Isolation and chemical characterization of two distinct “link proteins” from bovine nasal cartilage proteoglycan complex

Bovine nasal cartilage proteoglycan aggregates have been dissociated and separated by dissociative density gradient centrifugation into proteoglycan subunits and “link fraction”. The latter contained mainly the two “link proteins” as shown by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two “link proteins” were then separated by preparative gel electrophoresis under dissociative conditions. Molecular weight and amino acid composition of both proteins are presented.     

Isoforms of corneal keratan sulfate proteoglycan

Bovine corneal keratan sulfate proteoglycan was found to contain three major protein components. Two proteins (37 and 25 kDa) were released from the proteoglycan by endo-beta-galactosidase, N-glycanase, or chemical deglycosylation. A smaller protein (20 kDa), not covalently linked to keratan sulfate, co-purified with the proteoglycan by conventional and high performance ion exchange chromatography, by ethanol precipitation, and by affinity purification on columns of monoclonal antibody to keratan sulfate, but could be separated from the proteoglycan by gel filtration chromatography in dissociative agents. The three proteins produced different fragmentation patterns on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after digestion with V8 protease, and each had unique two-dimensional tryptic peptide maps. The N-terminal amino acid sequence of the core proteins differed. In addition, the proteoglycans containing these proteins differed in molecular size, suggesting different levels of glycosylation of the two core proteins. Similarity of the core proteins was suggested by similar amino acid composition, similarities in tryptic maps, and antigenic cross-reactivity. Corneal keratan sulfate proteoglycan, therefore, seems to occur in two different, but related, forms whose core proteins may represent members of a homologous family.     

Preparative electrophoresis on agarose submerged gels of two aggregating proteoglycan monomers from articular cartilage

Analytical electrophoresis on polyacrylamide-agarose gels of aggregating proteoglycan monomers from baboon articular cartilage produces two distinct bands, corresponding to two different aggregating monomer populations. A preparative electrophoresis procedure is described for isolating the two monomers. Proteoglycans were extracted from young baboon articular cartilage in 4 M guanidinium chloride containing proteolysis inhibitors and aggregated after hyaluronic acid addition. The aggregates were separated from non-aggregated proteoglycans by isopycnic centrifugation, followed by gel chromatography on Sepharose CL-2B. The monomers of the aggregates were obtained by isopycnic centrifugation under dissociative conditions. Two monomers were separated by preparative electrophoresis on 0.8 % agarose submerged gels. Approximately 60 % of the proteoglycans were recovered from the gel using a freeze-squeeze procedure. Aliquots of the separated monomers gave single bands when submitted to analytical polyacrylamide-agarose gel electrophoresis. Their migration and appearance were similar to that of the two bands present in the non separated preparation of monomers.     

Immunological studies of bovine nasal cartilage proteoglycan "link proteins".

Bovine nasal cartilage proteoglycan aggregates are dissociated and separated by density gradient centrifugation in 4 M guanidine into proteoglycan subunit (PGS) and glycoprotein link (GPL) fractions, the latter containing hyaluronic acid and "link proteins" responsible for aggregate formation. It was previously concluded on the basis of immunodiffusion studies that GPL has two antigenic components, one in common with PGS and one specific for the link proteins. However, in the present study it was found that antisera to PGS, which should lack link proteins, reacted with both "subunit" and "link" components of GPL, and antisera to fragments of PGS derived from the hyaluronic acid-binding portion of the molecule reacted preferentially with the link component. Reduction and alkylation of GPL led to modification of the reactions of both anti-GPL and anti-PGS sera with its link component. These immunodiffusion results indicate that the proteoglycan subunit and the link proteins are immunologically related and suggest that the link proteins may be identical with and derived from the hyaluronic acid binding portion of the proteoglycan subunit.     

A comparative study of the binding of cartilage link protein and the hyaluronate-binding region of the cartilage proteoglycan to hyaluronate-substituted Sepharose gel.

