Paul G. Hitchen

Mass spectrometry in the analysis of N-linked and O-linked glycans.

Mass spectrometry (MS) continues to play a vital role in defining the structures of N-glycans and O-glycans in glycoproteins via glycomic and glycoproteomic methodologies. The former seeks to define the total N-glycan and/or O-glycan repertoire in a biological sample whilst the latter is concerned with the analysis of glycopeptides. Recent technical developments have included improvements in tandem mass spectrometry (MS/MS and MS(n)) sequencing methodologies, more sensitive methods for analysing sulfated and polysialylated glycans and better procedures for defining the sites of O-glycosylation. New tools have been introduced to assist data handling and publicly accessible databases are being populated with glycomics data. Progress is exemplified by recent research in the fields of glycoimmunology, reproductive glycobiology, stem cells, bacterial glycosylation and non-mucin O-glycosylation.

Phase variation of a β-1,3 galactosyltransferase involved in generation of the ganglioside GM1-like lipo-oligosaccharide of Campylobacter jejuni

Ganglioside mimicry by Campylobacter jejuni lipo‐oligosaccharide (LOS) is thought to be a critical factor in the triggering of the Guillain–Barré and Miller–Fisher syndrome neuropathies after C. jejuni infection. The combination of a completed genome sequence and a ganglioside GM1‐like LOS structure makes C. jejuni NCTC 11168 a useful model strain for the identification and characterization of the genes involved in the biosynthesis of ganglioside‐mimicking LOS. Genome analysis identified a putative LOS biosynthetic cluster and, from this, we describe a putative gene (ORF Cj1139c), which we have termed wlaN, with a significant level of similarity to a number of bacterial glycosyltransferases. Mutation of this gene in C. jejuni NCTC 11168 resulted in a LOS molecule of increased electrophoretic mobility, which also failed to bind cholera toxin. Comparison of LOS structural data from wild type and the mutant strain indicated lack of a terminal β‐1,3‐linked galactose residue in the latter. The wlaN gene product was demonstrated unambiguously as a β‐1,3 galactosyltransferase responsible for converting GM2‐like LOS structures to GM1‐like by in vitro expression. We also show that the presence of an intragenic homopolymeric tract renders the expression of a functional wlaN gene product phase variable, resulting in distinct C. jejuni NCTC 11168 cell populations with alternate GM1 or GM2 ganglioside‐mimicking LOS structures. The distribution of wlaN among a number of C. jejuni strains with known LOS structure was determined and, for C. jejuni NCTC 12500, similar wlaN gene phase variation was shown to occur, so that this strain has the potential to synthesize a GM1‐like LOS structure as well as the ganglioside GM2‐like LOS structure proposed in the literature.

Functional analysis of the Campylobacter jejuni N‐linked protein glycosylation pathway

We describe in this report the characterization of the recently discovered N‐linked glycosylation locus of the human bacterial pathogen Campylobacter jejuni, the first such system found in a species from the domain Bacteria. We exploited the ability of this locus to function in Escherichia coli to demonstrate through mutational and structural analyses that variant glycan structures can be transferred onto protein indicating the relaxed specificity of the putative oligosaccharyltransferase PglB. Structural data derived from these variant glycans allowed us to infer the role of five individual glycosyltransferases in the biosynthesis of the N‐linked heptasaccharide. Furthermore, we show that C. jejuni‐ and E. coli‐derived pathways can interact in the biosynthesis of N‐linked glycoproteins. In particular, the E. coli encoded WecA protein, a UDP‐GlcNAc: undecaprenylphosphate GlcNAc‐1‐phosphate transferase involved in glycolipid biosynthesis, provides for an alternative N‐linked heptasaccharide biosynthetic pathway bypassing the requirement for the C. jejuni‐derived glycosyltransferase PglC. This is the first experimental evidence that biosynthesis of the N‐linked glycan occurs on a lipid‐linked precursor prior to transfer onto protein. These findings provide a framework for understanding the process of N‐linked protein glycosylation in Bacteria and for devising strategies to exploit this system for glycoengineering.

Multiple N-acetyl neuraminic acid synthetase (neuB) genes in Campylobacter jejuni: identification and characterization of the gene involved in sialylation of lipo-oligosaccharide.

