Study of galactoglucomannans from the seed of <Emphasis Type="Italic">Cercis canadensis</Emphasis> L.

Galactoglucomannans were isolated by selective precipitation from aqueous and alkaline extracts of endosperm and hulls of Cercis canadensis, a member of the family Fabaceae. Their monosaccharide composition (Man : Gal : Glu) was as follows: 10.4 : 0.9 : 1 (polysaccharide from the endosperm) and 4.5 : 0.9 : 1 (polysaccharide from the hulls). The identity of IR spectra was indicative of the similarity of their structure. Analysis of the structure of the galactoglucomannan from endosperm by ¹³C NMR spectroscopy showed that its main chain consisted of 1,4--D-manno- and 1,4--D-glucopyranose. Part of the mannose residues in the chain were substituted at C6 with single residues of -D-galactopyranose. Galactoglucomannans are located in different parts of the seed and implement different functions.__________Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 3, 2005, pp. 324–329.Original Russian Text Copyright © 2005 by Mestechkina, Egorov, Anulov, Shcherbukhin.     

Purification and structural characterization of a new water-soluble neutral polysaccharide GLP-F1-1 from Ganoderma lucidum

A water-soluble neutral polysaccharide (GLP-F1-1) was isolated from the fruiting bodies of Ganoderma lucidum by DEAE Sepharose Fast Flow and Sephacryl S-500 High Resolution Chromatography. The neutral polysaccharide had an average molecular weight (Mw) of approximately 2.5×10(6) kDa. GC analysis showed that this polysaccharide was mainly composed of glucose and galactose in the molar ratio of 34:1. 1H and 13C NMR spectroscopy in combination with GC-MS technique indicated that the new polysaccharide had a backbone chain of 1,4-disubstituted-β-glucoseopyranose and 1,4,6-trisubstituted-β-glucoseopyranosyl, while the branched chains were mainly composed of 1,6-disubstituted-β-glucopyranosyl and 1,4-disubstituted-β-galactoseopyranosyl residues.     

Chemotypes significance of lichenized fungi by structural characterization of heteropolysaccharides from the genera Parmotrema and Rimelia

Galactoglucomannans were isolated from the lichenized fungi of the genus Parmotrema (Parmotrema austrosinense, Parmotrema delicatulum, Parmotrema mantiqueirense, Parmotrema schindlerii, and Parmotrema tinctorum and that of Rimelia (Rimelia cetrata and Rimelia reticulata) via successive hot alkaline extraction and precipitation with Fehling solution. The structure of each polysaccharide was investigated using 13C NMR and HSQC-DEPT spectroscopy, methylation analysis, and HPSEC-MALLS. The galactoglucomannans had a (1-->6)-linked main chain of alpha-Manp units, substituted preferentially at O-2 and O-4 by alpha-Galp and beta-Galp nonreducing end-units, respectively. The C-1 region of the 13C NMR spectra of these heteropolysaccharides is typical of the lichen species, and is an additional tool in lichenized fungi classification.     

Polysaccharide structure of tetrasporic red seaweed Tichocarpus crinitus

Sulfated polysaccharide isolated from tetrasporic plants of Tichocarpus crinitus was investigated. The polysaccharide was isolated by two methods: with water extraction at 80 °C (HT) and with a mild alkaline extraction (AE). The extracted polysaccharides were presented by non-gelling ones only, while galactose and 3,6-AG were the main monosaccharides, at the same time amount of 3,6-AG in AE polysaccharides was the similar to that of HT. According to methods of spectroscopy and mass spectrometry, the polysaccharide from tetrasporic T. crinitus contains main blocks of 1,3-linked β-d-galactopyranosyl-2,4-disulfates and 1,4-linked 3,6-anhydro-α-d-galactopyranosyl while 6-sulfated 4-linked galactopyranosyl resudies are randomly distributed along the polysaccharide chain. The alkaline treatment of HT polysaccharide results in obtaining polysaccharide with regular structure that composed of alternating 1,3-linked β-d-galactopyranosyl-2,4-disulfates and 1,4-linked 3,6-anhydro-α-d-galactopyranosyl residues. Native polysaccharide (HT) possessed both high anticoagulant and antiplatelet activity measured by fibrin clotting and platelet aggregation induced by collagen. This activity could be connected with peculiar chemical structure of HT polysaccharide which has high sulfation degree and contains also 3,6-anhydrogalactose in the polymer chain.     

