Interactions between chondroitin sulfate and concanavalin A

Chondroitin sulfate, the major extracellular matrix glycosaminoglycan, formed an insoluble complex with concanavalin A at pH 5.4 or below. Concanavalin A (500 μg/ml) reacted only with a relatively narrow concentration range of chondroitin sulfate (optimally between 5 and 50 μg/ml) at pH 5.4 in 0.05 M buffer. Similar precipitin-like interactions were seen between concanavalin A and hyaluronic acid or heparin. No precipitating complexes formed between concanavalin A and the glycosaminoglycans at these concentrations in physiological salt solutions (approx. 0.15 M) unless the pH was below 4.5. Precipitating self-aggregates of concanavalin A appeared to be promoted by chondroitin sulfate at pH 7.3, but no significant precipitation occurred between the reactants at this pH even at very high concentrations, nor did soluble complexes form as determined by affinity chromatography on Sephadex G-200 or fractionation on Bio-Gel P-200. Thus, binding between the lectin and glycosaminoglycans appeared to depend upon reversible non-specific electrostatic interactions observed only at low pH and low ionic strength. Stable interactions were not seen in experiments using physiologically balanced salts at near neutral pH.     

Polyelectrolyte complexes. 2. Interaction between collagen and polyanions

The electrostatic interactions that occur in connective tissue between polyanions and proteins have been studied in model systems by a technique involving a fluorescent probe, acridine orange. It was found that collagen bound more strongly than bovine serum albumin to the polyanions studied. At pH 3.0, collagen formed strong complexes of definite stoichiometry with chondroitin-4-sulphate, chondroitin-6-sulphate, heparin and polystyrene sulphonate that were stable in sodium chloride solution of 0.1 M. The complexes of collagen with hyaluronic acid, or carboxymethylcellulose were less stable. The effect of pH variations (3.0–9.0) on the binding was investigated. Critical electrolyte concentrations (NaCl) were determined for complexes of collagen with glycosaminoglycans that dissociated at salt concentrations below that at which collagen precipitates. The values obtained were, 0.1 M for hyaluronic acid, and 0.5 M for chondroitin sulphate.     

Inhibition of leucocytic lysosomal enzymes by glycosaminoglycans in vitro.

1. A lysosomal fraction was separated by density-gradient centrifugation from a highly purified human polymorphonuclear leucocyte suspension. 2. Some 23 different lysosomal enzymes were assayed for activity in the presence of various concentrations of glycosaminoglycans. 3. The 21 acid hydrolases assayed were strongly inhibited to different degrees by low (0-12 mmol/l) concentrations of glycosaminoglycans in a pH-dependent manner. Thus inhibitions were stronger below pH4.5, with activity returning to control values at about pH5.0. 4. On a molar basis, the inhibitory activity for the several glycosaminoglycans studied was: heparin greater than chondroitin sulphate greater than hyaluronic acid. 5. Once the glycosaminoglycan-acid hydrolase complex was formed, it was partially dissociated by slight elevations in the pH of the incubation medium, by increasing the ionic strength of the incubation medium, or by adding several cationic proteins (e.g. histone, protamine). 6. As leucocytic lysosomes contain large amounts of chondroitin sulphate, and have a strongly acid intragranular pH, we suggest that glycosaminoglycans may modify lysosomal function through the formation of complexes with lysosomal enzymes, by inhibiting the digestive activity of the acid hydrolases when the intralysosomal pH is below their pI.     

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.     

Formation of dermatan sulfate by cultured human skin fibroblasts. Effects of sulfate concentration on proportions of dermatan/chondroitin