N‐acetyl neuraminic acid (NANA) is a common constituent of Campylobacter jejuni lipo‐oligosaccharide (LOS). Such structures often mimic human gangliosides and are thought to be involved in the triggering of Guillain–Barré syndrome (GBS) and Miller–Fisher syndrome (MFS) following C. jejuni infection. Analysis of the C. jejuni NCTC 11168 genome sequence identified three putative NANA synthetase genes termed neuB1, neuB2 and neuB3. The NANA synthetase activity of all three C. jejuni neuB gene products was confirmed by complementation experiments in an Escherichia coli neuB‐deficient strain. Isogenic mutants were created in all three neuB genes, and for one such mutant (neuB1) LOS was shown to have increased mobility. C. jejuni NCTC 11168 wild‐type LOS bound cholera toxin, indicating the presence of NANA in a LOS structure mimicking the ganglioside GM1. This property was lost in the neuB1 mutant. Gas chromatography–mass spectrometry and fast atom bombardment–mass spectrometry analysis of LOS from wild‐type and the neuB1 mutant strain demonstrated the lack of NANA in the latter. Expression of the neuB1 gene in E. coli confirmed that NeuB1 was capable of in vitro NANA biosynthesis through condensation of N‐acetyl‐d‐mannosamine and phosphoenolpyruvate. Southern analysis demonstrated that the neuB1 gene was confined to strains of C. jejuni with LOS containing a single NANA residue. Mutagenesis of neuB2 and neuB3 did not affect LOS, but neuB3 mutants were aflagellate and non‐motile. No phenotype was evident for neuB2 mutants in strain NCTC 11168, but for strain G1 the flagellin protein from the neuB2 mutant showed an apparent reduction in molecular size relative to the wild type. Thus, the neuB genes of C. jejuni appear to be involved in the biosynthesis of at least two distinct surface structures: LOS and flagella.

Similarities and differences in the glycosylation mechanisms in prokaryotes and eukaryotes.

Recent years have witnessed a rapid growth in the number and diversity of prokaryotic proteins shown to carry N- and/or O-glycans, with protein glycosylation now considered as fundamental to the biology of these organisms as it is in eukaryotic systems. This article overviews the major glycosylation pathways that are known to exist in eukarya, bacteria and archaea. These are (i) oligosaccharyltransferase (OST)-mediated N-glycosylation which is abundant in eukarya and archaea, but is restricted to a limited range of bacteria; (ii) stepwise cytoplasmic N-glycosylation that has so far only been confirmed in the bacterial domain; (iii) OST-mediated O-glycosylation which appears to be characteristic of bacteria; and (iv) stepwise O-glycosylation which is common in eukarya and bacteria. A key aim of the review is to integrate information from the three domains of life in order to highlight commonalities in glycosylation processes. We show how the OST-mediated N- and O-glycosylation pathways share cytoplasmic assembly of lipid-linked oligosaccharides, flipping across the ER/periplasmic/cytoplasmic membranes, and transferring “en bloc” to the protein acceptor. Moreover these hallmarks are mirrored in lipopolysaccharide biosynthesis. Like in eukaryotes, stepwise O-glycosylation occurs on diverse bacterial proteins including flagellins, adhesins, autotransporters and lipoproteins, with O-glycosylation chain extension often coupled with secretory mechanisms.

Mechanism of Ca2+ and Monosaccharide Binding to a C-type Carbohydrate-recognition Domain of the Macrophage Mannose Receptor

Site-directed mutagenesis has been used to identify residues that ligate Ca2+ and sugar to the fourth C-type carbohydrate-recognition domain (CRD) of the macrophage mannose receptor. CRD-4 is the only one of the eight CRDs of the mannose receptor to exhibit detectable monosaccharide binding when expressed in isolation, and it is central to ligand binding by the receptor. CRD-4 requires two Ca2+ for sugar binding, like the CRD of rat serum mannose-binding protein (MBP-A). Sequence comparisons between the two CRDs suggest that the binding site for one Ca2+, which ligates directly to the bound sugar in MBP-A, is conserved in CRD-4 but that the auxiliary Ca2+ binding site is not. Mutation of the four residues at positions in CRD-4 equivalent to the auxiliary Ca2+ binding site in MBP-A indicates that only one, Asn728, is involved in ligation of Ca2+. Alanine-scanning mutagenesis was used to identify two other asparagine residues and one glutamic acid residue that are probably involved in ligation of the auxiliary Ca2+ to CRD-4. Sequence comparisons with other C-type CRDs suggest that the proposed binding site for the auxiliary Ca2+ in CRD-4 of the mannose receptor is unique. Evidence that the conserved Ca2+ in CRD-4 bridges between the protein and bound sugar in a manner analogous to MBP-A was obtained by mutation of one of the amino acid side chains at this site. Ring current shifts seen in the 1H NMR spectra of methyl glycosides of mannose, GlcNAc, and fucose in the presence of CRD-4 and site-directed mutagenesis indicate that a stacking interaction with Tyr729 is also involved in binding of sugars to CRD-4. This interaction contributes about 25% of the total free energy of binding to mannose. C-5 and C-6 of mannose interact with Tyr729, whereas C-2 of GlcNAc is closest to this residue, indicating that these two sugars bind to CRD-4 in opposite orientations. Sequence comparisons with other mannose/GlcNAc-specific C-type CRDs suggest that use of a stacking interaction in the binding of these sugars is probably unique to CRD-4 of the mannose receptor.

The immunologically distinct O antigens from Francisella tularensis subspecies tularensis and Francisella novicida are both virulence determinants and protective antigens.

