Regulation of chondroitin sulfate synthesis. Effect of beta-xylosides on synthesis of chondroitin sulfate proteoglycan, chondroitin sulfate chains, and core protein

Monolayer cultures of embryonic chick chondrocytes were incubated with 35SO42- in the presence and absence of 1.0 mM p-nitrophenyl-beta-d-xyloside for 2 days. The relative amounts of chondroitin sulfate proteoglycan and free polysaccharide chains were measured following gel filtration on Sephadex G-200. Synthesis of beta-xyloside-initiated polysaccharide chains was accompanied by an apparent decrease in chondroitin sulfate proteoglycan production by the treated cultures. When levels of cartilage-specific core protein were determined by a radioimmunoassay, similar amounts of core protein were found in both beta-xyloside and control cultures, indicating that decreased synthesis of core protein is not responsible for the observed decrease in chondroitin sulfate proteoglycan production. Activity levels of the chain-initiating glycosyltransferases (UDP-D-xylose: core protein xylosyltransferase and UDP-D-galactose:D-xylose galactosyltransferase) as well as the extent of xylosylation of core protein were found to be similar in cell extracts from both culture types. Furthermore, beta-xylosides did not inhibit the xylosyltransferase reaction in cell-free studies. In contrast, the beta-xylosides effectively competed with several galactose acceptors, including an enzymatically synthesized xylosylated core protein acceptor, in the first galactosyltransferase reaction.     

Inhibition of chondroitin and heparan sulfate biosynthesis in Chinese hamster ovary cell mutants defective in galactosyltransferase I

We have isolated five Chinese hamster ovary cell mutants defective in galactosyltransferase I (UDP-D-galactose:xylose beta-1,4-D-galactosyltransferase) and studied the effect of p-nitrophenyl-beta-D-xyloside supplementation on glycosaminoglycan biosynthesis in the mutant cells. Assays of galactosyltransferase I showed that the mutants contained less than 2% of the enzyme activity present in wild-type cells, and enzyme activity was additive in mixtures of mutant and wild-type cell extracts, suggesting that the mutations most likely defined the structural gene encoding the enzyme. Cell hybridization studies showed that the mutations in all five strains were recessive and that the mutants belonged to the same complementation group. The mutants contained wild-type levels of xylosyltransferase (UDP-D-xylose:core protein (serine) beta-D-xylosyltransferase), lactose synthase (UDP-D-galactose:N-acetyl-glucosaminide beta-1,4-D-galactosyltransferase), and lactosylceramide synthase (UDP-D-galactose:glucosylceramide beta-1,4-D-galactosyltransferase). Their sensitivity to lectin-mediated cytotoxicity was virtually identical to that of the wild-type, indicating that there were no gross alterations in glycoprotein or glycolipid compositions. Anion-exchange high performance liquid chromatography of 35S-glycosaminoglycans from one of the galactosyltransferase I-deficient mutants showed a dramatic reduction in both heparan sulfate and chondroitin sulfate, demonstrating that galactosyltransferase I is responsible for the formation of both glycosaminoglycans in intact cells. Surprisingly, the addition of 1 mM-p-nitrophenyl-beta-D-xyloside, a substrate for galactosyltransferase I, restored glycosaminoglycan synthesis in mutant cells. This finding suggested that another galactosyltransferase, possibly lactose synthase, can transfer galactose to xylose in intact cells.     

Biosynthesis of chondroitin sulfate: interaction between xylosyltransferase and galactosyltransferase

An affinity matrix consisting of the core protein of cartilage proteoglycan coupled to Sepharose was used to study the interaction between the glycosyltransferases which catalyze the first two reactions in the biosynthesis of chondroitin sulfate. Xylosyltransferase, for which the core protein is a substrate, is quantitatively adsorbed to the matrix. In contrast, UDP-galactose:xylose galactosyltransferase is not significantly adsorbed, but does bind to matrix which has been previously equilibrated with xylosyltransferase. By virtue of this enzyme-enzyme interaction, a 7-fold purification of galactosyltransferase can be obtained.     

