Heterogeneity of rat rib chondroitin sulfate and susceptibility to rat gastric chondrosulfatase

Cartilage chondroitin sulfate isolated directly from rat rib or from in vitro culture of rat rib constitutes a population of glycosaminoglycans which is heterogeneous with respect to size, degree of sulfation and content of N-acetylgalactosamine 4-sulfate. Fractions elute from Dowex-1 in order of increasing molecular size and degree of sulfation up to a certain limit. Unsulfated disaccharides and disulfated disaccharides are present in both the undersulfated chondroitin sulfate fractions and in the average or more representative chondroitin sulfate. A small content of disaccharide 6-sulfate is present in all fractions and appears to be an integral part of the chondroitin 4-sulfate molecules. Rat gastric chondrosulfatase hydrolyzes sulfate preferentially from the larger chondroitin 4-sulfate molecules, and the sulfate is removed primarily from the disaccharide 4-sulfate units.     

Separation and characterization of chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase from chick embryo cartilage

Two distinct sulfotransferases (chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase), which catalyzed transfer of sulfate to position 6 and position 4 of acetylgalactosamine residues of chondroitin, were extracted from epiphyseal cartilage of 14-day-old chick embryos and separated by gel chromatography on Sephacryl S-200 in the presence of 3 M guanidine-HCl. When the enzyme solutions containing 3 M guanidine-HCl were dialyzed against 0.02 M Tris-HCl, pH 7.2, containing 10% glycerol, chondroitin 4-sulfotransferase became almost insoluble, whereas chondroitin 6-sulfotransferase remained soluble. Endogenous acceptors for sulfate transfer were completely removed from both enzyme preparations. Addition of basic proteins and polyamines as well as Mn²⁺ to the incubation medium caused a stimulation of both sulfotransferases; the stimulation of chondroitin 6-sulfotransferase with these cations was higher than that of chondroitin 4-sulfotransferase. The K_m values for 3′-phosphoadenylyl sulfate of both enzymes were much smaller in the presence of protamine or spermine than in the presence of Mn²⁺. The two sulfotransferases differed in the requirement for sulfhydryl compounds; in the absence of sulfhydryl compounds, the activity of chondroitin 4-sulfotransferase was very low, whereas the activity of chondroitin 6-sulfotransferase was essentially unaffected. These observations indicate that at least two sulfotransferases are involved in the biosynthesis of chondroitin sulfate, and suggest that the production of the isomers of chondroitin sulfate in chondrocytes is affected by various factors such as the intracellular concentration of sulfhydryl compounds and basic substances.     

Tissue specific distribution of sulfated mucopolysaccharides in mammals

A comparative study on the distribution of sulfated mucopolysaccharides in several tissues of five mammalian species is reported. It is shown that each tissue has a characteristic composition differing from each other regarding the relative amount, type and molecular size of chondroitin sulfate A/C, chondroitin sulfate B and heparan sulfate. It is also shown that the same tissue from different mammals has the same types and proportions of sulfated mucopolysaccharides, but with different molecular weights. Exception to this rule was observed for the distribution of heparin which was present only in a few tissues of the five mammals studied.The possible involvement of the sulfated mucopolysaccharides in cell recognition and/or adhesiveness is discussed in view of this characteristic distribution.     

Substrate recognition by bacterial solute-binding protein is responsible for import of extracellular hyaluronan and chondroitin sulfate from the animal host

ABSTRACT

Glycosaminoglycans (GAGs) such as hyaluronan and chondroitin in animal extracellular matrices contain disaccharide-repeating units. In a Gram-negative pathogenic Streptobacillus moniliformis, which belongs to Fusobacteria phylum and resides in rodent oral cavities, the solute-binding protein (Smon0123)-dependent ATP-binding cassette transporter imports unsaturated hyaluronan/chondroitin disaccharides into the cytoplasm after GAG lyase-dependent depolymerization. Here we show substrate recognition of unsaturated hyaluronan disaccharide by Smon0123. Moreover, Smon0123 exhibited no affinity for unsaturated chondroitin disaccharides containing three sulfate groups, distinct from non-sulfated, mono-sulfated, and di-sulfated chondroitin disaccharides previously identified as substrates. Crystal structure of Smon0123 with unsaturated hyaluronan disaccharide demonstrates that several residues, including Trp284 and Glu410, are crucial for binding to unsaturated hyaluronan/chondroitin disaccharides, whereas arrangements of water molecules at binding sites are found to be substrate dependent through comparison with substrate-bound structures determined previously. These residues are well conserved in Smon0123-like proteins of fusobacteria, and probably facilitate the fusobacterial residence in hyaluronan-rich oral cavities.     

