Isolation and partial characterization of low sulfated chondroitin 4-sulfate-proteoglycan in bovine blood.

Bovine plasma low sulfated chondroitin sulfate-proteoglycan (34 microgram/ml plasma), accounting for the main component of acidic glycosaminoglycans in blood, has been purified by isoelectric precipitation, dissociation with 4 M guanidine chloride followed by DEAE-chromatography, Sephadex G-200 chromatography and by preparative polyacrylamide gel electrophoresis. The proteoglycan, having a molecular weight of approx. 44,000, is composed of about 77% protein and 23% glycosaminoglycan at a molar ratio of 1 : 1 which could be cleaved by alkaline treatment into each component. Amino acid analysis of the proteoglycan and its glycosylpeptide has shown that the material is derived from a different origin from other tissue proteoglycans, though the amino acid residues surrounding O-glycosidic linkage to serine residue are quite similar to that of cartilage proteoglycan. Characteristic features of plasma low sulfate chondroitin sulfate-proteoglycan are discussed, compared with tissue materials.     

Chondroitin sulfate at the plasma membranes of cultured fibroblasts

We have previously shown that in confluent human fibroblast cultures chondroitin sulfate proteoglycan is a component of the fibronectin-containing pericellular matrix fibers. In the present work the distribution of chondroitin sulfate was studied in subconfluent cell cultures using antibodies that bind to a chemically defined carbohydrate fragment of chondroitinase ABC-modified chondroitin sulfate proteoglycan. Using immunofluorescence microscopy, we observed, in addition to the fibrillar matrix staining, chondroitin sulfate diffusely distributed at the cell surface. In indirect immunoferritin electron microscopy this staining corresponded to patchy binding of ferritin close (24 nm) to the outer aspect of the plasma membrane. The patchy organization appeared uniform in all cell surfaces. The cell surface chondroitin sulfate could not be removed from the plasma membrane by agents that dissociate electrostatic interactions. These data show that in fibroblasts chondroitin sulfate is a component of the outer aspect of the plasma membrane, and raise the possibility of an integral plasma membrane chondroitin sulfate proteoglycan.     

Studies on the polydispersity and heterogeneity of cartilage proteoglycans. Identification of 3 proteoglycan structures in bovine nasal cartilage.

1. Three chondroitin sulphate components were isolated from adult bovine nasal cartilage after treatment with alkaline NaB3H. Average molecular weights of 13000, 18 600 and 28 000 were obtained for chondroitin sulphate species representing 10, 52 and 38% (w/w) of the total chondroitin sulphate respectively. Each chondroitin sulphate pool has a narrow molecular-weight distribution. 2. A proteoglycan subunit preparation, isolated from one nasal cartilage by extraction and density-gradient fractionation in dissociative solvents, partitioned on a CSCl density gradient according to size and composition. Variation of proteoglycan molecular weight across the gradient was directly related to the average chondrotin sulphate chain length, which in turn reflected the relative proportion of the three chondroitin sulphate pools in each proteoglycan fraction. Consideration of proteoglycan molecular parameters, compositions and behaviour on sedimentation leads to a proposal that nasal cartilage contains 3 distinct proteoglycan pools, each of which has a constant number of chondroitin sulphate side chains of different average molecular weight. 3. Molecular-weight distribution parameters for these proteoglycan preparations indicate that all serine residues on the protein core capable of initiating chondroitin sulphate biosynthesis are occupied and that proteoglycan polydispersity results directly from the polydispersity of the attached chondroitin sulphate component.     

Isolation and characterization of developmentally regulated chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of brain identified with monoclonal antibodies

