Chondroitin Sulfate Oligosaccharides Research Articles

A fingerprinting method for chondroitin/dermatan sulfate and hyaluronan oligosaccharides

A previously published method for the analysis of glycosaminoglycan disaccharides by high pH anion exchange chromatography (Midura,R.J., Salustri,A., Calabro,A., Yanagishita,M. and Hascall,V.C. (1994), Glycobiology,4, 333-342) has been modified and calibrated for chondroitin and dermatan sulfate oligosaccharides up to hexasaccharide in size and hyaluronan oligosaccharides up to hexadecasaccharide. For hyaluronan oligosaccharides chain length controls elution position; however, for chondroitin and dermatan sulfate oligosaccharides elution times primarily depend upon the level of sulfation, although chain length and hence charge density plays a role. The sulfation position of GalNAc residues within an oligosaccharide is also important in determining its elution position. Compared to 4-sulfation a reducing terminal 6-sulfate retards elution; however, when present on an internal GalNAc residue it is the 4-sulfate containing oligosaccharide which elutes later. These effects allow discrimination between oligosaccharides differing only in the position of GalNAc sulfation. Using this simple methodology, a Dionex CarboPac PA-1 column with NaOH/NaCl eluents and detection by absorbance at 232 nm, a quantitative analytical fingerprint of a chondroitin/dermatan sulfate chain may be obtained, allowing a determination of the abundance of chondroitin sulfate, dermatan sulfate, and hyaluronan along with an analysis of structural features with a linear response to approximately 0.1 nmol. The method may readily be calibrated using either commercial disaccharides or the di- and tetrasaccharide products of a limit digest of commercial chondroitin sulfate by chondroitin ABC endolyase. Commercially available and freshly prepared shark, whale, bovine, and human cartilage chondroitin sulfates have been examined by this methodology and we have confirmed that freshly isolated shark cartilage CS contains significant amounts of the biologically important GlcA2Sbeta(1-3)GalNAc6S structure.

Preliminary investigation into the purification, NMR analysis, and molecular modelling of chondroitin sulphate epitopes

Electrophoretic methods are outlined for the rapid purification and analysis of chondroitin sulphate oligosaccharides on a milligram scale. Isomeric impurities however, exist within specific sized oligosaccharides. Detailed 1H and 13C NMR data for the chondroitin sulphate disaccharides Δ 4HexA(1-3)GalNAc6SO − 3 and Δ 4HexA(1–3)GalNAc4SO − 3 (prepared from shark chondroitin sulphate) are reported. Two-dimensional NMR methods have been employed in the assignment of spectra. Preliminary models of these disaccharides are proposed from molecular mechanics conformational searching and molecular dynamics procedures. This study provides 1H and 13C NMR reference data which will be useful in the investigation of larger chondroitin sulphate oligosaccharides, such as those prepared by the electrophoretic methods mentioned above.

A non-reducing terminal fragment from tracheal cartilage keratan sulphate chains contains α(2-3)-linked N-acetylneuraminic acid

Keratan sulphate chains were isolated from bovine tracheal ring cartilage (15-18-month-old animals) after papain digestion of the tissue followed by ethanol fractionation, chondroitinase ABC digestion and alkaline borohydride reduction. The keratan sulphate chains were further purified by anion-exchange chromatography on a Pharmacia Mono-Q column in order to remove any contaminating chondroitin sulphate and O-linked oligosaccharides. The chains were then treated with keratanase and the digest was subjected to alkaline borohydride reduction, producing oligosaccharides with galactitol at their reducing ends. The reduced digest was chromatographed on a Nucleosil 5 SB anion-exchange column and individual oligosaccharides were isolated. One of these, oligosaccharide (I), was shown by 500 MHz 1H-n.m.r. spectroscopy to have the following structure: NeuAc alpha 2-3Gal beta 1-4GlcNAc(6SO4) beta 1-3Gal-ol (I) The structure of this oligosaccharide shows that keratan sulphate chains from bovine tracheal ring cartilage may be terminated with N-acetylneuraminic acid linked alpha (2-3) to an unsulphated galactose. Keratan sulphate chains were also isolated from bovine femoral head cartilage (15-18-month-old animals) using an identical protocol, but with keratanase which was subsequently shown to have sialidase activity. This yielded oligosaccharide (II), the unsialyated version of (I): Gal beta 1-4GlcNAc(6SO4) beta 1-3Gal-ol (II).

Proteoglycan biosynthesis in chondrocytes: protein A-gold localization of proteoglycan protein core and chondroitin sulfate within Golgi subcompartments.

