Metabolic glycoengineering (MGE) is an established method to incorporate chemical reporter groups into cellular glycans for subsequent bioorthogonal labeling. The method has found broad application for the visualization and isolation of glycans allowing their biological roles to be probed. Furthermore, targeting of drugs to cancer cells that present high concentrations of sialic acids on their surface is an attractive approach. We report the application of a labeling reaction using 1,2-diamino-4,5-methylenedioxybenzene for the quantification of sialic acid derivates after MGE with various azide- and alkene-modified ManNAc, GlcNAc, and GalNAc derivatives. We followed the time course of sialic acid production and were able to detect sialic acids modified with the chemical reporter group - not only after addition of ManNAc derivatives to the cell culture. A cyclopropane-modified ManNAc derivative, being a model for the corresponding cyclopropene analog, which undergoes fast inverse-electron-demand Diels-Alder reactions with 1,2,4,5-tetrazines, resulted in the highest incorporation efficiency. Furthermore, we investigated whether feeding the cells with natural and unnatural ManNAc derivative results in increased levels of sialic acids and found that this is strongly dependent on the investigated cell type and cell fraction. For HEK 293T cells, a strong increase in free sialic acids in the cell interior was found, whereas cell-surface sialic acid levels are only moderately increased.

Human beta-hexosaminidase A (Hex A) (alphabeta) is composed of two subunits whose primary structures are approximately 60% identical. Deficiency of either subunit results in severe neurological disease due to the storage of GM2 ganglioside; Tay-Sachs disease, alpha deficiency, and Sandhoff disease, beta deficiency. Whereas both subunits contain active sites only the alpha-site can efficiently bind negatively charged 6-sulfated hexosamine substrates and GM2 ganglioside. We have recently identified the alphaArg(424) as playing a critical role in the binding of 6-sulfate-containing substrates, and betaAsp(452) as actively inhibiting their binding. To determine if these same residues affect the binding of the sialic acid moiety of GM2 ganglioside, an alphaArg(424)Gln form of Hex A was expressed and its kinetics analyzed using the GM2 activator protein:[3H]-GM2 ganglioside complex as a substrate. The mutant showed a approximately 3-fold increase in its K(m) for the complex. Next a form of Hex B (betabeta) containing a double mutation, betaAspLeu(453)AsnArg (duplicating the alpha-aligning sequences), was expressed. As compared to the wild type (WT), the mutant exhibited a >30-fold increase in its ability to hydrolyze a 6-sulfated substrate and was now able to hydrolyze GM2 ganglioside when the GM2 activator protein was replaced by sodium taurocholate. Thus, this alpha-site is critical for binding both types of negatively charge substrates.

The binding of monosaccharides and short peptides to lymphocyte receptors (human CD69 and rat NKR-P1A) was first reported in 1994 and then in a number of subsequent publications. Based on this observation, numerous potentially high-affinity saccharide ligands have been synthesized over the last two decades in order to utilize their potential in antitumor therapy. Due to significant inconsistencies in their reported binding properties, we decided to re-examine the interaction between multiple ligands and CD69 or NKR-P1A. Using NMR titration and isothermal titration calorimetry we were unable to detect the binding of the tested ligands such as N-acetyl-D-hexosamines and oligopeptides to both receptors, which contradicts the previous observations published in more than twenty papers over the last fifteen years.

A divalent cation-independent lectin-HOL-18, with cytotoxic activity against leukemia cells, was purified from a demosponge, Halichondria okadai. HOL-18 is a 72 kDa tetrameric lectin that consists of four non-covalently bonded 18 kDa subunits. Hemagglutination activity of the lectin was strongly inhibited by chitotriose (GlcNAcβ1-4GlcNAcβ1-4GlcNAc), fetuin and mucins from porcine stomach and bovine submaxillary gland. Lectin activity was stable at pH 4-12 and temperatures lower than 60 °C. Frontal affinity chromatography with 16 types of pyridylaminated oligosaccharides indicated that the lectin had an affinity for N-linked complex-type and sphingolipid-type oligosaccharides with N-acetylated hexosamines and neuramic acid at the non-reducing termini. The lectin killed Jurkat leukemia T cells and K562 erythroleukemia cells in a dose- and carbohydrate-dependent manner.

