Structure of fructans from excised leaves of New Zealand flax

The accumulation of total water-soluble carbohydrate, and specifically sucrose and fructan, by excised leaves of Phormium tenax and P. cookianum (family Phormiaceae J. G. Agardh, order Asparagales) was investigated. Total water-soluble carbohydrate content of excised leaves of P. tenax and P. cookianum increased during 48 h of continuous illumination at an average rate of 1.3 and 0.9 mg g(-1) fresh weight leaf per hour, respectively. The sucrose content of excised leaves increased throughout the experimental period. The fructan content of excised leaves of P. tenax increased slightly throughout the experimental period, whilst that of P. cookianum was variable and showed no overall change. Chemical and spectroscopic analysis of the fructans obtained from the two Phormium species showed that they were similar to each other and contained mostly 1-linked and terminal fructofuranosyl (Fruf) residues, together with smaller amounts of 6-linked Fruf, 1,6-branched Fruf, terminal and 6-linked glucopyranosyl residues. Separation of the fructans by thin-layer and high-performance anion-exchange chromatography revealed the presence of a complex mixture of fructo-oligosaccharides and higher molecular weight fructan. The branched structure of the fructans isolated from excised leaves of Phormium resembles that of fructans and fructo-oligosaccharides isolated from some related species within the order Asparagales (Agave vera cruz, Cordyline australis and Urginea maritima), but is distinct from the linear structure of fructans from others (Allium cepa and Asparagus officinalis). The structural heterogeniety of fructans within both the order Asparagales and superorder Liliiflorae may be a useful chemotaxonomic aid.     

Isolation and characterization of water-soluble prebiotic compounds from Australian and New Zealand plants

The water-soluble carbohydrates (WSCs) extracted from the underground parts (rhizome) of Arthropodium cirratum (Rengarenga lily extract); third order branches of Cordyline australis (Cabbage tree extract); a seaweed, Undaria pinnatifida (Undaria extract), and exudates from Acacia pycnantha (Acacia extract) were investigated. Extracts of Rengarenga lily, Cabbage tree, Undaria, and Acacia contained 576, 250, 275 and 794 g/kg DM WSCs, respectively. Constituent sugar analysis by gas–liquid chromatography (GLC) showed that extracts of Rengarenga lily and Cabbage tree contained predominantly fructose and glucose (82–95%). The analysis also revealed that Acacia extract contained mainly galactose (78%) and arabinose (22%) while Undaria extract, contained fucose (55%) and galactose (44%). Thin-layer chromatography (TLC) showed that, on the basis of R_F values, fructan composition of Rengarenga lily extract and Cabbage tree extract was different. Cabbage tree extract contained 45% (w/w) fructans while Rengarenga lily extract contained 65% (w/w) fructans. High performance size-exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS) showed that the extracts had varying weight average molecular weight due to differences in the average chain length of the major carbohydrates. Data for the amino acid compositions differed considerably depending on the type of extract. Water-soluble carbohydrate extracts prepared from the four plant sources gave a wide range of WSC (250–794 g/kg DM) due to the different proportions of structural material in different species. It is not known how these differences will impact on animal production, if diets are supplemented with the extracts.     

Construction of chimeric glucansucrases for analyzing substrate-binding regions that affect the structure of glucan products

