Cloning, expression and characterisation of a novel mollusc α-1,2-Fucosyltransferase from Crassostrea gigas (CgFUT2).

Glycans containing fucose play crucial roles in cell biology, particularly in recognition processes. In humans, fucose found in H-blood group antigens is recognized by various pathogens, thereby influencing host-pathogen interactions. However, in invertebrate biology the specific functions of these modifications and the corresponding glycosyltransferases are not fully elucidated.

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).

Expression and Characterisation of the First Snail-Derived UDP-Gal: Glycoprotein-N-acetylgalactosamine β-1,3-Galactosyltransferase (T-Synthase) from .

UDP-Gal: glycoprotein-N-acetylgalactosamine β-1,3-galactosyltransferase (T-synthase, EC 2.4.1.

Synthetic fragments of plant polysaccharides as tools for cell wall biology.

A sustainable bioeconomy that includes increased agricultural productivity and new technologies to convert renewable biomass to value-added products may help meet the demands of a growing world population for food, energy and materials. The potential use of plant biomass is determined by the properties of the cell walls, consisting of polysaccharides, proteins, and the polyphenolic polymer lignin. Comprehensive knowledge of cell wall glycan structure and biosynthesis is therefore essential for optimal utilization.

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.

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.

Utilization of different MurNAc sources by the oral pathogen Tannerella forsythia and role of the inner membrane transporter AmpG.

Background: The Gram-negative oral pathogen Tannerella forsythia strictly depends on the external supply of the essential bacterial cell wall sugar N-acetylmuramic acid (MurNAc) for survival because of the lack of the common MurNAc biosynthesis enzymes MurA/MurB. The bacterium thrives in a polymicrobial biofilm consortium and, thus, it is plausible that it procures MurNAc from MurNAc-containing peptidoglycan (PGN) fragments (muropeptides) released from cohabiting bacteria during natural PGN turnover or cell death. There is indirect evidence that in T.

Infrared and Raman spectra of lignin substructures: Dibenzodioxocin.

Vibrational spectroscopy is a very suitable tool for investigating the plant cell wall in situ with almost no sample preparation. The structural information of all different constituents is contained in a single spectrum. Interpretation therefore heavily relies on reference spectra and understanding of the vibrational behavior of the components under study.

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.

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.

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.

Pyruvate Substitutions on Glycoconjugates.

Glycoconjugates are the most diverse biomolecules of life. Mostly located at the cell surface, they translate into cell-specific "barcodes" and offer a vast repertoire of functions, including support of cellular physiology, lifestyle, and pathogenicity. Functions can be fine-tuned by non-carbohydrate modifications on the constituting monosaccharides.

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.

Synthesis of an Undecasaccharide Featuring an Oligomannosidic Heptasaccharide and a Bacterial Kdo-lipid A Backbone for Eliciting Neutralizing Antibodies to Mammalian Oligomannose on the HIV-1 Envelope Spike.

Lipooligosaccharides (LOS) from the bacterium Rhizobium radiobacter Rv3 are structurally related to antigenic mammalian oligomannoses on the HIV-1 envelope glycoprotein spike that are targets for broadly neutralizing antibodies. Here, we prepared a hybrid structure of viral and bacterial epitopes as part of a vaccine design strategy to elicit oligomannose-specific HIV-neutralizing antibodies using glycoconjugates based on the Rv3 LOS structure. Starting from a KdoGlcNAc tetrasaccharide precursor, a central orthogonally protected mannose trichloroacetimidate donor was coupled to OH-5 of the innermost Kdo residue.

Insights into Heptosyltransferase I Catalysis and Inhibition through the Structure of Its Ternary Complex.

Heptosyltransferase I (WaaC) is a highly conserved glycosyltransferase found in Gram-negative bacteria that transfers a heptose residue onto the endotoxin inner core structure (ReLPS) of the outer membrane. Knockouts of WaaC have decreased virulence and increased susceptibility to antibiotics, making WaaC a potential drug target. While previous studies have elucidated the structure of the holoenzyme and a donor analog complex, no information on the binding mode of the acceptor has been available so far.

Functional Characterization of Enzymatic Steps Involved in Pyruvylation of Bacterial Secondary Cell Wall Polymer Fragments.

Various mechanisms of protein cell surface display have evolved during bacterial evolution. Several Gram-positive bacteria employ S-layer homology (SLH) domain-mediated sorting of cell-surface proteins and concomitantly engage a pyruvylated secondary cell-wall polymer as a cell-wall ligand. Specifically, pyruvate ketal linked to β-D-ManNAc is regarded as an indispensable epitope in this cell-surface display mechanism.

Core Richness of N-Glycans of Caenorhabditis elegans: A Case Study on Chemical and Enzymatic Release.

Despite years of research, the glycome of the model nematode Caenorhabditis elegans is still not fully understood. Certainly, data over the years have indicated that this organism synthesizes unusual N-glycans with a range of galactose and fucose modifications on the ManGlcNAc core region. Previously, up to four fucose residues were detected on its N-glycans, despite these lacking the fucosylated antennae typical of many other eukaryotes; some of these fucose residues are capped with hexose residues as shown by the studies of us and others.

Synthesis of 3-- and 4--(2-aminoethylphosphono) derivatives of methyl l--α-d--heptopyranoside.

Abstract: Phosphoethanolamine derivatives of the bacterial saccharide l--d--heptose have been prepared using a phosphoramidite-based coupling reaction at position 4 of a side-chain-protected 2,3--orthoester methyl heptoside and at position 3 of a 3,4-diol heptoside, respectively. Global deprotection afforded the corresponding 2-aminoethylphosphodiester derivatives as substrates for crystallographic and binding studies with lectins and antibodies targeting the inner core structure of bacterial lipopolysaccharides.

The gymnosperm cytochrome P450 CYP750B1 catalyzes stereospecific monoterpene hydroxylation of (+)-sabinene in thujone biosynthesis in western redcedar.

Western redcedar (WRC; Thuja plicata) produces high amounts of oxygenated thujone monoterpenoids associated with resistance against herbivore feeding, particularly ungulate browsing. Thujones and other monoterpenoids accumulate in glandular structures in the foliage of WRC. Thujones are produced from (+)-sabinene by sabinol and sabinone.