An efficient methodology for the selective substitution of both terminal positions (C6 and C6') in 1',2,3,3',4,4'-hexa-O-benzylsucrose with different unsaturated monosaccharide units is presented. Such a highly functionalized intermediate was cyclized under RCM conditions to afford a macrocyclic derivative containing a 31-membered ring in 26% yield.

Xylan, a prominent component of cellulosic biomass, has a high potential for degradation into reducing sugars, and subsequent conversion into bioethanol. This process requires a range of xylanolytic enzymes. Among them, β-xylosidases are crucial, because they hydrolyze more glycosidic bonds than any of the other xylanolytic enzymes. They also enhance the efficiency of the process by degrading xylooligosaccharides, which are potent inhibitors of other hemicellulose-/xylan-converting enzymes. On the other hand, the β-xylosidase itself is also inhibited by monosaccharides that may be generated in high concentrations during the saccharification process. Structurally, β-xylosidases are diverse enzymes with different substrate specificities and enzyme mechanisms. Here, we review the structural diversity and catalytic mechanisms of β-xylosidases, and discuss their inhibition by monosaccharides.

New strategies for immune modulation have shown real promise in regenerative medicine as well as the fight against autoimmune diseases, allergies, and cancer. Dendritic cells (DCs) are gatekeepers of the immune system and their ability in shaping the adaptive immune responses makes DCs ideal targets for immune modulation. Carbohydrates are abundant in different biological systems and are known to modulate DC phenotype and function. However, how simple monosaccharides instruct DC function is less well understood. In this study, we used a combinatorial array of immobilized monosaccharides to investigate how they modulate DC phenotype and function and crucially the impact of such changes on downstream adaptive immune responses. Our data show that a selection of monosaccharides significantly suppress lipopolysaccharide-induced DC activation as evidenced by a reduction in CD40 expression, IL-12 production, and indoleamine 2,3-dioxygenase activity, while inducing a significant increase in IL-10 production. These changes are indicative of the induction of an anti-inflammatory or regulatory phenotype in DCs, which was further confirmed in DC-T cell co-cultures where DCs cultured on the 'regulatory' monosaccharide-coated surfaces were shown to induce naïve T cell polarization toward regulatory phenotype. Our data also highlighted a selection of monosaccharides that are able to promote mixed Treg and Th17 cell differentiation, a T cell phenotype expected to be highly immune suppressive. These data show the potential immunomodulatory effects of immobilized monosaccharides in priming DCs and skewing T cell differentiation toward an immune-regulatory phenotype. The ability to fine-tune immune responses using these simple carbohydrate combinations (e.g. as coatings for existing materials) can be utilized as novel tools for immune modulation with potential applications in regenerative medicine, implantable medical devices, and wound healing where reduction of inflammatory responses and maintaining immune homeostasis are desirable.

The conformational properties of carbohydrates can contribute to protein structure directly through covalent conjugation in the cases of glycoproteins and proteoglycans and indirectly in the case of transmembrane proteins embedded in glycolipid-containing bilayers. However, there continue to be significant challenges associated with experimental structural biology of such carbohydrate-containing systems. All-atom explicit-solvent molecular dynamics simulations provide a direct atomic resolution view of biomolecular dynamics and thermodynamics, but the accuracy of the results depends on the quality of the force field parametrization used in the simulations. A key determinant of the conformational properties of carbohydrates is ring puckering. Here, we applied extended system adaptive biasing force (eABF) all-atom explicit-solvent molecular dynamics simulations to characterize the ring puckering thermodynamics of the ten common pyranose monosaccharides found in vertebrate biology (as represented by the CHARMM carbohydrate force field). The results, along with those for idose, demonstrate that the CHARMM force field reliably models ring puckering across this diverse set of molecules, including accurately capturing the subtle balance between 4C1 and 1C4 chair conformations in the cases of iduronate and of idose. This suggests the broad applicability of the force field for accurate modeling of carbohydrate-containing vertebrate biomolecules such as glycoproteins, proteoglycans, and glycolipids.