A panel of monoclonal antibodies prepared to the chondroitin sulfate proteoglycans of rat brain was used for their immunocytochemical localization and isolation of individual proteoglycan species by immunoaffinity chromatography. One of these proteoglycans (designated 1D1) consists of a major component with an average molecular size of 300 kDa in 7-day brain, containing a 245-kDa core glycoprotein and an average of three 22-kDa chondroitin sulfate chains. A 1D1 proteoglycan of approximately 180 kDa with a 150-kDa core glycoprotein is also present at 7 days, and by 2-3 weeks postnatal this becomes the major species, containing a single 32-kDa chondroitin 4-sulfate chain. The concentration of 1D1 decreases during development, from 20% of the total chondroitin sulfate proteoglycan protein (0.1 mg/g brain) at 7 days postnatal to 6% in adult brain. A 45-kDa protein which is recognized by the 8A4 monoclonal antibody to rat chondrosarcoma link protein copurifies with the 1D1 proteoglycan, which aggregates to a significant extent with hyaluronic acid. A chondroitin/keratan sulfate proteoglycan (designated 3H1) with a size of approximately 500 kDa was isolated from rat brain using monoclonal antibodies to the keratan sulfate chains. The core glycoprotein obtained after treatment of the 3H1 proteoglycan with chondroitinase ABC and endo-beta-galactosidase decreases in size from approximately 360 kDa at 7 days to approximately 280 kDa in adult brain. In 7-day brain, the proteoglycan contains three to five 25-kDa chondroitin 4-sulfate chains and three to six 8.4-kDa keratan sulfate chains, whereas the adult brain proteoglycan contains two to four chondroitin 4-sulfate chains and eight to nine keratan sulfate chains, with an average size of 10 kDa. The concentration of 3H1 increases during development from 3% of the total soluble proteoglycan protein at 7 days to 11% in adult brain, and there is a developmental decrease in the branching and/or sulfation of the keratan sulfate chains. A third monoclonal antibody (3F8) was used to isolate a approximately 500-kDa chondroitin sulfate proteoglycan comprising a 400-kDa core glycoprotein and an average of four 28-kDa chondroitin sulfate chains. In the 1D1 and 3F8 proteoglycans of 7-day brain, 20 and 33%, respectively, of the chondroitin sulfate is 6-sulfated, whereas chondroitin 4-sulfate accounts for greater than 96% of the glycosaminoglycan chains in the adult brain proteoglycans.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hemostatic properties and serum lipoprotein binding of a heparan sulfate proteoglycan from bovine aorta

The biologic properties of two major proteoglycans of bovine aorta, heparan sulfate proteoglycan and chondroitin sulfate-dermatan sulfate proteoglycan were compared. The heparan sulfate proteoglycan was isolated either by elastase digestion or by 4.0 M guanidine hydrochloride extraction, of aorta tissue, fractionated by CsCl isopycnic centrifugation and purified by chondroitinase ABC treatment. The first method resulted in considerably greater yield (about 70% of the total heparan sulfate proteoglycan of the tissue) than the second procedure (12% of total). The chondroitin sulfate-dermatan sulfate proteoglycan was obtained by 4.0 M guanidine-HCl extraction of aorta tissue followed by CsCl isopycnic centrifugation. The chemical composition of both heparan sulfate proteoglycan preparations was similar. Unlike the chondroitin sulfate-dermatan sulfate proteoglycan, which eluted in the void volume of Sepharose CL-6B column, the heparan sulfate proteoglycan preparations were each resolved into a high molecular weight fraction (kav = 0.18 and 0.13) and a low molecular weight fraction (kav = 0.47 and 0.36). The heparan sulfate proteoglycan preparations exhibited significantly more potent anticoagulant and platelet aggregation inhibitory activities than the chondroitin sulfate-dermatan sulfate proteoglycan. The protein core of the proteoglycan molecules did not seem to be essential for their hemostatic properties. The complex forming ability of the heparan sulfate proteoglycan with serum low density lipoproteins (LDL) was much less than that of chondroitin sulfate-dermatan sulfate proteoglycan in the presence and absence of Ca2+. Interaction between heparan sulfate proteoglycan and LDL was also much more sensitive to changes in the ionic strength of the medium than that of chondroitin sulfate-dermatan sulfate proteoglycan and the lipoprotein. Since the total sulfate content of both proteoglycans is almost similar, the smaller molecular size and hence the lower overall charge density of the heparan sulfate proteoglycan appears to be partly responsible for its low affinity for LDL. The differences in biologic properties of the two proteoglycans might have implications in the pathophysiology of cardiovascular diseases.     

