Optical characteristics of carboxyl group in relation to the circular dichroic properties and dissociation constants of glycosaminoglycans

Circular dichroism studies of glycosaminoglycan including chemically transformed heparins at various pH values reveal that carboxyl chromophore plays an important role in the dichroic behavior of the polymers. With decreasing pH, iduronic acid-containing glycosaminoglycans show increased negative ellipticity near 220 nm whereas the polymers containing glucuronic acid display enhanced negative dichroism near 230 nm and decreased negative dichroism around 210 nm. The pH-dependent optical properties have been utilized to determine the pK_a values of uronic acid moieties. The acid strengths of the iduronic acid-containing glycosaminoglycans are inherently smaller than those of corresponding glucuronic acid-containing polymers. Glycosaminoglycans in which the amino sugars are linked with iduronic acid display a very weak n → π^* amide transition, or none. The rotational strength at 210 nm of these polymers is largely due to iduronic acid moieties. The CD variations above 200 nm with change in pH do not indicate any major conformational transition of the molecules but the difference between dermatan sulfate and heparin can be attributed to difference either in iduronic acid conformation or in intersaccharide linkages.     

Improved analytical method for the hexuronic acids at the reducing terminals of glycosaminoglycans

When endo-uronidases act on glycosaminoglycans, the reaction products have hexuronic acid residues at the reducing terminals. An analytical method for hexuronic acids at the reducing terminals was devised for hyaluronate oligosaccharides having hexuronic acid residues at the reducing terminals.The procedure is as follows: Hexuronic acid residues at the reducing terminals of hyaluronate oligosaccharides were tritiated with reduction using NaB[³H]₄ and the products were hydrolyzed with trifluoroacetic acid and nitrous acid. As a result, the tritiated and reduced hexuronic acid residues, that is aldonic acids, were liberated from the reducing terminals. After passing them through anion and cation ion-exchange resins, the aldonic acids were lactonized. The lactones were developed on paper chromatography, and their radioactivities determined on the paper.The method is also useful for discrimination between glucoronic acid and iduronic acid at the reducing terminals of glycosaminoglycans.     

Optical characteristics of carboxyl group in relation to the circular dichroic properties and dissociation constants of glycosaminoglycans.

Circular dichroism studies of glycosaminoglycans including chemically transformed heparins at various pH values reveal that carboxyl chromophore plays an important role in the dichroic behavior of the polymers. With decreasing pH, iduronic acid-containing glycosaminoglycans show increased negative ellipticity near 220 nm whereas the polymers containing glucuronic acid display enhanced negative dichroism near 230 nm and decreased negative dichroism around 210 nm. The pH-dependent optical properties have been utilized to determine the pKa values of uronic acid moieties. The acid strengths of the iduronic acid-containing glycosaminoglycans are inherently smaller than those of corresponding glucuronic acid-containing polymers. Glycosaminoglycans in which the amino sugars are linked with iduronic acid display a very weak n leads to pi* amide transition, or none. The rotational strength at 210 nm of these polymers is largely due to iduronic acid moieties. The CD variations above 200 nm with change in pH do not indicate any major conformational transition of the molecules but the difference between dermatan sulfate and heparin can be attributed to difference either in iduronic acid conformation or in intersaccharide linkages.     

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.     

Binding and internalization of exogenous glycosaminoglycans in weakly and highly metastatic rhabdomyosarcoma cells

The fate of exogenous glycosaminoglycans in cultures of strongly (RMS 0) and weakly (RMS 8) metastatic rat rhabdomyosarcoma cells was studied. The time course and concentration dependence of binding and internalization of the radiolabeled sulfated glycosaminoglycans were determined. Weakly metastatic cells took up heparin, heparan and dermatan sulfates into their pericellular compartment at a higher rate than the strongly metastatic RMS 0 cells. The RMS 8 cells exhibited about two times more binding sites for these iduronic acid containing glycosaminoglycans, and internalized higher amounts of them than the RMS 0 cells. The uptake of the chondroitin sulfate into the peri- and intracellular compartments of both cell types was about 5-15% of that of the other glycosaminoglycans studied. The specificity of displacement of the pericellular heparin and dermatan sulfate by the unlabeled glycosaminoglycans indicates the involvement of specific structural features of the polysaccharide chains in the interactions of glycosaminoglycans with the surface of rhabdomyosarcoma cells, beside ionic forces due to the polyanionic character of the glycosaminoglycans. Heparin and heparan sulfate degradation products, mainly large oligosaccharides, were recovered from the surface of RMS 0 cells but were absent on the surface of the RMS 8 cells. About 30% of the internalized heparin and heparan sulfate was present in the partially degraded form in both cell types. Oligosaccharides derived from glycosaminoglycans were not released into the medium. The decrease in the amount of iduronic acid containing glycosaminoglycans internalized by the highly invasive cells seems to be correlated with an increased cell-associated degradation and with an apparent loss of glycosaminoglycan binding sites on the cell surface.     