Hungateiclostridium thermocellum ATCC 27405 is a promising bacterium for consolidated bioprocessing with a robust ability to degrade lignocellulosic biomass through a multienzyme cellulosomal complex. The bacterium uses the released cellodextrins, glucose polymers of different lengths, as its primary carbon source and energy. In contrast, the bacterium exhibits poor growth on monosaccharides such as fructose and glucose. This phenomenon raises many important questions concerning its glycolytic pathways and sugar transport systems. Until now, the detailed mechanisms of H. thermocellum adaptation to growth on hexose sugars have been relatively poorly explored. In this study, adaptive laboratory evolution was applied to train the bacterium in hexose sugars-based media, and genome resequencing was used to detect the genes that got mutated during adaptation period. RNA-seq data of the first culture growing on either fructose or glucose revealed that several glycolytic genes in the Embden-Mayerhof-Parnas pathway were expressed at lower levels in these cells than in cellobiose-grown cells. After seven consecutive transfer events on fructose and glucose (~42 generations for fructose-adapted cells and ~40 generations for glucose-adapted cells), several genes in the EMP glycolysis of the evolved strains increased the levels of mRNA expression, accompanied by a faster growth, a greater biomass yield, a higher ethanol titer than those in their parent strains. Genomic screening also revealed several mutation events in the genomes of the evolved strains, especially in those responsible for sugar transport and central carbon metabolism. Consequently, these genes could be applied as potential targets for further metabolic engineering to improve this bacterium for bio-industrial usage.

High enzyme loading is a major economic bottleneck for the commercial processing of pretreated lignocellulosic biomass to produce fermentable sugars. Optimizing the enzyme cocktail for specific types of pretreated biomass allows for a significant reduction in enzyme loading without sacrificing hydrolysis yield. This is especially important for alkaline pretreatments such as Ammonia fiber expansion (AFEX) pretreated corn stover. Hence, a diverse set of hemicellulases supplemented along with cellulases is necessary for high recovery of monosaccharides.

Fluorinated sugar-1-phosphates are of emerging importance as intermediates in the chemical and biocatalytic synthesis of modified oligosaccharides, as well as probes for chemical biology. Here we present a systematic study of the activity of a wide range of anomeric sugar kinases (galacto- and N-acetylhexosamine kinases) against a panel of fluorinated monosaccharides, leading to the first examples of polyfluorinated substrates accepted by this class of enzymes. We have discovered four new N-acetylhexosamine kinases with a different substrate scope, thus expanding the number of homologs available in this subclass of kinases. Lastly, we have solved the crystal structure of a galactokinase in complex with 2-deoxy-2-fluorogalactose, giving insight into changes in the active site that may account for the specificity of the enzyme toward certain substrate analogs.

Saccharides in our diet are major sources of carbon for the formation of biomass such as proteins, lipids, nucleic acids and glycans. Among the dietary monosaccharides, glucose occupies a central role in metabolism, but human blood contains regulated levels of other monosaccharides as well. Their influence on metabolism and how they are utilized have not been explored thoroughly. Applying metabolic flux analysis on glycan synthesis can reveal the pathways that supply glycosylation precursors and provide a snapshot of the metabolic state of the cell. In this study, we traced the incorporation of six 13C uniformly labeled monosaccharides in the N-glycans, O-glycans and glycosphingolipids of both pluripotent and neural NTERA-2 cells. We gathered detailed isotopologue data for hundreds of glycoconjugates using mass spectrometry methods. The contributions of de novo synthesis and direct incorporation pathways for glucose, mannose, fructose, galactose, N-acetylglucosamine and fucose were determined based on their isotope incorporation. Co-feeding studies revealed that fructose incorporation is drastically decreased by the presence of glucose, while mannose and galactose were much less affected. Furthermore, increased sialylation slowed down the turnover of glycans, but fucosylation attenuated this effect. Our results demonstrated that exogenous monosaccharide utilization can vary markedly depending on the cell differentiation state and monosaccharide availability, and that the incorporation of carbons can also differ among different glycan structures. We contend that the analysis of metabolic isotope labeling of glycans can yield new insights about cell metabolism.

