Differences in the distribution of O-sulphate groups of cell-surface and secreted heparan sulphate produced by human neuroblastoma cells in culture

Confluent cultures of a human neuroblastoma cell line (CHP100) were incubated for 48 h with d-[1-³H]glucosamine and sodium [³⁵S]sulphate. Radioactive glycosaminoglycans were analysed in the growth medium, rapid trypsin digest of the cell monolayer and a 1% (w/v) Triton/0.5 M NaOH extract of the final cell pellet. Sulphated glycosaminoglycans co-chromatographed when eluted by NaCL gradient from DEAE-cellulose. The medium contained mainly chondroitin sulphates, whereas the cell surface was enriched in heparan sulphate. Heparan sulphate was isolated as chondroitinase ABC-resistant material and treated with nitrous acid. Analysis of the scission products on Bio-Gel P-10 yielded fragments varying in size from single disaccharides to glycans consisting of nine disaccharide units. Cell-surface and medium heparan sulphate had respectively 52% and 54% N-sulphated glucosamine residues distributed in similar patterns along the polymer chain. The N:O-sulphate ratio of neuroblastoma heparan sulphate was 1.1:1. Analysis by high-voltage electrophoresis of di- and tetrasaccharide products produced by nitrous acid treatment showed that the distribution of $\text{‘O’-}\text{sulphate}$ groups differed strikingly between heparan sulphates from the medium and cell-surface compartments. A di-O-sulphated tetrasaccharide was identified in both heparan sulphate species. The absence of detectable amounts of ³⁵[S]sulphate associated with fragments larger than tetrasaccharide supports the close topographical association of N-sulphate and O-sulphate groups.     

Effects of hyaluronate and heparan sulphate on collagen-fibronectin interactions

Interactions of fibronectin and glycosaminoglycans and the involvement of heparan sulphate and hyaluronate in fibronectin-collagen interactions have been studied by affinity chromatography. Partially periodate-oxidized glycosaminoglycans were coupled to adipic acid dihydrazide-substituted agarose. The elution of fibronectin was performed by using increasing concentrations of NaCl. Of the copolymeric glycosaminoglycans, heparin and self-associating heparan sulphates display the highest affinity towards fibronectin while hyaluronic acid and chondroitin 6-sulphate do not bind fibronectin. Competitive release experiments suggest the existence of common binding sites for copolymeric glycosaminoglycans on the fibronectin backbone. Heparan sulphate favours the formation of collagen-fibronectin complexes at low molarity, while hyaluronate is ineffective at low concentrations and prevents the formation of complexes when present at concentrations > 1 mg ml^?1. It is suggested that heparan sulphate promotes the formation of complexes which bind with fibronectin thus producing steric changes that increase the affinity for collagen, while hyaluronate prevents the binding of fibronectin to collagen by a steric exclusion mechanism.     

A rapid method for seperation and identification of several hexuronic acids and hexuronic acid-containing oligosaccharides.

Five naturally occurring hexuronic acids and several hexuronic acid-containing oligosaccharides were separated and identified by high voltage paper electrophoresis, using one of the following buffers. (i) Pyridine-acetic acid-water (1:10:89, by volume), the pH of which was adjusted to 2.3–3.5 with 98% formic acid. (ii) Pyridine-water (1:90, by volume), the pH of which was adjusted to 3.5–4.0 with glacial acetic acid. The best separation of the five hexuronic acids and heparin disaccharides was observed at pH 2.7 after electrophoresis for 180 min at 100 V/cm. At pH 3 l-gulosyluronic acid-l-guluronic acid could be easily isolated from an acid hydrolysate of alginate by the present method.     

Interactions of cartilage proteoglycans with hyaluronate. Inhibition of the interaction by modified oligomers of hyaluronate.

