Carboxyl Reduction Research Articles

Characterisation of secreted polysaccharides and (glyco)proteins from suspension cultures of Pyrus communis

High molecular weight material recovered from the culture filtrate of cell suspension cultured Pyrus communis was composed of 81% carbohydrate, 13% protein and 5% inorganic material. This material was separated into three fractions (one neutral (Fraction A) and two acidic (Fractions B and C)), by anion-exchange chromatography on DEAE-Sepharose CL-6B using a gradient of imidazole–HCl at pH 7.0. The monosaccharide and linkage composition of each fraction was determined after carboxyl reduction of uronic acid residues. From the combined results of the carbohydrate analyses, we conclude that the high molecular weight extracellular material consists of three major and two minor polysaccharides: a (fucogalacto)xyloglucan (36%) in the unbound neutral Fraction A; a type II arabinogalactan (as an arabinogalactan-protein, 29%) and an acidic (glucurono)arabinoxylan (2%) in Fraction B; and a galacturonan (33%) and a trace of heteromannan in Fraction C. The main amino acids in the proteins were Glx, Thr, Ser, Hyp/Pro and Gly. Further separation of Fraction B by solvent partition, SDS–PAGE and analysis by LC-MS/MS identified the major proteins as two chitanases, two thaumatin-like proteins, a β-1,3-glucanase, an extracellular dermal glycoprotein and a pathogenesis-related protein.

The binding of human betacellulin to heparin, heparan sulfate and related polysaccharides

Recombinant human betacellulin binds strongly to heparin, requiring of the order of 0.8 M NaCl for its elution from a heparin affinity matrix. This is in complete contrast to the prototypic member of its cytokine superfamily, epidermal growth factor, which fails to bind to the column at physiological pH and strength. We used a well-established heparin binding ELISA to demonstrate that fucoidan and a highly sulfated variant of heparan sulfate compete strongly for heparin binding. Low sulfated heparan sulfates and also chondroitin sulfates are weaker competitors. Moreover, although competitive activity is reduced by selective desulfation, residual binding to extensively desulfated heparin remains. Even carboxyl reduction followed by extensive desulfation does not completely remove activity. We further demonstrate that both hyaluronic acid and the E. coli capsular polysaccharide K5, both of which are unsulfated polysaccharides with unbranched chains of alternating N-acetylglucosamine linked beta(1-4) to glucuronic acid, are also capable of a limited degree of competition with heparin. Heparin protects betacellulin from proteolysis by LysC, but K5 polysaccharide does not. Betacellulin possesses a prominent cluster of basic residues, which is likely to constitute a binding site for sulfated polysaccharides, but the binding of nonsulfated polysaccharides may take place at a different site.

Semi-synthetic heparin derivatives: chemical modifications of heparin beyond chain length, sulfate substitution pattern and N-sulfo/ N-acetyl groups

The glycosaminoglycan heparin is a polyanionic polysaccharide most recognized for its anticoagulant activity. Heparin binds to cationic regions in hundreds of prokaryotic and eukaryotic proteins, termed heparin-binding proteins. The endogenous ligand for many of these heparin-binding proteins is a structurally similar glycosaminoglycan, heparan sulfate (HS). Chemical and biosynthetic modifications of heparin and HS have been employed to discern specific sequences and charge-substitution patterns required for these polysaccharides to bind specific proteins, with the goal of understanding structural requirements for protein binding well enough to elucidate the function of the saccharide–protein interactions and/or to develop new or improved heparin-based pharmaceuticals. The most common modifications to heparin structure have been alteration of sulfate substitution patterns, carboxyl reduction, replacement N-sulfo groups with N-acetyl groups, and chain fragmentation. However, an accumulation of reports over the past 50 years describe semi-synthetic heparin derivatives obtained by incorporating aliphatic, aryl, and heteroaryl moieties into the heparin structure. A primary goal in many of these reports has been to identify heparin-derived structures as new or improved heparin-based therapeutics. Presented here is a perspective on the introduction of non-anionic structural motifs into heparin structure, with a focus on such modifications as a strategy to generate novel reduced-charge heparin-based bind-and-block antagonists of HS–protein interactions. The chemical methods employed to synthesize such derivatives, as well as other unique heparin conjugates, are reviewed.

