Acid-containing Polysaccharides Research Articles

Nature's many mechanisms for the degradation of oligosaccharides.

Recent work on the mechanistic elucidation of the polysaccharide lyases, the [small alpha]-1,4-glucan lyases, and the Family 4 glycosidases have demonstrated that nature has evolved to use elimination steps for the degradation of oligosaccharides. The polysaccharide lyases (E.C. 4.2.2.-) have been shown to cleave uronic acid-containing polysaccharides via a stepwise E1cB mechanism. The mechanism of the alpha-1,4-glucan lyases (E.C. 4.2.2.13) is similar to the Family 31 glycosidases, forming a covalent glycosyl-enzyme intermediate, which is subsequently cleaved by an E1-like E2 mechanism. Meanwhile, the Family 4 glycosidases (E.C. 3.2.1.6) are suggested to undergo an oxidation-elimination-addition-reduction sequence. These three groups of enzymes are examples of stark contrast to the vast number of well-characterized glycosidases (E.C. 3.2.1.-), which utilize either the direct or double displacement mechanisms as proposed by Koshland over 50 years ago.

Adsorption behavior of oxidized galactomannans onto amino-terminated surfaces and their interaction with bovine serum albumin

Galactomannans extracted from Cassia fastuosa (CF) and Leucaena leucocephala (LL) seeds were purified and oxidized with 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) to form the uronic acid-containing polysaccharides CFOX with a degree of oxidation (DO) of 0.22 and LLOX with DO of 0.66, respectively. The adsorption behavior of CFOX and LLOX onto amino-terminated surfaces was studied by means of ellipsometric measurements. The influence of pH and ionic strength on the adsorption was investigated. A strong dependence of pH on the adsorbed amount of CFOX and LLOX was observed. At pH 4, there was a maximum in the adsorbed amount caused by strong electrostatic attraction between the substrate and the oxidized galactomannans. On the other hand, no ionic strength effect on the adsorption of CFOX and LLOX was observed. The immobilization of bovine serum albumin onto LLOX and CFOX was studied as a function of pH. LLOX proved to be a more attractive substrate for BSA than CFOX, indicating that the favorable interactions between the carboxylate groups and the BSA segments have driven the adsorption process. At the isoelectric point of BSA a maximum in the adsorbed amount was found for both surfaces.

Development of a high-performance anion-exchange chromatography with pulsed-amperometric detection based quantification assay for pneumococcal polysaccharides and conjugates

A method, using high-performance anion-exchange chromatography with pulsed-amperometric detection (HPAEC–PAD), has been developed to determine the concentrations of Streptococcus pneumoniae capsular polysaccharides and polysaccharide conjugates used in formulating a conjugate vaccine for the prevention of pneumococcal infections. In an effort to determine optimum hydrolysis conditions for the analysis, pneumococcal polysaccharides were subjected to three different hydrolysis methods: trifluoroacetic acid (TFA) hydrolysis, methanolysis followed by TFA hydrolysis, or hydrofluoric acid (HF) hydrolysis followed by TFA hydrolysis. For quantification purposes, best results were obtained by methanolysis followed by TFA hydrolysis for uronic acid-containing polysaccharides, and by TFA hydrolysis for all the others. For the quantification of all the polysaccharides (from native to conjugated forms), a monosaccharide reference mixture (Rha, Gal and GlcA) hydrolyzed along with the samples can be used as standards for routine analysis. This is much more convenient than to hydrolyze a well-characterized reference polysaccharide (necessary standard only for type 1 capsular polysaccharide). This method is rapid, very sensitive (less than 10 μg of polysaccharide is required), and may replace advantageously the currently used colorimetric assays used to determine polysaccharides content. Moreover, it can be readily adapted for use with other bacterial polysaccharide preparations as well.