The hyaluronate-binding proteins from bovine nasal cartilage, i.e. the hyaluronate-binding region of the proteoglycan and the link protein, were labelled with 125I and separated from each other by gel chromatography. The proteins were characterized by molecular-weight determinations and their purity was established by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and immunodiffusion. The binding properties of the two proteins by hyaluronate-substituted Sepharose gel were compared. It was found that both proteins behaved similarly. They bound with the same efficiency to the gel, they showed the same time course of binding, had slightly different pH optima for binding and both proteins had a decreasing affinity for the gel with increasing ionic strength. The binding to the gel could be inhibited by soluble hyaluronate, and the minimum size of a hyaluronate oligosaccharide required for inhibition was in both cases a decasaccharide (only even-numbered oligosaccharides were tested). The proteins did not show any co-operative binding in the system tested, which could be explained by the large number of binding sites in the hyaluronate-substituted gel. Binding constants for the protein-hyaluronate interaction were estimated. A value of 1.3 x 10(7) M-1 was obtained for the hyaluronate-binding region of the proteoglycan, in agreement with literature data. The corresponding value for the link protein was 0.7 x 10(7) M-1.     

The isolation and characterization of the link proteins from proteoglycan aggregates of bovine nasal cartilage.

A preparation containing the link proteins may be obtained from bovine nasal cartilage by extraction with 4 M guanidine hydrochloride and by equilibrium density gradient centrifugations of the extract as commonly employed in the isolation of proteoglycan monomers. In the present paper, protein-rich proteoglycans have been removed from such a preparation to give purified link proteins by chromatography on Sepharose CL-6B in 1% sodium dodecyl sulfate. The individual link proteins, which in order of increasing electrophoretic mobility are termed link proteins 1, 2, and 3, have been separated and isolated in a subsequent preparative gel electrophoresis step. The link proteins present in largest amount, link proteins 1 and 2, have essentially the same amino acid compositions, and following partial digestion with the V8 protease from Staphylococcus aureus and analytical electrophoresis in sodium dodecyl sulfate, their peptide patterns closely resemble each other. Therefore,it is probable that link proteins 1 and 2 are structurally similar. Link protein 1 contains more carbohydrate than link protein 2 (9.5% and 3.0%, respectively) and it is suggested that the major difference between them is in carbohydrate content.     

Radioimmunoassay of the link proteins associated with bovine nasal cartilage proteoglycan.

Link proteins from bovine nasal cartilage have been purified by preparative polyacrylamide gel electrophoresis in sodium dodecyl sulfate (Baker, J.R., and Caterson, B. (1979) J. Biol. Chem. 254, 2387-2393) and used to raise antisera in rabbits. A sensitive radioimmunoassay procedure utilizing binding of 125I-labeled antigen . antibody complexes to Protein A of Staphylococcus aureus has served to demonstrate the specificity of the antisera for the link proteins. The lack of reactivity with proteoglycan fractions indicates that link proteins and proteoglycan do not share antigenic determinants. This result is in accord with published cyanogen bromide peptide cleavage data (Baker, J.R., and Caterson B. (1977) Biochem. Biophys. Res. Commun. 77, 1-10) which showed proteoglycan and link protein to be structurally dissimilar. The radioimmunoassay procedure has been used to quantitate small amounts of link protein which remain associated with proteoglycan after purification by equilibrium density gradient centrifugation in 4 M guanidine HCl and by gel chromatography in sodium dodecyl sulfate.     

Characterization of link protein(s) from human intervertebral-disc tissues.