A gene that encodes dextransucrase S (dsrS) from Leuconostoc mesenteroides NRRL B-512F encodes a glucansucrase dextransucrase S (DSRS) which mainly produces water-soluble glucan (dextran), while the dsrT5 gene derived from dsrT of the B-512F strain encodes an enzyme dextransucrase T5 (DSRT5), which mainly produces water-insoluble glucan. Tyr340-Asn510 of DSRS and Tyr307-Asn477 of DSRT5 (Site 1), Lys696-Gly768 of DSRS and Lys668-Gly740 of DSRT5 (Site 2), and Asn917-Lys1131 of DSRS and Asn904-Lys1118 of DSRT5 (Site 3) were exchanged and six different chimeric enzymes were constructed. Water-soluble glucan produced by recombinant DSRS was composed of 64% 6-linked glucopyranoside (Glcp), 9% 3,6-linked Glcp, and 13% 4-linked Glcp. Water-insoluble glucan produced by recombinant DSRT5 was composed of 47% 6-linked Glcp and 43% 3-linked Glcp. All of the chimeric enzymes produced glucans different from the ones produced by their parental enzymes. Some of the glucans produced by chimeric enzymes were extremely changed. The Site 1 chimeric enzyme of DSRS (STS1) produced water-soluble glucan composed mostly of 6-linked Glcp. That of DSRT5 (TST1) produced water-insoluble glucan composed mostly of 4-linked Glcp. The Site 3 chimeric enzyme of DSRS (STS3) produced mainly water-insoluble glucan, DSRT5 (TST3) produced mainly water-soluble glucans, and all of the glucan fractions consisted of 3-Glcp, 4-Glcp, and 6-Glcp. The amounts of the three linkages in the water-soluble glucan produced by TST3 were about 1:1:1. Site 1 was assumed to be important for making or avoiding making alpha-1,4 linkages, while Site 3 was assumed to be important for determining the kinds of glucosyl linkages made.     

具有生物活性的川牛膝果聚糖的结构

从传统中药川牛膝(Cyathula offcinalis kuan)中分离提取到了一种具有生物活性的多糖RCP.核磁共振、甲基化分析、还原裂解和GC-MS分析揭示了RCP是一高度分支的果聚糖,它以(2→1)连接为骨架,其上有大量的(2→6)连接的分支,且属于新蔗果三糖系列.在93.17%果糖残基中,24.15%是末端果糖,26.24%是1-连接果糖,20.46%是6-连接果糖.在6.83%的葡萄糖残基中,2.14%是末端葡萄糖,4.69%是6-连接葡萄糖.RCP的平均聚合度是15.     

Changes in linkage pattern of glucan products induced by substitution of Lys residues in the dextransucrase

Dextransucrase S (DSRS) is the only active glucansucrase that has been found in Leuconostoc mesenteroides NRRL B-512F strain. Native DSRS produces mainly 6-linked glucopyranosyl residue (Glcp), while Escherichia coli recombinant DSRS was observed to produce a glucan consisting of 70% 6-linked Glcp and 15% 3,6-Glcp. Lys residues were introduced at the N-terminal end of the core domain by site-directed mutagenesis. In glucans produced by the one-point mutants T350K and S455K, the amount of 6-linked Glcp was increased to about 85% of the total glucan produced, more similar in structure to native B-512F dextran. The double mutant T350K/S455K produced adhesive, water-insoluble glucan with 77% 6-linked Glcp, 8% 3,6-linked Glcp and 4% 2,6-linked Glcp. The T350K/S455K mutant exhibited a 10-fold increase in glucosyltransferase activity over those of the parental DSRS-His(6) and its T350K and S455K mutants. This is the first report demonstrating a change in the properties of a dextransucrase or a related glucosyltransferase through simple site-directed mutagenesis to create 2,6-linked Glcp.     

Chemical structures of acholeplasmal lipoglycans and their relation to biological interactions

The partial characterization of the structure of the lipoglycan (LG) from Acholeplasma axanthum is added to the previous complete structural analysis of the lipoglycan from A. granularum. The terminal sequence of A. axanthum LG is Glcp(beta 1----2)-Glcp(beta 1----2)-Glcp(beta 1----6)-; of A. granularum Glcp(beta 1----2)-Glcp(alpha 1----4)-Glcp(beta 1----4)-. These specific residues define the major antigenic determinants of the LG as determined by blockage of hemagglutination of LG coated erythrocytes by specific oligosaccharides and binding of radiolabeled LG to specific immunoglobulins. The binding of LG to mammalian cells occurs by an interaction between specific eucaryotic cell receptors and the internal sequence of the oligosaccharide chain of LG. Size and sugar chains of LG rather than fatty acid residues appears to define the binding site on the LG.     