Determination of the distribution of constituent disaccharide units within the chain near the linkage region of shark-cartilage chondroitin sulfate C

A method for analyzing the distribution of constituent disaccharide units within the chain near the linkage region of chondroitin sulfate has been developed. The method consists of (a) chemical modification of the reducing terminal residue in the polysaccharide by a 2-(2,4-dinitrophenylamino)ethylamino (DNP-AEA) group, (b) controlled fragmentation of the DNP-AEA-labeled polysaccharide with chondroitinase AC-I, followed by separation of the digestion products into the DNP-AEA-labeled fragments and unlabeled fragments on octyl-Sepharose CL-4B gel, (c) fractionation of the DNP-AEA-labeled fragments into fractions having different chain-lengths of Sephadex G-100 (superfine), and (d) determination of the disaccharide unit composition of the de-dinitrophenylated products (AEA-labeled fragments) by the method combining chondroitinase AC-II treatment with HPLC analysis. A preparation of shark cartilage chondroitin sulfate C, which has been characterized well with regard to molecular species (M_r 48 000; average number of repeating disaccharide units (dp_av) 93–94; consisting of chondroitin 6-sulfated 22.5%, disulfate (D type) 10.3%, and nonsulfated units 0.4%), was analyzed by the above method. On the basis of the data obtained, distribution features of the dissacharide units within the chain near the linkage region of the polysaccharide (dp_av 27) were estimated. It was, however, difficult to propose a final primary sequence of the polysaccharide chain, although there was a definite trend towards an enrichment of 4-sulfated and nonsulfated dissacharide residues in the area close to the linkage region (dp_av 3–9 or 11). This was apparent together with an enrichment of 6-sulfated and disulfated disaccharide residues in the area distant from the linkage region (dp_av 11 of 13–27).     

Structural characterization of the bovine tracheal chondroitin sulfate chains and binding of Plasmodium falciparum-infected erythrocytes

Sequestration of Plasmodium falciparum-infected red blood cells (IRBCs) in the human placenta is mediated by chondroitin 4-sulfate (C4S). A cytoadherence assay using chondroitin sulfate proteoglycans (CSPGs) is widely used for studying C4S-IRBC interactions. Bovine tracheal chondroitin sulfate A (CSA) preparation lacking a major portion of core protein has been frequently used for the assay. Here the CSPG purified from bovine trachea and CSA were assessed for IRBC binding and the CS chains studied in detail for structure-activity relationship. The IRBCs bound at significantly higher density to the CSPG than CSA. The CS chains of CSPG/CSA are heterogeneous with varying levels of 4- and 6-sulfates, which are distributed such that approximately 80% of the 4-sulfated disaccharides are present as single and blocks of two or three separated by one to three 6-sulfated disaccharides. The remainder of the 4-sulfated disaccharides is present in blocks composed of 4-12 units, separated by 6-sulfated disaccharides. In the IRBC adherence inhibition analysis, CSA fragments with 88%-92% 4-sulfate were significantly less inhibitory than the intact CSA, indicating that the regions consisting of shorter 4-sulfated blocks efficiently bind IRBCs despite the presence of relatively high levels of 6-sulfate. This is because the 6-sulfated disaccharides have unsubstituted C-4 hydroxyls that are crucial for IRBC binding. The presence of high levels of 6-sulfate, however, significantly interfere with the IRBC binding activity of CSA, which otherwise would more efficiently bind IRBCs. Thus our study revealed the distribution pattern of 4- and 6-sulfate in bovine tracheal CSA and structural basis for IRBC binding.     

