<i>Escherichia coli</i> O86 O-Antigen Biosynthetic Gene Cluster and Stepwise Enzymatic Synthesis of Human Blood Group B Antigen Tetrasaccharide

Yi, Wen; Shao, Jianguo; Zhu, Lizhi; Li, Mei; Singh, Manorama; Lu, Yuquan; Lin, Steven; Li, Hanfen; Ryu, Kang; Shen, Jie; Guo, Hongjie; Yao, Qian; Bush, A.; Wang, Peng George

doi:10.1021/ja045021y

Cited by 103 publications

(77 citation statements)

References 15 publications

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“…Several species of bacteria are able to express HBGA-like sugars on their lipopolysaccharide O-chains (20,27); therefore, it is possible that rNVLP might have bound to lipopolysaccharide (LPS) or some other component of natural bacteria bound to veins (2). Our preliminary results suggest this is not the case, but we cannot yet eliminate this as a possible mechanism of binding to components in RE or on the leaf surface.…”

Section: Discussionmentioning

confidence: 81%

Binding of Virus-Like Particles of Norwalk Virus to Romaine Lettuce Veins

Gandhi

Mandrell

Tian

2010

Appl Environ Microbiol

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Section: Discussionmentioning

confidence: 81%

Binding of Virus-Like Particles of Norwalk Virus to Romaine Lettuce Veins

Gandhi

Mandrell

Tian

2010

Appl Environ Microbiol

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“…Moreover, several antigens are common to human and bacterial cells. The E. coli O86 antigen contains the human blood group B trisaccharide epitope (12). The glycoprotein obtained in our in vitro assay therefore contains a human epitope.…”

Section: Discussionmentioning

confidence: 93%

Extreme Substrate Promiscuity of the Neisseria Oligosaccharyl Transferase Involved in Protein O-Glycosylation

Faridmoayer

Fentabil

Haurat

et al. 2008

Journal of Biological Chemistry

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111

122

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Neisseria meningitidis PglL belongs to a novel family of bacterial oligosaccharyltransferases (OTases) responsible for O-glycosylation of type IV pilins. Although members of this family are widespread among pathogenic bacteria, there is little known about their mechanism. Understanding the O-glycosylation process may uncover potential targets for therapeutic intervention, and can open new avenues for the exploitation of these pathways for biotechnological purposes. In this work, we demonstrate that PglL is able to transfer virtually any glycan from the undecaprenyl pyrophosphate (UndPP) carrier to pilin in engineered Escherichia coli and Salmonella cells. Surprisingly, PglL was also able to interfere with the peptidoglycan biosynthetic machinery and transfer peptidoglycan subunits to pilin. This represents a previously unknown post-translational modification in bacteria. Given the wide range of glycans transferred by PglL, we reasoned that substrate specificity of PglL lies in the lipid carrier. To test this hypothesis we developed an in vitro glycosylation system that employed purified PglL, pilin, and the lipid farnesyl pyrophosphate (FarPP) carrying a pentasaccharide that had been synthesized by successive chemical and enzymatic steps. Although FarPP has different stereochemistry and a significantly shorter aliphatic chain than the natural lipid substrate, the pentasaccharide was still transferred to pilin in our system. We propose that the primary roles of the lipid carrier during O-glycosylation are the translocation of the glycan into the periplasm, and the positioning of the pyrophosphate linker and glycan adjacent to PglL. The unique characteristics of PglL make this enzyme a promising tool for glycoengineering novel glycan-based vaccines and therapeutics.Bacterial surface components are frequently composed of, or decorated with, carbohydrates. Among these glycosylated components are the type IV pili, hair-like structures protruding from the bacterial surface, mainly formed by a single protein generically named pilin (1). Type IV pili are important for host cell adhesion and virulence. Furthermore, pilins are O-glycosylated in diverse pathogenic bacteria, including Neisseria meningitidis and N. gonorrhoea. The glycan moieties in both species consist of short oligosaccharides, up to three sugar residues in length. Several pilin glycosylation (pgl) genes have been identified in N. meningitidis encoding for glycosyltransferases and sugar-modifying enzymes that are required for the biosynthesis of the oligosaccharides (2). However, how the glycans are transferred to pilin has been just recently described. Power et al. identified in a gene in N. meningitidis containing the Wzy_C PFAM domain (PF04932), a signature of enzymes that participate in O antigen biosynthesis, and which is also present in the PilO oligosaccharyltransferase (OTase) involved in pilin glycosylation in Pseudomonas aeruginosa 1244 (3). Inactivation of this gene, named pglL, resulted in an increase in the electrophoretic mobility of pilin, c...

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“…Genes involved in O-antigen biosynthesis are normally clustered between galF and gnd in E. coli and Shigella and are classified into three different groups: (i) nucleotide sugar synthesis genes; (ii) glycosyltransferase genes; and (iii) O-antigen processing genes, including the flippase gene (wzx) and polymerase gene (wzy). With few exceptions (25,27,35), most of the glycosyltransferases that synthesize E. coli O antigens have not been characterized.…”

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confidence: 99%

Characterization of Two β-1,3-Glucosyltransferases fromEscherichia coliSerotypes O56 and O152

Brockhausen

Liu

et al. 2008

J Bacteriol

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The O antigens of outer membrane-bound lipopolysaccharides (LPS) in gram-negative bacteria are oligosaccharides consisting of repeating units with various structures and antigenicities. The O56 and O152 antigens of Escherichia coli both contain a Glc-␤1-3-GlcNAc linkage within the repeating unit. We have cloned and identified the genes (wfaP in O56 and wfgD in O152) within the two O-antigen gene clusters that encode glucosyltransferases involved in the synthesis of this linkage. A synthetic substrate analog of the natural acceptor substrate undecaprenol-pyrophosphate-lipid [GlcNAc-␣-PO 3 -PO 3 -(CH 2 ) 11 -O-phenyl] was used as an acceptor and UDP-Glc as a donor substrate to demonstrate that both wfgD and wfaP encode glucosyltransferases. Enzyme products from both glucosyltransferases were isolated by high-pressure liquid chromatography and analyzed by nuclear magnetic resonance. The spectra showed the expected Glc-␤1-3-GlcNAc linkage in the products, confirming that both WfaP and WfgD are forms of UDP-Glc: GlcNAc-pyrophosphate-lipid ␤-1,3-glucosyltransferases. Both WfaP and WfgD have a DxD sequence, which is proposed to interact with phosphate groups of the nucleotide donor through the coordination of a metal cation, and a short hydrophobic sequence at the C terminus that may help to associate the enzymes with the inner membrane. We showed that the enzymes have similar properties and substrate recognition. They both require a divalent cation (Mn 2؉ or Mg 2؉ ) for activity, are deactivated by detergents, have a broad pH optimum, and require the pyrophosphate-sugar linkage in the acceptor substrate for full activity. Substrates lacking phosphate or pyrophosphate linked to GlcNAc were inactive. The length of the aliphatic chain of acceptor substrates also contributes to the activity.

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Escherichia coli O86 O-Antigen Biosynthetic Gene Cluster and Stepwise Enzymatic Synthesis of Human Blood Group B Antigen Tetrasaccharide

Cited by 103 publications

References 15 publications

Binding of Virus-Like Particles of Norwalk Virus to Romaine Lettuce Veins

Binding of Virus-Like Particles of Norwalk Virus to Romaine Lettuce Veins

Extreme Substrate Promiscuity of the Neisseria Oligosaccharyl Transferase Involved in Protein O-Glycosylation

Characterization of Two β-1,3-Glucosyltransferases fromEscherichia coliSerotypes O56 and O152

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