Proteoglycan aggregates (A1) were prepared from the anulus fibrosus, nucleus pulposus and cartilage-endplate tissues of postnatal (0-6-month-old)-and young-adult (20-30-year-old)-human intervertebral discs. The A1 fractions from young-adult disc contained a greater proportion of non-aggregating proteoglycans than did postnatal tissues. After dissociative CsCl-density-gradient fractionation of the A1, more than 90% of the uronic acid was found in the postnatal A1D1, whereas only 60-80% of the hexuronate was present in the A1D1 isolated from young-adult disc tissues. These results indicated that more lower-buoyant-density proteoglycans occur in the young-adult disc. Link-protein-rich fractions (A1D3) were subjected to SDS/polyacrylamide-gel electrophoresis and immunolocation analyses using monoclonal antibodies specific for epitopes on link protein or proteoglycan. Under non-reducing conditions, the major link protein present in postnatal disc tissues was link protein 1. By contrast, all three link proteins (1, 2 and 3) were detected in young-adult tissues, with the smaller link protein 3 predominating. Analyses of the A1D3 fractions under reducing conditions also indicated the presence of link-protein-degradation peptides (Mr approx. 26,000) from young-adult disc tissues, but not from postnatal tissues. Sequential Sepharose CL-6B and Sephacryl S-300 chromatography in 4 M-guanidinium chloride was employed to separate the link proteins of the A1D3 fraction from protein-rich proteoglycan. Immunolocation analyses indicated that postnatal samples contained no detectable contaminating proteoglycan fragments. However, young-adult link-protein preparations could not be separated from hyaluronic acid-binding region and other proteoglycan fragments by means of these chromatographic procedures. The studies indicate that, compared with hyaline articular cartilage, degraded link protein and proteoglycan accumulate at an early age in young-adult disc tissues. These partially degraded proteoglycan aggregate components may significantly alter the biomechanical properties of disc tissues.     

The isolation of “link proteins” from bovine nasal cartilage

Methods of isolating the water insoluble ‘link proteins’ from preparations of bovine nasal cartilage proteoglycan aggregates have been investigated. Upon chromatography on Sepharose 4B in 0.1% sodium dodecyl sulfate, the ‘link proteins’ are found in a discrete, included peak, whereas the bulk of the proteoglycan emerges at the void volume. Some low molecular weight proteoglycan is associated with the ‘link proteins’. An alternative procedure, i.e. chromatography on DEAE-cellulose in sodium dodecyl sulfate, has also been examined. In 0.1% sodium dodecyl sulfate, proteoglycan monomer is not absorbed by the column, whereas ‘link proteins’ are. Subsequently, the ‘link proteins’ with a minor fraction of the proteoglycan are eluted with 1.0% sodium dodecyl sulfate. Both procedures serve to separate the ‘link proteins’ from the bulk of proteoglycan present in an aggregate preparation, but additional steps are necessary to achieve homogeneity. Thus a ‘link protein’ preparation, fractionated from proteoglycan aggregate by equilibrium density gradient centrifugation under dissociative conditions, can be finally purified by chromatography on DEAE-cellulose in sodium dodecyl sulfate.     

Isolation and characterization of a link protein from bovine aorta proteoglycan aggregate

Proteoglycan aggregates were isolated from bovine aorta by extraction with 0.5 M guanidine hydrochloride in the presence of proteinase inhibitors and purified by isopycnic CsCl centrifugation. The bottom two-fifths (A1) of the gradient contained 30% of proteoglycans in the aggregated form. The aggregate had 14.8% protein and 20.4% hexuronic acid with hyaluronic acid, dermatan sulfate and chondroitin sulfates in a proportion of 18:18:69. A link protein-containing fraction was isolated from the bottom two-fifths by dissociative CsCl isopycnic centrifugation. The link protein that floated to the top one-fifth of the gradient was purified by chromatography on Sephadex G-200 in the presence of 4 M guanidine hydrochloride. It moved as a single band in SDS-polyacrylamide gel electrophoresis with a molecular weight of 49 000. The amino acid composition of link protein resembled that of link protein from cartilage, but was strikingly different from that of the protein core of the proteoglycan monomer. The neutral sugar content of link protein was 3.5% of dry weight. Galactose, mannose and fucose constituted 21, 62 and 16%, respectively of the total neutral sugars. In aggregation studies the link protein was found to interact with both proteoglycan monomer and hyaluronic acid. Oligosaccharides derived from hyaluronic acid decreased the viscosity of link protein-free aggregates of proteoglycan and hyaluronic acid but not of link-stabilized aggregates, demonstrating that the link protein increases the stability of proteoglycan aggregates.     