The introduction of unnatural chemical moieties into glycosaminoglycans (GAGs) has enormous potential to facilitate studies of the mechanism and application of these critical, widespread molecules. Unnatural N-acetylhexosamine analogs were metabolically incorporated into the capsule polysaccharides of Escherichia coli and Bacillus subtilis via bacterial metabolism. Targeted metabolic labeled hyaluronan and the precursors of heparin and chondroitin sulfate were obtained. The azido-labeled polysaccharides (purified or in capsules) were reacted with dyes, via bioorthogonal chemistry, to enable detection and imaging. Site-specific introduction of fluorophores directly onto cell surfaces affords another choice for observing and quantifying bacteria in vivo and in vitro. Furthermore, azido-polysaccharides retain similar biological properties to their natural analogs, and reliable and predictable introduction of functionalities, such as fluorophores, onto azido-N-hexosamines in the disaccharide repeat units provides chemical tools for imaging and metabolic analysis of GAGs in vivo and in vitro.

In recent years, oxidative stress and free radicals have been implicated in impaired wound healing. Ocimum sanctum (O. sanctum), a plant widely used in Ayurveda, possesses anti-inflammatory and antioxidant properties. The present study was undertaken to assess the potential of alcoholic and aqueous extracts in wound healing in Wistar albino rats. The rats were divided into five groups of six animals each. Group 1 is normal wounded control and the other four groups were treated with two different doses each of alcoholic and aqueous extract of O. sanctum. The wound healing parameters were evaluated by using incision, excision and dead space wounds in extract-treated rats and controls. Both the doses of alcoholic and aqueous extract significantly increased wound breaking strength, hydroxyproline, hexuronic acid, hexosamines, superoxide dismutase, catalase, reduced glutathione and significantly decreased percentage of wound contraction and lipid peroxidation when compared with the control group. The results suggest that O. sanctum has antioxidant properties, which may be responsible and favorable for faster wound healing and this plant extract may be useful in the management of abnormal healing and hypertropic scars.

T cell development requires the coordinated rearrangement of T cell receptor (TCR) gene segments and the expression of either αβ or γδ TCR. However, whether and how de novo synthesis of nutrients contributes to thymocyte commitment to either lineage remains unclear. Here, we find that T cell-specific deficiency in glutamine:fructose-6-phosphate aminotransferase 1 (GFAT1), the rate-limiting enzyme of the de novo hexosamine biosynthesis pathway (dn-HBP), attenuates hexosamine levels, blunts N-glycosylation of TCRβ chains, reduces surface expression of key developmental receptors, thus impairing αβ-T cell ontogeny. GFAT1 deficiency triggers defects in N-glycans, increases the unfolded protein response, and elevates  γδ-T cell numbers despite reducing γδ-TCR diversity. Enhancing TCR expression or PI3K/Akt signaling does not reverse developmental defects. Instead, dietary supplementation with the salvage metabolite, glucosamine, and an α-ketoglutarate analogue partially restores αβ-T cell development in GFAT1T-/- mice, while fully rescuing it in ex vivo fetal thymic organ cultures. Thus, dn-HBP fulfils, while salvage nutrients partially satisfy, the elevated demand for hexosamines during early T cell development.

Bifidobacterium breve C50 secretes a lipoprotein associated with glucose, acting in an aggregating form (>600kDa) as an agonist of TLR2/6. Similar lipoproteins were sought for in bifidobacteria. In silico, the closest homology was shown with a Bifidobacterium longum protein containing CHAP and lipobox domains. Two strains secreted aggregates whose peptides sequences aligned with the mined protein. C16:0 and C18:0 fatty acids detected in the aggregates further supported a lipoprotein structure. Glucose and mannose detected by gas chromatography were likely ligands of the lipoprotein. The binding of aggregates to galectin-1 indicated that hexosamines and galactose surrounded them. However, unlike B. breve C50, aggregate secreted by B. longum CBi0703 was unable to bind TLR2/6 likely because of a more hydrophobic structure. In gnotobiotic mice, the intake of B. longum aggregate induced, in splenic dendritic cells, the expression of genes involved in antigen presentation. A positive correlation between the number of dendritic cells and CD4(+)CD25(+) cells was observed in mice receiving these aggregates. In conclusion, B. longum secretes a lipoprotein forming aggregates which may influence dendritic and CD4(+)CD25(+) cell interactions independently of the TLR2/6 pathway.