Cell membrane protein glycosylation is dependent on the metabolic state of the cell as well as exogenous nutrients available. Although the metabolism and interconversion of monosaccharides have been well-studied, their incorporation into cell surface glycans and their corresponding glycoproteins remains relatively unknown. In this study, we developed a method to investigate quantitatively the incorporation pathways of dietary saccharides into specific glycans and glycoproteins on the cell membrane by treating intestinal Caco-2 and hepatic KKU-M213 cells with 13C-labeled monosaccharides and characterizing the resulting cell surface glycans and glycopeptides by LC-MS/MS. Time-course studies using uniformly labeled glucose revealed that the rate of incorporation was both glycan-specific and protein-dependent. Comparative studies using different dietary saccharides and multiple cell lines revealed the variance of monosaccharide utilization and interconversion in different tissues and organisms. The robust isotope-labeling and glycan profiling methods can provide a useful tool for differentiating glycosylation pathways and enhance the understanding of how dietary sugar intake affects health.

Reef-building corals thrive in oligotrophic environments due to their possession of endosymbiotic algae. Confined to the low pH interior of the symbiosome within the cell, the algal symbiont provides the coral host with photosynthetically fixed carbon. However, it remains unknown how carbon is released from the algal symbiont for uptake by the host. Here we show, using cultured symbiotic dinoflagellate, Breviolum sp., that decreases in pH directly accelerates the release of monosaccharides, that is, glucose and galactose, into the ambient environment. Under low pH conditions, the cell surface structures were deformed and genes related to cellulase were significantly upregulated in Breviolum. Importantly, the release of monosaccharides was suppressed by the cellulase inhibitor, glucopyranoside, linking the release of carbon to degradation of the agal cell wall. Our results suggest that the low pH signals the cellulase-mediated release of monosaccharides from the algal cell wall as an environmental response in coral reef ecosystems.

One of the primary functions of the mucosal barrier, found lining epithelial cells, is to serve as a first-line of defense against microbial pathogens. The major structural components of mucus are heavily glycosylated proteins called mucins. Mucins are key components of the innate immune system as they aid in the clearance of pathogens and can decrease pathogen virulence. It has also been recently reported that individual mucins and derived glycans can attenuate the virulence of the human pathogen Pseudomonas aeruginosa Here, we show data indicating that mucins not only play a role in host defense but that they can also be subverted by P. aeruginosa to cause disease. We found that the mucin MUL-1 and mucin-derived monosaccharides N-acetyl-galactosamine and N-acetylglucosamine are required for P. aeruginosa killing of Caenorhabditis elegans We also found that the defective adhesion of P. aeruginosa to human lung alveolar epithelial cells, deficient in the mucin MUC1, can be reversed by the addition of individual monosaccharides. The monosaccharides identified in this study are found in a wide range of organisms where they act as host factors required for bacterial pathogenesis. While mucins in C. elegans lack sialic acid caps, which makes their monosaccharides readily available, they are capped in other species. Pathogens such as P. aeruginosa that lack sialidases may rely on enzymes from other bacteria to utilize mucin-derived monosaccharides.IMPORTANCE One of the first lines of defense present at mucosal epithelial tissues is mucus, which is a highly viscous material formed by mucin glycoproteins. Mucins serve various functions, but importantly they aid in the clearance of pathogens and debris from epithelial barriers and serve as innate immune factors. In this study, we describe a requirement of host monosaccharides, likely derived from host mucins, for the ability of Pseudomonas aeruginosa to colonize the intestine and ultimately cause death in Caenorhabditis elegans We also demonstrate that monosaccharides alter the ability of bacteria to bind to both Caenorhabditis elegans intestinal cells and human lung alveolar epithelial cells, suggesting that there are conserved mechanisms underlying host-pathogen interactions in a range of organisms. By gaining a better understanding of pathogen-mucin interactions, we can develop better approaches to protect against pathogen infection.

