The application of chondro-2-sulfatase for identification of the products generated from chondroitin sulfate isomers by high-performance liquid chromatography

A specific chondroitin sulfate-lyase, chondro-2-sulfatase, was first used for identification of the unsaturated disaccharide constituents (delta Di-S) generated from variously sulfated chondroitin sulfate and dermatan sulfate isomers by a high-performance liquid chromatographic (HPLC) method. delta Di-S generated from oversulfated chondroitin sulfate and dermatan sulfate isomers following digestion with chondroitinases were further digested by the chondro-2-sulfatase, which led to the release of one sulfate from a specific 2-position of the uronic acid residue, as judged with the new HPLC system using a resin made from a sulfonized styrene-divinylbenzene copolymer. It was also found that the chondro-2-sulfatase digests not only delta Di-S with the structure of D-uronic acid 2 sulfate 1-3-N-acetyl-D-galactosamine but also other sulfated delta Di-S with partially the same constituents, i.e., unsaturated di-sulfated disaccharide B, unsaturated di-sulfated disaccharide D or G, and unsaturated tri-sulfated disaccharide.     

Biosynthesis of glycosaminoglycans in peritoneal macrophages from the guinea pig

The majority of glycosaminoglycans synthezied in peritoneal macrophages from the guinea pig in vitro were secreted into culture medium. The secreted glycosaminoglycans were reduced in size with alkali treatment, indicating that the glycosaminoglycanas existed in the form of proteoglycans. After the glycosaminoglycans were digested with chondroitinase AC and ABC, the high voltage paper electrophoretic analysis and the descending paper chromatographic analysis indicated the presence of a considerable amount of unsaturated disulfated disaccharides. Based on the enzymatic assay with chondro-4- and 6-sulfatase, the positions of sulfation in the disulfated disaccharide have been identified as the 4- and 6-position of N-acetylgalactosamine, Moreover, the results of the ion-exchange chromatography and the chondroitinase AC and ABC digestion indicate that ΔDi-diS_E derived from dermatan sulfate. This suggests that peritoneal macrophages are capable of synthesizing oversulfated proteodermatan sulfate as main component. The proportion of synthesized oversulfated dermatan sulfate to the total glycosaminoglycans was independent of the incubation time, and the distribution of oversulfated dermatan sulfate in cell and incubation medium also did not change. After exposure of macrophages to Escherichia coli for 15 min, the incorporation of [³⁵S]sulfate and [³H]glucosamine into the glycosaminoglycans was increased by about 40% with no significant change in the proportion of synthesized oversulfated dermatan sulfate, but the relese of glycosaminoglycans into the culture medium remains essentially unchanged. The difference of the existence of oversulfated dermatan sulfate is not yet understood.     

A high-performance liquid chromatography for constituent disaccharides of chondroitin sulfate and dermatan sulfate isomers

An improved high-performance liquid chromatography for unsaturated disaccharides prepared from chondroitin sulfate and dermatan sulfate isomers was developed using an ion-exchange resin made from a sulfonized styrene-divinylbenzene copolymer. By this newly devised method, it was found that the retention times of representative unsaturated disaccharides are very unique and appear in the following order: unsaturated 6-sulfated, nonsulfated, and 4-sulfated disaccharides. The content of the individual unsaturated disaccharides could be measured at similar sensitivities with ultraviolet absorbance. Sensitive and unique retention times as well as good resolution were found for various unsaturated disulfated disaccharides. The new microassay method by HPLC can be used to determine chondroitin sulfate and dermatan sulfate isomers in amounts as small as 100 ng to 8 micrograms. The practicality of this method was verified by application to the separation and quantitation of chondroitin sulfate and dermatan sulfate isomers from human coronary arteries.     

High-performance liquid chromatographic identification of eight constitutional disaccharides from heparan sulfate isomers digested with heparitinases

Identification with specific heparan sulfate-lyases, heparitinase I and heparinase of the constitutional unsaturated disaacharide (ΔDi-S_HS) derived from heparan sulfate (HS) isomers and heparin was achieved using high-performance liquid chromatography (HPLC) with a sulfonated styrene-divinylbenzene copolymer. Eight ΔDi-S_HS products derived from HS isomers were identified. Enzymatic digestion with heparitinase I and heparinase converts heterogeneous sulfated HS isomers and heparin into different ΔDi-S_HS. The practical application of these enzymes was examined using specific enzymes and HPLC. In a patient with Hurler syndrome, eight individual Δi-S_HS were identified in urinary HS isomers.     

