A combinatorial DNA assembly approach to biosynthesis of N-linked glycans in E. coli.

Glycoengineering of recombinant glycans and glycoconjugates is a rapidly evolving field. However, the production and exploitation of glycans has lagged behind that of proteins and nucleic acids. Biosynthetic glycoconjugate production requires the coordinated cooperation of three key components within a bacterial cell: a substrate protein, a coupling oligosaccharyltransferase, and a glycan biosynthesis locus. While the acceptor protein and oligosaccharyltransferase are the products of single genes, the glycan is a product of a multigene metabolic pathway. Typically, the glycan biosynthesis locus is cloned and transferred en bloc from the native organism to a suitable Escherichia coli strain. However, gene expression within these pathways has been optimized by natural selection in the native host and is unlikely to be optimal for heterologous production in an unrelated organism. In recent years, synthetic biology has addressed the challenges in heterologous expression of multigene systems by deconstructing these pathways and rebuilding them from the bottom up. The use of DNA assembly methods allows the convenient assembly of such pathways by combining defined parts with the requisite coding sequences in a single step. In this study, we apply combinatorial assembly to the heterologous biosynthesis of the Campylobacter jejuni  N-glycosylation (pgl) pathway in E. coli. We engineered reconstructed biosynthesis clusters that faithfully reproduced the C. jejuni heptasaccharide glycan. Furthermore, following a single round of combinatorial assembly and screening, we identified pathway clones that outperform glycan and glycoconjugate production of the native unmodified pgl cluster. This platform offers a flexible method for optimal engineering of glycan structures in E. coli.

Development of a novel glycoengineering platform for the rapid production of conjugate vaccines.

Conjugate vaccines produced either by chemical or biologically conjugation have been demonstrated to be safe and efficacious in protection against several deadly bacterial diseases. However, conjugate vaccine assembly and production have several shortcomings which hinders their wider availability. Here, we developed a tool, Mobile-element Assisted Glycoconjugation by Insertion on Chromosome, MAGIC, a novel biotechnological platform that overcomes the limitations of the current conjugate vaccine design method(s). As a model, we focused our design on a leading bioconjugation method using N-oligosaccharyltransferase (OTase), PglB. The installation of MAGIC led to at least twofold increase in glycoconjugate yield via MAGIC when compared to conventional N-OTase based bioconjugation method(s). Then, we improved MAGIC to (a) allow rapid installation of glycoengineering component(s), (b) omit the usage of antibiotics, (c) reduce the dependence on protein induction agents. Furthermore, we show the modularity of the MAGIC platform in performing glycoengineering in bacterial species that are less genetically tractable than the commonly used Escherichia coli. The MAGIC system promises a rapid, robust and versatile method to develop vaccines against serious bacterial pathogens. We anticipate the utility of the MAGIC platform could enhance vaccines production due to its compatibility with virtually any bioconjugation method, thus expanding vaccine biopreparedness toolbox.

The Type B Flagellin of Hypervirulent Clostridium difficile Is Modified with Novel Sulfonated Peptidylamido-glycans.

Glycosylation of flagellins is a well recognized property of many bacterial species. In this study, we describe the structural characterization of novel flagellar glycans from a number of hypervirulent strains of C. difficile We used mass spectrometry (nano-LC-MS and MS/MS analysis) to identify a number of putative glycopeptides that carried a variety of glycoform substitutions, each of which was linked through an initial N-acetylhexosamine residue to Ser or Thr. Detailed analysis of a LLDGSSTEIR glycopeptide released by tryptic digestion, which carried two variant structures, revealed that the glycopeptide contained, in addition to carbohydrate moieties, a novel structural entity. A variety of electrospray-MS strategies using Q-TOF technology were used to define this entity, including positive and negative ion collisionally activated decomposition MS/MS, which produced unique fragmentation patterns, and high resolution accurate mass measurement to allow derivation of atomic compositions, leading to the suggestion of a taurine-containing peptidylamido-glycan structure. Finally, NMR analysis of flagellin glycopeptides provided complementary information. The glycan portion of the modification was assigned as α-Fuc3N-(1→3)-α-Rha-(1→2)-α-Rha3OMe-(1→3)-ß-GlcNAc-(1→)Ser, and the novel capping moiety was shown to be comprised of taurine, alanine, and glycine. This is the first report of a novel O-linked sulfonated peptidylamido-glycan moiety decorating a flagellin protein.