[3H,35S]Dermatan/chondroitin sulfate glycosaminoglycans produced during culture of fibroblasts in medium containing varying concentrations of sulfate were tested for their susceptibility to chondroitin ABC lyase and chondroitin AC lyase. Chondroitin ABC lyase completely degraded [3H]hexosamine-labeled and [35S] sulfate-labeled dermatan/chondroitin sulfate to disaccharides. Chondroitin AC lyase treatment of the labeled glycosaminoglycans produced different results. With this enzyme, dermatan/chondroitin sulfate formed at high concentrations of sulfate yielded small glycosaminoglycans and larger oligosaccharides but almost no disaccharide. This indicated that the dermatan/chondroitin sulfate co-polymer contained mostly iduronic acid with only an occasional glucuronic acid. As the medium sulfate concentration was progressively lowered, there was a concomitant increase in the susceptibility to degradation by chondroitin AC lyase. Thus, the labeled glycosaminoglycans formed at the lowest concentration of sulfate yielded small oligosaccharides including substantial amounts of disaccharide. The smaller chondroitin AC lyase-resistant [3H,35S]dermatan/chondroitin sulfate oligosaccharides were analyzed by gel filtration. Results indicated that, in general, the iduronic acid-containing disaccharide residues present in the undersulfated [3H,35S]glycosaminoglycan were sulfated, whereas the glucuronic acid-containing disaccharide residues were non-sulfated. This work confirms earlier reports that there is a relationship between epimerization and sulfation. Moreover, it demonstrates that medium sulfate concentration is critical in determining the proportions of dermatan to chondroitin (iduronic/glucuronic acid) produced by cultured cells.     

Highly sulfated glycosaminoglycans inhibit aggrecanase degradation of aggrecan by bovine articular cartilage explant cultures.

The catabolism of 35S-labeled aggrecan and loss of tissue glycosaminoglycans was investigated using bovine articular cartilage explant cultures maintained in medium containing 10(-6) M retinoic acid or 40 ng/ml recombinant human interleukin-1alpha (rHuIL-1alpha) and varying concentrations (1-1000 microg/ml) of sulfated glycosaminoglycans (heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate) and calcium pentosan polysulfate (10 microg/ml). In addition, the effect of the sulfated glycosaminoglycans and calcium pentosan polysulfate on the degradation of aggrecan by soluble aggrecanase activity present in conditioned medium was investigated. The degradation of 35S-labeled aggrecan and reduction in tissue levels of aggrecan by articular cartilage explant cultures stimulated with retinoic acid or rHuIL-1alpha was inhibited by heparin and heparan sulfate in a dose-dependent manner and by calcium pentosan polysulfate. In contrast, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate did not inhibit the degradation of 35S-labeled aggrecan nor suppress the reduction in tissue levels of aggrecan by explant cultures of articular cartilage. Heparin, heparan sulfate and calcium pentosan polysulfate did not adversely affect chondrocyte metabolism as measured by lactate production, incorporation of [35S]-sulfate or [3H]-serine into macromolecules by articular cartilage explant cultures. Furthermore, heparin, heparan sulfate and calcium pentosan polysulfate inhibited the proteolytic degradation of aggrecan by soluble aggrecanase activity. These results suggest that highly sulfated glycosaminoglycans have the potential to influence aggrecan catabolism in articular cartilage and this effect occurs in part through direct inhibition of aggrecanase activity.     

Complexing of fibronectin glycosaminoglycans and collagen

Collagen-fibronectin complexes, formed by binding of fibronectin to gelatin or collagen insolubilized on Sepharose, were found to bind 20–40% of radioactivity in [³⁵S]heparin. Fibronectin attached directly to Sepharose also bound [³⁵S]heparin, while gelatin-Sepharose without fibronectin did not. Unlabeled heparin and highly sulfated heparan sulfate efficiently inhibited the binding of [³⁵S]heparin, hyaluronic acid and dermatan sulfate were slightly inhibitory, while chondroitin sulfates and heparan sulfate with a low sulfate content did not inhibit.The interaction of heparin with fibronectin bound to gelatin resulted in complexes which required higher concentrations of urea to dissociate than complexes of fibronectin and gelatin alone. Heparin as well as highly sulfated heparan sulfate and hyaluronic acid brought about agglutination of plastic beads coated with gelatin when fibronectin was present. Neither fibronectin nor glycosaminoglycans alone agglutinated the beads.It is proposed that the multiple interactions of fibronectin, collagen and glycosaminoglycans revealed in these assays could play a role in the deposition of these substances as an insoluble extracellular matrix. Alterations of the quality or quantity of any one of these components could have important effects on cell surface interactions, including the lack of cell surface fibronectin in malignant cells.     

