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Sample records for monogalactosyl octasaccharides xxlg

  1. Anti-herpes simplex virus activities of monogalactosyl diglyceride and digalactosyl diglyceride from Clinacanthus nutans, a traditional Thai herbal medicine

    Institute of Scientific and Technical Information of China (English)

    Sirada Pongmuangmul; Supaporn Phumiamorn; Phanchana Sanguansermsri; Nalin Wongkattiya; Ian Hamilton Fraser; Donruedee Sanguansermsri

    2016-01-01

    Objective: To evaluate the monogalactosyl diglyceride(MGDG) and digalactosyl diglyceride(DGDG) from Clinacanthus nutans(C. nutans) for their in vitro antiviral activities against herpes simplex virus type 1(HSV-1) and type 2(HSV-2) by plaque reduction assay.Methods: MGDG and DGDG were extracted with chloroform from C. nutans leaves.MGDG and DGDG were separated from chloroform crude extract using column chromatography, characterized by thin layer chromatography and quantified by high performance liquid chromatography. The anti HSV-1 and 2 activity against pre-treatment and posttreatment of the compounds was evaluated using plaque reduction assay. The cytotoxicity of the extract and the compounds on Vero cells were performed by MTT assay.Results: MGDG and DGDG obtained by column chromatography showed identical profiles as standard MGDG and standard DGDG using thin layer chromatography and high performance liquid chromatography. MGDG and DGDG from C. nutans showed 100%inhibition of HSV-1 replication at the post step of infection at noncytotoxic concentration with IC50 values of 36.00 and 40.00 mg/m L, and HSV-2 at 41.00 and 43.20 mg/mL,respectively. Moreover, MGDG and DGDG from C. nutans were demonstrated to have antiherpes simplex activity at the same level as standard synthetic compounds. In contrast, pretreatment of Vero cells with MGDG and DGDG before HSV-1 and HSV-2 infection did not show inhibitory effect against these viruses. MGDG and DGDG exhibited antiviral activity against HSV-1 with selectivity index of 26.00 and 23.00 and HSV-2 of 23.30 and 21.30.Conclusions: MGDG and DGDG from C. nutans, a traditional Thai herbal medicine illustrated inhibitory activity against HSV-1 and HSV-2, probably by inhibiting the late stage of multiplication, suggesting their promising use as anti-HSV agents.

  2. Continuous measurement of galactolipid hydrolysis by pancreatic lipolytic enzymes using the pH-stat technique and a medium chain monogalactosyl diglyceride as substrate.

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    Amara, Sawsan; Lafont, Dominique; Fiorentino, Brice; Boullanger, Paul; Carrière, Frédéric; De Caro, Alain

    2009-10-01

    Galactolipids are the main lipids from plants and galactolipases play a major role in their metabolism. These enzymes were however poorly studied so far and only few assays have been developed. A specific and continuous galactolipase assay using synthetic medium chain monogalactosyl diacylglycerol (MGDG) as substrate was developed using the pH-stat technique and recombinant human (rHPLRP2) and guinea pig (rGPLRP2) pancreatic lipase-related protein 2 as model enzymes. PLRP2s are the main enzymes involved in the digestion of galactolipids in the gastrointestinal tract. Monogalactosyl di-octanoylglycerol was mixed with bile salt solutions by sonication to form a micellar substrate before launching the assay. The nature of the bile salt and the bile salt to MGDG ratio were found to significantly affect the rate of MGDG hydrolysis by rHPLRP2 and rGPLRP2. The maximum galactolipase activity of both enzymes was recorded with sodium deoxycholate (NaDC) and at a NaDC to MGDG ratio of 1.33 and at basic pH values (8.0-9.0). The maximum rates of hydrolysis were obtained using a MGDG concentration of 10(-2) M and calcium chloride was found to be not necessary to obtain the maximum of activity. Under these conditions, the maximum turnovers of rGPLRP2 and rHPLRP2 on mixed NaDC/MGDG micelles were found to be 8000+/-500 and 2800+/-60 micromol/min/mg (U/mg), respectively. These activities are in the same order of magnitude as the activities on triglycerides of lipases and they are the highest specific activities ever reported for galactolipases. For the sake of comparison, the hydrolysis of mixed bile salt/MGDG micelles was also tested using other pancreatic lipolytic enzymes and only native and recombinant human carboxyl ester hydrolase were found to display significant but lower activities (240+/-17 and 432+/-62 U/mg, respectively) on MGDG.

  3. Neoglycoconjugate of Tetrasaccharide Representing One Repeating Unit of the Streptococcus pneumoniae Type 14 Capsular Polysaccharide Induces the Production of Opsonizing IgG1 Antibodies and Possesses the Highest Protective Activity As Compared to Hexa- and Octasaccharide Conjugates

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    Ekaterina A. Kurbatova

    2017-06-01

    Full Text Available Identifying protective synthetic oligosaccharide (OS epitopes of Streptococcus pneumoniae capsular polysaccharides (CPs is an indispensable step in the development of third-generation carbohydrate pneumococcal vaccines. Synthetic tetra-, hexa-, and octasaccharide structurally related to CP of S. pneumoniae type 14 were coupled to bovine serum albumin (BSA, adjuvanted with aluminum hydroxide, and tested for their immunogenicity in mice upon intraperitoneal prime-boost immunizations. Injections of the conjugates induced production of opsonizing anti-OS IgG1 antibodies (Abs. Immunization with the tetra- and octasaccharide conjugates stimulated the highest titers of the specific Abs. Further, the tetrasaccharide ligand demonstrated the highest ability to bind OS and CP Abs. Murine immune sera developed against tetra- and octasaccharide conjugates promoted pathogen opsonization to a higher degree than antisera against conjugated hexasaccharide. For the first time, the protective activities of these glycoconjugates were demonstrated in mouse model of generalized pneumococcal infections. The tetrasaccharide conjugate possessed the highest protective activities. Conversely, the octasaccharide conjugate had lower protective activities and the lowest one showed the hexasaccharide conjugate. Sera against all of the glycoconjugates passively protected naive mice from pneumococcal infections. Given that the BSA-tetrasaccharide induced the most abundant yield of specific Abs and the best protective activity, this OS may be regarded as the most promising candidate for the development of conjugated vaccines against S. pneumoniae type 14 infections.

  4. Structural determination of novel sulfated octasaccharides isolated from chondroitin sulfate of shark cartilage and their application for characterizing monoclonal antibody epitopes.

    Science.gov (United States)

    Deepa, Sarama S; Yamada, Shuhei; Fukui, Shigeyuki; Sugahara, Kazuyuki

    2007-06-01

    Twelve octasaccharide fractions were obtained from chondroitin sulfate C derived from shark cartilage after hyaluronidase digestion. Their sugar and sulfate composition was assigned by matrix-assisted laser desorption ionization time of flight mass spectrometry. The sequences were determined at low picomole amounts by a combination of enzymatic digestions with high-performance liquid chromatography, and were composed of disaccharide building units including O [GlcUAbeta1-3GalNAc], C [GlcUAbeta1-3GalNAc(6S)], A [GlcUAbeta1-3GalNAc(4S)], and/or D [GlcUA(2S)beta1-3GalNAc(6S)], where 2S, 4S, and 6S represent 2-O-, 4-O-, and 6-O-sulfate, respectively. As many as 24 different sequences including minor ones were revealed, exhibiting a high degree of structural diversity reflecting the enormous heterogeneity of the parent polysaccharides. Nineteen of them were novel, with the other four reported previously as unsaturated counterparts obtained after digestion with chondroitinase. Microarrays of these structurally defined octasaccharide fractions were prepared using low picomole amounts of their lipid-derivatives to investigate the binding specificity of four commercial anti-chondroitin sulfate antibodies CS-56, MO-225, 2H6, and LY111. The results revealed that multiple unique sequences were recognized by each antibody, which implies that the common conformation shared by the multiple primary sequences in the intact chondroitin sulfate chains is important as an epitope for each monoclonal antibody. Comparison of the specificity of the tested antibodies indicates that CS-56 and MO-225 specifically recognize octasaccharides containing an A-D tetrasaccharide sequence, whereas 2H6 and LY111 require a hexasaccharide as a minimum size for their binding, and prefer sequences with A- and C-units such as C-C-A-C (2H6) or C-C-A-O, C-C-A-A, and C-C-A-C (LY111) for strong binding but require no D-unit.

  5. A facile and effective synthesis of alpha-(1-->6)-linked mannose di-, tri-, tetra-, hexa-, octa-, and dodecasaccharides, and beta-(1-->6)-linked glucose di-, tri-, tetra-, hexa-, and octasaccharides using sugar trichloroacetimidates as the donors and unprotected or partially protected glycosides as the acceptors.

    Science.gov (United States)

    Zhu, Y; Kong, F

    2001-05-08

    Reaction of 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl trichloroimidate with allyl alpha-D-mannopyranoside in the presence of TMSOTf selectively gave allyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl-(1-->6)-alpha-D-mannopyranoside through an orthoester intermediate. Benzoylation of 3, followed by deallylation, and then trichloroimidation afforded the disaccharide donor 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroimidate, while benzoylation of 3 followed by selective removal of acetyl groups yielded the disaccharide acceptor allyl alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranoside. Coupling of 5 with 6 gave the tetrasaccharide allyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranoside, which were converted into the tetrasaccharide donor 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroimdate and the tetrasaccharide acceptor allyl alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranoside, respectively, by the same strategies as used for conversion of 3 into 5 and 6. Condensation of 5 with 13 gave the hexasaccharide 14, while condensation of 12 with 13 gave the octasaccharide 17. Dodecasaccharide 21 was obtained by the coupling of 12 with the octasaccharide acceptor 20. Similar strategies were used for the syntheses of beta-(1-->6)-linked glucose di-, tri-, tetra-, hexa-, and octamers. Deprotection of the oligosaccharides in ammonia-saturated methanol yielded the free alpha-(1-->6)-linked mannosyl and beta-(1-->6)-linked glucosyl oligomers.

