Characterization of exogenous bacterial oligosaccharyltransferases in Escherichia coli reveals the potential for O-linked protein glycosylation in Vibrio cholerae and Burkholderia thailandensis

Glycobiology. 2012 Jul;22(7):962-74. doi: 10.1093/glycob/cws059. Epub 2012 Mar 5.

Abstract

Bacterial protein glycosylation systems from varying species have been functionally reconstituted in Escherichia coli. Both N- and O-linked glycosylation pathways, in which the glycans are first assembled onto lipid carriers and subsequently transferred to acceptor proteins by an oligosaccharyltransferase (OTase), have been documented in bacteria. The identification and characterization of novel OTases with different properties may provide new tools for engineering glycoproteins of biotechnological interest. In the case of OTases involved in O-glycosylation (O-OTases), there is very low sequence homology between those from different bacterial species. The Wzy_C signature domain common to these enzymes is also present in WaaL ligases; enzymes involved in lipopolysaccharide biosynthesis. Therefore, the identification of O-OTases using solely bioinformatic methods is problematic. The hypothetical proteins BTH_I0650 from Burkholderia thailandensis E264 and VC0393 from Vibrio cholerae N16961 contain the Wzy_C domain. In this work, we demonstrate that both proteins have O-OTase activity and renamed them PglL(Bt) and PglL(Vc), respectively, similar to the Neisseria meningitidis counterpart (PglL(Nm)). In E. coli, PglL(Bt) and PglL(Vc) display relaxed glycan and protein specificity. However, effective glycosylation depends upon a specific combination of the protein acceptor, glycan and O-OTase analyzed. This knowledge has important implications in the design of glycoconjugates and provides novel tools for use in glycoengineering applications. The codification of enzymatically active O-OTase in the genomes of members of the Vibrio and Burkholderia genera suggests the presence of still unknown O-glycoproteins in these organisms, which might have a role in bacterial physiology or pathogenesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosamine / analogs & derivatives
  • Acetylglucosamine / metabolism
  • Amino Acid Sequence
  • Bacterial Proteins / biosynthesis*
  • Bacterial Proteins / genetics
  • Burkholderia / enzymology*
  • Burkholderia / genetics
  • Carbohydrate Conformation
  • Carbohydrate Sequence
  • Cloning, Molecular
  • Escherichia coli
  • Glycoproteins / biosynthesis
  • Glycosylation
  • Hexosyltransferases / biosynthesis*
  • Hexosyltransferases / genetics
  • Membrane Proteins / biosynthesis*
  • Membrane Proteins / genetics
  • Molecular Sequence Data
  • Peptide Mapping
  • Polysaccharides / chemistry
  • Polysaccharides / metabolism
  • Protein Processing, Post-Translational*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Vibrio cholerae / enzymology*
  • Vibrio cholerae / genetics

Substances

  • Bacterial Proteins
  • Glycoproteins
  • Membrane Proteins
  • Polysaccharides
  • N-acetylbacillosamine
  • Hexosyltransferases
  • dolichyl-diphosphooligosaccharide - protein glycotransferase
  • Acetylglucosamine