Role of Glycosyltransferases Modifying Type B Flagellin of Emerging Hypervirulent Clostridium difficile Lineages and Their Impact on Motility and Biofilm Formation.

Clostridium difficile is the principal cause of nosocomial infectious diarrhea worldwide. The pathogen modifies its flagellin with either a type A or type B O-linked glycosylation system, which has a contributory role in pathogenesis. We study the functional role of glycosyltransferases modifying type B flagellin in the 023 and 027 hypervirulent C. difficile lineages by mutagenesis of five putative glycosyltransferases and biosynthetic genes. We reveal their roles in the biosynthesis of the flagellin glycan chain and demonstrate that flagellar post-translational modification affects motility and adhesion-related bacterial properties of these strains. We show that the glycosyltransferases 1 and 2 (GT1 and GT2) are responsible for the sequential addition of a GlcNAc and two rhamnoses, respectively, and that GT3 is associated with the incorporation of a novel sulfonated peptidyl-amido sugar moiety whose structure is reported in our accompanying paper (Bouché, L., Panico, M., Hitchen, P., Binet, D., Sastre, F., Faulds-Pain, A., Valiente, E., Vinogradov, E., Aubry, A., Fulton, K., Twine, S., Logan, S. M., Wren, B. W., Dell, A., and Morris, H. R. (2016) J. Biol. Chem. 291, 25439-25449). GT2 is also responsible for methylation of the rhamnoses. Whereas type B modification is not required for flagellar assembly, some mutations that result in truncation or abolition of the glycan reduce bacterial motility and promote autoaggregation and biofilm formation. The complete lack of flagellin modification also significantly reduces adhesion of C. difficile to Caco-2 intestinal epithelial cells but does not affect activation of human TLR5. Our study advances our understanding of the genes involved in flagellar glycosylation and their biological roles in emerging hypervirulent C. difficile strains.

Clinical characteristics of eosinophilic COPD versus COPD patients with a history of asthma.

Eosinophilic COPD appears to be a distinct patient subgroup with an increased corticosteroid response. Eosinophilic COPD has been labelled as part of the asthma COPD overlap syndrome (ACOS). We compared the clinical characteristics of eosinophilic COPD patients (without any clinical history of asthma) and COPD patients with a childhood history of asthma. COPD patients with asthma were characterised by more allergies and more exacerbations, but less eosinophilic inflammation. While terms such as "ACOS" are used to "lump" patients together, we report distinct differences between eosinophilic COPD and COPD patients with asthma, and propose that these groups should be split rather than lumped.

Glycoproteomic studies of IgE from a novel hyper IgE syndrome linked to PGM3 mutation.

Glycans serve as important regulators of antibody activities and half-lives. IgE is the most heavily glycosylated antibody, but in comparison to other antibodies little is known about its glycan structure function relationships. We therefore describe the site specific IgE glycosylation from a patient with a novel hyper IgE syndrome linked to mutations in PGM3, which is an enzyme involved in synthesizing UDP-GlcNAc, a sugar donor widely required for glycosylation. A two-step method was developed to prepare two IgE samples from less than 1 mL of serum collected from a patient with PGM3 mutation and a patient with atopic dermatitis as a control subject. Then, a glycoproteomic strategy was used to study the site-specific glycosylation. No glycosylation was found at Asn264, whilst high mannose glycans were only detected at Asn275, tri-antennary glycans were exclusively observed at Asn99 and Asn252, and non-fucosylated complex glycans were detected at Asn99. The results showed similar glycosylation profiles between the two IgE samples. These observations, together with previous knowledge of IgE glycosylation, imply that IgE glycosylation is similarly regulated among healthy control, allergy and PGM3 related hyper IgE syndrome.

Z-band alternatively spliced PDZ motif protein (ZASP) is the major O-linked ß-N-acetylglucosamine-substituted protein in human heart myofibrils.