Interaction of sulfated glycosaminoglycans with lectins

The sulfated glycosaminoglycans, such as keratan sulfate and chitin sulfate having 3-hydroxy free N-acetyl-beta-D-glucosaminyl residues as constituents, reacted with wheat germ agglutinin and Solanum tuberosum agglutinin by sugar-specific interaction. The glycosaminoglycans showed different inhibitory activities to the hemagglutination reaction of these lectins and keratan sulfate and its modified products formed insoluble complexes with both of the lectins at pH 7.0 in physiological saline solutions (0.15 M NaCl). S. tuberosum agglutinin was precipitated within a particularly narrow concentration range of keratan sulfate, and the formation of a soluble complex was observed by gel chromatography. These interactions were specifically inhibited by N,N'-diacetylchitobiose but not by 2 M NaCl. The specific interactions of the glycosaminoglycans with S. tuberosum agglutinin were confirmed by their ultraviolet difference spectra with two peaks at 285 and 298 nm attributable to the tryptophan residues in the binding site of the agglutinin. It was also found that S. tuberosum agglutinin and wheat germ agglutinin have different binding specificities. The presence of sulfate groups in either keratan sulfate or chitin sulfate did not interfere with their specific interactions with S. tuberosum agglutinin as strongly as with wheat germ agglutinin. The N-acetylneuraminic acid residues in keratan sulfate were found to be receptor sites for wheat germ agglutinin but not for S. tuberosum agglutinin.     

Spectroscopic properties of complexes of acridine orange with glycosaminoglycans II. Aggregated complexes-evidence for long-range order.

Aggregated complexes of acridine orange with dermatan and chondroitin sulfates have been studied in aqueous solution by absorption and circular dichroism spectroscopy. Aggregation was found to be favored at high-dye and glycosaminoglycan concentrations, and in solutions where anionic sites of the glycosaminoglycan are effectively complexed with dye. The aggregates can be removed from solution by centrifugation at 27,000 × g for 1 hr or by filtration through a membrane containing pores of 0.1 μm diameter. The aggregated complexes exhibit large-magnitude-ellipticity circular dichroism bands. In addition, the circular dichroism spectrum observed for a solution containing aggregated acridine orange/chondroitin 4-sulfate complexes is nearly a mirror image of that obtained for aggregated acridine orange/dermatan sulfate complexes. Cooperative alterations (sharp transitions) in the circular dichroism ellipticities of the aggregates occur at elevated temperatures, and result in spectroscopically distinct aggregates upon cooling. The circular dichroism properties and temperature effects are attributed to a supramolecular ordering of acridine orange/glycosaminoglycan complexes within the aggregates, which can be reorganized to a more stable form at high temperatures. Mixed aggregates, containing two different glycosaminoglycans, can be formed. The circular dichroism properties of the mixed aggregates also indicate the existence of long-range order in the arrangement of the complexes. Mixed aggregates containing dermatan sulfate and either chondroitin 4-sulfate or chondroitin 6-sulfate resemble pure dermatan sulfate aggregates in circular dichroism characteristics.     

Specificity and affinity of binding of herpes simplex virus type 2 glycoprotein B to glycosaminoglycans.

Herpes simplex virus type 2 (HSV-2) interacts with cell surface glycosaminoglycans during virus attachment. Glycoprotein B of HSV-2 can potentially mediate the interaction between the virion and cell surface glycosaminoglycans. To determine the specificity, kinetics, and affinity of these interactions, we used plasmon resonance-based biosensor technology to measure HSV-2 glycoprotein binding to glycosaminoglycans in real time. The recombinant soluble ectodomain of HSV-2 gB (gB2) but not the soluble ectodomain of HSV-2 gD bound readily to biosensor surfaces coated with heparin. The affinity constants (Kds) were determined for gB2 (Kd = 7.7 x 10(-7) M) and for gB2 deltaTM (Kd = 9.9 x 10(-7) M), a recombinant soluble form of HSV-2 gB in which only its transmembrane domain has been deleted. gB2 binding to the heparin surface was competitively inhibited by low concentrations of heparin (50% effective dose [ED50] = 0.08 microg/ml). Heparan sulfate and dermatan sulfate glycosaminoglycans have each been suggested as cell surface receptors for HSV. Our biosensor analyses showed that both heparan sulfate and dermatan sulfate inhibited gB2 binding (ED50 = 1 to 5 microg/ml), indicating that gB2 interacts with both heparin-like and dermatan sulfate glycosaminoglycans. Chondroitin sulfate A, in contrast, inhibited gB2 binding to heparin only at high levels (ED50 = 65 microg/ml). The affinity and specificity of gB2 binding to glycosaminoglycans demonstrated in these studies support its role in the initial binding of HSV-2 to cells bearing heparan sulfate or dermatan sulfate glycosaminoglycans.     

