Feghhi-Najafabadi Saba, Shafiee Fatemeh
Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences, Isfahan, Iran.
Iran J Biotechnol. 2022 Jan 1;20(1):e3021. doi: 10.30498/ijb.2021.278685.3021. eCollection 2022 Jan.
Interleukin-6 (IL-6) has undeniable roles in inflammatory processes due to autoimmune diseases. In this regard, soluble receptors are considered a potential approach to mitigate its inflammatory effects and modulate its physiological effects by reducing the IL-6 binding to cell surface-specific receptors.
This study aimed to produce IL-6 receptor (IL-6R) in soluble form with enhanced affinity to IL-6 without signal transduction ability.
The 3D structure of IL-6R with the selective mutations for enhancing the IL-6 binding, with minimum ability to signal transduction (mIL-6R), was predicted using Modeller 9.19. This mutated form was docked to IL-6 and gp130 (a part of the native IL-6 receptor involved in signal transduction) by the HADDOCK2.2 web server. The expression of mIL-6R was performed in BL21 (DE3), using pTWIN-1 plasmid as its linkage to the Ssp Intein. IMPACT system manual was used to purify the protein at 25 °C overnight. Next, ELISA was performed to compare the affinity of mutated and native IL-6R to IL-6. Finally, A549 cells were used to compare the inhibition of cytotoxic effects of native and mutated IL-6R.
In the , results established the stability of mutant's structure with more and less affinity to IL-6 and gp130, respectively. The expression and purification results showed bands of about 50 and 23 kDa, representing the correct size of the Intein1-mIL-6R fusion protein and cleavaged mIL-6R in SDS-PAGE, respectively. Furthermore, a significant enhancement in the affinity of mutated IL-6R to IL-6 was observed compared to the native receptor. Finally, A549 cells showed more cytotoxic effects followed by treating with mutated IL-6R in comparison to cells treated with native soluble IL-6R.
The recombinant production of a mutated form of IL-6R with the potential ability to antagonize the IL-6 inflammatory effects confirmed with in was successfully performed for the first time to create a new drug candidate for suppressing the inflammatory effects of IL-6.
白细胞介素-6（IL-6）在自身免疫性疾病引发的炎症过程中发挥着不可忽视的作用。在这方面，可溶性受体被视为一种潜在方法，可通过减少IL-6与细胞表面特异性受体的结合来减轻其炎症作用并调节其生理效应。
本研究旨在生产具有增强的与IL-6亲和力且无信号转导能力的可溶性形式的IL-6受体（IL-6R）。
使用Modeller 9.19预测具有增强IL-6结合能力且信号转导能力最小的选择性突变的IL-6R（mIL-6R）的三维结构。通过HADDOCK2.2网络服务器将这种突变形式与IL-6和gp130（天然IL-6受体中参与信号转导的一部分）对接。使用pTWIN-1质粒作为与Ssp内含肽的连接体，在BL21（DE3）中进行mIL-6R的表达。使用IMPACT系统手册在25°C下过夜纯化蛋白质。接下来，进行酶联免疫吸附测定（ELISA）以比较突变型和天然型IL-6R与IL-6的亲和力。最后，使用A549细胞比较天然型和突变型IL-6R对细胞毒性作用的抑制情况。
结果确定了突变体结构的稳定性，其对IL-6和gp130的亲和力分别较高和较低。表达和纯化结果显示，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）中，约50 kDa和23 kDa的条带分别代表内含肽1-mIL-6R融合蛋白和切割后的mIL-6R的正确大小。此外，与天然受体相比，观察到突变型IL-6R与IL-6的亲和力显著增强。最后，与用天然可溶性IL-6R处理的细胞相比，A549细胞在用突变型IL-6R处理后显示出更多的细胞毒性作用。
首次成功进行了具有拮抗IL-6炎症作用潜在能力的突变型IL-6R的重组生产，以创建一种用于抑制IL-6炎症作用的新候选药物。