Amphiregulin-dependent proliferation of cultured human keratinocytes: Autocrine growth,the effects of exogenous recombinant cytokine,and apparent requirement for heparin-like glycosaminoglycans

Amphiregulin, a member of the epidermal growth factor family with heparin binding affinity, functions as a natural regulator of keratinocyte growth. Autocrine signaling by amphiregulin and the effects of exogenous recombinant cytokine were studied in serum-free cultures of human neonatal keratinocytes. A metabolic inhibitor of proteoglycan sulfation was used to assess the role of cellular heparin-like glycosaminoglycans in amphiregulin-dependent growth. Keratinocytes plated at >10³ cells/cm² grew in an autocrine manner in the absence of exogenous epidermal growth factor or amphiregulin. Incubation of keratinocytes with an amphiregulin-blocking antibody indicated that ～70% of autocrine growth is mediated by endogenous amphiregulin. Proliferation potential in the presence of recombinant human amphiregulin was dose dependent and saturable and above ～1 ng/ml was comparable to that achieved with similar concentrations of epidermal growth factor. Sodium chlorate, which blocks glycosaminoglycan sulfation, reversibly inhibited epidermal growth factor-dependent proliferation by 42%, exogenous amphiregulin-dependent proliferation by 75%, and autocrine growth by 95%; concurrent incubation with 1-100 μg/ml heparin partially reversed this inhibition. Exogenous heparin in the absence of chlorate, however, nearly completely inhibited growth under autocrine conditions and in the presence of recombinant amphiregulin. Structure-function studies indicate that the polymerization level, high sulfate group density, and possibly iduronic acid content of heparin-like moieties correlate with their inhibitory activity. Collectively, these observations indicate that amphiregulin is the major autocrine factor for keratinocytes and demonstrate that exogenous amphiregulin is an effective growth promoting factor with molar potency similar to that of epidermal growth factor. Autocrine and paracrine signaling by amphiregulin may require cellular heparin-like glycosaminoglycans, presumably as matrix or membrane proteoglycans, whereas soluble glycosaminoglycans inhibit signaling, possibly by competing for cytokine binding. © 1994 wiley-Liss, Inc.     

The determination of iduronic acid and glucuronic acid in heparins by a new technique: Re-evaluation of the variable hexuronic acid composition of mammalian heparins

A new technique for the quantitative determination of the uronic acid components of heparin is described. Heparin was deamination with organic nitrite and methanolyzed. The monomeric derivatives of uronic acids were quantitated by gas chromotography. Depolymerization to monomeric units appeared essentially complete as judged from the yield of uronic acid derivatives. By applying the method the relative contents of iduronic acid and glucuronic acid in a number of heparin samples were estimated. In all samples examined the content of iduronic acid was larger than that of glucuronic acid. A species specific difference in the iduronic acid to glucuronic acid ration of heparin was noted. Noticeable difference of this ratio was observed also between different organs of a species of animal and among heparin fractions obtained from an organ.     