Synthesis of chondroitin sulfate D and heparin proteoglycans in murine lymph node-derived mast cells. The dependence on fibroblasts

Proteoglycans synthesized in cultured mast cells derived from horse serum-immunized lymph node cells were analyzed. Treatment of the 35S-proteoglycans extracted from these cells with either chondroitinase ABC or AC resulted in 95% +/- 7% and 84% +/- 7%, respectively (mean +/- S.E., n = 3), of the radioactivity associated with disaccharides eluting in the included volume of PD-10. The 35S-proteoglycans were not hydrolyzed by nitrous acid elimination treatment. The chondroitinase ABC-generated disaccharides were analyzed by aminocyano high performance liquid chromatography. 35S-Disaccharides eluted in a major peak at a retention time of 8.1 min, corresponding to the disaccharide of chondroitin 4-sulfate proteoglycan (delta Di-4S), and a second peak at 12 min, corresponding to the disaccharide of chondroitin sulfate D proteoglycan (delta Di-diSD). Further treatment with chondro-4-sulfatase did not affect the retention time of the disaccharide corresponding to delta Di-diSD whereas this peak disappeared after the digested proteoglycan was treated either by chondro-6-sulfatase or by both sulfatases. Therefore, this disaccharide was identified as chondroitin sulfate D. Quantification of the radiolabeled disaccharides showed that delta Di-diSD contributed 20% +/- 2% (n = 3) of the total sulfated disaccharides of the chondroitin sulfate of these cultured cells. The role of fibroblasts in inducing the shift of chondroitin sulfate D into heparin proteoglycan in these mast cells was also investigated by using three types of monolayers: mouse embryonic skin fibroblasts (MESF), rat embryonic skin fibroblasts (RESF), and 3T3 fibroblasts. 35S-Proteoglycans that were extracted from the lymph node-derived mast cells cultured for 30 days on MESF and on 3T3 fibroblast monolayers were 93% +/- 4% and 30% +/- 7% (n = 3) susceptible to nitrous acid elimination, respectively. No degradation by nitrous acid was observed in 35S-proteoglycans extracted from cells cultured on RESF monolayer. Since the MESF was found to be the most potent monolayer in the induction of heparin synthesis, the kinetics of changes in the synthesis of proteoglycan types were determined in lymph node-derived mast cells cultured on MESF for up to 30 days. It was found that the synthesis of chondroitin sulfate gradually declined whereas that of heparin starting between 4 and 7 days after plating gradually increased. From the 17th day on, only the synthesis of heparin was detected.     

Highly sulfated proteochondroitin sulfates synthesized in vitro by rat glomeruli

A previous report from this laboratory (Kobayashi, S., Oguri, K., Kobayashi, K. and Okayama, M. (1983) J. Biol. Chem. 258, 12051-12057) indicated that isolated rat glomeruli synthesized three species of sulfated glycoconjugates in vitro, namely, sulfated glycoproteins, proteoheparan sulfates and proteochondroitin sulfates. In the present study, the proteochondroitin sulfates, which showed the greatest incorporation of [35S]sulfate among the three sulfated glycoconjugates, were isolated and characterized. Radiolabeled tissue proteochondroitin sulfates were clearly separated on Sepharose CL-6B into three components with partition coefficients (Kd) of 0.16, 0.22 and 0.58, and medium proteochondroitin sulfates were separated into two components with Kd values of 0.33 and 0.62. When the chondroitin sulfate chains released by alkaline borohydride treatment were analyzed by digestion with chondroitinase AC-II, chondroitinase ABC, chondro-6-sulfatase and chondro-4-sulfatase, the results showed that all the samples contained glucuronosyl-N-acetylgalactosamine (chondroitinase AC-II-susceptible sequences, 72-86%) and iduronosyl-N-acetylgalactosamine (chondroitinase ABC-susceptible sequences, 14-28%), containing 4-sulfated N-acetylgalactosamine (50-70%) and 4,6-disulfated N-acetylgalactosamine (30-50%). On two-dimensional electrophoresis on cellulose acetate, all samples gave a single spot which closely coincided with chondroitin sulfate E of squid cartilage in electrophoretic mobility. These results indicated that the chains were highly sulfated chondroitin sulfates containing glucuronic acid and iduronic acid residues.     