Purification and some properties of UDP-xylosyltransferase of rat ear cartilage

UDP-xylosyltransferase (UDP-D-xylose:proteoglycan core protein beta-D-xylosyltransferase EC 2.4.2.26) initiates the formation of chondroitin sulfate in the course of proteoglycan biosynthesis. The enzyme catalyzes the transfer of D-xylose from UDP-D-xylose to specific serine residues in the core protein. A procedure for purification of xylosyltransferase from rat ear cartilage was developed which includes ammonium sulfate fractionation, chromatography on heparin-agarose, on Sephacryl S300 and finally a substrate affinity chromatography applying the dodeca peptide Q-E-E-E-G-S-G-G-G-Q-G-G. The specific activity of the purified enzyme was about 420 mU per mg protein. The purification factor was about 26.000 with 27% yield. In SDS-polyacrylamide gel electrophoresis, the highly purified enzyme is homogeneous and yields only a single distinct band of 78 kDa. An apparent molecular mass of 71 kDa was determined for the native enzyme. These data suggest a monomeric structure for the enzyme. Xylosyltransferase activity was found to depend essentially on the presence of divalent metal ions. The K(m) value for UDP-D-xylose was determined to 6.5 micromol/l and for the dodeca peptide Q-E-E-E-G-S-G-G-G-Q-G-G as xylose acceptor to 8 micromol/l.     

The effect of hormones on synthesis of the region linking chondroitin sulfate to protein

1. 1. Particulate fractions of costal cartilage from young rats are capable of catalyzing the formation of the first two monosaccharide units of the chondroitin sulfate-protein linkage region.

2. 2. Hormonal imbalance has been shown to influence the activity of the glycosyltransferases responsible for the sequential transfer of xylose and galactose from UDPxylose and UDPgalactose, respectively, in the formation of the linkage region.

3. 3. The activity of xylosyltransferase was found to be decreased in costal cartilage of diabetic, thyroidectomized and hypophysectomized rats, but not in rats injected with either testosterone or hydrocortisone. In the latter two treatment groups, galactosyltransferase activity was decreased only in the group receiving hydrocorsitone.

4. 4. The combined results of this and previous studies suggest that decreased levels of chondroitin sulfate in diabetic, thyroidectomized and hypophysectomized animals are due to interference in the synthesis of the linkage region of the proteoglycan at the xylosyltransferase level whereas hydrocortisone acts primarily at the level of the galactosyltransferase.

Identification of chondroitin sulfate E in human lung mast cells

Human lung mast cells (HLMC) enriched up to 99% purity by counter current elutriation and density gradient centrifugation were labeled with 35S-sulfate to determine cell-associated proteoglycans. The 35S-labeled proteoglycans were extracted by the addition of detergent and 4 M guanidine-HCl, and separated from unincorporated precursor by Sephadex G-50 chromatography. 35S-Proteoglycans chromatographed over Sepharose 4B with a Kav of 0.48. 35S-Glycosaminoglycans separated from the parent 35S-proteoglycans by beta-elimination and chromatographed over Sepharose 4B with a Kav of 0.63. Characterization of 35S-proteoglycans by chondroitin ABC lyase treatment revealed approximately 36% of the proteoglycan to be composed of chondroitin sulfates. Analysis by HPLC of component disaccharides liberated by chondroitin ABC lyase using an amino-cyano-substituted silica column indicated that the chondroitin sulfates consisted of the monosulfated A disaccharide (GlcUA----GaINAc4SO4) (75%) and the over-sulfated E disaccharide (GlcUA----GaINAc4,6-diSO4) (25%). Nitrous acid/heparinase-susceptible heparin proteoglycans accounted for approximately 62% of the total 35S-proteoglycans present in the HLMC. Proteoglycans remaining after exposure of the original proteoglycan extract to either heparinase or chondroitin ABC lyase were of similar size, suggesting that the majority of heparin and chondroitin sulfate glycosaminoglycans were on separate protein cores. Proteoglycans extracted from HLMC were protease insensitive. Hence, in addition to heparin proteoglycans, HLMC synthesize a hitherto unrecognized quantity of chondroitin sulfate E proteoglycans.     

Biosynthesis of chondroitin 4-sulfate-proteoglycan by a transplantable rat chondrosarcoma.

