Carboxyl-reduced Polysaccharide Research Articles

Structural Features and Anti-Complement Activity of an Acidic Polysaccharide from Forsythia suspensa

Background: The complement system is an important host defence mechanism against the invasion of foreign materials. However, excessive or uncontrolled activation of the complement system might lead to severe complement-mediated disorders which are harmful to human bodies. There are still no desirable therapeutic compounds available on the market for complement inhibition. In recent years, researches have been focused on natural polysaccharides to modulate of the immune system because of their immunomodulatory activities and safety. In this study, Fs-8-ba2, a homogeneous acidic polysaccharide (MW ca. 7.2 kDa) was isolated from Forsythia suspensa and its anti-complementary effect was investigated. Method: The major primary structural features of Fs-8-ba2 were elucidated using HPGPC, IR, absolute configuration, component analysis and methylation analysis, periodate oxidation, carboxyl reduction partial acid hydrolysis, and NMR spectroscopy, etc. To identify the anti-complementary activity of Fs-8-ba2, the hemolytic assays through the classical pathway (CP) and the alternative pathway (AP) of complement system in vitro were taken. Results: The backbone of Fs-8-ba2 was composed of 7 homogalacturonan (HG) and 2 rhamnogalacturonan (RG-I) moieties with the side chains attached at O-4 of 1,2,4-linked α-L-Rha in the RG-I moieties. The anti-complement assay showed that the native acidic polysaccharide (Fs-8-ba2) possessed stronger inhibitory effect on the complement activation than the carboxyl-reduced polysaccharide (Fs-8-ba2re) through the classical (IC50: 0.311 ± 0.020 mg/mL vs. 3.292 ± 0.032 mg/mL) and alternative pathways (IC50: 0.218 ± 0.015 mg/mL vs. no activity). It indicated that the anti-complement effect of pectic polysaccharide was related to GalA content (carboxyl group). Preliminary mechanism studies by using complement-depleted sera indicated that Fs-8-ba2 selectively interacted with C1q, C1r, C1s, C2, C3 and C9, but not C4 and C5. Conclusion: These results suggested that Fs-8-ba2, an acidic polysaccharide, could be of potential benefits in the treatment of the complement-mediated diseases.

Structure of the O-polysaccharide of Hafnia alvei strain PCM 1189 that has hexa- to octasaccharide repeating units owing to incomplete glucosylation

The O-polysaccharide of Hafnia alvei PCM 1189 consists of d-glucose, d-galactose, d-GalNAc and d-GlcA and lacks the strict regularity. The intact and carboxyl-reduced polysaccharides as well as oligosaccharides obtained by partial acid hydrolysis were studied by chemical and enzymatic analyses, methylation and NMR spectroscopy. The following structure was established for the O-polysaccharide, which is built up of branched hexa- to octasaccharide repeating units differing in the number of lateral glucose residues: ▪ where the glucose residues shown in italics are nonstoichiometric substituents. The repeating units include also a minor O-acetyl group, whose position was not determined.

Structure of a galactoarabinan-rich pectic polysaccharide of native and fermented blackgram ( Phaseolus mungo)

Two homogeneous galactoarabinans containing ∼15% d-galacturonic acid were isolated and characterized from native and fermented blackgram. Linkage analysis indicated highly branched structures consisting of a (1→5)-linked l-arabinan backbone additionally being substituted with l-arabinofuranose, d-galactopyranose, d-galactopyranosyluronic acid and l-rhamnopyranose residues; a structure reminiscent of pectic polysaccharides. Analysis of carboxyl-reduced polysaccharides showed that the galacturonic acid residues are all essentially (1→4)-linked. The fermented polysaccharide had a relatively higher degree of branching which was manifested in its overall higher viscosity and as a result better product making qualities. The uronic acid carboxyls were at least partly involved in maintaining the solution viscosity and functional property attributes, because the carboxyl-reduced polysaccharide was devoid of both these characteristics.

