Intact Proteoglycan Research Articles

Asymmetric distribution of sites with high affinity for antithrombin III in rat skin heparin proteoglycans.

The distribution of sites with high affinity for antithrombin III in [35S]heparin proteoglycans from rat skin was studied by affinity chromatography of the intact proteoglycans (Mr congruent to 1 x 10(6)) and degradation products. Unfractionated proteoglycan and proteoglycan fractions with low affinity and high affinity separated on antithrombin III-agarose were treated with alkali, releasing heparin chains (Mr congruent to 1 x 10(5)). Each chain preparation was fractionated on antithrombin III-agarose into fractions with low affinity and high affinity. Unfractionated proteoglycan and proteoglycan fractions with low affinity and high affinity were incubated with rat serum at pH 6.0, which gave products of similar size to commercial heparins (Mr congruent to 1 x 10(4)) termed heparin fragments. Each fragment preparation was fractionated on antithrombin III-agarose, yielding fractions with no affinity, low affinity, and high affinity, 40% of the proteoglycan preparation had low affinity, containing 4% high affinity chains and 7% high affinity fragments. The high affinity proteoglycan fraction yielded 40% high affinity chains and 22% high affinity fragments. The data show that the distribution of binding sites with high affinity for antithrombin III in heparin proteoglycans is highly asymmetric. Therefore, the concept that polymer modification reactions occurring during heparin biosynthesis, which must be involved in the formation of high affinity binding sites, occur in a random way must be reappraised.

High molecular weight proteoglycans biosynthesized in culture by pigeon aortas

The properties of aortic proteoglycans synthesized in vitro were examined to demonstrate synthesis of intact proteoglycans by aortic tissue in culture and to compare labeling and synthetic rates of two different populations of proteoglycan. Following 3, 6, or 9 h of incubation in medium containing [ 35S]sodium sulfate and [ 3H]serine, the tissue was extracted with 4.0 M guanidine hydrochloride containing protease inhibitors. Extracts were chromatographed on Sepharose CL-4B and subjected to buoyant density centrifugation under dissociative conditions. Radioactive precursors were incorporated into two major populations of aortic proteoglycan, one of high molecular weight eluting near the void volume of Sepharose CL-4B (Protooglycan I) and one of lower molecular weight (Proteoglycan II) having a K av of 0.40–0.44. The radioactively labeled proteoglycans were localized at densities 1.50–1.56 g/ml (Preparation 1) and 1.43–1.49 g/ml (Preparation 2) following CsCl buoyant density centrifugation. Both proteoglycan populations had increased incorporation of 35S and 3H over time. At all times the lower molecular weight proteoglycan had a higher specific activity (dpm 35S and 3H/μg hexuronic acid). At 3, 6, and 9 h, the specific activity of Proteoglycan II was 8.2-, 6.7- and 3.0-fold higher than Proteoglycan I using 35S and 13.0-, 8.1- and 2.7-fold higher using 3H, suggesting different synthetic rates for the two proteoglycans. The results illustrate synthesis of intact proteoglycans during short-term artery culture. The proteoglycan types have size and buoyant density characteristics as described for artery, but based upon changes in specific activity ratios, the two proteoglycan populations differ in rates of synthesis.

Proteoglycans: their structure, interactions and molecular organization in cartilage.