  6. Conformational studies on five octasaccharides isolated from chondroitin sulfate using NMR spectroscopy and molecular modeling

    NARCIS (Netherlands)

    Blanchard, V.; Chevalier, F.; Imberty, A.; Leeflang, B.R.; Sugahara, K.; Kamerling, J.P.

    2007-01-01

    Chondroitin sulfate proteoglycans (CS-PG) are involved in the regulation of the central nervous system in vertebrates due to their presence on cell surfaces and in the extracellular matrix of tissues. The CS moieties are built up from repeating -4)GlcA(β 1-3)GalNAc(β 1- disaccharide units, partly O-

  7. Anti-herpes simplex virus activities of monogalactosyl diglyceride and digalactosyl diglyceride from Clinacanthus nutans, a traditional Thai herbal medicine

    Directory of Open Access Journals (Sweden)

    Sirada Pongmuangmul

    2016-03-01

    Conclusions: MGDG and DGDG from C. nutans, a traditional Thai herbal medicine illustrated inhibitory activity against HSV-1 and HSV-2, probably by inhibiting the late stage of multiplication, suggesting their promising use as anti-HSV agents.

  8. Modular Synthesis of Heparin-Related Tetra-, Hexa- and Octasaccharides with Differential O-6 Protections: Programming for Regiodefined 6-O-Modifications

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    Marek Baráth

    2015-04-01

    Full Text Available Heparin and heparan sulphate (H/HS are important members of the glycosaminoglycan family of sugars that regulate a substantial number of biological processes. Such biological promiscuity is underpinned by hetereogeneity in their molecular structure. The degree of O-sulfation, particularly at the 6-position of constituent D-GlcN units, is believed to play a role in modulating the effects of such sequences. Synthetic chemistry is essential to be able to extend the diversity of HS-like fragments with defined molecular structure, and particularly to deconvolute the biological significance of modifications at O6. Here we report a synthetic approach to a small matrix of protected heparin-type oligosaccharides, containing orthogonal D-GlcN O-6 protecting groups at programmed positions along the chain, facilitating access towards programmed modifications at specific sites, relevant to sulfation or future mimetics.

  9. Lipid profiling of cyanobacteria Synechococcus sp. PCC 7002 using two-dimensional liquid chromatography with quadrupole time-of-flight mass spectrometry.

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    Shan, Yabing; Liu, Yiqun; Yang, Li; Nie, Honggang; Shen, Sensen; Dong, Chunxia; Bai, Yu; Sun, Qing; Zhao, Jindong; Liu, Huwei

    2016-10-01

    Glycerolipid is a main component of membranes in oxygenic photosynthetic organisms. Up to now, the majority of publication in this area has focused on the physiological functions of glycerolipids and lipoprotein complexes in photosynthesis, but the study on the separation and identification of glycerolipids in thylakoid membrane in cyanobacteria is relatively rare. Here we report a new method to separate and identify five photosynthetic glycerolipid classes, including monoglucosyl diacylglycerol, monogalactosyl diacylglycerol, digalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol, and phosphatidylglycerol, in cyanobacteria Synechococcus sp. PCC 7002 by two-dimensional (normal- and reversed-phase) liquid chromatography online coupled to quadrupole time-of-flight mass spectrometry. Over twice as many lipid species were detected by our method compared to the previously reported methods. Ten new odd-chain fatty acid glycerolipids were discovered for the first time. Moreover, complete separation of isomers of monogalactosyl diacylglycerol and monoglucosyl diacylglycerol was achieved. According to the tandem mass spectrometry results, we found that the head group of monoglucosyl diacylglycerols was not as stable as that of monogalactosyl diacylglycerols, which might explain why the organism chose monogalactosyl diacylglycerols and digalactosyl diacylglycerols instead of monoglucosyl diacylglycerols as the main content of the photosynthetic membranes in the history of evolution. This work will benefit further research on the physiological function of glycerolipids.

  10. Regulation of succinoglycan and galactoglucan biosynthesis in Sinorhizobium meliloti.

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    Becker, Anke; Rüberg, Silvia; Baumgarth, Birgit; Bertram-Drogatz, Peter Alexander; Quester, Ingmar; Pühler, Alfred

    2002-05-01

    Sinorhizobium meliloti (Rhizobium meliloti) 2011 has the ability to produce the two acidic exopolysaccharides succinoglycan (EPS I) and galactoglucan (EPS II). EPS I is a branched heteropolysaccharide composed of octasaccharide repeating units, whereas EPS II is a linear heteropolysaccharide consisting of disaccharide subunits. The exo-exs and exp gene clusters are involved in the biosynthesis of EPSI and EPSII, respectively. EPSI and EPSII biosynthesis genes are differentially expressed resulting in a complex regulation of EPS production in S. meliloti. The phosphate concentration was identified as an important factor affecting the expression of exp genes.

  11. Trans-α-xylosidase and trans-β-galactosidase activities, widespread in plants, modify and stabilize xyloglucan structures.

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    Franková, Lenka; Fry, Stephen C

    2012-07-01

    Cell-wall components are hydrolysed by numerous plant glycosidase and glycanase activities. We investigated whether plant enzymes also modify xyloglucan structures by transglycosidase activities. Diverse angiosperm extracts exhibited transglycosidase activities that progressively transferred single sugar residues between xyloglucan heptasaccharide (XXXG or its reduced form, XXXGol) molecules, at 16 μM and above, creating octa- to decasaccharides plus smaller products. We measured remarkably high transglycosylation:hydrolysis ratios under optimized conditions. To identify the transferred monosaccharide(s), we devised a dual-labelling strategy in which a neutral radiolabelled oligosaccharide (donor substrate) reacted with an amino-labelled non-radioactive oligosaccharide (acceptor substrate), generating radioactive cationic products. For example, 37 μM [Xyl-³H]XXXG plus 1 mM XXLG-NH₂ generated ³H-labelled cations, demonstrating xylosyl transfer, which exceeded xylosyl hydrolysis 1.6- to 7.3-fold, implying the presence of enzymes that favour transglycosylation. The transferred xylose residues remained α-linked but were relatively resistant to hydrolysis by plant enzymes. Driselase digestion of the products released a trisaccharide (α-[³H]xylosyl-isoprimeverose), indicating that a new xyloglucan repeat unit had been formed. In similar assays, [Gal-³H]XXLG and [Gal-³H]XLLG (but not [Fuc-³H]XXFG) yielded radioactive cations. Thus plants exhibit trans-α-xylosidase and trans-β-galactosidase (but not trans-α-fucosidase) activities that graft sugar residues from one xyloglucan oligosaccharide to another. Reconstructing xyloglucan oligosaccharides in this way may alter oligosaccharin activities or increase their longevity in vivo. Trans-α-xylosidase activity also transferred xylose residues from xyloglucan oligosaccharides to long-chain hemicelluloses (xyloglucan, water-soluble cellulose acetate, mixed-linkage β-glucan, glucomannan and arabinoxylan). With

  12. Influence of Fucoidans on Hemostatic System

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    Mikhail V. Kiselevskiy

    2013-07-01

    Full Text Available Three structurally different fucoidans from the brown seaweeds Saccharina latissima (SL, Fucus vesiculosus (FV, and Cladosiphon okamuranus (CO, two chemically modified fucoidans with a higher degree of sulfation (SL-S, CO-S, and a synthetic totally sulfated octasaccharide (OS, related to fucoidans, were assessed on anticoagulant and antithrombotic activities in different in vitro experiments. The effects were shown to depend on the structural features of the compounds tested. Native fucoidan SL with a degree of sulfation (DS of 1.3 was found to be the most active sample, fucoidan FV (DS 0.9 demonstrated moderate activity, while the polysaccharide CO (DS 0.4 was inactive in all performed experiments, even at high concentrations. Additional introduction of sulfate groups into fucoidan SL slightly decreased the anticoagulant effect of SL-S, while sulfation of CO, giving rise to the preparation CO-S, increased the activity dramatically. The high level of anticoagulant activity of polysaccharides SL, SL-S, and CO-S was explained by their ability to form ternary complexes with ATIII-Xa and ATIII-IIa, as well as to bind directly to thrombin. Synthetic per-O-sulfated octasaccharide OS showed moderate anticoagulant effect, determined mainly by the interaction of OS with the factor Xa in the presence of ATIII. Comparable tendencies were observed in the antithrombotic properties of the compounds tested.

  13. Xyloglucan Antibodies Inhibit Auxin-Induced Elongation and Cell Wall Loosening of Azuki Bean Epicotyls but Not of Oat Coleoptiles 1

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    Hoson, Takayuki; Masuda, Yoshio; Sone, Yoshiaki; Misaki, Akira

    1991-01-01

    Polyclonal antibodies were raised in rabbits against isoprimeverose (Xyl1Glc1), xyloglucan heptasaccharides (Xyl3Glc4), and octasaccharides (Gal1Xyl3Glc4). Antibodies specific for hepta- and octasaccharides suppressed auxin-induced elongation of epicotyl segments of azuki bean (Vigna angularis Ohwi and Ohashi cv Takara). These antibodies also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time and the relaxation rate of the cell walls) of azuki segments. However, none of the antibodies influenced auxin-induced elongation or cell wall loosening of coleoptile segments of oat (Avena sativa L. cv Victory). Auxin caused a decrease in molecular mass of xyloglucans in the cell walls of azuki epicotyls and oat coleoptiles. The antibodies inhibited such a change in molecular mass of xyloglucans in both species. Preimmune serum exhibited little or no inhibitory effect on auxin-induced elongation, cell wall loosening, or breakdown of xyloglucans. The results support the view that the breakdown of xyloglucans is associated with the cell wall loosening responsible for auxin-induced elongation in dicotyledons. The view does not appear to be applicable to poaceae, because the inhibition of xyloglucan breakdown by the antibodies did not influence auxin-induced elongation or cell wall loosening of oat coleoptiles. ImagesFigure 1Figure 2 PMID:16668221

  14. Xyloglucan antibodies inhibit auxin-induced elongation and cell wall loosening of azuki bean epicotyls but not of oat coleoptiles.