Revised Structure of the Capsular Polysaccharide of Klebsiella Serotype 15

Summary The capsular polysaccharide from Klebsiella Type 15 was found to contain D-galactose, D-glucose, and D-glucuronic acid in the ratios 4:1:1. Methylation analysis of the native and carboxyl-reduced polysaccharide provided informaton about the linkages of different sugar residues in the repeating unit. Partial acid hydrolysis of the polysaccharides gave three acidic and one neutral oligomers whose structures were established. The sequence of different sugar moieties were established from the structures of oligomers and from the Smith degradation of the native antigen. The anomeric configurations of different sugar residues were established with the help of 1 H and 13 C NMR spectral analysis of the native polysaccharides, the aldobliouronic acid and the glyceryl glycoside of the trisaccharide from Smith degradation together with the findings of enzymic hydrolysis. Based on all these results, the hexasaccharide repeating unit 1 was assigned to the K-15 polysaccharide. The results of the inhibition study of several oligomers related to K15 antigen on the homologous immune system support this structure.{fx505-01}

Structure of water-soluble acidic polysaccharides isolated from the bark of Ceiba pentandra var. indica

The water-soluble polysaccharide fraction, isolated from the bark of C. pentandra var. indica by delignification, contained l-rhamnose, d-xylose, l-arabinose, d-glucose, d-galactose, d-glucuronic acid, and d-galacturonic acid. Fractionation using Cetavlon and ethanol gave two acidic polysaccharides, each containing l-rhamnose, d-glucuronic acid, and d-galacturonic acid with mol. wts. of 1.318 × 10 6 and 1.445 × 10 5. On the basis of the results of methylation analysis of the native and carboxyl-reduced polysaccharides, partial hydrolysis, oxidation with periodate and chromium trioxide, and 1H-n.m.r. spectroscopy, the two acidic polysaccharides were concluded to have the following repeating unit. ▪

Isolation and characterization of the gel-forming polygalacturonide from seeds of Ficus awkeotsang.

To examine the spontaneous gel-forming property of the polysaccharide of seeds of Ficus awkeotsang Makino, seeds were extracted sequentially with cold and hot water, and then aqueous ammonium oxalate. The major acidic polysaccharide ([α]25D+239°; DE = 63.6%; M.W. 3.4 × 105), which was responsible for gel-formation, was obtained from the surface of heat-treated seeds and found to be composed mainly of galacturonic acid (96%). On fractionation of the major acidic polysaccharide by anion-exchange column chromatography, three kinds of methyl esterified components were obtained (P-1, DE=69.3%; P-2, 49.2% and P-3, 22.8%). Methylation analysis of the carboxylreduced polysaccharide indicated that the acidic polysaccharide is an essentially linear polysaccharide containing α-(1→4)-linked D-galacturonic acid residues and devoid of L-rhamnose residues, which confirmed it to be a polygalacturonide. Comparison of the IR spectra and the degrees of methyl esterification of the polygalacturonide extracted from heat-treated and heat-untreated seeds suggested that the methyl ester groups in the polygalacturonide must have been released during the seed extraction at room temperature. As a model test, furthermore, when the acid polysaccharide prepared from heat-treated seeds was incubated with pectinesterase from lemon peel in the presence of 1mM CaCl2-2H2O, the gel-forming behavior observed was closely similar to the spontaneous gel-formation of the awkeotsang extract. These results may support the possibility that the high amount of calcium ions (4.0-5.6 %) in an aqueous extract of the seeds contributes to the egg-box formation, by being placed between the de-esterified structurally regular polygalacturonide chains.

Polysaccharides from the roots of the marsh mallow (Althaea officinalis L., var. Rhobusta): structural features of an acidic polysaccharide

An acidic heteropolysaccharide isolated from the mucilage of the roots of the marsh mallow ( Althaea officinalis L., var. Rhobusta) via its insoluble barium salt contained d-galactose, l-rhamnose, d-glucuronic acid, and d-galacturonic acid in the molar ratios 1.2:1.0:1.0:1.0. It was homogenous on free-boundary electrophoresis and in the anlytical ultracentrifuge, and it had M w = 26,700, M n = 23,900. Partial acid hydrolysis and analyses of the methylated and the methylated, carboxyl-reduced polysaccharide indicated that the polymer backbone is composed of (1→4)-linked d-galactopyranuronic acid and (1→2)-linked l-rhamnopyranose units in the ratio of 1:1. Each d-galacturonic unit carries a single β- d-glucopyranuronic residue linked to C-3, and each l-rhamnopyranose unit carries d-galactopyranose residues, mainly as non-reducing terminals linked to C-4. A smal number of presumably short chains of (1→4)-linked d-galactopyranose units are also involved in branching.

