Efficient Enzymatic Ligation of Inhibitor Cystine Knot Spider Venom Peptides: Using Sortase A To Form Double-Knottins That Probe Voltage-Gated Sodium Channel NaV1.7,Bioconjugate Chemistry

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Efficient Enzymatic Ligation of Inhibitor Cystine Knot Spider Venom Peptides: Using Sortase A To Form Double-Knottins That Probe Voltage-Gated Sodium Channel NaV1.7
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2018-08-27 00:00:00 , DOI: 10.1021/acs.bioconjchem.8b00505
Akello J. Agwa ₁ , Linda V. Blomster ₁ , David J. Craik ₁ , Glenn F. King ₁ , Christina I. Schroeder ₁

Affiliation

Gating modifier toxins from spider venom are disulfide-rich peptides that typically comprise a stabilizing inhibitor cystine knot (ICK). These knottin peptides are being pursued as therapeutic leads for a range of conditions linked to transmembrane proteins. Recently, double-knottin peptides discovered in spider venom and produced by recombinant expression have provided insights into the pharmacology of transmembrane channels. Here, we use chemoenzymatic ligation to produce double-knottins to probe the effect of bivalent modulation on the voltage-gated sodium channel subtype 1.7 (Na_V1.7), which is implicated in pain signaling. Monovalent knottins were oxidatively folded and then biochemically conjugated using sortase A, to form double-knottins. The structural integrity of the peptides was confirmed using NMR, and fluorescence-based activity assays provided evidence suggesting that coincubated monovalent and bivalent knottins can cooperatively modulate Na_V1.7. We anticipate that double-knottins will provide novel tools for enhancing our understanding of, and design strategies for, therapeutically relevant voltage-gated ion channels.

中文翻译：

抑制剂胱氨酸结蜘蛛毒液肽的高效酶促连接：使用分选酶A形成双Knottins，探测电压门控钠通道Na _V 1.7

来自蜘蛛毒的门控修饰剂毒素是富含二硫键的肽，通常包含稳定抑制剂胱氨酸结（ICK）。这些结蛋白肽正在寻求作为与跨膜蛋白有关的一系列疾病的治疗先导。最近，在蜘蛛毒液中发现并通过重组表达产生的双-knottin肽为跨膜通道的药理学提供了见识。在这里，我们使用化学酶促连接产生双结蛋白，以探究二价调制对电压门控钠通道亚型1.7（Na _V1.7），这与疼痛信号有关。将一价结蛋白氧化折叠，然后使用分选酶A生化偶联，形成双结蛋白。使用NMR证实了肽的结构完整性，基于荧光的活性测定提供了证据，表明共同孵育的单价和二价结蛋白可以协同调节Na _V 1.7。我们预计，双结蛋白将提供新颖的工具，以增强我们对与治疗相关的电压门控离子通道的理解和设计策略。

更新日期：2018-08-27

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