The activities and subcellular distribution of five glycosyltransferases involved in the biosynthesis of chondroitin sulfate by a transplantable rat chondrosarcoma were compared with the activities and distribution of the corresponding enzymes of normal embryonic rat and chick cartilage.Two important differences were found: 1) UDP-d-xylose:core protein β-d-xylosyltransferase was found in concentrations 10–15 times higher in the chondrosarcoma, and 2) all five glycosyltransferases were found to be more soluble in the chondrosarcoma. More than 90% of the xylosyltransferase activity could be extracted from the tumor without rupturing cells. This transferase exhibited optimal activity in solutions of 0.25 m KCl. The K_m for the exogenous protein acceptor obtained by Smith degradation of bovine chondroitin sulfate-proteoglycan was 300 μg/ml; the K_m for Ser-Gly-Gly, 30 mm. The activity of xylosyltransferase was maximal at pH 6.5 and was dependent upon the presence of Mg²⁺ or Mn²⁺. The K_m for UDP-xylose was 5 × 10^?5, m. In view of the extraordinarily high level of xylosyltransferase activity found in the chondrosarcoma the authenticity of the xylosyl transfer reaction was verified by chemical characterization of [¹⁴C]xylose-labeled products.     

A study on heterogeneity in molecular species of shark cartilage chondroitin sulfate C. Fractionation of the polysaccharide on Sepharose CL-4B in the presence of high concentrations of ammonium sulfate

Shark cartilage chondroitin sulfate C was fractionated by chromatography on Sepharose CL-4B-2.5 to 1.5M ammonium sulfate in 10mM hydrochloric acid at 4 degrees. Both unit-disaccharide composition and molecular-size distribution clearly affected the fractionation. Comparison of this fractionation with the fractionation on Sepharose 6B gel in 0.2M sodium chloride revealed that the former is distinctly superior to the latter. The fractionation on Sepharose CL-4B in the presence of ammonium sulfate also showed that the chondroitin sulfate C molecules having a larger molecular size contain generally more chondroitin 6-sulfate units (as major constituent) and less chondroitin disulfate units (D type, as minor constituent) than those having a smaller molecular size).     

Deglycosylation of chondroitin sulfate proteoglycan by hydrogen fluoride in pyridine

The original deglycosylation procedure using HF/pyridine has been modified for maximal removal of carbohydrate from chondroitin sulfate proteoglycan, with minimal alteration of the core protein. Gas-liquid chromatography analysis after treatment for various times showed that 95% of xylose and mannose and 70-85% of other sugars were removed within 30 min, indicating that almost all chondroitin sulfate chains and about 80% of N- and O-linked oligosaccharides were removed. In contrast to the loss of carbohydrate, no change in amino acid composition or loss of immunoreactivity occurred. Longer treatment of up to 16 h resulted in little additional removal of carbohydrate, but did cause a significant decrease in solubility and recovery of the deglycosylated product. Optimal removal of xylose residues after about 1 h was also shown by maximal acceptor activity of the product in a xylosyltransferase assay. Rapid removal of the HF reagent by vacuum evacuation and ion-exchange chromatography, coupled with the reduced time of treatment allowed recovery of an intact, homogenous protein core that is amenable to structural and sequence studies.     

The structures of N- and O-glycosidic carbohydrate chains of a chondroitin sulfate proteoglycan isolated from the media of the human aorta