Oligomers of hyaluronic acid were prepared by digestion of hyaluronic acid from rooster combs with testicular hyaluronidase (hyaluronate 4-glycanohydrolase, EC 3.2.1.35), leech head hyaluronidase (hyaluronate 3-glycanohydrolase, EC 3.2.1.36), and with fungal hyaluronidase (hyaluronate lyase from Streptomyces hyalurolyticus). The oligomers were fractionated by gel permeation, using Sephadex G-50. Oligomers isolated after incubation of the hyaluronic acid with the testicular hyaluronidase were further modified. To prepare oligomers with N-acetylglucosamine at both ends, terminal nonreducing glucuronic acid residues were removed with beta-glucuronidase. Reducing terminal N-acetylglucosamine residues were removed by reaction under mildly alkaline conditions. The reducing terminal N-acetylglucosamine residues were also reduced with sodium borohydride to form N-acetylglucosaminitol. The potentials of the various oligosaccharides to bind to the proteoglycan from bovine nasal septum cartilage were estimated by determining their effectiveness as inhibitors of the proteoglycan-hyaluronate interaction. The present study shows that, to bind maximally to the proteoglycan, the hyaluronate oligosaccharide must be at least 10 sugar residues in length and be terminated at the nonreducing and reducing ends with a glucuronate residue and an N-acetylglucosamine residue, respectively. Sugar residues extended beyond this basic decasaccharide, do not interact with the hyaluronate binding site on the proteoglycan.     

A specific, sensitive method for the determination of hyaluronate.

A sensitive and specific assay for hyaluronate was devised. Hyaluronate contained in biological mixtures was digested with a commercially available microbial hyaluronate lyase. The β-Δ4,5-eneglucopyranuronic acid residues contained at the nonreducing termini of the resulting oligosaccharides were oxidized with periodic acid to yield, among other products, formyl pyruvic acid. The latter compound reacted with thiobarbituric acid to yield a chromophore with an absorption maximum at 549 nm. Optimal conditions for quantitative assay of hyaluronate are described.     

Automated hypoiodite oxidation of carbohydrates

An automated system is described for the hypoiodite oxidation of aldoses and substituted aldoses to the corresponding aldonic acids. Automated determination of the glyoxylic acid and formaldehyde obtained on oxidation with periodate enables the 3-O-, 4-O-, and 6-O-substituted aldonic acids to be distinguished. The method is applied to the analysis of oligosaccharides in column eluates.     

Structural characterisation of oligosaccharides obtained by Fenton-type radical depolymerisation of dermatan sulfate

The contamination of heparin in 2008 brought to the attention of health authorities an urgent need for structural characterisation of low molecular weight heparins and other glycosaminoglycans (GAGs) intended for clinical applications. Potentially harmful compounds can be introduced into these preparations as contaminants of the original material or as by-products of the depolymerisation process. Radical depolymerisation is one of the methods used for fractionations of GAGs. We report here on the results of the Fenton-type radical depolymerisation of dermatan sulfate (DS) by hydrogen peroxide in the presence of Cu²⁺ cations. A low molecular fraction of the reaction mixture was investigated by a combination of 2D ¹H,¹³C HSQC, 2D HSQC-TOCSY and 2D HMBC experiments at 800 MHz. The analysis of the spectra revealed the formation of oligosaccharides with structures corresponding to the native DS sequence and containing almost exclusively GalNAc4S as the reducing end monosaccharide. In addition, oligosaccharides containing a C-4 sulfated N-acetylgalactosaminic acid in place of the reducing end GalNAc4S were identified. This open chain monosaccharide represents a non-native DS structure.     

A gas chromatographic method for the determination of aldose and uronic Acid constituents of plant cell wall polysaccharides