The intracellular pathway of cartilage proteoglycan biosynthesis was investigated in isolated chondrocytes using a protein A-gold electron microscopy immunolocalization procedure. Proteoglycans contain a protein core to which chondroitin sulfate and keratan sulfate chains and oligosaccharides are added in posttranslational processing. Specific antibodies have been used in this study to determine separately the distribution of the protein core and chondroitin sulfate components. In normal chondrocytes, proteoglycan protein core was readily localized only in smooth-membraned vesicles which co-labeled with ricin, indicating them to be galactose-rich medial/trans-Golgi cisternae, whereas there was only a low level of labeling in the rough endoplasmic reticulum. Chondroitin sulfate was also localized in medial/trans-Golgi cisternae of control chondrocytes but was not detected in other cellular compartments. In cells treated with monensin (up to 1.0 microM), which strongly inhibits proteoglycan secretion (Burditt, L.J., A. Ratcliffe, P. R. Fryer, and T. Hardingham, 1985, Biochim. Biophys. Acta., 844:247-255), there was greatly increased intracellular localization of proteoglycan protein core in both ricin-positive vesicles, and in ricin-negative vesicles (derived from cis-Golgi stacks) and in the distended rough endoplasmic reticulum. Chondroitin sulfate also increased in abundance after monensin treatment, but continued to be localized only in ricin-positive vesicles. The results suggested that the synthesis of chondroitin sulfate on proteoglycan only occurs in medial/trans-Golgi cisternae as a late event in proteoglycan biosynthesis. This also suggests that glycosaminoglycan synthesis on proteoglycans takes place in a compartment in common with events in the biosynthesis of both O-linked and N-linked oligosaccharides on other secretory glycoproteins.

Influence of Constituents of Proteoglycans on Type II Collagen Fibrillogenesis

Turbimetry was used to study the influence of glycosaminoglycans of cartilage proteoglycans on type II collagen fibrillogenesis. The monosaccharides, D-glucuronate, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine and D-galactose, all decreased the rate of the fibril formation. D-glucuronate had the strongest effect. The influence of chondroitin sulphate on type II collagen fibrillogenesis depended on the pH of the final solution, the length of the chondroitin sulphate chains and the concentration of chondroitin sulphate. At pH = 7.3 all chondroitin sulphate preparations decreased the rate of fibrillogenesis, while at pH = 6.9 and lower fibrillogenesis was stimulated by chondroitin sulphate (whale/shark), chondroitin 4-sulphate (whale) and chondroitin 6-sulphate (shark). The chain length of these three appeared to be longer than the chain length of chondroitin sulphate (human) and chondroitin sulphate oligosaccharides (whale/shark). At high concentrations (more than 3 mg/ml) fibril formation was less strongly retarded by keratan sulphate (human) than by chondroitin sulphate. At low concentrations a slight stimulation was observed in the presence of keratan sulphate. Glycosaminoglycans did not bind to collagen fibrils. At 0.5 mg/ml chondroitin 4-sulphate had a large solubilizing effect on fibrils compared to chondroitin 6-sulphate. Fibrillogenesis of type II collagen is in many aspects not comparable with fibrillogenesis of type I collagen.

Rabbit antibodies to degraded and intact glycosaminoglycans which are naturally occurring and present in arthritic rabbits.

Using enzyme-linked immunosorbent assays and radioimmunoassays employing chondroitinase ABC-treated rabbit cartilage proteoglycan, we have shown that approximately one-third of the outbred New Zealand white rabbits we have examined possess naturally occurring antibodies which react with oligosaccharides of hyaluronic acid (independently of chain length) bearing saturated and 4,5-unsaturated glucuronosyl residues at the nonreducing ends. Such antibodies were also found in a similar proportion of rabbits with an experimental inflammatory arthritis. There was a preferential reactions in the majority of sera with unsaturated oligosaccharides of hyaluronic acid. One serum (R64) reacted only with unsaturated oligosaccharides of hyaluronic acid. Sera reacted also with unsaturated (never saturated) oligosaccharides of chondroitin 4-sulfate and with chondroitin 6-sulfate, particularly when chondroitin sulfate oligosaccharides remained bound to a proteoglycan core protein. Reactions were also observed to both unsaturated and saturated oligosaccharides of chondroitin. Some of these sera also reacted with intact hyaluronic acid and chondroitin but never with intact chondroitin sulfate. The antibodies were present in the IgG fraction of four sera studied and in the IgM fraction of one of these sera: they bound through the F(ab')2 region of the molecule. These observations suggest that, in some rabbits, humoral immunity to hyaluronic acid and/or chondroitin sulfate bound to core protein can develop after these reactive glycosaminoglycans have been degraded by eliminases or hydrolases produced by naturally occurring bacteria and rabbit cells, respectively. Immunological studies of proteoglycans and hyaluronic acid treated with eliminases and hydrolases employing rabbit antisera, and possibly those from other species, should be evaluated in the light of these observations.