We studied O-linked ß-N-acetylglucosamine (O-GlcNAc) modification of contractile proteins in human heart using SDS-PAGE and three detection methods: specific enzymatic conjugation of O-GlcNAc with UDP-N-azidoacetylgalactosamine (UDP-GalNAz) that is then linked to a tetramethylrhodamine fluorescent tag and CTD110.6 and RL2 monoclonal antibodies to O-GlcNAc. All three methods showed that O-GlcNAc modification was predominantly in a group of bands ~90 kDa that did not correspond to any of the major myofibrillar proteins. MALDI-MS/MS identified the 90-kDa band as the protein ZASP (Z-band alternatively spliced PDZ motif protein), a minor component of the Z-disc (about 1 per 400 α-actinin) important for myofibrillar development and mechanotransduction. This was confirmed by the co-localization of O-GlcNAc and ZASP in Western blotting and by immunofluorescence microscopy. O-GlcNAcylation of ZASP increased in diseased heart, being 49 ± 5% of all O-GlcNAc in donor, 68 ± 9% in end-stage failing heart, and 76 ± 6% in myectomy muscle samples (donor versus myectomy p < 0.05). ZASP is only 22% of all O-GlcNAcylated proteins in mouse heart myofibrils.

Latrophilin 1 and its endogenous ligand Lasso/teneurin-2 form a high-affinity transsynaptic receptor pair with signaling capabilities.

Latrophilin 1 (LPH1), a neuronal receptor of α-latrotoxin, is implicated in neurotransmitter release and control of presynaptic Ca(2+). As an "adhesion G-protein-coupled receptor," LPH1 can convert cell surface interactions into intracellular signaling. To examine the physiological functions of LPH1, we used LPH1's extracellular domain to purify its endogenous ligand. A single protein of ∼275 kDa was isolated from rat brain and termed Lasso. Peptide sequencing and molecular cloning have shown that Lasso is a splice variant of teneurin-2, a brain-specific orphan cell surface receptor with a function in neuronal pathfinding and synaptogenesis. We show that LPH1 and Lasso interact strongly and specifically. They are always copurified from rat brain extracts. Coculturing cells expressing LPH1 with cells expressing Lasso leads to their mutual attraction and formation of multiple junctions to which both proteins are recruited. Cells expressing LPH1 form chimerical synapses with hippocampal neurons in cocultures; LPH1 and postsynaptic neuronal protein PSD-95 accumulate on opposite sides of these structures. Immunoblotting and immunoelectron microscopy of purified synapses and immunostaining of cultured hippocampal neurons show that LPH1 and Lasso are enriched in synapses; in both systems, LPH1 is presynaptic, whereas Lasso is postsynaptic. A C-terminal fragment of Lasso interacts with LPH1 and induces Ca(2+) signals in presynaptic boutons of hippocampal neurons and in neuroblastoma cells expressing LPH1. Thus, LPH1 and Lasso can form transsynaptic complexes capable of inducing presynaptic Ca(2+) signals, which might affect synaptic functions.

Agl16, a thermophilic glycosyltransferase mediating the last step of N-Glycan biosynthesis in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius.

Recently, the S-layer protein of Sulfolobus acidocaldarius was shown to be N-linked with a tribranched hexasaccharide, composed of Man2Glc1GlcNAc2 and a sulfated sugar called sulfoquinovose. To identify genes involved in the biosynthesis and attachment of this glycan, markerless in-frame deletions of genes coding for predicted glycosyltransferases were created. The successful deletion of agl16, coding for a glycosyltransferase, resulted in the S-layer protein and archaellins having reduced molecular weights, as visualized by Coomassie staining or immunoblotting. This analysis indicated a change in the N-glycan composition. Nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses confirmed that the glycan of the S-layer protein from the agl16 deletion mutant was a pentasaccharide, which was missing a terminal hexose residue. High-performance liquid chromatography (HPLC) analyses of the hydrolyzed N-glycan indicated that the missing hexose is a glucose residue. A physiological characterization of the agl16 deletion mutant revealed a significant effect on the growth at elevated salt concentrations. At 300 mM NaCl, the doubling time of the Δagl16 mutant was increased 2-fold compared to that of the background strain. Furthermore, the incomplete glycan structure of the Δagl16 deletion strain affected the assembly and function of the archaellum, as exemplified by semisolid Gelrite plate analysis, in which the motility is decreased according to the N-glycan size.

Identification of neutrophil granule glycoproteins as Lewis(x)-containing ligands cleared by the scavenger receptor C-type lectin.