Distribution of sulphate and iduronic acid residues in heparin and heparan sulphate

1. A method was developed for determination of the uronic acid composition of heparin-like glycosaminoglycans. Polymers or oligosaccharides are degraded to monosaccharides by a combination of acid hydrolysis and deamination with HNO₂. The resulting uronic acid monosaccharides (accounting for about 70% of the uronic acid contents of the starting materials) are isolated and converted into the corresponding aldono-1,4-lactones, which are separated by g.l.c. The calculated ratios of glucuronic acid/iduronic acid are reproducible within 5%. 2. Samples of heparin from pig intestinal mucosa (molar ratio of sulphate/disaccharide unit, 2.40) and heparan sulphate from human aorta (sulphate/disaccharide ratio, 0.46) were subjected to uronic acid analysis. l-Iduronic acid constituted 77% and 19% respectively of the total uronic acid contents. 3. The correlation between the contents of sulphate and iduronic acid indicated by this finding also applied to the fractionated deamination products of the two polymers. The sulphated fragments varied in size from disaccharide to octasaccharide (or larger) and showed sulphate/disaccharide molar ratios in the range of 0.05–2.0. The proportion of iduronic acid increased with increasing ester sulphate contents of the oligosaccharides. 4. Previous studies on the biosynthesis of heparin in a cell-free system have shown that l-iduronic acid residues are formed by C-5 epimerization of d-glucuronic acid units at the polymer level; the process requires concomitant sulphation of the polymer. The results obtained in the present structural study conform to these findings, and suggest further that similar mechanisms may operate in the biosynthesis of heparan sulphate. The epimerization reaction appears to be linked to the sulphation of hydroxyl groups but does not seem to require sulphation of the target uronic acid residues. The significance of sulphamino groups in relation to the formation of iduronic acid is unknown.     

A simple and rapid electrophoretic method for the separation of glucuronic acid and iduronic acid

A new electrophoretic method using Titan III cellulose acetate plates has been developed for the separation and quantitation of glucuronic acid and iduronic acid. This method is quite simple, and glucuronic acid and iduronic acid can be separated within 50 min. This method was applied to the analyses of uronic acids in chondroitin sulfates A and C, and dermatan sulfate.     

Gas chromatographic assay of iduronic and glucuronic acids as aldonic acid butaneboronates

A gas chromatographic procedure has been developed for the analysis of uronic acids as aldonic acid butaneboronates. With these derivatives, aldoses frequently accompanying uronic acids in polysaccharide hydrolyzates are readily separated and measured. The method has been applied to the assay of iduronic and glucuronic acid released by enzymes associated with various mucopolysaccharidoses.     

Optical properties of Cu(II) complexes with heparin and related glycosaminoglycans.

Absorption and circular dichroism measurements have been carried out to obtain information regarding the stability and the nature of complexes between Cu(II) and heparin, and between Cu(II) and related glycosaminoglycans. In the presence of Cu(II), all glycosaminoglycans, except keratan sulfate, show a characteristic absorption band near 237 nm, which we assign to charge-transfer bands involving ligands to metal ion. From the absorbance values, the formation constants of Cu(II)-heparin and Cu(II)-(itN)-desulfated heparin have been determined to be approximately 1 × 10⁴ and 2 × 10² mol^?1, respectively. The large difference in the stability constant values is attributed to the difference in the charge density of the polymers, and to involvement of more than one ligand in the case of heparin. The CD characteristics of the Cu(II)-heparin complex suggest that both carboxyl and sulfamino groups are involved as ligands. The appearance of extrinsic CD bands in heparin, heparan sulfate, dermatan sulfate, and N-desulfated heparin at pH > 5 is ascribed to asymmetry of chelate rings. Absence of CD change in chondroitin sulfate and N-desulfated heparin (pH < 5) in the presence of Cu(II) suggests that only the carboxyl group is involved in those complexes. The differences either in iduronic acid conformation (C-1 vs. 1-C) or in intersaccharide linkages between dermatan sulfate and heparin (or heparan sulfate) are revealed in the difference CD spectra between the complexes and the polymers. The change in the intrinsic Cotton effect on complex formation is accounted for as a change in spatial orientation of the ligand groups rather than as a major conformational change of the polymers.     

Composition and distribution of glycosaminoglycans in cultures of human normal and malignant glial cells.