Structural investigation of a fucoidan containing a fucose-free core from the brown seaweed, Hizikia fusiforme

A fucoidan, obtained from the hot-water extract of the brown seaweed, Hizikia fusiforme, was separated into five fractions by DEAE Sepharose CL-6B and Sepharose CL-6B column chromatography. All five fractions contained predominantly fucose, mannose and galactose and also contained sulfate groups and uronic acid. The fucoidans had MWs from 25 to 950 kDa. The structure of fraction F32 was investigated by desulfation, carboxyl-group reduction, partial hydrolysis, methylation analysis and NMR spectroscopy. The results showed that the sugar composition of F32 was mainly fucose, galactose, mannose, xylose and glucuronic acid; sulfate was 21.8%, and the MW was 92.7 kDa. The core of F32 was mainly composed of alternating units of →2)-α- d-Man(1→ and →4)-β- d-GlcA(1→, with a minor portion of →4)-β- d-Gal(1→ units. The branch points were at C-3 of →2)-Man-(1→, C-2 of →4)-Gal-(1→ and C-2 of →6)-Gal-(1→. About two-thirds of the fucose units were at the nonreducing ends, and the remainder were (1→4)-, (1→3)- and (1→2)-linked. About two-thirds of xylose units were at the nonreducing ends, and the remainder were (1→4)-linked. Most of the mannose units were (1→2)-linked, and two-thirds of them had a branch at C-3. Galactose was mainly (1→6)-linked. The absolute configurations of the sugar residues were α- d-Man p, α- l-Fuc p, α- d-Xyl p, β- d-Gal p and β- d-Glc pA. Sulfate groups in F32 were at C-6 of →2,3)-Man-(1→, C-4 and C-6 of →2)-Man-(1→, C-3 of →6)-Gal-(1→, C-2, C-3 or C-4 of fucose, while some fucose had two sulfate groups. There were no sulfate groups in either the GlcA or xylose residues.

Structure and serological characteristics of the capsular K4 antigen of Escherichia coli O5:K4:H4, a fructose-containing polysaccharide with a chondroitin backbone

The chemical structure of the K4-specific capsular polysaccharide (K4 antigen) of Escherichia coli O5:K4:H4 was elucidated by composition, carboxyl reduction periodate oxidation methylation nuclear-magnetic-resonance spectroscopy and enzymatic cleavage. The polysaccharide consists of a backbone with the structure → 3)-β-D-glucuronyl-(1,4)-β-D-N-acetylgalactosaminyl(1- to which β-fructofuranose is linked at C-3 of glucuronic acid. Mild acid hydrolysis liberated fructose and converted the K4 antigen into a polysaccharide which has the same structure as chondroitin. The defructosylated polysaccharide was a substrate for hyaluronidase and chondroitinase. The serological reactivity of the K4 polysaccharide was markedly reduced after defructosylation.

Structural characterization, degree of esterification and some gelling properties of Krueo Ma Noy ( Cissampelos pareira) pectin

Pectins extracted from Krueo Ma Noy ( Cissampelos pareira) leaves mainly consisted of galacturonic acid with trace amount of neutral sugars. The dominant structure of Krueo Ma Noy pectin was established as a 1,4-linked α- d-galacturonan by a combination of carboxyl reduction and methylation analysis, and confirmed by FT-IR spectroscopy. The degree of esterification of Krueo Ma Noy pectins was 41.7 and 33.7% for crude and dialyzed pectins, respectively. Krueo Ma Noy pectin has an average molecular weight of 55 kDa, radius of gyration of 15.2 nm and intrinsic viscosity of 2.3 dl/g. Krueo Ma Noy pectin exhibited gelling properties in aqueous solutions at 0.5% (w/v) at 5 °C. Gels were formed at concentrations of 1.0% (w/v) and above even at room temperature. The gel strength, melting point, and melting enthalpy of Krueo Ma Noy pectin increased with polysaccharide concentration.