Isolation of UDP-N-acetylgalactosamine-6-sulfate sulfatase from quail oviduct and its action on chondroitin sulfate.

A sulfatase, which liberates sulfate from UDP-N-acetylgalactosamine-6-sulfate (the nucleotide occurring in quail egg white at high concentration), has been isolated from quail oviduct. The tissue also contained sulfatase activities for UDP-N-acetylgalactosamine-4-sulfate and nitrocatechol sulfate but these activities were removed from the 6-sulfatase fraction during purification. The UDP-N-acetylgalactosamine-6-sulfate sulfatase appears to be very closely related to a sulfatase activity for the non-reducing N-acetylgalactosamine-6-sulfate end group in chondroitin sulfate, $\text{i.}\text{e.}$ the two activities could not be separated from each other by various fractionation procedures and were affected in a parallel fashion by mild heating. The results, coupled with those of earlier studies on UDP-N-acetylgalactosamine-4-sulfate in hen oviduct, suggest that in avian oviducts a sulfation/desulfation system may exist wherein sulfated sugar nucleotides and sulfated glycosaminoglycans are involved as alternative or competitive substrates.     

Hyaluronic acid and chondroitin sulfate unsaturated disaccharides analysis by high-performance liquid chromatography and fluorimetric detection with dansylhydrazine

A system capable of resolving all the known unsaturated nonsulfated, mono- and disulfated disaccharides derived from chondroitin sulfate samples, dermatan sulfate, and hyaluronic acid after their derivatization with dansylhydrazine and separation by HPLC and fluorimetric detection is reported. This method was found superior to others in that unsaturated disaccharides can be separated with good resolution in about 50 min in an isocratic solvent with a sensitivity greater than about 50 pmol (approx 20-30 ng) and linearity from 50 to 500 pmol. The system was applied to the analysis of various chondroitin sulfate samples, including highly sulfated species and dermatan sulfate, and also to a defructosylated polysaccharide with a chondroitin backbone purified from Escherichia coli U1-41. Excellent agreement was obtained with traditional compositional analysis performed by anion-exchange HPLC separation and UV absorption at 230 nm.     

Heparitinases facilitate separation of disaccharides of heparan sulfate isomers in human arteries using high-performance liquid chromatography

The constituents of heparan sulfate isomers in human arteries were analysed at the disaccharide unit by high-performance liquid chromatography. Heparitinases I and II facilitated differentiation of six unsaturated disaccharides from heparan sulfate isomers. The variously sulfated disaccharide components of these heparan sulfate isomers were detected after digestion with heparitinases I and II. The heparan sulfate isomers in the aorta and pulmonary arteries were found to consist of various disaccharide units. These heparan sulfate isomers in the arteries are apparently formed during the process of aging and may influence arterial matrix components.     

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.     

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.     

Enzymatic sulfation of gastrin in rat gastric mucosa

An enzyme which catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to gastrin (G17) was identified in rat gastric mucosal cells. The enzyme activity was detected in the 105,000xg supernatant fraction. Formation of gastrin sulfate was shown by using 125I-gastrin and non-radioactive PAPS. The product was sensitive to acid hydrolysis, arylsulfatase treatment and removed by gastrin antibody, but not changed by treatments with chondro-4-sulfatase and chondro-6-sulfatase. The product had a molecular weight of 2050 daltons, close to the molecular weight of G17 sulfate, and, therefore, indicating the sulfated product is not APS derived from the degradation of PAPS. The enzyme activity showed a Km value of 5 microM for PAPS and a pH optimum of 6.0. The activity was not detected in the liver preparation.     

Preparation of unsaturated disaccharides by eliminative cleavage of heparin and heparan sulfate with heparitinases.

1. Six kinds of unsaturated disaccharides were prepared by enzymatic digestion of heparin and heparan sulfate with heparitinases I0 and IV, and subsequent column chromatography. They were identified by HPLC showing good separation from each other. 2. The content of each unsaturated disaccharide fraction was determined colorimetrically, and found to range from 130.7 to 722.3 mumol. 3. Molecular extinction coefficient of each unsaturated disaccharide was calculated from absorbance at a wavelength of around 230 nm where a peak appeared on the ultraviolet spectrum of each disaccharide solution at pH 2. The values varied from 6000 to 6600.     