Structure of the capsular polysaccharide of Klebsiella serotype K40

The capsular polysaccharide from Klebsiella Serotype K40 contains d-galactose, d-mannose, l-rhamnose, and d-glucuronic acid in the ratios of 4:1:1:1. Methylation analysis of the native and carboxyl-reduced polysaccharide provided information about the glycosidic linkages in the repeating unit. Degradation of the permethylated polymer with base established the identity of the sugar unit preceding the glycosyluronic acid residue. The modes of linkages of different sugar residues were further confirmed by Smith degradation and partial hydrolysis of the K40 polysaccharide. The anomeric configurations of the different sugar residues were determined by oxidation of the peracetylated native and carboxyl-reduced polysaccharide with chromium trioxide. Based on all of these results, the heptasaccharide structure 1 was assigned to the repeating unit of the K40 polysaccharide. ▪

Structure of the Klebsiella type 10 capsular polysaccharide

The capsular polysaccharide from Klebsiella Type 10 was found to contain d-galactose, d-glucose, d-mannose, and d-glucuronic acid in the ratios 3:1:1:1. Acid hydrolysis of the polysaccharide gave one aldobiouronic acid, one aldotriouronic acid, one aldotetraouronic acid, and two neutral disaccharides the structures of which were established. The native and carboxyl-reduced polysaccharide have been subjected as appropriate to methylation analysis and Smith degradation. Degradation of the methylated polysaccharide with base established the identity of the sugar unit preceding the glucosyluronic acid residue. The anomeric configurations of the sugar residues were determined by oxidation of the acetylated native and carboxyl-reduced polysaccharides with chromium trioxide. Based on these studies, the hexasaccharide structure 1 has been assigned to the repeating unit of the K-10 polysaccharide. ▪

Structure of the extracellular polysaccharide produced by the bacterium Alcaligenes (ATCC 31555) species

The extracellular anionic polysaccharide produced by the bacterium Alcaligenes (ATCC 31555) contains l-mannose, l-rhamnose, d-glucose, and d-glucuronic acid in the molar ratios 1.0:4.5:3.1:2.3. Analysis of the methylated and methylated, carboxyl-reduced polysaccharide indicated terminal non-reducing rhamnose and mannose, (1→4)-linked rhamnose, (1→3)- and (1→3,1→4)-linked glucose, and (1→4)-linked glucuronic acid to be present in the ratios 1.0:0.8:2.1:2.2:2.0:2.2. Partial acid hydrolysis and base-catalysed β-elimination gave a series of oligosaccharides that were isolated as their alkylated alditol derivatives by reverse-phase h.p.l.c. and characterised by f.a.b.-m.s., e.i.-m.s., and 1H-n.m.r. spectroscopy. The repeating unit 1, excluding O-acyl groups, is proposed. ▪

Structural characterization of oligosaccharides isolated from the pectic polysaccharide rhamnogalacturonan II