Changes in sulfated mucopolysaccharide composition of mammalian tissues during growth and in cancer tissues

The distribution of sulfated mucopolysaccharides in different tissues during growth and in cancer tissues is reported. It is shown that most of the tissues of 1 day-old rats and rabbits contain chondroitin sulfate A/C, chonroitin sulfate B and heparan sulfate in about the same proportions, whereas in adult animals chondroitin sulfate A/C decreases in concentration or disappears. Changes in the relative proportions of chondroitin sulfate B and heparan sulfate were also observed in most of the tissues. In rats, these changes occur in the first 25 days of extrauterine development. A great increase of chondoitin sulfate A/C was observed in human tumors of different origins when compared with the normal adjacent tissues. Changes in the relative proportions of chondroitin sulfate B and heparan sulfate were also observed in most of the tumors analysed. The possible role of chondroitin sulfate A/C in cell division is discussed in view of the present findings.     

High-performance liquid chromatographic determinations of disaccharides resulting from enzymatic degradation of isomeric chondroitin sulfates.

The use of Whatman Partisil-10 PAC and Altex LiChrosorb NH₂ bonded phase columns for the high-performance liquid chromatographic separation of the disaccharides obtained from digestion of isomeric chondroitin sulfates with chondroitinases has been investigated. The substituted unsaturated disaccharides in the enzymatic degradation mixtures undergo rapid isocratic separations on both bonded stationary phases. A complete separation can be established within 10 min. The development of these procedures has expedited enzymatic studies of isomeric chondroitin sulfates.The rate of depolymerization of chondroitin sulfates by the action of chondroitinases based on quantitative high-performance liquid chromatographic separations and detection of the disaccharide products was studied.     

Buffer induced modification of 6-sulfated disaccharide in chondroitin lyase digestions

High-performance liquid chromatographic analyses of chondroitin lyase AC or ABC hydrolysates revealed unexpected high content of material coeluting with the nonsulfated disaccharide 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyranosyl uronic acid)-d-galactose. Incubation of a commercial preparation of the 6-sulfated disaccharide, 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyranosyl uronic acid)-6-O-sulfo-d-galactose with “enriched Tris buffer” generated material coeluting with nonsulfated disaccharide. The amount of material exhibiting this anomalous chromatographic behavior was proportional to the amount of 6-sulfated disaccharide added to the incubation mixture. This suggested a precursor/product relationship between the 6-sulfated disaccharide and the anomalous peak. The result was specific for the 6-sulfated disaccharide: incubation of the 4-sulfated disaccharide, 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyranosyl uronic acid)-4-O-sulfo-d-galactose, with enriched Tris buffer did not generate material with anomalous chromatographic properties. When [³⁵S]sulfate labeled cartilage glycosaminoglycans were hydrolyzed with chondroitin lyases, some of the radioactivity coeluted with the nonsulfated disaccharide. Thus, buffer-induced modification of 6-sulfated disaccharide was not caused by hydrolysis of ester sulfate. Although the proportion of the 6-sulfated disaccharide which was recovered in the anomalous peak was constant for incubations done simultaneously, incubations done at different times gave variable results. Thus, control incubations of 6-sulfated disaccharide with chondroitinase buffer must be included with each reaction series to allow correction for the proportion of the material eluting with nonsulfated disaccharide which is actually 6-sulfated.     

Occurrence of a unique fucose-branched chondroitin sulfate in the body wall of a sea cucumber

The sulfated polysaccharides in the body wall of the sea cucumber occur as three fractions that differ markedly in molecular mass and chemical composition. The fraction containing a high molecular mass component has a high proportion of fucose and small amounts of galactose and amino sugars, whereas another fraction contains primarily a sulfated fucan. The third fraction (F-2), which represents the major portion of the sea cucumber-sulfated polysaccharides, contains approximately equimolar quantities of glucuronic acid, N-acetyl galactosamine, and fucose, and has a sulfate content higher than that in the other two fractions. The structure of fraction F-2 was examined in detail. This polysaccharide has an unusual structure composed of a chondroitin sulfate-like core, containing side chain disaccharide units of sulfated fucopyranosyl linked to approximately half of the glucuronic acid moieties through the O-3 position of the acid. These unusual fucose branches obstruct the access of chondroitinases to the chondroitin sulfate core of F-2. However, after partial acid hydrolysis, which removes the sulfated fucose residues from the polymer, fraction F-2 is degraded by chondroitinases into 6-sulfated and nonsulfated disaccharides.     