The term ‘proteoglycan’ was introduced in 1967 (see Balazs. 1970) to describe the family of molecules in which glycosaminoglycan chains are linked to protein. However. their rate of diffusion into textbooks of biochemistry has been rather slow, and I will therefore begin this presentation with a general introduction, which I hope will set the basis for their structural identity and indicate how their properties may be of importance in a broad biological context, before describing in more detail studies with proteoglycans from cartilage. Glycosaminoglycans are characteristic components of the extracellular space of vertebrate tissues. They are long unbranched polysaccharide chains which contain many acidic, carboxylate and/or sulphate groups. They do not normally occur as free chains in uiuo, but as proteoglycans, in which many like chains are linked by the reducing end of the terminal sugar residue to a protein. A whole technology was developed for the isolation, identification and quantification of glycosaminoglycan chains before their attachment to proteins was clearly established (Roden et al., 1972). Proteoglycan research thus began as a specialized area of carbohydrate chemistry. It is now apparent that in understanding the biological role of these macromolecules, the structure and interactions of the proteoglycans cannot be assessed by the analysis of the glycosaminoglycan chains alone. However, there are many difficulties in isolating and purifying intact proteoglycans, and it is only in recent years that a beginning has been made in exploring the full diversity of proteoglycan structure. There are seven types of glycosaminoglycan. on the basis of different repeating disaccharide units, which occur in mammalian tissues (Table I ) (see Muir & Hardingham, 1975). With the exception of hyaluronate, they have all been reported to occur linked to protein in different types of proteoglycan. The linkage to protein is via a neutral trisaccharide (Gal-Gal-Xyl) at the reducing end of the glycosaminoglycan chain, and xylose forms an 0-glycosidic bond with a serine residue in the protein. Keratan sulphate contains no neutral trisaccharide, but is linked via an 0-glycosidic bond between N-acetylgalactosamine to The Sixteenth Colworth Medal Lecture

On the structure of proteoglycan core

Core protein from bovine nasal proteoglycan has been obtained by cyanogen bromide cleavage and by removal of most of the glycosaminoglycan side chains by hydrogen fluoride treatment. Amino acid analysis of the cyanogen bromide fragment shows it to consist mainly of proline, serine, glycine and glutamic acid (glutamine). End-group analyses of the fragment and HF stripped core reveal and N-terminal residue to be valine in each case. The stripped core has been subjected to sequencing and some sequential information is presented. Based upon the amino acid analysis, sequence information and other properties, conformation analysis indicates that the most likely conformation is that of a flexible extended chain containing β-turns. The existence of a common N-terminal residue indicates that it is the C-terminal region which lies in the region of the hyaluronic acid backbone in intact proteoglycan. Furthermore, enzymatic cleavage of core protein which occurs in proteoglycan turnover, aging and degenerative diseases, probably does not occur by a stepwise cleavage from the N terminus of proteoglycan but by a more drastic degradation process.

Immunochemical analysis of cartilage proteoglycans. Antigenic determinants of substructures.

Antibodies were raised in rabbits by injection of cartilage proteoglycan monomers, isolated hyaluronic acid-binding region, polysaccharide-peptides prepared by trypsin digestion of proteoglycans and link-protein. The rabbits injected with the proteoglycan monomers made antibodies reacting with the intact proteoglycan. The antiserum contained antibodies specific for, and also reacting with, the isolated hyaluronic acid-binding region and the keratan sulphate-rich region. In addition there were probably antibodies reacting with other structures of the proteoglycan monomer. When isolated hyaluronic acid-binding region was used for immunization the antibodies obtained reacted specifically with the hyaluronic acid-binding region. The antibodies obtained from rabbits immunized with the polysaccharide-peptides reacted with the proteoglycan monomers and showed a reaction identical with that of the chondroitin sulphate-peptides isolated after trypsin digestion of proteoglycans. The antibodies prepared with the link-protein as the antigen reacted only with the link-protein and not with any preparation from the proteoglycan monomer. Neither did any of the antisera raised against the proteoglycan monomer or its substructures react with the link-protein. Separately it was shown that the peptide 'maps' prepared from trypsin digests of the link-protein and the hyaluronic acid-binding region were different. Therefore it appears that the link-protein is not structurally related to the proteoglycan or the hyaluronic acid-binding region. Digestion of proteoglycan monomers or isolated hyaluronic acid-binding region with trypsin did not destroy the antigenic sites of the hyaluronic acid-binding region. In contrast trypsin digests of previously reduced and alkylated preparations did not react with the anti-(hyaluronic acid-binding region). The trypsin digests, however, reacted with both the antibodies directed against the chondroitin sulphate-peptides and those against the keratan sulphate-peptides. Trypsin digestion of the link-proteins destroyed the antigenic site and the reactivity with the antibodies. By combining immunoassay of proteoglycan preparations before and after trypsin digestion it is feasible to quantitatively determine its substructures by using the antisera described above.