Immunofluorescence Studies of Chondroitin Sulfate Proteoglycan Biosynthesis: The Use of Monoclonal Antibodies

Several monoclonal antibodies which recognize different antigenic determinants of chondroitin sulfate proteoglycan were used to study chondroitin sulfate proteoglycan biosynthesis in chicken chondrocyte cultures. The intracellular sites of synthesis and processing and extracellular deposition in matrix were localized by double immunofluorescence reactions. One rat monoclonal antibody, S103L, which recognizes an antigenic determinant of the core protein of the chicken cartilage chondroitin sulfate proteoglycan monomer, was used to identify both extracellular chondroitin sulfate proteoglycan and intracellular compartments containing chondroitin sulfate proteoglycan precursors. Intracellular staining with S103L was localized to perinuclear regions, and, in some chondrocytes, to a few other cytoplasmic vesicles as well. When chondrocytes were not fed for several days, intracellular chondroitin sulfate proteoglycan precursors were accumulated in larger compartments distributed throughout the cytoplasm. Polyclonal chondroitin sulfate proteoglycan antibodies displayed similar staining characteristics. In contrast, several of the monoclonal antibodies, including the rat monoclonals S11D and P100D, and the mouse monoclonals 1-B-5, 3-B-3 and 9-A-2, did not recognize native chondroitin sulfate proteoglycan, but reacted only with chondroitinase ABC-digested (and/or hyaluronidase-digested) chondroitin sulfate proteoglycan. These antibodies were particularly useful in the demonstration of the extracellular codistribution of chondroitin sulfate proteoglycan with either type II collagen or fibronectin. In other experiments, the monoclonal antibodies to chondroitin sulfate proteoglycan served to demonstrate that the perinuclear subset of intracellular compartments is uniquely involved in the addition of chondroitin sulfate oligosaccharides to the chondroitin sulfate proteoglycan core protein. Lastly, using the mouse monoclonal 5-D-4, which recognizes keratan sulfate determinants, the perinuclear region was identified as the site for keratan sulfate addition. Results suggest heterogeneity of keratan sulfate synthesis at the level of individual chondrocytes, even for cells apparently containing equivalent amounts of intracellular chondroitin sulfate proteoglycan.

Release of chromaffin granule glycoproteins and proteoglycans from potassium-stimulated PC12 pheochromocytoma cells.

Cultured PC12 pheochromocytoma cells were labeled with [3H]glucosamine, and the glycoproteins and proteoglycans released following potassium-induced depolarization were fractionated and characterized. Exposure of PC12 cells for 20 min to a high concentration of potassium (51.5 mM in Krebs-Ringers-HEPES buffer) results in an approximately sixfold increase in the release of labeled glycoproteins and proteoglycans, compared to incubation in physiological levels of potassium (6 mM). The released complex carbohydrates include chromogranins, dopamine beta-hydroxylase, and two chondroitin sulfate/heparan sulfate proteoglycan fractions, which together account for 7.4% of the soluble cell radioactivity. The chromogranins contained galactosyl(beta 1 leads to 3)N-acetylgalactosamine, as well as several mono- and disialyl O-glycosidically-linked oligosaccharides, and the tetrasaccharide AcNeu(alpha 2 leads to 3)Gal(beta 1 leads to 3)[AcNeu(alpha 2 leads to 6)] GalNAcol, obtained by alkaline borohydride treatment of the chromogranin glycopeptides, accounted for almost half of the total chromogranin labeling. The proteoglycan fractions varied in their relative proportions of chondroitin sulfate (23-68%), heparan sulfate (16-23%), and glycoprotein oligosaccharides (16-54%), which are of the tri- and tetraantennary and O-glycosidic types. As previously found in the case of proteoglycans from bovine chromaffin granules, the more acidic species has a considerably higher proportion of carbohydrate in the form of sulfated glycosaminoglycans.

The presence of a cartilage-like proteoglycan in the adult human meniscus.

Proteoglycans were extracted from the adult human meniscus under dissociative conditions and purified by CsCl-density-gradient centrifugation. The preparations of highest density contained proteoglycan that possessed the ability to interact with hyaluronic acid, was of large subunit size and was composed of chondroitin sulphate, keratan sulphate and sialic acid-containing oligosaccharides. This 'cartilage-like' proteoglycan also exhibited subunit and aggregate structures analogous to those of hyaline-cartilage proteoglycans when examined by electron microscopy. However, the composition of this proteoglycan was more comparable with proteoglycans from immature cartilage than from age-matched cartilage. The preparations from lower density, which were enriched in dermatan sulphate, contained smaller proteoglycan that was not able to interact with hyaluronic acid. This non-aggregating proteoglycan may be structurally distinct from the 'cartilage-like' proteoglycan, which does not contain dermatan sulphate.