Structure of the O-polysaccharide of Proteus mirabilis O38 containing 2-acetamidoethyl phosphate and N-linked d-aspartic acid

The O-antigen of Proteus mirabilis O38 was found to be unique among bacterial polysaccharides and to have the following structure: where d-Qui4N(Ac- d-Asp) is 4-( N-acetyl- d-aspart-4-ylamino)-4,6-dideoxy- d-glucose and AcEtn P is 2-acetamidoethyl phosphate. Neither of these entities have been hitherto found in natural polysaccharides. Structural studies were performed using 1D and 2D NMR spectroscopy, including experiments run in an H 2O/D 2O mixture to reveal correlations for NH protons. In addition, dephosphorylation, carboxyl reduction and selective cleavages were applied. Solvolysis of the polysaccharide with anhydrous HF gave an α- d-GlcNAc-(1→3)- d-Qui4N(Ac- d-Asp) disaccharide. Solvolysis with trifluoromethanesulfonic (triflic) acid afforded d-GlcNAc6(AcEtn P), thus showing the suitability of this reagent for the preparation of phosphorylated sugar derivatives.

Structural and rheological properties of polysaccharides from mango ( Mangifera indica L.) pulp

The structure and rheological properties of water-soluble polysaccharides from industrialized mango pulp were investigated. Soluble fraction (SF) 2 was heterogeneous on high performance size exclusion chromatography, giving two peaks as determined by multi-angle laser light scattering and refractive index detectors. The presence of starch in SF2 was demonstrated by a positive iodine reaction and by 13C nuclear magnetic resonance (NMR) spectroscopy. The presence of pectic polysaccharides was shown by a calorimetric method, 13C-NMR spectroscopy and carboxyl reduction. The main pectic polysaccharide was polygalacturonic acid; type I rhamnogalacturonan was also detected. Analysis of the rheological properties of SF2 showed a pseudoplastic behavior up to 3 g l −1. ‘Creep and recovery’ tests and analysis performed under a dynamic state revealed a weak gel character for solutions at concentrations of 15, 20 and 30 g l −1.

Structure of the O-specific polysaccharide of Vibrio cholerae O9 containing 2-acetamido-2-deoxy- d-galacturonic acid

The O-specific polysaccharide (OPS) was isolated by mild-acid degradation of the lipopolysaccharide of Vibrio cholerae O9 and studied by carboxyl reduction, sugar and methylation analyses, Smith degradation, and two-dimensional NMR spectroscopy, including COSY, TOCSY, NOESY, and H-detected 1H, 13C HMQC experiments. The following structure of the pentasaccharide-repeating unit of the OPS was established:

An immunologically active arabinogalactan from Tinospora cordifolia

An arabinogalactan of mean M r 2.2×10 6 has been isolated from the dried stems of Tinospora cordifolia and examined by methylation analysis, partial hydrolysis and carboxyl reduction. Purified polysaccharide showed polyclonal mitogenic activity against B-cells, their proliferation did not require macrophages.

Structural elucidation of a novel exopolysaccharide produced by a mucoid clinical isolate of Burkholderia cepacia. Characterization of a trisubstituted glucuronic acid residue in a heptasaccharide repeating unit.

The structure of the exopolysaccharide (EPS) produced by a clinical isolate of Burkholderia cepacia isolated from a patient with fibrocystic lung disease has been investigated. By means of methylation analyses, carboxyl reduction, partial depolymerization by fuming HCl and chemical degradations such as Smith degradation, lithiumethylenediamine degradation and beta-elimination, supported by GC/MS and NMR spectroscopic analyses, the repeat unit of the EPS has been identified and was shown to correspond to the acidic branched heptasaccharide with the following structure: [formula: see text]. This partially acetylated acidic polymer, distinguished by the presence of the less usual D-isomer of rhamnose and of a trisubstituted glucuronic acid residue, could represent the main EPS produced by this bacterial species.