A large Mr chondroitin sulfate proteoglycan was extracted from the media of human aorta under dissociative conditions and purified by density-gradient centrifugation, ion-exchange chromatography, and gel filtration chromatography. Removal of a contaminating dermatan sulfate proteoglycan was accomplished by reduction, alkylation and rechromatography on the gel filtration column. After chondroitinase ABC treatment, the proteoglycan core was separated from a residual heparan sulfate proteoglycan by a third gel filtration chromatography step. As assessed by radioimmunoassay, the isolated proteoglycan core was free of link protein, but possessed epitopes that were recognized by antisera against the hyaluronic acid binding region of bovine cartilage proteoglycan as well as those that were weakly recognized by anti-keratan sulfate antisera. Following beta-elimination of the protein core, the liberated low Mr oligosaccharides were partially resolved by Sephadex G-50 chromatography, and their primary structure was determined by 500-MHz1H NMR spectroscopy in combination with compositional sugar analysis. The N-glycosidic carbohydrate chains, which were obtained as glycopeptides, were all biantennary glycans containing NeuAc and Fuc; microheterogeneity in the NeuAc----Gal linkage was detected in one of the branches. The N-glycosidic glycans have the following overall structure: (Formula: see text). The majority of the O-glycosidic carbohydrate chains bound to the protein core were found to be of the mucin type. They were obtained as glycopeptides and oligosaccharide alditols, and possessed the following structures: NeuAc alpha(2----3)Gal beta(1----3)GalNAc-ol, [NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----6)]GalNAc-ol, and NeuAc alpha-(2----3) Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)] GalNAc-ol. The remainder of the O-glycosidic carbohydrate chains bound to the isolated proteoglycan were the hexasaccharide link regions of the chondroitin sulfate chains that remained after chondroitinase ABC treatment of the native molecule. These latter glycans, which were obtained as oligosaccharide alditols, had the following structure (with GalNAc free of sulfate or containing sulfate bound at either C-4 or C-6): delta 4,5GlcUA beta(1----3)GalNAc beta(1----4)GlcUA beta(1----3)Gal beta(1----3)Gal beta(1----4)Xyl-ol.     

Distribution of keratan sulfate in cartilage proteoglycans.

After chondroitinase digestion of bovine nasal and tracheal cartilage proteoglycans, subsequent treatment with trypsin or trypsin followed by chymotrypsin yielded two major types of polypeptide-glycosaminoglycan fragments which could be separated by Sepharose 6B chromatography. One fragment, located close to the hyaluronic acid-binding region of the protein core, had a high relative keratan sulfate content. This fragment contained about 60% of the total keratan sulfate, but less than 10% of the total chondroitin sulfate present in the original proteoglycan preparation. The weight average molecular weight of the keratan sulfate-enriched fragment was 122,000, as determined by sedimentation equilibrium centrifugation. The chemical and physical data indicate that this fragment contains an average of 10 to 15 keratan sulfate chains, if the average molecular weight of individual chains is assumed to be about 8,000, and about 5 chondroitin sulfate chains attached to a peptide of about 20,000 daltons. The other population of fragments was derived from the other end of the proteoglycan molecule, the chondroitin sulfate-enriched region, and contained mainly chondroitin sulfate chains. About 90% of the total chondroitin sulfate, but only 20 to 30% of the total keratan sulfate was recovered in these fragments. On the average, approximately 5 chondroitin sulfate chains and 1 keratan sulfate chain could be linked to the same peptide. Another 10 to 20% of the total keratan sulfate, originally found in or near the hyaluronic acid-binding region, was not separated from the chondroitin sulfate-enriched fragments. Hydroxylamine could be used to liberate a large molecular size, chondroitin sulfate-enriched fragment (Kav 0.54 on Sepharose 2B) from the proteoglycan aggregates. The remainder of the protein core, containing the keratan sulfate-enriched region, was bound to hyaluronic acid with the link proteins and recovered in the void volume on the Sepharose 2B column.     

Membrane-associated chondroitin sulfate proteoglycans of human lung fibroblasts

Cultured human fetal lung fibroblasts produce some chondroitin sulfate proteoglycans that are extracted as an aggregate in chaotropic buffers containing 4 M guanidinium chloride. The aggregated proteoglycans are excluded from Sepharose CL4B and 2B, but become included, eluting with a Kav value of 0.53 from Sepharose CL4B, when Triton X-100 is included in the buffer. Conversely, some of the detergent-extractable chondroitin sulfate proteoglycans can be incorporated into liposomes, suggesting the existence of a hydrophobic membrane-intercalated chondroitin sulfate proteoglycan fraction. Purified preparations of hydrophobic chondroitin sulfate proteoglycans contain two major core protein forms of 90 and 52 kD. A monoclonal antibody (F58-7D8) obtained from the fusion of myeloma cells with spleen cells of BALB/c mice that were immunized with hydrophobic proteoglycans recognized the 90- but not the 52-kD core protein. The epitope that is recognized by the antibody is exposed at the surface of cultured human lung fibroblasts and at the surface of several stromal cells in vivo, but also at the surface of Kupffer cells and of epidermal cells. The core proteins of these small membrane-associated chondroitin sulfate proteoglycans are probably distinct from those previously identified in human fibroblasts by biochemical, immunological, and molecular biological approaches.     

