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  • The EMBO Journal (2006) 25, 1957 - 1966
  • doi:10.1038/sj.emboj.7601087

Published online: 13 April 2006

Definition of the bacterial N-glycosylation site consensus sequence

Michael Kowarik1, N Martin Young2, Shin Numao1, Benjamin L Schulz1, Isabelle Hug1, Nico Callewaert1,3,a, Dominic C Mills1, David C Watson2, Marcela Hernandez1,b, John F Kelly2, Michael Wacker1,c and Markus Aebi1

  1. Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zürich, Switzerland
  2. Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada
  3. The Zürich Glycomics Initiative (GlycoInit), Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, Zürich, Switzerland

Correspondence to:

Markus Aebi, Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology Zurich, ETH Hönggerberg, 8093 Zürich, Switzerland. Tel.: +41 1 632 6413; Fax: +41 1 632 1375; E-mail: aebi@micro.biol.ethz.ch

aPresent address: Cytos Biotechnology AG, Wagistrasse 25, 8952 Schlieren, Switzerland

bPresent address: GlycoVaxyn AG, Einsiedlerstrasse 31, 8820 Wädenswil, Switzerland

cPresent address: Unit for Molecular Glycobiology, Department for Molecular Biomedical Research, Ghent University and VIB, 9052 Ghent-Zwijnaarde, Belgium

Received 7 November 2005; Accepted 21 March 2006

The Campylobacter jejuni pgl locus encodes an N-linked protein glycosylation machinery that can be functionally transferred into Escherichia coli. In this system, we analyzed the elements in the C. jejuni N-glycoprotein AcrA required for accepting an N-glycan. We found that the eukaryotic primary consensus sequence for N-glycosylation is N terminally extended to D/E-Y-N-X-S/T (Y, Xnot equalP) for recognition by the bacterial oligosaccharyltransferase (OST) PglB. However, not all consensus sequences were N-glycosylated when they were either artificially introduced or when they were present in non-C. jejuni proteins. We were able to produce recombinant glycoproteins with engineered N-glycosylation sites and confirmed the requirement for a negatively charged side chain at position -2 in C. jejuni N-glycoproteins. N-glycosylation of AcrA by the eukaryotic OST in Saccharomyces cerevisiae occurred independent of the acidic residue at the -2 position. Thus, bacterial N-glycosylation site selection is more specific than the eukaryotic equivalent with respect to the polypeptide acceptor sequence.

  • Keywords:

    • Campylobacter jejuni,
    • consensus sequence,
    • N-glycosylation,
    • periplasm,
    • oligosaccharyltransferase