 receptors. Therefore, while the MIT-like ACTX family appears to adopt the ancestral disulfide-directed β-hairpin protein fold of MIT1, a motif believed to be shared by other AVIT family peptides, variations in the amino acid sequence and surface charge result in a loss of activity on prokineticin receptors.     

A model genetic system for testing the in vivo function of peptide toxins

We have developed a model genetic system for analyzing the function of peptide toxins from animal venoms. We engineered and propagated strains of Drosophila melanogaster expressing heat-inducible transgenes encoding either kappa-ACTX-Hv1c or omega-ACTX-Hv1a, two insect-specific neurotoxic peptides found in the venom of the Australian funnel-web spider Hadronyche versuta. Heat induction of transgene expression for 20 min was sufficient to kill all transgenic flies, indicating that the ion channels targeted by these toxins are viable insecticide targets. The unusual phenotype of flies induced to express omega-ACTX-Hv1a recapitulates that of a hypomorphic allele of the high-voltage-activated calcium channel Dmca1D, suggesting that this is likely to be the target of omega-ACTX-Hv1a.     

Evidence for a high molecular weight pre-robustoxin molecule in the venom of the male Sydney funnel-web spider (Atrax robustus)

Robustoxin is the lethal polypeptide toxin in Atrax robustus venom. A monoclonal antibody was produced using synthetic, unfolded robustoxin conjugated to keyhole limpet haemocyanin as the immunogen. This monoclonal antibody did not protect newborn mice against challenge with the crude venom of the male Sydney funnel-web spider, but did slightly prolong their survival time. Western blotted crude venom of the male Sydney funnel-web spider showed two monoclonal antibody binding bands. One band at low M_r corresponded to robustoxin (M_r 4854), while the other higher M_r band (approximately 37,000) may be due to a pre-robustoxin molecule.     

Discovery and characterization of a family of insecticidal neurotoxins with a rare vicinal disulfide bridge

We have isolated a family of insect-selective neurotoxins from the venom of the Australian funnel-web spider that appear to be good candidates for biopesticide engineering. These peptides, which we have named the Janus-faced atracotoxins (J-ACTXs), each contain 36 or 37 residues, with four disulfide bridges, and they show no homology to any sequences in the protein/DNA databases. The three-dimensional structure of one of these toxins reveals an extremely rare vicinal disulfide bridge that we demonstrate to be critical for insecticidal activity. We propose that J-ACTX comprises an ancestral protein fold that we refer to as the disulfide-directed beta-hairpin.     

间斑寇蛛(L.tredecimguttatus)室内饲养及活体采毒方法的初步探讨

间斑寇蛛Latrotectus tredecimguttatus是目前已知毒性最强的蜘蛛之一,为了解决间斑寇蛛毒素研究中毒素来源有限的问题,本实验室开展了间斑寇蛛的室内人工饲养与采毒方法的研究,着重探索影响室内饲养间斑寇蛛生长发育、成活率及繁殖力的主要因素及活体采毒方法.结果表明,温度、湿度和食物种类等多种因素都影响湖南一带室内饲养间斑寇蛛的效果,但相对而言,空气的相对湿度是最重要的因素.与同批次的雌、雄蛛交配比较,利用前批次雌蛛与相隔30天左右的后批次雄蛛交配更有利于产卵率的提高.间斑寇蛛幼蛛度过适当长度的"卵袋期"对于以后的成活与生长发育,尤其是早期的成活与生长发育来说是必要的.尽管有多种方法可用来进行间斑寇蛛的活体采毒,但电刺激采毒法是最佳的活体采毒方法.     

Amino acid sequence of versutoxin, a lethal neurotoxin from the venom of the funnel-web spider Atrax versutus.

The complete amino acid sequence of versutoxin, a lethal neurotoxic polypeptide isolated from the venom of male and female funnel-web spiders of the species Atrax versutus, was determined. Sequencing was performed in a gas-phase protein sequencer by automated Edman degradation of the S-carboxymethylated toxin and fragments of it produced by reaction with CNBr. Versutoxin consisted of a single chain of 42 amino acid residues. It was found to have a high proportion of basic residues and of cystine. The primary structure showed marked homology with that of robustoxin, a novel neurotoxin recently isolated from the venom of another funnel-web-spider species, Atrax robustus.     

In vivo actions of atraxin, a protein neurotoxin from the venom glands of the funnel-web spider (Atrax robustus)

The effects of atraxin, a neurotoxic protein from the venom glands of the funnel-web spider (Atrax robustus), have been studied in anaesthetized monkeys. At doses of 70 and 80 micrograms kg-1 i.v., atraxin caused respiratory disturbances (dyspnoea and apnoea), and profound alterations in heart rate and blood pressure. These doses also caused salivation, lachrymation, skeletal muscle fasciculation and an elevation in body temperature. Concurrent increases in firing were recorded from the phrenic nerve and from respiratory and other skeletal muscles. It is concluded that atraxin produces the same syndrome in primates as that observed with whole milked male funnel-web venom.     

Genomic organization and cloning of novel genes encoding toxin-like peptides of three superfamilies from the spider Orinithoctonus huwena

The bird spider Ornithoctonus huwena is one of the most venomous spiders in China. Its venom is a mixture of various compounds with diverse bioactivities. Ninety proteins and 47 peptides have been identified, and 67 cDNA sequences encoding different toxin precursors have been cloned. However, the genomic DNA of them is seldom reported. To characterize the genomic DNA structure of huwentoxins, the genomic DNA encoding toxins of three superfamilies were cloned by using sequence specific or partially degenerate primers based on their cDNA sequences. An unexpected finding was that the intron was lacking in the genomic sequences of three superfamilies. The genomic DNA information has predictive value for better understanding the relationship of spider toxin evolution. In addition, we have cloned and analyzed 19 novel genes encoding toxin-like precursors by using the genomic DNA of the spider O. huwena.     

