Global Identification of Prokaryotic Glycoproteins Based on an <em>Escherichia coli</em> Proteome Microarray

Glycosylation is one of the most abundant protein posttranslational modifications. Protein glycosylation plays important roles not only in eukaryotes but also in prokaryotes. To further understand the roles of protein glycosylation in prokaryotes, we developed a lectin binding assay to screen glycoproteins on an Escherichia coli proteome microarray containing 4,256 affinity-purified E.coli proteins. Twenty-three E.coli proteins that bound Wheat-Germ Agglutinin (WGA) were identified. PANTHER protein classification analysis showed that these glycoprotein candidates were highly enriched in metabolic process and catalytic activity classes. One sub-network centered on deoxyribonuclease I (sbcB) was identified. Bioinformatics analysis suggests that prokaryotic protein glycosylation may play roles in nucleotide and nucleic acid metabolism. Fifteen of the 23 glycoprotein candidates were validated by lectin (WGA) staining, thereby increasing the number of validated E. coli glycoproteins from 3 to 18. By cataloguing glycoproteins in E.coli, our study greatly extends our understanding of protein glycosylation in prokaryotes.

糖基化（Glycosylation）是最为普遍的蛋白质翻译后修饰（protein posttranslational modifications）类型之一。蛋白质糖基化不仅在真核生物中发挥关键作用，在原核生物中同样具有重要功能。为进一步阐明蛋白质糖基化在原核生物中的生物学功能，我们开发了凝集素结合检测方法，在包含4256种经亲和纯化的大肠杆菌（Escherichia coli）蛋白质的大肠杆菌蛋白质组微阵列上开展糖蛋白筛选。最终鉴定得到23种可结合小麦胚芽凝集素（Wheat-Germ Agglutinin，WGA）的大肠杆菌蛋白质。PANTHER蛋白质分类分析显示，这些候选糖蛋白显著富集于代谢过程与催化活性功能类别中。我们还鉴定得到一个以脱氧核糖核酸酶I（deoxyribonuclease I，sbcB）为核心的蛋白质子网络。生物信息学分析结果表明，原核生物蛋白质糖基化可能参与核苷酸与核酸代谢过程。在23种候选糖蛋白中，有15种通过凝集素（WGA）染色实验得到验证，由此将已验证的大肠杆菌糖蛋白数量从3种提升至18种。本研究通过对大肠杆菌糖蛋白进行系统编目，极大拓展了学界对原核生物蛋白质糖基化的认知。