A major problem in determining the composition of plant cell wall polysaccharides has been the lack of a suitable method for accurately determining the amounts of galacturonic and glucuronic acids in such polymers. A gas chromatographic method for aldose analysis has been extended to include uronic acids. Cell wall polysaccharides are depolymerized by acid hydrolysis followed by treatment with a mixture of fungal polysaccharide-degrading enzymes. The aldoses and uronic acids released by this treatment are then reduced with NaBH₄ to alditols and aldonic acids, respectively. The aldonic acids are separated from the alditols with Dowex-1 (acetate form) ion exchange resin, which binds the aldonic acids. The alditols, which do not bind, are washed from the resin and then acetylated with acetic anhydride to form the alditol acetate derivatives. The aldonic acids are eluted from the resin with HCl. After the resin has been removed, the HCl solution of the aldonic acids is evaporated to dryness, converting the aldonic acids to aldonolactones. The aldonolactones are reduced with NaBH₄ to the corresponding alditols, dried and acetylated. The resulting alditol acetate mixtures produced from the aldoses and those from the uronic acids are analyzed separately by gas chromatography. This technique has been used to determine the changes in composition of Red Kidney bean (Phaseolus vulgaris) hypocotyl cell walls during growth, and to compare the cell wall polysaccharide compositions of several parts of bean plants. Galacturonic acid is found to be a major component of all the cell wall polysaccharides examined.     

Autolysis of isolated cell walls of Bacillus licheniformis N.C.T.C. 6346 and Bacillus subtilis Marburg strain 168. Separation of the products and characterization of the mucopeptide fragments

1. Cell walls were isolated from Bacillus licheniformis N.C.T.C. 6346 and Bacillus subtilis Marburg strain 168 trp grown on casein hydrolysate into exponential phase. Autolysis was carried out and the soluble products, separated by chromatography on DEAE-cellulose, from the two wall preparations are broadly similar in composition and are in agreement with autolysis proceeding with hydrolysis of amide bonds between l-alanine and N-acetylmuramic acid residues in the mucopeptide components. 2. Peptides originating from the mucopeptide components were isolated and shown to be a monomer peptide, l-alanyl-d-glutamyl-meso-diaminopimelic acid and a dimer peptide containing two monomer peptides linked through a residue of d-alanine. Approximately one amide group is present for each equivalent tripeptide unit and is probably substituted on diaminopimelic acid residues. 3. Oligosaccharides originating from the mucopeptide components were isolated and after hydrolysis contained almost equimolar amounts of glucosamine and muramic acid and only very small amounts of amino acids. The number-average chain length, estimated by the release of non-reducing end groups of N-acetylglucosamine with exo-beta-N-acetylglucosaminidase, is approximately ten hexosamine residues for oligosaccharides isolated from either organism. The oligosaccharides are polydisperse. 4. N-Acetylglucosamine residues are the only reducing terminals detectable in the oligosaccharides isolated from B. subtilis or B. licheniformis cell-wall autolysates. The number-average chain lengths of the oligosaccharides were determined by estimation of the content of these residues and are higher than those found by enzymic assay. Possible reasons for the discrepancy are discussed.     

Oxidation of mannosyl oligosaccharides by hydroxyl radicals as assessed by electrospray mass spectrometry

The hydroxyl radicals are widely implicated in oxidation of carbohydrates during biological and industrial processes being responsible for their structural modifications and causing functional damage. The identification of intermediate oxidation products is hampered by a lack of reliable sensible methods for their detection. In this study, the oxidation of two models of galactomannans (Man₃ and GalMan₂) has been studied in reaction with hydroxyl radical generated by Fenton reaction. The oxidation patterns were assessed using preparative ligand-exchange/size-exclusion chromatography (LEX/SEC) coupled with tandem electrospray mass spectrometry (ESI-MS/MS). This allowed the identification of derived oligosaccharides (OS) containing hexuronic, hexonic, pentonic and erythronic acid residues and neutral OS bearing hydroperoxy, hydrated carbonyl moieties and residues from pyranosyl ring cleavage. The depolymerization products have been also detected upon oxidation of oligomers. This study allowed developing a simple, effective ‘fingerprinting’ protocol for detecting the damage done to mannans by oxidative radicals.     