The glycosaminoglycans of human cultured normal glial and malignant glioma cells were studied. [35S]Sulphate or [3H]glucosamine added to the culture medium was incorporated into glycosaminoglycans; labelled glycosaminoglycans were isolated by DEAE-cellulose chromatography or gel chromatography. A simple procedure was developed for measurement of individual sulphated glycosaminoglycans in cell-culture fluids. In normal cultures the glycosaminoglycans of the pericellular pool (trypsin-susceptible material), the membrane fraction (trypsin-susceptible material of EDTA-detached cells) and the substrate-attached material consisted mainly of heparan sulphate. The intra- and extra-cellular pools showed a predominance of dermatan sulphate. The net production of hyaluronic acid was low. The accumulation of 35S-labelled glycosaminoglycans in the extracellular pool was essentially linear with time up to 72h. The malignant glioma cells differed in most aspects tested. The total production of glycosaminoglycans was much greater owing to a high production of hyaluronic acid and hyaluronic acid was the major cell-surface-associated glycosaminoglycan in these cultures. Among the sulphated glycosaminoglycans chondroitin sulphate, rather than heparan sulphate, was the predominant species of the pericellular pool. This was also true for the membrane fraction and substrate-attached material. Furthermore, the accumulation of extracellular 35S-labelled glycosaminoglycans was initially delayed for several hours and did not become linear with time until after 24 h of incubation. The glioma cells produced little dermatan sulphate and the dermatan sulphate chains differed from those of normal cultures with respect to the distribution of iduronic acid residues. The observed differences between normal glial and malignant glioma cells were not dependent on cell density; rather they were due to the malignant transformation itself.     

The transfer of platelet factor 4 from its proteoglycan carrier to natural and synthetic polymers

1. Transfer of dansyl-platelet factor 4 complexed with a series of glycosaminoglycans to heparin has been detected and studied by measuring changes in the anisotropy of the dansyl fluorescence. The protein was most easily transferred from chondroitin sulphate and least easily from heparan sulphaet. 2. The transfer of the dye-labelled protein from its biological chondroitin 4-sulphate proteoglycan carrier to natural and synthetic anionic polymers was similarly followed. The transfer to heparin and dermatan sulphate was shown to be the same whether 3 mM Ca²⁺ or 8 mM EGTA was present in the solution. 3. The shapes of the binding curves of the dansyl-factor to the polymers have been compared at I = 0.4 M. 4. The observed changes in anisotropy of dye fluorescence have been correlated with the charge density and the stereochemistry of the charged groups of the natural polymers. Large complexes are observed with polymers of high negative charge/weight ratios. Less charged polymers containing disaccharide units of iduronic acid and glucosamine N-sulphate will also form large complexes at I = 0.15 M. 5. It is demonstrated that the release of a platelet factor 4 proteoglycan complex in vivo would result in the transfer of the protein to heparin, moderate quantities of either dermatan or heparan sulphates would not prevent this transfer.     

Separation of glycopeptides derived from brain glycoproteins by gel filtration.

Glycopeptides obtained from rat brain by proteolytic digestion with papain have been separated from glycosaminoglycans by means of gel filtration. The glycosaminoglycans appear in the void volume, whereas the glycopeptides are retarded. Glycopeptides of groups A+B (Brunngraber et al., 1973) (MW = 3800-500) C+D (MW = 2000) which were partially resolved by the method, were identified in the elution profile. Nucleic acids, also solubilized by papain, are eluted together with the glycosaminoglycans.     

Characterisation of oligosaccharides from the chondroitin/dermatan sulphates: H and C NMR studies of oligosaccharides generated by nitrous acid depolymerisation

The polymers chondroitin sulphate and dermatan sulphate have been fragmented by an anhydrous hydrazine/nitrous acid procedure. The resulting disaccharides from the polymer repeat sequences were reduced with NaBH₄ and purified by ion exchange chromatography. Whereas enzymatic depolymerisation leads to the loss of the distinction between glucuronic and iduronic acids of CS and DS in the resultant disaccharides, nitrous acid depolymerisation retains these structures. Complete ¹H and ¹³C NMR data have been derived for the major components which were shown to have the structures: GlcA-(β1→3)-anTal6S-ol (I) and l-IdoA-(α1→3)-anTal4S-ol (II), where anTal-ol represents (2,5)anhydro-d-talitol and 6S/4S represent O-ester sulphate groups at C-6/C-4 sites.     