Human Colonic Bacterial Degradability of Dietary Fibres from Sea-Lettuce (Ulva sp)

Sea-lettuce (Ulva sp) is one of the commonly consumed seaweeds which contains 16·5% of water-soluble and 13·3% insoluble dietary fibres. Since physiological effects of fibres are partly related to their colonic bacteria fermentability, Ulva sp and its constitutive soluble and insoluble fibres were incubated with faecal bacteria in an in vitro batch fermenter system. After 24 h of incubation, 32·0±0·4%, 25·9±0·4% and 50·9±7·4% of Ulva, soluble and insoluble fibres constitutive sugars, respectively, were degraded. Consequently, Ulva and its soluble fibre, ulvan, are poorly fermented by colonic bacteria. The constitutive sugars, rhamnose and glucuronate and the aldobiouronate β-D-glucuronosyluronate-(1,4)-L-rhamnose of the glucuronoxylorhamnan sulphate present in the soluble fibre are highly fermented. Chemical desulphation and/or carboxyl group reduction did not modify this fermentation behaviour. Thus, the particular chemical structure of ulvan is responsible for the resistance of this polysaccharide and of Ulva to colonic bacterial fermentation. As a physiological consequence of this particular behaviour, consumption of dietary fibres from sea-lettuce could be expected to act mainly as bulking agents with little effect on nutrient metabolism due to colonic bacterial fermentation products (short-chain fatty acids). © 1997 SCI.

Structure of fucose branches in the glycosaminoglycan from the body wall of the sea cucumber Stichopus japonicus

Fucose-branched chondroitin sulfate E was prepared from the body wall of sea cucumber Stichopus japonicus. The purified glycosaminoglycan (GAG) was chemically desulfated, followed by carboxyl reduction. Intact, desulfated, and desulfated/carboxyl-reduced GAG fractions were subjected to per- O-methylation. GC-MS analyses of the resultant partially methylated alditol acetates demonstrated that the fucose branch is formed by two fucopyranosyl residues linked glycosidically through position (1 → 3), and that the fucose branch and glucuronic acid are almost equimolar. In addition, it was elucidated that about 20% of the branches stretch from O-3 position of a glucuronic acid moiety of the core chondroitin sulfate polymer, while remaining fucose branches are postulated to protrude from O-4 and/or O-6 position(s) of a N-acetylgalactosamine moiety. This fucose branch was also confirmed to be highly sulfated according to six kinds of substitution pattern in methylation analysis.

Structure of the O-specific polysaccharide of Hafnia alvei PCM 1222 containing 2-aminoethyl phosphate

The O-specific polysaccharide of H. alvei strain PCM 1222 has a branched hexasaccharide repeating unit containing d-galactose, l-rhamnose, d-ribose, d-galacturonic acid, and 2-acetamido-2-deoxy- d-glucose in the ratios 1:2:1:1:1, as well as 2-aminoethyl phosphate (EtNP) and O-acetyl groups in nonstoichiometric amounts. The polysaccharide was modified by carboxyl reduction, O-deacetylation, and dephosphorylation with 48% hydrofluoric acid, the last reaction being accompanied by removal of the lateral residue of β-galactofuranose. The modified polysaccharides were studied by methylation analysis and 1H and 13C NMR spectroscopy, including 2D correlation spectroscopy (COSY), H-detected 1H, 13C and 1H, 31P heteronuclear multiple-quantum coherence (HMQC), 1D NOE, 2D rotating-frame NOE spectroscopy (ROESY), and 2D combined total correlation spectroscopy (TOCSY) and ROESY (TORO). The following structure of the O-deacetylated polysaccharide was established: ▪ In different batches of the polysaccharide, the content of EtNP varied from 0.35 to 0.55 and that of the O-acetyl groups from 0.05 to 0.4 per repeating unit. It was tentatively suggested that the O-acetyl group is located at position 4 of a rhamnosyl residue.

