Functional Characterization of the Initiation Enzyme of S-Layer Glycoprotein Glycan Biosynthesis in
            <i>Geobacillus stearothermophilus</i>
            NRS 2004/3a

Steiner, Kerstin; Novotny, René; Patel, Kinnari B.; Vinogradov, Evgenij; Whitfield, Chris; Valvano, Miguel A.; Messner, Paul; Schäffer, Christina

doi:10.1128/jb.01592-06

Cited by 48 publications

(52 citation statements)

References 54 publications

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“…This protein is the prototype of the PHPT family, a large family of prokaryotic proteins involved in the initiation reaction for O antigen and exopolysaccharide synthesis (Valvano, 2003), and also in the biosynthesis of the glycan moiety attached to bacterial glycoproteins (Steiner et al, 2007). The predicted topology of WbaP revealed important differences from WecA, which belongs to another large family of initiating enzymes in prokaryotes and eukaryotes that catalyse the transfer of Nacetylhexosamine-phosphate onto isoprenoid lipid intermediates (Valvano, 2003).…”

Section: Discussionmentioning

confidence: 99%

“…It has no known homologues in eukaryotic cells and lacks amino acid sequence similarity with members of the PNPT family. WbaP shows high sequence similarity with proteins involved in the synthesis of capsule exopolysaccharides in Xanthomonas campestris (Katzen et al, 1998), Erwinia amylovora (Bugert & Geider, 1995), Streptococcus pneumoniae (Cartee et al, 2005), Klebsiella pneumoniae (Arakawa et al, 1995;Drummelsmith & Whitfield, 1999) and E. coli K-12 (Stevenson et al, 1996) among others, and also with proteins involved in S-layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus (Steiner et al, 2007). The WbaP protein is a large hydrophobic and basic protein with a predicted mass of 56 kDa and a pI of 9.…”

Section: Introductionmentioning

confidence: 99%

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Distinct functional domains of the Salmonella enterica WbaP transferase that is involved in the initiation reaction for synthesis of the O antigen subunit

et al. 2008

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WbaP is a membrane enzyme that initiates O antigen synthesis in Salmonella enterica by catalysing the transfer of galactose 1-phosphate (Gal-1-P) onto undecaprenyl phosphate (Und-P). WbaP possesses at least three predicted structural domains: an N-terminal region containing four transmembrane helices, a large central periplasmic loop, and a C-terminal domain containing the last transmembrane helix and a large cytoplasmic tail. In this work, we investigated the contribution of each region to WbaP function by constructing a series of mutant WbaP proteins and using them to complement O antigen synthesis in DwbaP mutants of S. enterica serovars Typhi and Typhimurium. Truncated forms of WbaP lacking the periplasmic loop exhibited altered chain-length distributions in O antigen polymerization, suggesting that this central domain is involved in modulating the chain-length distribution of the O polysaccharide. The N-terminal and periplasmic domains were dispensable for complementation of O antigen synthesis in vivo, suggesting that the C-terminal domain carries the sugar-phosphate transferase activity. However, despite the fact that they complemented the synthesis of O antigen in the DwbaP mutant in vivo, membrane extracts containing WbaP derivatives without the N-terminal domain failed to transfer radioactive Gal from UDP-Gal into a lipid-rich fraction. These results suggest that the N-terminal region of WbaP, which contains four transmembrane domains, is essential for the insertion or stability of the protein in the bacterial membrane. We propose that the domain structure of WbaP enables this protein not only to function in the transfer of Gal-1-P to Und-P but also to establish critical interactions with additional proteins required for the correct assembly of O antigen in S. enterica.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Distinct functional domains of the Salmonella enterica WbaP transferase that is involved in the initiation reaction for synthesis of the O antigen subunit

et al. 2008

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“…Nonreducing terminal modifications analogous to those terminating the E. coli O8 and O9a O-PSs have also been identified on the S-layer glycans of a number of species of Bacillaceae (131). Others have already noted obvious similarities in the biosynthesis of some S-layer glycans and ABC transporter-dependent O-PSs (102,134,149). The ABC transporters from these S-layer glycan systems have separate TMD and NBD polypeptides resembling Wzm and Wzt, respectively (102).…”