An Antioxidant Acidic Polysaccharide from Cuscuta chinensis

An acidic polysaccharide, H2, was isolated from the alkali-extract CHC of seeds of Cuscuta chinensis Lam. with the molecular weight more than 1.0×106. Chemical and spectroscopic studies led to the structure determination as follows: the backbone chain consists of 1,6-linked-β- D Galp, 1,4-linked-β- D Galp, 1,4-linked-β- D GalA and 1,2- or 1,4-linked-β- L Rhap having branching points at position O-3 of some 1,6-linked-β- D Galp residues (one among eight) and O-4 or O-2 of 1,2- or 1,4-linked-β- L Rhap residues to terminal β-D-galactopyranose. The side chains composed of terminal Galp, 1,6-linked-β- D Galp, 1,4-linked β- D Galp and 1,3,6-linked-β- D Galp also linked at position O-3 of 1,6-linked-β- D Galp residues in the backbone chain. β- L -arabinofuranosyl and terminal β- L -rhamnopyranosyl residues existed in the periphery of this polysaccharide linked to O-3 of 1,6-linked-β- D Galp residues in the backbone chain and the side chains. The polysaccharide H2 increased significantly the survival rate of PC12 cells indicating that it had protective effects against H2O2 insult.     

Structure of Hemicellulose Isolated from Rice Endosperm Cell Wall : Mode of Linkages and Sequences in Xyloglucan, β-Glucan and Arabinoxylan

A hemicellulosic polysaccharide, which was homogeneous on sedimentation analysis and also on electrophoresis, was isolated from the rice endosperm cell walls by the combination of alkaline extraction, ion exchange chromatography and iodine complex formation. It is composed of arabinose, xylose and glucose (molar ratio, 1.0: 2.0: 5.7) together with a small amount of galactose and rhamnose. Methylation analysis, Smith degradation and fragmentation with cellulase showed that this polysaccharide is composed of three distinct polysaccharide moieties i.e., xyloglucan, β-glucan and arabinoxylan. The xyloglucan consists of β-(1→4)-linked glucan back bone and short side chains of single xylose units or galactosylxylose both attached to C-6 of the glucose residues. The β-glucan contains both (1 →3)-and (1→4)-linkages similarly to the other cereal β-glucans, but differ from them in containing the blocks of (1→3)-linked glucose residues in the chain. The arabinoxylan has a highly branched structure, in which 78% of (1→4)-linked xylose residues have short side chains of arabinose at C-3 position.

On the basis of these findings, the interconnection of these polysaccharide moieties is discussed.     

Structural study of bergenan, a pectin from Bergenia crassifolia

A pectin polysaccharide named bergenan was isolated from the freshly collected leaves of the leather bergenia Bergenia crassifolia by extraction with an aqueous solution of ammonium oxalate. The main component of its carbohydrate chain was shown to be the residues of D-galacturonic acid (about 80%). In addition, the polysaccharide contains residues of galactose, arabinose, and rhamnose; their total content is less than 15%. It was shown that the bergenan samples from bergenia leaves collected at different vegetation periods (from July to September) do not substantially differ either in monosaccharide composition or in the viscosity of aqueous solutions they form. The results of enzymatic hydrolysis by alpha-1,4-galacturonase (pectinase), partial acidic hydrolysis, NMR spectroscopy, and methylation with subsequent analysis of the results by GC-MS indicate that the bergenan macromolecule contains the regions of a linear alpha--1,4-D-galactopyranosyluronan and rhamnogalacturonan-I (RG-1). Galacturonan responds for a greater part of the macromolecule. A considerable amount of its constituent galacturonic acid residues are present as methyl esters. The side chains in RG-I are attached to the rhamnopyranose residues of the main carbohydrate chain by 1,4-link and are composed of the residues of terminal arabinofuranose and galactopyranose, 1,5-linked (-arabinofuranose, and 1,4-and 1,6-linked beta-galactopyranose. The branching points of the side chains of the RG-I molecule are 3,4- and 3,6-di-O-substituted galactose residues.     