Protective effect of a polysaccharide from stem of Codonopsis pilosula against renal ischemia/reperfusion injury in rats

In this study, we purified a homogeneous polysaccharide (S-CPPA1) with a molecular weight (Mw) of 133.2kDa from the stem of Codonopsis pilosula for the first time. Gas chromatography (GC) analysis identified that S-CPPA1 contained glucose, galactose, and arabinose with a molar ratio of 10.5:3.4:1.7, along with a trace of mannose. Methylation analysis suggested S-CPPA1 was a branched polysaccharide, with five glucosidic linkage forms, namely (1→4)-linked Glcp (residue A), (1→6)-linked Galp (residue B), (1→2,6)-linked Glcp (residue C), (1→5)-linked Araf (residue D), and non-reducing terminal (1→)-linked Glcp (residue E). The protective effect of S-CPPA1 on kidney ischemia/reperfusion (I/R) injury was also evaluated. Blood urea nitrogen (BUN), creatinine and TNF-α levels, as well as lactate dehydrogenase (LDH) and alanine transaminase (AST) activities were elevated in the I/R group as compared to the sham group. On the other hand, S-CPPA1 treatment reversed all these biochemical indices, as well as histopathological alterations, which were induced by I/R. The findings imply that S-CPPA1 plays a causal role in the protection against I/R-induced renal injury and its renoprotective effect is probably mediated by inhibiting the proinflammatory cytokine TNF-α release.     

Structural characterization of a 2-O-acetylglucomannan from Dendrobium officinale stem

A heteropolysaccharide obtained from an aqueous extract of dried stem of Dendrobium officinale Kimura and Migo by anion-exchange chromatography and gel-permeation chromatography, was investigated by chemical techniques and NMR spectroscopy, and is demonstrated to be a 2-O-acetylglucomannan, composed of mannose, glucose, and arabinose in 40.2:8.4:1 molar ratios. It has a backbone of (1-->4)-linked beta-d-mannopyranosyl residues and beta-d-glucopyranosyl residues, with branches at O-6 consisting of terminal and (1-->3)-linked Manp, (1-->3)-linked Glcp, and a small proportion of arabinofuranosyl residues at the terminal position. The acetyl groups are substituted at O-2 of (1-->4)-linked Manp and Glcp. The main repeating unit of the polysaccharides is reported.     

Characterization of a novel dextran produced by Gluconobacter oxydans DSM 2003

A novel water-soluble dextran was synthesized from maltodextrin by cell-free extract of Gluconobacter oxydans DSM 2003. The dextran was purified by size exclusion chromatography, and the structure was determined by Fourier transform infrared spectroscopy, nuclear magnetic resonance, and gas chromatography-mass spectrometer. Based on the spectral data, we found that the dextran contained only D-glucose residues. The ratio of nonreducing end glucopyranosyl (Glcp) to 6-linked Glcp to 4,6-linked Glcp was estimated to be 8.62:78.79:12.59 by methylation analysis. This result indicated the existence of a small proportion of α(1,4) branches in α(1,6) glucosyl linear chains. Here, we reported the first time a novel dextran was synthesized by G. oxydans DSM 2003.     

Determination of the structure and degree of polymerisation of fructans from Echinacea purpurea roots

Highly water soluble fructans have been isolated from Echinacea purpurea (L.) Moench. roots by hot water extraction and precipitation at three different ethanol concentrations (80% v/v, 60% v/v and 40% v/v). The structure of the fructans has been characterised by three analytical methods: GC of silylated oxime derivatives and partially methylated alditol acetates, respectively, as well as 13C NMR analysis. The mean degree of polymerisation (mean DP) of each fructan has been determined by the glucose/fructose ratio. E. purpurea fructans represent linear inulin-type fructans with almost exclusively beta-(2-->1)-linked fructosyl units, terminal glucose and terminal fructose. Small proportions of beta-(2-->1,2-->6)-linked branch point residues were detected. The mean DP of the fructan fractions depends on the ethanol concentration used for precipitation: the lower the ethanol concentration the higher the mean DP. Corresponding results were found with all of the three analytical methods: 80% ethanol-insoluble fructan from E. purpurea shows an average mean DP of 35, 60% ethanol-insoluble fructan of 44 and 40% ethanol-insoluble fructan of 55. The applied methods provide sufficient sensitivity to determine not only the composition and structure but also the mean degree of polymerisation of fructans.     