Initiation of chondroitin sulfate biosynthesis: a kinetic analysis of UDP-D-xylose: core protein beta-D-xylosyltransferase

The nature of the primary signals important for the addition of xylose to serines on the core protein of the cartilage chondroitin sulfate proteoglycan has been investigated. The importance of consensus sequence elements (Acidic-Acidic-Xxx-Ser-Gly-Xxx-Gly) in the natural acceptor was shown by the significant decrease in acceptor capability of peptide fragments derived by digestion of deglycosylated core protein with Staphylococcus aureus V8 protease, which cleaves within the consensus sequence, compared to the similar reactivity of trypsin-derived peptide fragments, in which consensus sequences remain intact. A comparison of the acceptor efficiencies (Vmax/Km) of synthetic peptides containing the proposed xylosylation consensus sequence and the natural acceptor (deglycosylated core protein) was then made by use of the in vitro xylosyltransferase assay. The two types of substrates were found to have nearly equivalent acceptor efficiencies and to be competitive inhibitors of each other's acceptor capability, with Km = Kiapparent. These results suggest that the artificial peptides containing the consensus sequence are analogues of individual substitution sites on the core protein and allowed the kinetic mechanism of the xylosyltransferase reaction to be investigated, with one of the artificial peptides as a model substrate. The most probable kinetic mechanism for the xylosyltransferase reaction was found to be an ordered single displacement with UDP-xylose as the leading substrate and the xylosylated peptide as the first product released. This represents the first reported formal kinetic mechanism for this glycosyltransferase and the only one reported for a nucleotide sugar:protein transferase.     

Purification and characterization of glycosyltransferases involved in anthocyanin biosynthesis in cell-suspension cultures ofDaucus carota L.

The major anthocyanins accumulated by an Afghan cultivar ofDaucus carota L. are cyanidin 3-(xylosylglucosylgalactosides) acylated with sinapic or ferulic acid. The formation of the branched triglycoside present as a common structural element requires an ordered sequence of glycosylation events. Two of these enzymic glycosylation reactions have been detected in protein preparations from carrot cell-suspension cultures. The first step is a galactosyl transfer catalyzed by UDP-galactose: cyanidin galactosyltransferase (CGT) resulting in cyanidin 3-galactoside. The putative second step is the formation of cyanidin 3-(xylosylgalactoside) catalyzed by UDP-xylose: cyanidin 3-galactoside xylosyltransferase (CGXT). Both enzyme activities were characterized from crude protein preparations. The CGT was purified 526-fold from the cytosolic fraction of UV-irradiated cell cultures by ion-exchange chromatography on diethylaminoethyl (DEAE)-Sephacel, affinity chromatography on Blue Sepharose CL-6B, gel permeation chromatography on Sephadex G-75 and elution from the gel matrix after non-dissociating PAGE. Its molecular mass was estimated by SDS-PAGE and by calibrated gel permeation chromatography on Sephadex G-75. In both cases a molecular mass of 52 kDa was determined, indicating that the native protein is a monomer of 52 kDa. The galactosyl transfer and the xylosyl transfer are presumed to be catalyzed by separate enzymes.Abbreviations CGT UDP-galactose: cyanidin galactosyltransferase - CGXT UDP-xylose: cyanidin 3-galactoside xylosyltrans-ferase - DEAE diethylaminoethyl This study was supported by a grant from the Deutsche Forschun-gsgemeinschaft and a fellowship (W.E.G.) from the Land Baden-Württemberg. The skilful technical assistance of Johannes Madlung is gratefully acknowledged.     

Isolation and characterization of chondroitin sulfate proteoglycans from porcine thoracic aorta

A chondroitin sulfate proteoglycan fraction was prepared from the 3 M MgCl2 extract of porcine aortas by DEAE-cellulose chromatography, followed by gel filtration through Sepharose CL-4B. Affinity chromatography of the fraction with antithrombin III-agarose yielded two chondroitin sulfate proteoglycans of a non-binding (proteoglycan IA) and binding (proteoglycan IB) nature. Proteoglycans IA and IB were different from each other in molecular size, in proportion of the protein relative to the polysaccharide portion, and in size of the chondroitin sulfate chain. They were also distinguished immunochemically. These data indicate that the intima-media of the aorta contains at least two distinct species of chondroitin sulfate proteoglycan.     

