The Cystine Knot Is Responsible for the Exceptional Stability of the Insecticidal Spider Toxin ω-Hexatoxin-Hv1a.
The Cystine Knot Is Responsible for the Exceptional Stability of the Insecticidal Spider Toxin ω-Hexatoxin-Hv1a.
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DOI：
10.3390/toxins7104366
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发表时间：
2015-10-26
期刊：
影响因子：
4.2
通讯作者：
King GF
中科院分区：
文献类型：
--
作者：
Herzig V;King GF
抑制剂胱氨酸结（ICK）是一种不寻常的三硫键结构，其中一个二硫键将由另外两个二硫键桥以及蛋白质主链的中间部分形成的环一分为二。含有ICK基序的肽由于二硫键提供的交叉支撑，通常被认为具有高水平的热稳定性、化学稳定性和酶稳定性。支持这一论点的实验研究很少，特别是对于蜘蛛毒液毒素而言，它们代表了ICK肽的最大多样性。我们以ω - 六毒素 - Hv1a（Hv1a），一种来自致命的澳大利亚漏斗网蜘蛛的杀虫毒素，作为一个模型系统来研究胱氨酸结对ICK肽稳定性的贡献。我们表明，Hv1a在高达75°C的温度、低至1的pH值以及各种有机溶剂中都具有高度稳定性。此外，Hv1a对蛋白酶K的消化以及在昆虫血淋巴和人血浆中孵育时都具有高度抗性。我们证明ICK基序对Hv1a的显著稳定性至关重要，当二硫键被消除时，肽的稳定性会大幅降低。因此，这项研究表明ICK基序显著提高了蜘蛛毒液肽的化学和热稳定性，并使它们具有高水平的蛋白酶抗性。这项研究还为Hv1a作为生物杀虫剂的储存和使用条件提供了指导。
The inhibitor cystine knot (ICK) is an unusual three-disulfide architecture in which one of the disulfide bonds bisects a loop formed by the two other disulfide bridges and the intervening sections of the protein backbone. Peptides containing an ICK motif are frequently considered to have high levels of thermal, chemical and enzymatic stability due to cross-bracing provided by the disulfide bonds. Experimental studies supporting this contention are rare, in particular for spider-venom toxins, which represent the largest diversity of ICK peptides. We used ω-hexatoxin-Hv1a (Hv1a), an insecticidal toxin from the deadly Australian funnel-web spider, as a model system to examine the contribution of the cystine knot to the stability of ICK peptides. We show that Hv1a is highly stable when subjected to temperatures up to 75 °C, pH values as low as 1, and various organic solvents. Moreover, Hv1a was highly resistant to digestion by proteinase K and when incubated in insect hemolymph and human plasma. We demonstrate that the ICK motif is essential for the remarkable stability of Hv1a, with the peptide’s stability being dramatically reduced when the disulfide bonds are eliminated. Thus, this study demonstrates that the ICK motif significantly enhances the chemical and thermal stability of spider-venom peptides and provides them with a high level of protease resistance. This study also provides guidance to the conditions under which Hv1a could be stored and deployed as a bioinsecticide.