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Assay Methods for the Glycosyltransferases Involved in Synthesis of Bacterial Polysaccharides

  • Tasnim Abukar
  • Nakita Buenbrazo
  • Bettina Janesch
  • Laura Kell
  • Warren WakarchukEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1954)

Abstract

Glycans play many important roles in bacterial biology and the complexity of the glycan structures requires biochemical assays in place to help characterize the biosynthetic pathways. Our focus has been on the use of enzymes from pathogens which make molecular mimics of host glycans. We have been examining glycosyltransferases that make strategic linkages in biologically active glycans which can be also exploited for potential therapeutic glycoconjugate synthesis. This chapter will provide details on assays for a variety of bacterial glycosyltransferases that we and others have used for the characterization of pathogen glycoconjugate biosynthetic pathways, and for the in vitro synthesis of human-like glycans produced by bacterial pathogens. The methods presented here should enable other assays to be developed for new pathway characterization.

Key words

Lipopolysaccharide Sialyltransferase BODIPY Enzyme assay Synthetic acceptor 

Notes

Acknowledgements

We thank Dr. Hong-Ming Chen, University of British Columbia, for the gift of BODIPY-NHS. We thank Sussex Research Chemicals, for the gift of azido-sugars. The work was supported by grants to WW from GlycoNet doi:  https://doi.org/10.13039/501100009056, and BJ was supported by an Erwin Schrödinger Fellowship.