Solution structures of the cis- and trans-Pro30 isomers of a novel 38-residue toxin from the venom of Hadronyche Infensa sp. that contains a cystine-knot motif within its four disulfide bonds

The primary sequence and three-dimensional structure of a novel peptide toxin isolated from the Australian funnel-web spider Hadronyche infensa sp. is reported. ACTX-Hi:OB4219 contains 38 amino acids, including eight-cysteine residues that form four disulfide bonds. The connectivities of these disulfide bonds were previously unknown but have been unambiguously determined in this study. Three of these disulfide bonds are arranged in an inhibitor cystine-knot (ICK) motif, which is observed in a range of other disulfide-rich peptide toxins. The motif incorporates an embedded ring in the structure formed by two of the disulfides and their connecting backbone segments penetrated by a third disulfide bond. Using NMR spectroscopy, we determined that despite the isolation of a single native homologous product by RP-HPLC, ACTX-Hi:OB4219 possesses two equally populated conformers in solution. These two conformers were determined to arise from cis/trans isomerization of the bond preceding Pro30. Full assignment of the NMR spectra for both conformers allowed for the calculation of their structures, revealing the presence of a triple-stranded antiparallel beta sheet consistent with the inhibitor cystine-knot (ICK) motif.     

Synthesis and characterization of a cyclooctapeptide analogue of ω-agatoxin IVB enhancing the activity of CaV2.1 calcium channels activity in cultured hippocampal neurons

The structure of the toxin ω-agatoxin IVB, extracted from the venom of funnel-web spider Agelenopsis aperta, is an important lead structure when considering the design of modulators of synaptic transmission which largely involves P/Q-type (CaV2.1) voltage gated calcium channels (VGCC) at central synapses. Focusing on the loop 2 of the ω-agatoxin IVB that seems to be the most preeminent interacting domain of the toxin with the CaV2.1 VGCC, cyclooctapeptides mimicking this loop were synthesized. While (14)Trp is essential for the binding of the neurotoxin to the CaV2.1 VGCC, the substitution of the (12)Cys for a glycidyl residue led to a cyclooctapeptide named EP14 able to enhance CaV2.1 VGCC-associated currents measured with patch-clamp recordings and to evoke ω-agatoxin IVA-sensitive intracellular Ca(2+) increase as measured by fura-2 spectrofluoroimaging. Furthermore, this cyclooctapeptide was able to potentiate spontaneous excitatory synaptic transmission in a network of cultured hippocampal neurons, consistent with the activation of presynaptic VGCC by EP14. In addition, this peptide did not affect cell survival measured with the MTT assay. Therefore, such new cyclopeptidic structures are potential good candidates for synthesis of new agents aimed at the restoration deficient excitatory synaptic transmission.     

Spider toxins selectively block calcium currents in Drosophila

Toxins from spider venom, originally purified for their ability to block synaptic transmission in Drosophila, are potent and specific blockers of Ca2+ currents measured in cultured embryonic Drosophila neurons using the whole-cell, patch-clamp technique. Differential actions of toxins from two species of spiders indicate that different types of Drosophila neuronal Ca2+ currents can be pharmacologically distinguished. Hololena toxin preferentially blocks a non-inactivating component of the current, whereas Plectreurys toxin blocks both inactivating and non-inactivating components. These results suggest that block of a non-inactivating Ca2+ current is sufficient to block neurotransmitter release at Drosophila neuromuscular junction.     

Immunization with a synthetic robustoxin derivative lacking disulphide bridges protects against a potentially lethal challenge with funnel-web spider (<Emphasis Type="Italic">Atrax robustus</Emphasis>) venom

The venom of male Atrax robustus spiders is potentially lethal to primates. These spiders have been responsible for a number of human deaths. Robustoxin is the lethal toxin in the venom. It is a highly cross-linked polypeptide that has 42 amino acid residues and four disulphide bridges. If these bridges are broken, the resulting polypeptide is nontoxic. Robustoxin was chemically synthesized with all of its eight cysteine residues protected with acetamidomethyl groups in order to avoid formation of disulphide bridges. The resulting derivative was co-polymerized with keyhole limpet haemocyanin. Two Macaca fascicularis monkeys were immunized with this conjugate. The monkeys were challenged, under anaesthesia, with a potentially lethal dose of male A. robustus crude venom. Both monkeys showed some minor symptoms of intoxication but recovered fully with no adverse after-effects. Immunization with the same immunogen, in the absence of keyhole limpet haemocyanin, did not protect a third monkey. The N-terminal 23 amino acid peptide derived from the sequence of robustoxin was synthesized and conjugated with ovalbumin. A fourth monkey was immunized with this conjugate. However, it was not protected against challenge. The implications of these results for the preparation of synthetic peptide vaccines are discussed.     

An experimental study of the effects of spider venom on animals

The effects of venom of spiders from the families Pisauridae, Argyronetidae, and Araneidae on different animals (worms, mollusks, arthropods, fishes, and mammals) were studied. The animals of different classes varied in their sensitivity to spider venom. The animals that can be a potential prey were the most sensitive. The venom of spider females was more efficient than that of males. The spiders were found to be able to kill five victims in sequence; the most effective action of venom was on the first two ones. The venom regenerates in 1.5–2.0 hours.