Functional Characterization of Lpt3 and Lpt6, the Inner-Core Lipooligosaccharide Phosphoethanolamine Transferases from Neisseria meningitidis
Functional Characterization of Lpt3 and Lpt6, the Inner-Core Lipooligosaccharide Phosphoethanolamine Transferases from Neisseria meningitidis
复制标题
DOI：
10.1128/jb.00558-09
复制
发表时间：
2010-01-01
影响因子：
3.2
通讯作者：
Richards, James C.
中科院分区：
文献类型：
--
作者：
Wenzel, Cory Q.;St Michael, Frank;Richards, James C.
脑膜炎奈瑟氏菌的脂寡糖（LOS）含有庚糖（Hep）残基，其在3（3-PEtn）和/或6（6-PEtn）位被磷酸乙醇胺（PEtn）修饰。对N.脑膜炎，这是建议编码所需的HepII 3-和6-PEtn转移酶，分别克隆和过表达的C-末端多组氨酸标记的融合蛋白在大肠杆菌中，并发现本地化的内膜，基于蔗糖密度梯度离心。通过镍螯合层析从TritonX-100溶解的膜中纯化Lpt 3-His（6）和Lpt 6-His（6），用3-PEtn和6-PEtn特异性单克隆抗体对酶反应进行斑点印迹分析，最终证明Lpt 3和Lpt 6分别是磷脂酰乙醇胺依赖性LOS HepII 3-和6-PEtn转移酶，并且这两种酶都能够将PEtn转移到完全酰化的LOS和脱-O-酰化的（de-O-Ac）LOS。使用毛细管电泳-质谱法（MS）的进一步酶促研究表明，Lpt 3和Lpt 6都能够将PEtn转移到分别在HepII的6位和3位已经含有PEtn的de-O-Ac LOS分子，这表明在体外产生3，6-二-PEtn LOS部分中没有PEtn添加的强制顺序。
The lipooligosaccharide (LOS) of Neisseria meningitidis contains heptose (Hep) residues that are modified with phosphoethanolamine (PEtn) at the 3 (3-PEtn) and/or 6 (6-PEtn) position. The lpt3 (NMB2010) and lpt6 (NMA0408) genes of N. meningitidis, which are proposed to encode the required HepII 3- and 6-PEtn transferases, respectively, were cloned and overexpressed as C-terminally polyhistidine-tagged fusion proteins in Escherichia coli and found to localize to the inner membrane, based on sucrose density gradient centrifugation. Lpt3-His(6) and Lpt6-His(6) were purified from Triton X-100-solubilized membranes by nickel chelation chromatography, and dot blot analysis of enzymatic reactions with 3-PEtn- and 6-PEtn-specific monoclonal antibodies demonstrated conclusively that Lpt3 and Lpt6 are phosphatidylethanolamine-dependent LOS HepII 3- and 6-PEtn transferases, respectively, and that both enzymes are capable of transferring PEtn to both fully acylated LOS and de-O-acylated (de-O-Ac) LOS. Further enzymatic studies using capillary electrophoresis-mass spectrometry (MS) demonstrated that both Lpt3 and Lpt6 are capable of transferring PEtn to de-O-Ac LOS molecules already containing PEtn at the 6 and 3 positions of HepII, respectively, demonstrating that there is no obligate order of PEtn addition in the generation of 3,6-di-PEtn LOS moieties in vitro.