Isolation and identification of glycosaminoglycans associated with purified nuclei from rat liver

Nuclei isolated from rat liver were purified extensively and then subjected to extraction of glycosaminoglycans by the conventional method with a slight modification including the treatments with amylase nucleases, (DNAase and RNAase), and sialidase in addition to the pronase treatment. The nuclear glycosaminoglycan fraction thus prepared was subjected to chromatography on Dowex 1-X2 (Cl^?) and electrophoresis before or after digestion with specific enzymes such as Streptomyces hyaluronidase, chondroitinase ABC and AC. These results together with the results of chemical analyses have revealed that the purified nuclei from rat liver contain glycosaminoglycans equivalent to 0.2–0.3 μg hexuronic acid per mg DNA. A major component of the nuclear glycosaminoglycans has been identified as hyaluronic acid, while a minor component as chondroitin sulfate A (or C). Preliminary investigations have shown that most of the nuclear glycosaminoglycans are associated with the chromatin fraction.     

Asparaginyl-N-acetylgalactosamine. Linkage unit of halobacterial glycosaminoglycan

The cell surface glycoprotein of Halobacteria contains two different types of sulfated saccharides: hexuronic acid-containing oligosaccharides linked to the protein via asparaginylglucose, and a serially repeated saccharide unit containing amino sugars that resembles the animal glycosaminoglycans. Here we report that 1) the sulfated repeating unit saccharide is linked to the cell surface glycoprotein via asparaginyl-N-acetylgalactosamine, 2) the amino acid sequence surrounding this linkage region is -Asn-Ala-Ser-, and thus in agreement with the acceptor sequence ASN-X-Thr(Ser) common to all eucaryotic N-glycosidically bound saccharides determined so far; 3) in addition to galactose, galacturonic acid, N-acetylglucosamine, and N-acetylgalactosamine, the methylated hexuronic acid 3-O-methylgalacturonic acid occurs as a stoichiometric constituent of the sulfated building block of the glycosaminoglycan chain.     

Oxidation of lactose with bromine

Oxidation of lactose by bromine in an aqueous buffered solution was conducted as a model experiment to examine the glycosidic linkage cleavage occurring during the oxidation of oligosaccharides and polysaccharides. The resulting oxidation products, after reduction with sodium borodeuteride, were characterized by GLC-MS analyses of the per-O-methyl or per-O-Me3Si derivatives. Most of the products were carboxylic acids, of which lactobionic acid was major. Minor products, identified after partial fractionation on a BioGel P-2 column, comprised oxalic acid; glyceric acid; threonic and erythronic acids; tartaric acid; lyxonic, arabinonic, and xylonic acids; galactonic and gluconic acids; galactosylerythronic acid; galactosylarabinonic acid; galactosylarabinaric acid; galacturonosylarabinonic acid; and galactosylglucaric acid. No keto acids were identified. Galactose was detected as 1-deuteriogalactitol, the presence of which, together with the C6 aldonic acids, supported a galactosidic bond cleavage. Galactosylarabinonic acid was the major constituent (7.5%) among minors, and others constituted 0.2-3.7% of the principal lactobionic acid. These products together comprised 29% of the lactobionic acid, more than half (17%) of which were accounted for by the galactosidic linkage cleavage, supporting the significant decrease in molecular weight seen earlier in the bromine-oxidized polysaccharides by glycosidic cleavage.     

Mode of action of glycoside hydrolase family 5 glucuronoxylan xylanohydrolase from Erwinia chrysanthemi

The mode of action of xylanase A from a phytopathogenic bacterium, Erwinia chrysanthemi, classified in glycoside hydrolase family 5, was investigated on xylooligosaccharides and polysaccharides using TLC, MALDI-TOF MS and enzyme treatment with exoglycosidases. The hydrolytic action of xylanase A was found to be absolutely dependent on the presence of 4-O-methyl-D-glucuronosyl (MeGlcA) side residues in both oligosaccharides and polysaccharides. Neutral linear beta-1,4-xylooligosaccharides and esterified aldouronic acids were resistant towards enzymatic action. Aldouronic acids of the structure MeGlcA(3)Xyl(3) (aldotetraouronic acid), MeGlcA(3)Xyl(4) (aldopentaouronic acid) and MeGlcA(3)Xyl(5) (aldohexaouronic acid) were cleaved with the enzyme to give xylose from the reducing end and products shorter by one xylopyranosyl residue: MeGlcA(2)Xyl(2), MeGlcA(2)Xyl(3) and MeGlcA(2)Xyl(4). As a rule, the enzyme attacked the second glycosidic linkage following the MeGlcA branch towards the reducing end. Depending on the distribution of MeGlcA residues on the glucuronoxylan main chain, the enzyme generated series of shorter and longer aldouronic acids of backbone polymerization degree 3-14, in which the MeGlcA is linked exclusively to the second xylopyranosyl residue from the reducing end. Upon incubation with beta-xylosidase, all acidic hydrolysis products of acidic oligosaccharides and hardwood glucuronoxylans were converted to aldotriouronic acid, MeGlcA(2)Xyl(2). In agreement with this mode of action, xylose and unsubstituted oligosaccharides were essentially absent in the hydrolysates. The E. chrysanthemi xylanase A thus appears to be an excellent biocatalyst for the production of large acidic oligosaccharides from glucuronoxylans as well as an invaluable tool for determination of the distribution of MeGlcA residues along the main chain of this major plant hemicellulose.     