Interaction of cartilage proteoglycans with hyaluronic acid

AbstractMost proteoglycans are present in hyaline cartilage matrices as aggregates with as many as 100 molecules, each with average molecular weight of about 2 × 106, bound through specific, noncovalent interactions to individual strands of hyaluronic acid (HA). The interactions with HA are mediated by the HA‐binding region of the core protein, which is located at one end of each of the interactive proteoglycans. A fragment of the core protein, average molecular weight of about 6 × 104, which contains the HA‐binding site, can be isolated in an active form from trypsin digests of proteoglycan aggregates. The “active” HA‐binding site in this preparation interacts strongly with HA‐10 but weakly with HA‐8, (oligomers of HA derived from partial digests of HA with testicular hyaluronidase); HA‐9 derived from β‐glucuronidase digestion of HA‐10 also interacts strongly. No polysaccharide other than HA has been found to interact. Christner, Brown, and Dziewiatkowski (personal communication) modified the carboxyls on glucuronic acid groups in mixture of HA‐10 to HA‐30, and they found that the interaction with proteoglycan no longer occurred if about 60% of the total carboxyls were (a) methyl esterified, (b) reduced to glucose, or (c) substituted with glycine in amide linkage. Saponification of the methyl esters restored activity. Dansylation of lysine residues in the HA‐binding region preparation abolished binding activity. However, when the dansylation reaction was done in the presence of HA, the HA‐binding activity was protected. Acetylation of the same residues did not abolish binding activity but did prevent subsequent inactivation by dansylation. Hardingham, Ewins, and Muir (Biochem J 157:127–143, 1976) studied the effect of various amino acid modifiers on the interaction of intact proteoglycans with HA and showed that reaction of arginine residues with low concentrations of 2,3‐butanedione was particularly effective in destroying binding. In sum, the data above suggests that the HA‐binding region (a) contains accessible arginine residues necessary for activity, (b) contains lysine residues near the binding site which, when substituted with bulky groups such as dansyl, but not acetyl, sterically block interaction, and (c) requires a length of HA with at least 4.5 repeat disaccharides containing 3, and possibly 4, unmodified glucuronic acid carboxyls for interaction. The possible relevance of proteoglycan‐hyaluronic acid interaction to the observations that hyaluronic acid specifically inhibits proteogly can synthesis by cultured chondrocytes is discussed.

Characterization of the fragments obtained by enzymic and alkaline degradation of rice-bran proteoglycans

Repeated digestion of the proteoglycan B from rice bran with Pronase and hemicellulase yielded two types of glycopeptides. The first group, obtained from the third enzymic digest, was a type of low molecular-weight glycopeptide and was fractionated into two subtractions by paper electrophoresis. The second group, obtained from the fourth enzymic digest, was a mixture of glycopeptides of relatively high molecular-weight and was fractionated into three subfractions by gel filtration on Sephadex G-75. Alkaline degradation of these glycopeptides or the intact proteoglycan A yielded a sugar-amino acid compound, whose structure was established as O-α- L-arabinofuranosyl-hydroxyproline. These results indicate that the carbohydrate-protein linkage of the rice-bran proteoglycan is an O-glycosyl linkage between L-arabinose and hydroxyproline.

The nature of the protein moieties of cartilage proteoglycans of pig and ox.

Proteoglycans extracted with 4M-guanidinium chloride from pig laryngeal cartilage and bovine nasal septum were purified by density-gradient centrifugation in CsCl under 'associative' followed by 'dissociative' conditions [Hascall & Sajdera (1969) J. Biol. Chem. 244, 2384-2396]. Proteoglycans were then digested exhaustively with testicular hyaluronidase, which removed about 80% of the chondroitin sulphate. The hyaluronidase was purified until no proteolytic activity was detectable under the conditions used for digestion. The resulting 'core' proteins of both species were fractionated by a sequence of gel-chromatographic procedures which gave four major fractions of decreasing hydrodynamic size. Those that on electrophoresis penetrated 5.6% (w/v) polyacrylamide gels migrated as discrete bands whose mobility increased with decreasing hydrodynamic size. The unfractionated 'core' proteins had the same N-terminal amino acids as the intact proteoglycan, suggesting that no peptide bonds had been cleaved during hyaluronidase digestion. Alanine predominated as the N-terminal residue in all the fractions of both species. Fractions were analysed for amino acid, amino sugar, uronic acid and neutral sugar compositions. In pig 'core' proteins, the glutamic acid content increased significantly with hydrodynamic size, but in bovine 'core' proteins this trend was less marked. Significant differences in amino acid composition between fractions suggested that in each species there was more than one variety of proteoglycan. The molar proportions of xylose to serine destroyed on alkaline beta-elimination were equivalent in most fractions, indicating that the serine residues destroyed were attached to the terminal xylose of chondroitin sulphate chains. The ratio of serine residues to threonine residues destroyed on beta-elimination, was similar in all fractions of both species. Since the fractions of smallest hydrodynamic size contained less keratan sulphate than those of larger size, it implies that in the former the keratan sulphate chains were shorter than in the latter.