Malaria, the world's most devastating parasitic disease, is transmitted between humans by mosquitoes of the Anopheles genus. An. gambiae is the principal malaria vector in Sub-Saharan Africa. The C-type lectins CTL4 and CTLMA2 cooperatively influence Plasmodium infection in the malaria vector Anopheles. Here we report the purification and biochemical characterization of CTL4 and CTLMA2 from An. gambiae and An. albimanus. CTL4 and CTLMA2 are known to form a disulfide-bridged heterodimer via an N-terminal tri-cysteine CXCXC motif. We demonstrate in vitro that CTL4 and CTLMA2 intermolecular disulfide formation is promiscuous within this motif. Furthermore, CTL4 and CTLMA2 form higher oligomeric states at physiological pH. Both lectins bind specific sugars, including glycosaminoglycan motifs with β1-3/β1-4 linkages between glucose, galactose and their respective hexosamines. Small-angle x-ray scattering data supports a compact heterodimer between the CTL domains. Recombinant CTL4/CTLMA2 is found to function in vivo, reversing the enhancement of phenol oxidase activity in dsCTL4-treated mosquitoes. We propose these molecular features underline a common function for CTL4/CTLMA2 in mosquitoes, with species and strain-specific variation in degrees of activity in response to Plasmodium infection.

The transfer of toxic cyanobacterial Microcystis blooms from freshwater to estuaries constitutes a serious environmental problem worldwide that is expected to expand in scale and intensity with anthropogenic and climate change. The formation and maintenance of Microcystis in colonial form is conditioned to the presence of extracellular polymeric substances (EPS). In this study, we attempted to better understand how the mucilaginous colonial form of Microcystis evolves under environmental stress conditions. In particular, we studied and compared the production and the composition of EPS fractions (attached and free) from natural colonies of a Microcystis bloom and from a unicellular M. aeruginosa strain under salinity and nutrient stress (representing a land-sea continuum). Our results highlighted a greater production of EPS from the natural colonies of Microcystis than the unicellular one under nutrient and combined stress conditions dominated by the attached form. In comparison to the unicellular Microcystis, EPS produced by the colonial form were characterized by high molecular weight polysaccharides which were enriched in uronic acids and hexosamines, notably for the free fraction in response to increased salinities. This complex extracellular matrix gives the cells the ability to aggregate and allows the colonial cyanobacterial population to cope with osmotic shock.

Recently, we isolated two marine strains, F1T and F21T, which together with Kiritimatiella glycovorans L21-Fru-ABT are the only pure cultures of the class Kiritimatiellae within the phylum Verrucomicrobiota. Here, we present an in-depth genome-guided characterization of both isolates with emphasis on their exopolysaccharide synthesis. The strains only grew fermentatively on simple carbohydrates and sulfated polysaccharides. Strains F1T, F21T and K. glycovorans reduced elemental sulfur, ferric citrate and anthraquinone-2,6-disulfonate during anaerobic growth on sugars. Both strains produced exopolysaccharides during stationary phase, probably with intracellularly stored glycogen as energy and carbon source. Exopolysaccharides included N-sulfated polysaccharides probably containing hexosamines and thus resembling glycosaminoglycans. This implies that the isolates can both degrade and produce sulfated polysaccharides. Both strains encoded an unprecedently high number of glycoside hydrolase genes (422 and 388, respectively), including prevalent alpha-L-fucosidase genes, which may be necessary for degrading complex sulfated polysaccharides such as fucoidan. Strain F21T encoded three putative glycosaminoglycan sulfotransferases and a putative sulfate glycosaminoglycan biosynthesis gene cluster. Based on phylogenetic and chemotaxonomic analyses, we propose the taxa Pontiella desulfatans F1T gen. nov., sp. nov. and Pontiella sulfatireligans F21T sp. nov. as representatives of the Pontiellaceae fam. nov. within the class Kiritimatiellae.