Distribution of chondroitin sulfate in cartilage proteoglycans under associative conditions.

Proteoglycan aggregates and proteoglycan subunits were extracted from bovine articular cartilage with guanidine-HC1 folowed by fractionation by equilibrium centrifugation in cesium chloride density gradients. The distribution of chondroitin sulfates (CS) in the cartilage proteoglycans was studied at the disaccharide level by digestion with chondroitinases. In the proteoglycan aggregate fraction, it was observed that the proportion of 4-sulfated disaccharide units to total CS increased from the bottom to the top fractions, whereas that of 6-sulfated disaccharide units was in the reverse order. Thus, the ratio of 4-sulfated disaccharide units to 6-sulfated disaccharide units increased significantly with decreasing density. The proportion of non-sulfated disaccharide units to total CS tended to increase with increasing density. These data indicate a polydisperse distribution of CS chains, under the conditions used here, in proteoglycan aggregates from bovine articular cartilage.     

Isolation and structural studies of heparan sulfates and chondroitin sulfates from three species of molluscs

The isolation and some structural features of heparan sulfates and chondroitin sulfates from three species of molluscs (Pomacea sp., Tagelus gibbus, and Anomalocardia brasiliana) are reported. It is shown that heparan sulfates with structural similarities to the ones found in mammals are present in the three molluscs analyzed. All the heparan sulfates were degraded by heparitinases I and II to four distinct unsaturated disaccharides with the same properties as the ones present in heparan sulfates of mammalian origin. This suggests that these four disaccharide units are maintained through the evolution. Furthermore, the proportion of these units varied in the heparan sulfates according to the species of origin. The chondroitin sulfates, on the other hand, exhibit different structural features according to the species of origin. For instance, by the action of chondroitinase AC, 4- and nonsulfated disaccharides are produced from Pomacea chondroitin, whereas 4- and 6-sulfated disaccharides are formed from Tagelus and Anomalocardia. The possible role of these compounds in cell recognition and/or adhesiveness is discussed in view of the present findings.     

Sulfation of chondroitin sulfate in human articular cartilage. The effect of age, topographical position, and zone of cartilage on tissue composition.

The chondroitin ABC lyase digestion products of normal human femoral condyle articular cartilage and of purified aggrecan were analyzed for their mono- and nonsulfated disaccharide composition. Changes in the total tissue chemistry were most pronounced during the period from birth to 20 years of age, when the -[GlcAbeta,3GalNAc6]- disaccharide content increased from approximately 50% to 85% of the total disaccharide content and there was a concomitant decrease in the content of the 4-sulfated disaccharide. In general, the disaccharide content of the deeper layers of immature cartilage were richer in the 4-sulfated residue than the upper regions of the tissue. As the tissue aged and decreased in thickness, the disaccharide composition became more evenly 6-sulfated. The newly synthesized chondroitin sulfate chains had a similar composition to the endogenous chains and also underwent the same age and zonal changes. The monoclonal antisera 3B3(+) and 2B6(+) were used to immunolocalize the unsaturated 6- and 4-sulfated residues generated at the reducing termini of the chondroitin sulfate chains by digestion with chondroitin ABC lyase, and these analyses indicated that the sulfation pattern at this position did not necessarily reflect the internal disaccharide composition of the chains. In summary, the sulfation pattern of chondroitin sulfate disaccharides from human normal articular cartilage varies with the age of the specimen, the position (topography) on the joint surface, and the zone of cartilage analyzed. Furthermore, these changes in composition are a consequence of both extracellular, post-translational processing of the core protein of aggrecan and changes in the sulfotransferase activity of the chondrocyte.     

Sulfated mucopolysaccharides from normal Swiss 3T3 cell line and its tumorigenic mutant ST1: possible role of chondroitin sulfates in neoplastic transformation.