Large effects of preparative techniques on lymphocyte cyclic AMP content.

When a cell suspension is formed by disruption of a pig lymph node into medium, large and transient increases in intracellular cyclic AMP occur. Similar effects are observed when pig lymphocytes are centrifuged and the cell pellets resuspended, or when the cells are subjected to rapid temperature changes. These observations define the conditions required to manipulate the cells while maintaining a stable cyclic AMP concentration. Under these conditions, neither concanavalin A nor ionophore A23187 at mitogenic concentrations have any early effect on cyclic AMP in pig lymphocytes, but small increases in cyclic AMP (less than 2-fold) were observed at supramitogenic concentrations of concanavalin A (50 microgram/ml) or A23187 (500nM). Mouse thymocytes show qualitatively similar but much smaller changes in cyclic AMP concentration in response to experimental manipulations, and no response to mitogenic or supramitogenic concentrations of concanavalin A below the cytotoxic value.     

Glycosaminoglycan production by bovine aortic endothelial cells cultured in sulfate-depleted medium

Bovine aortic endothelial cells were cultured in medium containing [3H]glucosamine and concentrations of [35S]sulfate ranging from 0.01 to 0.31 mM. While the amount of [3H]hexosamine incorporated into chondroitin sulfate and heparan sulfate was constant, decreasing concentrations of sulfate resulted in lower [35S]sulfate incorporation. Sulfate concentrations greater than 0.11 mM were required for maximal [35S]sulfate incorporation. Chondroitin sulfate was particularly affected so that the sulfate to hexosamine ratio in [3H]chondroitin [35S]sulfate dropped considerably more than the sulfate to hexosamine ratio in [3H] heparan [35S]sulfate. Sulfate concentration had no effect on the ratio of chondroitin 4-sulfate to chondroitin 6-sulfate. The ratios of sulfate to hexosamine in cell-associated glycosaminoglycans were essentially identical with the ratios in media glycosaminoglycans at all sulfate concentrations. DEAE-cellulose chromatography confirmed that sulfation of chondroitin sulfate was particularly sensitive to low sulfate concentrations. While cells incubated in medium containing 0.31 mM sulfate produced chondroitin sulfate which eluted later than heparan sulfate, cells incubated in medium containing less than 0.04 mM sulfate produced chondroitin sulfate which eluted before heparan sulfate and near hyaluronic acid, indicating that many chains were essentially unsulfated. At intermediate concentrations of sulfate, chondroitin sulfate was found in very broad elution patterns suggesting that most did not fit an "all or nothing" mechanism. Heparan sulfate produced at low concentrations of sulfate eluted with narrower elution patterns than chondroitin sulfate, and there was no indication of any "all or nothing" sulfation.     

Altered expression of glycosaminoglycans in metastatic 13762NF rat mamma adenocarcinoma cells

