Xylose Residues Research Articles

Synthesis of Fluorophore-Tagged Xylosides That Prime Glycosaminoglycan Chains

Biosynthesis and functions of glycosaminoglycan (GAG) chains are complex and remain elusive. To better understand the factors that regulate the biosynthesis and functions, fluorophore-tagged xylosides carrying two different linkages between fluorophore and xylose residue were synthesized and evaluated for their ability to prime GAG chains such as heparan sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS) in various cell lines. These in vitro studies resulted in the identification of fluorophore-tagged xylosides that prime high molecular weight GAG chains. Primed GAG chains carrying a fluorophore group has several advantages for studying the factors that regulate the biosynthesis, analyzing intact fine structures at low detection limits, and setting the stage for studying structure–function relations of GAG chains of cellular origin.

Structural elucidation of an asparagine-linked oligosaccharide from the hyperthermophilic archaeon, Pyrococcus furiosus

An oligosaccharide chain attached to the asparagine residue in a structurally defined peptide was produced by an in vitro oligosaccharide-transfer reaction, using membrane fractions that contained the oligosaccharyltransferase from the hyperthermophilic archaeon, Pyrococcus furiosus. The chemical structure of the N-glycan was elucidated by sugar analysis, NMR spectroscopy, and MS spectrometry, which revealed the structure. [Display omitted] The shorter glycan structures lacking one or two xylose residues were also transferred by the P. furiosus oligosaccharyltransferase. The archaeal N-glycans are known to exhibit a high degree of structural variation. The structure of the P. furiosus N-glycan is novel and unique. The present data will be useful for structural and functional studies of the P. furiosus oligosaccharyltransferase.

Rapid mass spectrometric analysis of a novel fucoidan, extracted from the brown alga Coccophora langsdorfii.

The novel highly sulfated (35%) fucoidan fraction Cf2 , which contained, along with fucose, galactose and traces of xylose and uronic acids was purified from the brown alga Coccophora langsdorfii. Its structural features were predominantly determined (in comparison with fragments of known structure) by a rapid mass spectrometric investigation of the low-molecular-weight fragments, obtained by “mild” (5 mg/mL) and “exhaustive” (maximal concentration) autohydrolysis. Tandem matrix-assisted laser desorption/ionization mass spectra (MALDI-TOF/TOFMS) of fucooligosaccharides with even degree of polymerization (DP), obtained by “mild” autohydrolysis, were the same as that observed for fucoidan from Fucus evanescens, which have a backbone of alternating (1 → 3)- and (1 → 4) linked sulfated at C-2 and sometimes at C-4 of 3-linked α-L-Fucp residues. Fragmentation patterns of oligosaccharides with odd DP indicated sulfation at C-2 and at C-4 of (1 → 3) linked α-L-Fucp residues on the reducing terminus. Minor sulfation at C-3 was also suggested. The “exhaustive” autohydrolysis allowed us to observe the “mixed” oligosaccharides, built up of fucose/xylose and fucose/galactose. Xylose residues were found to occupy both the reducing and nonreducing termini of FucXyl disaccharides. Nonreducing galactose residues as part of GalFuc disaccharides were found to be linked, possibly, by 2-type of linkage to fucose residues and were found to be sulfated, most likely, at position C-2.

Evidence That GH115 α-Glucuronidase Activity, Which Is Required to Degrade Plant Biomass, Is Dependent on Conformational Flexibility

The microbial degradation of the plant cell wall is an important biological process that is highly relevant to environmentally significant industries such as the bioenergy and biorefining sectors. A major component of the wall is glucuronoxylan, a β1,4-linked xylose polysaccharide that is decorated with α-linked glucuronic and/or methylglucuronic acid (GlcA/MeGlcA). Recently three members of a glycoside hydrolase family, GH115, were shown to hydrolyze MeGlcA side chains from the internal regions of xylan, an activity that has not previously been described. Here we show that a dominant member of the human microbiota, Bacteroides ovatus, contains a GH115 enzyme, BoAgu115A, which displays glucuronoxylan α-(4-O-methyl)-glucuronidase activity. The enzyme is significantly more active against substrates in which the xylose decorated with GlcA/MeGlcA is flanked by one or more xylose residues. The crystal structure of BoAgu115A revealed a four-domain protein in which the active site, comprising a pocket that abuts a cleft-like structure, is housed in the second domain that adopts a TIM barrel-fold. The third domain, a five-helical bundle, and the C-terminal β-sandwich domain make inter-chain contacts leading to protein dimerization. Informed by the structure of the enzyme in complex with GlcA in its open ring form, in conjunction with mutagenesis studies, the potential substrate binding and catalytically significant amino acids were identified. Based on the catalytic importance of residues located on a highly flexible loop, the enzyme is required to undergo a substantial conformational change to form a productive Michaelis complex with glucuronoxylan.