    Science.gov (United States)

    Hoson, T; Masuda, Y; Sone, Y; Misaki, A

    1991-06-01

    Polyclonal antibodies were raised in rabbits against isoprimeverose (Xyl(1)Glc(1)), xyloglucan heptasaccharides (Xyl(3)Glc(4)), and octasaccharides (Gal(1)Xyl(3)Glc(4)). Antibodies specific for hepta- and octasaccharides suppressed auxin-induced elongation of epicotyl segments of azuki bean (Vigna angularis Ohwi and Ohashi cv Takara). These antibodies also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time and the relaxation rate of the cell walls) of azuki segments. However, none of the antibodies influenced auxin-induced elongation or cell wall loosening of coleoptile segments of oat (Avena sativa L. cv Victory). Auxin caused a decrease in molecular mass of xyloglucans in the cell walls of azuki epicotyls and oat coleoptiles. The antibodies inhibited such a change in molecular mass of xyloglucans in both species. Preimmune serum exhibited little or no inhibitory effect on auxin-induced elongation, cell wall loosening, or breakdown of xyloglucans. The results support the view that the breakdown of xyloglucans is associated with the cell wall loosening responsible for auxin-induced elongation in dicotyledons. The view does not appear to be applicable to poaceae, because the inhibition of xyloglucan breakdown by the antibodies did not influence auxin-induced elongation or cell wall loosening of oat coleoptiles.

  15. Cell wall carbohydrates from fruit pulp of Argania spinosa: structural analysis of pectin and xyloglucan polysaccharides.

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    Aboughe-Angone, Sophie; Nguema-Ona, Eric; Ghosh, Partha; Lerouge, Patrice; Ishii, Tadashi; Ray, Bimalendu; Driouich, Azeddine

    2008-01-14

    Isolated cell walls of Argania spinosa fruit pulp were fractionated into their polysaccharide constituents and the resulting fractions were analysed for monosaccharide composition and chemical structure. The data reveal the presence of homogalacturonan, rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II) in the pectic fraction. RG-I is abundant and contains high amounts of Ara and Gal, indicative of an important branching in this polysaccharide. RG-II is less abundant than RG-I and exists as a dimer. Structural characterisation of xyloglucan using enzymatic hydrolysis, gas chromatography, MALDI-TOF-MS and methylation analysis shows that XXGG, XXXG, XXLG and XLLG are the major subunit oligosaccharides in the ratio of 0.6:1:1.2:1.6. This finding demonstrates that the major neutral hemicellulosic polysaccharide is a galacto-xyloglucan. In addition, Argania fruit xyloglucan has no XUFG, a novel xyloglucan motif recently discovered in Argania leaf cell walls. Finally, the isolation and analysis of arabinogalactan-proteins showed that Argania fruit pulp is rich in these proteoglycans.

  16. Structural investigation of hemicellulosic polysaccharides from Argania spinosa: characterisation of a novel xyloglucan motif.

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    Ray, Bimalendu; Loutelier-Bourhis, Corinne; Lange, Catherine; Condamine, Eric; Driouich, Azeddine; Lerouge, Patrice

    2004-01-22

    Hemicellulose polymers were isolated from Argania spinosa leaf cell walls by sequential extractions with alkali. The structure of the two main polymers, xylan and xyloglucan, was investigated by enzyme degradation with specific endoglycosidases followed by analysis of the resulting fragments by high performance anion exchange chromatography (HPAEC) and matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS). The results show that A. spinosa xylan is composed of a beta-(1-->4)-linked-D-xylopyranose backbone substituted with 4-O-methyl-D-glucuronic acid residues. Xyloglucan oligosaccharide subunits were generated by treatment with an endo-(1-->4)-beta-D-glucanase of the xyloglucan-rich hemicellulosic fractions. MALDI-TOF mass spectra and HPAE-PAD chromatography of the pool of endoglucanase-generated xyloglucan oligomers indicated that A. spinosa cell wall contains a XXXG-type xyloglucan. In addition to XXXG, XXFG, XLXG/XXLG, XLFG fragments previously characterised in various plants, a second group of XXXG-type fragments was detected. The primary structure of the major subunit was determined by a combination of sugar analysis, methylation analysis, post-source decay (PSD) fragment analysis of MALDI-TOF MS and 1H NMR spectroscopy. This fragment, termed XUFG, contains a novel beta-D-Xylp-(1-->2)-alpha-D-Xylp side chain linked to C-6 of the second glucose unit from the nonreducing end of the cellotetraose sequence.

  17. Strong IgG antibody responses to Borrelia burgdorferi glycolipids in patients with Lyme arthritis, a late manifestation of the infection.

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    Jones, Kathryn L; Seward, Robert J; Ben-Menachem, Gil; Glickstein, Lisa J; Costello, Catherine E; Steere, Allen C

    2009-07-01

    In this study, the membrane lipids of B. burgdorferi were separated into 16 fractions; the components in each fraction were identified, and the immunogenicity of each fraction was determined by ELISA using sera from Lyme disease patients. Only the 2 glycolipids, acylated cholesteryl galactoside (ACG, BbGL-I) and monogalactosyl diacylglycerol (MgalD, BbGL-II), were immunogenic. Early in the infection, 24 of 84 patients (29%) who were convalescent from erythema migrans and 19 of the 35 patients (54%) with neuroborreliosis had weak IgG responses to purified MgalD, and a smaller percentage of patients had early responses to synthetic ACG. However, almost all of 75 patients with Lyme arthritis, a late disease manifestation, had strong IgG reactivity with both glycolipids. Thus, almost all patients with Lyme arthritis have strong IgG antibody responses to B. burgdorferi glycolipid antigens.

  18. Isolation and purification of feruloylated oligosaccharides from cell walls of sugar-beet pulp.

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    Ralet, M C; Thibault, J F; Faulds, C B; Williamson, G

    1994-10-17

    Cell walls from sugar-beet pulp contain some feruloyl groups linked to the pectic neutral side-chains. Enzymic as well as chemical hydrolysis of the pulp yielded a series of feruloylated oligosaccharides, which have been purified by Sephadex LH-20 and Biogel P-2 chromatography in aqueous solvents. Feruloylated arabinose di-, tri-, hexa-, hepta-, and octa-saccharides as well as feruloylated galactose disaccharides were obtained after hydrolysis of the pulp with a mixture of fungal carbohydrases (Driselase). Feruloylated arabinose and galactose monosaccharides were obtained through mild acid hydrolyses. Both arabinose and galactose residues in the side-chains are feruloylated, 50-55% of the feruloyl groups being linked to arabinose residues and 45-50% to galactose residues. It is concluded that 1 out of 56 arabinose residues and 1 out of 16 galactose residues present as pectic side-chains in sugar-beet pulp carry a feruloyl group.

  19. Structural and functional peculiarities of the lipopolysaccharide of Azospirillum brasilense SR55, isolated from the roots of Triticum durum.

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    Boyko, Alevtina S; Konnova, Svetlana A; Fedonenko, Yulia P; Zdorovenko, Evelina L; Smol'kina, Olga N; Kachala, Vadim V; Ignatov, Vladimir V

    2011-10-20

    Azospirillum brasilense SR55, isolated from the rhizosphere of Triticum durum, was classified as serogroup II on the basis of serological tests. Such serogroup affiliation is uncharacteristic of wheat-associated Azospirillum species. The lipid A of A. brasilense SR55 lipopolysaccharide contained 3-hydroxytetradecanoic, 3-hydroxyhexadecanoic, hexadecanoic and octadecenoic fatty acids. The structure of the lipopolysaccharide's O polysaccharide was established, with the branched octasaccharide repeating unit being represented by l-rhamnose, l-3-O-Me-rhamnose, d-galactose and d-glucuronic acid. The SR55 lipopolysaccharide induced deformations of wheat root hairs. The lipopolysaccharide was not involved in bacterial cell aggregation, but its use to pretreat wheat roots was conducive to cell adsorption. This study shows that Azospirillum bacteria can utilise their own lipopolysaccharide as a carbon source, which may give them an advantage in competitive natural environments.

  20. Positional distribution of fatty acids on the glycerol backbone during the biosynthesis of glycerolipids in Ectocarpus fasciculatus

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The biosynthesis of glycolipids in E. fasciculatus was studied by 14C label and chase. The fatty acids in sulphoquinovosyl diacylglycerol (SQDG) were almost 16-car- bon and 18-carbon ones. In addition to the two fatty acids, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) contained 8.5 mol% and 31.0 mol% of eicosapentaenoic acid (20∶5), respectively, and this fatty acid was usually distributed in the sn-1 position of the glycerol backbone. When plants were incubated with [2-14C] acetate, differences existed in the positional distribution of the labeled fatty acids in sn-1 and sn-2 among the three glycerolipids. In SQDG, 14C-labeled fatty acids were distributed uniformly in the sn-1 and sn-2 positions. In DGDG, 14C-labeled fatty acids were mainly distributed in the sn-2 position. In MGDG, the radioactivity of fatty acids in sn-1 position was far greater than that in sn-2 position after a 30 min pulse label, and the difference in radioactivity between the two positions decreased rapidly. The above results indicated that differences in the positional distribution of 14C-labeled fatty acids between sn-1 and sn-2 positions might be related to 20∶5 and the biosynthesis of DGDG. Our results also suggested that E. fasciculatus had the same DGDG biosynthetic pathway as that in higher plants and galactosyl transferase was selective for MGDG.