The scavenger receptor C-type lectin (SRCL) is a glycan-binding receptor that has the capacity to mediate endocytosis of glycoproteins carrying terminal Lewis(x) groups (Galß1-4(Fucα1-3)GlcNAc). A screen for glycoprotein ligands for SRCL using affinity chromatography on immobilized SRCL followed by mass spectrometry-based proteomic analysis revealed that soluble glycoproteins from secondary granules of neutrophils, including lactoferrin and matrix metalloproteinases 8 and 9, are major ligands. Binding competition and surface plasmon resonance analysis showed affinities in the low micromolar range. Comparison of SRCL binding to neutrophil and milk lactoferrin indicates that the binding is dependent on cell-specific glycosylation in the neutrophils, as the milk form of the glycoprotein is a much poorer ligand. Binding to neutrophil glycoproteins is fucose-dependent, and mass spectrometry-based glycomic analysis of neutrophil and milk lactoferrin was used to establish a correlation between high affinity binding to SRCL and the presence of multiple clustered terminal Lewis(x) groups on a heterogeneous mixture of branched glycans, some with poly N-acetyllactosamine extensions. The ability of SRCL to mediate uptake of neutrophil lactoferrin was confirmed using fibroblasts transfected with SRCL. The common presence of Lewis(x) groups in granule protein glycans can thus target granule proteins for clearance by SRCL. PCR and immunohistochemical analysis confirm that SRCL is widely expressed on endothelial cells and thus represents a distributed system that could scavenge released neutrophil glycoproteins both locally at sites of inflammation or systemically when they are released in the circulation.

Glycoproteomic characterization of recombinant mouse α-dystroglycan.

α-Dystroglycan (DG) is a key component of the dystrophin-glycoprotein complex. Aberrant glycosylation of the protein has been linked to various forms of congenital muscular dystrophy. Unusually α-DG has previously been demonstrated to be modified with both O-N-acetylgalactosamine and O-mannose initiated glycans. In the present study, Fc-tagged recombinant mouse α-DG was expressed and purified from human embryonic kidney 293T cells. α-DG glycopeptides were characterized by glycoproteomic strategies using both nano-liquid chromatography matrix-assisted laser desorption ionization and electrospray tandem mass spectrometry. A total of 14 different peptide sequences and 38 glycopeptides were identified which displayed heterogeneous O-glycosylation. These data provide new insights into the complex domain-specific O-glycosylation of α-DG.

The Tip of Brucella O-Polysaccharide Is a Potent Epitope in Response to Brucellosis Infection and Enables Short Synthetic Antigens to Be Superior Diagnostic Reagents.

Brucellosis is a global disease and the world's most prevalent zoonosis. All cases in livestock and most cases in humans are caused by members of the genus Brucella that possess a surface O-polysaccharide (OPS) comprised of a rare monosaccharide 4-deoxy-4-formamido-D-mannopyranose assembled with α1,2 and α1,3 linkages. The OPS of the bacterium is the basis for serodiagnostic tests for brucellosis. Bacteria that also contain the same rare monosaccharide can induce antibodies that cross-react in serological tests. In previous work we established that synthetic oligosaccharides, representing elements of the Brucella A and M polysaccharide structures, were excellent antigens to explore the antibody response in the context of infection, immunisation and cross reaction. These studies suggested the existence of antibodies that are specific to the tip of the Brucella OPS. Sera from naturally and experimentally Brucella abortus-infected cattle as well as from cattle experimentally infected with the cross-reactive bacterium Yersinia enterocolitica O:9 and field sera that cross react in conventional serological assays were studied here with an expanded panel of synthetic antigens. The addition of chemical features to synthetic antigens that block antibody binding to the tip of the OPS dramatically reduced their polyclonal antibody binding capability providing conclusive evidence that the OPS tip (non-reducing end) is a potent epitope. Selected short oligosaccharides, including those that were exclusively α1,2 linked, also demonstrated superior specificity when evaluated with cross reactive sera compared to native smooth lipopolysaccharide (sLPS) antigen and capped native OPS. This surprising discovery suggests that the OPS tip epitope, even though common to both Brucella and Y. enterocolitica O:9, has more specific diagnostic properties than the linear portion of the native antigens. This finding opens the way to the development of improved serological tests for brucellosis.

N-glycosylation in Archaea: on the coordinated actions of Haloferax volcanii AglF and AglM.