Extraction and purification of polysaccharides from pine medicinal mushroom, Tricholoma matsutake (higher Basidiomycetes) fruit bodies.

Ultrasound-assisted extraction, purification, and characterization of antioxidant polysaccharides from Tricholoma matsutake were carried out. On the basis of the results of a single-factor test, three independent and main variables, including extraction time (X1: 130-170 s), ultrasonic power (X2: 340-380 W), and ratio of water to raw material (X3: 45-55 mL/g) were studied by Box-Behnken design. The optimum extraction conditions are as follows: extraction time 160 s, ultrasonic power 365 W, and ratio of water to raw material 53.5 mL/g. Under the optimized conditions, the yield of T. matsutake polysaccharides (TMP) was 7.97±0.31%. The crude TMP was purified by DEAE-cellulose 52 chromatography and Sephadex G-100 chromatography to afford two fractions, TMP-1 and TMP-2. The crude TMP contained 85.76% carbohydrates, 3.57% proteins, and 0.13% uronic acids. The constituent monosaccharides were predominantly glucose, galactose, mannose, xylose, fucose, and glucuronic acid residues.

Structure elucidation and immunomodulatory activity in vitro of a xylan from roots of Cudrania tricuspidata

To discover new immunomodulator in the food industry, a water-soluble polysaccharide, CTPB1 was obtained from roots of Cudrania tricuspidata (Carr.) Bur. The average molecular weight of this polysaccharide was estimated to be 4.0×104Da. Monosaccharide composition analysis indicated that the only neutral sugar present in this polysaccharide was xylose. From methylation analysis and 1H and 13C NMR spectroscopy, the structure of CTPB1 was deduced to be (4-O-methyl-d-glucurono)-d-xylan, with the 4-O-methyl-d-glucopyranosyluronic acid group linked to O-2 of a (1→4)-β-d-xylan. The repeating unit of CTPB1 is composed of one non-reducing terminal residue of 4-O-methyl-d-glucuronic acid on every four xylose residues. Immunomodulatory activity assays in vitro showed that CTPB1 may induce the proliferation of mouse splenocytes. It was suggested that CTPB1 could be a potential immunostimulant used in the food industry.

Engineering the Xylan Utilization System in Bacillus subtilis for Production of Acidic Xylooligosaccharides

Xylans are the predominant polysaccharides in hemicelluloses and an important potential source of biofuels and chemicals. The ability of Bacillus subtilis subsp. subtilis strain 168 to utilize xylans has been ascribed to secreted glycoside hydrolase family 11 (GH11) and GH30 endoxylanases, encoded by the xynA and xynC genes, respectively. Both of these enzymes have been defined with respect to structure and function. In this study, the effects of deletion of the xynA and xynC genes, individually and in combination, were evaluated for xylan utilization and formation of acidic xylooligosaccharides. Parent strain 168 depolymerizes methylglucuronoxylans (MeGXn), releasing the xylobiose and xylotriose utilized for growth and accumulating the aldouronate methylglucuronoxylotriose (MeGX3) with some methylglucuronoxylotetraose (MeGX4). The combined GH11 and GH30 activities process the products generated by their respective actions on MeGXn to release a maximal amount of neutral xylooligosaccharides for assimilation and growth, at the same time forming MeGX3 in which the internal xylose is substituted with methylglucuronate (MeG). Deletion of xynA results in the accumulation of β-1,4-xylooligosaccharides with degrees of polymerization ranging from 4 to 18 and an average degree of substitution of 1 in 7.2, each with a single MeG linked α-1,2 to the xylose penultimate to the xylose at the reducing terminus. Deletion of the xynC gene results in the accumulation of aldouronates comprised of 4 or more xylose residues in which the MeG may be linked α-1,2 to the xylose penultimate to the nonreducing xylose. These B. subtilis lines may be used for the production of acidic xylooligosaccharides with applications in human and veterinary medicine.