  1. Combined Effects of Ozone and Drought on the Physiology and Membrane Lipids of Two Cowpea (Vigna unguiculata (L.) Walp) Cultivars

    Science.gov (United States)

    Moura Rebouças, Deborah; Maia De Sousa, Yuri; Bagard, Matthieu; Costa, Jose Helio; Jolivet, Yves; Fernandes De Melo, Dirce; Repellin, Anne

    2017-01-01

    The interactive effects of drought and ozone on the physiology and leaf membrane lipid content, composition and metabolism of cowpea (Vigna unguiculata (L.) Walp.) were investigated in two cultivars (EPACE-1 and IT83-D) grown under controlled conditions. The drought treatment (three-week water deprivation) did not cause leaf injury but restricted growth through stomatal closure. In contrast, the short-term ozone treatment (130 ppb 12 h daily during 14 day) had a limited impact at the whole-plant level but caused leaf injury, hydrogen peroxide accumulation and galactolipid degradation. These effects were stronger in the IT83-D cultivar, which also showed specific ozone responses such as a higher digalactosyl-diacylglycerol (DGDG):monogalactosyl-diacylglycerol (MGDG) ratio and the coordinated up-regulation of DGDG synthase (VuDGD2) and ω-3 fatty acid desaturase 8 (VuFAD8) genes, suggesting that membrane remodeling occurred under ozone stress in the sensitive cultivar. When stresses were combined, ozone did not modify the stomatal response to drought and the observed effects on whole-plant physiology were essentially the same as when drought was applied alone. Conversely, the drought-induced stomatal closure appeared to alleviate ozone effects through the reduction of ozone uptake. PMID:28273829

  2. A Cultivated Form of a Red Seaweed (Chondrus crispus, Suppresses β-Amyloid-Induced Paralysis in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Jatinder Singh Sangha

    2015-10-01

    Full Text Available We report here the protective effects of a methanol extract from a cultivated strain of the red seaweed, Chondrus crispus, against β-amyloid-induced toxicity, in a transgenic Caenorhabditis elegans, expressing human Aβ1-42 gene. The methanol extract of C. crispus (CCE, delayed β-amyloid-induced paralysis, whereas the water extract (CCW was not effective. The CCE treatment did not affect the transcript abundance of amy1; however, Western blot analysis revealed a significant decrease of Aβ species, as compared to untreated worms. The transcript abundance of stress response genes; sod3, hsp16.2 and skn1 increased in CCE-treated worms. Bioassay guided fractionation of the CCE yielded a fraction enriched in monogalactosyl diacylglycerols (MGDG that significantly delayed the onset of β-amyloid-induced paralysis. Taken together, these results suggested that the cultivated strain of C. crispus, whilst providing dietary nutritional value, may also have significant protective effects against β-amyloid-induced toxicity in C. elegans, partly through reduced β-amyloid species, up-regulation of stress induced genes and reduced accumulation of reactive oxygen species (ROS.

  3. Methyl Jasmonate Reduces Water Stress in Strawberry.

    Science.gov (United States)

    Wang

    1999-11-01

    The effect of methyl jasmonate (MJ) on changes of oxygen-scavenging enzyme activities and membrane lipid composition was studied in strawberry leaves under water stress. Under water stress, MJ treatment reduced the increase of peroxidase (EC 1.11.1.7; POD) activity, maintained higher catalase (EC 1.11.1.6; CAT) and superoxide dismutase (EC 1.15.1.1; SOD) activities, and ascorbic acid content. In addition, MJ treatment reduced transpiration and membrane-lipid peroxidation as expressed by malondialdehyde (MDA) content, lessened the reduction of membrane lipids, glycolipids [monogalactosyl diglyceride (MGDG), digalactosyl diglyceride (DGDG)], and phospholipids [phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylinositol (PI)]. In water-deficit conditions, MJ treatment also alleviated the decline in the degree of fatty acid unsaturation and the ratio of linolenic (18:3) to linoleic acid (18:2). These results indicate that MJ treatment appears to alter the metabolism of strawberry plants rendering the tissue better able to withstand water stress.

  4. Valorization of Lipids from Gracilaria sp. through Lipidomics and Decoding of Antiproliferative and Anti-Inflammatory Activity

    Science.gov (United States)

    da Costa, Elisabete; Melo, Tânia; Moreira, Ana S. P.; Bernardo, Carina; Helguero, Luisa; Ferreira, Isabel; Cruz, Maria Teresa; Rego, Andreia M.; Domingues, Pedro; Calado, Ricardo; Abreu, Maria H.; Domingues, Maria Rosário

    2017-01-01

    The lipidome of the red seaweed Gracilaria sp., cultivated on land-based integrated multitrophic aquaculture (IMTA) system, was assessed for the first time using hydrophilic interaction liquid chromatography-mass spectrometry and tandem mass spectrometry (HILIC–MS and MS/MS). One hundred and forty-seven molecular species were identified in the lipidome of the Gracilaria genus and distributed between the glycolipids classes monogalactosyl diacylglyceride (MGDG), digalactosyl diacylglyceride (DGDG), sulfoquinovosyl monoacylglyceride (SQMG), sulfoquinovosyl diacylglyceride (SQDG), the phospholipids phosphatidylcholine (PC), lyso-PC, phosphatidylglycerol (PG), lyso-PG, phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatic acid (PA), inositolphosphoceramide (IPC), and betaine lipids monoacylglyceryl- and diacylglyceryl-N,N,N-trimethyl homoserine (MGTS and DGTS). Antiproliferative and anti-inflammatory effects promoted by lipid extract of Gracilaria sp. were evaluated by monitoring cell viability in human cancer lines and by using murine macrophages, respectively. The lipid extract decreased cell viability of human T-47D breast cancer cells and of 5637 human bladder cancer cells (estimated half-maximal inhibitory concentration (IC50) of 12.2 μg/mL and 12.9 μg/mL, respectively) and inhibited the production of nitric oxide (NO) evoked by the Toll-like receptor 4 agonist lipopolysaccharide (LPS) on the macrophage cell line RAW 264.7 (35% inhibition at a concentration of 100 μg/mL). These findings contribute to increase the ranking in the value-chain of Gracilaria sp. biomass cultivated under controlled conditions on IMTA systems. PMID:28257116

  5. N-glycan abnormalities in children with galactosemia.

    Science.gov (United States)

    Coss, Karen P; Hawkes, Colin P; Adamczyk, Barbara; Stöckmann, Henning; Crushell, Ellen; Saldova, Radka; Knerr, Ina; Rubio-Gozalbo, Maria E; Monavari, Ardeshir A; Rudd, Pauline M; Treacy, Eileen P

    2014-02-07

    Galactose intoxication and over-restriction in galactosemia may affect glycosylation pathways and cause multisystem effects. In this study, we describe an applied hydrophilic interaction chromatography ultra-performance liquid chromatography high-throughput method to analyze whole serum and extracted IgG N-glycans with measurement of agalactosylated (G0), monogalactosylated (G1), and digalactosylated (G2) structures as a quantitative measure of galactose incorporation. This was applied to nine children with severe galactosemia (genotype Q188R/Q188R) and one child with a milder variant (genotype S135L/S135L). The profiles were also compared with those obtained from three age-matched children with PMM2-CDG (congenital disorder of glycosylation type Ia) and nine pediatric control samples. We have observed that severe N-glycan assembly defects correct in the neonate following dietary restriction of galactose. However, treated adult galactosemia patients continue to exhibit ongoing N-glycan processing defects. We have now applied informative galactose incorporation ratios as a method of studying the presence of N-glycan processing defects in children with galactosemia. We identified N-glycan processing defects present in galactosemia children from an early age. For G0/G1, G0/G2, and (G0/G1)/G2 ratios, the difference noted between galactosemia patients and controls was found to be statistically significant (p = 0.002, 0.01, and 0.006, respectively).

  6. Trienoic fatty acids and plant tolerance of temperature

    Directory of Open Access Journals (Sweden)

    Routaboul Jean-Marc

    2002-01-01

    Full Text Available The biophysical reactions of light harvesting and electron transport during photosynthesis take place in a uniquely constructed bilayer, the thylakoid. In all photosynthetic eukaryotes, the complement of atypical glycerolipid molecules that form the foundation of this membrane are characterised by sugar head-groups and a very high level of unsaturation in the fatty acids that occupy the central portion of the thylakoid bilayer. alpha-linolenic (18:3 or a combination of 18:3 and hexadecatrienoic (16:3 acids typically account for approximately two-thirds of all thylakoid membrane fatty acids and over 90% of the fatty acids of monogalactosyl diacylglycerol, the major thylakoid lipid [1, 2]. The occurrence of trienoic fatty acids as a major component of the thylakoid membrane is especially remarkable since these fatty acids form highly reactive targets for active oxygen species and free radicals, which are often the by-products of oxygenic photosynthesis. Photosynthesis is one of the most temperature-sensitive functions of plant [3, 4]. There remains a widespread belief that these trienoic fatty acids might have some crucial role in plants to be of such universal occurrence, especially in photosynthesis tolerance of temperature [5].

  7. Lipid content and fatty acid composition of green algae Scenedesmus obliquus grown in a constant cell density apparatus

    Science.gov (United States)

    Choi, K. J.; Nakhost, Z.; Barzana, E.; Karel, M.

    1987-01-01

    The lipids of alga Scenedesmus obliquus grown under controlled conditions were separated and fractionated by column and thin-layer chromatography, and fatty acid composition of each lipid component was studied by gas-liquid chromatography (GLC). Total lipids were 11.17%, and neutral lipid, glycolipid and phospholipid fractions were 7.24%, 2.45% and 1.48% on a dry weight basis, respectively. The major neutral lipids were diglycerides, triglycerides, free sterols, hydrocarbons and sterol esters. The glycolipids were: monogalactosyl diglyceride, digalactosyl diglyceride, esterified sterol glycoside, and sterol glycoside. The phospholipids included: phosphatidyl choline, phosphatidyl glycerol and phosphatidyl ethanolamine. Fourteen fatty acids were identified in the four lipid fractions by GLC. The main fatty acids were C18:2, C16:0, C18:3(alpha), C18:1, C16:3, C16:1, and C16:4. Total unsaturated fatty acid and essential fatty acid compositions of the total algal lipids were 80% and 38%, respectively.

