title: Generation of free oligosaccharides from bacterial protein N-linked glycosylation systems.
abstract: All Campylobacter species are capable of N-glycosylating their proteins and releasing the same oligosaccharides into the periplasm as free oligosaccharides (fOS). Previously, analysis of fOS production in Campylobacter required fOS derivatization or large culture volumes and several chromatography steps prior to fOS analysis. In this study, label-free fOS extraction and purification methods were developed and coupled with quantitative analysis techniques. Our method follows three simple steps: (1) fOS extraction from the periplasmic space, (2) fOS purification using silica gel chromatography followed by porous graphitized carbon purification and (3) fOS analysis and accurate quantitation using a combination of thin-layer chromatography, mass spectrometry, NMR, and high performance anion exchange chromatography with pulsed amperometric detection. We applied our techniques to analyze fOS from C. jejuni, C. lari, C. rectus, and C. fetus fetus that produce different fOS structures. We accurately quantified fOS in Campylobacter species that ranged from 7.80 (±0.84) to 49.82 (±0.46) nmoles per gram of wet cell pellet and determined that the C. jejuni fOS comprises 2.5% of the dry cell weight. In addition, a novel di-phosphorylated fOS species was identified in C. lari. This method provides a sensitive and quantitative method to investigate the genesis, biology and breakdown of fOS in the bacterial N-glycosylation systems.
authors.affiliation: Alberta Glycomics Center and Department of Biological Sciences, University of Alberta, Canada.
authors.forename: Ritika
authors.initials: R
authors.lastname: Dwivedi
authors.forename: Harald
authors.initials: H
authors.lastname: Nothaft
authors.forename: Bela
authors.initials: B
authors.lastname: Reiz
authors.forename: Randy M
authors.initials: RM
authors.lastname: Whittal
authors.forename: Christine M
authors.initials: CM
authors.lastname: Szymanski
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D016123:Campylobacter jejuni; D006031:Glycosylation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D009844:Oligosaccharides
keywords: NMR; campylobacter; free oligosaccharides; mass spectrometry; protein glycosylation