A high-performance liquid chromatography approach for isolation and sequencing of chondroitin sulfate oligosaccharides

Chondroitin-4-SO 4 from whale cartilage and chondroitin-6-SO 4 from shark cartilage were digested with hyaluronidase and the resulting tetra-, hexa-, octa-, and decasaccharides were purified in two stages. In Stage I the oligosaccharides from each digest were separated according to size by gel filtration on Sephadex G-50 and by subsequent preparative paper chromatography of their Na-[ 3H]borohydride-reduced derivatives. The Stage I oligosaccharides were mixtures of oligosaccharides having the same degrees of polymerization but differing in their proportions of 4-sulfated and 6-sulfated N-acetyl- d-galactosamine residues. The Stage I 3H-labeled oligosaccharides were further purified by high-performance liquid chromatography on a Partisil-10 SAX anion-exchange column to give relatively homogeneous Stage II products. The most prominent Stage II oligosaccharide obtained from each of the Stage I mixtures was assayed for the percentages of the 4- and 6-sulfated disaccharides at its reducing terminal and its nonreducing terminal, and in its internal positions using a newly developed high-performance liquid chromatography procedure for analysis of the saturated and unsaturated disaccharides released when the purified oligosaccharide is treated with bacterial chondroitinase. The tetra- and hexasaccharides obtained from the two starting materials were completely 4-sulfated (whale cartilage chondroitin SO 4) or 6-sulfated (shark cartilage chondroitin SO 4), but the octa- and decasaccharides, which appeared to be chromatographically homogeneous, contained both 4- and 6-sulfated N-acetyl- d-galactosamine residues.

Sequential degradation of a chondroitin sulphate trisaccharide by lysosomal enzymes from embryonic-chick epiphysial cartilage.

The disulphated trisaccharide D-N-acetylgalactosamine sulphate-beta-D-glucuronic acid-beta-D-N-acetylgalactosamine sulphate prepared from 35S- or 14C-labelled chondroitin sulphate was incubated with a preparation of lysosomal enzymes from embryonic-chick epiphysial cartilage. Degradation was demonstrated by analysis of the reaction products. By use of the appropriate intermediate products as substrates, in conjunction with specific enzyme inhibitors, it was shown that the degradation proceeded sequentially from the non-reducing end. It was initiated by sulphatase (preferentially hydrolysing sulphate ester groups at the 6-position), followed by beta-N-acetylgalactosaminidase and beta-glucuronidase, converting the substrate into monosaccharides and inorganic sulphate. The latter enzyme preferentially attacked disaccharides carrying their sulphate ester group at C-4 of the hexosamine residue. Generation of chondroitin sulphate oligosaccharides may occur by the action of an endoglycosidase, previously demonstrated in embryonic-chick cartilage. Endo- and exo-enzymes may thus form a functional unit in lysosomal degradation of chondroitin sulphate.

Automated ion-exchange chromatography of uronic acids and uronic acid containing oligosaccharides

An automated anion-exchange chromatographic procedure for the separation of uronic acids and uronic acid containing oligosaccharides is described. The method has been applied, in particular, to the separation and quantitation of l-iduronic and d-glucuronic acid in hydrolyzates of dermatan sulfate, and the hydrolysis conditions required for optimum yield of uronic acids have been determined. Whereas uronic acids and aldobiuronic acids were successfully separated by elution with formic acid, hyaluronic acid oligosaccharides and, especially, the highly charged chondroitin sulfate oligosaccharides were more conveniently resolved in a chloride system by elution with a nonlinear LiCl gradient. This system was also employed for the separation of unsaturated and saturated disaccharides obtained by chondroitinase digestion of chondroitin and chondroitin sulfate. The effluent from the columns was analyzed by the automated orcinolsulfuric acid method of the Technicon sugar chromatography system.

Isolation of keratosulfate from chondromucoprotein of bovine nasal septa

Chondromucoprotein has usually been regarded as containing chondroitin sulfate as the only polysaccharide component. We have recently been studying its composition and structure with the aim of elucidating the nature of the protein-carbohydrate linkage. Enzymatic degradation has allowed the isolation of a small fragment containing amino acids, hexose, and chondroitin sulfate oligosaccharides. In addition, however, a fraction has been obtained with the properties of keratosulfate, with which this paper will mainly be concerned. Chondromucoprotein was prepared from fresh cartilage ( Malawista and Schubert, 1958), and proteinpolysaccharide-light (PP-L) was isolated by ultracentrifugation ( Gerber, Franklin and Schubert, 1960). Analytical values for PP-L are given in Table I.