Potentiating Activity of GmhA Inhibitors on Gram-Negative Bacteria.

Inhibition of the biosynthesis of bacterial heptoses opens novel perspectives for antimicrobial therapies. The enzyme GmhA responsible for the first committed biosynthetic step catalyzes the conversion of sedoheptulose 7-phosphate into d--d--heptose 7-phosphate and harbors a Zn ion in the active site. A series of phosphoryl- and phosphonyl-substituted derivatives featuring a hydroxamate moiety were designed and prepared from suitably protected ribose or hexose derivatives.

Molecular modelling and site-directed mutagenesis provide insight into saccharide pyruvylation by the Paenibacillus alvei CsaB enzyme.

Pyruvylation is a biologically versatile but mechanistically unexplored saccharide modification. 4,6-Ketal pyruvylated N-acetylmannosamine within bacterial secondary cell wall polymers serves as a cell wall anchoring epitope for proteins possessing a terminal S-layer homology domain trimer. The pyruvyltransferase CsaB from Paenibacillus alvei served as a model to investigate the structural basis of the pyruvyltransfer reaction by a combination of molecular modelling and site-directed mutagenesis together with an enzyme assay using phosphoenolpyruvate (PEP; donor) and synthetic β-D-ManNAc-(1 → 4)-α-D-GlcNAc-diphosphoryl-11-phenoxyundecyl (acceptor).

Site-Specific Multi-Functionalization of the Carrier Protein CRM by Disulfide Rebridging for Conjugate Vaccine Development.

Conjugation of an antigen to a carrier protein is widely used for vaccine development. To develop the next generation of conjugate vaccines, we describe here a method for the controlled multi-functionalization of the widely employed carrier protein CRM with a carbohydrate-based antigen and an immune potentiator. The approach is based on the selective reduction of one of the disulfides of CRM followed by disulfide rebridging employing an appropriately functionalized dibromopyridazinedione.

Antigen binding by conformational selection in near-germline antibodies.

Conformational flexibility in antibody-combining sites has been hypothesized to facilitate polyspecificity toward multiple unique epitopes and enable the limited germline repertoire to match an overwhelming diversity of potential antigens; however, elucidating the mechanisms of antigen recognition by flexible antibodies has been understandably challenging. Here, multiple liganded and unliganded crystal structures of the near-germline anticarbohydrate antibodies S25-2 and S25-39 are reported, which reveal an unprecedented diversity of complementarity-determining region H3 conformations in apparent equilibrium. These structures demonstrate that at least some germline or near-germline antibodies are flexible entities sensitive to their chemical environments, with conformational selection available as an evolved mechanism that preserves the inherited ability to recognize common pathogens while remaining adaptable to new threats.

A Lead-Based Fragment Library Screening of the Glycosyltransferase WaaG from .

Glucosyl transferase I (WaaG) in catalyzes the transfer of an α-d-glucosyl group to the inner core of the lipopolysaccharide (LPS) and plays an important role in the biogenesis of the outer membrane. If its activity could be inhibited, the integrity of the outer membrane would be compromised and the bacterium would be susceptible to antibiotics that are normally prevented from entering the cell. Herein, three libraries of molecules (A, B and C) were docked in the binding pocket of WaaG, utilizing the docking binding affinity as a filter to select fragment-based compounds for further investigations.

The S-layer homology domains of Paenibacillus alvei surface protein SpaA bind to cell wall polysaccharide through the terminal monosaccharide residue.

Self-assembling (glyco)protein surface layers (S-layers) are ubiquitous prokaryotic cell-surface structures involved in structural maintenance, nutrient diffusion, host adhesion, virulence, and other processes, which makes them appealing targets for therapeutics and biotechnological applications as biosensors or drug delivery systems. However, unlocking this potential requires expanding our understanding of S-layer properties, especially the details of surface-attachment. S-layers of Gram-positive bacteria often are attached through the interaction of S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs).

Synthetic Neoglycoconjugates of Hepta- and Nonamannoside Ligands for Eliciting Oligomannose-Specific HIV-1-Neutralizing Antibodies.

Oligomannose-type glycans on the spike protein of HIV-1 constitute relevant epitopes to elicit broadly neutralizing antibodies (bnAbs). Herein we describe an improved synthesis of α- and β-linked hepta- and nonamannosyl ligands that were subsequently converted into BSA and CRM neoglycoconjugates. We assembled the ligands from anomeric 3-azidopropyl spacer glycosides from select 3-O-protected thiocresyl mannoside donors.

Achieving Absolute Molar Lipid Concentrations: A Phospholipidomics Cross-Validation Study.