Unnatural monosaccharides such as azidosugars that can be metabolically incorporated into cellular glycans are currently used as a major tool for glycan imaging and glycoproteomic profiling. As a common practice to enhance membrane permeability and cellular uptake, the unnatural sugars are per-O-acetylated, which, however, can induce a long-overlooked side reaction, non-enzymatic S-glycosylation. Herein, we develop 1,3-di-esterified N-azidoacetylgalactosamine (GalNAz) as next-generation chemical reporters for metabolic glycan labeling. Both 1,3-di-O-acetylated GalNAz (1,3-Ac2GalNAz) and 1,3-di-O-propionylated GalNAz (1,3-Pr2GalNAz) exhibit high efficiency for labeling protein O-GlcNAcylation with no artificial S-glycosylation. Applying 1,3-Pr2GalNAz in mouse embryonic stem cells (mESCs), we identify ESRRB, a critical transcription factor for pluripotency, as an O-GlcNAcylated protein. We show that ESRRB O-GlcNAcylation is important for mESC self-renewal and pluripotency. Mechanistically, ESRRB is O-GlcNAcylated by O-GlcNAc transferase at serine 25, which stabilizes ESRRB, promotes its transcription activity and facilitates its interactions with two master pluripotency regulators, OCT4 and NANOG.

In order to reduce sucrose content in jujube juice and prepare a jujube juice beverage rich in rare sugars, jujube juice was used as raw material for multienzyme catalysis in this study. The effects of single factors such as substrate, pH, DPE and L-RI addition ratio, enzyme treatment temperature, and metal ions on sucrose conversion and D-allulose formation in jujube juice were investigated. Changes in glucose, D-allulose, and D-allose contents in jujube juice before and after enzyme conversion were analyzed by high-performance liquid chromatography (HPLC). The results showed that 'Xiangfenmuzao' was more suitable for subsequent double enzyme coupling reactions in different varieties of jujube juice at different periods. Factors such as pH, DPE and L-RI enzyme ratio, temperature, and treatment time had significant effects on sucrose conversion and D-allulose production in 'Xiangfenmuzao' juice (p < 0.05). When the ratio of DPE and L-RI was 1:10, pH was 7.5, and the temperature was 60 °C for 7 h, the fructose content in the full-red stage jujube juice of 'Xiangfenmuzao' and 'Jinsixiaozao' decreased gradually, and the final yield was about 53%. The yield of D-allulose was about 29%, and the yield of D-allulose was about 17%. In this study, DPE and L-RI were used to treat whole red jujube juice, which could effectively reduce sucrose content in jujube juice and obtain a functional jujube juice beverage that is low in calories and rich in rare sugar.

Brassinolide (BL) represents brassinosteroids (BRs)-a group of phytohormones that are essential for plant growth and development. Brassinazole (Brz) is as a synthetic inhibitor of BRs' biosynthesis. In the present study, the responses of Wolffia arrhiza to the treatment with BL, Brz, and the combination of BL with Brz were analyzed. The analysis of BRs and Brz was performed using LC-MS/MS. The photosynthetic pigments (chlorophylls, carotenes, and xanthophylls) levels were determined using HPLC, but protein and monosaccharides level using spectrophotometric methods. The obtained results indicated that BL and Brz influence W. arrhiza cultures in a concentration-dependent manner. The most stimulatory effects on the growth, level of BRs (BL, 24-epibrassinolide, 28-homobrassinolide, 28-norbrassinolide, catasterone, castasterone, 24-epicastasterone, typhasterol, and 6-deoxytyphasterol), and the content of pigments, protein, and monosaccharides, were observed in plants treated with 0.1 µM BL. Whereas the application of 1 µM and 10 µM Brz caused a significant decrease in duckweed weight and level of targeted compounds. Application of BL caused the mitigation of the Brz inhibitory effect and enhanced the BR level in duckweed treated with Brz. The level of BRs was reported for the first time in duckweed treated with BL and/or Brz.