Biosynthesis of cartilage proteoglycan and link protein by articular chondrocytes from immature and mature rabbits

Chondrocytes from immature and mature rabbits have been compared in biosynthetic studies with [3H] leucine and [35S]sulfate as precursors. The time course of incorporation of [3H]leucine into general protein, proteoglycan monomer core protein, and link protein and of [35S]sulfate into proteoglycan monomer has been examined. Proteoglycan monomer was isolated from the high buoyant density (p greater than 1.60) fractions of dissociative CsCl gradients and link protein by immunoprecipitation with antibody 8A4 followed by gel electrophoresis. Results based on the period of linear isotope incorporation showed that mature cells synthesize protein at about 40% of the rate of immature cells and both proteoglycan and link protein at about 20% of the rate of immature cells. The labeling rates obtained suggest that immature cells synthesize an approximate 1:1 molar ratio of link protein to proteoglycan monomer, and for mature cells this ratio is about 0.8:1. While cell layer retention of newly synthesized proteoglycan was markedly lower in mature relative to immature cell cultures, link protein retention was high in both immature and mature cultures; this finding provides an explanation for our previous observation (Plaas, A. H. K., and Sandy, J. D. (1984) Biochem, J. 220, 337-340) that link-free monomer accumulates in the medium of mature but not immature cultures. The link protein synthesized by both ages of cells and isolated from cell layer or medium was a single major species of apparent molecular mass 48-51 kDa. The results suggest that mature chondrocytes are less efficient than immature chondrocytes in the coordinated assembly of link-stabilized proteoglycan aggregates in this culture system.     

The interaction of link proteins with proteoglycan monomers in the absence of hyaluronic acid

Cartilage proteoglycan aggregates contain three classes of interacting components: proteoglycan monomers, hyaluronic acid and link proteins. Direct evidence is presented for a link protein to proteoglycan monomer association which hitherto has only been presumed to occur. Thus, when mixtures of purified link proteins and proteoglycan monomers were subjected to ultracentrifugation or gel chromatography under ‘associative’ conditions, link proteins were found to fractionate with proteoglycan.     

Isolation of dermatan sulfate proteoglycans from mature bovine articular cartilages

Two species of dermatan sulfate proteoglycans, called DS-PGI and DS-PGII, have been isolated from mature bovine articular cartilages. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis at low ionic strength in 0.01 M phosphate the dermatan sulfate proteoglycans appeared as a single polydisperse species whose molecular weight ranged from 80,000 to 140,000. The dermatan sulfate proteoglycans eluted as a single peak on Sepharose CL-4B chromatography in 4 M guanidine hydrochloride and showed no tendency to separate into two components. Following chondroitinase AC and ABC digestion, a core protein was obtained whose molecular weight was 45,000. However, what appeared to be a single dermatan sulfate proteoglycan was consistently separated into two species of distinctly different mobilities by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at high ionic strength in 0.375 M Tris. The molecular weight of the smaller species (DS-PGII) ranged from 87,000 to 120,000. The molecular weight of the larger species (DS-PGI) ranged from 165,000 to 285,000. DS-PGI self-associates in 0.375 M Tris, while DS-PGII does not. This phenomenon was exploited to separate DS-PGI and DS-PGII by preparative electrophoresis on 5 to 20% gradient slab gels. The immunological identities of the individual species, DS-PGI and DS-PGII, were examined by enzyme-linked immunosorbent assay using polyclonal antiserum to cartilage-specific proteoglycan monomer from bovine articular cartilage and polyclonal and monoclonal antibodies to DS-PGII. The polyclonal antiserum to cartilage-specific proteoglycan monomer did not react with DS-PGI or DS-PGII, indicating that DS-PGI and DS-PGII possess different core proteins from cartilage-specific proteoglycan monomer. Polyclonal and monoclonal antibodies raised against the mixture of DS-PGI and DS-PGII reacted strongly with DS-PGII, but weakly or not at all with DS-PGI. These results suggest that DS-PGI and DS-PGII possess different core proteins and may represent two different species of dermatan sulfate proteoglycans.     