ABSTRACT We have determined the sequence of the gene cluster encoding the O antigen in Francisella novicida and compared it to the previously reported O-antigen cluster in Francisella tularensis subsp. tularensis. Immunization with purified lipopolysaccharide (LPS) from F. tularensis subsp. tularensis or F. novicida protected against challenge with Francisella tularensis subsp. holarctica and F. novicida, respectively. The LPS from F. tularensis subsp. tularensis did not confer protection against challenge with F. novicida, and the LPS from F. novicida did not confer protection against challenge with F. tularensis subsp. holarctica. Allelic replacement mutants of F. tularensis subsp. tularensis or F. novicida which failed to produce O antigen were attenuated, but exposure to these mutants did not induce a protective immune response. The O antigen of F. tularensis subsp. tularensis appeared to be important for intracellular survival whereas the O antigen of F. novicida appeared to be critical for serum resistance and less important for intracellular survival.

Structural characterization of lipo‐oligosaccharide (LOS) from Yersinia pestis: regulation of LOS structure by the PhoPQ system

The two‐component regulatory system PhoPQ has been shown to regulate the expression of virulence factors in a number of bacterial species. For one such virulence factor, lipopolysaccharide (LPS), the PhoPQ system has been shown to regulate structural modifications in Salmonella enterica var Typhi‐murium. In Yersinia pestis, which expresses lipo‐oligosaccharide (LOS), a PhoPQ regulatory system has been identified and an isogenic mutant constructed. To investigate potential modifications to LOS from Y. pestis, which to date has not been fully characterized, purified LOS from wild‐type plague and the phoP defective mutant were analysed by mass spectrometry. Here we report the structural characterization of LOS from Y. pestis and the direct comparison of LOS from a phoP mutant. Structural modifications to lipid A, the host signalling portion of LOS, were not detected but analysis of the core revealed the expression of two distinct molecular species in wild‐type LOS, differing in terminal galactose or heptose. The phoP mutant was restricted to the expression of a single molecular species, containing terminal heptose. The minimum inhibitory concentration of cationic antimicrobial peptides for the two strains was determined and compared with the wild‐type: the phoP mutant was highly sensitive to polymyxin. Thus, LOS modification is under the control of the PhoPQ regulatory system and the ability to alter LOS structure may be required for survival of Y. pestis within the mammalian and/or flea host.

Inactivation of Corynebacterium glutamicum NCgl0452 and the role of MgtA in the biosynthesis of a novel mannosylated glycolipid involved in lipomannan biosynthesis.

Mycobacterium tuberculosis PimB has been demonstrated to catalyze the addition of a mannose residue from GDP-mannose to a monoacylated phosphatidyl-myo-inositol mannoside (Ac1PIM1) to generate Ac1PIM2. Herein, we describe the disruption of its probable orthologue Cg-pimB and the chemical analysis of glycolipids and lipoglycans isolated from wild type Corynebacterium glutamicum and the C. glutamicum::pimB mutant. Following a careful analysis, two related glycolipids, Gl-A and Gl-X, were found in the parent strain, but Gl-X was absent from the mutant. The biosynthesis of Gl-X was restored in the mutant by complementation with either Cg-pimB or Mt-pimB. Subsequent chemical analyses established Gl-X as 1,2-di-O-C16/C18:1-(α-d-mannopyranosyl)-(1→4)-(α-d-glucopyranosyluronic acid)-(1→3)-glycerol (ManGlcAGroAc2) and Gl-A as the precursor, GlcAGroAc2. In addition, C. glutamicum::pimB was still able to produce Ac1PIM2, suggesting that Cg-PimB catalyzes the synthesis of ManGlcAGroAc2 from GlcAGroAc2. Isolation of lipoglycans from C. glutamicum led to the identification of two related lipoglycans. The larger lipoglycan possessed a lipoarabinomannan-like structure, whereas the smaller lipoglycan was similar to lipomannan (LM). The absence of ManGlcA-GroAc2 in C. glutamicum::pimB led to a severe reduction in LM. These results suggested that ManGlcAGroAc2 was further extended to an LM-like molecule. Complementation of C. glutamicum::pimB with Cg-pimB and Mt-pimB led to the restoration of LM biosynthesis. As a result, Cg-PimB, which we have assigned as MgtA, is now clearly defined as a GDP-mannose-dependent α-mannosyltransferase from our in vitro analyses and is involved in the biosynthesis of ManGlcAGroAc2.

Identification of AglE, a Second Glycosyltransferase Involved in N Glycosylation of the Haloferax volcanii S-Layer Glycoprotein

Archaea, like Eukarya and Bacteria, are able to N glycosylate select protein targets. However, in contrast to relatively advanced understanding of the eukaryal N glycosylation process and the information being amassed on the bacterial process, little is known of this posttranslational modification in Archaea. Toward remedying this situation, the present report continues ongoing efforts to identify components involved in the N glycosylation of the Haloferax volcanii S-layer glycoprotein. By combining gene deletion together with mass spectrometry, AglE, originally identified as a homologue of murine Dpm1, was shown to play a role in the addition of the 190-Da sugar subunit of the novel pentasaccharide decorating the S-layer glycoprotein. Topological analysis of an AglE-based chimeric reporter assigns AglE as an integral membrane protein, with its N terminus and putative active site facing the cytoplasm. These finding, therefore, contribute to the developing picture of the N glycosylation pathway in Archaea.