Separate effects of exogenous hyaluronic acid on proteoglycan synthesis and deposition in pericellular matrix by cultured chick embryo limb chondrocytes

The effects of exogenous hyaluronic acid on cell cultures of chick embryo limb chondrocytes are reported in this paper. The evidence shows that exogenous hyaluronic acid (HA) can both depress the incorporation of ³⁵SO₄ into glycosaminoglycans and cause a displacement of newly synthesized proteoglycan from the cell layer to the culture medium. The results demonstrate that these two effects are mediated by distinct mechanisms. The displacement effect has a rapid onset (by 2 hr) while the effect of exogenous HA on ³⁵SO₄ incorporation has a long latency (12 hr). The displacement effect is produced by a lower concentration (5 μg/ml) of hyaluronate oligomers than the effect on ³⁵SO₄ incorporation (50 μg/ml). In addition, displacement is produced only by hyaluronate oligomers that are decasaccharides or larger. The depression of ³⁵SO₄ incorporation is produced by tetrasaccharides as well as high molecular weight HA. In fact tetrasaccharides can depress ³⁵SO₄ incorporation without causing the displacement effect.     

Exploration of the action pattern of Streptomyces hyaluronate lyase using high-resolution capillary electrophoresis

Hyaluronic acid was treated exhaustively with a hyaluronate lyase (hyaluronidase, EC 4.2.2.1) from Streptomyces hyalurolyticus to obtain a tetrasaccharide and a hexasaccharide product in a molar ratio of 1 to 1.2. The tetrasaccharide product was fluorescently labeled at the reducing end by reductive amination with 7-amino 1,3-naphthalene disulfonic acid (AGA) and the structure of the conjugate was determined spectroscopically. Partial treatments of hyaluronic acid with hyaluronate lyase afforded complex mixtures of oligosaccharides that were similarly fluorescently labeled. These labeled oligosaccharide mixtures were analyzed using high-resolution capillary electrophoresis. The resulting electropherograms showed the content of each hyaluronic acid derived oligosaccharide, having a degree of polymerization (dp) from 4 to 50, throughout the enzymatic reaction. Computer simulation studies gave comparable kinetic profiles suggesting that hyaluronate lyase exhibits a random endolytic action pattern. Interestingly, oligosaccharides of certain size (dp) were under-represented in these oligosaccharide mixtures suggesting that linkages at spacings of 10 to 12 saccharide units are somewhat resistant to this enzyme. The cause of this resistance might be the result of secondary or higher order structural features present in the hyaluronic acid polymer.     

The determination of aldonic acids and 2-C-methylaldonic acids

Specific, spectrophotometric methods are described for the determination of glyoxylic acid from aldonic acids and pyruvic acid from 2-C-methylaldonic acids, which allow their determination in admixture. Confirmation of the classification of these aldonic acids is obtained by ion-exchange chromatography of the products of periodate oxidation.     