Glycans of cell surface glycoproteins are involved in the regulation of cell migration, growth, and differentiation. N-acetyl-glucosaminyltransferase V (GnT-V) transfers N-acetyl-d-glucosamine to form β1,6-branched N-glycans, thus playing a crucial role in the biosynthesis of glycoproteins. This study reveals the distinct expression of GnT-V in STRO-1 and CD-146 double-positive dental pulp stem cells (DPSCs). Furthermore, we investigated three types of hexosamines and their N-acetyl derivatives for possible effects on the osteogenic differentiation potential of DPSCs. Our results showed that exogenous d-glucosamine (GlcN), N-acetyl-d-glucosamine (GlcNAc), d-mannosamine (ManN), and acetyl-d-mannosamine (ManNAc) promoted DPSCs' early osteogenic differentiation in the absence of osteogenic supplements, but d-galactosamine (GalN) or N-acetyl-galactosamine (GalNAc) did not. Effects include the increased level of TGF-β receptor type I, activation of TGF-β signaling, and increased mRNA expression of osteogenic differentiation marker genes. The hexosamine-treated DPSCs showed an increased mineralized matrix deposition in the presence of osteogenic supplements. Moreover, the level of TGF-β receptor type I and early osteogenic differentiation were abolished in the DPSCs transfected with siRNA for GnT-V knockdown. These results suggest that GnT-V plays a critical role in the hexosamine-induced activation of TGF-β signaling and subsequent osteogenic differentiation of DPSCs.

Analyses of energy metabolism in human cancer have been difficult because of rapid turnover of the metabolites and difficulties in reducing time for collecting clinical samples under surgical procedures. Utilization of xenograft transplantation of human-derived colon cancer HCT116 cells in spleens of superimmunodeficient NOD/SCID/IL-2Rγ(null) (NOG) mice led us to establish an experimental model of hepatic micrometastasis of the solid tumor, whereby analyses of the tissue sections collected by snap-frozen procedures through newly developed microscopic imaging mass spectrometry (MIMS) revealed distinct spatial distribution of a variety of metabolites. To perform intergroup comparison of the signal intensities of metabolites among different tissue sections collected from mice in fed states, we combined matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-TOF-IMS) and capillary electrophoresis-mass spectrometry (CE-MS), to determine the apparent contents of individual metabolites in serial tissue sections. The results indicated significant elevation of ATP and energy charge in both metastases and the parenchyma of the tumor-bearing livers. To note were significant increases in UDP-N-acetyl hexosamines, and reduced and oxidized forms of glutathione in the metastatic foci versus the liver parenchyma. These findings thus provided a potentially important method for characterizing the properties of metabolic systems of human-derived cancer and the host tissues in vivo.

Bacteria have developed a unique strategy to survive in extreme environmental conditions through the synthesis of an extracellular polymeric matrix conferring upon the cells a protective microenvironment. The main structural component of this complex network constitutes high-molecular weight hydrophilic macromolecules, namely exopolysaccharides (EPS). EPS composition with the presence of particular chemical features may closely be related to the specific conditions in which bacteria evolve. Deep-sea hydrothermal vent bacteria have already been shown to produce EPS rich in hexosamines and uronic acids, frequently bearing some sulfate groups. Such a particular composition ensures interesting functional properties, including biological activities mimicking those known for glycosaminoglycans (GAG). The aim of the present study was to go further into the exploration of the deep-sea hydrothermal vent IFREMER (French Research Institute for Exploitation of the Sea) collection of bacteria to discover new strains able to excrete EPS endowed with GAG-like structural features. After the screening of our whole collection containing 692 strains, 38 bacteria have been selected for EPS production at the laboratory scale. EPS-producing strains were identified according to 16S rDNA phylogeny. Chemical characterization of the obtained EPS highlighted their high chemical diversity with the presence of atypical compositional patterns. These EPS constitute potential bioactives for a number of biomedical applications, including regenerative medicines and cancer treatment.