Section: Other Systems With Nbds Containing Putative Cbmsmentioning

confidence: 95%

ABC Transporters Involved in Export of Cell Surface Glycoconjugates

Cuthbertson

Kos

Whitfield

2010

Microbiol Mol Biol Rev

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178

152

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SUMMARY Complex glycoconjugates play critical roles in the biology of microorganisms. Despite the remarkable diversity in glycan structures and the bacteria that produce them, conserved themes are evident in the biosynthesis-export pathways. One of the primary pathways involves representatives of the ATP-binding cassette (ABC) transporter superfamily. These proteins are responsible for the export of a wide variety of cell surface oligo- and polysaccharides in both Gram-positive and Gram-negative bacteria. Recent investigations of the structure and function of ABC transporters involved in the export of lipopolysaccharide O antigens have revealed two fundamentally different strategies for coupling glycan polymerization to export. These mechanisms are distinguished by the presence (or absence) of characteristic nonreducing terminal modifications on the export substrates, which serve as chain termination and/or export signals, and by the presence (or absence) of a discrete substrate-binding domain in the nucleotide-binding domain polypeptide of the ABC transporter. A bioinformatic survey examining ABC exporters from known oligo- and polysaccharide biosynthesis loci identifies conserved nucleotide-binding domain protein families that correlate well with themes in the structures and assembly of glycans. The familial relationships among the ABC exporters generate hypotheses concerning the biosynthesis of structurally diverse oligo- and polysaccharides, which play important roles in the biology of bacteria with different lifestyles.

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“…[42] Western immunoblotting using anti-SgsE antibody and anti-pgl-glycan antibody was done as described elsewhere. [43] Anti-SgsE antibody was available in our laboratory from previous studies and anti-pgl-glycan antibody was kindly provided by Prof. Markus Aebi (ETH Zurich, Switzerland). N-terminal sequencing of blotted protein bands that have been excised from the polyvinylidene fluoride (PVDF) membrane after visualization with Coomassie Blue R-250 staining reagent was performed by Prof. Herbert Lindner, University of Innsbruck, Austria.…”

Section: General Methodsmentioning

confidence: 99%

Recombinant Glycans on an S‐Layer Self‐Assembly Protein: A New Dimension for Nanopatterned Biomaterials

Steiner

Hanreich

Kainz

et al. 2008

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Crucial biological phenomena are mediated through carbohydrates that are displayed in a defined manner and interact with molecular scale precision. We lay the groundwork for the integration of recombinant carbohydrates into a "biomolecular construction kit" for the design of new biomaterials, by utilizing the self-assembly system of the crystalline cell surface (S)-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a. SgsE is a naturally O-glycosylated protein, with intrinsic properties that allow it to function as a nanopatterned matrix for the periodic display of glycans. By using a combined carbohydrate/protein engineering approach, two types of S-layer neoglycoproteins are produced in Escherichia coli. Based on the identification of a suitable periplasmic targeting system for the SgsE self-assembly protein as a cellular prerequisite for Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts protein glycosylation, and on engineering of one of the natural protein O-glycosylation sites into a target for N-glycosylation, the heptasaccharide from the AcrA protein of Campylobacter jejuni and the O7 polysaccharide of E. coli are co-or post-translationally transferred to the S-layer protein by the action of the oligosaccharyltransferase PglB. The degree of glycosylation of the Slayer neoglycoproteins after purification from the periplasmic fraction reaches completeness. Electron microscopy reveals that recombinant glycosylation is fully compatible with the S-layer protein self-assembly system. Tailor-made ("functional") nanopatterned, self-assembling neoglycoproteins may open up new strategies for influencing and controlling complex biological systems with potential applications in the areas of biomimetics, drug targeting, vaccine design, or diagnostics.

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Functional Characterization of the Initiation Enzyme of S-Layer Glycoprotein Glycan Biosynthesis in Geobacillus stearothermophilus NRS 2004/3a

Cited by 48 publications

References 54 publications

Distinct functional domains of the Salmonella enterica WbaP transferase that is involved in the initiation reaction for synthesis of the O antigen subunit

Distinct functional domains of the Salmonella enterica WbaP transferase that is involved in the initiation reaction for synthesis of the O antigen subunit

ABC Transporters Involved in Export of Cell Surface Glycoconjugates

Recombinant Glycans on an S‐Layer Self‐Assembly Protein: A New Dimension for Nanopatterned Biomaterials

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