The isolation of collagen-associated proteoglycan from bovine nasal cartilage and its preferential interaction with α2 chains of type I collagen

A collagen complex from bovine nasal cartilage was prepared by extraction of the tissue with 3M-MgCl2 solutions, by using two different procedures. When it was compared with calf skin acid-soluble tropocollagen by polyacrylamide-gel electrophoresis, the 3M-MgCl2-soluble cartilage collagen in the complex appeared to be predominantly type I in nature, consisting of both alpha1 and alpha2 chains. The soluble cartilage collagens were digested with purified bacterial collagenase, and the soluble digests were fractionated on Sepharose 4B. Hydroxyproline-free proteoglycan was isolated in the excluded volume of the column eluate, and this was found to be an aggregate which could be dissociated to link proteins and proteoglycan subunit by equilibrium-density-gradient centrifugation in a CsCl-4M-guanidinium chloride gradient. Interaction with calf skin-soluble tropocollagen was studied by CM-cellulose chromatography. The link-protein system did not interact, but proteoglycan from the bottom of the gradient did interact. In addition, when proteoglycan subunit was allowed to interact with collagen, there was a preferential binding to the alpha2 and beta12 components, and this effect was also observed with the proteoglycan material obtained from the collagenase digests of 3M-MgCl2-soluble cartilage collagen complexes. However, specificity for alpha2 and beta12 chains was not exhibited by chondroitin sulphate glycosaminoglycan, and it is therefore concluded that preference for alpha2 and beta12 chains is a function of the intact proteoglycan structure.

The effect of D-xylose, beta-D-xylosides and beta-D-galactosides on chondroitin sulphate biosynthesis in embryonic chicken cartilage.

The incorporation of [3H]acetate into chondroitin sulphate was used as a measure of the rate of synthesis of this polysaccharide in whole tibias and femurs of embryonic chicken cartilage in vitro. The incorporation is inhibited by puromycin and by cycloheximide, but the inhibition is relieved by the addition of D-xylose, beta-D-xylosides and beta-D-galactosides to the incubation medium. Beta-D-Xylosides can stimulate the incorporation to 300% of that of controls incubated in the absence of cycloheximide or puromycin, D-Xylose, beta-D-xylosides and beta-D-galactosides appear to act as artificial initiators of chondroitin sulphate synthesis and enable polysaccharide-chain synthesis to be studied as an event separate from the synthesis of intact proteoglycan.

Interactions of an intact proteoglycan and its fragments with basic homopolypeptides in dilute aqueous solution.

The interactions between a proteoglycan and cationic polypeptides have been investigated by the use of circular-dichroism spectroscopy. The interaction produces an induced conformational change for poly(l-arginine) and poly(l-lysine), similar to the effects previously reported for mucopolysaccharide-polypeptide mixtures. For bovine nasal septum proteoglycan, the interactions are similar to those for chondroitin 4-sulphate, which comprises approximately 63% of the total polysaccharide. The results also suggest that the interactions produce a conformational change in the protein core. Similar studies for the Smith-degradation product show that the protein core can adopt a substantial alpha-helical content and is capable of interactions with poly-(l-arginine). The interactions for chondroitin sulphate ;doublets' are significantly different from those for the separated chains, indicating that the arrangement of the polysaccharide side chains in pairs (and larger groups) along the protein backbone contributes to the interaction properties of the intact proteoglycan.