  8. Effect of post harvest radiation processing and storage on the volatile oil composition and glucosinolate profile of cabbage.

    Science.gov (United States)

    Banerjee, Aparajita; Variyar, Prasad S; Chatterjee, Suchandra; Sharma, Arun

    2014-05-15

    Effect of radiation processing (0.5-2 kGy) and storage on the volatile oil constituents and glucosinolate profile of cabbage was investigated. Among the volatile oil constituents, an enhancement in trans-hex-2-enal was noted on irradiation that was attributed to the increased liberation of precursor linolenic acid mainly from monogalactosyl diacyl glycerol (MGDG). Irradiation also enhanced sinigrin, the major glucosinolate of cabbage that accounted for the enhanced allyl isothiocyanate (AITC) in the volatile oils of the irradiated vegetable. During storage the content of trans-hex-2-enal increased immediately after irradiation and then returned to the basal value within 24h while the content of sinigrin and AITC increased post irradiation and thereafter remained constant during storage. Our findings on the enhancement in potentially important health promoting compounds such as sinigrin and AITC demonstrates that besides extending shelf life and safety, radiation processing can have an additional advantage in improving the nutritional quality of cabbage. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Valorization of Lipids from Gracilaria sp. through Lipidomics and Decoding of Antiproliferative and Anti-Inflammatory Activity.

    Science.gov (United States)

    da Costa, Elisabete; Melo, Tânia; Moreira, Ana S P; Bernardo, Carina; Helguero, Luisa; Ferreira, Isabel; Cruz, Maria Teresa; Rego, Andreia M; Domingues, Pedro; Calado, Ricardo; Abreu, Maria H; Domingues, Maria Rosário

    2017-03-02

    The lipidome of the red seaweed Gracilaria sp., cultivated on land-based integrated multitrophic aquaculture (IMTA) system, was assessed for the first time using hydrophilic interaction liquid chromatography-mass spectrometry and tandem mass spectrometry (HILIC-MS and MS/MS). One hundred and forty-seven molecular species were identified in the lipidome of the Gracilaria genus and distributed between the glycolipids classes monogalactosyl diacylglyceride (MGDG), digalactosyl diacylglyceride (DGDG), sulfoquinovosyl monoacylglyceride (SQMG), sulfoquinovosyl diacylglyceride (SQDG), the phospholipids phosphatidylcholine (PC), lyso-PC, phosphatidylglycerol (PG), lyso-PG, phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatic acid (PA), inositolphosphoceramide (IPC), and betaine lipids monoacylglyceryl- and diacylglyceryl-N,N,N-trimethyl homoserine (MGTS and DGTS). Antiproliferative and anti-inflammatory effects promoted by lipid extract of Gracilaria sp. were evaluated by monitoring cell viability in human cancer lines and by using murine macrophages, respectively. The lipid extract decreased cell viability of human T-47D breast cancer cells and of 5637 human bladder cancer cells (estimated half-maximal inhibitory concentration (IC50) of 12.2 μg/mL and 12.9 μg/mL, respectively) and inhibited the production of nitric oxide (NO) evoked by the Toll-like receptor 4 agonist lipopolysaccharide (LPS) on the macrophage cell line RAW 264.7 (35% inhibition at a concentration of 100 μg/mL). These findings contribute to increase the ranking in the value-chain of Gracilaria sp. biomass cultivated under controlled conditions on IMTA systems.

  10. Galactosemia, a single gene disorder with epigenetic consequences.

    LENUS (Irish Health Repository)

    Coman, David J

    2010-03-01

    Long-term outcomes of classic galactosemia (GAL) remain disappointing. It is unclear if the complications result mainly from prenatal-neonatal toxicity or persistent glycoprotein and glycolipid synthesis abnormalities. We performed gene expression profiling (T transcriptome) to characterize key-altered genes and gene clusters of four patients with GAL with variable outcomes maintained on a galactose-restricted diet, compared with controls. Significant perturbations of multiple cell signaling pathways were observed including mitogen-activated protein kinase (MAPK) signaling, regulation of the actin cytoskeleton, focal adhesion, and ubiquitin mediated proteolysis. A number of genes significantly altered were further investigated in the GAL cohort including SPARC (osteonectin) and S100A8 (S100 calcium-binding protein). The whole serum N-glycan profile and IgG glycosylation status of 10 treated patients with GAL were compared with healthy control serum and IgG using a quantitative high-throughput analytical HPLC platform. Increased levels of agalactosylated and monogalactosylated structures and decreases in certain digalactosylated structures were identified in the patients. The persistent abnormal glycosylation of serum glycoproteins seen with the microarray data indicates persisting metabolic dyshomeostasis and gene dysregulation in "treated" GAL. Strict restriction of dietary galactose is clearly life saving in the neonatal period; long-term severe galactose restriction may contribute to ongoing systemic abnormalities.

  11. Chemoenzymatic synthesis of hydrophobic glycoprotein: synthesis of saposin C carrying complex-type carbohydrate.

    Science.gov (United States)

    Hojo, Hironobu; Tanaka, Hiromasa; Hagiwara, Masashi; Asahina, Yuya; Ueki, Akiharu; Katayama, Hidekazu; Nakahara, Yuko; Yoneshige, Azusa; Matsuda, Junko; Ito, Yukishige; Nakahara, Yoshiaki

    2012-11-02

    The complex-type N-linked octasaccharide oxazoline having LacNAc as the nonreducing end sugar was efficiently synthesized using the benzyl-protected LacNAc, mannose, and β-mannosyl GlcNAc units as key building blocks. To achieve a highly β-selective glycosylation with the LacNAc unit, the N-trichloroacetyl group was used for the protection of the amino group in the LacNAc unit. After complete assembly of these units and deprotection, the obtained free sugar was successfully derivatized into the corresponding sugar oxazoline. On the other hand, the N-acetylglucosaminylated saposin C, a hydrophobic lipid-binding protein, was chemically synthesized by the native chemical ligation reaction. On the basis of the previous results related to the synthesis of the nonglycosylated saposin C, the O-acyl isopeptide structure was introduced to the N-terminal peptide thioester carrying GlcNAc to improve its solubility toward aqueous organic solvents. The ligation reaction efficiently proceeded with the simultaneous O- to N-acyl shift at the O-acyl isopeptide moiety. After the removal of the cysteine-protecting group and folding, saposin C carrying GlcNAc was successfully obtained. The synthetic sugar oxazoline was then transferred to this glycoprotein using the mutant of endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) (glycosynthase), and the saposin C carrying the complex-type nonasaccharide was successfully obtained.

  12. NMR-based conformation and dynamics of a tetrasaccharide-repeating sulfated fucan substituted by different counterions.

    Science.gov (United States)

    Soares, Paulo A G; Queiroz, Ismael N L; Santos, Gustavo R C; Mourão, Paulo A S; Pomin, Vitor H

    2016-11-01

    The sulfated fucan from the sea urchin Lytechinus variegatus is composed of the repetitive sequence [-3)-α-l-Fucp-4( OSO3-)-(1-3)-α-l-Fucp-2,4-di( OSO3-)-(1-3)-α-l-Fucp-2( OSO3-)-(1-3)-α-l-Fucp-2( OSO3-)-(1-]n . Conformation (of rings and chains) and dynamics of this tetrasaccharide-repeating sulfated fucan substituted by Na(+) , Ca(2+) , and Li(+) as counterions have been examined through experiments of liquid-state nuclear magnetic resonance spectroscopy. Scalar coupling and nuclear Overhauser effect (NOE)-based data have confirmed that all composing units occur as (1) C4 chair conformer regardless of the cation type, unit position within the repeating sequence, and sulfation type. Chain conformation determined by NOE signal pattern assisted by molecular modeling for a theoretical octasaccharide has shown a similar linear 3D structure for the three differently substituted forms. Data derived from spin-relaxation measurements have indicated a contribution of counterion type to dynamics. The calcium-based preparation has shown the highest mobility while the sodiated one showed the lowest mobility. The set of results from this work suggests that counterion type can affect the physicochemical properties of the structurally well-defined sulfated fucan. The counterion effect seems to impact more on the structural mobility than on average conformation of the studied sulfated glycan in solution.

  13. NMR study of hydroxy and amide protons in hyaluronan polymers.

    Science.gov (United States)

    Nestor, Gustav; Sandström, Corine

    2017-02-10

    Hyaluronan (HA) is an important and well characterized glycosaminoglycan with high viscosity and water-retaining capacity. Nonetheless, it is not fully understood whether conformational properties of the easily characterized HA oligomers can be transferred to HA polymers. To investigate possible differences in hydration, hydrogen bonding and flexibility between HA polymers and oligomers, hydroxy and amide protons of HA polymers were studied by solution-state and high-resolution magic angle spinning (HR-MAS) NMR spectroscopy. Measurements of chemical shifts, temperature coefficients and NOEs in HA polymers revealed that the NMR data are very similar compared to the interior of a HA octasaccharide, supporting transient hydrogen bond interactions across the β(1→3) and β(1→4) glycosidic linkages. However, differences in NOEs suggested a cis-like orientation between NH and H2 in the HA polymer. The lack of concentration dependence of the hydroxy proton chemical shifts suggests that there are no direct inter-chain interactions involving hydroxy protons at the concentrations investigated.

