The separation of chondroitin sulfate disaccharides and hyaluronan oligosaccharides by capillary zone electrophoresis

We have developed techniques for the separation of unsulfated (2-acetamido-2-deoxy-3-O-(4-deoxy-alpha-L-threo- hex-4-enopyranosyluronicacid)-D-galactose and -D-glucose), monosulfated (2-acetamido-2-deoxy-3- O-(4-deoxy-2-O-sulfo-alpha-L-threo-hex-4-enopyranosyluronic acid)-D-galactose and 2-acetamido-2-deoxy-3-O-(4-deoxy-alpha-L-threo-hex- 4-enopyranosyluronic acid)-4-sulfo-D-galactose and -6-sulfo-D-galactose),disulfated (2-acetamido-2-deoxy-3-O-(4-deoxy-2-O-sulfo-alpha-L-threo-hex-4- enopyranosyluronic acid)-4-sulfo-D-galactose and -6-sulfo-D-galactose and 2-acet-amido-2-deoxy-3-O-(4-deoxy-alpha-L-threo-hex-4-enopy- ranosyluronic acid)-4,6-di-O-sulfo-D-galactose), and trisulfated (2-acetamido-2-deoxy-3-O-(4-deoxy-2-O- sulfo-alpha-L-threo-hex-4-enopyranosyluronic acid)-4,6-di-O-sulfo-D-galactose) isomers of chondroitin using capillary zone electrophoresis. In addition, it is possible to separate oligomers of hyaluronan by similar protocols. These techniques represent a rapid, sensitive, and reproducible technique for the assay of these molecules from digests of connective tissues.     

Analysis by high-performance liquid chromatography of hyaluronic acid and chondroitin sulfates

The use of high-performance liquid chromatography for the quantification of glycosaminoglycan disaccharides has been hampered by the inability to isocratically resolve the chondroitinase digestion products 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-glucose (delta Di-HA) and 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (delta Di-OS). To overcome this limitation, we have developed a solvent system capable of resolving delta Di-HA, delta Di-OS, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (delta Di-6S), and 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose (delta Di-4S). Integrator responses were linear from 1 microgram down to 25 ng for delta Di-HA, delta Di-OS, and delta Di-4S and down to 100 ng for delta Di-6S. This method was used to examine changes in the content of urinary hyaluronic acid and chondroitin sulfates isolated from normal individuals and from patients with Lowe Syndrome, Werner Syndrome, and Hutchinson-Gilford Progeria Syndrome. We confirmed that the HPLC method gave results comparable to colorimetric methods.     

Isolation and chemical study of the glycosaminoglycans from squid cornea

1. Oversulphated chondroitin sulphate (ca 93% of tissue glycosaminoglycans) with average molecular weight 72,500, chondroitin sulphate (5%) and small amounts of lowsulphated chondroitin sulphate were isolated from squid cornea. 2. The sulphation pattern of oversulphated chondroitin sulphate was delta di-4S (52%), delta di-diSD (28%), delta di-6S (9%) and delta di-OSCS (11%) and that of chondroitin sulphate 49, 1, 20 and 30% respectively. 3. All glycosaminoglycans contained neutral monosaccharides, glucose being the predominant neutral monosaccharide in oversulphated chondroitin sulphate and chondroitin sulphate and fucose in low-sulphated chondroitin sulphate. 4. Although L-iduronic acid was not detected, the digestion of oversulphated chondroitin sulphate with chondroitinases ABC and AC gave unexpected results.     

Comparative study on glycosaminoglycans synthesized in peripheral and peritoneal polymorphonuclear leucocytes from guinea pigs.