Structural studies of the capsular polysaccharide of a non-neoformans Cryptococcus species identified as C. laurentii, which was reclassified as Cryptococcus flavescens, from a patient with AIDS

Cryptococcus flavescens, a strain originally identified as C. laurentii, was isolated from the cerebrospinal fluid of an AIDS patient, and the soluble capsular polysaccharide of the yeast was investigated. Glucuronoxylomannan (GXM) was obtained from C. flavescens under conditions similar to those used to obtain C. neoformans polysaccharide. However, the GXM differed from C. neoformans polysaccharide in the decreased O-acetyl group content. The structure of GXM was determined by methylation analysis, partial acid hydrolysis, NMR analyses, and controlled Smith degradation. These analyses indicated that GXM has the following structure: an alpha-(1-->3)-D-mannan backbone with side chains of beta-D-glucuronic acid residues bound to the C-2 position of the mannose residue. The C-6 position of the mannose is substituted with D-man-beta-(1-->4)-D-xyl-beta-(1--> disaccharide. Furthermore, the existence of side chains containing more than two xylose residues was suggested. This mannosylxylose side chain is a novel structure in polysaccharides of C. neoformans and other Cryptococcus species.     

Structural studies of the pectic polysaccharide from duckweed Lemna minor L

The pectic polysaccharide of duckweed Lemna minor L. termed lemnan (LM) was shown to contain the ramified, "hairy" region. Using partial acid hydrolysis and Smith degradation followed by NMR spectroscopy of the fragments obtained, some structural features of the hairy region of LM were elucidated. Partial acid hydrolysis of LM afforded the crude polysaccharide fraction LMH that was separated into two polysaccharide fractions: LMH-1 and LMH-2. In addition, the oligosaccharide fraction LMH-3 contained 97% D-apiose was obtained from the supernatant. A further more rigorous acidic hydrolysis of LMH led to the crude polysaccharide fraction LMHR which was separated in to two fractions: LMHR-1 and LMHR-2. Smith degradation of LMH afforded the polysaccharide fragment LMHS differed in low contents of apiose residues. Unfortunately, NMR-spectroscopy failed to provide significant evidence concerning the structure of LMH-1 due to the complexity of the macromolecule. The structure of the 1H/13C-NMR spectroscopy including the correlation 2D NMR spectroscopy. As a result, alpha-1,4-D-galactopyranosyluronan was confirmed to be the main constituent of the LM backbone. In addition, the ramified, "hairy" region of the macromolecule appeared to contain segments consisting of residues of terminal and beta-1,5-linked apiofuranose, terminal and alpha-1,5-linked arabinofuranose, terminal and beta-1,3- and beta-1,4- linked galactopyranose, the terminal and beta-1,4-linked xylopyranose, and beta-1,4-linked 2-mono-O-methyl xylopyranose. Analytical and NMR-spectral data of LMHS confirmed the presence of considerable amounts of the non-oxidized of 1,4-linked D-galactopyranosyl uronic acid residues. Thus, some side chains of the ramified region of lemnan appeared to attach to D-galactopyranosyl uronic acid residues of the backbone.     