Chemical composition and 13C NMR spectroscopic characterisation of ulvans from Ulva (Ulvales, Chlorophyta)

The chemical composition and structures of several ulvan extracts isolated from various Ulva species were studied. They were all composed mainly of rhamnose, glucuronic acid, xylose, glucose and sulphate with smaller amounts of iduronic acid and traces of galactose. Proteins were also present, most likely as contaminants. Precise quantification of the uronic acid content by chemical-enzymatic hydrolysis coupled to HPAEC-PAD analysis and by colorimetry was not achieved, most likely due to the incomplete hydrolysis of glucuronan segments, inadequate HPAEC-pulsed-amperometric response factor for iduronic acid and to a possible differential colorimetric response of the two uronic acids. ¹³C NMR spectroscopic investigation of different ulvans demonstrated that they were all based on ulvanobiuronic acid 3-sulphate A and B repeating units [β-D-Glc pA-(1->4)-α-L-Rhap3S and α-L-IdopA-(1->4)-α-L-Rha p3S, respectively] as well as contiguous β 1->4 linked D-glucuronic acids possibly occurring either in ulvan or as a separate glucuronan. Marked variations in the content of the repeating structures were seen among the different samples. However, due to the limited number of samples studied, no conclusion was reached concerning the effects of species and ecophysiological conditions on the chemistry of ulvan. This revised version was published online in June 2006 with corrections to the Cover Date.     

Structural determinants in streptococcal unsaturated glucuronyl hydrolase for recognition of glycosaminoglycan sulfate groups

Pathogenic Streptococcus agalactiae produces polysaccharide lyases and unsaturated glucuronyl hydrolase (UGL), which are prerequisite for complete degradation of mammalian extracellular matrices, including glycosaminoglycans such as chondroitin and hyaluronan. Unlike the Bacillus enzyme, streptococcal UGLs prefer sulfated glycosaminoglycans. Here, we show the loop flexibility for substrate binding and structural determinants for recognition of glycosaminoglycan sulfate groups in S. agalactiae UGL (SagUGL). UGL also degraded unsaturated heparin disaccharides; this indicates that the enzyme released unsaturated iduronic and glucuronic acids from substrates. We determined the crystal structures of SagUGL wild-type enzyme and both substrate-free and substrate-bound D175N mutants by x-ray crystallography and noted that the loop over the active cleft exhibits flexible motion for substrate binding. Several residues in the active cleft bind to the substrate, unsaturated chondroitin disaccharide with a sulfate group at the C-6 position of GalNAc residue. The sulfate group is hydrogen-bonded to Ser-365 and Ser-368 and close to Lys-370. As compared with wild-type enzyme, S365H, S368G, and K370I mutants exhibited higher Michaelis constants toward the substrate. The conversion of SagUGL to Bacillus sp. GL1 UGL-like enzyme via site-directed mutagenesis demonstrated that Ser-365 and Lys-370 are essential for direct binding and for electrostatic interaction, respectively, for recognition of the sulfate group by SagUGL. Molecular conversion was also achieved in SagUGL Arg-236 with an affinity for the sulfate group at the C-4 position of the GalNAc residue. These residues binding to sulfate groups are frequently conserved in pathogenic bacterial UGLs, suggesting that the motif "R-//-SXX(S)XK" (where the hyphen and slash marks in the motif indicate the presence of over 100 residues in the enzyme and parentheses indicate that Ser-368 makes little contribution to enzyme activity) is crucial for degradation of sulfated glycosaminoglycans.     

Relationship between binding activity of 67Ga and low sulfated acid glycosaminoglycans

The sulfate content of acid glycosaminoglycan (AGAG) extracted from granuloma which had been produced by turpentine oil was inversely proportional to the amount of ⁶⁷Ga accumulation in the granuloma. Additionally, the lowest sulfation occurred in granuloma at a peak of inflammation when the uptake of ⁶⁷Ga had reached a maximum. On the basis of electrophoretic pattern, sulfate content, and specific optical rotation, it was concluded that acid glycosaminoglycans obtained from granuloma are mainly composed of chondroitin sulfate-A, -B, and desulfated heparin, while heparan sulfate was a minor component. From in vitro assays, desulfated acid glycosaminoglycans, especially desulfated-heparin and desulfated-heparan sulfate, were found to have a high affinity to ⁶⁷Ga. These results suggest that low- or de-sulfation of AGAG is related to the accumulation of ⁶⁷Ga in inflammatory lesions such as granuloma. Moreover, these results suggest that ⁶⁷Ga does not bind to glycosaminoglycans via sulfuric acid residues.     