Glycosaminoglycans synthesized in polymorphonuclear (PMN) leucocytes isolated from blood (peripheral PMN leucocytes) and in those induced intraperitoneally by the injection of caseinate (peritoneal PMN leucocytes) were compared. Both peripheral and peritoneal PMN leucocytes were incubated in medium containing [35S]sulphate and [3H]glucosamine. Each sample obtained after incubation was separated into cell, cell-surface and medium fractions by trypsin digestion and centrifugation. The glycosaminoglycans secreted from peripheral and peritoneal PMN leucocytes were decreased in size by alkali treatment, indicating that they existed in the form of proteoglycans. Descending paper chromatography of the unsaturated disaccharides obtained by the digestion of glycosaminoglycans with chondroitinase AC and chondroitinase ABC identified the labelled glycosaminoglycans of both the cell and the medium fractions in peripheral PMN leucocytes as 55-58% chondroitin 4-sulphate, 16-19% chondroitin 6-sulphate, 16-19% dermatan sulphate and 6-8% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found only in the medium fraction. In peritoneal PMN leucocytes there is a difference in the composition of glycosaminoglycans between the cell and the medium fractions; the cell fraction was composed of 60% chondroitin 4-sulphate, 5.5% chondroitin 6-sulphate, 16.8% dermatan sulphate and 13.9% heparan sulphate, whereas the medium fraction consisted of 24.5% chondroitin 4-sulphate, 28.2% chondroitin 6-sulphate, 33.7% dermatan sulphate and 10% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found in the cell, cell-surface and medium fractions. On the basis of enzymic assays with chondro-4-sulphatase and chondro-6-sulphatase, the positions of sulphation in the disulphated disaccharides were identified as 4- and 6-positions of N-acetylgalactosamine. Most of the 35S-labelled glycosaminoglycans synthesized in peripheral PMN leucocytes were retained within cells, whereas those in peritoneal PMN leucocytes were secreted into the culture medium. Moreover, the amount of glycosaminoglycans in peritoneal PMN leucocytes was significantly less than that in peripheral PMN leucocytes. Assay of lysosomal enzymes showed that these activities in peritoneal PMN leucocytes were 2-fold higher than those in peripheral PMN leucocytes.     

Determination of the chondroitin sulfate disaccharides in dog and horse plasma by HPLC using chondroitinase digestion,precolumn derivatization,and fluorescence detection

A sensitive and selective HPLC method for the determination of the disaccharides of chondroitin sulfate in horse and dog plasma was validated. Chondroitin sulfate is degraded by chondroitinase ABC to three primary unsaturated disaccharides, (1) 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose, (2) 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose, and (3) 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose, when treated with chondroitinase. Plasma samples (0.5 ml) were treated with 50 mU of chondroitinase ABC in 50 microl of 1 mM sodium phosphate buffer (pH 7.0) at 37 degrees C for 6 h. The samples were extracted with 25% trifluoroacetic acid in ethanol. The resultant samples were derivatized with 1% dansylhydrazine in ethanol at 40 degrees C for 3 h. The chromatographic conditions consisted of fluorescence detection (excitation at 350 nm and emission at 530 nm), mu-Bondapack NH(2) (300 x 3.9 mm), and mobile phase of acetonitrile:100 mM acetate buffer, pH 5.6 (76:24), pumped at 1.0 ml/min. The standard curves for each chondroitin disaccharide showed linearity over the selected concentration range (r > or = 0.99). The intraday percentage relative standard deviation was < or =9.5% and the interday precision was < or =6.9% or less. The relative intraday and interday error ranged from -7.3 to 6.6% for each chondroitin disaccharide in the plasma. The extraction recovery was found to be in the range of 90-96%. The validated method accurately quantitated the disaccharides of chondroitin sulfate after administration to dogs and horses.     

Isolation and characterization of proteoglycans synthesized by mouse osteoblastic cells in culture during the mineralization process.

Proteoglycans in mineralized (0.5 M-EDTA/4 M-guanidinium chloride-extractable) and non-mineralized (4 M-guanidinium chloride-extractable) matrices synthesized by a mouse osteoblastic-cell line MC3T3-E1 were characterized at different phases of mineralization in vitro. Cell cultures were labelled with [35S]sulphate and either [3H]glucosamine or 3H-labelled amino acids. At the mineralization phase a large majority of proteoglycans were extracted with 4 M-guanidinium chloride (G extract), and at least five species of labelled proteoglycans were identified; dermatan sulphate proteoglycans (DSPG), apparent Mr approx. 120,000 and 70,000), heparan sulphate proteoglycans (HSPG, apparent Mr approx. 200,000 and 120,000) and DS chains with very little core protein. DSPGs weakly bound to an octyl-Sepharose CL-4B column and HSPGs bound more tightly, whereas DS chains did not bind to the column. Amounts of labelled proteoglycans extracted with 0.5 M-EDTA/4 M-guanidinium chloride (EDTA extract) were much less than those in G extract. Although the predominant species in the EDTA extract were comparable with the DS or DSPGs in the G extract, none of them bound to octyl-Sepharose CL-4B, indicating their lack of hydrophobicity. At the nonmineralizing phase a large chondroitin sulphate proteoglycan (Mr greater than 600,000) was found in the matrix in addition to the five proteoglycan species similar to those at the mineralization phase. Although DS chains at the early phase were similar in size to those at the mineralization phase, the ratio of 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulpho-D-galactose to 2-acetamido-2-deoxy-3-O-(beta-D-gluculo-4-enepyranosyluronic acid)-6-O-sulpho-D-galactose was less than that at the mineralization phase. These results agree with those of previous studies performed in vivo and suggest that alteration in the synthesis of proteoglycans is involved in the mineralization process. They also suggest that at the osteoblastic mineralization front proteoglycans undergo partial degradation and lose their hydrophobicity.     