The sulfated mucopolysaccharide composition of normal Swiss 3T3 cell line and its tumorigenic mutant ST1 is reported. It is shown that chondroitin sulfate B and heparitin sulfate are the sulfated mucopolysaccharides of the normal 3T3 line whereas chondroitin sulfate A and heparitin sulfate are the major ones of the ST1 variant. Degradation of the chondroitin sulfates derived from both cell lines with chondroitinases B and ABC have shown that they contain only 4-sulfated disaccharides differing from each other by the type of uronic acid residue. It is also shown that the chondroitin sulfate A from the tumorigenic variant is mostly located at the cell surface whereas the chondroitin sulfate B from the normal line is less accessible to trypsinization. A relative increase of chondroitin sulfate A was also observed in 3T3 that had lost contact inhibition after successive subcultures, and in the 3T6 cell line. These combined results are in agreement with the earlier proposal that glucuronic acid-containing chondroitin sulfate plays a role in the stimulation of cell division in neoplastic and embryonic tissues.     

Characterization of a heparan sulfate and a peculiar chondroitin 4-sulfate proteoglycan from platelets. Inhibition of the aggregation process by platelet chondroitin sulfate proteoglycan

A high molecular weight chondroitin sulfate proteoglycan (Mr 240,000) is released from platelet surface during aggregation induced by several pharmacological agents. Some details on the structure of this compound are reported. beta-Elimination with alkali and borohydride produces chondroitin sulfate chains with a molecular weight of 40,000. The combined results indicate a proteoglycan molecule containing 5-6 chondroitin sulfate chains and a protein core rich in serine and glycine residues. Degradation with chondroitinase AC shows that a 4-sulfated disaccharide is the only disaccharide released from this chondroitin sulfate, characterizing it as a chondroitin 4-sulfate homopolymer. It is shown that this proteoglycan inhibits the aggregation of platelets induced by ADP. Analysis of the sulfated glycosaminoglycans not released during aggregation revealed the presence of a heparan sulfate in the platelets. Degradation by heparitinases I and II yielded the four disaccharide units of heparan sulfates: N,O-disulfated disaccharide, N-sulfated disaccharide, N-acetylated 6-sulfated disaccharide, and N-acetylated disaccharide. The possible role of the sulfated glycosaminoglycans on cell-cell interaction is discussed in view of the present findings.     

Characterization of a low-sulfated chondroitin sulfate from the body of Viviparus ater (mollusca gastropoda). Modification of its structure by lead pollution

A chondroitin sulfate was purified from the body of Viviparus ater (Mollusca gastropoda) and analyzed for molecular mass, constituent disaccharides, and structure by 1H NMR and 1H 2D NMR. A quite unique glycosaminoglycan species was isolated having a high molecular mass (greater than 45,000) and low charge density, about 0.60, due to the presence of 42% non-sulfated disaccharide, 5% 6-sulfated disaccharide, 48% 4-sulfated disaccharide, and 5% 4,6-disulfated disaccharide. Specimens of Mollusca were also submitted to lead exposure for different times, and the effect on chondroitin sulfate structure was studied. After 96 h treatment a strong decrease in chondroitin sulfate content was observed with a significant modification of its structure producing a more desulfated polymer, in particular in position 4 of the galactosamine unit. Simultaneously, the amount of unsaturated non-sulfated disaccharide increased with an overall decrease of the charge density.     

High-performance liquid chromatography and on-line mass spectrometry detection for the analysis of chondroitin sulfates/hyaluronan disaccharides derivatized with 2-aminoacridone