Rhamnogalacturonan II (RG-II) is a structurally complex pectic ( d-galactosyl-uronic acid-rich) polysaccharide that is present in the primary (growing) cell-walls of higher plants. RG-II is composed of ∼60 glycosyl residues. The isolation and structural characterization of 23 oligosaccharide fragments of the residue of RG-II that remained after removal of hepta- and di-saccharides by partial hydrolysis with acid are reported. In order to obtain the oligosaccharide fragments characterized herein, the carboxyl groups of RG-II were dideuterio-reduced, and the carboxyl-reduced polysaccharide was per- O-methylated. The per- O-methylated polysaccharide was fragmented by partial hydrolysis with acid, producing partially O-methylated oligosaccharides. These derivatized oligosaccharides were reduced, to afford a mixture of partially O-methylated oligoglycosyl-alditols, which was then per- O-methylated. The structures of the resulting per- O-methylated oligoglycosylalditols were determined by chemical-ionization mass spectrometry, electron-impact mass spectrometry, fast-atom-bombardment mass spectrometry, 1H-n.m.r. spectroscopy, and analysis of corresponding, partially O-acetylated, partially O-methylated alditols. Seventeen of the oligosaccharides isolated from RG-II were parts of a single heptasaccharide, namely.

Structural studies of plant gum from sap of the lac tree, Rhus vernicifera

The plant gum isolated from sap of the lac tree, Rhus vernicifera (China), was separated into two fractions having mol. wt. 84,000 and 27,700 by aqueous-phase gel-permeation chromatography. The fractions contain d-galactose (65 mol%), 4- O-methyl- d-glucuronic acid (24 mol%), d-glucuronic acid (3 mol%), l-arabinose (4 mol%), and l-rhamnose (3 mol%). Smith degradation of the carboxyl-reduced polysaccharides gives products of halved molecular weight, and these consist of a β-(1→3)-linked galactopyranan main chain and side chains made up of galactopyranose residues. Peripheral groups, such as α- d-Gal p-, α- d-Gal p-(1→6)-β- d-Gal p-, 4- O-methyl-β- d-Glc pA-, and 4- O-methyl-β- d-Glc pA-(1→6)-β- d-Gal p-, are attached to this interior core through β-(1→3)- or β-(1→6)-linkages.

Binding of cryptococcal polysaccharide to Cryptococcus neoformans.

Radioiodinated cryptococcal polysaccharide was used to study binding of the soluble polysaccharide to encapsulated and non-encapsulated cryptoccoci. Binding of polysaccharide to non-encapsulated cryptococci occurred rapidly over a 30-min period and was largely complete after 2 h. Bound, labeled polysaccharide was slowly eluted from Cryptococcus neoformans after the addition of unlabeled polysaccharide, indicating reversibility of binding. Non-encapsulated cryptococci bound polysaccharide in two ways. Specific binding to the yeast was saturable by ca. 82 ng of polysaccharide per 10(6) yeast cells. Nonspecific binding also occurred which was not saturable under the conditions used in our experiments. Phagocytosis of the non-encapsulated yeast strain was inhibited when the specific binding was ca. 50% saturated. Binding of polysaccharide to an encapsulated strain showed nonspecific, nonsaturable binding, but little specific binding occurred. Presumably the specific binding sites were saturated in the encapsulated strain. Polysaccharides obtained from a hypocapsular mutant (A61) and a normally encapsulated strain competed effectively with labeled serotype D polysaccharide for binding sites on non-encapsulated cryptococci and had identical phagocytosis-inhibiting properties. Similarly, polysaccharides from all four cryptococcal serotypes competed effectively with labeled serotype D polysaccharide for binding sites on the non-encapsulated strain, and all four polysaccharides inhibited phagocytosis of non-encapsulated Cryptococcus neoformans. Unmodified, de-O-acetylated, carboxyl-reduced, periodate-oxidized and reduced (polyalcohol), and Smith-degraded polysaccharides competed with labeled polysaccharide for binding sites on the cell. The unmodified, de-O-acetylated and carboxyl-reduced polysaccharides inhibited phagocytosis of non-encapsulated cells, but the polyalcohol and Smith product were unable to inhibit phagocytosis.