Characterization of a polysaccharide component of lipopolysaccharide from Pseudomonas aeruginosa IID 1008 (ATCC 27584) as D-rhamnan

Structural studies were carried out on a rhamnose-rich polysaccharide isolated from the O-polysaccharide fraction of lipopolysaccharide in Pseudomonas aeruginosa IID 1008 (ATCC 27584) after destruction of the major O-specific chain by alkaline treatment. The isolated polysaccharide contained rhamnose, 3-O-methyl-6-deoxyhexose, glucose, xylose, alanine, galactosamine and phosphorus in a molar ratio of 67:6.9:4.3:2.1:1.1:1.0:4.1. Data from analysis involving Smith degradation, methylation, 1H-NMR spectroscopy and optical rotation measurement showed that the polysaccharide was built up of three moieties, a rhamnan chain composed of about 70 D-rhamnose residues, the core chain and an oligosaccharide chain comprising 3-O-methyl-6-deoxyhexose, xylose, rhamnose and probably glucose. The repeating unit of the rhamnan chain was indicated to have the following structure:----3)D-Rha(alpha 1----3)D-Rha(alpha 1----2)D-Rha(alpha 1----. This structure is identical with that proposed previously for the repeating unit of the side chain of lipopolysaccharide from plant pathogenic bacteria Pseudomonas syringae pv. morsprunorum C28 [Smith, A.R.W., Zamze, S.E., Munro, S.M., Carter, K. J. and Hignett, R.C. (1985) Eur. J. Biochem. 149, 73-78].     

Acetylated methylmannose polysaccharide of Streptomyces.

A polysaccharide composed of 3-O-methyl-D-mannose and D-mannose in a molar ratio of approximately 10:1 and containing 3 to 4 esterified acetyl residues has been isolated from Streptomyces griseus. This acetylated methylmannose polysaccharide (AMMP) is similar to the methylmannose polysaccharide (MMP) of Mycobacterium smegmatis (Gray, G. R., and Ballou, C. E. (1971) J. Biol. Chem. 246, 6835-6842) in its size and composition, the absence of acidic or basic groups, and the lack of a reducing end. It is different, however, in its content of esterified acetyl residues, and it is slightly different in its structure and in its gel filtration properties. The structure of AMMP has been established by proton magnetic resonance spectroscopy, and by combinations of methylation analysis and Smith degradation utilizing non-radioactively labeled polysaccharide and [3H]methyl-labeled polysaccharide obtained from cells grown in the presence of L-[methyl-3H]methionine. It is concluded that AMMP is a linear, nonreducing, neutral polysaccharide composed of a terminal D-mannose residue linked alpha(1 leads to 4) to a chain of 10 consecutive alpha(1 leads to 4)-linked 3-O-methyl-D-mannose residues. The reducing terminal 3-O-methyl-D-mannose residue exists, at least in part, as its alpha-methyl glycoside. The positions of attachment of the ester residues have not been established.     

Human low-molecular-mass kininogen. Amino-acid sequence of the light chain; homology with other protein sequences

The chemical structure of the polysaccharide moiety of the lipopolysaccharide Rhodopseudomonas sphaeroides ATCC 17023 was established. Mild acetic acid hydrolysis of isolated lipopolysaccharide, followed by preparative high-voltage paper electrophoresis afforded three oligosaccharides. They were characterized by chemical and physicochemical studies to be: GlcA(alpha 1----4)dOclA8P, Thr(6') GlcA(alpha 1----4)GlcA and GlcA(alpha 1----4)dOclA, where GlcA is D-glucuronic acid and dOc1A is 3-deoxy-D-manno-octulosonic acid. Carboxyl-reduction of the lipopolysaccharide followed by acid hydrolysis gave a trisaccharide: GlcA(alpha 1----4)Glc(alpha 1----4)Glc, showing the presence of three residues of glucuronic acids in the O-specific chain and indicating that only two of them are reducible by NaBH4. The linkage between the polysaccharide and lipid A was shown to be through a single 1,4-linked residue of dOc1A attached by a 2,6'-linkage to the lipid A moiety.     

