Structure of the N-Linked Glycan Present on Multiple Glycoproteins in the Gram-negative Bacterium, Campylobacter jejuni

N Martin Young,Jean-Robert Brisson,John Kelly,David C Watson,Luc Tessier,Patricia H Lanthier,Harold C Jarrell,Nicolas Cadotte,Frank St Michael,Erika Aberg,Christine M Szymanski

doi:10.1074/jbc.m206114200

N Martin Young, Jean-Robert Brisson + Show 9 more

Open Access

https://doi.org/10.1074/jbc.m206114200

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Abstract

Mass spectrometry investigations of partially purified Campylobacter jejuni protein PEB3 showed it to be partially modified with an Asn-linked glycan with a mass of 1406 Da and composed of one hexose, five N-acetylhexosamines and a species of mass 228 Da, consistent with a trideoxydiacetamidohexose. By means of soybean lectin affinity chromatography, a mixture of glycoproteins was obtained from a glycine extract, and two-dimensional gel proteomics analysis led to the identification of at least 22 glycoproteins, predominantly annotated as periplasmic proteins. Glycopeptides were prepared from the glycoprotein mixture by Pronase digestion and gel filtration. The structure of the glycan was determined by using nano-NMR techniques to be GalNAc-alpha1,4-GalNAc-alpha1,4-[Glcbeta1,3-]GalNAc-alpha1,4-GalNAc-alpha1,4-GalNAc-alpha1,3-Bac-beta1,N-Asn-Xaa, where Bac is bacillosamine, 2,4-diacetamido-2,4,6-trideoxyglucopyranose. Protein glycosylation was abolished when the pglB gene was mutated, providing further evidence that the enzyme encoded by this gene is responsible for formation of the glycopeptide N-linkage. Comparison of the pgl locus with that of Neisseria meningitidis suggested that most of the homologous genes are probably involved in the biosynthesis of bacillosamine.

Highlights

Glycosylation of proteins was once considered to be a eukaryotic phenomenon, but it is clear that it is widespread in both the Archaea and Eubacteria domains [1, 2]
We have undertaken a comprehensive analysis of the C. jejuni NCTC 11168 glycoprotein complement, and in this report we present data on the identification of individual glycoproteins and structural characterization of the N-linked glycan moiety, which is common to all these glycoproteins, by mass spectrometry and NMR spectroscopy
PEB3 was purified from the extract by cation exchange chromatography, and re-fractionated on the same column, using a shallower NaCl gradient, resulting in the PEB3 appearing in three fractions (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Bacterial Strains and Plasmids—C. jejuni NCTC 11168 was routinely grown on Mueller Hinton agar under microaerophilic conditions (10% CO2, 5% O2, 85% N2) at 37 °C. The column eluate was monitored for UV absorbance at 280 nm, and fractions were examined by SDS-PAGE analysis [18] in Mini Protean II slab gels (Bio-Rad Laboratories). Analysis of Tryptic Peptides—Selected fractions were digested overnight at 37 °C with modified trypsin (Promega) in 50 mM ammonium bicarbonate and analyzed by capillary LC-tandem mass spectrometry using a capillary high-performance liquid chromatography system (CapLC, Waters) coupled with a Q-TOF2 hybrid quadrupole time-offlight mass spectrometer (Micromass). The fraction containing the glycopeptide was infused at a flow rate of 1 ␮l/min into the microelectrospray interface of the Q-TOF2 mass spectrometer. The peptide extracts from the intense protein spots were analyzed by MALDI-TOF MS using a M@LDI-LR mass spectrometer (Micromass). Membranes were probed with a 1:500 dilution of HS: serotyping serum followed by a 1:5000 dilution of goat anti-rabbit antiserum (SigmaAldrich) and developed with nitro blue tetrazolium chloride/5-bromo-4chloro-3-indolyl phosphate (Roche Molecular Biochemicals)

RESULTS

Putative periplasmic protein

DISCUSSION