Immunoregulation and antioxidant activities of a novel acidic polysaccharide from Radix Paeoniae Alba

Radix Paeoniae Alba is widely used in Chinese traditional medicine to treat various diseases such as gastrointestinal disorders, immunomodulatory, cancer, and other diseases. In this paper, a novel acidic polysaccharide RPAPS purified from Radix Paeoniae Alba was evaluated for its structural features and potential of immunomodulatory and antioxidant activities. RPAPS (molecular weight: 1.0× 105 Da) was mainly composed of α-(1 → 4)-Glcp, α-Arap, α-Galp, α-Rhap, β-D-Glcp, α-(1 → 6)-linked Glcp and GalA. Immunological tests indicated that RPAPS could improve RAW264.7 phagocytic activity and LPS-induced splenocyte proliferation. For antioxidant activities, RPAPS showed reducing power and DPPH scavenging activity in dose dependent. Moreover, RPAPS could significantly protect the PC12 cells from H2O2 damage. These data implied polysaccharides RPAPS had the potential to be novel natural antioxidative and immunopotentiating agents for using in functional foods or medicine.     

Phosphoribosylation of xanthine by extracts from insect cells

Using a sensitive TLC method, we have detected the production of xanthine monophosphate (XMP) from [¹⁴C]xanthine by mosquito cell extracts incubated in the presence of phosphoribosyl pyrophosphate and a phosphatase inhibitor. Extracts from both cultured Aedes albopictus cells, and from intact Aedes aegypti mosquitoes contained activity; particularly high activity was found in extracts from adult male mosquitoes. XMP-producing activity was at least 4-fold higher in extracts from cultured mosquito cells than in extracts from Drosophila melanogaster Kc cells or Spodoptera frugiperda (Lepidoptera) cells.     

High molecular weight constituents from roots of Echinacea pallida: an arabinogalactan-protein and an arabinan

This investigation shows structural features of two macromolecules from roots of Echinacea pallida (Nutt.) Nutt: an arabinogalactan-protein (AGP) and an arabinan. The arabinogalactan-protein was precipitated with beta-glucosyl Yariv reagent from a high molecular weight fraction. Investigations of the neutral sugar composition revealed Gal (52.1% w/w) and Ara (38.2% w/w) in a ratio of 1.4:1, accompanied by Glc (6.9% w/w) and Rha (2.8% w/w). The content of uronic acids was 6.2%. Mild acid hydrolysis detects Ara and Glc being located at the periphery of the molecule. Linkage analyses and NMR spectroscopy revealed a backbone of the polysaccharide mainly consisting of 3-linked and 3,6-linked Galp-residues. Side chains are composed of 3,6-linked or 6-linked Galp terminating in 5-linked Araf, terminal Araf, Glcp and GlcAp. The protein part (3.9% w/w) of the AGP is rich in Hyp, Ser, Ala, Thr, Glu, Asp and Gly. The amount of Hyp was determined by a colorimetric method and found to be (0.65% (w/w) of the AGP, which is in good agreement with the result obtained by amino acid hydrolysis (0.67% w/w). The arabinan was isolated from the supernatant of the Yariv precipitation on the basis of solubility in EtOH (80%). It mainly consists of Ara (85.8%). Linkage analyses and NMR spectroscopy indicate a highly branched molecule, consisting of 3,5-linked, 5-linked and terminal Araf-residues in equal amounts.     

Neu5Acalpha3Gal is part of the Helicobacter pylori binding epitope in polyglycosylceramides of human erythrocytes.