A difference in the expression and metabolism of sulfated glycosaminoglycans between rat mammary tumor cells derived from a primary tumor and those from its metastatic lesions has been observed. Cells from the primary tumor possessed about equal quantities of chondroitin sulfate and heparan sulfate on their cell surfaces but released fourfold more chondroitin sulfate than heparan sulfate into their medium. In contrast, cells from distal metastatic lesions expressed approximately 5 times more heparan sulfate than chondroitin sulfate in both medium and cell surface fractions. This was observed to be the result of differential synthesis of the glycosaminoglycans and not of major structural alterations of the individual glycosaminoglycans. The degree of sulfation and size of heparan sulfate were similar for all cells examined. However, chondroitin sulfate, observed to be only chondroitin 4-sulfate, from the metastases-derived cells had a smaller average molecular weight on gel filtration chromatography and showed a decreased quantity of sulfated disaccharides upon degradation with chondroitin ABC lyase compared to the primary tumor derived cells. Major qualitative or quantitative alterations were not observed for hyaluronic acid among the various 13762NF cells. The metabolism of newly synthesized sulfated glycosaminoglycans was also different between cells from primary tumor and metastases. Cells from the primary tumor continued to accumulate glycosaminoglycans in their medium over a 72-h period, while the accumulation of sulfated glycosaminoglycans in the medium of metastases-derived cells showed a plateau after 18-24 h. A pulse-chase kinetics study demonstrated that both heparan sulfate and chondroitin sulfate were degraded by the metastases-derived cells, whereas the primary tumor derived cells degraded only heparan sulfate and degraded it at a slower rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complexes of heparin proteoglycans, chondroitin sulfate E proteoglycans, and [3H]diisopropyl fluorophosphate-binding proteins are exocytosed from activated mouse bone marrow-derived mast cells

The predominant [3H]diisopropyl fluorophosphate (DFP)-binding proteins that are released from the secretory granules of activated mouse bone marrow-derived mast cells (BMMC) are demonstrated to have an isoelectric point of approximately 9.1 and to be complexed to proteoglycans. Upon Sepharose CL-2B chromatography of the supernatants of calcium ionophore-activated BMMC, 67-78% of the total exocytosed [3H]DFP-binding proteins co-eluted in the excluded volume of the column as a greater than 1 X 10(7) Mr complex bound to 4-7% of the total exocytosed proteoglycans. The remainder of the exocytosed proteoglycans, which filtered in the included volume of the gel filtration column with a Kav of 0.66, contained chondroitin sulfate E glycosaminoglycans. After dissociation of the large Mr complexes of [3H]DFP-binding proteins-proteoglycans with 5 M NaCl and removal of the proteins via phenyl-Sepharose chromatography, the proteoglycans filtered from the Sepharose CL-2B column as a single peak with a Kav of 0.66. The susceptibility of 24-59% and 36-76% of the glycosaminoglycans in the large Mr complex to degradation by nitrous acid and chondroitinase ABC, respectively, indicated the presence of proteoglycans that contained heparin and chondroitin sulfate glycosaminoglycans. Disaccharide analysis revealed that the chondroitin sulfate in the high Mr complex was chondroitin sulfate E. Following chondroitinase ABC treatment of the large Mr complex, the residual heparin proteoglycans filtered on Sepharose CL-4B under dissociative conditions with the same Kav as the original, untreated proteoglycans. Thus, the protein-proteoglycan complexes that are exocytosed from activated mouse BMMC contain approximately equal amounts of proteoglycans of comparable size that bear either predominantly heparin or predominantly chondroitin sulfate E glycosaminoglycans. The demonstration of these secreted complexes indicates that the intragranular protease-resistant heparin and chondroitin sulfate E proteoglycans in the T cell factor-dependent BMMC bind serine proteases throughout the activation-secretion response.     

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)     

Microelectrophoresis studies of the binding of glycosaminoglycans to phosphatidylcholine liposomes.

The binding of the glycosaminoglycans (GAG) chondroitin sulfate and heparin and the homologous molecule dextran sulfate to multilamellar dimyristoyl phosphatidylcholine (DMPC), dilaureyl phosphatidylcholine (DLPC) and egg lecithin liposomes was investigated by microelectrophoresis measurements. Drastic changes of the zeta potential of the liposomes to negative values indicate the binding of the highly anionic macromolecules. Binding depends strongly on Ca2+ and NaCl concentrations in the medium and does not occur in the absence of Ca2+. The adsorption is saturated at concentrations of about 0.1 mg/ml chondroitin sulfate and heparin and 0.01 mg/ml dextran sulfate. In the gel state of the phospholipid bilayer more GAG can associate with the surface compared to the fluid state.     

Purification of hyaluronidase from human placenta.