Morphine Metabolism in the Opium Poppy and Its Possible Physiological Function: BIOCHEMICAL CHARACTERIZATION OF THE MORPHINE METABOLITE, BISMORPHINE

We identified a novel metabolic system of morphine in the opium poppy (Papaver somniferum L.). In response to stress, morphine is quickly metabolized to bismorphine consisting of two morphine units, followed by accumulation in the cell wall. This bismorphine binds predominantly to pectins, which possess high galacturonic acid residue contents, through ionical bonds. Our newly developed method using artificial polysaccharides demonstrated that bismorphine bridges are formed between the two amino groups of bismorphine and the carboxyl groups of galacturonic acid residues, resulting in cross-linking of galacturonic acid-containing polysaccharides to each other. The ability of bismorphine to cross-link pectins is much higher than that of Ca2+, which also acts as a cross-linker of these polysaccharides. Furthermore, we confirmed that cross-linking of pectins through bismorphine bridges leads to resistance against hydrolysis by pectinases. These results indicated that production of bismorphine is a defense response of the opium poppy. Bismorphine formation is catalyzed by anionic peroxidase that pre-exists in the capsules and leaves of opium poppies. The constitutive presence of morphine, together with bismorphine-forming peroxidase, enables the opium poppy to rapidly induce the defense system.

Intestinal degradation of dietary fibre in green beans-effects of microwave treatments

Intestinal degradation of dietary fibre in blanched and microwaved green beans was studied by using a rat experimental model. Content and composition of dietary fibre as well as molecular weight distribution of water-soluble polysaccharides (WSP) were analysed. There was a solubilization and a shift towards lower Mw of mainly uronic acid-containing polysaccharides with repeated microwave treatment in the raw material. Thus, the apparent Mw of water-soluble polysaccharides decreased from ~1 550 000 to ~300 000. After the beans had been digested the Mw of the WSP was significantly reduced, to ~100 000, and the differences in Mw seen between various processed raw materials had been evened out. After fermentation the Mw of the WSP decreased further ~10 times. Fibre fermentability was high (~90%) and similar for the various processed beans.

Structure and Composition of Biological Slimes on Paper and Board Machines

Biological slimes (biofilms) collected from the wet end of paper and board machines were examined by electron microscopy and analyzed for fatty acid composition, neutral sugar composition, and ATP. Electron microscopy revealed minuscule prokaryotic organisms (diameter, 0.2 to 0.4 mum). Larger cells morphologically resembling Sphaerotilus and Leptothrix spp. were found in slimes from machines using recycled fiber or unbleached pulp. The bacteria were embedded in a slimy matrix and often contained reserve materials microscopically resembling poly-beta-hydroxybutyrate and glycogen. Fatty acid analysis of the slimes revealed bacterial signature fatty acids in concentrations equivalent to the presence of 2 x 10 to 2.6 x 10 (average, 7 x 10) bacterial cells (live and dead) per g (dry weight) of slime. The slimes contained several known components of bacterial polysaccharides in addition to glucose, indicating that the slime body consisted of bacterial polysaccharides. The slimes contained uronic acids equivalent to a binding capacity of 12.5 to 50 mumol of divalent cations per g (dry weight) of slime. The uronic acid-containing polysaccharides may be responsible for the accumulation of heavy metals in the slime. Calculation of the ATP contents of the slimes resulted in an estimate of 5 x 10 cells per g (dry weight) of slime when calibrated with pure bacterial cultures isolated from the slimes. From electron micrographs, an estimate ranging from 1 x 10 to 1.5 x 10 (average, 4 x 10) cells per g (dry weight) of slime was obtained.

