Discovery and Structure of a Potent and Highly Specific Blocker of Insect Calcium Channels

Wang, Xiuhong; Connor, Mark; Wilson, David T.; Wilson, Harry I.; Nicholson, Graham M.; Smith, Ross; Shaw, Douglas J.; Mackay, Joel P.; Alewood, Paul F.; Christie, MacDonald J.; King, Glenn F.

doi:10.1074/jbc.m105206200

Cited by 83 publications

(62 citation statements)

References 35 publications

(33 reference statements)

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“…8. Ensemble of 20 NMR-derived structures of x-ACTX-Hv2a, a specific blocker of insect voltage-gated calcium channels [64]. (A) In this view, the structures have been overlaid to minimize the backbone rmsd over all 45 residues; (B) An overlay over the backbone atoms of residues 3-32 only reveals that the furball in part (A) results from inclusion of the highly disordered C-terminal region in the rmsd calculation.…”

Section: How Does One Look At An Nmr Structure?mentioning

confidence: 99%

Macromolecular NMR spectroscopy for the non‐spectroscopist

et al. 2011

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NMR spectroscopy is a powerful tool for studying the structure, function and dynamics of biological macromolecules. However, non‐spectroscopists often find NMR theory daunting and data interpretation nontrivial. As the first of two back‐to‐back reviews on NMR spectroscopy aimed at non‐spectroscopists, the present review first provides an introduction to the basics of macromolecular NMR spectroscopy, including a discussion of typical sample requirements and what information can be obtained from simple NMR experiments. We then review the use of NMR spectroscopy for determining the 3D structures of macromolecules and examine how to judge the quality of NMR‐derived structures.

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Section: How Does One Look At An Nmr Structure?mentioning

confidence: 99%

Macromolecular NMR spectroscopy for the non‐spectroscopist

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“…124 At low toxin doses, the paralysis is reversible, with affected insects recovering in 4-5 h, but injection of a second bolus prior to recovery from the first dose is lethal. 139 Whether recovery from paralysis results from clearance of the toxin, turnover of affected channels, or some other mechanism remains to be determined.…”

Section: Peptide Toxins That Block Insect Ca V 2 Channelsmentioning

confidence: 99%

“…ω-ACTX-Hv2a is the prototypic member of a family of 42-45-residue insecticidal peptides isolated from the venom of Australian funnel-web spiders. 139 The toxin is also lethal to ticks, 129 but it causes no adverse effects when injected into newborn mice. 139 Injection of ω-ACTX-Hv2a into insects induces i nstantaneous paralysis, 139 which contrasts with the slow onset of paralysis following injection of ω-ACTX-Hv1a.…”

Section: Peptide Toxins That Block Insect Ca V 2 Channelsmentioning

confidence: 99%

“…139 The toxin is also lethal to ticks, 129 but it causes no adverse effects when injected into newborn mice. 139 Injection of ω-ACTX-Hv2a into insects induces i nstantaneous paralysis, 139 which contrasts with the slow onset of paralysis following injection of ω-ACTX-Hv1a. 124 At low toxin doses, the paralysis is reversible, with affected insects recovering in 4-5 h, but injection of a second bolus prior to recovery from the first dose is lethal.…”

Section: Peptide Toxins That Block Insect Ca V 2 Channelsmentioning

confidence: 99%

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Peptide toxins that selectively target insect Na_Vand Ca_Vchannels

King¹,

Escoubas²,

Nicholson³

2008

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Numerous metazoans express venoms for the purpose of defense, competitor deterrence or prey capture. Peptide neurotoxins are particularly well represented in the venoms of arachnids, cnidarians and mollusks and these toxins often possess high affinity and specificity for particular classes of ion channels. Some of these toxins have become the defining pharmacology for certain vertebrate ion channel subtypes. Unfortunately, due to differences in the structure, pharmacology and ion selectivity of insect voltage-gated sodium (Na V ) and calcium (Ca V ) channels compared with their vertebrate counterparts, these peptide toxins have proven less useful for the characterization of insect ion channels. Despite these disparities in channel structure and function, the armament of peptide toxins that specifically modulate the activity of insect ion channels is slowly expanding.