Culture from mouse bone marrow of a subclass of mast cells possessing a distinct chondroitin sulfate proteoglycan with glycosaminoglycans rich in N-acetylgalactosamine-4,6-disulfate

A differentiated population of cells with metachromatically staining granules and surface IgE receptors was obtained from mouse bone marrow cultured for 2 weeks in the presence of conditioned medium derived from concanavalin A-stimulated splenocytes. The cells were found to incorporate large amounts of [35S]sulfate into an intracellular 35S-labeled proteoglycan of Mr approximately 200,000 containing a maximum of seven glycosaminoglycan side chains (Mr = 25,000). After chondroitinase ABC treatment of density gradient-purified [3H] serine-labeled proteoglycan, the resulting core was Mr approximately 26,000 as assessed by gel filtration. Two-dimensional cellulose acetate electrophoresis of beta-eliminated 35S-labeled glycosaminoglycan revealed a single type of glycosaminoglycan that migrated at the position of oversulfated chondroitin sulfate E from squid cartilage. Chondroitinase ABC degradation of the 35S-labeled glycosaminoglycan yielded two cleavage products in approximately equal molar amounts which co-migrated in both descending paper chromatography and high voltage paper electrophoresis with a monosulfated disaccharide, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose, and a disulfated disaccharide, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-6-di-O-sulfo-D-galactose. The release of some free [35S]sulfate from the oversulfated disaccharide with either chondro-4-sulfatase or chondro-6-sulfatase and the complete desulfation by their combined action established that the oversulfated disaccharide contained N-acetylgalactosamine-4,6-disulfate. The 35S]labeled proteoglycan of these unique IgE receptor-bearing and histamine-containing cells, therefore, is composed of chondroitin sulfate E rather than heparin glycosaminoglycan, and thus is the first identification of such an intracellular localized proteoglycan in a mammalian cell.     

The disaccharides formed by deaminative cleavage of N-deacetylated glycosaminoglycans.

Chondroitin 4-sulphate, chondroitin 6-sulphate, dermatan sulphate and keratan sulphate were N-deacetylated by treatment with hydrazine and then cleaved with HNO2 at pH 4.0, and the resulting products were reduced with NaB3H4. This reaction sequence cleaved the glycosaminoglycans at their N-acetyl-D-glucosamine or N-acetyl-D-galactosamine residues, which were converted into 3H-labelled 2,5-anhydro-D-mannitol (AManR) or 2,5-anhydro-D-talitol (ATalR) residues respectively. The end-labelled disaccharides, composed of D-glucuronic acid (GlcA), L-iduronic acid (IdoA) or D-galactose (Gal) and one of the anhydrohexitols, were identified as follows: both chondroitin 4-sulphate and chondroitin 6-sulphate gave GlcA----ATalR(4-SO4), GlcA----ATalR(6-SO4), IdoA----ATalR (4-SO4) and GlcA(2-SO4)----ATalR(6-SO4); dermatan sulphate gave IdoA----ATalR(4-SO4), GlcA----ATalR(4-SO4), GlcA----ATalR(6-SO4)----IdoA(2-SO4)ATalR(4-SO4) and IdoA----ATalR (4,6-diSO4); keratan sulphate gave Gal(6-SO4)----AManR(6-SO4), Gal----AManR(6-SO4), Gal(6-SO4)----AManR and Gal----AManR. Several additional disaccharides were generated by treatment of the uronic acid-containing disaccharides with hydrazine to epimerize their uronic acid residues at C-5. A number of these disaccharides were found to be substrates for lysosomal sulphatases and glycuronidases. Methods were developed for the separation of all of the disaccharide products by h.p.l.c. The rate of N-deacetylation of chondroitin 4-sulphate by hydrazinolysis was significantly lower than the rate of N-deacetylation of chondroitin 6-sulphate or chondroitin. Dermatan sulphate was N-deacetylated at an intermediate rate. The relative amounts of disaccharides obtained from chondroitin 4-sulphate, chondroitin 6-sulphate and dermatan sulphate under optimum hydrazinolysis/deamination conditions were comparable with the amounts of the corresponding products released from the polymers by chondroitinase treatment.     