Carbohydrate analysis of water-soluble uronic acid-containing polysaccharides with high-performance anion-exchange chromatography using methanolysis combined with TFA hydrolysis is superior to four other methods

Sulfuric acid hydrolysis according to the Saeman procedure, TFA hydrolysis, and methanolysis combined with TFA hydrolysis were compared for the hydrolysis of water-soluble uronic acid-containing polysaccharides originating from fungi, plants, and animals. The constituent sugar residues released were subsequently analyzed by either conventional GLC analysis of alditol acetates or high-performance anion-exchange chromatography with pulsed-amperometric detection. It was shown that TFA hydrolysis alone is not sufficient for complete hydrolysis. Sulfuric acid hydrolysis of these polysaccharides resulted in low recoveries of 6-deoxysugar residues. Best results were obtained by methanolysis combined with TFA hydrolysis. Methanolysis with 2 m HCl prior to TFA hydrolysis resulted in complete liberation of monosaccharides from pectic material and from most fungal and animal polysaccharides tested. Any incomplete hydrolysis could be assessed easily by HPAEC, by the detection of characteristic oligomeric products, which is difficult using alternative methods currently in use. Methanolysis followed by TFA hydrolysis of 20 μg water-soluble uronic acid containing polysaccharides and subsequent analysis of the liberated sugar residues by HPAEC allowed us to determine the carbohydrate composition of these polysaccharides rapidly and accurately in one assay without the need for derivatization.

High performance liquid chromatographic strategy for the analysis of exopolysaccharides extracted from pathogenic bacteria.

A high performance liquid chromatographic (HPLC) method has been developed to permit the rapid comparison of acidic polysaccharides of diverse compositions and the sensitive determination of their constituents. It is based on two combined analyses of the polysaccharide hydrolysates--a separation of the released compounds by ion-moderated partition chromatography with UV detection at two wavelengths and a separation of the sugar dansylhydrazine derivatives by reversed phase chromatography. The former permits identification and quantitation of uronic and carboxylic acids, the latter permits more sensitive and specific determination of the neutral aldoses. Some bacterial exopolysaccharides have been used to demonstrate the validity of this HPLC procedure for the chemical characterization of uronic acid-containing polysaccharides. This method appears to be useful for studying capsular polysaccharides, which are involved in the evasion of phagocytosis by pathogenic bacteria.

Purification and Characterization of a Novel Lyase from Cellulomonas sp. that Degrades Fusarium and Gibberella Acidic Polysaccharides.

A Cellulomonas sp. isolated from soil produced a novel lyase that degraded the acidic polysaccharide of Fusarium sp. M7-1 with the formation of mannose and O-beta-D-mannopyranosyl-(1----2)-D-mannose. DEAE-Toyopearl 650M column chromatography showed three lyase activity peaks (fractions I, II, and III). The major fraction was purified to homogeneity by polyacrylamide gel electrophoresis analysis, and its molecular weight was 74,000. The optimum pH was 6.5 to 8.0 and the stable pH range was 6.0 to 8.0. The purified enzyme did not degrade glucuronic or galacturonic acid-containing polysaccharides such as chondroitin, hyaluronic acid, pectin, or pectic acid. However, the purified enzyme specifically degraded various Fusarium and Gibberella acidic polysaccharides, and unsaturated sugars were produced with the release of mannose and O-beta-D-mannopyranosyl-(1----2)-D-mannose. These results suggest that the acidic polysaccharides derived from Fusarium and Gibberella have similar structures.

Characterization of the saccharide composition of heteropolysaccharides by pyrolysis-capillary gas chromatography-mass spectrometry

The saccharide composition of a number of heteropolysaccharides has been successfully characterized by pyrolysis-gas chromatography. In addition, small residues of carbohydrate impurities in samples can be easily identified. The success of pyrolysis-gas chromatography to differentiate among carbohydrates was established by previous studies which found that rapid pyrolysis initiates glycosidic cleavage of saccharide units through transglycosidation thereby forming intact fragments which retain the stereoconfiguration of the saccharide, i.e., anhydro sugars. The high molecular weight product mixtures are resolved on a proper capillary column and the individual anhydro sugars identified by mass spectrometry. Bacterial and plant polysaccharides were selected having various compositions, consisting of hexoses, pentoses, deoxy sugars and uronic acids. The composition of a highly sulfated polysaccharide was also examined. It was found that uronic acid-containing polysaccharides require that the carboxylate functional group be protonated in order to obtain structurally-significant pyrolysis products.