Intracellular parasites, such as Leishmania spp, must acquire suitable carbon sources from the host cell in order to replicate. Here we present evidence that intracellular amastigote stages of Leishmania exploit amino sugars in the phagolysosome of mammalian macrophages as a source of carbon and energy. L. major parasites are capable of using N-acetylglucosamine and glucosamine as primarily carbon sources and contain key enzymes required for conversion of these sugars to fructose-6-phosphate. The last step in this pathway is catalyzed by glucosamine-6-phosphate deaminase (GND), which was targeted to glycosomes via a canonical C-terminal targeting signal when expressed as a GFP fusion protein. Mutant parasites lacking GND were unable to grow in medium containing amino sugars as sole carbohydrate source and rapidly lost viability, concomitant with the hyper-accumulation of hexosamine-phosphates. Expression of native GND, but not a cytosolic form of GND, in Δgnd parasites restored hexosamine-dependent growth, indicating that toxicity is due to depletion of glycosomal pools of ATP. Non-lethal increases in hexosamine phosphate levels in both Δgnd and wild type parasites was associated with a defect in promastigote metacyclogenesis, suggesting that hexosamine phosphate levels may influence parasite differentiation. Promastigote and amastigote stages of the Δgnd mutant were unable to replicate within macrophages and were either completely cleared or exhibited reduced lesion development in highly susceptible Balb/c mice. Our results suggest that hexosamines are a major class of sugars in the macrophage phagolysosome and that catabolism of scavenged amino sugars is required to sustain essential metabolic pathways and prevent hexosamine toxicity.

Corynebacterium glutamicum is used for the million-ton-scale production of food and feed amino acids such as L-glutamate and L-lysine and has been engineered for production of carotenoids such as lycopene. These fermentation processes are based on sugars present in molasses and starch hydrolysates. Due to competing uses of starch and sugars in human nutrition, this bacterium has been engineered for utilization of alternative feedstocks, for example, pentose sugars present in lignocellulosic and hexosamines such as glucosamine (GlcN) and N-acetyl-D-glucosamine (GlcNAc). This study describes strain engineering and fermentation using N-acetyl-D-muramic acid (MurNAc) as non-food-competing feedstock. To this end, the genes encoding the MurNAc-specific PTS subunits MurP and Crr and the etherase MurQ from Escherichia coli K-12 were expressed in C. glutamicumΔnanR. While MurP and MurQ were required to allow growth of C. glutamicumΔnanR with MurNAc, heterologous Crr was not, but it increased the growth rate in MurNAc minimal medium from 0.15 h-1 to 0.20 h-1. When in addition to murP-murQ-crr the GlcNAc-specific PTS gene nagE from C. glycinophilum was expressed in C. glutamicumΔnanR, the resulting strain could utilize blends of GlcNAc and MurNAc. Fermentative production of the amino acids L-glutamate and L-lysine, the carotenoid lycopene, and the L-lysine derived chemicals 1,5-diaminopentane and L-pipecolic acid either from MurNAc alone or from MurNAc-GlcNAc blends was shown. MurNAc and GlcNAc are the major components of the bacterial cell wall and bacterial biomass is an underutilized side product of large-scale bacterial production of organic acids, amino acids or enzymes. The proof-of-concept for valorization of MurNAc reached here has potential for biorefinery applications to convert non-food-competing feedstocks or side-streams to valuable products such as food and feed additives.

Prostate cancer is the second most common cause of cancer-associated deaths in men and signalling via a transcription factor called androgen receptor (AR) is an important driver of the disease. Androgen treatment is known to affect the expression and activity of other oncogenes including receptor tyrosine kinases (RTKs). In this study we report that AR-positive prostate cancer cell-lines express 50% higher levels of enzymes in the hexosamine biosynthesis pathway (HBP) than AR-negative prostate cell-lines. HBP produces hexosamines that are used by endoplasmic reticulum and golgi enzymes to glycosylate proteins targeted to plasma-membrane and secretion. Inhibition of O-linked glycosylation by ST045849 or N-linked glycosylation with tunicamycin decreased cell viability by 20%. In addition, tunicamycin inhibited the androgen-induced expression of AR target genes KLK3 and CaMKK2 by 50%. RTKs have been shown to enhance AR activity and we used an antibody array to identify changes in the phosphorylation status of RTKs in response to androgen stimulation. Hormone treatment increased the activity of Insulin like Growth Factor 1-Receptor (IGF-1R) ten-fold and this was associated with a concomitant increase in the N-linked glycosylation of the receptor, analyzed by lectin enrichment experiments. Glycosylation is known to be important for the processing and stability of RTKs. Inhibition of N-linked glycosylation resulted in accumulation of IGF-1R pro-receptor with altered mobility as shown by immunoprecipitation. Confocal imaging revealed that androgen induced plasma-membrane localization of IGF-1R was blocked by tunicamycin. In conclusion we have established that the glycosylation of IGF-1R is necessary for the full activation of the receptor in response to androgen treatment and that perturbing this process can break the feedback loop between AR and IGF-1R activation in prostate cells. Achieving similar results selectively in a clinical setting will be an important challenge in the future.