Differences between easy- and difficult-to-mill chickpea (Cicer arietinumL.) genotypes. Part III: free sugar and non-starch polysaccharide composition

Parts I and II of this series of papers identified several associations between the ease of milling and the chemical compositions of different chickpea seed fractions. Non-starch polysaccharides were implicated; hence, this study examines the free sugars and sugar residues. Difficult milling is associated with: (1) lower glucose and xylose residues (less cellulose and xyloglucans) and more arabinose, rhamnose and uronic acid in the seed coat, suggesting a more flexible seed coat that resists cracking and decortication; (2) a higher content of soluble and insoluble non-starch polysaccharide fractions in the cotyledon periphery, supporting a pectic polysaccharide mechanism comprising arabinogalacturonan, homogalacturonan, rhamnogalalcturonan, and glucuronan backbone structures; (3) higher glucose and mannose residues in the cotyledon periphery, supporting a lectin-mediated mechanism of adhesion; and (4) higher arabinose and glucose residues in the cotyledon periphery, supporting a mechanism involving arabinogalactan-proteins. This series has shown that the chemical composition of chickpea does vary in ways that are consistent with physical explanations of how seed structure and properties relate to milling behaviour. Seed coat strength and flexibility, pectic polysaccharide binding, lectins and arabinogalactan-proteins have been implicated. Increased understanding in these mechanisms will allow breeding programmes to optimise milling performance in new cultivars.

Exploring the N-glycosylation Pathway in Chlamydomonas reinhardtii Unravels Novel Complex Structures

Chlamydomonas reinhardtii is a green unicellular eukaryotic model organism for studying relevant biological and biotechnological questions. The availability of genomic resources and the growing interest in C. reinhardtii as an emerging cell factory for the industrial production of biopharmaceuticals require an in-depth analysis of protein N-glycosylation in this organism. Accordingly, we used a comprehensive approach including genomic, glycomic, and glycoproteomic techniques to unravel the N-glycosylation pathway of C. reinhardtii. Using mass-spectrometry-based approaches, we found that both endogenous soluble and membrane-bound proteins carry predominantly oligomannosides ranging from Man-2 to Man-5. In addition, minor complex N-linked glycans were identified as being composed of partially 6-O-methylated Man-3 to Man-5 carrying one or two xylose residues. These findings were supported by results from a glycoproteomic approach that led to the identification of 86 glycoproteins. Here, a combination of in-source collision-induced dissodiation (CID) for glycan fragmentation followed by mass tag-triggered CID for peptide sequencing and PNGase F treatment of glycopeptides in the presence of (18)O-labeled water in conjunction with CID mass spectrometric analyses were employed. In conclusion, our data support the notion that the biosynthesis and maturation of N-linked glycans in the endoplasmic reticulum and Golgi apparatus occur via a GnT I-independent pathway yielding novel complex N-linked glycans that maturate differently from their counterparts in land plants.

Structure and Biological Action of Cladolosides B1, B2, C, C1, C2 and D, Six New Triterpene Glycosides from the Sea Cucumber Cladolabes schmeltzii

Six new nonsulfated triterpene glycosides, cladolosides B1 (1), B2 (2), C (3), C1 (4), C2 (5) and D (6) and known holotoxin A1 (7) have been isolated from the tropical Indo-West Pacific sea cucumber Cladolabes schmeltzii (Cladolabinae, Sclerodactylidae, Dendrochirotida). Structures of the glycosides were elucidated by 2D NMR spectroscopy and mass-spectrometry. Glycosides 1 and 2 have pentasaccharide branched carbohydrate moieties and differ from each other by the aglycone structures. Compounds 3-6 are hexaosides; 3-5 contain identical carbohydrate moieties with two terminal 3-O-methylglucose residues and glucose as the fifth sugar unit and differ from each other in the structures of their aglycones. Cladoloside D (6) has a new variant of carbohydrate chain with xylose and glucose residues as fifth and sixth monosaccharides, correspondingly. All of the substances demonstrated rather strong cytotoxic and hemolytic effects.