Structure of a polysaccharide containing galactose and galacturonic acid from Rhizobium meliloti. Characterization and partial purification of a 2-O-methyltransferase.

The teichuronic acid type polysaccharide found in Rhizobium meliloti which is associated with sensitivity to phage 16B and is formed in the inner membranes from UDP-galactose and UDP-galacturonic acid (Ugalde, R. A., Coira, J. A., and Brill, W. J. (1986) J. Bacteriol. 168, 270-275) has been studied further. Results of acid hydrolysis, periodate oxidation, and borohydride reduction show that this polysaccharide contains the repetitive unit -galacturonosyl(1-3)galactosyl(1-4-). A soluble enzyme was found to catalyze the transfer of methyl groups from S-adenosylmethionine to position 2 of the galacturonosyl residue. The enzyme requires Mn2+ or Mg2+, its pH optimum is 8.2, and the apparent Km for S-adenosylmethionine is 2.7 microM. The teichuronic acid type polysaccharide bound to a trichloroacetic acid-insoluble cell residue is a substrate for the methyltransferase; however, the polysaccharide released from the trichloroacetic acid-insoluble portion by mild acid treatment is no longer methylated. Other soluble galacturonic acid-containing polysaccharides were not used as substrates. The methyltransferase and the polysaccharide acceptor are both found in R. meliloti strain 102F51. Spontaneously arising mutants resistant to phage 16B do not form teichuronic acid but are transferase-positive. Other strains of R. meliloti as well as Agrobacterium tumefaciens and Escherichia coli cells do not form teichuronate and have no transferase.

Degradation of glucuronic acid-containing exopoly-saccharides from rhizobium by the hakomori methylation procedure

The uronic acid-containing polysaccharides secreted by some fast-growing, nodulating and non-nodulating strains of Rhizobium underwent marked degradation when methylated by the Hakomori procedure. The polysaccharide produced by Rh. meliloti, which lacks uronic acid, was not degraded.

Structure of the Primary Cell Walls of Suspension-Cultured Rosa glauca Cells

Cell walls of suspension-cultured cells of Rosa glauca were fractionated by two different extraction procedures. The first involved a stepwise fractionation scheme based on alkaline extraction. The second took advantage of the powerful cellulose solvent system N-methylmorpholine N-oxide/dimethyl sulfoxide which is capable of solubilizing whole cell walls. From the analytical composition of each solubilized fraction and of the corresponding residues, the fate of each type of cell wall polysaccharide constituent was followed at each step of the extraction scheme and the mode of action of the extractant was interpreted. Although the two fractionation procedures were very different, they yielded very similar cellulosic complex residues and extracts, thus delimiting two blocks of polysaccharides in the cell wall. The cellulose residues still comprised uronic acid-containing polysaccharides and hemicelluloses in association with cellulose. Graded acid hydrolysis provided evidence for the central role of a homogalacturonan core interconnecting xyloglucans and arabinogalactans. A tentative model showing the possible interaction existing between the constituent polysaccharides still associated to cellulose after alkaline extraction is presented. Hydrogen bonding between xyloglucan and cellulose is confirmed, and glycosidic linkages between xyloglucans and pectic polymers are suggested.

Structure, conformation and immunology of sialic acid-containing polysaccharides of human pathogenic bacteria

Abstract

The 13C-N.M.R. spectra of methyl (methyl O-methyl-α- d-glucopyranosid)uronates

13C-N.m.r. spectra of all of the methyl ethers of methyl (methyl α- d-glucopyranosid)uronate have been interpreted. The data can be used as an aid in the analysis of 13C-n.m.r. spectra of α- d-glucopyranosyluronic acid-containing polysaccharides.

