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).

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.

The Tip of 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 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.

Quantitative Analyses Reveal Novel Roles for Glycosylation in a Major Enteric Bacterial Pathogen.

In eukaryotes, glycosylation plays a role in proteome stability, protein quality control, and modulating protein function; however, similar studies in bacteria are lacking. Here, we investigate the roles of general protein glycosylation systems in bacteria using the enteropathogen as a well-defined example. By using a quantitative proteomic strategy, we were able to monitor changes in the proteome when glycosylation is disrupted.

Mechanisms involved in the activation of C/EBPα by small activating RNA in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is generally accompanied by high mortality and low cure rate. CCAAT enhancer-binding proteins (CEBPs) are transcriptional regulators that play a key role in maintaining liver function. Altered expression of C/EBPα and C/EBPβ occurs in many tumours including HCC.

Clinical characteristics of COPD patients with tidal expiratory flow limitation.

We have used impulse oscillometry to identify COPD patients with tidal expiratory flow limitation (EFL), which is a measurement related to small airway disease. We report that 37.4% of COPD patients had EFL; these patients had multiple clinical characteristics of more severe disease including lower forced expiratory volume in 1 second values, greater hyperinflation, reduced exercise performance, and increased small airway impairment.

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.

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.

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.

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.

Analyzing the modification of the Shewanella oneidensis MR-1 flagellar filament.

The unsheathed flagellar filament of Shewanella oneidensis MR-1 is composed of two highly homologous flagellins, FlaA, and the major structural unit, FlaB. We identified a gene cluster, SO_3261-SO_3265 (now sfmABCDE), that is required for the formation of a fully functional filament and for motility. The predicted function of the corresponding gene products strongly indicated a role in flagellin modification.

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.

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

We studied O-linked β-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.

Gene function hypotheses for the Campylobacter jejuni glycome generated by a logic-based approach.

Increasingly, experimental data on biological systems are obtained from several sources and computational approaches are required to integrate this information and derive models for the function of the system. Here, we demonstrate the power of a logic-based machine learning approach to propose hypotheses for gene function integrating information from two diverse experimental approaches. Specifically, we use inductive logic programming that automatically proposes hypotheses explaining the empirical data with respect to logically encoded background knowledge.

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.

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.

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 (Galβ1-4(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.

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.

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.

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.

Systems analysis of bacterial glycomes.

Bacteria produce an array of glycan-based structures including capsules, lipo-oligosaccharide and glycosylated proteins, which are invariably cell-surface-located. For pathogenic bacteria, such structures are involved in diverse roles in the life cycle of the bacterium, including adhesion, colonization, avoidance of predation and interactions with the immune system. Compared with eukaryotes, bacteria produce huge combinatorial variations of glycan structures, which, coupled to the lack of genetic data, has previously hampered studies on bacterial glycans and their role in survival and pathogenesis.

Lipopolysaccharide from Burkholderia thailandensis E264 provides protection in a murine model of melioidosis.

Burkholderia thailandensis is a less virulent close relative of Burkholderia pseudomallei, a CDC category B biothreat agent. We have previously shown that lipopolysaccharide (LPS) extracted from B. pseudomallei can provide protection against a lethal challenge of B.

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.

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.

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.