Glycosaminoglycan synthesis in mouse mastocytoma

The glycosaminoglycan synthesis in Furth solid mastocytoma tissue has been studied. Approx. 10% of the polysaccharide isolated after incubation in vitro with [(14)C]-glucosamine was digestible with chondroitinase ABC and the product of digestion was identified as 2-acetamido-2-deoxy-3-O-(beta-d-gluco-4-enepyranosyluronic acid)-4-O-sulpho-d-galactose. Similarly, labelling of polysaccharide in vivo with (35)SO(4) (2-) followed by isolation of mast-cell fractions by density-gradient centrifugation on colloidal silica revealed the presence of a polysaccharide which migrated as did chondroitin sulphate on electrophoresis in barium acetate. Chondroitinase ABC produced the same digestion product as before. Finally, the presence of the UDP-N-acetylgalactosamine-chondroitin 6-sulphate hexasaccharide N-acetylgalactosaminyltransferase previously implicated in chondroitin sulphate biosynthesis was demonstrated in microsomal particles from fractions of purified mast cells.     

Determination of endogenous glycosaminoglycans derived disaccharides in human plasma by HPLC: validation and application in a clinical study

SB-424323 is a new, orally active anti-thrombotic agent presently in phase-II clinical development, with limited hemorrhagic risk and a unique mechanism of action involving the induction of glycosaminoglycans (GAGs) biosynthesis. The objective of the present study was to develop a simple and rapid high performance liquid chromatography (HPLC) method for determination of endogenous GAGs derived disaccharides in plasma samples from a phase-II clinical study of SB-424323. Sample preparation was a simple heat treatment of the diluted plasma followed by digestion of endogenous GAGs with chondroitinase ABC to yield unsaturated disaccharides, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (DeltaDi-0S), 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose (DeltaDi-4S), and 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (DeltaDi-6S). These disaccharides were recovered and purified using centrifugal filtration through a filter with 3000 molecular weight cut-off along with externally added internal standard 2-acetamido-2-deoxy-3-O-(2-O-sulfo-beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (DeltaDi-UA2S). A gradient reverse phase HPLC separation was developed on a Waters Symmetry C(18) column (4.6 mm x 150 mm, 5 microm) with a gradient mobile phase system consisting of 0.8 mM tetrabutylammonium hydrogen sulfate and 2mM sodium chloride and acetonitrile at a flow rate of 1.0 mL/min. The eluate was monitored with an ultraviolet detector set at 230 nm. Plasma standard curves were linear (r(2)> or =0.994) in the concentration range 1.0-20 microg/mL with a lower limit of quantification (LLOQ) of 1.0 microg/mL for each of the disaccharide. The mean measured quality control (QC) concentrations for the disaccharides deviated from the nominal concentrations in the range of -8.92 to 5.61% and -16.3 to 16.7%, for inter and intra-day, respectively. The inter and intra-day precision in the measurement of QC samples, were in the range of 3.21 to 18.2% relative standard deviation (R.S.D.) and 0.32 to 20.9% R.S.D., respectively. The inter and intra-day precision in the measurement of endogenous GAGs derived disaccharides in human control plasma, were in the range of 5.8 to 15.9% R.S.D. and 1.17 to 7.74% R.S.D., respectively. Stability of the processed samples was confirmed up to 48 h in the auto-sampler. The method is simple, reliable, and easily adaptable to analysis of large number of samples under logistics of a clinical study. The present method has been used to investigate the GAGs levels in the plasma of patients in a phase II clinical study of SB-424323.     

Purification and characterization of human platelet proteoglycan.