Structural studies on heparan sulphate from human lung fibroblasts. Characterization of oligosaccharides obtained by selective periodate oxidation of d-glucuronic acid residues followed by scission in alkali

1. ³H- and ³⁵S-labelled heparan sulphate was isolated from monolayers of human lung fibroblasts and subjected to degradations by (a) deaminative cleavage and (b) periodate oxidation/alkaline elimination. Fragments were resolved by gel- and ion-exchange-chromatography. 2. Deaminative cleavage of the radioactive glycan afforded mainly disaccharides with a low content of ester-sulphate and free sulphate, indicating that a large part (approx. 80%) of the repeating units consisted of uronosyl-glucosamine-N-sulphate. Blocks of non-sulphated [glucuronosyl-N-acetyl glucosamine] repeats (3–4 consecutive units) accounted for the remainder of the chains. 3. By selective oxidation of glucuronic acid residues associated with N-acetylglucosamine, followed by scission in alkali, the radioactive glycan was degraded into a series of fragments. The glucuronosyl-N-acetylglucosamine-containing block regions yielded a compound N-acetylglucosamine–R, where R is the remnant of an oxidized and degraded glucuronic acid. Periodate-insensitive uronic acid residues were recovered in saccharides of the general structure glucosamine–(uronic acid–glucosamine)_n–R. 4. Further degradations of these saccharides via deaminative cleavage and re-oxidations with periodate revealed that iduronic acid may be located in sequences such as glucosamine-N-sulphate→iduronic acid→N-acetylglucosamine. Occasionally the iduronic acid was sulphated. Blocks of iduronic acid-containing repeats may contain up to five consecutive units. Alternating arrangements of iduronic acid- and glucuronic acid-containing repeats were also observed. 5. ³H- and ³⁵S-labelled heparan sulphates from sequential extracts of fibroblasts (medium, EDTA, trypsin digest, dithiothreitol extract, cell-soluble and cell-insoluble material) afforded similar profiles after both periodate oxidation/alkaline elimination and deaminative cleavage.     

Biosynthesis of proteoglycans by rat embryo parietal yolk sacs in organ culture

The embryonic rat parietal yolk sac has been previously shown to synthesize a number of basement membrane glycoconjugates including type IV procollagen, laminin, and entactin. In this study, parietal yolk sacs were isolated from 14.5-day rat embryos and incubated in organ culture for 4-7 h with [35S]sulfate, [3H] glucosamine, and/or 3H-labeled amino acids, and the newly synthesized proteoglycans were characterized. The major [35S]sulfate-labeled macromolecule represented approximately 90% of the medium and 80% of the tissue radioactivity. It also represented nearly 80% of the total [3H]glucosamine-labeled glycosaminoglycans. After purification by sequential ion-exchange chromatography and isopycnic CsCI density gradient ultracentrifugation, size-exclusion high-performance liquid chromatography showed a single species with an estimated Mr of 8-9 X 10(5). The intact proteoglycan did not form aggregates in the presence of exogenous hyaluronic acid or cartilage aggregates. Alkaline borohydride treatment released glycosaminoglycan chains with Mr of 2.0 X 10(4) which were susceptible to chondroitinase AC II and chondroitinase ABC digestion. Analysis by high-performance liquid chromatography of the disaccharides generated by chondroitinase ABC digestion revealed that chondroitin 6-sulfate was the predominant isomer. The uronic acid content of the glycosaminoglycans was 92% glucuronic acid and 8% iduronic acid, and the hexosamine content was 96% galactosamine and 4% glucosamine. No significant amounts of N- or O-linked oligosaccharides were detected. Deglycosylation of the proteoglycan with chondroitinase ABC in the presence of protease inhibitors revealed a protein core with an estimated Mr of 1.25-1.35 X 10(5). These results indicated that the major proteoglycan synthesized by the 14.5-day rat embryo parietal yolk sac is a high-density chondroitin sulfate containing small amounts of copolymeric dermatan sulfate. Hyaluronic acid and minor amounts of heparan sulfate proteoglycan were also detected.