Wang Shuo, Czuchry Diana, Liu Bin, Vinnikova Anna N, Gao Yin, Vlahakis Jason Z, Szarek Walter A, Wang Lei, Feng Lu, Brockhausen Inka
Department of Medicine and Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada.
TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin, China.
J Bacteriol. 2014 Sep;196(17):3122-33. doi: 10.1128/JB.01698-14. Epub 2014 Jun 23.
Escherichia coli displays O antigens on the outer membrane that play an important role in bacterial interactions with the environment. The O antigens of enterohemorrhagic E. coli O104 and O5 contain a Galβ1-3GalNAc disaccharide at the reducing end of the repeating unit. Several other O antigens contain this disaccharide, which is identical to the mammalian O-glycan core 1 or the cancer-associated Thomsen-Friedenreich (TF) antigen. We identified the wbwC genes responsible for the synthesis of the disaccharide in E. coli serotypes O104 and O5. To functionally characterize WbwC, an acceptor substrate analog, GalNAcα-diphosphate-phenylundecyl, was synthesized. WbwC reaction products were isolated by high-pressure liquid chromatography and analyzed by mass spectrometry, nuclear magnetic resonance, galactosidase and O-glycanase digestion, and anti-TF antibody. The results clearly showed that the Galβ1-3GalNAcα linkage was synthesized, confirming WbwCECO104 and WbwCECO5 as UDP-Gal:GalNAcα-diphosphate-lipid β1,3-Gal-transferases. Sequence analysis revealed a conserved DxDD motif, and mutagenesis showed the importance of these Asp residues in catalysis. The purified enzymes require divalent cations (Mn(2+)) for activity and are specific for UDP-Gal and GalNAc-diphosphate lipid substrates. WbwC was inhibited by bis-imidazolium salts having aliphatic chains of 18 to 22 carbons. This work will help to elucidate mechanisms of polysaccharide synthesis in pathogenic bacteria and provide technology for vaccine synthesis.
大肠杆菌在外膜上展示O抗原，这些抗原在细菌与环境的相互作用中发挥重要作用。肠出血性大肠杆菌O104和O5的O抗原在重复单元的还原端含有一个Galβ1-3GalNAc二糖。其他几种O抗原也含有这种二糖，它与哺乳动物O-聚糖核心1或癌症相关的汤姆森-弗里德赖希（TF）抗原相同。我们鉴定出了负责在大肠杆菌血清型O104和O5中合成该二糖的wbwC基因。为了从功能上表征WbwC，合成了一种受体底物类似物GalNAcα-二磷酸-苯基十一烷基。通过高压液相色谱分离WbwC反应产物，并通过质谱、核磁共振、半乳糖苷酶和O-聚糖酶消化以及抗TF抗体进行分析。结果清楚地表明合成了Galβ1-3GalNAcα连接，证实WbwCECO104和WbwCECO5为UDP-Gal:GalNAcα-二磷酸-脂质β1,3-半乳糖基转移酶。序列分析揭示了一个保守的DxDD基序，诱变表明这些天冬氨酸残基在催化中的重要性。纯化的酶需要二价阳离子（Mn(2+)）来发挥活性，并且对UDP-Gal和GalNAc-二磷酸脂质底物具有特异性。WbwC受到具有18至22个碳原子脂肪链的双咪唑盐的抑制。这项工作将有助于阐明病原菌多糖合成的机制，并为疫苗合成提供技术。
