Characterization of N-linked protein glycosylation in Helicobacter pullorum

Abstract

The first bacterial N-linked glycosylation system was discovered in Campylobacter jejuni, and the key enzyme involved in the coupling of glycan to asparagine residues within the acceptor sequon of the glycoprotein is the oligosaccharyltransferase PglB. Emerging genome sequence data have revealed that pglB orthologues are present in a subset of species from the Deltaproteobacteria and Epsilonproteobacteria, including three Helicobacter species: H. pullorum, H. canadensis, and H. winghamensis. In contrast to C. jejuni, in which a single pglB gene is located within a larger gene cluster encoding the enzymes required for the biosynthesis of the N-linked glycan, these Helicobacter species contain two unrelated pglB genes (pglB1 and pglB2), neither of which is located within a larger locus involved in protein glycosylation. In complementation experiments, the H. pullorum PglB1 protein, but not PglB2, was able to transfer C. jejuni N-linked glycan onto an acceptor protein in Escherichia coli. Analysis of the characterized C. jejuni N-glycosylation system with an in vitro oligosaccharyltransferase assay followed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry demonstrated the utility of this approach, and when applied to H. pullorum, PglB1-dependent N glycosylation with a linear pentasaccharide was observed. This reaction required an acidic residue at the -2 position of the N-glycosylation sequon, as for C. jejuni. Attempted insertional knockout mutagenesis of the H. pullorum pglB2 gene was unsuccessful, suggesting that it is essential. These first data on N-linked glycosylation in a second bacterial species demonstrate the similarities to, and fundamental differences from, the well-studied C. jejuni system.

FIG. 1.

Schematic representation of the C. jejuni pgl gene N-glycosylation locus and orthologues present in H. pullorum. The percent values below open reading frames are the levels of identity between amino acid sequences encoded by H. pullorum and C. jejuni pgl gene orthologues and between H. pullorum PglB1 and PglB2. Arrows representing genes are not to scale.

FIG. 2.

Complementation analysis of H. pullorum pglB1. (A) The C. jejuni Cj0114 gene product with four potential N-glycosylation sites (boldface type) within C. jejuni N-glycosylation sequons (italic). (B) The C. jejuni Cj0114 gene on plasmid pET0114 and the site-directed mutants indicated were expressed in E. coli cells along with either the complete C. jejuni pgl locus on plasmid pACYCpgl containing all genes required for biosynthesis and the transfer of the C. jejuni heptasaccharide (46) or a version (pACYCpglB::kan) lacking PglB function (24). The functional complementation of C. jejuni PglB function was tested by coexpressing the H. pullorum pglB1 gene on plasmid pMLHP1. The hexahistidine-tagged Cj0114 protein and glycosylated forms of decreased mobility were detected with anti-His antibodies.

FIG. 3.

In vitro analysis of C. jejuni oligosaccharyltransferase activity. (A) Detergent-solubilized C. jejuni membrane preparations were assayed for oligosaccharyltransferase activity with peptides as indicated. Following Tricine-SDS-PAGE, the highest-mobility band in each lane is the unmodified peptide, indicated with an asterisk, and decreased-mobility bands are presumed derived glycosylated forms. The decreased mobility of the AQNAT-containing peptide relative to those of DQNAT- and DQQAT-containing peptides is due to the loss of a negatively charged residue. The C. jejuni NCTC 11168- and corresponding pglB knockout mutant-derived membrane preparations were mixed in a 1:1 ratio. (B) Products of the oligosaccharyltransferase assay with membrane preparations from wild-type strain C. jejuni NCTC 11168 and the corresponding insertional knockout mutants in the pglH, pglJ, and pglA genes.

FIG. 4.

In vitro analysis of H. pullorum oligosaccharyltransferase activity. Detergent-solubilized membrane preparations from H. pullorum, H. pullorum pglB1::kan, H. pullorum pglDEF::kan, and E. coli pMAF10 (7) were assayed for oligosaccharyltransferase activity with fluorescent peptides as indicated.

FIG. 5.

MALDI-MS/MS analysis of C. jejuni and H. pullorum N-linked glycans. (A) MS/MS spectrum of the m/z 3,015 precursor ion generated following incubation of C. jejuni membranes with a biotin-labeled fluorescent peptide (FITC-ADQNATAK-biotin, with a mass of 1,587 Da). Fragment ions resulting from the sequential loss of sugar residues are indicated in the spectrum. In both C. jejuni (A)- and H. pullorum (B)-derived spectra, a peak corresponding to the y ion of the biotinylated peptide lacking FITC was present at m/z 1,199. (B) MS/MS spectrum of the m/z 2,645 precursor ion generated following incubation of FITC-ADQNATAK-biotin with H. pullorum membranes. The peaks labeled with an asterisk (m/z 1,384 and 2,627) are likely generated by dehydration. The peak labeled with a cross at m/z 1,182, 17 Da less than the m/z 1,199 peak, is characteristic of the fragmentation of a side-chain amide bond of an N-linked glycan. a.u., arbitrary units. (C) The N-linked glycan structure of the C. jejuni heptasaccharide (51) is consistent with the fragmentation pattern of the glycopeptide as observed for A. (D) N-linked glycan structure of the H. pullorum-generated glycopeptide inferred from the fragmentation pattern in B.