Integration of a phenolic-acid recovery step in the CaCCO process for efficient fermentable-sugar recovery from rice straw

An advanced sugar-platform bioprocess for lignocellulosic feedstocks by adding a phenolic-acid (PA: p-coumaric acid and ferulic acid) recovery step to the CaCCO process was designed. For efficient PA extraction, pretreatment was 95°C for 2h, producing a yield of 7.30 g/kg-dry rice straw (65.2% of total ester-linked PAs) with insignificant effects on saccharification. PAs were readily recovered in solution during the repeated washings of solids, and the glucose yield, after 72-h saccharification of the washed solids, was significantly improved from 65.9% to 70.3-72.7%, suggesting the removal of potential enzyme inhibitors. The promotion of xylose yield was insignificant, probably due to 13.1-17.8% loss of xylose residues after washing(s). This new bioprocess, termed the SRB (simultaneous recovery of by-products)-CaCCO process, would effectively produce fermentable sugars and other valuables from feedstocks, strengthening the platform in both economic and environmental terms.

Microwave superheated water and dilute alkali extraction of brewers’ spent grain arabinoxylans and arabinoxylo-oligosaccharides

Microwave superheated water extractions (MWE) were performed to evaluate the feasibility of this technology for quantitative recovery of the arabinoxylans (AX) or arabinoxylo-oligosaccharides (AXOS) from brewers’ spent grain (BSG). The AX+AXOS yield increased with the increase of the temperature in the range from 140 to 210°C during 2min. The higher temperatures promoted depolymerisation, debranching, and deesterification of the polysaccharides, with formation of brown products. The conditions that promote a compromise between the yield and the structure obtained, minimizing the thermal degradation of the fractions extracted by MWE are the following: (1) 140°C, to remove the residual starch mixed with β-glucans; (2) Suspension of the residue left in water and treated at 180°C; (3) suspension of the residue in 0.1M KOH and treated at 180°C. Using this sequential procedure, it was possible to extract 62% of BSG AX+AXOS, presenting degrees of polymerization ranging between 7 and 24 xylose residues, and a degree of phenolic acids esterification between 5 and 21%. The structural variability obtained by MWE allows defining specific types of compounds for different applications and uses depending on the extraction conditions used.

Co-immobilization of fungal endo-xylanase and -L-arabinofuranosidase in glyoxyl agarose for improved hydrolysis of arabinoxylan

Plant cell-wall arabinoxylans have a complex structure that requires the action of a pool of debranching (arabinofuranosidases) and depolymerizing enzymes (endo-xylanase). Two Aspergillus nidulans strains over-secreting endo-xylanase and arabinofuranosidase were inoculated in defined 2% maltose-minimum medium resulting in the simultaneously production of these enzymes. To study the synergistic hydrolysis was used arabinoxylan with 41% of arabinose and 59% of xylose residues. Thus, it was adopted different approaches to arabinoxylan hydrolysis using immobilized arabinofuranosidase and endo-xylanase: (i) endo-xylanase immobilized on glyoxyl agarose; (ii) arabinofuranosidase immobilized on glyoxyl agarose; (T1) hydrolysis of arabinoxylan with arabinofuranosidase immobilized on glyoxyl agarose for debranching, followed by a second hydrolysis with endo-xylanase immobilized on glyoxyl agarose; (T2) hydrolysis using (i) and (ii) simultaneously; and (T3) hydrolysis of arabinoxylan with endo-xylanase and arabinofuranosidase co-immobilized on glyoxyl agarose. It was concluded that arabinoxylan hydrolysis using two derivatives simultaneously (T2) showed greater hydrolytic efficiency and consequently a higher products yield. However, the hydrolysis with multi-enzymatic derivative (T3) results in direct release of xylose and arabinose from a complex substrate as arabinoxylan, which is a great advantage as biotechnological application of this derivative, especially regarding the application of biofuels, since these monosaccharides are readily assimilable for fermentation and ethanol production.