  14. Heparin sodium compliance to USP monograph: structural elucidation of an atypical 2.18 ppm NMR signal.

    Science.gov (United States)

    Mourier, Pierre A J; Guichard, Olivier Y; Herman, Fréderic; Viskov, Christian

    2012-01-01

    The ¹H nuclear magnetic resonance (NMR) acceptance criteria in the new heparin US Pharmacopeia (USP) monograph do not take into account potential structural modifications responsible for any extra signals observed in ¹H NMR spectra, some purified heparins may be non-compliant under the proposed new USP guidelines and incorrectly classified as unsuitable for pharmaceutical use. Heparins from the "ES" source, containing an extra signal at 2.18 ppm, were depolymerized under controlled conditions using heparinases I, II, and III. The oligosaccharides responsible for the 2.18 ppm signal were enriched using orthogonal chromatographic techniques. After multiple purification steps, we obtained an oligosaccharide mixture containing a highly enriched octasaccharide bearing the structural modification responsible for the extra signal. Following heparinase I depolymerization, a pure tetrasaccharide containing the fingerprint structural modification was isolated for full structural determination. Using 1D and 2D ¹H NMR spectroscopy, the structural moiety responsible for the extra signal at 2.18 ppm was identified as an acetyl group on the heparin backbone, most likely resulting from a very minor manufacturing process side reaction that esterifies the uronic acid at position 3. Such analytical peculiarity has always been present in this heparin source and it was used safety over the years. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Single-molecule interrogation of a bacterial sugar transporter allows the discovery of an extracellular inhibitor

    Science.gov (United States)

    Kong, Lingbing; Harrington, Leon; Li, Qiuhong; Cheley, Stephen; Davis, Benjamin G.; Bayley, Hagan

    2013-08-01

    Capsular polysaccharides form the outermost protective layer around many Gram-negative bacteria. Antibiotics aimed directly at weakening this layer are not yet available. In pathogenic Escherichia coli E69, a protein, Wza, forms a pore in the outer membrane that transports K30 capsular polysaccharide from its site of synthesis to the outside of the cell. This therefore represents a prospective antibiotic target. Here we test a variety of grommet-like mimics of K30 capsular polysaccharide on wild-type Wza and on mutant open forms of the pore by electrical recording in planar lipid bilayers. The most effective glycomimetic was the unnatural cyclic octasaccharide octakis(6-deoxy-6-amino)cyclomaltooctaose (am8γCD), which blocks the α-helix barrel of Wza, a site that is directly accessible from the external medium. This glycomimetic inhibited K30 polysaccharide transport in live E. coli E69. With the protective outer membrane disrupted, the bacteria can be recognized and killed by the human immune system.

  16. Synthesis of heparin-like oligosaccharides on polymer supports.

    Science.gov (United States)

    Ojeda, Rafael; Terentí, Olimpia; de Paz, José-Luis; Martín-Lomas, Manuel

    2004-01-01

    The biological functions of a variety of proteins are regulated by heparan sulfate glycosaminoglycans. In order to facilitate the elucidation of the molecular basis of glycosaminoglycan-protein interactions we have developed syntheses of heparin-like oligosaccharides on polymer supports. A completely stereoselective strategy previously developed by us for the synthesis of these oligosaccharides in solution has been extended to the solid phase using an acceptor-bound approach. Both a soluble polymer support and a polyethylene glycol-grafted polystyrene resin have been used and different strategies for the attachment of the acceptor to the support have been explored. The attachment of fully protected disaccharide building blocks to a soluble support through the carboxylic group of the uronic acid unit by a succinic ester linkage, the use of trichloroacetimidates as glycosylating agents and of a functionalized Merryfield type resin for the capping process allowed for the construction of hexasaccharide and octasaccharide fragments containing the structural motif of the regular region of heparin. This strategy may facilitate the synthesis of glycosaminoglycan oligosaccharides by using the required building blocks in the glycosylation sequence.

  17. The structure of the carbohydrate backbone of the lipopolysaccharide of Pectinatus frisingensis strain VTT E-79104.

    Science.gov (United States)

    Vinogradov, Evgeny; Li, Jianjun; Sadovskaya, Irina; Jabbouri, Said; Helander, Ilkka M

    2004-06-22

    The structure of the carbohydrate backbone of the lipopolysaccharide from Pectinatus frisingensis strain VTT E-79104 was analyzed using chemical degradations, NMR spectroscopy, mass spectrometry, and chemical methods. The LPS contains two major structural variants, differing in the presence or absence of an octasaccharide fragment. The largest structure of the carbohydrate backbone of the LPS, that could be deduced from experimental results, consists of 20 monosaccharides arranged in a nonrepetitive sequence: [carbohydrate structure: see text] where R is H or 4-O-Me-alpha-L-Fuc-(1-2)-4-O-Me-beta-Hep-(1-3)-alpha-GlcNAc-(1-2)-beta-Man-(1-3)-beta-ManNAc-(1-4)-alpha-Gal-(1-4)-beta-Hep-(1-3)-beta-GalNAc-(1- where Hep is a residue of D-glycero-D-galacto-heptose; all monosaccharides have the D-configuration except for 4-O-Me-L-Fuc and L-Ara4N. This structure is architecturally similar to the oligosaccharide system reported previously in P. frisingensis VTT E-82164 LPS, but differs from the latter in composition and also in the size of the outer region.

  18. Impact of sulfation pattern on the conformation and dynamics of sulfated fucan oligosaccharides as revealed by NMR and MD.

    Science.gov (United States)

    Queiroz, Ismael N L; Wang, Xiaocong; Glushka, John N; Santos, Gustavo R C; Valente, Ana P; Prestegard, James H; Woods, Robert J; Mourão, Paulo A S; Pomin, Vitor H

    2015-05-01

    Sulfated fucans from sea urchin egg jelly express well-defined chemical structures that vary with species. This species specificity regulates the sperm acrosome reaction, a critical step to assure intra-specific fertilization. In addition, these polysaccharides are involved in other biological activities such as anticoagulation. Although sulfation patterns are relevant to the levels of response in both activities, conformation and dynamics of these glycans are also contributing factors. However, data about these features of sulfated fucans are very rare. To address this, we have employed nuclear magnetic resonance experiments combined with molecular dynamics on structurally defined oligosaccharides derived from two sulfated fucans. The results have indicated that the oligosaccharides are flexible in solution. Ring conformation of their composing units displays just the (1)C4 chair configuration. In a particular octasaccharide, composed of two tetrasaccharide sequences, inter-residual hydrogen bonds play a role to decrease dynamics in these repeating units. Conversely, the linking disaccharide [-3)-α-L-Fucp-2(OSO3(-))-(1-3)-α-L-Fucp-4(OCO3(-))-(1-] located right between the two tetrasaccharide units has amplified motions suggested to be promoted by electrostatic repulsion of sulfates on opposite sides of the central glycosidic bond. This conjunction of information about conformation and dynamics of sulfated fucan oligosaccharides provides new insights to explain how these glycans behave free in solution and influenced by sulfation patterns. It may also serve for future studies concerning structure-function relationship of sulfated fucans, especially those involving sea urchin fertilization and anticoagulation.

  19. HABA-based ionic liquid matrices for UV-MALDI-MS analysis of heparin and heparan sulfate oligosaccharides.

    Science.gov (United States)

    Przybylski, Cedric; Gonnet, Florence; Bonnaffé, David; Hersant, Yael; Lortat-Jacob, Hugues; Daniel, Regis

    2010-02-01

    Polysulfated carbohydrates such as heparin (HP) and heparan sulfate (HS) are not easily amenable to usual ultraviolet matrix-assisted laser desorption/ionization-mass spectrometry (UV-MALDI)-MS analysis due to the thermal lability of their O- and N-SO(3) moieties, and their poor ionization efficiency with common crystalline matrices. Recently, ionic liquid matrices showed considerable advantages over conventional matrices for MALDI-MS of acidic compounds. Two new ionic liquid matrices (ILMs) based on the combination of 2-(4-hydroxyphenylazo)benzoic acid (HABA) with 1,1,3,3-tetramethylguanidine and spermine were evaluated in the study herein. Both ILMs were successfully applied to the analysis of synthetic heparin oligosaccharides of well-characterized structures as well as to heparan sulfate-derived oligosaccharides from enzymatic depolymerization. HABA-based ILMs showed improved signal-to-noise ratio as well as a decrease of fragmentation/desulfation processes and cation exchange. Sulfated oligosaccharides were detected with higher sensitivity than usual crystalline matrices, and their intact fully O- and N-sulfated species [M-Na](-) were easily observed on mass spectra. MALDI-MS characterization of challenging analytes such as heparin octasaccharide carrying 8-O and 4 N-sulfo groups, and heparin octadecasulfated dodecasaccharide was successfully achieved.

  20. Valorization of Lipids from Gracilaria sp. through Lipidomics and Decoding of Antiproliferative and Anti-Inflammatory Activity

    Directory of Open Access Journals (Sweden)

    Elisabete da Costa

    2017-03-01

    Full Text Available The lipidome of the red seaweed Gracilaria sp., cultivated on land-based integrated multitrophic aquaculture (IMTA system, was assessed for the first time using hydrophilic interaction liquid chromatography-mass spectrometry and tandem mass spectrometry (HILIC–MS and MS/MS. One hundred and forty-seven molecular species were identified in the lipidome of the Gracilaria genus and distributed between the glycolipids classes monogalactosyl diacylglyceride (MGDG, digalactosyl diacylglyceride (DGDG, sulfoquinovosyl monoacylglyceride (SQMG, sulfoquinovosyl diacylglyceride (SQDG, the phospholipids phosphatidylcholine (PC, lyso-PC, phosphatidylglycerol (PG, lyso-PG, phosphatidylinositol (PI, phosphatidylethanolamine (PE, phosphatic acid (PA, inositolphosphoceramide (IPC, and betaine lipids monoacylglyceryl- and diacylglyceryl-N,N,N-trimethyl homoserine (MGTS and DGTS. Antiproliferative and anti-inflammatory effects promoted by lipid extract of Gracilaria sp. were evaluated by monitoring cell viability in human cancer lines and by using murine macrophages, respectively. The lipid extract decreased cell viability of human T-47D breast cancer cells and of 5637 human bladder cancer cells (estimated half-maximal inhibitory concentration (IC50 of 12.2 μg/mL and 12.9 μg/mL, respectively and inhibited the production of nitric oxide (NO evoked by the Toll-like receptor 4 agonist lipopolysaccharide (LPS on the macrophage cell line RAW 264.7 (35% inhibition at a concentration of 100 μg/mL. These findings contribute to increase the ranking in the value-chain of Gracilaria sp. biomass cultivated under controlled conditions on IMTA systems.

