TBK1的O-GlcNAc修饰促进LPS诱导的BV-2小胶质细胞炎性介质的释放

O-Glc NAc modification of TBK1 increases LPS-induced inflammatory mediator release from BV-2 microglia

目的:研究TANK结合激酶1(TANK-binding kinase 1,TBK1)O-连接的N乙酰葡萄糖胺(O-Glc NAc)修饰对脂多糖(lipopolysaccharide,LPS)诱导的小胶质细胞炎性介质释放的影响。方法:选取小鼠小胶质细胞株BV-2,利用免疫沉淀方法检测TBK1的O-Glc NAc修饰状况;构建野生型TBK1和丝氨酸266号糖基化位点突变的TBK1即S266A质粒。用酶联免疫吸附分析法(enzyme-linked immunosorbent assay,ELISA)检测BV-2细胞中炎症因子的分泌。结果:LPS诱导BV-2中TBK1的蛋白表达。TBK1存在O-Glc NAc修饰。过表达野生型TBK1能增加LPS诱导的小胶质细胞中炎性介质肿瘤坏死因子α(tumor necrosis factorα,TNF-α)和干扰素γ(interferonγ,IFN-γ)的释放;N-乙酰氨基葡萄糖苷酶抑制剂PUGNAc能增强TBK1的该效应。而过表达S266A能抑制LPS诱导的小胶质细胞炎性介质的释放。结论:TBK1通过丝氨酸266号位的O-Glc NAc修饰增强LPS诱导的小胶质细胞炎性介质的释放。

Objective： To study the effect of O-GlcNAc modification of TANK-binding kinase I（TBK1） on the lipopolysacc- haride（LPS）-induced release of inflammatory mediators from microglia. Methods： The O-GlcNAcylation of TBK1 in BV-2 mouse microglia ceils was detected by immunoprecipitation. The wild-type（WT） and S266A TBK1 mutant were constructed. enzyme-linked immunosorbent assay（ELISA） was used to detect the secretion of inflammatory mediators in BV-2 cells. Results： LPS induced the protein expression of TBK1 in BV-2. O-GlcNAc modification occurred in TBK1. Overexpression of WT TBK1 increased the release of inflammatory mediators including tumor necrosis factor ot（TNF-ct） and interferon γ（IFN-γ） in LPS-stimulated microglia. This effect of TBK1 was enhanced by PUGNAc, an O-GlcNAease inhibitor. Overexpression of TBK1 mutant $266A inhibited release of inflammatory mediators. Conclusion： The O-GleNAeylation of TBK1 on S266A enhances the release of inflammatory mediators from microglia stimulated by LPS.