Negative Regulation of Notch Signaling by Xylose

The Notch signaling pathway controls a large number of processes during animal development and adult homeostasis. One of the conserved post-translational modifications of the Notch receptors is the addition of an O-linked glucose to epidermal growth factor-like (EGF) repeats with a C-X-S-X-(P/A)-C motif by Protein O-glucosyltransferase 1 (POGLUT1; Rumi in Drosophila). Genetic experiments in flies and mice, and in vivo structure-function analysis in flies indicate that O-glucose residues promote Notch signaling. The O-glucose residues on mammalian Notch1 and Notch2 proteins are efficiently extended by the addition of one or two xylose residues through the function of specific mammalian xylosyltransferases. However, the contribution of xylosylation to Notch signaling is not known. Here, we identify the Drosophila enzyme Shams responsible for the addition of xylose to O-glucose on EGF repeats. Surprisingly, loss- and gain-of-function experiments strongly suggest that xylose negatively regulates Notch signaling, opposite to the role played by glucose residues. Mass spectrometric analysis of Drosophila Notch indicates that addition of xylose to O-glucosylated Notch EGF repeats is limited to EGF14–20. A Notch transgene with mutations in the O-glucosylation sites of Notch EGF16–20 recapitulates the shams loss-of-function phenotypes, and suppresses the phenotypes caused by the overexpression of human xylosyltransferases. Antibody staining in animals with decreased Notch xylosylation indicates that xylose residues on EGF16–20 negatively regulate the surface expression of the Notch receptor. Our studies uncover a specific role for xylose in the regulation of the Drosophila Notch signaling, and suggest a previously unrecognized regulatory role for EGF16–20 of Notch.

Chemical and structural characterization of alkaline‐extractable hemicelluloses from various eucalyptus species

ABSTRACTEucalyptus species are currently one of the main feedstock for pulping and papermaking industry in China. In the present study, alkali‐extractable hemicelluloses were isolated from different eucalyptus species (Eucalyptus camaldulensis, E. urophylla × grandis, and E. urophylla × E. tereticornis) at mild conditions prior to pulping. Structural characterization of these hemicellulosic polymers based on monosaccharide, molecular weight, Fourier transform infrared, 1H, 13C, and two‐dimensional heteronuclear single quantum coherence nuclear magnetic resonance analysis revealed that these alkali‐extractable polysaccharides shared the common structure composed of the (1→4)‐linked‐β‐D‐xylopyranosyl backbone with 4‐O‐methyl‐α‐D‐glucuronic acid attached to O‐2 of the xylose residues. The potential structures of the alkali‐extractable hemicelluloses were proposed based on the comprehensive analysis. The well‐characterized structures of these hemicelluloses could enlarge the industrial application of these hemicelluloses from the Eucalyptus species in a biorefinery process. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 130: 2390–2398, 2013

An arabino(glucurono)xylan isolated from immunomodulatory active hemicellulose fraction of Salvia officinalis L.

From the aerial parts of sage (Salvia officinalis L.) an arabino-(4-O-methyl-glucurono)-xylan (AGX) was isolated by alkaline extraction followed by precipitation with barium hydroxide solution. Polymer was isolated from sage as a light brown polysaccharide material of molecular mass (Mp) 84,000. Compositional analyses of sage AGX revealed xylose (81%), arabinose (10%), glucuronic acid (8%) and small amounts of hexoses (1%). Linkage sugar analyses showed the (1→4)-linked xylopyranosyl backbone with low degree of substitution (9–10%) at O-2 and O-3. Arabinofuranose residues were found as the terminal, 1,3-, 1,5- and 1,3,5-linked. NMR structural analyses of acidic oligomers, generated by partial acidic hydrolysis of AGX, confirmed a substitution of xylose residues by glucuronic acid and its 4-O-methyl derivate at O-2 at an average on every fourteenth xylose residue. NMR and FT-IR measurements, as well as a high negative optical rotation confirmed the β configuration of glycosidic linkages in AGX backbone.