  1. Solution NMR characterization of chemokine CXCL8/IL-8 monomer and dimer binding to glycosaminoglycans: structural plasticity mediates differential binding interactions.

    Science.gov (United States)

    Joseph, Prem Raj B; Mosier, Philip D; Desai, Umesh R; Rajarathnam, Krishna

    2015-11-15

    Chemokine CXCL8/interleukin-8 (IL-8) plays a crucial role in directing neutrophils and oligodendrocytes to combat infection/injury and tumour cells in metastasis development. CXCL8 exists as monomers and dimers and interaction of both forms with glycosaminoglycans (GAGs) mediate these diverse cellular processes. However, very little is known regarding the structural basis underlying CXCL8-GAG interactions. There are conflicting reports on the affinities, geometry and whether the monomer or dimer is the high-affinity GAG ligand. To resolve these issues, we characterized the binding of a series of heparin-derived oligosaccharides [heparin disaccharide (dp2), heparin tetrasaccharide (dp4), heparin octasaccharide (dp8) and heparin 14-mer (dp14)] to the wild-type (WT) dimer and a designed monomer using solution NMR spectroscopy. The pattern and extent of binding-induced chemical shift perturbation (CSP) varied between dimer and monomer and between longer and shorter oligosaccharides. NMR-based structural models show that different interaction modes coexist and that the nature of interactions varied between monomer and dimer and oligosaccharide length. MD simulations indicate that the binding interface is structurally plastic and provided residue-specific details of the dynamic nature of the binding interface. Binding studies carried out under conditions at which WT CXCL8 exists as monomers and dimers provide unambiguous evidence that the dimer is the high-affinity GAG ligand. Together, our data indicate that a set of core residues function as the major recognition/binding site, a set of peripheral residues define the various binding geometries and that the structural plasticity of the binding interface allows multiplicity of binding interactions. We conclude that structural plasticity most probably regulates in vivo CXCL8 monomer/dimer-GAG interactions and function.

  2. Sulfation of p-nitrophenyl-N-acetyl-beta-D-galactosaminide with a microsomal fraction from cultured chondrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Habuchi, O.; Conrad, H.E.

    1985-10-25

    Chick embryo chondrocyte microsomes containing intact Golgi vesicles took up 3'-phosphoadenosine-5'-phospho(TVS)sulfate ((TVS)PAPS) in a time- and temperature-dependent, substrate-saturable manner. When (TVS)PAPS and p-nitrophenyl-N-acetyl-beta-D-galactosaminide (pNP-GalNAc) were added to the incubation in the absence of detergent, the microsomes catalyzed the transfer of sulfate from (TVS)PAPS to pNP-GalNAc to form pNP-GalNAc-6-TVSO4. The apparent Km values for PAPS in the uptake and the pNP-GalNAc sulfation reactions were 2 X 10(-7) and 2 X 10(-6) M, respectively. The sulfation of pNP-GalNAc by the microsomal preparation was inhibited by detergent. The microsomal fraction also catalyzed the transfer of sulfate from (TVS)PAPS to oligosaccharides prepared from chondroitin. However, in contrast to the sulfation of pNP-GalNAc, the rate of sulfation of these oligosaccharides was low in the absence of detergent and was markedly stimulated when detergent was added. Sulfation of pNP-GalNAc by the freeze-thawed microsomes was inhibited when the octasaccharide prepared from chondroitin was present in the reaction mixture. As the PAPS that had been internalized in the microsomal vesicles was consumed in the sulfation of pNP-GalNAc, more (TVS)PAPS was taken up and the sulfated pNP-GalNAc was released from the vesicles. These observations suggest that pNP-GalNAc may serve as a model membrane-permeable substrate for study of the 6-sulfo-transferase reaction involved in sulfation of chondroitin sulfate in intact Golgi vesicles.

  3. Influence of heparin mimetics on assembly of the FGF.FGFR4 signaling complex.

    Science.gov (United States)

    Saxena, Krishna; Schieborr, Ulrich; Anderka, Oliver; Duchardt-Ferner, Elke; Elshorst, Bettina; Gande, Santosh Lakshmi; Janzon, Julia; Kudlinzki, Denis; Sreeramulu, Sridhar; Dreyer, Matthias K; Wendt, K Ulrich; Herbert, Corentin; Duchaussoy, Philippe; Bianciotto, Marc; Driguez, Pierre-Alexandre; Lassalle, Gilbert; Savi, Pierre; Mohammadi, Moosa; Bono, Françoise; Schwalbe, Harald

    2010-08-20

    Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF signaling as they promote FGF.FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM(6)), octasaccharide (HM(8)), and decasaccharide (HM(10)), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM(8) and HM(10) are significantly more potent than HM(6) in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1.FGFR4 signaling. To understand the molecular basis for these differential activities in FGF1/2.FGFR4 signaling, we used NMR spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence of both a secondary FGF1.FGFR4 interaction site and a direct FGFR4.FGFR4 interaction site thus supporting the formation of the symmetric mode of FGF.FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM(8) relative to HM(6) in stimulating FGF2.FGFR4 signaling correlates with the higher affinity of HM(8) to bind and dimerize FGF2. Notably FGF2.HM(8) exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF.FGFR dimerization model, which incorporates the differential ability of HM to dimerize FGFs.

  4. Inhibition of allergic airway responses by heparin derived oligosaccharides: identification of a tetrasaccharide sequence

    Directory of Open Access Journals (Sweden)

    Ahmed Tahir

    2012-01-01

    Full Text Available Abstract Background Previous studies showed that heparin's anti-allergic activity is molecular weight dependent and resides in oligosaccharide fractions of Objective To investigate the structural sequence of heparin's anti-allergic domain, we used nitrous acid depolymerization of porcine heparin to prepare an oligosaccharide, and then fractionated it into disaccharide, tetrasaccharide, hexasaccharide, and octasaccharide fractions. The anti-allergic activity of each oligosaccharide fraction was tested in allergic sheep. Methods Allergic sheep without (acute responder and with late airway responses (LAR; dual responder were challenged with Ascaris suum antigen with and without inhaled oligosaccharide pretreatment and the effects on specific lung resistance and airway hyperresponsiveness (AHR to carbachol determined. Additional inflammatory cell recruitment studies were performed in immunized ovalbumin-challenged BALB/C mice with and without treatment. Results The inhaled tetrasaccharide fraction was the minimal effective chain length to show anti-allergic activity. This fraction showed activity in both groups of sheep; it was also effective in inhibiting LAR and AHR, when administered after the antigen challenge. Tetrasaccharide failed to modify the bronchoconstrictor responses to airway smooth muscle agonists (histamine, carbachol and LTD4, and had no effect on antigen-induced histamine release in bronchoalveolar lavage fluid in sheep. In mice, inhaled tetrasaccharide also attenuated the ovalbumin-induced peribronchial inflammatory response and eosinophil influx in the bronchoalveolar lavage fluid. Chemical analysis identified the active structure to be a pentasulfated tetrasaccharide ([IdoU2S (1→4GlcNS6S (1→4 IdoU2S (1→4 AMan-6S] which lacked anti-coagulant activity. Conclusions These results demonstrate that heparin tetrasaccharide possesses potent anti-allergic and anti-inflammatory properties, and that the domains responsible for anti

  5. Glycosylation and epitope mapping of the 5T4 glycoprotein oncofoetal antigen.

    Science.gov (United States)

    Shaw, David M; Woods, Andrew M; Myers, Kevin A; Westwater, Caroline; Rahi-Saund, Veena; Davies, Michael J; Renouf, David V; Hounsell, Elizabeth F; Stern, Peter L

    2002-01-01

    The human 5T4 oncofoetal antigen is a focus for development of several antibody-directed therapies on the basis of the murine monoclonal antibody against 5T4 (mAb5T4), which recognizes a conformational epitope. 5T4 molecules are highly N-glycosylated transmembrane glycoproteins whose extracellular domain contains two regions of leucine-rich repeats (LRRs) and associated flanking regions, separated by an intervening hydrophilic sequence. Using a series of deletion and mutated cDNA constructs as well as chimaeras with the murine homologue, we have mapped the mAb5T4 epitope to the more membrane-proximal LRR2 or its flanking region. Analysis of the glycosylation of the seven consensus Asp-Xaa-Ser/Thr sites was consistent with all of the sites being glycosylated. A combination of two high-mannose chains (predominantly octasaccharide) and five mostly sialylated bi-, tri- and tetra-antennary complex chains with minor quantities of core fucose were detected. The two glycosylation sites, which are the most likely to have predominantly high-mannose chains, are in the only two regions that show significant differences between the human and the 81% identical mouse sequence. A site-directed mutation, which abolished glycosylation at one of these sites (position 192), did not alter antigenicity. The other, which is nearest to the N-terminus in the human, has an Asn-Leu-Thr to Asn-Leu-Leu conversion in the mouse, so cannot be glycosylated in the latter species. The large complex glycosylation at the other sites is likely to influence the antigenicity and tertiary structure generating the 5T4 epitope. PMID:11903056

  6. Conformational changes of ovine α-1-proteinase inhibitor: The influence of heparin binding

    Science.gov (United States)

    Gupta, Vivek Kumar; Gowda, Lalitha R.