Like Eukarya and Bacteria, Archaea are also capable of performing N-glycosylation. In the halophilic archaeon Haloferax volcanii, N-glycosylation is mediated by the products of the agl gene cluster. In the present report, this gene cluster was expanded to include an additional sequence, aglM, shown to participate in the biosynthesis of hexuronic acids contained within a pentasaccharide decorating the S-layer glycoprotein, a reporter H. volcanii glycoprotein. In response to different growth conditions, changes in the transcription profile of aglM mirrored changes in the transcription profiles of aglF, aglG and aglI, genes encoding confirmed participants in the H. volcanii N-glycosylation pathway, thus offering support to the hypothesis that in H. volcanii, N-glycosylation serves an adaptive role. Following purification, biochemical analysis revealed AglM to function as a UDP-glucose dehydrogenase. In a scoupled reaction with AglF, a previously identified glucose-1-phosphate uridyltransferase, UDP-glucuronic acid was generated from glucose-1-phosphate and UTP in a NAD(+)-dependent manner. These experiments thus represent the first step towards in vitro reconstitution of the archaeal N-glycosylation process.

AglP is a S-adenosyl-L-methionine-dependent methyltransferase that participates in the N-glycosylation pathway of Haloferax volcanii.

While pathways for N-glycosylation in Eukarya and Bacteria have been solved, considerably less is known of this post-translational modification in Archaea. In the halophilic archaeon Haloferax volcanii, proteins encoded by the agl genes are involved in the assembly and attachment of a pentasaccharide to select asparagine residues of the S-layer glycoprotein. AglP, originally identified based on the proximity of its encoding gene to other agl genes whose products were shown to participate in N-glycosylation, was proposed, based on sequence homology, to serve as a methyltransferase. In the present report, gene deletion and mass spectrometry were employed to reveal that AglP is responsible for adding a 14 Da moiety to a hexuronic acid found at position four of the pentasaccharide decorating the Hfx. volcanii S-layer glycoprotein. Subsequent purification of a tagged version of AglP and development of an in vitro assay to test the function of the protein confirmed that AglP is a S-adenosyl-L-methionine-dependent methyltransferase.

Glycoproteomic characterization of carriers of the CD15/Lewisx epitope on Hodgkin's Reed-Sternberg cells.

BACKGROUND: The Lewisx trisaccharide, also referred to as the CD15 antigen, is a diagnostic marker used to distinguish Hodgkin's lymphoma from other lymphocytic cancers. However, the role of such fucosylated structures remains poorly understood, in part because carriers of Lewisx structures on Hodgkin's Reed-Sternberg cells have not been identified. METHODS: GalMBP, an engineered carbohydrate-recognition protein that binds selectively to oligosaccharides with paired terminal galactose and fucose residues, has been used in conjunction with proteomic and glycomic analysis to identify glycoprotein carriers of Lewisx and related glycan structures in multiple Hodgkin's Reed-Sternberg cell lines. RESULTS: Multiple glycoproteins that bind to GalMBP and carry CD15/Lewisx have been identified in a panel of six Reed-Sternberg cell lines. The most commonly identified Lewisx-bearing glycoproteins are CD98hc, which was found in all six cell lines tested, and intercellular adhesion molecule-1 and DEC-205, which were detected in five and four of the lines, respectively. Thus, several of the most prominent cell adhesion molecules on the lymphomas carry this characteristic glycan epitope. In addition, the Hodgkin's Reed-Sternberg cell lines can be grouped into subsets based on the presence or absence of less common Lewisx-bearing glycoproteins. CONCLUSIONS: CD98 and intercellular adhesion molecule-1 are major carriers of CD15/Lewisx on Reed-Sternberg cells. Binding of DC-SIGN and other glycan-specific receptors to the Lewisx epitopes on CD98 and intercellular adhesion molecule-1 may facilitate interaction of the lymphoma cells with lymphocytes and myeloid cells in lymph nodes.

AglJ adds the first sugar of the N-linked pentasaccharide decorating the Haloferax volcanii S-layer glycoprotein.

