Structural, genetic and functional characterization of the flagellin glycosylation process in Helicobacter pylori
Structural, genetic and functional characterization of the flagellin glycosylation process in Helicobacter pylori
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DOI：
10.1046/j.1365-2958.2003.03527.x
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发表时间：
2003-06-01
影响因子：
3.6
通讯作者：
Logan, SM
中科院分区：
文献类型：
--
作者：
Schirm, M;Soo, EC;Logan, SM
对幽门螺杆菌复杂极性鞭毛的质谱分析表明FlaA和FlaB蛋白均被假氨基酸（Pse 5Ac 7Ac，5，7-二乙酰氨基-3，5，7，9-四脱氧-L-甘油-L-甘露-非-酮糖酸）后修饰。与弯曲杆菌不同，螺杆菌中鞭毛糖基化在同种型或糖型分布中显示出很少的异质性，尽管所有糖基化位点以与弯曲杆菌中发现的类似的方式位于蛋白单体的中心核心区域。生物信息学分析揭示了5个基因（HP 0840、HP 0178、HP 0326 A、HP 0326 B、HP 0114）与先前报道的参与运动、鞭毛糖基化或多糖生物合成的其他原核生物基因同源。在螺杆菌（HP 0178，HP 0326 A，HP 0326 B，HP 0114）中插入突变这些同源物中的四个导致非运动表型，没有结构鞭毛丝，并且通过Western免疫印迹仅检测到少量的鞭毛蛋白。然而，鞭毛蛋白结构基因的mRNA水平不受每个突变的影响。鉴于组合的生物信息学和结构证据表明这些基因产物在聚糖生物合成中的作用，随后的研究集中在相应基因产物的功能表征上。设计了一种新的方法，以确定生物合成的糖核苷酸前体细胞内代谢池的父母和同基因突变体使用毛细管电泳-电喷雾质谱（CE-ESMS）和前体离子扫描。HP 0326 A、HP 0326 B和HP 0178基因产物直接参与Pse的核苷酸活化形式CMP-Pse的生物合成。来自HP 0326 A和HP 0326 B等基因突变体的胞质提取物的质谱分析揭示了单乙酰氨基三脱氧己糖UDP糖核苷酸前体和二乙酰氨基三脱氧己糖UDP糖核苷酸前体的积累。
Mass spectrometry analyses of the complex polar flagella from Helicobacter pylori demonstrated that both FlaA and FlaB proteins are post-translationally modified with pseudaminic acid (Pse5Ac7Ac, 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-ulosonic acid). Unlike Campylobacter , flagellar glycosylation in Helicobacter displays little heterogeneity in isoform or glycoform distribution, although all glycosylation sites are located in the central core region of the protein monomer in a manner similar to that found in Campylobacter . Bioinformatic analysis revealed five genes (HP0840, HP0178, HP0326A, HP0326B, HP0114) homologous to other prokaryote genes previously reported to be involved in motility, flagellar glycosylation or polysaccharide biosynthesis. Insertional mutagenesis of four of these homologues in Helicobacter (HP0178, HP0326A, HP0326B, HP0114) resulted in a non-motile phenotype, no structural flagella filament and only minor amounts of flagellin protein detectable by Western immunoblot. However, mRNA levels for the flagellin structural genes remained unaffected by each mutation. In view of the combined bioinformatic and structural evidence indicating a role for these gene products in glycan biosynthesis, subsequent investigations focused on the functional characterization of the respective gene products. A novel approach was devised to identify biosynthetic sugar nucleotide precursors from intracellular metabolic pools of parent and isogenic mutants using capillary electrophoresis-electrospray mass spectrometry (CE-ESMS) and precursor ion scanning. HP0326A, HP0326B and the HP0178 gene products are directly involved in the biosynthesis of the nucleotide-activated form of Pse, CMP-Pse. Mass spectral analyses of the cytosolic extract from the HP0326A and HP0326B isogenic mutants revealed the accumulation of a mono- and a diacetamido trideoxyhexose UDP sugar nucleotide precursor.