Immunochemistry of cartilage proteoglycan. Immunodiffusion and gel-electrophoretic studies

Cartilage proteoglycan is thought to be composed of subunits, core proteins with covalently attached sulphated polysaccharide side chains, which form aggregates by non-covalent association with a link protein. The new technique of non-disruptive extraction followed by fractionation in caesium chloride gradients provides a useful means of preparing relatively pure proteoglycan aggregate, subunit and link fractions. Immunological studies of these fractions led to the identification of an antigen associated with the proteoglycan subunit which was common to several species and to the demonstration of additional species-specific antigens in aggregate and link fractions derived from bovine nasal cartilage. Polyacrylamide-gel electrophoresis with sodium dodecyl sulphate of bovine proteoglycan aggregate and link fractions gave two protein bands in the gels and a protein-polysaccharide band at the origin; subunit fractions gave only the band at the origin. These results are consistent with the current concept of cartilage proteoglycan structure.     

Protein metabolism of Drosophila melanogaster male accessory glands—I: Characterization of secretory proteins

The electrophoretic properties of male accessory gland proteins of Drosophila melanogaster were studied in both the native and the denatured state. The molecular weights and the isoelectric points were determined. In addition, the relative abundance of individual fractions was measured. More than 40 protein bands were observed on one-dimensional dissociative gels, approx. 85 proteins were separated on two-dimensional gels. Secretions and epithelia of both the accessory gland and the ductus ejaculatorius each contain a distinct complement of proteins. The majority of accessory secretion proteins are basic. In the native state they are only soluble with difficulty. In the presence of urea, however, 21 fractions can be separated by one-dimensional electrophoresis with a low-pH buffer system. For two-dimensional separation non-equilibrium pH gel electrophoresis gave best results. All but one of the proteins of the ductus ejaculatorius secretion are acidic. The epithelial proteins were found to be acidic.     

Isolation of enamelinlike proteins from blue shark (Prionace glauca) enameloid

A sequential dissociative extraction scheme was used to extract proteins from developing Blue Shark enameloid. The first extraction solution (4 M guanidine HC1) solubilized the polypeptides, mainly collagenous, not closely associated with the hydroxyapatite. The next extraction solution (4 M guanidine HC1, 0.5 M ethylenediaminetetraacedic acid (EDTA] solubilized the proteins more closely associated with the tooth mineral component. After extraction, the proteins were separated and isolated with gel electrophoresis. Protein molecular weights were determined and selected proteins were isolated for amino acid composition analysis. The two proteins isolated were tested for mammalian enamel protein antigenic determinants by a "Dot" immunobinding assay. The isolated proteins were enamelinlike by extraction criteria and amino acid composition. Further, the two proteins share antigenic determinants with mammalian enamel proteins.     