Structure of the acidic extracellular gelling polysaccharide produced by Pseudomonas elodea

The gelling polysaccharide secreted by the bacterium Pseudomonas elodea contains l-rhamnose, d-glucose, and d-glucuronic acid in the molar ratios 1:2:1. Methylation analysis of native and carboxyl-reduced polysaccharide indicated (1→4)-Rha p, (1→3)-Glc p, (1→4)-Glc p, and (1→4)-Glc pA to be present in the ratios 1:1:1:1. Graded acid hydrolysis gave a series of acidic oligosaccharides that were isolated by ion-exchange chromatography and fractionated by gel-filtration. The purified oligosaccharides were analysed and characterised as their methylated alditol derivatives by e.i.-m.s. and c.i.-m.s., and also examined by fast-atom-bombardment (f.a.b.)-m.s. The tetrasaccharide repeating-unit 1, excluding acetyl groups, is proposed. ▪

The capsule of Cryptococcus neoformans passively inhibits phagocytosis of the yeast by macrophages

We examined the mechanism by which cryptococcal capsular polysaccharide inhibits phagocytosis of Cryptococcus neoformans by macrophages. O-Acetyl and carboxyl groups are major structural features of serotype D polysaccharide. Serotype D capsular polysaccharide or encapsulated whole cryptococci were de-O-acetylated by alkaline hydrolysis with 0.1 M NaOH. Carboxyl groups were reduced by treatment with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide followed by reduction with sodium borohydride. De-O-acetylated or carboxyl-reduced polysaccharides had phagocytosis-inhibiting properties that did not differ appreciably from the untreated polysaccharide; thus, neither the O-acetyl nor the carboxyl groups were essential phagocytosis-inhibiting determinants. Antiserum specific for these noninhibitory groups was obtained by adsorption of cryptococcal antiserum with de-O-acetylated or carboxyl-reduced whole cells to produce antiserum enriched respectively with antibody specific for the O-acetyl and carboxyl groups. These adsorbed antisera showed opsonic activity for the untreated yeast that was similar to the unadsorbed antiserum when these antisera were compared at identical levels of precipitating antibody. We also examined the ability of Fab' fragments of opsonic IgG to reduce the amount of anticapsular IgG needed to opsonize the yeast. No synergy was noted between Fab' fragments and undigested IgG in opsonization. These results are consistent with a passive mechanism for inhibition of phagocytosis. The capsule does not directly modulate phagocytic function, but instead, presents a surface that is not recognized by the phagocyte. This absence of recognition is corrected by opsonizing antibody specific for any surface determinant on the capsule.

Structural studies of an acidic polysaccharide from the mucin secreted by Drosera capensis

The polysaccharide of the mucin secreted by the leaves of Drosera capensis is composed of l-arabinose, d-xylose, d-galactose, d-mannose, and d-glucuronic acid in the molar ratio of 3.6:1.0:4.9:8.4:8.2. For structural elucidation, methylation analysis using g.l.c. and g.l.c.-m.s. was performed on the native, the carboxyl-reduced, and the degraded polysaccharides. Partial hydrolysis, periodate oxidation, chromium trioxide oxidation, and uronic acid degradation were also performed on the native and carboxyl-reduced polysaccharides. Partial hydrolysis of the native and carboxyl-reduced polysaccharides gave various oligosaccharides that were characterized and suggest a structure containing a d-glucurono- d-mannan backbone having a repeating unit → 4)-β- d-Glc pA-(1 → 2)-α- d-Man p-(1 →. l-Arabinose and d-xylose are present as nonreducing furanosyl and pyranosyl end-groups, respectively, both attached to O-3 of d-glucuronic acid residues of the backbone. d-Galactose is present as non-reducing pyranosyl end-group linked to O-3 of d-mannose residues.

The capsule of cryptococcus neoformans passively inhibits phagocytosis of the yeast by macrophages.

