Two Protein Lysine Methyltransferases Methylate Outer Membrane Protein B from Rickettsia
Two Protein Lysine Methyltransferases Methylate Outer Membrane Protein B from Rickettsia
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DOI：
10.1128/jb.01379-12
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发表时间：
2012-12-01
影响因子：
3.2
通讯作者：
Ching, Wei-Mei
中科院分区：
文献类型：
--
作者：
Abeykoon, Amila H.;Chao, Chien-Chung;Ching, Wei-Mei
普氏立克次体是流行性斑疹伤寒的病原体，是一种潜在的生物威胁因子。其外膜蛋白B（Omp B）是一种免疫显性抗原，起保护性包膜和粘附素的作用。立克次体OmpB中赖氨酸残基甲基化与细菌毒力之间的相关性的观察表明OmpB甲基化的酶系统的重要性。然而，没有立克次体赖氨酸甲基转移酶的特点。立克次体基因组DNA序列的生物信息学分析确定了推定的赖氨酸甲基转移酶。合成潜在的甲基转移酶的基因，克隆，并在大肠杆菌中表达，表达蛋白通过镍-次氮基三乙酸（Ni-NTA）亲和层析纯化。通过将放射性标记的S-腺苷甲硫氨酸甲基掺入OmpB的重组片段中来分析纯化蛋白的甲基转移酶活性。两种推定的重组甲基转移酶（rRP 789和rRP 027 -028）甲基化重组OmpB片段。rRP 789的比活性比rRP 027 -028高10- 30倍。使用针对三甲基赖氨酸的特异性抗体的Western印迹分析表明，rRP 789和rRP 027 -028都催化重组OmpB片段的三甲基化。液相色谱-串联质谱（LC/MS-MS）分析表明，rRP 789催化赖氨酸的单、二和三甲基化，而rRP 027 -028仅催化三甲基化。据我们所知，rRP 789和rRP 027 -028是来自革兰氏阴性菌的外膜蛋白的第一个生化表征的赖氨酸甲基转移酶。立克次体赖氨酸甲基转移酶的产生和表征为研究OmpB的甲基化机制、甲基化对OmpB结构和功能的影响以及基于甲基化OmpB的诊断检测和疫苗候选物的开发提供了新的工具。
Rickettsia prowazekii, the etiologic agent of epidemic typhus, is a potential biological threat agent. Its outer membrane protein B (OmpB) is an immunodominant antigen and plays roles as protective envelope and as adhesins. The observation of the correlation between methylation of lysine residues in rickettsial OmpB and bacterial virulence has suggested the importance of an enzymatic system for the methylation of OmpB. However, no rickettsial lysine methyltransferase has been characterized. Bioinformatic analysis of genomic DNA sequences of Rickettsia identified putative lysine methyltransferases. The genes of the potential methyltransferases were synthesized, cloned, and expressed in Escherichia coli, and expressed proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The methyltransferase activities of the purified proteins were analyzed by methyl incorporation of radioactively labeled S-adenosylmethionine into recombinant fragments of OmpB. Two putative recombinant methyltransferases (rRP789 and rRP027-028) methylated recombinant OmpB fragments. The specific activity of rRP789 is 10- to 30-fold higher than that of rRP027-028. Western blot analysis using specific antibodies against trimethyl lysine showed that both rRP789 and rRP027-028 catalyzed trimethylation of recombinant OmpB fragments. Liquid chromatography-tandem mass spectrometry (LC/MS-MS) analysis showed that rRP789 catalyzed mono-, di-, and trimethylation of lysine, while rRP027-028 catalyzed exclusively trimethylation. To our knowledge, rRP789 and rRP027-028 are the first biochemically characterized lysine methyltransferases of outer membrane proteins from Gram-negative bacteria. The production and characterization of rickettsial lysine methyltransferases provide new tools to investigate the mechanism of methylation of OmpB, effects of methylation on the structure and function of OmpB, and development of methylated OmpB-based diagnostic assays and vaccine candidates.