Deglycosylation of chondroitin sulfate proteoglycan and derived peptides

In order to define the domain structure of proteoglycans as well as identify primary amino acid sequences specific for attachment of the various carbohydrate substituents, reliable techniques for deglycosylating proteoglycans are required. In this study, deglycosylation of cartilage chondroitin sulfate proteoglycan (CSPG) with minimal core protein cleavage was accomplished by digestion with chondroitinase ABC and keratanase, followed by treatment with anhydrous HF in pyridine. Nearly complete deglycosylation of secreted proteoglycan was verified within 45 min of HF treatment by loss of incorporated [3H]glucosamine label from the proteoglycan as a function of time of treatment, as well as by direct analysis of carbohydrate content and xylosyltransferase acceptor activity of unlabeled core protein preparations. The deglycosylated CSPG preparations were homogeneous and of high molecular weight (approximately 370,000). Comparison of the intact deglycosylated core protein preparations with newly synthesized unprocessed precursors (apparent Mr approximately 360,000) suggested that extensive proteolytic cleavage of the core protein did not occur during normal intracellular processing. Furthermore, peptide patterns generated after clostripain digestion of core protein precursor and of deglycosylated secreted proteoglycan were comparable. With the use of the clostripain digestion procedure, peptides were produced from unlabeled proteoglycan, and two predominant peptides from the most highly glycosylated regions (the chondroitin sulfate rich regions of the proteoglycan) were isolated, characterized, and deglycosylated. These peptides were found to follow similar kinetics of deglycosylation and to acquire xylose acceptor activity comparable to the intact core protein.     

Characterization of chondroitin sulfate isolated from trypsin-chymotrypsin digests of cartilage proteoglycans

Proteoglycans from bovine tracheal cartilage were digested with trypsin and chymotrypsin by procedures similar to those described by Mathews (Biochem. J.125, 37 (1971)). Chondroitin sulfate-peptide fragments in the digest were precipitated with cetylpyridinium chloride and subsequently fractionated on a preparative Sepharose 6B column. The fragments, which emerged from the column as a broad peak, were divided into five fractions. Rechromatography of these fractions on an analytical Sepharose 6B column indicated that they had K_av values from 0.17 (fraction 1) to 0.62 (fraction 5). The weight average molecular weight values obtained by meniscus depletion equilibrium centrifugation were 193,000, 126,000, 80,000, 46,000, and 23,000 for fractions 1 to 5, respectively. Values for the molecular weights and for the limiting viscosity numbers, [η], of the fractions were used to determine estimates for α of 0.40–0.46 and for K of 0.43–0.88 in the equation [η] = K·M_v^α. These values for α are consistent with a branched structure for the chondroitin sulfate fractions. Papain digests of each of the fractions were chromatographed on Sephadex G-200. The observed distributions of the monomer chains released by this protease were almost the same for each sample, which indicates that the individual chondroitin sulfate chains in all of the original fractions had nearly the same average molecular weights. The data in sum indicate that peptide fragments which contain from 1 to 8 polysaccharide chains are released when the proteoglycans are digested with trypsin-chymotrypsin.Analytical data indicated that all fractions contained 3–11% of their polysaccharide as keratan sulfate. This indicates either that about 50% of the keratan sulfate chains in the original proteoglycan molecules are located in close proximity to the chondroitin sulfate chains or that some peptides contain large numbers of keratan sulfate chains. Proteoglycan preparations which differed by a factor of about 6 in their ratio of chondroitin sulfate to protein yielded very similar elution patterns on Sepharose 6B after trypsin-chymotrypsin digestion.     

Isolation and partial characterization of low sulfated chondroitin 4-sulfate-proteoglycan in bovine blood.