Hyaluronidase [EC 3.2.1.35] was isolated from human placenta and purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography and gel filtration on Sephadex G-150. Its isoelectric point was at pH 5.2 and the molecular weight was 7 X 10(4) based on Sephadex G-200 gel filtration data. This enzyme was very stable at temperatures below 30 degree, but was almost completely inactivated at 60degree within 30 min. Its optimum pH was 3.9, a characteristic property of a lysosomal hyaluronidase. The Michaelis constant was 1.18 x 10(-1) mg per ml with purified hyaluronate. This enzyme depolymerized hyaluronate, chondroitin, chondroitin 4-sulfate and 6-sulfate, and the end product formed from hyaluronate was tetrasaccharide. Its biological diffusing activity was statistically significant on intracutaneous injection of 1.86 mU of the hyaluronidase into the back skine of a rabbit.     

Disaccharide analysis and molecular mass determination to microgram level of single sulfated glycosaminoglycan species in mixtures following agarose-gel electrophoresis.

The separation of sulfated glycosaminoglycans in mixtures by agarose-gel electrophoresis and the recovery of single polysaccharide bands has been applied to the characterization of polysaccharides extracted from tissues without previous purification of single species. Sulfated glycosaminoglycans, heparin with its two components, slow-moving and fast-moving, heparan sulfate, dermatan sulfate, and chondroitin sulfate, were separated to microgram level by conventional agarose-gel electrophoresis. After their separation, they were fixed in the agarose-gel matrix by precipitation in a cetyltrimethylammonium bromide solution, making them visible on a dark background. After recovery of gel containing the fixed bands, high temperatures (90 degrees C for 15 min) were necessary to dissolve the gel matrix, and a solution of NaCl (3 M) was used to release sulfated polysaccharides from the complex with cetyltrimethylammonium. After precipitation of glycosaminoglycans in the presence of ethanol, the recovery of slow-moving heparin, fast-moving heparin, heparan sulfate, dermatan sulfate, and chondroitin sulfate was from 1 to 10 microg, with a percentage greater than 45% and a purity above 90%. Sulfated glycosaminoglycans in mixtures recovered from gel matrix as single species were evaluated for purity and characterized for unsaturated disaccharides after treatment with bacterial lyases (heparinases for heparin and heparan sulfate samples, and chondroitinases for dermatan sulfate and chondroitin sulfate) and molecular mass. Bovine lung and heart Glycosaminoglycans were extracted and separated into single species by agarose-gel electrophoresis and recovered from gel matrix after treatment in cetyltrimethylammonium solution. Unsaturated disaccharides pattern, the sulfate to carboxyl ratio, and the molecular mass of each single polysaccharide species were determined.     

The cell surface proteoglycan from mouse mammary epithelial cells bears chondroitin sulfate and heparan sulfate glycosaminoglycans

The cell surface proteoglycan fraction isolated by mild trypsin treatment of NMuMG mouse mammary epithelial cells contains largely heparan sulfate, but also 15-24% chondroitin sulfate glycosaminoglycans. We conclude that this fraction contains a unique hybrid proteoglycan bearing both heparan sulfate and chondroitin sulfate glycosaminoglycans because (i) the proteoglycan behaves as a single species by sizing, ion exchange and collagen affinity chromatography, and by isopycnic centrifugation, even in the presence of 8 M urea or 4 M guanidine hydrochloride, (ii) the behavior of the chondroitin sulfate in these separation techniques is affected by heparan sulfate-specific probes and vice versa, and (iii) proteoglycan core protein bearing both heparan sulfate and chondroitin sulfate is recognized by a single monoclonal antibody. Removal of both types of glycosaminoglycan reduces the proteoglycan to a core protein of approximately 53 kDa. The proteoglycan fraction is heterogeneous in size, largely due to a variable number and/or length of the glycosaminoglycan chains. We estimate that one or two chondroitin sulfate chains (modal Mr of 17,000) exist on the proteoglycan for every four heparan sulfate chains (modal Mr of 36,000). Synthesis of these chains is reportedly initiated on an identical trisaccharide that links the chains to the same amino acid residues on the core protein. Therefore, some regulatory information, perhaps residing in the amino acid sequence of the core protein, must determine the type of chain synthesized at any given linkage site. Post-translational addition of these glycosaminoglycans to the protein may provide information affecting its ultimate localization. It is likely that the protein is directed to specific sites on the cell surface because of the ability of the glycosaminoglycans to recognize and bind extracellular components.