Freshly prepared platelets were shown to contain glycosaminoglycans equivalent to 530 micrograms of hexuronate/10(11) platelets. When the platelets were extracted with 4 M-guanidinium chloride containing proteinase inhibitors, and the extract was dialysed extensively against 7 M-urea solution, almost all of proteoglycan was recovered in the urea-soluble fraction. The proteoglycan was purified from the urea-soluble fraction with a yield of 47% by DEAE-Sephacel chromatography, CsCl-density-gradient centrifugation, Bio-Gel A-15m gel filtration and then rechromatography on DEAE-Sephacel. The purified proteoglycan contained 30% glucuronic acid, 32% N-acetylgalactosamine, 14% sulphate and 15% protein. Serine, glutamic acid, glycine, aspartic acid and leucine accounted for 64% of the total amino acids. The Mr of the proteoglycan was assessed to be approx. 136000 by sedimentation-equilibrium methods. The galactosaminoglycan released by alkaline-borohydride treatment of the proteoglycan was converted stoichiometrically into 4-sulphated unsaturated disaccharide by digestion with chondroitinase AC-II, indicating that the galactosaminoglycan was fully sulphated chondroitin 4-sulphate. The apparent Mr of the chondroitin sulphate was assessed to be 28000 by gel filtration on Bio-Gel A-0.5m (KD 0.18). On two-dimensional electrophoresis on a cellulose acetate membrane, the chondroitin sulphate gave a single compact spot co-migrating with a reference chondroitin sulphate, indicating that the chondroitin sulphate chains were homogeneous in both length and charge density. On the basis of these results, the proteoglycan in human platelets was concluded to be a macromolecule of Mr 136000 containing four chondroitin 4-sulphate chains each with the apparent Mr of 28000.     

Identification of N-sulphated disaccharide units in heparin-like polysaccharides.

1. Preparations of heparin and heparan sulphate were degraded with HNO2. The resulting disaccharides were isolated by gel chromatography, reduced with either NaBH4 or NaB3H4 and were then fractionated into non-sulphated, monosulphated and disulphated species by ion-exchange chromatography or by paper electrophoresis. The non-sulphated disaccharides were separated into two, and the monosulphated disaccharides into three, components by paper chromatography. 2. The uronic acid moieties of the various non- and mono-sulphated disaccharides were identified by means of radioactive labels selectively introduced into uronic acid residues (3H and 14C in D-glucuronic acid, 14C only in L-iduronic acid units) during biosynthesis of the polysaccharide starting material. Labelled uronic acids were also identified by paper chromatography, after liberation from disaccharides by acid hydrolysis or by glucuronidase digestion. Similar procedures, applied to disaccharides treated with NaB3H4, indicated 2,5-anhydro-D-mannitol as reducing terminal unit. On the basis of these results, and the known positions and configurations of the glycosidic linkages in heparin, the two non-sulphated disaccharides were identified as 4-O-(beta-D-glucopyranosyluronic acid)-2,5-anhydro-D-mannitol and 4-O-(alpha-L-idopyranosyluronic acid)-2,5-anhydro-D-mannitol. 3. The three monosulphated [1-3H]anhydromannitol-labelled disaccharides were subjected to Smith degradation or to digestion with homogenates of human skin fibroblasts, and the products were analysed by paper electrophoresis. The results, along with the 1H n.m.r. spectra of the corresponding unlabelled disaccharides, permitted the allocation of O-sulphate groups to various positions in the disaccharides. These were thus identified as 4-O-(beta-D-glucopyranosyl-uronic acid)-2,5-anhydro-D-mannitol 6-sulphate, 4-O-(alpha-L-idopyranosyluronic acid)-2,5-anhydro-D-mannitol 6-sulphate and 4-O-(alpha-L-idopyranosyluronic acid 2-sulphate)-2,5-anhydro-D-mannitol. The last-mentioned disaccharide was found to be a poor substrate for the iduronate sulphatase of human skin fibroblasts, as compared with the disulphated species, 4-O-(alpha-L-idopyranosyluronic acid 2-sulphate)-2,5-anhydro-D-mannitol 6-sulphate. 4. The identified [1-3H]anhydromannitol-labelled disaccharides were used as reference standards in a study of the disaccharide composition of heparins and heparan sulphates. Low N-sulphate contents, most pronounced in the heparin sulphates, were associated with high ratios of mono-O-sulphated/di-O-sulphated (N-sulphated) disaccharide units, and in addition, with relatively large amounts of 2-sulphated L-iduronic acid residues bound to C-4 of N-sulpho-D-glucosamine units lacking O-sulphate substituents.     

Crystallization and some properties of chondroitinase from Arthrobacter aurescens.

