N-linked protein glycosylation systems operate in species from all three domains of life. The model bacterial N-linked glycosylation system from Campylobacter jejuni is encoded by pgl genes present at a single chromosomal locus. This gene cluster includes the pglB oligosaccharyltransferase responsible for transfer of glycan from lipid carrier to protein. Although all genomes from species of the Campylobacter genus contain a pgl locus, among the related Helicobacter genus only three evolutionarily related species (H. pullorum, H. canadensis and H. winghamensis) potentially encode N-linked protein glycosylation systems. Helicobacter putative pgl genes are scattered in five chromosomal loci and include two putative oligosaccharyltransferase-encoding pglB genes per genome. We have previously demonstrated the in vitro N-linked glycosylation activity of H. pullorum resulting in transfer of a pentasaccharide to a peptide at asparagine within the sequon (D/E)XNXS/T. In this study, we identified the first H. pullorum N-linked glycoprotein, termed HgpA. Production of histidine-tagged HgpA in the background of insertional knockout mutants of H. pullorum pgl/wbp genes followed by analysis of HgpA glycan structures demonstrated the role of individual gene products in the PglB1-dependent N-linked protein glycosylation pathway. Glycopeptide purification by zwitterionic-hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry identified six glycosites from five H. pullorum proteins, which was consistent with proteins reactive with a polyclonal antiserum generated against glycosylated HgpA. This study demonstrates functioning of a H. pullorum N-linked general protein glycosylation system.

中文翻译：

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白痢螺杆菌N-连接蛋白糖基化系统的功能分析。

N-连接蛋白糖基化系统在生命的所有三个领域的物种中运作。来自空肠弯曲杆菌的模型细菌 N-连接糖基化系统由存在于单个染色体基因座的 pgl 基因编码。该基因簇包括负责将聚糖从脂质载体转移到蛋白质的 pglB 寡糖基转移酶。尽管来自弯曲杆菌属物种的所有基因组都包含一个 pgl 基因座，但在相关的螺杆菌属中，只有三个进化相关的物种（H.pullorum、H.canadensis 和 H.winghamensis）可能编码 N 连接蛋白糖基化系统。螺杆菌推定的 pgl 基因分散在五个染色体基因座中，每个基因组包括两个推定的寡糖基转移酶编码 pglB 基因。我们之前已经证明了白痢菌的体外 N-连接糖基化活性，导致五糖转移到序列子 (D/E)XNXS/T 内天冬酰胺的肽上。在这项研究中，我们鉴定了第一个白痢菌 N 连接糖蛋白，称为 HgpA。在白痢 pgl/wbp 基因的插入敲除突变体的背景下产生组氨酸标记的 HgpA，然后分析 HgpA 聚糖结构，证明了单个基因产物在 PglB1 依赖性 N 连接蛋白糖基化途径中的作用。通过两性离子-亲水相互作用液相色谱与串联质谱联用的糖肽纯化鉴定了来自五种白藜芦醇蛋白的六种糖位点，这与与针对糖基化 HgpA 产生的多克隆抗血清反应的蛋白质一致。