Standardization is essential in lipidomics and part of a huge community effort. However, with the still ongoing lack of reference materials, benchmarking quantification is hampered. Here, we propose traceable lipid class quantification as an important layer for the validation of quantitative lipidomics workflows.

A Combination of Structural, Genetic, Phenotypic and Enzymatic Analyses Reveals the Importance of a Predicted Fucosyltransferase to Protein -Glycosylation in the Bacteroidetes.

Diverse members of the Bacteroidetes phylum have general protein -glycosylation systems that are essential for processes such as host colonization and pathogenesis. Here, we analyzed the function of a putative fucosyltransferase (FucT) family that is widely encoded in Bacteroidetes protein -glycosylation genetic loci. We studied the FucT orthologs of three Bacteroidetes species-, , and .

Potential of Inulin-Fructooligosaccharides Extract Produced from Red Onion ( var. (Metz) Mansf.) as an Alternative Prebiotic Product.

Red onion is a popular ingredient in many Thai dishes and has recently been promoted for commercial cultivation. In this study, inulin-fructooligosaccharides (inulin-FOSs) were extracted from red onions in a simplified extraction method. The extract contained 24.

Assaying CsaB-Catalysed Ketalpyruvyltransfer to Saccharides by Measurement of Phosphate Release.

Ketalpyruvyltransferases belong to a widespread but little investigated class of enzymes, which utilise phosphoenolpyruvate (PEP) for the pyruvylation of saccharides. Pyruvylated saccharides play pivotal biological roles, ranging from protein binding to virulence. Limiting factors for the characterisation of ketalpyruvyltransferases are the availability of cognate acceptor substrates and a straightforward enzyme assay.

Determination of modification degree of polysialylated therapeutic proteins using H-NMR spectroscopy.

Water soluble polymers and their derivatives bound to proteins can dramatically favor the biological activity of new drugs and vaccines. Quantification of the modification degree of the protein is crucial during the development and licensing phase and later in order to monitor the industrial production process and to match product specification. In this work, we describe an innovative way to measure directly the modification degree of polysialylated proteins using proton NMR (Nuclear Magnetic Resonance) spectroscopy.

The Structural Difference of Isobaric -Glycans of Two Microalgae Samples Reveals Taxonomic Distance.

Microalgae of the clade are extensively investigated as an environmentally friendly source of renewable biofuels and high-value nutrients. In addition, essentially unprocessed serves as wholesome food additive. A recent study on 80 commercial preparations revealed an unexpected variety of protein-linked -glycan patterns with unprecedented structural features, such as the occurrence of arabinose.

Rhamnogalacturonan II: Chemical Synthesis of a Substructure Including α-2,3-Linked Kdo*.

The synthesis of a fully deprotected Kdo-containing rhamnogalacturonan II pentasaccharide is described. The strategy relies on the preparation of a suitably protected homogalacturonan tetrasaccharide backbone, through a post-glycosylation oxidation approach, and its stereoselective glycosylation with a Kdo fluoride donor.

A glycoside analog of mammalian oligomannose formulated with a TLR4-stimulating adjuvant elicits HIV-1 cross-reactive antibodies.

The occurrence of oligomannose-specific broadly neutralizing antibodies (bnAbs) has spurred efforts to develop immunogens that can elicit similar antibodies. Here, we report on the antigenicity and immunogenicity of a CRM-conjugate of a previously reported oligomannose mimetic. Oligomannose-specific bnAbs that are less dependent on interactions with the HIV envelope protein sequence showed strong binding to the glycoconjugates, with affinities approximating those reported for their cognate epitope.

Serum alpha-mannosidase as an additional barrier to eliciting oligomannose-specific HIV-1-neutralizing antibodies.

Oligomannose-type glycans on HIV-1 gp120 form a patch that is targeted by several broadly neutralizing antibodies (bnAbs) and that therefore is of interest to vaccine design. However, attempts to elicit similar oligomannose-specific bnAbs by immunizing with oligomannosidic glycoconjugates have only been modestly successful so far. A common assumption is that eliciting oligomannose-specific bnAbs is hindered by B cell tolerance, resulting from the presented oligomannosides being sensed as self molecules.

The N-glycans of Chlorella sorokiniana and a related strain contain arabinose but have strikingly different structures.

The many emerging applications of microalgae such as Chlorella also instigate interest in their ability to conduct protein modifications such as N-glycosylation. Chlorella vulgaris has recently been shown to equip its proteins with highly O-methylated oligomannosidic N-glycans. Two other frequently occurring species names are Chlorella sorokiniana and Chlorella pyrenoidosa-even though the latter is taxonomically ill defined.