We examined the mechanism by which cryptococcal capsular polysaccharide inhibits phagocytosis of Cryptococcus neoformans by macrophages. O-Acetyl and carboxyl groups are major structural features of serotype D polysaccharide. Serotype D capsular polysaccharide or encapsulated whole cryptococci were de-O-acetylated by alkaline hydrolysis with 0.1 M NaOH. Carboxyl groups were reduced by treatment with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide followed by reduction with sodium borohydride. De-O-acetylated or carboxyl-reduced polysaccharides had phagocytosis-inhibiting properties that did not differ appreciably from the untreated polysaccharide; thus, neither the O-acetyl nor the carboxyl groups were essential phagocytosis-inhibiting determinants. Antiserum specific for these noninhibitory groups was obtained by adsorption of cryptococcal antiserum with de-O-acetylated or carboxyl-reduced whole cells to produce antiserum enriched respectively with antibody specific for the O-acetyl and carboxyl groups. These adsorbed antisera showed opsonic activity for the untreated yeast that was similar to the unadsorbed antiserum when these antisera were compared at identical levels of precipitating antibody. We also examined the ability of Fab' fragments of opsonic IgG to reduce the amount of anticapsular IgG needed to opsonize the yeast. No synergy was noted between Fab' fragments and undigested IgG in opsonization. These results are consistent with a passive mechanism for inhibition of phagocytosis. The capsule does not directly modulate phagocytic function, but instead, presents a surface that is not recognized by the phagocyte. This absence of recognition is corrected by opsonizing antibody specific for any surface determinant on the capsule.

Structure of the capsular polysaccharide of Klebsiella type 73 ( Enterobacter aerogenes)

The capsular polysaccharide from Klebsiella Type 73 ( Enterobacter aerogenes) was found to contain equimolar amounts of d-galactose, d-glucose, l-rhamnose, and d-glucuronic acid. Acid hydrolysis of the polysaccharide gave one aldobio- and one aldotrio-uronic acid, whose structures were established by acid hydrolysis and by methylation analysis. The anomeric configurations of the different sugar residues were determined from the specific rotations of the polysaccharide and the aldobio- and aldotrio-uronic acids, and also by oxidation of the native and the carboxyl-reduced polysaccharide with chromium trioxide. Methylation analysis of the polysaccharide provided information about the linkages of the different sugar residues. Based on all of these results, the structure assigned to the repeating unit of the polysaccharide is as follows. ▪

Structural studies of the methylated, acidic polysaccharide associated with coccoliths of Emiliania huxleyi (lohmann) kamptner

For the structure analysis of the methylated, acidic polysaccharide associated with the coccoliths of the alga Emiliania huxleyi (Lohmann) Kamptner, the native, the carboxyl-reduced, and the desulphated, carboxyl-reduced polysaccharides have been submitted to methylation analysis. Graded hydrolysis with acid, uronic acid degradation, and periodate oxidation/partial hydrolysis with acid in conjunction with methylation analysis were also applied. The results of the various degradation procedures have led to a proposed structure for the average unit of the polysaccharide. The mannan backbone consists of at least 80% of (1→3)-linked d-mannosyl residues and has many side-chains. The mannan backbone carries ester-bound sulphate groups, terminal d-ribosyl or l-arabinosyl groups, and short side-chains of two or three d-galactosyluronic acid residues. There is also a complex side-chain composed of d-xylose and l-rhamnose, wherein the branched rhamnosyl residues are substituted by 3- O-methyl- d-xylose, 2,3-di- O-methyl- l-rhamnose, a di-( d-galactosyl-uronic acid)- d-xylosyl unit, and an oligosaccharide. The oligosaccharide is composed of 2,3-di- O-methyl- l-rhamnose, 3- O-methyl- d-galacturonic acid, 6- O-methyl- l-mannose, d-galacturonic acid, l-mannose, and l-rhamnose.

The structure of the capsular polysaccharide (K5 antigen) of urinary-tract-infective Escherichia coli 010:K5:H4. A polymer similar to desulfo-heparin.

The capsular polysaccharide was isolated from Escherichia coli 010:K5:H4; it could not be obtained from a uncapsulated (K5-) mutant. It contains N-acetylglucosamine and glucuronic acid in a molar ratio of 1:1. Acid hydrolysis of the acidic polysaccharide as well as Smith degradation and degradation by deamination of the carboxyl-reduced polysaccharide suggested that the polysaccharide is composed of a disaccharide repeating unit. The data obtained by methylation analysis and nuclear magnetic resonance spectroscopy indicated that the repeating sequence of the capsular polysaccharide is the 4-beta-glucuronyl-1,4-alpha-N-acetylglucosaminyl unit. This structure is similar to that of desulfo-heparin.