Department for Environment, Food and Rural Affairs, Food and Environmental Research Agency, Sand Hutton, United Kingdom.
PLoS One. 2012;7(6):e39389. doi: 10.1371/journal.pone.0039389. Epub 2012 Jun 22.
The spider-venom peptide ω-hexatoxin-Hv1a (Hv1a) targets insect voltage-gated calcium channels, acting directly at sites within the central nervous system. It is potently insecticidal when injected into a wide variety of insect pests, but it has limited oral toxicity. We examined the ability of snowdrop lectin (GNA), which is capable of traversing the insect gut epithelium, to act as a "carrier" in order to enhance the oral activity of Hv1a.
METHODOLOGY/PRINCIPAL FINDINGS: A synthetic Hv1a/GNA fusion protein was produced by recombinant expression in the yeast Pichia pastoris. When injected into Mamestra brassicae larvae, the insecticidal activity of the Hv1a/GNA fusion protein was similar to that of recombinant Hv1a. However, when proteins were delivered orally via droplet feeding assays, Hv1a/GNA, but not Hv1a alone, caused a significant reduction in growth and survival of fifth stadium Mamestra brassicae (cabbage moth) larvae. Feeding second stadium larvae on leaf discs coated with Hv1a/GNA (0.1-0.2% w/v) caused ≥ 80% larval mortality within 10 days, whereas leaf discs coated with GNA (0.2% w/v) showed no acute effects. Intact Hv1a/GNA fusion protein was delivered to insect haemolymph following ingestion, as shown by Western blotting. Immunoblotting of nerve chords dissected from larvae following injection of GNA or Hv1a/GNA showed high levels of bound proteins. When insects were injected with, or fed on, fluorescently labelled GNA or HV1a/GNA, fluorescence was detected specifically associated with the central nerve chord.
CONCLUSIONS/SIGNIFICANCE: In addition to mediating transport of Hv1a across the gut epithelium in lepidopteran larvae, GNA is also capable of delivering Hv1a to sites of action within the insect central nervous system. We propose that fusion to GNA provides a general mechanism for dramatically enhancing the oral activity of insecticidal peptides and proteins.
蜘蛛毒液肽 ω-六毒素-Hv1a（Hv1a）靶向昆虫电压门控钙通道，直接作用于中枢神经系统内的部位。当将其注入到各种害虫中时，它具有很强的杀虫作用，但口服毒性有限。我们研究了能够穿透昆虫肠上皮的雪花莲凝集素（GNA）作为“载体”的能力，以增强 Hv1a 的口服活性。
方法/主要发现：通过在毕赤酵母 Pichia pastoris 中的重组表达生产了合成的 Hv1a/GNA 融合蛋白。当将其注入到小菜蛾幼虫中时，Hv1a/GNA 融合蛋白的杀虫活性与重组 Hv1a 相似。但是，当通过液滴喂养试验经口给予蛋白质时，Hv1a/GNA 而不是单独的 Hv1a 导致第五龄期小菜蛾（菜蛾）幼虫的生长和存活率显着降低。用 Hv1a/GNA（0.1-0.2％w/v）涂覆的叶片圆盘喂养第二龄幼虫在 10 天内引起≥80％的幼虫死亡，而涂覆 GNA（0.2％w/v）的叶片圆盘没有引起急性作用。摄取后，通过 Western blotting 显示完整的 Hv1a/GNA 融合蛋白被递送到昆虫血淋巴中。用 GNA 或 Hv1a/GNA 注射后从幼虫解剖的神经索的免疫印迹显示结合的蛋白质水平很高。当昆虫注射或喂食荧光标记的 GNA 或 Hv1a/GNA 时，荧光特异性地与中央神经索相关联。
结论/意义：除了介导 Hv1a 在鳞翅目幼虫的肠上皮中的转运外，GNA 还能够将 Hv1a 递送到昆虫中枢神经系统中的作用部位。我们提出，融合到 GNA 为显著增强杀虫肽和蛋白质的口服活性提供了一种通用机制。
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