In cancer cells, glutaminolysis is the primary source of biosynthetic precursors, fueling the TCA cycle with glutamine-derived α-ketoglutarate. The enhanced production of α-ketoglutarate is critical to cancer cells as it provides carbons for the TCA cycle to produce glutathione, fatty acids, and nucleotides, and contributes nitrogens to produce hexosamines, nucleotides, and many nonessential amino acids. Efforts to inhibit glutamine metabolism in cancer using amino acid analogs have been extensive. l-γ-Methyleneglutamine was shown to be of considerable biochemical importance, playing a major role in nitrogen transport in Arachis and Amorpha plants. Herein we report for the first time an efficient synthetic route to l-γ-methyleneglutamine and its amide derivatives. Many of these l-γ-methyleneglutamic acid amides were shown to be as efficacious as tamoxifen or olaparib at arresting cell growth among MCF-7 (ER+/PR+/HER2-), and SK-BR-3 (ER-/PR-/HER2+) breast cancer cells at 24 or 72 h of treatment. Several of these compounds exerted similar efficacy to olaparib at arresting cell growth among triple-negative MDA-MB-231 breast cancer cells by 72 h of treatment. None of the compounds inhibited cell growth in benign MCF-10A breast cells. Overall, N-phenyl amides and N-benzyl amides, such as 3, 5, 9, and 10, arrested the growth of all three (MCF-7, SK-BR-3, and MDA-MB-231) cell lines for 72 h and were devoid of cytotoxicity on MCF-10A control cells; N-benzyl amides with an electron withdrawing group at the para position, such as 5 and 6, inhibited the growth of triple-negative MDA-MB-231 cells commensurate to olaparib. These compounds hold promise as novel therapeutics for the treatment of multiple breast cancer subtypes.

β-hexosaminidase is an enzyme responsible for the degradation of gangliosides, glycans, and other glycoconjugates containing β-linked hexosamines that enter the lysosome. GM2 gangliosidoses, such as Tay-Sachs and Sandhoff, are lysosomal storage disorders characterized by β-hexosaminidase deficiency and subsequent lysosomal accumulation of its substrate metabolites. These two diseases result in neurodegeneration and early mortality in children. A significant difference between these two disorders is the accumulation in Sandhoff disease of soluble oligosaccharide metabolites that derive from N- and O-linked glycans. In this paper we describe our results from a longitudinal biochemical study of a feline model of Sandhoff disease and an ovine model of Tay-Sachs disease to investigate the accumulation of GM2/GA2 gangliosides, a secondary biomarker for phospholipidosis, bis-(monoacylglycero)-phosphate, and soluble glycan metabolites in both tissue and fluid samples from both animal models. While both Sandhoff cats and Tay-Sachs sheep accumulated significant amounts of GM2 and GA2 gangliosides compared to age-matched unaffected controls, the Sandhoff cats having the more severe disease, accumulated larger amounts of gangliosides compared to Tay-Sachs sheep in their occipital lobes. For monitoring glycan metabolites, we developed a quantitative LC/MS assay for one of these free glycans in order to perform longitudinal analysis. The Sandhoff cats showed significant disease-related increases in this glycan in brain and in other matrices including urine which may provide a useful clinical tool for measuring disease severity and therapeutic efficacy. Finally, we observed age-dependent increasing accumulation for a number of analytes, especially in Sandhoff cats where glycosphingolipid, phospholipid, and glycan levels showed incremental increases at later time points without signs of peaking. This large animal natural history study for Sandhoff and Tay-Sachs is the first of its kind, providing insight into disease progression at the biochemical level. This report may help in the development and testing of new therapies to treat these disorders.