脆弱拟杆菌岩藻糖基化蛋白组学研究

Proteomic study of the fucosylated glycoproteins in Bacteroides fragilis

目的对脆弱拟杆菌岩藻糖基化蛋白(FGPs)进行标记、富集并进行蛋白质组研究,构建脆弱拟杆菌FGPs数据库,以探索FGPs在拟杆菌属细菌生长、生存及与宿主共生关系中的作用。方法通过单糖代谢标记、生物正交反应和链霉亲和素Pull down富集脆弱拟杆菌FGPs,并通过串联质谱和数据库搜索鉴定蛋白,利用接合转移和Western Blot验证候选FGPs岩藻糖基化。结果成功鉴定筛选得到135个脆弱拟杆菌FGPs,验证了候选蛋白Q5L9P7的岩藻糖基化。GO功能注释及富集分析显示FGPs大多数为膜蛋白。脆弱拟杆菌FGPs岩藻糖基化对不同的多糖营养条件的响应存在明显差异。结论岩藻糖基化可能是拟杆菌响应外源多糖营养的关键因素,为探索FGPs在拟杆菌生长、生存过程中的生物学作用提供理论依据。

Objective To construct the fucosylated glycoproteins(FGPs)database in Bacteroides fragilis(B.fragilis)by labeling,enriching and identifying the FGPs;and explore the roles of FGPs in the growth,survival and symbiosis with the host of Bacteroides based on the GO enrichment analysis.Methods The FGPs were labeled by metabolic labeling and bio-orthogonal reactions,and enriched by streptavidin pull down.Then tandem mass spectrometry and database searching were used to identify the enriched FGPs.The fucosylation of candidate FGPs were validated by E.coli-to-Bacteroides matings and Western blot.Results Totally 135 FGPs in B.fragilis were successfully identified and screened.The fucosylation of candidate FGPs Q5L9P7 was verified.The GO enrichment analysis demonstrated that the identified FGPs were mainly membrane proteins.The expression and fucosylation of FGPs in B.fragilis under different polysaccharide nutrition conditions were significantly different.Conclusion The fucosylation of Bacteroides might act as a key factor for Bacteroides to respond to exogenous polysaccharide nutrition,and this study provided a theoretical basis for exploring the biological role of FGPs fucosylation in the growth and survival of Bacteroides.