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“…Pireneitega luctuosa 4.047 Na v LD 50 2350 pmol/g (S. litura) (Corzo et al, 2000) δ-AMATX-PI1b Pireneitega luctuosa 4.124 Na v LD 50 5870 pmol/g (S. litura) (Corzo et al, 2000;Corzo et al, 2005) δ-AMATX-PI1c Pireneitega luctuosa 3.934 Na v LD 50 3130 pmol/g (S. litura) (Corzo et al, 2000) δ-AMATX-PI1d Pireneitega luctuosa 4.062 Na v LD 50 > 11,030 pmol/g (S. litura) (Corzo et al, 2000) ω-hexatoxin-Hv2a Hadronyche versuta 4.484 HVA Ca v PD 50 160 ± 9 pmol/g (A. domestica) (Wang et al, 2001) EC 50~1 39 pmol (bee brain neurons) (Wang et al, 2001) µ-hexatoxin-Mg1a Macrothele gigas 14.188 Na v LD 50 17,600 pmol/g (Spodoptera litura) (Corzo et al, 2003) µ-hexatoxin-Mg2a Macrothele gigas…”

Section: δ-Amatx-pi1amentioning

confidence: 99%

Engineering insect-resistant plants by transgenic expression of an insecticidal spider-venom peptide

Alam¹

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The insecticidal spider-venom peptide ω-hexatoxin-Hv1a (Hv1a) from the Australian Blue Mountains funnel-web spider Hadronyche versuta is one of the most potent insect-specific neurotoxins isolated to date. Hv1a blocks voltage-gated calcium channels in the insect central nervous system, a mechanism quite distinct from existing chemical insecticides. It induces a slow-onset paralysis that precedes death in a taxonomically wide range of insects. Hv1a's broad spectrum of target insects, novel mode of action, and absence of toxicity to vertebrates makes the Hv1a gene an attractive tool for generating insectresistant transgenic crops.The oral activity of Hv1a can be enhanced by coupling it to the plant lectin Galanthus nivalis agglutinin (GNA) or with the minor capsid protein of pea enation mosaic virus (CP).Recombinant fusions of Hv1a with GNA were produced using the Pichia pastoris expression system to study the intrinsic insecticidal activity of Hv1a-GNA and GNA-Hv1a fusion proteins. By using injection bioassays with houseflies, we found that the intrinsic insecticidal activity of Hv1a was maintained when it was fused to GNA. Moreover, feeding bioassays with diamondback moth larvae revealed that fusion of Hv1a to GNA, in either orientation, enhances its oral insecticidal activity.In order to generate transgenic plants expressing Hv1a alone or fused to GNA or CP, transformation vectors were constructed by ligating synthesised genes in the pAOV binary vector with the constitutive Cauliflower Mosaic Virus 35S promoter (35S) or the phloem tissue-specific Arabidopsis thaliana SUCROSE TRANSPORTER 2 (SUC2) promoter.Homozygous transgenic Arabidopsis were generated using the floral dip method of Agrobacterium-mediated plant transformation and subsequent herbicide selection. PCR of genomic DNA and western blotting were used to confirm integration of the transgenes and protein expression in the transgenic Arabidopsis respectively. Initial experiments revealed a very high level of mortality of Helicoverpa armigera larvae on wild-type plants due to the presence of endogenous glucosinolates, which masked the insecticidal effects of the transgenes Thus, a new set of transgenic plants was generated using an Arabidopsis cyp79B2 cyp79B myb28 myb29 quadruple mutant that lacks endogenous glucosinolates.Bioassays revealed that H. armigera larvae had a lower level of survival and retarded growth when fed on leaves of transgenic Arabidopsis expressing Hv1a toxins under 35S promoter control compared with those fed on gluc-null control plants. Moreover, larval mortality was higher for plants expressing Hv1a/GNA fusions than those expressing Hv1a or GNA alone. The highest larval mortality, lowest larval weight gain, and lowest level of leaf damage were observed for larvae fed on plants expressing GNA-Hv1a. Mortality was extremely high (~90%) for larvae fed on GNA-Hv1a plants for 15 days. The resistance to cotton bollworms conferred by expression of GNA-Hv1a in transgenic Arabidopsis highlights the potential of Hv1a transgenes as an ...

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Discovery and Structure of a Potent and Highly Specific Blocker of Insect Calcium Channels

Cited by 83 publications

References 35 publications

Macromolecular NMR spectroscopy for the non‐spectroscopist

Macromolecular NMR spectroscopy for the non‐spectroscopist

Peptide toxins that selectively target insect Na_Vand Ca_Vchannels

Engineering insect-resistant plants by transgenic expression of an insecticidal spider-venom peptide

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Discovery and Structure of a Potent and Highly Specific Blocker of Insect Calcium Channels

Cited by 83 publications

References 35 publications

Macromolecular NMR spectroscopy for the non‐spectroscopist

Macromolecular NMR spectroscopy for the non‐spectroscopist

Peptide toxins that selectively target insect NaVand CaVchannels

Engineering insect-resistant plants by transgenic expression of an insecticidal spider-venom peptide

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Product

Resources

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Peptide toxins that selectively target insect Na_Vand Ca_Vchannels