Nonceliac gluten sensitivity (NCGS) is a recently defined clinical entity characterized by intestinal and extraintestinal symptoms associated with gluten ingestion in individuals in whom celiac disease (CD) or wheat allergy (WA) has been excluded. Despite its name and definition, gluten has been shown to precipitate symptoms in only 16-30% of these patients. In addition to gluten, other components of wheat, including fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs), alpha-amylase trypsin inhibitors (ATIs) and wheat germ agglutinin have been implicated in the causation of the symptoms of NCGS, with FODMAPs garnering the most attention. We present a review of the existing literature evaluating the role of FODMAPs in NCGS symptomatology.

Non-enzymatic saccharide sensors are of great interest in diagnostics, but their non-selectivity limits their practical diagnostic abilities. In this study, we investigated the electrochemical oxidation of monosaccharides at nanoporous gold (NPG) catalysts with different contributions of surface crystallographic orientations. Fructose elicited no clear electrochemical response, but glucose, galactose, and mannose produced clear oxidative current. The onset potentials for oxidation of these saccharides depended on the surface atomic structure of the NPG. The oxidation potential was approximately 100 mV less positive at the Au(100)-enhanced NPG than at the Au(111)-enhanced NPG. Furthermore, the voltammetric responses significantly differed among the saccharides. Galactose was oxidized at less positive potential and exhibited a higher current response than the other saccharides. This tendency was enhanced in the presence of chloride ions. These features enabled the selective and sensitive detection of galactose at an NPG electrode without enzymes under physiological conditions. A linear range of 10 μM to 1.8 mM was obtained in the calibration plot, which was comparable to those in previously reported enzymatic galactose sensors. Thus, we demonstrated that controlling the crystallographic orientation on the nanostructured electrode surface is useful in developing electrochemical sensors.

In cholera diarrhoea, the pituitary gland produces a 60-kDa protein known as antisecretory factor (ASF) which reverses intestinal secretion induced by the cholera toxin. We show here that ASF-like proteins are produced in the rat during intestinal secretion triggered by intake of a 500 mg dose of mannose, sorbitol, glycine or alanine. All the ASF-like proteins reversed cholera secretion, and all were of a similar size. However, they differed in charge: mannose and sorbitol induced a protein with an isoelectric point of 4.5; glycine induced two proteins, one with a pI of 6.3, the other of 7.7; and alanine induced two proteins, one with a pI of 6.3, the other of 9.4. Antibodies against naturally occurring ASF from porcine pituitary gland neutralized ASF induced by cholera toxin and two of the amino acid-induced proteins, while the sugar-induced protein(s) did not cross-react. All the proteins showed affinity to agarose and were dissociated again with methyl alpha-D-glucoside. A single peroral dose of cholera toxin or sorbitol induced antisecretory proteins which persisted in the pituitary gland for only 1-3 days. Seven treatments gave a sustained response, the protein induced by cholera toxin persisting for over 2 months, and that induced by sorbitol about 1 month.