Crystal transition between hydrate and anhydrous (1→3)-β-d-xylan from Penicillus dumetosus

The crystal structures of hydrated and anhydrous (1→3)-β-d-xylan and the crystal transition between them were investigated. Highly crystalline samples of (1→3)-β-d-xylan were prepared from a siphoneous green alga, Penicillus dumetosus. The crystal structure was analyzed using synchrotron X-ray diffraction and solid-state 13C NMR spectroscopy. The hydrated form was converted to the anhydrous form with contraction in both the a-axis and c-axis directions, and the crystallinity decreased considerably at the same time. The crystal transition between hydrated and anhydrous (1→3)-β-d-xylan was monitored using synchrotron X-ray diffraction under controlled relative humidity. The transition took place at certain transition points, and was accompanied by a large hysteresis. From the difference in the weight and unit-cell volume at the transition points, the number of water molecules in the hydrated form was estimated as one per xylose residue.

Synthesis and Assessment of Glycosaminoglycan Priming Activity of Cluster-xylosides for Potential Use as Proteoglycan Mimetics

One of the distinct structural features of many proteoglycans (PGs) is the presence of two or more glycosaminoglycan (GAG) side chains covalently linked to a core protein. Previous studies have shown that the synergistic biological activity of multiple GAG chains, as found in the majority of PGs, cannot be accomplished by the sum of the activities of individual GAG chains. To delineate the biological significance of GAG valency, a number of cluster-xylosides carrying two, three, or four xylose residues on the same scaffold were synthesized using click chemistry. Assessment of cluster-xylosides for their GAG chain priming activity in a cellular system revealed that these cluster-xylosides prime multiple GAG chains per scaffold. Multivalent GAG chains, produced by cluster-xylosides, can better mimic PGs as they carry two or more GAG chains attached to a core protein and therefore can be used as molecular probes to examine the biological significance of GAG multivalency in model organisms.

Structures and Biological Activities of Typicosides A1, A2, B1, C1 and C2, Triterpene Glycosides from the Sea Cucumber Actinocucumis typica

Five new minor triterpene glycosides, typicosides A1 (1), A2 (2), B1 (3), C1 (4) and C2 (5), along with two known glycosides, intercedenside A and holothurin B3, have been isolated from the sea cucumber Actinocucumis typica. Structures of the glycosides were elucidated by 2D NMR spectroscopy andMS. Glycosides 1-5 are linear mono- and disulfated tetraosides differing from each other in both aglycone structures and monosaccharide composition of the carbohydrate chains. Typicosides A1 (1) and A2 (2) have identical monosulfated carbohydrate moieties with a xylose residue as the third monosaccharide unit and differ from each other in aglycon structures. Typicoside B1 (3) has glucose as the third monosaccharide residue. Typicosides C1 (4) and C2 (5) contain the same disulfated carbohydrate chains and differ from each other in structures of aglycone side chains. Antifungal activity of glycosides 1-5 against three species of fungi along with cytotoxic activity against mouse spleen lymphocytes and mouse Ehrlich carcinoma cells (ascite form), as well as hemolytic activities against mouse erythrocytes have been studied. All new glycosides, except for typicoside C1 (4), containing a hydroxy-group in the aglycone side chain, demonstrate rather strong hemolytic and cytotoxic activities.

Incomplete Elongation of the Chondroitin Sulfate Linkage Region on Aggrecan and Response to Interleukin-1β

Aggrecan is the prominent proteoglycan in cartilage and is modified with approximately 100 chondroitin sulfate (CS) chains through a tetrasaccharide linkage structure. In osteoarthritis (OA), the viscoelastic properties of cartilage are compromised on both the quantity and integrity of aggrecan core protein expressed as well as reduced overall CS chain length. Herein, we postulated that chronic low-level inflammation may also contribute to OA progression by promoting regulatory mechanisms in early CS biosynthesis that yield incomplete linkage structures on aggrecan. To test this idea, chondrocytes extracted from human tali were cultured in alginate beads and challenged with 5 ng/mL IL-1β as a model for chronic inflammation leading to OA progression. Novel mass spectrometry-based methods were devised to detect and quantify partially elongated linkage structures relative to control cultures. The total mole fraction of unelongated xylose residues per aggrecan was significantly less (p = 0.03) after IL-1β treatment compared to control cultures, with unelongated xylose residues constituting between 6% and 12% of the fraction of total CS measured. A portion (<1%) of the partially elongated linkage structures was found to be either phosphorylated or sulfated. These results establish quantitative mass spectrometry as a very sensitive and effective platform for evaluating truncated proteoglycan linkage structures. Our observations using this method suggest a possible role for aberrant linkage structure elongation in OA progression.