High affinity of a fucosylated chondroitin sulfate for plasma low density lipoprotein

Factors that influence the binding of sulfated polysaccharides to plasma low density lipoprotein (LDL) were investigated. Among the naturally occurring polysaccharides tested, a fucosylated chondroitin sulfate from an echinoderm exhibited the strongest interaction with LDL. Defucosylation and desulfation totally abolished the interaction with LDL while reduction of carboxyl groups had little effect. These data indicate that the sulfated fucose branches are essential for binding of fucosylated chondroitin sulfate to LDL. In addition, there was a positive correlation between the binding to LDL and increasing length of the sulfated polysaccharide chains. The possibility of a practical use of this fucosylated chondroitin sulfate for the binding of LDL is discussed.

Structural study and serological characterisation of the O-specific polysaccharide of Hafnia alvei PCM 1185, another Hafnia O-antigen that contains [formula omitted

The O-specific polysaccharide of H. alvei strain PCM 1185 contains d-glucose, d-glucuronic acid, 2-acetamido-2-deoxy- d-glucose, and 3,6- dideoxy-3-[(R)-3- hydroxybutyramido]- d-glucose (Qui3NAcyl) in the ratios 2:1:1:1, as well as O-acetyl groups. On the basis of sugar and methylation analyses of the polysaccharide before and after chemical modifications ( O-deacetylation, carboxyl reduction, Smith degradation), as well as 1H and 13C NMR spectroscopy, including 1D sequential, selective spin-decoupling, 2D homonuclear, and 13C, 1H heteronuclear correlation spectroscopy (COSY), and 2D rotating-frame NOE spectroscopy, it was found that the polysaccharide has a pentasaccharide repeating unit with the following structure: ▪ with O-acetyl groups present in nonstoichiometric amounts, mainly at position 2 of GlcA and position 6 of GlcNAc or lateral Glc. Serological study showed that H. alvei strain PCM 1185 can be placed in a new serotype D and that an O-acetyl group can be a part of its epitope.

Differences in the susceptibility of herpes simplex virus types 1 and 2 to modified heparin compounds suggest serotype differences in viral entry

Although heparan sulfate (HS) serves as an initial receptor for the binding of both herpes simplex virus type 1 (HSV-1) and HSV-2 to cell surfaces, the two serotypes differ in epidemiology, cell tropism, and ability to compete for viral receptors in vitro. These observations are not necessarily contradictory and can be explained if the two serotypes recognize different structural features of HS. To compare the specific features of HS important for the binding and infection of HSV-1 and HSV-2, we took advantage of structural similarities between heparin and cell surface HS and compared the abilities of chemically modified heparin compounds to inhibit plaque formation. We found that the antiviral activity of heparin for both serotypes was independent of anticoagulant activity. Moreover, specific negatively charged regions of the polysaccharide, including N sulfations and the carboxyl groups, are key structural features for interactions of both HSV-1 and HSV-2 with cell surfaces since N desulfation or carboxyl reduction abolished heparin's antiviral activity. In contrast, 6-O sulfations and 2-,3-O sulfations are important determinants primarily for HSV- 1 infection. The O-desulfated heparins had little or no inhibitory effect on HSV-1 infection but inhibited HSV-2 infection. Using a series of intertypic recombinant mutant viruses, we found that susceptibility to O-desulfated heparins can be transferred to HSV-1 by the gene for glycoprotein C of HSV-2 (gC-2). This supports the notion that the envelope glycoproteins of HSV-1 and HSV-2 interact with different affinities for different structural features of heparin. To determine if the modified heparin compounds inhibited plaque formation by competing with cell surface HS for viral attachment, binding studies were also performed. As anticipated, most compounds inhibited binding and plaque formation in parallel. However, several compounds inhibited the binding of HSV-1 to cells during the initial attachment period at 4 degrees C; this inhibitory effect was reversed when the cells and inoculum were shifted to 37 degrees C. This temperature-dependent differential response to modified heparin compounds was evident primarily when glycoprotein C of HSV-1 (gC-1) was present in the virion envelope. Minimal temperature-dependent differences were seen for HSV-1 with gC-1 deleted and for HSV-2. These results suggest differences in the interactions of HSV-1 and HSV-2 with cell surface HS that may influence cell tropism.

Structure of the O-specific polysaccharide of Acinetobacter baumannii O5 containing 2-acetamido-2-deoxy-D-galacturonic acid.