Synthesis of -glycosyl phosphonate derivatives of 4-amino-4-deoxy-α-ʟ-arabinose.

The incorporation of basic substituents into the structurally conserved domains of cell wall lipopolysaccharides has been identified as a major mechanism contributing to antimicrobial resistance of Gram-negative pathogenic bacteria. Inhibition of the corresponding enzymatic steps, specifically the transfer of 4-amino-4-deoxy-ʟ-arabinose, would thus restore the activity of cationic antimicrobial peptides and several antimicrobial drugs. -glycosidically-linked phospholipid derivatives of 4-amino-4-deoxy-ʟ-arabinose have been prepared as hydrolytically stable and chain-shortened analogues of the native undecaprenyl donor.

Zwitterionic Phosphodiester-Substituted Neoglycoconjugates as Ligands for Antibodies and Acute Phase Proteins.

Zwitterionic modifications of glycans, such as phosphorylcholine and phosphoethanolamine, are known from a range of prokaryotic and eukaryotic species and are recognized by mammalian antibodies and pentraxins; however, defined saccharide ligands modified with these zwitterionic moieties for high-throughput studies are lacking. In this study, we prepared and tested example mono- and disaccharides 6-substituted with either phosphorylcholine or phosphoethanolamine as bovine serum albumin neoglycoconjugates or printed in a microarray format for subsequent assessment of their binding to lectins, pentraxins, and antibodies. C-Reactive protein and anti-phosphorylcholine antibodies bound specifically to ligands with phosphorylcholine, but recognition by concanavalin A was abolished or decreased as compared with that to the corresponding nonzwitterionic compounds.

Are there specific antibodies against Neu5Gc epitopes in the blood of healthy individuals?

Strong discrepancies in published data on the levels and epitope specificities of antibodies against the xenogenic N-glycolyl forms of sialoglycans (Hanganutziu-Deicher Neu5Gcɑ2-3Galβ1-4Glc and related antigens) in healthy donors prompted us to carry out a systematic study in this area using the printed glycan array and other methods. This article summarizes and discusses our published and previously unpublished data, as well as publicly available data from the Consortium for Functional Glycomics. As a result, we conclude that (1) the level of antibodies referred to as anti-Neu5Gc in healthy individuals is low; (2) there are antibodies that seem to interact with Neu5Gc-containing epitopes, but in fact they recognize internal fragments of Neu5Gc-containing glycans (without sialic acids), which served as antigens in the assays used and; (3) a population capable of interacting specifically with Neu5Gc (it does not bind the corresponding NAc analogs) does exist, but it binds the monosaccharide Neu5Gc better than the entire glycans containing it.

Molecular Conformations of Di-, Tri-, and Tetra--(2→8)-Linked Sialic Acid from NMR Spectroscopy and MD Simulations.

By using molecular dynamics simulations with an efficient enhanced sampling technique and in combination with nuclear magnetic resonance (NMR) spectroscopy quantitative structural information on α -2,8-linked sialic acids is presented. We used a bottom-up approach to obtain a set of larger ensembles for tetra- and deca-sialic acid from model dimer and trimer systems that are in agreement with the available J-coupling constants and nuclear Overhauser effects. The molecular dynamic (MD) simulations with enhanced sampling are used to validate the force field used in this study for its further use.

2-O-Benzyloxycarbonyl protected glycosyl donors: a revival of carbonate-mediated anchimeric assistance for diastereoselective glycosylation.

By reviving an old idea, we demonstrate that alkoxycarbonyl groups can be used in glycosylation reactions to achieve full stereocontrol through participation of a carbonate moiety at O-2. Various benzyloxycarbonyl-protected glycosyl donors were prepared and used for efficient 1,2-trans glycosylation of base-labile compounds and the synthesis of glycosyl esters.

Synthetic Phosphodiester-Linked 4-Amino-4-deoxy-l-arabinose Derivatives Demonstrate that ArnT is an Inverting Aminoarabinosyl Transferase.

4-Amino-4-deoxy-l-arabinopyranose (Ara4N) residues have been linked to antibiotic resistance due to reduction of the negative charge in the lipid A and core regions of the bacterial lipopolysaccharide (LPS). To study the enzymatic transfer of Ara4N onto lipid A, which is catalysed by the ArnT transferase, we chemically synthesised a series of anomeric phosphodiester-linked lipid Ara4N derivatives containing linear aliphatic chains as well as E- and Z-configured monoterpene units. Coupling reactions were based on sugar-derived H-phosphonates, followed by oxidation and global deprotection.