Sialic Acid-Containing Polysaccharides of Neisseria meningitidis and Escherichia coli Strain Bos-12: Structure and Immunology

A polysaccharide antigenically related to that of group C Neisseria meningitidis was isolated from Escherichia coli strain Bos-12 (O48: K91:NM). Like the polysaccharides of groups B and CN. meningitidis, the Bos-12 antigen was shown to be a pure polymer of sialic acid. The 13C-nuclear magnetic resonance studies of the groups B and C polysaccharides indicated that the former consists of units of sialic acid joined in alpha-2 leads to 8 linkages, whereas the latter contains sialic acid residues linked by alpha-2 leads to 9 glycosidic bonds. Chemical and nuclear magnetic resonance studies of the polysaccharide of E. coli strain Bos-12 established that it is a heteropolymer containing both alpha-2 leads to 8 and alpha-2 leads to 9 linkages. Physical parameters including partial specific volume, reduced viscosity, diffusion and sedimentation coefficients, shape, weight-average molecular weight, and the Stokes radius of the polysaccharides of groups A, B, and C N. meningitidis have been determined. The results indicate that the polysaccharides are highly asymmetric and exist in solution as rigid rods; aggregates are formed by the association of these rods. The polysaccharides appear to have "restricted" length.

Determination of the linkages in some methylated, sialic acid-containing, meningococcal polysaccharides by mass spectrometry

The application of gas-liquid chromatography-mass spectrometric (g.l.c.-m.s.) analysis to a number of sialic acid-containing polysaccharides of meningococcal origin has been studied. Methylation of these polysaccharides by the Hakomori conditions resulted in both O- and N-methylation. Methanolysis of the methylated polysaccharides from serogroup C [(2→9)-linked], colominic acid [(2→8)-linked], and serogroups Y and W-135 [both (1→4)-linked], yielded the respective 4,7,8,4,7,9-, and 7,8,9-tri- O-methyl derivatives of methyl N-acetyl- N-methyl-β- D-neuraminate methyl glycoside. As model compounds, methyl N-acetyl-4,7,8,9-tetra- O-methyl-α- D-neuraminate methyl glycoside and its N-methyl derivative were also synthesized. All of the methylated derivatives could be identified on the basis of their typical fragmentation-patterns, indicating that this method is applicable to the determination of the position of linkages to sialic acid residues in biopolymers.

The circular dichroic spectra of several sialic acid-containing polysaccharides isolated from neisseria meningitidis

The circular dichroic spectra of the acid and sodium salt forms of several sialic acid-containing homo- and hetero-polysaccharides have been measured. The spectra are shown to be influenced by the state of ionization of the carboxyl groups contained in the sialic acid, the location within the individual sialic acid residues of the inter-saccharide linkages, and changes in the configuration of hydroxyl groups remote to the carboxyl group of the sialic acid.

Modification of polysaccharides containing uronic acid residues

Klebsiella Type 47 capsular polysaccharide has side chains attached to the main chain via D-glucuronic acid residues. The side chains have been removed to yield an essentially linear polysaccharide by the following sequence of reactions: ( 1) substitution of hydroxyl and car☐yl groups with methyl vinyl ether; ( 2) β-elimination by treatment with base; ( 3) removal of modified uronic acid residues and protecting groups by mild acid hydrolysis. The possibility of modifying other uronic acid-containing polysaccharides by this method is discussed.

Comparative immunochemical behavior of acid polysaccharides of Cryptococcus laurentii, Tremella mesenterica and of oxidized cellulose with a type II antipneumococcal serum

The immunochemical behavior of native and partially dexylosylated glucuronic acid-containing polysaccharides of Cryptococcus laurentii (NRRL Y-1401) and of haploid cells of Tremella mesenterica (Y-6151) was examined with Type II anti-pneumococcal horse serum. Cryptococcal polysaccharide and its derivatives cross reacted while the corresponding Tremella preparations did not. The specificity of cross reactive antibodies precipitating with C. laurentii polysaccharide and oxidized cellulose (Oxycel) was examined by quantitative hapten inhibition and compared with the homologous S II-anti-S II system. Differences were found in the relative ability of sodium glucuronate and p-nitrophenyl-β- d-glucuronide to inhibit precipitation of anti-S II antibodies of C. laurentii, Oxycel and Type II pneumococcal polysaccharide.