Structural studies of a mucilage from Abroma augusta root bark

The structure of an acidic polysaccharide isolated from Abroma augusta root bark was determined by sugar and methylation analyses and high resolution ¹H- and ¹³C-NMR spectroscopy. The main chain of the polysaccharide was composed of 1,2-linked - -rhamnopyranose and 1,4- or 1,3-linked - -galacturonic acid residues. The terminal β- -glucuronic acid residue was attached to the 3- and/or 4-position of the - -galacturonic acid residue.     

Antigenic bacterial polysaccharides. 36. A study of the structure of the O-specific polysaccharide chain of the lipopolysaccharide of Pseudomonas fluorescens IMV 2763 (biostrain G)

Chemical and serological characterization of the Pseudomonas fluorescens IMV 2763 (biovar G) lipopolysaccharide was carried out. The O-specific polysaccharide chain of the lipopolysaccharide is composed of D-mannose, 6-deoxy-L-talose, N-acetyl-D-galactosamine and O-acetyl groups in the ratio of approximately 2:1:1:1. The polysaccharide is branched and a half of residues of 6-deoxytalose and monosubstituted mannose carry O-acetyl groups. On the basis of methylation, partial acid hydrolysis and 13C NMR analysis it was concluded that the repeating unit of the polysaccharide has the following structure: (formula; see text)     

Studies on the Non-starchy Polysaccharides of the Endosperm of Buckwheat

Water soluble polysaccharides from the buckwheat endosperm was fractionated by salting out and a DEAE-cellulose column (phosphate form) chromatography and the main component (polysaccharide A₁) was isolated as an ultracentrifugally and electrophoretically pure preparation.

The content of polysaccharide A₁ in the buckwheat endosperm was 0.1~0.2%.

Its water solution showed high viscosity and [α]_d was +39.4°. The molecular weight was 240,000~260,000.

Polysaccharide A₁ consisted of xylose, mannose, galactose and glucuronic acid. The hydrolysis of methylated polysaccharide A₁ gave 2,3,4-tri-O-methyl-xylose, 2,3,4,6-tetra-O-methyl-galactose, 2,4,6-tri-O-methyl-galactose, di-O-methyl-mannose and 4-O-methyl- and 5-O-methyl-glucuronic acid. These results suggested that the main chain of this polysaccharide consisted of glucuronic acid, mannose and galactose and the former two occupied branching position with xylose and galactose residues as nonreducing end.     

Isolation and characterization of polysaccharides from Tansy

Using extraction with 0.75% aqueous ammonium oxalate, the following polysaccharide fractions were isolated: tanacetans TVF, TVS, and TVR from floscules, sprouts, and roots, respectively, of Tanacetum vulgare L., spread throughout the European North of Russia. The sugar chain of tanacetan TVF consists of D-galacturonic acid (61.4%), arabinose (14.7%), galactose (10.2%), and rhamnose (3.7%) as the main constituents as well as xylose, glucose, mannose, apiose, and 2-O-methylxylose in trace amounts. Tanacetans TVS and TVR were shown to differ in the sugar quantitative composition. They contain 67 and 28% galacturonic acid, respectively. A partial acid hydrolysis of the tanacetan TVF gave a polysaccharide fragment TVF1, alpha-1,4-D-galacturonan (GalA 98.2%). Digestion with pectinase (alpha-1,4-D-polygalacturonase) resulted in fragment TVF3, containing residues of arabinose (27.1%) and galactose (17.3%). NMR spectroscopy allowed detection of the terminal residues of alpha-Araf and beta-Galp as well as of the residues of alpha-Araf substituted in 3,5- and 5-positions. Thus, tanacetan TVF was proved to be a pectic polysaccharide.     