Bovine plasma low sulfated chondroitin sulfate-proteoglycan (34 microgram/ml plasma), accounting for the main component of acidic glycosaminoglycans in blood, has been purified by isoelectric precipitation, dissociation with 4 M guanidine chloride followed by DEAE-chromatography, Sephadex G-200 chromatography and by preparative polyacrylamide gel electrophoresis. The proteoglycan, having a molecular weight of approx. 44,000, is composed of about 77% protein and 23% glycosaminoglycan at a molar ratio of 1 : 1 which could be cleaved by alkaline treatment into each component. Amino acid analysis of the proteoglycan and its glycosylpeptide has shown that the material is derived from a different origin from other tissue proteoglycans, though the amino acid residues surrounding O-glycosidic linkage to serine residue are quite similar to that of cartilage proteoglycan. Characteristic features of plasma low sulfate chondroitin sulfate-proteoglycan are discussed, compared with tissue materials.     

Purification and characterization of protease-resistant secretory granule proteoglycans containing chondroitin sulfate di-B and heparin-like glycosaminoglycans from rat basophilic leukemia cells

Proteoglycans were extracted from nuclease-digested sonicates of 10(9) rat basophilic leukemia (RBL-1) cells by the addition of 0.1% Zwittergent 3-12 and 4 M guanidine hydrochloride and were purified by sequential CsCl density gradient ultracentrifugation, DE52 ion exchange chromatography, and Sepharose CL-6B gel filtration chromatography under dissociative conditions. Between 0.3 and 0.8 mg of purified proteoglycan was obtained from approximately 1 g initial dry weight of cells with a purification of 200-800-fold. The purified proteoglycans had a hydrodynamic size range of Mr 100,000-150,000 and were resistant to degradation by a molar excess of trypsin, alpha-chymotrypsin, Pronase, papain, chymopapain, collagenase, and elastase. Amino acid analysis of the peptide core revealed a preponderance of Gly (35.4%), Ser (22.5%), and Ala (9.5%). Approximately 70% of the glycosaminoglycan side chains of RBL-1 proteoglycans were digested by chondroitinase ABC and 27% were hydrolyzed by treatment with nitrous acid. Sephadex G-200 chromatography of glycosaminoglycans liberated from the intact molecule by beta-elimination demonstrated that both the nitrous acid-resistant (chondroitin sulfate) and the chondroitinase ABC-resistant (heparin/heparan sulfate) glycosaminoglycans were of approximately Mr 12,000. Analysis of the chondroitin sulfate disaccharides in different preparations by amino-cyano high performance liquid chromatography revealed that 9-29% were the unusual disulfated disaccharide chondroitin sulfate di-B (IdUA-2-SO4----GalNAc-4-SO4); the remainder were the monosulfated disaccharide GlcUA----GalNAc-4-SO4. Subpopulations of proteoglycans in one preparation were separated by anion exchange high performance liquid chromatography and were found to contain chondroitin sulfate glycosaminoglycans whose disulfated disaccharides ranged from 9-49%. However, no segregation of subpopulations without both chondroitin sulfate di-B and heparin/heparan sulfate glycosaminoglycans was achieved, suggesting that RBL-1 proteoglycans might be hybrids containing both classes of glycosaminoglycans. Sepharose CL-6B chromatography of RBL-1 proteoglycans digested with chondroitinase ABC revealed that less than 7% of the molecules in the digest chromatographed with the hydrodynamic size of undigested proteoglycans, suggesting that at most 7% of the proteoglycans lack chondroitin sulfate glycosaminoglycans.(ABSTRACT TRUNCATED AT 400 WORDS)     

Variations in the composition of arterial wall isomeric chondroitin sulfate proteoglycans among different animal species

1. Isomeric chondroitin sulfate proteoglycans were extracted from human, bovine, swine and rabbit aortas by 4 M guanidine-HCl and were fractionated and purified by CsCl isopycnic centrifugation, Sepharose CL-4B gel filtration, DEAE-Sepharose ion-exchange chromatography and octyl-Sepharose hydrophobic interaction chromatography. 2. The molecular size and the composition of isomeric chondroitin sulfate proteoglycans varied among species. Variations were also noted in the composition and molecular weight of constituent glycosaminoglycan chains. 3. Observations made on chondroitinase ABC and chondroitinase AC digests of proteoglycans indicate that dermatan sulfate is linked to the core proteins through chondroitin sulfates.