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.

Non-lysosomal Degradation of Singly Phosphorylated Oligosaccharides Initiated by the Action of a Cytosolic Endo-β-N-acetylglucosaminidase

“…3A) (10, 13), providing conclusive evidence that the putative DLO-degrading enzyme is a DLO-pyrophosphatase that splits the pyrophosphate bond of DLOs producing mono-phosphorylated POSs. Although mono-phosphorylated POSs have been identified by MS in bacteria (30) or in milk oligosaccharides (31), to the best of our knowledge this is the first example of the use of MS for the identification of DLO-derived POSs of mammalian origin.…”

Swainsona Formosa (G.Don) Joy Thomp. Solvent Extractions Inhibit the Growth of a Panel of Pathogenic Bacteria

“…[9][10][11][12][13] Swainsonine induces toxicity by inhibiting α-mannosidase and mannosidase II activity, 10 which subsequently inhibits glycoprotein processing and maturation. Many pathogenic bacteria incorporate N-glycosylically linked surface proteins 11,12 and thus swainsonine may affect bacterial growth. However, this is yet to be adequately tested.…”

“…10 Interestingly, many pathogenic bacteria also contain N-glycosylates attached to their surface proteins. 11,12 Despite the ability of swainsonine to disrupt this process, there is a lack of studies examining the growth inhibitory properties of swainsonine, or of extracts containing this compound. S. formosa has also been reported to produce significant quantities of N,N-dimethyltryptamine (DMT; Figure 1c).…”

Demonstration of an oligosaccharide-diphosphodolichol diphosphatase activity whose subcellular localization is different than those of dolichyl-phosphate-dependent enzymes of the dolichol cycle

“…14), a formal demonstration that this type of reaction actually occurs has yet to be presented. In fact, DLO could be split by either a diphosphatase to yield OSP and DolP or a phospholipase-like enzyme to yield dolichol and an oligosaccharyl diphosphate (OSPP) intermediate that could subsequently be rapidly dephosphorylated to yield OSP (15). Finally, whether the biochemical profile of this in vitro activity is compatible with a role in DLO regulation has not been addressed.…”

“…The liberation of OSP from DLO could occur by either splitting of the diphosphate linkage by a diphosphatase or initial cleavage of the DolP linkage by a phospholipase-like enzyme followed by rapid conversion of OSPP to OSP (15). In order to look for a putative OSPP, Glc 3-0 [2-3 H]Man-5 GlcNAc 2 -PP-dolichol was incubated with HepG2 membranes under standard conditions in the presence of mannosidase and glucosidase inhibitors as described for Fig.…”