In this study, we developed an on-line reverse-phase high-performance liquid chromatography-electrospray ionization-mass spectrometry (RP-HPLC-ESI-MS) separation and structural characterization of hyaluronan (HA)/chondroitin sulfate (CS)/dermatan sulfate (DS) disaccharides released by enzymatic treatment and derivatized with 2-aminoacridone (AMAC), providing a high-resolution system also applicable by using a further fluorimetric detector (Fp) before ESI-MS spectral acquisition. Isomeric nonsulfated HA and CS/DS disaccharides, isomeric monosulfated and isomeric disulfated CS/DS disaccharides, and the trisulfated species were distinctly separated and unambiguously identified by their retention times and mass spectra in negative ionization mode. In general, no multiply charged ions were detected even for highly charged disaccharides, but the presence of desulfonated products for highly sulfated species due to the relative instability of sulfo groups was observed. RP-HPLC-ESI-MS of each AMAC disaccharide was found to be linear from 3 to 500 ng with very high coefficient of correlation values due to the high efficiency of separation and the sharp outline of the peaks. Various CS/DS samples were characterized for disaccharide composition, and minor oligomer species identified as GalNAcSO₄ at the nonreducing end of chains was observed as a common component of these macromolecules. Furthermore, purified endogenous normal human plasma CS disaccharides were also evaluated by means of RP-HPLC-(Fp)-ESI-MS.     

Isolation and characterization of a novel chondroitin sulfate from squid liver integument rich in N-acetylgalactosamine(4,6-disulfate) and glucuronate(3-sulfate) residues

Novel chondroitin sulfate (CS) chains with an average molecular mass of 79.6 kDa were purified from squid liver integument. A compositional analysis of the CS chains using chondroitinases (CSases) ABC and AC-I revealed a range of variably sulfated disaccharides with GlcAβ1→3GalNAc(6-sulfate), GlcAβ1→3GalNAc(4-sulfate), and GlcAβ1→3GalNAc(4,6-disulfate) as the major ones, significant amounts of rare 3-sulfated GlcA-containing disaccharides, and a small amount of nonsulfated GlcAβ1→3GalNAc. The CS chains exhibited neurite outgrowth-promoting activity toward embryonic mouse hippocampal neurons, which was abolished completely by digestion with CSase ABC or AC-I. Consequently, whether these CS chains interact with heparin-binding growth factors was tested in a BIAcore system. All of the growth factors exhibited concentration-dependent and specific binding. CS chains from squid liver integument, with their unique composition and strong biological activities, may be a good candidate for therapeutic application.     

On the structure of heparitin sulfates Analyses of the products formed from heparitin sulfates by two heparitinases and a heparinase from Flavobacterium heparinum

The analyses of the products formed from heparitin sulfates by the action of two heparitinases and a heparinase from Flavobacterium heparinum is reported. Heparitin sulfates A and B are degraded by heparitinase I yielding two disaccharides, one of them composed of N-acetylglucosamine and an unsaturated uronic, joined by α(1 → 4) linkage, and the other, with the same composition but with an O-sulfate at the hexosamine moiety. A third disaccharide is also formed from heparitin sulfate B, by the action of the same enzyme, composed of glucosamine N-sulfate and an unsaturated uronic acid joined probably by α(1 → 4) linkage. Besides these three disaccharides, heparitin sulfate B yields, by the action of heparitinase I, an oligosaccharide (with an average molecular weight of 6000) which is completely degraded by the heparitinase II yielding a disaccharide composed of glucosamine 2,6-disulfate and unsaturated uronic acid. All the disaccharides are further degraded by α-glycuronidase from Flavobacterium heparinum yielding the respective monosaccharides. Based on these and other analyses the possible structures of the heparitin sulfates are proposed.     

Chondroitinase C from Flavobacterium heparinum.

A chondroitinase that acts upon chondroitin sulfate C and hyaluronic acid was isolated from Flavobacterium heparinum. This enzyme was seperated from constitutional chondroitinase AC and an induced chondroitinase B also present in extracts of F. heparinum previously grown in the presence of chondroitin sulfates A, B or C. The enzyme acts upon chondroitin sulfate C producing tetrasaccharide plus an unsaturated 6-sulfated disaccharide (delta Di-6S), and upon hyaluronic acid producing unsaturated nonsulfated disaccharide (delta Di-OS). Chondroitin sulfate A is also degraded producing oligosaccharides and delta Di-6S but not delta Di-4S. The chondroitinase C is also distinguished from the chondroitinases B and AC by several properties, such as effect of ions, temperature for optimal activity, and susceptibility to increasing salt concentrations. The substrate specificity of the chondroitinase C is different from that of any other chondroitinase or hyaluronidase described so far.