Like the Eukarya and Bacteria, the Archaea also perform N glycosylation. Using the haloarchaeon Haloferax volcanii as a model system, a series of Agl proteins involved in the archaeal version of this posttranslational modification has been identified. In the present study, the participation of HVO_1517 in N glycosylation was considered, given its homology to a known component of the eukaryal N-glycosylation pathway and because of the genomic proximity of HVO_1517 to agl genes encoding known elements of the H. volcanii N-glycosylation process. By combining the deletion of HVO_1517 with mass spectrometric analysis of both dolichol phosphate monosaccharide-charged carriers and the S-layer glycoprotein, evidence was obtained showing the participation of HVO_1517, renamed AglJ, in adding the first hexose of the N-linked pentasaccharide decorating this reporter glycoprotein. The deletion of aglJ, however, did not fully prevent the attachment of a hexose residue to the S-layer glycoprotein. Moreover, in the absence of AglJ, the level of only one of the three monosaccharide-charged dolichol phosphate carriers detected in the cell was reduced. Nonetheless, in cells lacking AglJ, no further sugar subunits were added to the remaining monosaccharide-charged dolichol phosphate carriers or to the monosaccharide-modified S-layer glycoprotein, pointing to the importance of the sugar added through the actions of AglJ for proper N glycosylation. Finally, while aglJ can be deleted, H. volcanii surface layer integrity is compromised in the absence of the encoded protein.

Characterization of N-linked protein glycosylation in Helicobacter pullorum.

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.

Modification of the Campylobacter jejuni flagellin glycan by the product of the Cj1295 homopolymeric-tract-containing gene.

The Campylobacter jejuni flagellin protein is O-glycosylated with structural analogues of the nine-carbon sugar pseudaminic acid. The most common modifications in the C. jejuni 81-176 strain are the 5,7-di-N-acetylated derivative (Pse5Ac7Ac) and an acetamidino-substituted version (Pse5Am7Ac). Other structures detected include O-acetylated and N-acetylglutamine-substituted derivatives (Pse5Am7Ac8OAc and Pse5Am7Ac8GlnNAc, respectively). Recently, a derivative of pseudaminic acid modified with a di-O-methylglyceroyl group was detected in C. jejuni NCTC 11168 strain. The gene products required for Pse5Ac7Ac biosynthesis have been characterized, but those genes involved in generating other structures have not. We have demonstrated that the mobility of the NCTC 11168 flagellin protein in SDS-PAGE gels can vary spontaneously and we investigated the role of single nucleotide repeats or homopolymeric-tract-containing genes from the flagellin glycosylation locus in this process. One such gene, Cj1295, was shown to be responsible for structural changes in the flagellin glycoprotein. Mass spectrometry demonstrated that the Cj1295 gene is required for glycosylation with the di-O-methylglyceroyl-modified version of pseudaminic acid.

The S-layer glycoprotein of the crenarchaeote Sulfolobus acidocaldarius is glycosylated at multiple sites with chitobiose-linked N-glycans.

Glycosylation of the S-layer of the crenarchaea Sulfolobus acidocaldarius has been investigated using glycoproteomic methodologies. The mature protein is predicted to contain 31 N-glycosylation consensus sites with approximately one third being found in the C-terminal domain spanning residues L(1004)-Q(1395). Since this domain is rich in Lys and Arg and therefore relatively tractable to glycoproteomic analysis, this study has focused on mapping its N-glycosylation. Our analysis identified nine of the 11 consensus sequence sites, and all were found to be glycosylated. This constitutes a remarkably high glycosylation density in the C-terminal domain averaging one site for each stretch of 30-40 residues. Each of the glycosylation sites observed was shown to be modified with a heterogeneous family of glycans, with the largest having a composition Glc(1)Man(2)GlcNAc(2) plus 6-sulfoquinovose (QuiS), consistent with the tribranched hexasaccharide previously reported in the cytochrome b(558/566) of S. acidocaldarius. S. acidocaldarius is the only archaeal species whose N-glycans are known to be linked via the chitobiose core disaccharide that characterises the N-linked glycans of Eukarya.

Glycoproteomics: a powerful tool for characterizing the diverse glycoforms of bacterial pilins and flagellins.

With glycosylation now firmly established across both Archaeal and bacterial proteins, a wide array of glycan diversity has become evident from structural analysis and genomic data. These discoveries have been built in part on the development and application of mass spectrometric technologies to the bacterial glycoproteome. This review highlights recent findings using high sensitivity MS of the large variation of glycans that have been reported on flagellin and pilin proteins of bacteria, using both 'top down' and 'bottom up' approaches to the characterization of these glycoproteins. We summarize current knowledge of the sugar modifications that have been observed on flagellins and pilins, in terms of both the diverse repertoire of monosaccharides observed, and the assemblage of moieties that decorate many of these sugars.