Endurance athletes commonly experience lower gastrointestinal (GI) symptoms similar to those of irritable bowel syndrome (IBS). Previous research on the restriction of fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAP), a diet-based mitigation strategy initially developed for IBS, has shown promise for application in athlete populations. Athlete's dietary strategies surrounding exercise have not been formally assessed in relation to FODMAP content of foods or sports nutrition products. Additionally, the FODMAP content of athlete's habitual diets has not been examined in larger sample sizes. This research aims to investigate the FODMAP content of endurance athlete diets by examining these three areas, in conjunction with GI symptoms. Dietary habits surrounding exercise and GI symptoms were examined in 430 endurance athletes using a previously validated Endurance Athlete Questionnaire. A subset of athletes (n = 73) completed a FODMAP-specific food frequency questionnaire for habitual intake. The most commonly reported sports nutrition products were analyzed for FODMAP content using standardized analytical methods. Mean habitual intakes were compared to previous FODMAP studies and medians were compared between those with and without lower GI symptoms. Athletes commonly consumed high FODMAP foods during pre-race dinners and breakfasts, with over 60% reporting specific high FODMAP foods. More frequent nutrition product use, particularly solid, gel/gummy, and homemade products, was often related to increased frequency of GI symptoms. Of the sixteen commonly used sports nutrition products tested, seven were high FODMAP in one serving. All but one of the remaining products became high FODMAP when consumed in multiple servings, as is likely the case during endurance exercise. Average habitual FODMAP intake was 26.1 g (±15.9 g), similar to intakes classified as high FODMAP in previous research on FODMAPs and IBS or GI symptoms. Only 15.1% of athletes consumed a diet that would be considered low in FODMAP. Exploratory analyses showed higher intake of some FODMAP types among athletes exhibiting various lower GI symptoms. Overall, this study demonstrated that FODMAP intake by endurance athletes is high both surrounding exercise and habitually, and may be contributing to GI symptoms experienced during exercise. This information can be utilized when analyzing athlete diets and selecting foods to decrease GI symptoms.

The galectins are a family of β-galactoside-specific animal lectins, and have attracted much attention as novel regulators of the immune system. Galectin-10 is well-expressed in eosinophils, and spontaneously forms Charcot-Leyden crystals (CLCs), during prolonged eosinophilic inflammatory reactions, which are frequently observed in eosinophilic diseases. Although biochemical and structural characterizations of galectin-10 have been done, its biological role and molecular mechanism are still unclear, and few X-ray structures of galectin-10 in complex with monosaccharides/oligosaccharides have been reported. Here, X-ray structures of galectin-10 in complexes with seven monosaccharides are presented with biochemical analyses to detect interactions of galectin-10 with monosaccharides/oligosaccharides. Galectin-10 forms a homo-dimer in the face-to-face orientation, and the monosaccharides bind to the carbohydrate recognition site composed of amino acid residues from two galectin-10 molecules of dimers, suggesting that galectin-10 dimer likely captures the monosaccharides in solution and in vivo. d-Glucose, d-allose, d-arabinose, and D-N-acetylgalactosamine bind to the interfaces between galectin-10 dimers in crystals, and they affect the stability of molecular packing in crystals, leading to easy-dissolving of CLCs, and/or inhibiting the formation of CLCs. These monosaccharides may serve as effectors of G10 to form CLCs in vivo.

Grape stalks, an inedible lignocellulosic residue from winemaking and agro-industrial grape juice production, can be valorized as a source of bioactive compounds and as feedstock for the saccharification and bioconversion of soluble sugars. Solid-state fermentation (SSF) by six white-rot fungi was applied as pretreatment. Fiber composition, free radical scavenging activity, four ligninolytic, and three hydrolytic enzyme activities were determined. Saccharification kinetics, yield, and productivity were evaluated and complemented with scanning electron microscopy (SEM), high performance liquid chromatography (HPLC) quantification of monosaccharides, and principal component analysis (PCA). After SSF, the biomass exhibited a drastic free radical scavenging activity decrease and the main enzymes produced were manganese-dependent peroxidase and xylanase. Scanning electron microscopy revealed the erosion of cell walls, and PCA exhibited a negative correlation between saccharification, and neutral detergent fiber and acid detergent lignin. Phlebia rufa pretreated biomass gave the highest sugars yield and productivity, representing a nearly three-fold increase compared to untreated samples. Also, monosaccharides quantification revealed that the 1:1 ratio of glucose to the sum of xylose plus galactose changes to the value of 2:1 after pretreatment. In this work, and for the first time, P. rufa proved to be an effective pretreatment of grape stalks for the saccharification and further bioconversion into value-added chemicals. In addition, lignocellulolytic enzymes were also produced through SSF.