Unique glycosylation of three keratan sulfate proteoglycan isoforms

Recent work demonstrates isoforms of bovine corneal keratan sulfate proteoglycan containing structurally unique core proteins of 25 and 37 kDa (Funderburgh, J., and Conrad, G. (1990) J. Biol. Chem. 265, 8297-8303). In the current study, two forms (37A and 37B) of the 37-kDa protein were separated by ion-exchange chromatography after removal of keratan sulfate with endo-beta-galactosidase. Keratan sulfate linkage sites in core proteins were labeled with UDP-[3H]galactose using galactosyltransferase. Labeled proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by tryptic digestion and reversed-phase chromatography. The 37A protein has three keratan sulfate-linkage sites, and the 37B and 25-kDa proteins each contain one linkage site. Reversed-phase tryptic maps of the three proteins differed in total peptide profile and in glycosylated peptides labeled with periodate-[3H]-NaBH4. Tryptic mapping of the two 37-kDa isoforms after deglycosylation showed differences in total tryptic peptides, in peptides labeled with [14C]iodoacetic acid, and in peptides recognized by antibodies to a mixture of the 37-kDa cores. Antibody to a synthetic peptide with N-terminal sequence obtained from mixed 37-kDa cores reacted exclusively with the 37B isoform. These results show that bovine corneal keratan sulfate proteoglycan has three different core proteins each with distinct glycosylation and unique primary structure.     

A novel low-molecular weight chondroitin sulphate proteoglycan isolated from cartilage.

Proteoglycans were isolated from cartilage by extraction with 4M-guanidinium chloride followed by direct centrifugation in 4M-guanidinium chloride/CsCl at a low starting density, 1.34 g/ml. N-Ethylmaleimide was included in the extraction solvent as a precaution against contamination of proteoglycans with unrelated proteins mediated by disulphide exchange. A novel, discrete, low-buoyant-density proteoglycan (1.40--1.35 g/ml) was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Its proteoglycan nature was revealed by the shift in the molecular size observed on gel electrophoresis after treatment with chondroitinase ABC. The core protein was monodisperse. The proteoglycan was further purified by gel chromatography with and without addition of hyaluronate. The proteoglycan constitutes less than 2% (by weight) of the total extracted proteoglycans and is not capable of interacting with hyaluronate. The same proteoglycan was purified in larger quantities by sequential associative and dissociative CsCl-density-gradient centrifugation, zonal rate sedimentation in a sucrose gradient and gel chromatography on Sepharose CL-4B. The pure proteoglycan had a molecular weight of 76 300 determined by sedimentation-equilibrium centrifugation and an apparent partial specific volume of 0.59 ml/g. It contained about 25% protein (of dry weight) and had remarkably high contents of leucine and cysteine as compared with other proteoglycans. The proteoglycan contained two to three large chondroitin sulphate chains and some oligosaccharides.     

Chondroitin/dermatan sulfate proteoglycan in human fetal membranes. Demonstration of an antigenically similar proteoglycan in fibroblasts

A proteoglycan was isolated from fetal membranes which had been separated from human postpartum placenta. The glycosaminoglycan side chains (Mr = 55,000) were found to be composed of 75% chondroitin sulfate and 23% dermatan sulfate as determined by chondroitinase ABC or AC II digestion. NH2-terminal microsequencing of the intact proteoglycan revealed a single amino acid sequence of (sequence; see text) A rabbit antiserum raised against the intact proteoglycan reacted in sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting with Mr = 45,000 and 43,000 core polypeptides from chondroitinase-treated proteoglycan. Affinity-purified antibodies from this antiserum precipitated from human embryonic fibroblast culture fluid a proteoglycan which has an approximate Mr = 120,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This proteoglycan has on the average two polysaccharide side chains. As defined by chondroitinase digestion, these chains consist of 66% dermatan sulfate and 20% chondroitin sulfate. Digestion of the glycosaminoglycan with chondroitinase ABC converted the proteoglycan to a Mr = 45,000 major and a Mr = 43,000 minor core polypeptide. Tissue immunofluorescence localized the proteoglycan to interstitial matrices, suggesting that it is a product of mesenchymal cells. The methods we have devised for the purification of the fetal membrane proteoglycan in chemical amounts and the antibodies we have prepared against it will allow studies on the structural and functional properties of the proteoglycan and on the expression of immunologically cross-reactive proteoglycans by various cells and tissues.     

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.