A polysaccharide containing 2-acetamido-2-deoxy-D-glucose (GlcNAc), 2-acetamido-2-deoxy-L-fucose (FucNAc), and 2-acetamido-2-deoxy-D-galacturonic acid (GalNAcA) was isolated from an aqueous phenol extract of lipid-free, isolated cell walls of the reference strain for Acinetobacter baumannii serogroup O5, by mild acid hydrolysis of the extract and chromatography of the water-soluble products on Sephadex G-50. By means of NMR studies, methylation analysis, carboxyl reduction and chemical degradations, the repeating unit of the polymer was identified as a branched tetrasaccharide of the structure shown. The serologically active polymer is believed to correspond to the side chain of the O5 lipopolysaccharide: [table: see text]

Characterization of the lipopolysaccharides from Rhizobium meliloti strain 102F51 and its nonnodulating mutant WL113.

Lipopolysaccharides from the Rhizobium meliloti wild-type strain 102F51, which is effective in symbiosis with alfalfa, and from the nonnodulating mutant WL113, defective in root hair adhesion, derived thereof, were isolated and comparatively analyzed. Both preparations were composed of galactose, glucose, glucuronic acid, galacturonic acid, glucosamine, 3-deoxyheptulosaric acid, and 2-keto-3-deoxyoctonic acid as the major sugar constitutents. After a modified methylation analysis (consisting of the following consecutive steps: methylation, carboxyl reduction, remethylation, mild acid hydrolysis, reduction, and trideuterio-methylation), all of the 3-deoxyheptulosaric and some of the 2-keto-3-deoxyoctonic acid residues were converted into their corresponding 3-deoxyalditol derivatives, which carried trideuteriomethyl groups at positions C-2, C-4, and C-6. Another part of the permethylated 3-deoxyoctitol was also found as 2,5,6- and 2,6,8-tri-O-trideuteriomethyl derivatives. NMR data obtained with the separated oligosaccharides and the results of methylation analysis indicated that the majority of 2-keto-3-deoxyoctonate was present in the fraction of permethylated disaccharide alditols, namely as 6-O-CD3-alpha Glc(1-->5)3-deoxyoctitol, 6-O-CD3-beta GlcNMeAcyl(1-->4)-3-deoxyoctitol, and as the permethylated trisaccharide alditol, alpha GalA(1-->3)-[6-O-CD3]-beta-Glc(1-->5)-[4-O-CD3]-3-deoxyoctitol. The presence of trideuteriomethyl groups at C-4 of both 3-deoxyalditols and at C-6 of the glucosaminyl or glycosyl residues indicated the linkage points of the released acid-labile ketosidic substituents, such as 3-deoxyheptulosarate and 2-keto-3-deoxyoctonate, in these oligosaccharides. The main differences between the preparations from the wild-type 102F51 and its mutant strain WL 113 were found in the higher content (in strain 102F51) of the following oligosaccharides: alpha-glucuronosyl(1-->4)2-keto-3-deoxyoctonate and alpha-galacturonosyl-(1-->3)alpha-glucosyl-(1-->5)2-keto-3-deoxyoc tonate and in the decreased content of beta-glucosaminyl(1-->4)2-keto-3-deoxy-octonate.

Structure of the O-specific polysaccharide chain of Shigella boydii type 5 lipopolysaccharide: a repeated study

An acidic, partially O-acetylated O-specific polysaccharide was obtained by mild acid degradation of Shigella boydii type 5 lipopolysaccharide and studied by 1H and 13C NMR spectroscopy, including 2D COSY, 13C- 1H heteronuclear COSY, 1D NOE, and 2D ROESY experiments, and chemical methods (sugar and methylation analysis, O-deacetylation, carboxyl reduction, solvolysis with anhydrous HF, partial acid hydrolysis, Smith degradation). It was concluded that the polysaccharide has a hexasaccharide repeating unit of the following structure: ▪ with the degree of O-acetylation varying over 30–50%. The established structure differs from that proposed recently for the O-specific polysaccharide of the same S. boydii serotype [M.J. Albert et al., Carbohydr. Res., 265 (1994) 121–127].