Molecular Toxinology Laboratory, Department of Molecular Biology and Biotechnology, Tezpur University, Assam 784028, India.
Cellular, Molecular and Environmental Biotechnology Laboratory, Department of Molecular Biology and Biotechnology, Tezpur University, India.
Int J Biol Macromol. 2020 Oct 1;160:602-611. doi: 10.1016/j.ijbiomac.2020.05.169. Epub 2020 May 26.
Cysteine-Rich Secretory Proteins (CRISP) are widespread in snake venoms and known to target ion channels. More recently, CRISPs have been shown to mediate inflammatory responses. Involvement of potential receptor in CRISP-induced inflammatory reactions, however, remains unknown. A CRISP protein named as Nk-CRISP, was isolated from the venom of Naja kaouthia. The molecular mass of the purified protein was found to be ~25 kDa and the primary sequence was determined by MALDI TOF-TOF. The involvement of this protein in proinflammatory effects were evaluated in THP-1 macrophage-like cells. Nk-CRISP treated cells induced up-regulation of several inflammatory marker genes in dose dependent manner. Toll like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex are known to play crucial role in recognition of damage/pathogen-associated molecular patterns and activation of innate immune response. Therefore, we hypothesized that snake venom CRISP could also modulate the innate immune response via TLR4-MD2 complex. In-silico molecular docking study of cobra CRISP with TLR4-MD2 receptor complex reveals CRISP engages its cysteine-rich domain (CRD) to interact with complex. Inhibition of TLR4 signalling pathway using CLI-095 confirmed the role of TLR4 in Nk-CRISP induced inflammatory responses. Collectively, these findings imply that TLR4 initiates proinflammatory signalling following recognition of cobra CRISP and alteration of TLR4 receptor might improve or control CRISP induced inflammation.
半胱氨酸丰富分泌蛋白（CRISP）广泛存在于蛇毒中，已知其靶向离子通道。最近，CRISPs 已被证明可介导炎症反应。然而，潜在受体在 CRISP 诱导的炎症反应中的作用尚不清楚。一种名为 Nk-CRISP 的 CRISP 蛋白从 Naja kaouthia 的毒液中分离得到。纯化蛋白的分子量约为 25 kDa，通过 MALDI TOF-TOF 确定了其一级序列。该蛋白在 THP-1 巨噬样细胞中的促炎作用评估。Nk-CRISP 处理的细胞以剂量依赖的方式诱导几种炎症标志物基因的上调。Toll 样受体 4（TLR4）-髓样分化因子 2（MD2）复合物在识别损伤/病原体相关分子模式和激活先天免疫反应方面起着至关重要的作用。因此，我们假设蛇毒 CRISP 也可以通过 TLR4-MD2 复合物来调节先天免疫反应。眼镜蛇 CRISP 与 TLR4-MD2 受体复合物的计算机分子对接研究表明，CRISP 利用其富含半胱氨酸的结构域（CRD）与复合物相互作用。使用 CLI-095 抑制 TLR4 信号通路证实了 TLR4 在 Nk-CRISP 诱导的炎症反应中的作用。总之，这些发现表明，TLR4 在识别眼镜蛇 CRISP 后启动促炎信号，改变 TLR4 受体可能改善或控制 CRISP 诱导的炎症。
