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
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
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DOI：
10.1021/acs.bioconjchem.8b00505
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发表时间：
2018-10-01
影响因子：
4.7
通讯作者：
Schroeder, Christina, I
中科院分区：
文献类型：
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作者：
Agwa, Akello J.;Blomster, Linda, V;Schroeder, Christina, I
蜘蛛毒液中的门控修饰毒素是富含二硫键的肽，通常包含稳定抑制剂胱氨酸结 (ICK)。这些结蛋白肽正在被寻求作为一系列与跨膜蛋白相关疾病的治疗先导药物。最近，在蜘蛛毒液中发现并通过重组表达产生的双结肽为跨膜通道的药理学提供了见解。在这里，我们使用化学酶连接产生双结来探测二价调节对电压门控钠通道亚型 1.7 (Na(v)1.7) 的影响，该亚型与疼痛信号传导有关。单价结蛋白被氧化折叠，然后使用分选酶 A 进行生化缀合，形成双结蛋白。使用NMR证实了肽的结构完整性，并且基于荧光的活性测定提供了证据表明共孵育的单价和二价结蛋白可以协同调节Na(v)1.7。我们预计双结将为增强我们对治疗相关的电压门控离子通道的理解和设计策略提供新的工具。
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(v)1.7), 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(v)1.7. We anticipate that double-knottins will provide novel tools for enhancing our understanding of, and design strategies for, therapeutically relevant voltage-gated ion channels.