A strain of Arthrobacter aurescens which secretes a large amount of chondroitinase into a culture broth, was isolated from soil. The chondroitinase was purified 380-fold over culture broth in 24% yield and crystallized. Some properties of the purified enzyme were studied and described: thermal stability (below 45 degrees), pH stability (pH 4.9 to 7.4), optimum temperature (50 degrees), and optimum pH (pH 6.0). Chrondroitin sulfate A and C, chondroitin, and hyaluronic acid were split by the enzyme but dermatan sulfate could not be. The initial rates of enzymic degradation of chondroitin sulfate C, chondroitin, and hyaluronic acid were 1.1, 1.95, and 3.2, respectively, compared to that of chondroitin sulfate A. When the enzyme was allowed to act on chondroitin sulfate A and C, the reducing power and the ultraviolet absorption at 232 nm increased proportionally to the decrease in viscosity of the substrate solution. Finally these substrates were degraded to the extent of 100% to disaccharides. By the enzyme action the main products from chondroitin sulfate A and C were deta 4,5-unsaturated disaccharides, which were identified as 2-acetamido-2-deoxy-3-O-(Beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose and 2-acet-amido-2-deoxy-3-O-(Beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose by paper chromatography, ultraviolet absorption spectroscophy, and infrared spectroscopy. Thus it is suggested that the chondroitinase is a chondroitin sulfate A and C lyase, one of the hyaluronate lyases (EC 4.2.99.1).     

One- and two-dimensional 1H-NMR characterization of two series of sulfated disaccharides prepared from chondroitin sulfate and heparan sulfate/heparin by bacterial eliminase digestion.

The 1H-NMR spectra of eight unsaturated disaccharides obtained by bacterial eliminase digestion of chondroitin sulfate and of heparan sulfate/heparin were recorded in order to construct an NMR data base of sulfated oligosaccharides and to investigate the effects of sulfation on the proton chemical shifts. These shifts were assigned by two-dimensional HOHAHA (homonuclear Hartmann-Hahn) and COSY (correlation spectroscopy) methods. The results indicated the following. (1) Two sets of proton signals were observed, corresponding to the alpha and beta anomers of these disaccharides, except those containing N-sulfated GlcN (2-deoxy-2-amino-D-glucose), in which only one set of signals appeared, corresponding to the alpha anomer. (2) Signals of protons bound to an O-sulfated carbon atom and those bound to the immediately neighboring carbon atoms were shifted downfield by 0.4-0.7 and 0.07-0.3 ppm, respectively. (3) For the disaccharides containing the N-sulfated GlcN, the signals of the protons bound to C-2 and C-3 were shifted upfield by 0.6 and 0.15 ppm, respectively, but that of C-1 was shifted downfield by 0.25 ppm when compared with those of the corresponding N-acetylated disaccharides. (4) For the chondroitin sulfate disaccharides sulfated on the C-4 position of GalNAc (2-deoxy-2-N-acetylamino-D-galactose) or the C-2 position of delta GlcA (D-gluco-4-ene-pyranosyluronic acid), the signal of the H-3 proton of delta GlcA or the H-4 proton of GalNAc was shifted upfield by 0.1-0.15 ppm, indicating the steric interaction of the two sugar components. (5) These effects of sulfation on chemical shifts are additive.     

Characterisation of oligosaccharides from the chondroitin/dermatan sulphates: (1)H and (13)C NMR studies of oligosaccharides generated by nitrous acid depolymerisation

The polymers chondroitin sulphate and dermatan sulphate have been fragmented by an anhydrous hydrazine/nitrous acid procedure. The resulting disaccharides from the polymer repeat sequences were reduced with NaBH(4) and purified by ion exchange chromatography. Whereas enzymatic depolymerisation leads to the loss of the distinction between glucuronic and iduronic acids of CS and DS in the resultant disaccharides, nitrous acid depolymerisation retains these structures. Complete (1)H and (13)C NMR data have been derived for the major components which were shown to have the structures: GlcA-(β1→3)-anTal6S-ol (I) and L-IdoA-(α1→3)-anTal4S-ol (II), where anTal-ol represents (2,5)anhydro-D-talitol and 6S/4S represent O-ester sulphate groups at C-6/C-4 sites.     

Human liver glucuronate 2-sulphatase. Purification, characterization and catalytic properties.