Uncovering the Catalytic Direction of Chondroitin AC Exolyase: FROM THE REDUCING END TOWARDS THE NON-REDUCING END*

Glycosaminoglycans (GAGs) are polysaccharides that play vital functional roles in numerous biological processes, and compounds belonging to this class have been implicated in a wide variety of diseases. Chondroitin AC lyase (ChnAC) (EC 4.2.2.5) catalyzes the degradation of various GAGs, including chondroitin sulfate and hyaluronic acid, to give the corresponding disaccharides containing an Δ⁴-unsaturated uronic acid at their non-reducing terminus. ChnAC has been isolated from various bacteria and utilized as an enzymatic tool for study and evaluating the sequencing of GAGs. Despite its substrate specificity and the fact that its crystal structure has been determined to a high resolution, the direction in which ChnAC catalyzes the cleavage of oligosaccharides remain unclear. Herein, we have determined the structural cues of substrate depolymerization and the cleavage direction of ChnAC using model substrates and recombinant ChnAC protein. Several structurally defined oligosaccharides were synthesized using a chemoenzymatic approach and subsequently cleaved using ChnAC. The degradation products resulting from this process were determined by mass spectrometry. The results revealed that ChnAC cleaved the β1,4-glycosidic linkages between glucuronic acid and glucosamine units when these bonds were located on the reducing end of the oligosaccharide. In contrast, the presence of a GlcNAc-α-1,4-GlcA unit at the reducing end of the oligosaccharide prevented ChnAC from cleaving the GalNAc-β1,4-GlcA moiety located in the middle or at the non-reducing end of the chain. These interesting results therefore provide direct proof that ChnAC cleaves oligosaccharide substrates from their reducing end toward their non-reducing end. This conclusion will therefore enhance our collective understanding of the mode of action of ChnAC.     

Action of sweet-potato beta-amylase on phosphodextrin of potato starch

The specificity of sweet-potato beta-amylase in the vicinity of the phosphate ester groups was studied by determining the structures of the phosphorylated oligosaccharides (alpha-phosphodextrin and beta-limit-alpha-phosphodextrin) formed by its action on potato starch. The beta-limit-alpha-phosphodextrin was separated by chromatography on Dowex-1 (HCOO^?) resin into three fractions that were distinguishable by the d.p. and by the ratio of d-glucose 6-phosphate residues to total organic phosphate. Each fraction contained linear molecules having one phosphate ester group that was not located at the reducing or non-reducing terminals. The smallest phosphodextrin was 6²-phosphorylmaltotriose. It was deduced that beta-amylase hydrolysed (1→4)-α-d linkages from the non-reducing end until one or two d-glucosyl residues remained attached to the phosphorylated residue, depending on whether there was originally an odd or even number of glucosyl residues on the non-reducing side of the phosphorylated residue.     

Enzymatic and chemical methods for the generation of pure hyaluronan oligosaccharides with both odd and even numbers of monosaccharide units

Hyaluronan oligosaccharides display physiological activities not associated with the polymer and are widely used to characterize hyaluronan-binding proteins. They can also be used as biocompatible starting blocks for chemical derivatization. Here we present methods for generating milligram quantities of unusual odd- and even-numbered oligosaccharides, greatly increasing the diversity of reagents for use in such studies. These methods are based upon protocols from the 1960s, at which time it was very difficult to assess the stereochemical purity of the products. To address this, products were analyzed with modern high-field nuclear magnetic resonance spectroscopy. Alkaline beta-elimination conditions previously used to remove reducing-terminal N-acetylglucosamine residues in fact introduce a significant ( approximately 30%) level of stereoisomerism in the products by alkali-catalyzed keto-enol tautomerizations. Milder alkaline conditions were used to overcome this problem, reducing the contamination to <5%. The elimination by-products from this reaction were isolated and characterized, allowing the mechanism of alkaline degradation of hyaluronan to be investigated for the first time. beta-Glucuronidase was used to remove nonreducing-terminal glucuronic acid residues from oligosaccharides. Odd-numbered oligosaccharides with terminal glucuronic acid residues isolated from hyaluronidase digests are shown to originate from acid-catalyzed acetal hydrolysis during boiling denaturation and also have significant levels of stereochemical impurities.