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外文期刊>The biochemical journal
>Molecular analysis of the sea anemone toxin Av3 reveals selectivity to insects and demonstrates the heterogeneity of receptor site-3 on voltage-gated Na+ channels
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Molecular analysis of the sea anemone toxin Av3 reveals selectivity to insects and demonstrates the heterogeneity of receptor site-3 on voltage-gated Na+ channels
pAv3 is a short peptide toxin from the sea anemone iAnemonia viridis/i shown to be active on crustaceans and inactive on mammals. It inhibits inactivation of Nasubv/subs (voltage-gated Nasup+/sup channels) like the structurally dissimilar scorpion α-toxins and type I sea anemone toxins that bind to receptor site-3. To examine the potency and mode of interaction of Av3 with insect Nasubv/subs, we established a system for its expression, mutagenized it throughout, and analysed it in toxicity, binding and electrophysiological assays. The recombinant Av3 was found to be highly toxic to blowfly larvae (EDsub50/sub=2.65±0.46 pmol/100 mg), to compete well with the site-3 toxin LqhαIT (from the scorpion iLeiurus quinquestriatus/i) on binding to cockroach neuronal membranes (iK/isubi/sub=21.4±7.1 nM), and to inhibit the inactivation of iDrosophila melanogaster/i channel, DmNasubv/sub1, but not that of mammalian Nasubv/subs expressed in iXenopus/i oocytes. Moreover, like other site-3 toxins, the activity of Av3 was synergically enhanced by ligands of receptor site-4 (e.g. scorpion β-toxins). The bioactive surface of Av3 was found to consist mainly of aromatic residues and did not resemble any of the bioactive surfaces of other site-3 toxins. These analyses have portrayed a toxin that might interact with receptor site-3 in a different fashion compared with other ligands of this site. This assumption was corroborated by a D1701R mutation in DmNasubv/sub1, which has been shown to abolish the activity of all other site-3 ligands, except Av3. All in all, the present study provides further evidence for the heterogeneity of receptor site-3, and raises Av3 as a unique model for design of selective anti-insect compounds./p
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机译:> Av3是一种来自海葵 Anemonia viridis i>的短肽毒素,显示对甲壳动物有活性,对哺乳动物则无活性。它可抑制Na v sub> s(电压门控Na + sup>通道)的失活,如结构不同的蝎子α毒素和结合受体部位的I型海葵毒素。 3。为了检查Av3与昆虫Na v sub> s相互作用的效力和模式,我们建立了它的表达系统,对其进行了诱变,并在毒性,结合和电生理分析中进行了分析。发现重组蛋白Av3对蝇blow幼虫(ED 50 sub> = 2.65±0.46npmol / 100nmg)具有高毒性,可与三点毒素LqhαIT竞争(蝎子< (i> Leiurus quinquestriatus i>)对蟑螂神经元膜( K i> i sub> = 21.4±7.1nM)的结合,并抑制果蝇的果蝇通道DmNa v sub> 1,而不是非洲爪蟾卵母细胞中表达的哺乳动物Na v sub>通道。此外,与其他site-3毒素一样,Av3的活性通过受体site-4的配体(例如蝎子β-毒素)协同增强。发现Av3的生物活性表面主要由芳族残基组成,与其他site-3毒素的任何生物活性表面均不相似。这些分析描绘了一种毒素,与该位点的其他配体相比,它可能以不同的方式与受体位点3相互作用。 DmNa v sub> 1中的D1701R突变证实了这一假设,该突变已证明可以消除除Av3外的所有其他site-3配体的活性。总而言之,本研究为受体部位3的异质性提供了进一步的证据,并提出了Av3作为设计选择性抗昆虫化合物的独特模型。 p>
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