Aggregation of Cartilage Proteoglycans: II. OLIGOSACCHARIDE COMPETITORS OF THE PROTEOGLYCAN-HYALURONIC ACID INTERACTION

A protein-keratan sulfate core preparation isolated from a chondroitinase digest of bovine nasal proteoglycan chromatographed as a single peak (Kav = 0.65) on a Sepharose 2B column. Sepharose 2B elution patterns for mixtures of the core preparation with different proportions of hyaluronic acid indicated that slightly more than 70% of the core molecules could bind to hyaluronic acid. When the hyaluronic acid was saturated with core molecules, each core molecule occupied a minimum length of 8 to 10 hyaluronic acid disaccharides. Hyaluronic acid oligosaccharides were isolated by Sephadex G-50 chromatography of partial digests of hyaluronic acid with testicular hyaluronidase. Sepharose 2B chromatographic analyses showed that the interaction of reactive core molecules with larger hyaluronic acid molecules was strongly inhibited by HA10 (5 disaccharide repeat units) and by HA9 derived from a β-glucuronidase digest of HA10. HA8, smaller hyaluronic acid oligosaccharides and chondroitin were essentially ineffective in inhibiting the core-hyaluronic acid interaction. Low concentrations of HA10 effectively decreased the viscosity of a mixture of hyaluronic acid with intact proteoglycan, whereas higher concentrations of HA8, HA6, or HA4 caused much smaller decreases. Thus the binding site for hyaluronic acid in reactive core (or intact proteoglycan) molecules interacts strongly with HA10 or larger oligomers, while it only interacts weakly with HA4-HA8 oligomers. Chondroitin did not decrease the viscosity of such a mixture at all, showing that there is specificity for the configuration of the 4-hydroxyl in the glucosamine residues in the hyaluronic acid oligosaccharides.

Solvent-dependent Changes in Proteoglycan Subunit Conformation in Aqueous Guanidine Hydrochloride Solutions

Abstract Prior studies of the viscosity of the subunit proteoglycan obtained by dissociative extraction of bovine nasal septum cartilage suggested that the proteoglycan subunit molecule behaves as a rigid rod in aqueous solution over the range of 0.5 to 4.0 m guanidine hydrochloride. A more direct view of dimension and shape can be obtained from light-scattering and viscosity of bovine nasal cartilage proteoglycan subunit over the range from 0.05 to 4.0 m guanidine hydrochloride. The molecular weight, radius of gyration, second virial coefficient, and intrinsic viscosity were determined. The weight average molecular weight of the proteoglycan subunit was constant (2.3 x 105) at all guanidine hydrochloride concentrations. The radius of gyration decreased from a value of 1590 A in 0.05 m guanidine hydrochloride to 570 A in 4.0 m guanidine hydrochloride, and there were changes in the second virial coefficient indicating a major change in solvent-proteoglycan subunit interaction in the 0.15 to 4.0 m guanidine hydrochloride range. The second virial coefficient goes from a minimum value of -7.0 x 10-5 ml g-2 mole in 0.15 m guanidine hydrochloride to a value of 5.7 x 10-5 ml g-2 mole in 4.0 m guanidine hydrochloride. At concentrations of guanidine hydrochloride greater than 0.5 m, the viscosity was nearly constant. These data suggest that at low ionic strength in guanidine hydrochloride, the proteoglycan subunit is a rather rigid structure, but at approximately 0.3 m guanidine hydrochloride, a conformational change occurs, providing a more flexible structure which persists up to concentrations as high as 4.0 m guanidine hydrochloride. Over the same range, the separate chondroitin 4-sulfate chains show typical polyelectrolyte effects and a positive second virial coefficient ranging from +4.8 x 10-3 in 0.1 m guanidine hydrochloride to 0.6 x 10-3 in 3.0 m guanidine hydrochloride. This comparison indicates that the behavior of the intact proteoglycan subunit cannot be understood in terms of the properties of the isolated chondroitin 4-sulfate chains. It is likely that both the protein core itself and the packing of the polysaccharide chains along the core are involved in this difference.