The application of high-performance liquid chromatography in enzymatic assays of chondroitin sulfate isomers in normal human urine

A study of the urinary excretion of isomeric chondroitin sulfates in normal individuals by high-performance liquid chromatographic (HPLC) determinations of the unsaturated disaccharides produced by digestion with chondroitinases is described. The composition of the HPLC mobile phase was systematically varied in order to select the optimal conditions for separation.The data show that chondroitin 4-sulfate is the major component of the chondroitin sulfate isomers in normal urine, and that chondroitin 6-sulfate is a lesser component. It is also evident that dermatan sulfate is present in small quantities in normal urine.     

Analysis of unsaturated disaccharides from glycosaminoglycuronan by high-performance liquid chromatography

A rapid and simple analytical method for unsaturated disaccharide isomers formed by enzymatic digestion from hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, and heparin by high-performance liquid chromatography using an amine-bound silica column with a linear gradient of sodium dihydrogen phosphate was developed. The analyses were performed on isomers of two groups belonging to the chondroitin sulfate family and the heparin sulfate family. In both families, disaccharide isomers eluted in the order non-, mono-, di-, and trisulfated disaccharides by elevating salt concentrations. The method was applied to the analysis of constituent disaccharides of representative sulfated glycosaminoglycans, which proved that most constituents could be quantified separately. This method is advantageous in that enzymatic digests can be applied directly on a column without any pretreatment and good resolution of several disaccharides can be obtained by one chromatography.     

Structural characterization of chondroitin sulfate from sturgeon bone

Chondroitin sulfate (CS) was purified for the first time from the bones of sturgeon and analyzed to evaluate its structure and properties. A single polysaccharide was extracted from sturgeon bone in a concentration of 0.28-0.34% for dry tissue and characterized as CS. By means of specific chondroitinases and HPLC separation of generated unsaturated repeating disaccharides, this polymer was found to be composed of ∼55% of disaccharide monosulfated in position 6 of the GalNAc, ∼38% of disaccharide monosulfated in position 4 of the GalNAc, and ∼7% of nonsulfated disaccharide. The charge density was 0.93 and the ratio of 4:6 sulfated residues was equal to 0.69, a value confirmed by ¹³C NMR experiments. Chondroitinase B confirmed that the purified sturgeon CS contained mainly GlcA (>99.5%) as uronic acid. PAGE analysis showed a CS having a high molecular mass with an average value of 39,880 according to HPSEC values producing a weight average molecular weight (Mw) of 37,500. On the basis of the data collected, it is reasonable to assume that CS isolated from sturgeon bone might be potentially useful for scientific and pharmacological applications, making this bony fish, which is generally discarded after ovary collection, a useful source of this polymer. Finally, this newly identified source of CS would enable the production of this macromolecule having a particular repeating disaccharide composition, structure, and biological properties.     

Structural analysis of the linkage region oligosaccharides and unsaturated disaccharides from chondroitin sulfate using CarboPac PA1.

Swarm rat chondrosarcoma cell cultures were metabolically labeled with [35S]sulfate, [3H]glucose, or [3H]glucosamine. Chondroitin sulfate chains were isolated from purified aggrecan using alkaline borohydride treatment and Superose 6 chromatography. Various linkage region oligosaccharide alditols were derived from these chains using sequential chondroitinase digestions (ABC lyase followed by ACII lyase). They were then further processed by mercuric acetate treatment, which removed the 4,5-unsaturated uronosyl residue from the nonreducing end of the linkage, and then beta-galactosidase digestion which liberated the 2 galactose residues from the xylitol reducing terminus. Alkaline phosphatase digestions were performed to verify the presence of phosphate esters. All linkage region structures were isolated and identified using a combination of Progel-TSK G2500 and CarboPac PA1 chromatography steps in conjunction with monosaccharide analyses. This study revealed that chondroitin sulfate chains from aggrecan synthesized by rat chondrosarcoma cells in vitro have the following properties: 1) three out of every four of their linkage regions carry a phosphate ester on xylose, 2) nearly three out of every five chains begin the repeating disaccharide region with an unsulfated first disaccharide unit, 3) nearly twice as many nonphosphorylated chains have a sulfated first disaccharide than their phosphorylated counterparts, and 4) the vast majority of these chains do not contain sulfated galactose in their linkage regions. This report also describes a borohydride reduction procedure to confer alkali stability to the 3-substituted, unsaturated disaccharides derived from chondroitinase digests of chondroitin sulfate. Furthermore, a CarboPac PA1 method is demonstrated that separates these reduced disaccharides with exceptional resolution.     