Human glucuronate 2-sulphatase (GAS), which is involved in the degradation of the glycosaminoglycans heparan sulphate and chondroitin 6-sulphate, was purified almost 2,000,000-fold to homogeneity in 8% yield from liver with a four-step six-column procedure, which consists of a concanavalin A-Sepharose/Blue A-agarose coupled step, a DEAE-Sephacel/octyl-Sepharose coupled step, CM-Sepharose chromatography and gel-permeation chromatography. Although more than 90% of GAS activity had a pI of greater than 7.5, other forms with pI values of 5.8, 5.3, 4.7 and less than 4.0 were also present. The pI greater than 7.5 form of GAS had a native molecular mass of 63 kDa. SDS/polyacrylamide-gel-electrophoretic analysis resulted in two polypeptide subunits of molecular mass 47 and 19.5 kDa. GAS was active towards disaccharide substrates derived from heparin [O-(beta-glucuronic acid 2-sulphate)-(1----4)-O-(2,5)-anhydro[1-3H]mannitol 6-sulphate (GSMS)] and chondroitin 6-sulphate [O-(beta-glucuronic acid 2-sulphate-(1----3)-O-(2,5)-anhydro[1-3H]talitol 6-sulphate (GSTS)]. GAS activity towards GSMS and GSTS was at pH optima of 3.2 and 3.0 respectively with apparent Km values of 0.3 and 0.6 microM respectively and corresponding Vmax values of 12.8 and 13.7 mumol/min per mg of protein respectively. Sulphate and phosphate ions are potent inhibitors of enzyme activity. Cu2+ ions stimulated, whereas EDTA inhibited enzyme activity. It was concluded that GAS is required together with a series of other exoenzyme activities in the lysosomal degradation of glycosaminoglycans containing glucuronic acid 2-sulphate residues.     

Purification and characterisation of a minor low-sulphated dermatan sulphate-proteoglycan from ray skin.

A minor low-sulphated dermatan sulphate proteoglycan was isolated from ray skin by extraction with 2% sodium dodecyl sulphate, followed with ion-exchange chromatography, gel chromatography and density gradient centrifugation. The proteoglycan with a relative molecular mass (Mr) ranging from 70 to 120 kDa is composed of about two dermatan sulphate chains (Mr 33 kDa) bound on a protein core of Mr 27 kDa, and oligosaccharides consisting of uronic acids, hexosamines and neutral sugars. The major amino acids of the protein core were glycine (corresponding to about one-fourth of the total amino acids), serine, threonine, glutamic acid/glutamine, leucine and cysteine, together amounting to 56% of the total. The isolated proteoglycan does not interact with hyaluronic acid and does not form self-aggregates. Dermatan sulphate was rich in iduronic acid (62% of total uronic acid) and composed of non-sulphated (44%), and mono-sulphated disaccharides bearing esterified sulphate groups at positions C-4 (53%) or C-6 (3%) of the N-acetyl galactosamine. HPLC analysis of a pure preparation of dermatan sulphate, showed the presence of galactose and glucose possibly as branches on the dermatan sulphate chain.     

The proteoglycans of the canine intervertebral disc

The high-buoyant-density proteoglycans of the nucleus pulposus and annulus fibrosus of the beagle intervertebral disc have been isolated by CsCl density gradient ultracentrifugation. The sulphated proteoglycans were labelled in vivo with 35SO4, 24 h and 60 days prior to killing. The hydrodynamic size and aggregation of the 24 h, 60 day and resident (from hexuronic acid and hexosamine analysis) proteoglycan subunit populations were determined by Sepharose CL-2B chromatography in the presence or absence of excess hyaluronic acid. The hydrodynamic size of the keratan sulphate-proteoglycan core protein complexes were also determined by Sepharose CL-2B chromatography after chondroitinase ABC digestion of proteoglycans. When initially synthesised (24 h) or after 60 days, the percentage aggregation and hydrodynamic size of the proteoglycans derived from the annulus fibrosus were larger than those present in the nucleus pulposus. Hexosamine, hexuronic and protein determination of the high-buoyant-density fractions showed that the proteoglycans of the nucleus pulposus were richer in chondroitin sulphate than those in the annulus. However there was no difference in Mr of the chondroitin sulphate and keratan sulphate attached to the proteoglycans of the two disc regions, nor were differences detected by HPLC between the proportions of chondroitin 4-sulphate and chondroitin 6-sulphate present in these high-density fractions. In contrast, the low-buoyant-density (1.54 greater than p greater than 1.45) proteoglycan fractions and tissue residues remaining after 4 M GuHCl extraction were found to contain dermatan sulphate, suggesting the presence of a third proteoglycan species possibly associated with the collagen of the fibrocartilagenous matrix.     