Immunological relationships between proteoglycans of different hydrodynamic size from articular cartilage of foetal and mature pigs

Proteoglycans were extracted from articular and laryngeal cartilages of foetal and mature pigs. Antisera were prepared to extracts containing protoglycans whose size had been determined by gel chromatography. The smallest, which were separated on 6% agarose, gave a single precipitin line without needing prior digestion with hyaluronidase. Proteoglycans large enough to be excluded from 6% agarose failed to react in double diffusion and were inacapable of absorbing specific antibody unless first digested with hyaluronides, implying a difference in structure between proteoglycans of different size. After digestion, however, the larger proteoglycans effectively absorbed specific antibody and produced multiple precipitation line, consistent with the presence of several core proteins. One of these lines which developed more rapidly than the rest appeared towards the antibody well and showed identify with the determinant of the smallest proteoglycans. This determinant however, was not dissociated from the intact proteoglycans by treatment with quanidine · HCl. When the largest proteoglycans were digested with hyaluronidase and diffused against antiserum to purified small proteoglycans two precipitin lines were produced. The one nearer the antibody well showed complete identity with the determinant of the small proteoglycans, while the other, nearer the antigen well, fused with the above, suggesting that this determinant may also be amongst those on the core proteins of larger proteglycans.

The excretion and degradation of chondroitin 4-sulphate administered to guinea pigs as free chondroitin sulphate and as proteoglycan.

The excretion and degradation was studied of (35)S-labelled 4-chondroitin sulphate injected into guinea pigs in the form of proteoglycan isolated from cartilage and in the form of free chondroitin 4-sulphate prepared from the same proteoglycan by proteolysis. When the proteoglycan was injected there was a delay of about 15-20min before significant amounts or radioactivity were excreted, whereas after injection of chondroitin 4-sulphate a considerable amount of radioactivity was excreted within 10min and a much higher proportion of the radioactive dose was excreted in 1h or 24h compared with the proteoglycan. In both cases, however, a major part of the radioactivity was not excreted even in 24h. Sterile conditions were used to collect the radioactive material directly from the bladder. When chondroitin 4-sulphate was injected, the molecular sizes of injected and excreted materials were similar, as assessed by gel chromatography on Sephadex G-200, whereas when proteoglycan was injected the molecular size of the excreted labelled material was similar to that of the chondroitin 4-sulphate chains in the original proteoglycan. In neither case did the size of the excreted labelled material change with time over 1h, and low-molecular-weight labelled material was virtually absent. In contrast, when urine was collected for 24h without preservative the labelled material in it was extensively degraded after either the proteoglycan or chondroitin 4-sulphate had been given. Chondroitin 4-sulphate became similarly degraded when incubated with non-sterile urine, but not when the urine was passed through a bacterial filter, suggesting that degradation was caused by contaminating micro-organisms in the experiments in which urine was collected for 24 h. It is concluded that chondroitin 4-sulphate chains of about 18000 molecular weight can be excreted readily as such, whereas intact proteoglycans must be degraded to free glycosaminoglycans first, although both are taken up by the tissues more rapidly than they are excreted.

The antigenicity of cartilage proteoglycans: The relationship of the antigenic determinants present in Smith degraded and intact proteoglycans

In cartilage proteoglycans, chains of chondroitin sulphate are attached through a linkage region of neutral sugars to the protein core. Smith degradation, in which periodate oxidation is employed, cleaves the linkage at the terminal xylose residue, removing the entire chain without modifying the amino acids involved in the linkage. Rabbits were immunised with Smith degraded proteoglycan from bovine nasal cartilage, as well as with the intact proteoglycan and with proteoglycan from pig laryngeal cartilage. Cross reaction between all of the antigens and antisera showed that there was an antigenic determinant common to all three proteoglycans. Results obtained with the Smith degraded proteoglycan and its antiserum showed that the sugars of the linkage region and the chondroitin sulphate chains did not contribute to the expression of the species-common determinant, which must be due solely to amino acid sequence. Immunodiffusion and tanned-cell haemagglutination experiments showed that this species-common determinant was localised in that region of the core protein which in intact proteoglycans is resistant to trypsin and chymotrypsin.