Biosynthesis of lutropin in ovine pituitary slices: Incorporation of [35S]sulfate in carbohydrate units

Sulfate incorporation into carbohydrate of lutropin (LH) has been studied in sheep pituitary slices using H₂³⁵SO₄. Labeled ovine LH was purified to homogeneity by Sephadex G-100 and carboxymethyl-Sephadex chromatography from both the incubation medium and tissue extract. Autoradiography of the gel showed only two protein bands which comigrated with the α and β subunits of ovine LH in both the purified ovine LH and the immunoprecipitate obtained with LH-specific rabbit antiserum. Furthermore, [³⁵S]sulfate was also incorporated into several other proteins in addition to LH. The location of ³⁵SO₄^2? in the oligosaccharides of ovine LH was evidenced by its presence in the glycopeptides obtained by exhaustive Pronase digestion. The location and the point of attachment of sulfate in the carbohydrate unit were established by the isolation of 4-O-[³⁵S]sulfo-N-acetylhexosaminyl-glycerols and 4-O-[³⁵S]sulfo-N-acetylglucosaminitol from the Smith degradation products and by the release of ³⁵SO₄^2? by chondro-4-sulfatase. Thus, the present line of experimentation indicates the presence of sulfate on both the terminal N-acetylglucosamine and N-acetylgalactosamine in the oligosaccharide chains of the labeled ovine LH.     

An automated version of the periodate-thiobarbituric acid assay for analysis of delta-4,5 unsaturated uronic acids and its application to the assay of hyaluronic acids and chondroitin sulfates

An automated periodate-thiobarbituric acid assay of Δ-4,5 unsaturated uronic acids which avoids extraction of chromogen has been developed and applied to the analysis of hyaluronic acid and chondroitin sulfates in standard glycosaminoglycan mixtures and in biological samples following digestion with eliminase enzymes. Assay of hyaluronic acid was linear between 0.1 and 2.5 μg of uronic acid, when digested with hyaluronidase from S. hyalurolyticus and use directly in the automated procedure. The measurement of unsaturated disaccharide standards (25–100 μm) derived from chondroitin sulfates was also linear although the color yields were different. The proportions of chondroitin sulfate isomers were estimated by assay of the unsaturated chondroitin disaccharides which has been separated by thin-layer chromatography.     

Characterization of heparan sulfate from the unossified antler of Cervus elaphus

The antler is the most rapidly growing tissue in the animal kingdom. According to previous reports, antler glycosaminoglycans (GAGs) consist of all kinds GAGs except for heparan sulfate (HS). Chondroitin sulfate is the major antler GAG component comprising 88% of the total uronic acid content. In the current study, we have isolated HS from antler for the first time and characterized it based on both NMR spectroscopy and disaccharide composition analysis. Antler GAGs were isolated by protease treatment and followed by cetylpyridinium chloride precipitation. The sensitivity of antler GAGs to heparin lyase III showed that this sample contained heparan sulfate. After incubation of antler GAGs with chondroitin lyase ABC, the HS-containing fraction was recovered by ethanol precipitation. The composition of HS disaccharides in this fraction was determined by its complete depolymerization with a mixture of heparin lyase I, II, and III and analysis of the resulting disaccharides by the reversed-phase (RP) ion pairing-HPLC, monitored by the fluorescence detection using 2-cyanoacetamide as a post-column labeling reagent. Eight unsaturated disaccharides (DeltaUA-GlcNAc, DeltaUA-GlcNS, DeltaUA-GlcNAc6S, DeltaUA2S-GlcNAc, DeltaUA-GlcNS6S, DeltaUA2S-GlcNS, DeltaUA2S-GlcNAc6S, DeltaUA2S-GlcNS6S) were produced from antler HS by digestion with the mixture of heparin lyases. The total content of 2-O-sulfo disaccharide units in antler HS was higher than that of heparan sulfate from most other animal sources.