The sialic acid dependent binding by the human pathogen Helicobacter pylori to polyglycosylceramides of human erythrocytes was investigated. Polyglycosylceramides, complex glycosphingolipids with a branched N-acetyllactosamine core, were isolated from human erythrocytes, blood group O, and subfractionated after peracetylation by anion-exchange chromatography. Three subfractions were deacetylated, analysed by matrix-assisted laser desorption ionization-time of flight MS and 2D 1H NMR spectroscopy. The observed mass ranges were m/z = 3093-7622, 3968-7255 and 3459-7987 in the mass spectra of the first, second and third fractions, respectively. The observed ions agreed with the general formula Hex(x+2)HexNAcxFucyNeu5AczCer. Two-dimensional 1H total correlation spectra of the mixtures showed that the first fraction contained 3-linked sialic acid and the second and third fractions contained both 3-linked and 6-linked sialic acid. Thin-layer chromatogram binding assays using the lectins from Maackia amurensis, specific for Neu5Acalpha3Galbeta4GlcNAc, and Sambucus nigra, specific for Neu5Acalpha6Gal/GalNAc, were used to confirm this distribution. H. pylori recognized all three fractions in the binding assay, indicating that the 3-linked, rather than 6-linked, sialic acid is essential for binding.     

Chemical and biological properties of a highly branched β-glucan from edible mushroom Pleurotus sajor-caju

Hot aqueous extraction of the basidiocarps of the mushroom Pleurotus sajor-caju provided a cold water-soluble, gel-like glucan, which was characterized chemically, and its effects on RAW 264.7 cell line (mouse leukaemic monocyte macrophage) activation were determined. NMR spectroscopy, HPSEC, methylation analysis, and a controlled Smith degradation showed it to have a branched structure with a (1→3)-linked β-Glcp main-chain, substituted at O-6 by single-unit β-Glcp side-chains, on the average of two to every third residues of the backbone, with a molar mass of 9.75×10(5)gmol(-1). In macrophage cell culture, the β-glucan induced production of NO and the cytokines TNF-α, IL-1β, these effects being very similar as those of Escherichia coli serotype 0111:B4 Sigma-Aldrich lipopolysaccharide (LPS), although not modifying the response of LPS-activated macrophages. The results suggest that the (1→3), (1→6)-linked β-glucan from P. sajor-caju may have potential for immunological activities, although additional experiments are necessary for a better understanding of the mechanisms involved.     

Lactobacillus helveticus glycosyltransferases: from genes to carbohydrate synthesis

Bioactive carbohydrates are crucial in mediating essential biological processes, and their biosynthesis is an essential aspect to develop for a global view of their biological functions. Lactic acid bacteria display an array of diverse and complex carbohydrates and, therefore, are of particular interest. Here we present the identification of a novel exocellular polysaccharide structure and the corresponding gene cluster from Lactobacillus helveticus NCC2745. The development of a glycosyltransferase-specific enzymatic assay allowed the assignment of sugar specificities, which as a general approach will for the future permit a faster and more direct characterization of glycosyltransferase specificities. A model of the biosynthesis of the repeating unit is proposed. EpsE is a phosphoglucosyltransferase initiating the repeating unit biosynthesis by linking a glucose residue to a membrane-associated lipophilic acceptor. EpsF elongates the carbohydrate chain by forming an alpha(1,3)-Glcp linkage onto the first Glcp, whereas EpsG adds a backbone alpha(1,6)-Galp onto alpha-Glcp and EpsH attaches a alpha(1,6)-Glcp branch onto the first glucose residue. Finally, EpsI would add a beta(1,6)-Galp linkage onto alpha-Glcp terminating the sidechain and EpsJ would terminate the synthesis of the polysaccharides' repeating unit by forming a beta(1,3)-Galp linkage onto alpha-Galp.     

Fructan:fructan 1-fructosyltransferase,a key enzyme for biosynthesis of graminan oligomers in hardened wheat