Characteristics of the Protein-Keratan Sulfate Core and of Keratan Sulfate Prepared from Bovine Nasal Cartilage Proteoglycan

Abstract Protein-keratan sulfate core was isolated from bovine nasal cartilage proteoglycan after enzymatically removing chondroitin sulfate with chondroitinase AC from Flavobacterium heparinum. The core preparation exhibited a broad, approximately Gaussian distribution of buoyant densities in CsCl density gradients (between 1.43 and 1.58 g per ml with an average of 1.51 g per ml). Chemical and physical analyses of subfractions from the density gradient indicate that the core macromolecules have a wide polydispersity in molecular weights (350,000 to 550,000, with an average of 450,000). Molecules with larger molecular weights have greater buoyant densities and higher ratios of keratan sulfate to protein. The data suggest that the core molecules contain a protein (or combination of proteins) with a molecular weight of about 200,000 to which different amounts of keratan sulfate are attached. Differences in average keratan sulfate chain lengths and chain numbers appear to contribute to the polydispersity of the protein-keratan sulfate core. Keratan sulfate was isolated from the core preparation after proteolysis with papain and purification with DEAE-cellulose chromatography. Chemical and physical analyses of the keratan sulfate indicate that the polysaccharide exhibits a large polydispersity of molecular weights with a weight average molecular weight of about 8,500 and a z-average molecular weight of about 11,000, and that polysaccharide molecules with larger molecular weights have higher ratios of glucosamine to galactosamine. Alkali treatment of the keratan sulfate selectively destroys threonine and serine as it does for other skeletal keratan sulfates. After alkaline treatment the keratan sulfate chromatographs on Sephadex G-100 with a higher retention volume, Kd = 0.6, than that for the untreated preparation, Kd = 0.3, which indicates that the sizes of the molecules have been significantly reduced. The data are consistent with the hypothesis that the papain-prepared keratan sulfate consists of molecules which contain two polysaccharide chains, each with an average of about six repeat disaccharides, connected through a peptide bridge. Each chain probably contains a terminal sialic acid residue and is attached to the protein with a glycosidic bond between galactosamine and a threonine or serine residue. The high concentration of glutamic acid in the fraction suggests that the polysaccharide chains are also attached in some way to this amino acid. Such a model suggests that the intact proteoglycan macromolecules each contain about 60 keratan sulfate chains of variable lengths built onto the protein core structure.

Biosynthesis of Chondroitin Sulfate Proteoglycan

Abstract The chain-initiating reaction in chondroitin sulfate synthesis is catalyzed by a xylosyltransferase, which transfers xylose from UDP-xylose to a polypeptide acceptor. The enzyme has previously been detected in several cell-free tissue preparations, which also contain endogenous xylose acceptors. However, reliable quantitation of enzyme levels has not been possible, since such crude systems contain two unknown dependent variables, enzyme concentration and acceptor concentration. The purpose of the present work was (a) to develop exogenous acceptors which would permit accurate determination of enzyme levels and (b) at the same time gain some insight into the substrate specificity of the enzyme. Many small molecular weight compounds were tested as potential acceptors, including free serine, serine derivatives, and serine-containing peptides. One of the peptides had some acceptor activity, i.e. the tripeptide, Ser-Gly-Gly, although it had a rather high Km value of 20.0 mm. Considerably higher acceptor activity was observed with peptides derived from the chondroitin sulfate-protein linkage region by Smith degradation of a chondroitin sulfate peptidoglycan preparation, which had been isolated after trypsin digestion of the proteoglycan. Smith degradation of the entire proteoglycan molecule yielded the best acceptor obtained so far; this material had a Km of 0.064 mm, expressed in terms of concentration of serine residues. The intact proteoglycan was not an acceptor, nor were the products obtained by digestion with testicular hyaluronidase or bacterial chondroitinase, although about half of the serine residues in these three compounds are unsubstituted. Characterization of the [14C]xylosyl-labeled Smith-degraded proteoglycan showed that (a) xylose had been incorporated into the protein in an alkali-labile linkage from which it could be released as xylitol on treatment with alkaline borohydride, and that (b) exhaustive proteolysis yielded a compound with electrophoretic and chromatographic properties of xylosylserine. It is therefore concluded that xylose had been transferred to the serine hydroxyl groups which had been liberated by Smith degradation. Furthermore, the failure of the intact proteoglycan and the enzymatic digestion products to serve as acceptors suggests that glycosylation occurs specifically at certain serine sites of the proteoglycan molecule.