Functional analysis of the Helicobacter pullorum N-linked protein glycosylation system.

Adrian J Jervis,Rebecca Langdon,Brendan W Wren,Elizabeth Lord,Helen Frost,Alison G Wood,Joel A Cain,Dennis Linton,Stuart J Cordwell,Jonathan A Butler

doi:10.1093/glycob/cwx110

Abstract

N-linked protein glycosylation systems operate in species from all three domains of life. The model bacterial N-linked glycosylation system from Campylobacter jejuni is encoded by pgl genes present at a single chromosomal locus. This gene cluster includes the pglB oligosaccharyltransferase responsible for transfer of glycan from lipid carrier to protein. Although all genomes from species of the Campylobacter genus contain a pgl locus, among the related Helicobacter genus only three evolutionarily related species (H. pullorum, H. canadensis and H. winghamensis) potentially encode N-linked protein glycosylation systems. Helicobacter putative pgl genes are scattered in five chromosomal loci and include two putative oligosaccharyltransferase-encoding pglB genes per genome. We have previously demonstrated the in vitro N-linked glycosylation activity of H. pullorum resulting in transfer of a pentasaccharide to a peptide at asparagine within the sequon (D/E)XNXS/T. In this study, we identified the first H. pullorum N-linked glycoprotein, termed HgpA. Production of histidine-tagged HgpA in the background of insertional knockout mutants of H. pullorum pgl/wbp genes followed by analysis of HgpA glycan structures demonstrated the role of individual gene products in the PglB1-dependent N-linked protein glycosylation pathway. Glycopeptide purification by zwitterionic-hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry identified six glycosites from five H. pullorum proteins, which was consistent with proteins reactive with a polyclonal antiserum generated against glycosylated HgpA. This study demonstrates functioning of a H. pullorum N-linked general protein glycosylation system.

Highlights

In all three domains of life subsets of proteins are modified by the covalent attachment of sugars to asparagine residues within a conserved consensus sequon of N-X-S/T (Calo et al 2010; Nothaft and Szymanski 2010; Larkin and Imperiali 2011; Schwarz and Aebi; 2011; Eichler 2013)
H. pullorum N-linked protein glycosylation loci Orthologues of C. jejuni pglABCDEFHIJK genes that encode the well-characterised Nlinked protein glycosylation system are present in H. pullorum (Fig. 1) as well as the closely related species H. canadensis and H. winghamensis but not in other Helicobacter species
In contrast to C. jejuni and the majority of Campylobacter species where pgl genes are located in a single locus, in Helicobacter species these genes are present in five distinct loci

Summary

Introduction

In all three domains of life subsets of proteins are modified by the covalent attachment of sugars to asparagine residues within a conserved consensus sequon of N-X-S/T (Calo et al 2010; Nothaft and Szymanski 2010; Larkin and Imperiali 2011; Schwarz and Aebi; 2011; Eichler 2013). The second type of bacterial N-linked protein glycosylation system was discovered in Campylobacter jejuni (Szymanski et al 1999) and subsequently other related species from the Epsilon subdivision of the Proteobacteria (Nothaft et al 2010) In this system cytoplasmic assembly of an oligosaccharide on an isoprenoid lipid, is followed by transfer across the inner membrane and attachment onto proteins in the periplasm mediated by an integral membrane oligosaccharyltransferase (OTase). Five cytoplasmic glycosyltransferases (PglA, PglC, PglJ, PglH and PglI) assemble the heptasaccharide on the lipid carrier (Glover et al 2005; Linton et al 2006; Glover et al 2006) This is transported across the innermembrane and into the periplasm by the “flippase” PglK (Alaimo et al 2006) and transferred onto protein by the OTase, PglB (Wacker et al 2002; Glover et al 2005; Lizak et al 2011). The specificity of the C. jejuni PglB for lipid-linked oligosaccharides (LLOs) with an acetamido group on the C-2 carbon of the reducing end sugar and accessibility of the target sequon on the surface of the folded target protein, are the major limitations of this approach to generating N-linked glycoproteins of choice

Methods

Results

Conclusion