Division of Structural Biology & Bio-informatics, CSIR-Indian Institute of Chemical Biology, Kolkata, India.
Division of Structural Biology & Bio-informatics, CSIR-Indian Institute of Chemical Biology, Kolkata, India.
J Biol Chem. 2021 Oct;297(4):101198. doi: 10.1016/j.jbc.2021.101198. Epub 2021 Sep 15.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fulfills various physiological roles that are unrelated to its glycolytic function. However, to date, the nonglycolytic function of GAPDH in trypanosomal parasites is absent from the literature. Exosomes secreted from Leishmania, like entire parasites, were found to have a significant impact on macrophage cell signaling and function, indicating cross talk with the host immune system. In this study, we demonstrate that the Leishmania GAPDH (LmGAPDH) protein is highly enriched within the extracellular vesicles (EVs) secreted during infection. To understand the function of LmGAPDH in EVs, we generated control, overexpressed, half-knockout (HKO), and complement cell lines. HKO cells displayed lower virulence compared with control cells when macrophages and BALB/c mice were infected with them, implying a crucial role for LmGAPDH in Leishmania infection and disease progression. Furthermore, upon infection of macrophages with HKO mutant Leishmania and its EVs, despite no differences in TNFA mRNA expression, there was a considerable increase in TNF-α protein expression compared with control, overexpressed, and complement parasites as determined by ELISA, RT-PCR, and immunoblot data. In vitro protein translation studies suggest that LmGAPDH-mediated TNF-α suppression occurs in a concentration-dependent manner. Moreover, mRNA binding assays also verified that LmGAPDH binds to the AU-rich 3'-UTR region of TNFA mRNA, limiting its production. Together, these findings confirmed that the LmGAPDH contained in EVs inhibits TNF-α expression in macrophages during infection via posttranscriptional repression.
甘油醛-3-磷酸脱氢酶（GAPDH）具有多种与糖酵解功能无关的生理作用。然而，迄今为止，文献中缺乏 GAPDH 在原生动物寄生虫中的非糖酵解功能。从莱什曼原虫分泌的外泌体与完整寄生虫一样，被发现对巨噬细胞信号和功能有重大影响，表明与宿主免疫系统存在串扰。在这项研究中，我们证明了莱什曼 GAPDH（LmGAPDH）蛋白在感染期间分泌的细胞外囊泡（EVs）中高度富集。为了了解 LmGAPDH 在 EVs 中的功能，我们生成了对照、过表达、半敲除（HKO）和互补细胞系。与对照细胞相比，感染 HKO 细胞的巨噬细胞和 BALB/c 小鼠的毒力较低，这表明 LmGAPDH 在莱什曼原虫感染和疾病进展中起着至关重要的作用。此外，在用 HKO 突变的莱什曼原虫及其 EVs 感染巨噬细胞后，尽管 TNFA mRNA 表达没有差异，但与对照、过表达和互补寄生虫相比，TNF-α 蛋白表达显著增加，这通过 ELISA、RT-PCR 和免疫印迹数据确定。体外蛋白质翻译研究表明，LmGAPDH 介导的 TNF-α 抑制呈浓度依赖性。此外，mRNA 结合测定还验证了 LmGAPDH 结合 TNFA mRNA 的富含 AU 的 3'-UTR 区域，限制了其产生。总之，这些发现证实了 EVs 中包含的 LmGAPDH 通过转录后抑制在感染期间抑制巨噬细胞中 TNF-α 的表达。