References

  1. 1.
    Carlin AF, Uchiyama S, Chang Y-C et al (2009) Molecular mimicry of host sialylated glycans allows a bacterial pathogen to engage neutrophil Siglec-9 and dampen the innate immune response. Blood 113:3333–3336CrossRefGoogle Scholar
  2. 2.
    Harvey HA, Jennings MP, Campbell CA et al (2001) Receptor-mediated endocytosis of Neisseria gonorrhoeae into primary human urethral epithelial cells: the role of the asialoglycoprotein receptor. Mol Microbiol 42:659–672CrossRefGoogle Scholar
  3. 3.
    Pavliak V, Brisson JR, Michon F et al (1993) Structure of the sialylated L3 lipopolysaccharide of Neisseria meningitidis. J Biol Chem 268:14146–14152PubMedGoogle Scholar
  4. 4.
    Penner JL, Aspinall GO (1997) Diversity of lipopolysaccharide structures in Campylobacter jejuni. J Infect Dis 176:S135–S138CrossRefGoogle Scholar
  5. 5.
    Hood DW, Cox AD, Wakarchuk WW et al (2001) Genetic basis for expression of the major globotetraose-containing lipopolysaccharide from H. influenzae strain Rd (RM118). Glycobiology 11:957–967CrossRefGoogle Scholar
  6. 6.
    Fox KL, Cox AD, Gilbert M et al (2006) Identification of a bifunctional lipopolysaccharide sialyltransferase in Haemophilus influenzae: incorporation of disialic acid. J Biol Chem 281:40024–40032CrossRefGoogle Scholar
  7. 7.
    DeAngelis PL (2002) Microbial glycosaminoglycan glycosyltransferases. Glycobiology 12:9R–16RCrossRefGoogle Scholar
  8. 8.
    Monteiro MA (2001) Helicobacter pylori: a wolf in sheep's clothing: the glycotype families of Helicobacter pylori lipopolysaccharides expressing histo-blood groups: structure, biosynthesis, and role in pathogenesis. Adv Carbohydr Chem Biochem 57:99–158CrossRefGoogle Scholar
  9. 9.
    Bhattacharjee AK, Jennings HJ, Kenny CP et al (1975) Structural determination of the sialic acid polysaccharide antigens of Neisseria meningitidis serogroups B and C with carbon 13 nuclear magnetic resonance. JBiolChem 250:1926–1932Google Scholar
  10. 10.
    Lairson LL, Henrissat B, Davies GJ et al (2008) Glycosyltransferases: structures, functions, and mechanisms. Annu Rev Biochem 77:521–555CrossRefGoogle Scholar
  11. 11.
    Cantarel BL, Coutinho PM, Rancurel C et al (2009) The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res 37:D233–D238CrossRefGoogle Scholar
  12. 12.
    Greenfield LK, Richards MR, Li J et al (2012) Biosynthesis of the polymannose lipopolysaccharide O-antigens from Escherichia coli serotypes O8 and O9a requires a unique combination of single- and multiple-active site mannosyltransferases. J Biol Chem 287:35078–35091CrossRefGoogle Scholar
  13. 13.
    Kolkman MA, Wakarchuk W, Nuijten PJ et al (1997) Capsular polysaccharide synthesis in Streptococcus pneumoniae serotype 14: molecular analysis of the complete cps locus and identification of genes encoding glycosyltransferases required for the biosynthesis of the tetrasaccharide subunit. Mol Microbiol 26:197–208CrossRefGoogle Scholar
  14. 14.
    Wakarchuk W, Martin A, Jennings MP et al (1996) Functional relationships of the genetic locus encoding the glycosyltransferase enzymes involved in expression of the lacto-N-neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis. J Biol Chem 271:19166–19173CrossRefGoogle Scholar
  15. 15.
    Willis LM, Stupak J, Richards MR et al (2013) Conserved glycolipid termini in capsular polysaccharides synthesized by ATP-binding cassette transporter-dependent pathways in gram-negative pathogens. Proc Natl Acad Sci U S A 110:7868–7873CrossRefGoogle Scholar
  16. 16.
    Gilbert M, Karwaski MF, Bernatchez S et al (2002) The genetic bases for the variation in the lipo-oligosaccharide of the mucosal pathogen, Campylobacter jejuni. Biosynthesis of sialylated ganglioside mimics in the core oligosaccharide. J Biol Chem 277:327–337CrossRefGoogle Scholar
  17. 17.
    Chiu CP, Watts AG, Lairson LL et al (2004) Structural analysis of the sialyltransferase CstII from Campylobacter jejuni in complex with a substrate analog. Nat Struct Mol Biol 11:163–170CrossRefGoogle Scholar
  18. 18.
    Chiu CP, Lairson LL, Gilbert M et al (2007) Structural analysis of the alpha-2,3-sialyltransferase Cst-I from Campylobacter jejuni in apo and substrate-analogue bound forms. Biochemistry 46:7196–7204CrossRefGoogle Scholar
  19. 19.
    Lindhout T, Bainbridge CR, Costain WJ et al (2013) Biochemical characterization of a polysialyltransferase from Mannheimia haemolytica A2 and comparison to other bacterial polysialyltransferases. PLoS One 8:e69888CrossRefGoogle Scholar
  20. 20.
    Willis LM, Whitfield C (2013) Structure, biosynthesis, and function of bacterial capsular polysaccharides synthesized by ABC transporter-dependent pathways. Carbohydr Res 378:35–44CrossRefGoogle Scholar
  21. 21.
    Namdjou DJ, Chen HM, Vinogradov E et al (2008) A beta-1,4-galactosyltransferase from Helicobacter pylori is an efficient and versatile biocatalyst displaying a novel activity for thioglycoside synthesis. Chembiochem 9:1632–1640CrossRefGoogle Scholar
  22. 22.
    Gilbert M, Brisson JR, Karwaski MF et al (2000) Biosynthesis of ganglioside mimics in Campylobacter jejuni OH4384. Identification of the glycosyltransferase genes, enzymatic synthesis of model compounds, and characterization of nanomole amounts by 600-mhz (1)h and(13)c NMR analysis. J Biol Chem 275(6):3896–3906CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Tasnim Abukar
    • 1
  • Nakita Buenbrazo
    • 1
  • Bettina Janesch
    • 1
  • Laura Kell
    • 1
  • Warren Wakarchuk
    • 1
    Email author
  1. 1.Department of Chemistry and BiologyRyerson UniversityTorontoCanada

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