Fructans play important roles not only as a carbon source for survival under persistent snow cover but also as agents that protect against various stresses in overwintering plants. Complex fructans having both ß-(2,1)- and ß-(2,6)-linked fructosyl units accumulate in wheat (Triticum aestivum L.) during cold hardening. We detected fructan: fructan 1-fructosyltransferase (1-FFT; EC 2.4.1.100) activity for catalyzing the formation and extension of ß-(2,1)-linked fructans in hardened wheat tissues, cloned cDNAs (wft3 and wft4) of 1-FFT, and analyzed the enzymatic properties of a wft3 recombinant protein (Wft3m) produced by yeast. Wft3m transferred ß-(2,1)-linked fructosyl units to phlein, an extension of sucrose through ß-(2,6)-linked fructosyl units, as well as to inulin, an extension of sucrose through ß-(2,1)-linked fructosyl units, but could not efficiently synthesize long inulin oligomers. Incubation of a mixture of Wft3m and another recombinant protein of wheat, sucrose:fructan 6-fructosyltransferase (6-SFT), with sucrose and 1-kestotriose produced fructans similar to those that accumulated in hardened wheat tissues. The results demonstrate that 1-FFT produces branches of ß-(2,1)-linked fructosyl units to phlein and graminan oligomers synthesized by 6-SFT and contributes to accumulation of fructans containing ß-(2,1)- and ß-(2,6)-linked fructosyl units. In combination with sucrose:sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99) and 6-SFT, 1-FFT is necessary for fructan synthesis in hardened wheat.     

Structural studies of a phosphocholine substituted beta-(1,3);(1,6) macrocyclic glucan from Bradyrhizobium japonicum USDA 110.

In our previous in vivo 31P study of intact nitrogen-fixing nodules (Rolin, D.B., Boswell, R.T., Sloger, C., Tu, S.I. and Pfeffer, P.E., 1989 Plant Physiol. 89, 1238-1246), we observed an unknown phosphodiester. The compound was also observed in the spectra of isolated bacteroids as well as extracts of the colonizing Bradyrhizobium japonicum USDA 110. In order to characterize the phosphodiester in the present study, we took advantage of the relatively hydrophobic nature of the material and purified it by elution from a C-18 silica reverse-phase chromatography column followed by final separation on an aminopropyl silica HPLC column. Structural characterization of this compound with a molecular weight of 2271 (FAB mass spectrometry), using 13C-1H and 31P-1H heteronuclear 2D COSY and double quantum 2D phase sensitive homonuclear 1H COSY NMR spectra, demonstrated that the molecule contained beta-(1,3); beta-(1,6); beta-(1,3,6) and beta-linked non-reducing terminal glucose units in the ratio of 5:6:1:1, respectively, as well as one C-6 substituted phosphocholine (PC) moiety associated with one group of (1,3) beta-glucose residues. Carbohydrate degradation analysis indicated that this material was a macrocyclic glucan, (absence of a reducing end group) with two separated units containing three consecutively linked beta-(1,3) glucose residues and 6 beta-(1,6) glucose residues. The sequences of beta-(1,3)-linked glucose units contained a single non-reducing, terminal, unsubstituted glucose linked at the C-6 position and a PC group attached primarily to an unsubstituted C-6 position of a beta-(1,3)-linked glucose.     

Structural and immunological properties of arabinogalactan polysaccharides from pollen of timothy grass (Phleum pratense L.)

Extracts from pollen of timothy grass (Phleum pratense L.) contain up to 20% arabinogalactan proteins (AGPs). Separation of the AGP polysaccharide moieties by tryptic digestion, size exclusion chromatography (GPC), and reverse phase HPLC yielded arabinogalactan fractions AG-1 and AG-2 with molecular weights of approximately 15,000 and approximately 60,000Da, respectively. The backbones of both polysaccharides are composed of (1-->6)-linked beta-D-galactopyranosides with beta-D-GlcUAp or 4-O-Me-beta-D-GlcUAp at their terminal ends as revealed by chemical analysis, FT-IR, MALDI-MS, and NMR spectroscopy. AG-1 contains a small number of beta-l-Araf side chains while AG-2 possesses a variety of (1-->3)-linked units, which consist of beta-l-Araf-(1-->, alpha-l-Araf-(1-->3)-beta-l-Araf-(1-->, and alpha-l-Araf-(1-->5)-beta-l-Araf-(1--> as well as a small number of longer arabinogalactan side chains. In contrast to crude pollen extracts, the immunological properties of the arabinogalactan mixture reveal an IgG4 reactivity instead of IgE reactivity. Structural properties of timothy pollen arabinogalactan might thus influence the immune response.