    2008-11-01

    α-1-Proteinase inhibitor (α-1-PI), the archetypal serpin causes rapid, irreversible stoichiometric inhibition of redundant circulating serine proteases and is associated with emphysema, inflammatory response and maintenance of protease-inhibitor equilibrium in vascular and peri-vascular spaces. A homogenous preparation of heparin octasaccharide binds to ovine and human α-1-PI and enhances their protease inhibitory activity phenomenally. Size-exclusion chromatography and dynamic light scattering experiments reveal that ovine α-1-PI undergoes a decrease in the Stokes' radius upon heparin binding. A strong binding; characterizes this α-1-PI-heparin interaction as revealed by the binding constant ( Kα) 1.98 ± 0.2 × 10 -6 M and 2.1 ± 0.2 × 10 -6 M determined by fluorescence spectroscopy and equilibrium dialysis, respectively. The stoichiometry of heparin binding to ovine α-1-PI was 1.1 ± 0.2:1. The Stern-Volmer constants ( Ksv) for heparin activated ovine and human α-1-PI were found to be 5.13 × 10 -6 M and 5.67 × 10 -6 M, respectively, significantly higher than the native inhibitors. FTIR and CD spectroscopy project the systematic structural reorientations that α-1-PI undergoes upon heparin binding characterized by a decrease in α-helical content and a concomitant increase in β-turn and random coil elements. It is likely that these conformational changes result in the movement of the α-1-PI reactive site loop into an extended structure that is better poised to combat the cognate protease and accelerate the inhibition.

  7. The Impact of Chain Length and Flexibility in the Interaction between Sulfated Alginates and HGF and FGF-2.

    Science.gov (United States)

    Arlov, Øystein; Aachmann, Finn L; Feyzi, Emadoldin; Sundan, Anders; Skjåk-Bræk, Gudmund

    2015-11-09

    Alginate is a promising polysaccharide for use in biomaterials as it is biologically inert. One way to functionalize alginate is by chemical sulfation to emulate sulfated glycosaminoglycans, which interact with a variety of proteins critical for tissue development and homeostasis. In the present work we studied the impact of chain length and flexibility of sulfated alginates for interactions with FGF-2 and HGF. Both growth factors interact with defined sequences of heparan sulfate (HS) at the cell surface or in the extracellular matrix. Whereas FGF-2 interacts with a pentasaccharide sequence containing a critical 2-O-sulfated iduronic acid, HGF has been suggested to require a highly sulfated HS/heparin octasaccharide. Here, oligosaccharides of alternating mannuronic and guluronic acid (MG) were sulfated and assessed by their relative efficacy at releasing growth factor bound to the surface of myeloma cells. 8-mers of sulfated MG (SMG) alginate showed significant HGF release compared to shorter fragments, while the maximum efficacy was achieved at a chain length average of 14 monosaccharides. FGF-2 release required a higher concentration of the SMG fragments, and the 14-mer was less potent compared to an equally sulfated high-molecular weight SMG. Sulfated mannuronan (SM) was subjected to periodate oxidation to increase chain flexibility. To assess the change in flexibility, the persistence length was estimated by SEC-MALLS analysis and the Bohdanecky approach to the worm-like chain model. A high degree of oxidation of SM resulted in approximately twice as potent HGF release compared to the nonoxidized SM alginate. The release of FGF-2 also increased with the degree of oxidation, but to a lower degree compared to that of HGF. It was found that the SM alginates were more efficient at releasing FGF-2 than the SMG alginates, indicating a greater dependence on monosaccharide identity and charge orientation over chain flexibility and charge density.

  8. Structure and Mechanism of ORF36, an Amino Sugar Oxidizing Enzyme in Everninomicin Biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Vey, Jessica L.; Al-Mestarihi, Ahmad; Hu, Yunfeng; Funk, Michael A.; Bachmann, Brian O.; Iverson, T.M. (Vanderbilt)

    2010-12-07

    Everninomicin is a highly modified octasaccharide that belongs to the orthosomycin family of antibiotics and possesses potent Gram-positive antibiotic activity, including broad-spectrum efficacy against multidrug resistant enterococci and Staphylococcus aureus. Among its distinctive structural features is a nitro sugar, L-evernitrose, analogues of which decorate a variety of natural products. Recently, we identified a nitrososynthase enzyme encoded by orf36 from Micromonospora carbonacea var. africana that mediates the flavin-dependent double oxidation of synthetically generated thymidine diphosphate (TDP)-L-epi-vancosamine to the corresponding nitroso sugar. Herein, we utilize a five-enzyme in vitro pathway both to verify that ORF36 catalyzes oxidation of biogenic TDP-L-epi-vancosamine and to determine whether ORF36 exhibits catalytic competence for any of its biosynthetic progenitors, which are candidate substrates for nitrososynthases in vivo. Progenitors solely undergo single-oxidation reactions and terminate in the hydroxylamine oxidation state. Performing the in vitro reactions in the presence of {sup 18}O{sub 2} establishes that molecular oxygen, rather than oxygen from water, is incorporated into ORF36-generated intermediates and products and identifies an off-pathway product that correlates with the oxidation product of a progenitor substrate. The 3.15 {angstrom} resolution X-ray crystal structure of ORF36 reveals a tetrameric enzyme that shares a fold with acyl-CoA dehydrogenases and class D flavin-containing monooxygenases, including the nitrososynthase KijD3. However, ORF36 and KijD3 have unusually open active sites in comparison to these related enzymes. Taken together, these studies map substrate determinants and allow the proposal of a minimal monooxygenase mechanism for amino sugar oxidation by ORF36.

  9. Structure of the Receptor-Binding Protein of Bacteriophage Det7: a Podoviral Tail Spike in a Myovirus▿

    Science.gov (United States)

    Walter, Monika; Fiedler, Christian; Grassl, Renate; Biebl, Manfred; Rachel, Reinhard; Hermo-Parrado, X. Lois; Llamas-Saiz, Antonio L.; Seckler, Robert; Miller, Stefan; van Raaij, Mark J.

    2008-01-01

    A new Salmonella enterica phage, Det7, was isolated from sewage and shown by electron microscopy to belong to the Myoviridae morphogroup of bacteriophages. Det7 contains a 75-kDa protein with 50% overall sequence identity to the tail spike endorhamnosidase of podovirus P22. Adsorption of myoviruses to their bacterial hosts is normally mediated by long and short tail fibers attached to a contractile tail, whereas podoviruses do not contain fibers but attach to host cells through stubby tail spikes attached to a very short, noncontractile tail. The amino-terminal 150 residues of the Det7 protein lack homology to the P22 tail spike and are probably responsible for binding to the base plate of the myoviral tail. Det7 tail spike lacking this putative particle-binding domain was purified from Escherichia coli, and well-diffracting crystals of the protein were obtained. The structure, determined by molecular replacement and refined at a 1.6-Å resolution, is very similar to that of bacteriophage P22 tail spike. Fluorescence titrations with an octasaccharide suggest Det7 tail spike to bind its receptor lipopolysaccharide somewhat less tightly than the P22 tail spike. The Det7 tail spike is even more resistant to thermal unfolding than the already exceptionally stable homologue from P22. Folding and assembly of both trimeric proteins are equally temperature sensitive and equally slow. Despite the close structural, biochemical, and sequence similarities between both proteins, the Det7 tail spike lacks both carboxy-terminal cysteines previously proposed to form a transient disulfide during P22 tail spike assembly. Our data suggest receptor-binding module exchange between podoviruses and myoviruses in the course of bacteriophage evolution. PMID:18077713

  10. Comparison of glycerolipid biosynthesis in non-green plastids from sycamore (Acer pseudoplatanus) cells and cauliflower (Brassica oleracea) buds.

    Science.gov (United States)

    Alban, C; Joyard, J; Douce, R

    1989-05-01

    The availability of methods to fractionate non-green plastids and to prepare their limiting envelope membranes [Alban, Joyard & Douce (1988) Plant Physiol. 88, 709-717] allowed a detailed analysis of the biosynthesis of lysophosphatidic acid, phosphatidic acid, diacylglycerol and monogalactosyl-diacylglycerol (MGDG) in two different types of non-green starch-containing plastids: plastids isolated from cauliflower buds and amyloplasts isolated from sycamore cells. An enzyme [acyl-ACP (acyl carrier protein):sn-glycerol 3-phosphate acyltransferase) recovered in the soluble fraction of non-green plastids transfers oleic acid from oleoyl-ACP to the sn-1 position of sn-glycerol 3-phosphate to form lysophosphatidic acid. Then a membrane-bound enzyme (acyl-ACP:monoacyl-sn-glycerol 3-phosphate acyltransferase), localized in the envelope membrane, catalyses the acylation of the available sn-2 position of 1-oleoyl-sn-glycerol 3-phosphate by palmitic acid from palmitoyl-ACP. Therefore both the soluble phase and the envelope membranes are necessary for acylation of sn-glycerol 3-phosphate. The major difference between cauliflower (Brassica oleracea) and sycamore (Acer pseudoplatanus) membranes is the very low level of phosphatidate phosphatase activity in sycamore envelope membrane. Therefore, very little diacylglycerol is available for MGDG synthesis in sycamore, compared with cauliflower. These findings are consistent with the similarities and differences described in lipid metabolism of mature chloroplasts from 'C18:3' and 'C16:3' plants (those with MGDG containing C18:3 and C16:3 fatty acids). Sycamore contains only C18 fatty acids in MGDG, and the envelope membranes from sycamore amyloplasts have a low phosphatidate phosphatase activity and therefore the enzymes of the Kornberg-Pricer pathway have a low efficiency of incorporation of sn-glycerol 3-phosphate into MGDG. By contrast, cauliflower contains MGDG with C16:3 fatty acid, and the incorporation of sn-glycerol 3