幽门螺杆菌分子伴侣GroEL相互作用蛋白分析

Interacting proteins of Helicobacter pylori GroEL protein

摘要:

【目的】获得幽门螺杆菌(Helicobacter pylori, HP) GroEL结合蛋白质组构成谱，为进一步探究GroEL及其与相互作用蛋白在HP致病机制中的作用提供新思路。【方法】在构建HP GroEL原核表达重组大肠杆菌(Escherichia coli) BL21(DE3)^{pET-28a(+)-groEL}基础上，纯化带有His标签的GroEL蛋白，与HP全菌蛋白提取液共孵育后，利用Protein G磁珠和抗His标签抗体免疫沉淀法对复合物进行捕获，然后对复合物中GroEL及其结合的蛋白质进行质谱法鉴定，根据主要功能对其进行分类，并完成蛋白质相互关系网络分析。【结果】对GroEL蛋白捕获成分进行分析，共鉴定出59种可能与GroEL结合的蛋白质，其中包括19种代谢酶类(KatA、GltA和AhpC等参与氧化还原相关酶类7种，PepA、RocF和HtrA等肽酶5种，以及2种参与脂肪代谢酶、2种参与ATP合成酶、2种尿素酶和HP17_08079蛋白等)、15种外膜蛋白(黏附素BabA、SabA、HapA及其他膜蛋白等)、8种转录翻译相关蛋白(Tuf、RpoBC等)、5种分子伴侣(DnaK、GroES等)、3种细胞毒素相关蛋白(CagA等)、3种氧化还原相关蛋白(TrxA等)、2种信号转导蛋白(TlpB、TlpD)和4种功能尚不明确蛋白。蛋白质相互关系网络分析发现，可形成多个分簇，并以GroEL为网络的中心节点。外膜蛋白，特别是重要黏附素BabA、SabA、HapA等与GroEL的广泛关联，提示GroEL可能在HP与宿主胃黏膜上皮细胞相互作用中发挥重要作用。【结论】HP中GroEL结合蛋白质谱广泛，功能涉及HP代谢、转录翻译、氧化还原和黏附等，并参与HP生存适应、定殖及致病过程。GroEL有望成为一种新的研究HP致病性及发展感染干预策略的重要靶点。

Abstract:

[Objective] To obtain the composition spectrum of Helicobacter pylori (HP) GroEL binding proteins and provide new insights into the role of GroEL and its interacting proteins in the pathogenesis of H. pylori. [Methods] Based on the construction of H. pylori GroEL prokaryotic expression recombinant bacteria Escherichia coli BL21(DE3)^{pET-28a(+)-groEL}, the His-tagged GroEL protein was purified and incubated with H. pylori whole-cell protein extract. The complex was captured using Protein G magnetic beads and anti-His tag antibody immunoprecipitation, and the GroEL and its possible binding proteins were identified by mass spectrometry. The proteins were classified according to their main functions, and protein interaction network analysis was performed. [Results] A total of 59 proteins were identified as possible binding partners of GroEL, including 19 metabolic enzymes (7 of which are involved in oxidative stress, such as KatA, GltA, and AhpC; 5 peptidases, such as PepA, RocF, and HtrA; 2 enzymes involved in lipid metabolism; 2 enzymes involved in ATP synthesis; HP17_08079 and 2 urease enzymes), 15 outer membrane proteins (including adhesins BabA, SabA, HapA, and other membrane proteins), 8 transcription and translation-related proteins (such as Tuf and RpoBC), 5 molecular chaperones (such as DnaK and GroES), 3 cytotoxin-related proteins (such as CagA), 3 oxidative stress-related proteins (such as TrxA), 2 signal transduction proteins (TlpB and TlpD), and 4 proteins with unknown functions. Protein interaction analysis revealed that the entire network could form multiple clusters, with GroEL as the central node of the network. The extensive association of outer membrane proteins, particularly the important adhesins BabA, SabA, HapA, with GroEL suggests that GroEL may play an important role in the interaction between H. pylori and host gastric mucosal epithelial cells. [Conclusion] The spectrum of H. pylori GroEL binding proteins is extensive, and the related proteins are involved in H. pylori metabolism, transcription and translation, oxidation-reduction, and adhesion, participating in the survival, colonization, and pathogenic processes. GroEL is expected to become a novel important target for studying H. pylori pathogenicity and developing infection intervention strategies.