Functional complementation reveals the importance of intermolecular monomer interactions for Helicobacter pylori VacA vacuolating activity.
Functional complementation reveals the importance of intermolecular monomer interactions for Helicobacter pylori VacA vacuolating activity.
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功能互补揭示了分子间单体相互作用对于幽门螺杆菌 VacA 空泡活性的重要性。
DOI：
10.1046/j.1365-2958.2002.02818.x
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发表时间：
2002
影响因子：
3.6
通讯作者：
Blanke,StevenR
中科院分区：
文献类型：
--
作者：
Ye,Dan;Blanke,StevenR
幽门螺杆菌空泡毒素(VacA)可诱导敏感哺乳动物细胞的退行性空泡化。虽然越来越多的证据表明VacA从细胞内的作用部位进入细胞并发挥作用，但VacA介导细胞空泡化的生化机制尚未建立。在这项研究中，我们使用功能互补和生化方法来探索VacA的结构。VacA由两个离散的片段p37和p58组成，这两个片段都是空泡化活动所必需的。使用瞬时转染系统，我们表达了基因修饰形式的VacA，并确定了p37或p58中使毒素失活的突变。在p37区域有一个失活的单残基取代的VacA[VacA(P9A)]在功能上补充了第二个突变形式的VacA，在p58区域有一个失活的两个残基缺失[VacAΔ(346-347)]。VacA(P9A)和VacAΔ(346-347)也从空泡化的单层中免疫共沉淀，支持这两个失活突变体与反式功能直接相关的假说。当p37和p58作为单独的片段在HeLa细胞内表达时，它们直接相互作用，这表明p37-p58的相互作用促进了VacA单体的结合。总而言之，这些结果支持这样一个模型，即VacA的活性形式需要组装成两个或更多单体的络合物来阐述毒素功能。
TheHelicobacter pylorivacuolating cytotoxin (VacA) induces degenerative vacuolation of sensitive mammalian cell lines. Although evidence is accumulating that VacA enters cells and functions from an intracellular site of action, the biochemical mechanism by which VacA mediates cellular vacuolation has not been established. In this study, we used functional complementation and biochemical approaches to probe the structure of VacA. VacA consists of two discrete fragments, p37 and p58, that are both required for vacuolating activity. Using a transient transfection system, we expressed genetically modified forms of VacA and identified mutations in either p37 or p58 that inactivated the toxin. VacA with an inactivating single‐residue substitution in the p37 domain [VacA (P9A)] functionally complemented a second mutant form of VacA with an inactivating two‐residue deletion in the p58 domain [VacA Δ(346–347)]. VacA (P9A) and VacA Δ(346–347) also co‐immunoprecipitated from vacuolated monolayers, supporting the hypothesis that these two inactive mutants associate directly to functionin trans. p37 and p58 interact directly when expressed as separate fragments within HeLa cells, suggesting that p37–p58 inter‐actions facilitate VacA monomer associations. Collectively, these results support a model in which the active form of VacA requires assembly into a complex of two or more monomers to elaborate toxin function.