A conserved DGGK motif is essential for the function of the PglB oligosaccharyltransferase from Campylobacter jejuni

From National Research Council Canada

DOIResolve DOI: https://doi.org/10.1093/glycob/cwx067
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Name affiliation
  1. National Research Council Canada. Human Health Therapeutics
FormatText
TypeArticle
Journal titleGlycobiology
ISSN0959-6658
1460-2423
Volume27
Issue10
Pages978–989
SubjectCampylobacter jejuni; lipid-linked oligosaccharide; N-linked protein glycosylation; oligosaccharyltransferase; PglB
Abstract
In Campylobacter jejuni, the PglB oligosaccharyltransferase catalyzes the transfer of a heptasaccharide from a lipid donor to asparagine within the D/E-X1-N–X2-S/T sequon (X1,2 ≠ P) or releases this heptasaccharide as free oligosaccharides (fOS). Using available crystal structures and sequence alignments, we identified a DGGK motif near the active site of PglB that is conserved among all Campylobacter species. We demonstrate that amino acid substitutions in the aspartate and lysine residues result in loss of protein glycosylation in the heterologous Escherichia coli system. Similarly, complementation of a C. jejuni pglB knock-out strain with mutated pglB alleles results in reduced levels of N-linked glycoproteins and fOS in the native host. Analysis of the PglB crystal structures from Campylobacter lari and the soluble C-terminal domain from C. jejuni suggests a particularly important structural role for the aspartate residue and the two following glycine residues, as well as a more subtle, less defined role for the lysine residue. Limited proteolysis experiments indicate that conformational changes of wildtype PglB that are induced by the binding of the lipid-linked oligosaccharide are altered by changes in the DGGK motif. Related to these findings, certain Campylobacter species possess two PglB orthologues and we demonstrate that only the orthologue containing the DGGK motif is active. Combining the knowledge gained from the PglB structures and mutagenesis studies, we propose a function for the DGGK motif in affecting the binding of the undecaprenyl-pyrophosphate glycan donor substrate that subsequently influences N-glycan and fOS production.
Publication date
PublisherOxford University Press
Terms of use
LanguageEnglish
Peer reviewedYes
NPARC number23002458
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