Antigenic polysaccharides of bacteria of the genus Shigella. The structure of the polysaccharide chain of Shigella boydii type 14

A specific acidic polysaccharide has been isolated from the Shigella boydii type 14 antigenic lipopolysaccharide after mild hydrolysis followed by chromatography on Sephadex G-50. The polysaccharide consists of the D-glucuronic acid, 2-acetamido-2-deoxy-D-glucose and D-galactose residues in the ratio 1:1:3. From the results of methylation analysis and partial acid hydrolysis, the structure of the repeating unit of the specific polysaccharide was deduced as follows: (-6DGalp alpha 1-4DGlcAp beta 1-6DGalp beta 1-4DGalp beta 1-4DGlcNAcp beta 1-)n. The 13C NMR spectra of native and carboxyl-reduced polysaccharides, as well as of oligosaccharides produced by partial acid hydrolysis fully confirmed the proposed structure. The approach was suggested to determine the type of substitution of uronic acid moieties in polysaccharide chain by use of chromato-mass-spectrometry of acetylated methyl esters of partially methylated aldonic acids. Serological characteristics of Sh. boydii LPS type 14 and its modified derivatives are discussed.     

Chemical and physical properties of an arabinogalactan-peptide from wheat endosperm

1. An arabinogalactan-peptide from wheat endosperm was studied by using physicochemical techniques and some aspects of its chemical structure were determined. 2. The arabinogalactan-peptide is a non-associating, polydisperse macromolecule ([unk]=22000) which exhibits only minor non-ideal effects in aqueous solution. 3. Examination of the products of partial acid hydrolysis of the polysaccharide component showed that arabinose is present in the alpha-l-arabinofuranosyl configuration, and i.r.-absorption spectroscopy and optical-rotation studies suggest that the d-galactopyranose residues are linked by glycosidic linkages in the beta-anomeric configuration. 4. The arabinogalactan is linked to a peptide which represents 8% (w/w) of the arabinogalactan-peptide and which may be present as a molecular core. Partial degradation of the polymer by successive treatment with oxalic acid and NaOH showed that the linkage between polysaccharide and peptide involves galactose and hydroxyproline residues and is glycosidic in nature. A tentative model is proposed for the structure of the wheat endosperm arabinogalactan-peptide. 5. The subcellular location and function of the arabinogalactan-peptide is discussed in relation to previous work with related molecules.     

Insight of Endo-1,4-Xylanase II from Trichoderma reesei: Conserved Water-Mediated H-Bond and Ion Pairs Interactions

Endo-1,4-Xylanase II is an enzyme which degrades the linear polysaccharide beta-1,4-xylan into xylose. This enzyme shows highest enzyme activity around 55 °C, even without being stabilized by the disulphide bridges. A set of nine high resolution crystal structures of Xylanase II (1.11–1.80 Å) from Trichoderma reesei were selected and analyzed in order to identify the invariant water molecules, ion pairs and water-mediated ionic interactions. The crystal structure (PDB-id: 2DFB) solved at highest resolution (1.11 Å) was chosen as the reference and the remaining structures were treated as mobile molecules. These structures were then superimposed with the reference molecule to observe the invariant water molecules using 3-dimensional structural superposition server. A total of 37 water molecules were identified to be invariant molecules in all the crystal structures, of which 26 invariant molecules have hydrogen bond interactions with the back bone of residues and 21 invariant water molecules have interactions with side chain residues. The structural and functional roles of these water molecules and ion pairs have been discussed. The results show that the invariant water molecules and ion pairs may be involved in maintaining the structural architecture, dynamics and function of the Endo-1,4-Xylanase II.     

Structure of the repeating chain of the O-specific polysaccharide of Vibrio fluvialis

O-Specific polysaccharide chain of the Vibrio fluvialis lipopolysaccharide is built up of pentasaccharide repeating units, containing one N-acetyl-D-glucosamine and four L-rhamnose residues. The structure of the polysaccharide was elucidated using two-dimensional correlation 1H-NMR-spectroscopy, 13C-NMR-spectroscopy and nuclear Overhauser effect and confirmed by methylation analysis and selective cleavage of N-acetylglucosamine residues by the N-deacetylation-deamination method which yielded linear L-rhamnan representing the backbone of the polysaccharide. Thus, the repeating unit of the O-specific polysaccharide has the following structure: (formula; see text)