旋毛虫与小鼠 TLR2 结合蛋白 GGH 的鉴定及其免疫调节作用

Identification and immunomodulatory effects of mouse TLR2 binding protein GGH from Trichinella spiralis

摘要

[目的] 本文旨在探究旋毛虫肌肉幼虫排泄分泌蛋白(Trichinella spiralis muscle larvae excretory-secretory proteins, TsMESP)中叶酸 γ-谷氨酰水解酶(folate gamma-glutamyl hydrolase, GGH)结合小鼠 Toll 样受体 2(Toll-like receptor 2, TLR2)能力，以及结合后对小鼠巨噬细胞功能和下游通路的影响。[方法]通过 Pull-down 试验和质谱分析，筛选出与小鼠 TLR2 结合的 TsMESP，对其中 GGH 进行鉴定。构建表达 GGH 和 TLR2 胞外域的真核质粒，共转染 HEK293T 细胞，利用免疫共沉淀(Co-IP)和荧光共定位试验分析二者的结合情况。将重组旋毛虫 GGH 蛋白(rTsGGH)与小鼠巨噬细胞共孵育，研究其对细胞吞噬、活性氧(ROS)释放、一氧化氮(NO)分泌、细胞因子(IL-1β、IL-6 和 IL-10)和 TLR2、Myd88 蛋白表达的影响。[结果]筛选到 28 个与小鼠 TLR2 结合的旋毛虫候选蛋白。Co-IP 结果显示 GGH 和 TLR2 胞外域存在较强的共定位关系，共定位系数为 0.88。rTsGGH 能够显著促进巨噬细胞的吞噬、ROS 的释放和 NO 的分泌(P<0.05)，显著上调 TLR2、Myd88、IL-1β、IL-6 和 IL-10 的表达(P<0.05)。[结论]旋毛虫 GGH 是小鼠 TLR2 的结合蛋白，rTsGGH 通过 TLR2/Myd88 通路显著影响宿主免疫功能，是有效的免疫调节分子。

Abstract

[Objectives]: This study aimed to investigate whether the folate gamma-glutamyl hydrolase (GGH) inTrichinella spiralis muscle larvae excretory-secretory proteins (TsMESP) could bind to mouse Toll-like receptor 2 (TLR2) and its effects on macrophage function and downstream pathway. [Methods] Pull-down experiments combined with mass spectrometry analysis were used to screen TsMESP that bind to mouse TLR2, and the GGH among them was identified. Eukaryotic plasmids expressing the GGH and extracellular domain of TLR2 were constructed and co-transfected into HEK293T cells, and their binding was analyzed by immunoprecipitation. Co-IP and fluorescence co-localization assays. Recombinant Trichinella spiralis GGH protein (rTsGGH) was co-incubated with mouse macrophage to study its effects on cellular phagocytosis, reactive oxygen species (ROS) release, nitric oxide (NO) secretion, cytokine (IL-1β, IL-6, and IL-10), and TLR2, Myd88 protein expression. [Results] Twenty-eight candidate proteins that might bind to mouse TLR2 were screened fromTrichinella spiralis. Co-IP results showed that GGH and TLR2 extracellular domains had binding effect. Co-localization results showed that they had a strong co-localization relationship, and the co-localization coefficient was 0.88. rTsGGH could significantly promote macrophage phagocytosis, ROS release and NO secretion (P<0.05), significantly upregulate the expression of TLR2, Myd88, IL-1β, IL-6 and IL-10 (P<0.05). [Conclusions]Trichinella spiralis GGH is a binding protein of mouse TLR2. The rTsGGH upregulates host immune function through the TLR2/Myd88 pathway and is an effective immune regulatory molecule.

关键词

旋毛虫 / Toll 样受体 2 / 叶酸 γ-谷氨酰水解酶(GGH) / 结合蛋白 / 免疫共沉淀(Co-IP)

Key words

Trichinella spiralis / Toll-like receptor 2 (TLR2) / folate gamma-glutamyl hydrolase / binding protein / immunoprecipitation (Co-IP)

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基金资助

江苏省社会发展计划项目(BE2022848)