Disaccharide compositional analysis of heparin and heparan sulfate using capillary zone electrophoresis.

Capillary zone electrophoresis (CZE) was used to separate eight commercial disaccharide standards of the structure delta UA2X(1----4)-D-GlcNY6X (where delta UA is 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, GlcN is 2-deoxy-2-aminoglucopyranose, S is sulfate, Ac is acetate, X may be S, and Y is S or Ac). These eight disaccharides had been prepared from heparin, heparan sulfate, and derivatized heparins. A similar CZE method was recently reported for the analysis of eight chondroitin and dermatan sulfate disaccharides (A. Al-Hakim and R.J. Linhardt, Anal. Biochem. 195, 68-73, 1991). Two of the standard heparin/heparan sulfate disaccharides, having an identical charge of -2, delta UA2S(1----4)-D-GlcNAc and delta UA(1----4)-D-GlcNS, were not fully resolved using standard sodium borate/boric acid buffer. This buffer had proven effective in separating chondroitin/dermatan sulfate disaccharides of identical charge. Resolution of these two heparin/heparan sulfate disaccharides could be improved by extending the capillary length, preparing the buffer in 2H2O, or eliminating boric acid. Baseline resolution was achieved in sodium dodecyl sulfate in the absence of buffer. The structure and purity of each of the eight new commercial heparin/heparan sulfate disaccharide standards were confirmed using fast-atom-bombardment mass spectrometry and high-field 1H-NMR spectroscopy. Heparin and heparan sulfate were then depolymerized using heparinase (EC 4.2.2.7), heparin lyase II (EC 4.2.2.-), heparinitase (EC 4.2.2.8), and a combination of all three enzymes. CZE analysis of the products formed provided a disaccharide composition of each glycosaminoglycan. As little as 50 fmol of disaccharide could be detected by ultraviolet absorbance.     

Effect of low-density lipoproteins on the synthesis and secretion of proteoglycans by human endothelial cells in culture.

We studied the effect of low-density lipoproteins (LDL) on the synthesis and secretion of proteoglycans by cultured human umbilical-vein endothelial cells. Confluent cultures were incubated with [35S]sulphate or [3H]glucosamine in lipoprotein-deficient serum in the presence and in the absence (control) of LDL (100-400 micrograms/ml), and metabolically labelled proteoglycans in culture medium and cell layer were analysed. LDL increased accumulation of labelled proteoglycans in medium and cell fractions up to a concentration of 200 micrograms/ml. At this concentration of LDL the accumulations of proteoglycans in medium and cell layer were 65% and 32% respectively above control for 35S-labelled proteoglycans, and 55% and 28% respectively above control for 3H-labelled proteoglycans. At concentrations above this LDL was found to depress the accumulation of proteoglycans in medium and cell layer. Gel filtration on Sepharose CL-4B showed that in both control and LDL-treated cultures the cell layer contained a large (Kav. = 0) and a small (Kav. = 0.35) heparan sulphate proteoglycan, whereas the culture medium contained a large heparan sulphate proteoglycan (Kav. = 0) and a smaller isomeric chondroitin sulphate proteoglycan (control, Kav. = 0.35; LDL-treated, Kav. = 0.17). The relative increase in hydrodynamic size of the isomeric chondroitin sulphate proteoglycan (Mr 150,000 compared with 90,000) in the medium of cultures exposed to LDL was partly attributable to the larger size of the glycosaminoglycan side chains (Mr 39,000 compared with 21,000). The isomeric chondroitin sulphate proteoglycan in LDL-treated culture was relatively enriched in chondroitin 6-sulphate compared with that in control cultures (39% compared with 29%). Pulse-chase studies showed that LDL treatment did not alter the turnover rate of proteoglycans as compared with controls, implying that the elevation in proteoglycan accumulation in LDL-treated cultures was due to enhanced synthesis. These results demonstrate that LDL can modulate proteoglycan synthesis by cultured vascular endothelial cells, resulting in the secretion of a larger isomeric chondroitin sulphate proteoglycan enriched in chondroitin 6-sulphate.