Characterization of a chondroitin 4-sulfate proteoglycan carrier for heparin neutralizing activity (platelet factor 4) released from human blood platelets

Platelet heparin neutralizing activity (platelet factor 4) is released from human blood platelets by thrombin in the form of a high molecular weight proteoglycan-platelet factor 4 complex. This complex was partially purified by isoelectric precipitation and gel filtration. At high ionic strength (I = 0.75) the complex dissociates into the active component (mol. wt 29000) and the proteoglycan carrier. The components were separated by gel filtration and the proteoglycan further purified by Na₂SO₄ treatment. The molecular weight of the purified carrier was 59000. The carbohydrate moieties of the proteoglycan isolated after papain digestion and ion-echange chromatography were shown to consist of chondroitin 4-sulfate by chemical, physical and electrophoretic analysis. The multichain proteoglycan consists of four chondroitin 4-sulfate chains (mol. wt 12000) in covalent linkage to a single polypeptide. The molecular weight (350000) of the fully saturated proteoglycan carrier suggests that 4 moles of platelet factor 4 are bound per mole of proteoglycan and that the carrier occurs in the form of a dimer consisting of 8 moles of platelet factor 4 and 2 moles of proteoglycan. The isolated chondroitin 4-sulfate moieties combine with platelet factor 4 at a binding ratio of one mole of platelet factor 4 per carbohydrate chain. Heparin completely displaces platelet factor 4 from both the saturated proteoglycan and chondroitin 4-sulfate complexes. Heparitin sulfate, dermatan sulfate and chondroitin 6-sulfate also combine stoichiometrically with platelet factor 4 and are displaced by equimolar amounts of heparin. Hyaluronic acid did not combine with platelet factor 4. The relative binding capacities of glycosaminoglycans for platelet factor 4 were shown to be: heparin (100), heparitin sulfate (75), chondroitin 4-sulfate (50), dermatan sulfate (50), chondroitin 6-sulfate (50), and hyaluronic acid (o). Chondroitin 4-sulfate was identified as the major glycosaminoglycan in all platelet subcellular fractions; in addition, the soluble fraction contains a minor amount of hyaluronic acid. Subcellular distribution studies revealed that 55% of both the proteoglycan carrier and platelet factor 4 activity were localized in the “granule rich” fraction. This data together with the low recovery of both these components in the membrane fraction, suggest that they occur together as a complex within specific granules and are released in this form under physiologic conditions.     

Membrane-anchored proteoglycans of mouse macrophages: P388D1 cells express a syndecan-4–like heparan sulfate proteoglycan and a distinct chondroitin sulfate form

Proteoglycan accumulation by thioglycollate-elicited mouse peritoneal macrophages and a panel of murine monocyte-macrophage cell lines has been examined to determine whether these cells express plasma membrane-anchored heparan sulfate proteoglycans. Initially, cells were screened for heparan sulfate and chondroitin sulfate glycosaminoglycans after metabolic labeling with radiosulfate. Chondroitin sulfate is secreted to a variable extent by every cell type examined. In contrast, heparan sulfate is all but absent from immature pre-monocytes and is associated predominantly with the cell layer of mature macrophage-like cells. In the P388D1 cell line, the cell-associated chondroitin sulfate is largely present as a plasma membrane-anchored proteoglycan containing a 55 kD core protein moiety, which appears to be unique. In contrast, the cell-associated heparan sulfate is composed of a proteoglycan fraction and protein-free glycosaminoglycan chains, which accumulate intracellularly. A fraction of the heparan sulfate proteoglycan contains a lipophilic domain and can be released from cells following mild treatment with trypsin, suggesting that it is anchored in the plasma membrane. Isolation of this proteoglycan indicates that it is likely syndecan-4: it is expressed as a heparan sulfate proteoglycan at the cell surface, it is cleaved from the plasma membrane by low concentrations of trypsin, and it consists of a single 37 kD core protein moiety that co-migrates with syndecan-4 isolated from NMuMG mouse mammary epithelial cells. Northern analysis reveals that a panel of macrophage-like cell lines accumulate similar amounts of syndecan-4 mRNA, demonstrating that this proteoglycan is expressed by a variety of mature macrophage-like cells. Syndecan-1 mRNA is present only in a subset of these cells, suggesting that the expression of this heparan sulfate proteoglycan may be more highly regulated by these cells. © 1993 Wiley-Liss, Inc.     

Digestion of proteoglycan by Bacteroides thetaiotaomicron

It has been shown previously that Bacteroides thetaiotaomicron, a human colonic anaerobe, can utilize the tissue mucopolysaccharide chondroitin sulfate as a source of carbon and energy and that the enzymes involved in this utilization are all cell associated (A. A. Salyers and M. B. O'Brien, J. Bacteriol. 143:772-780, 1980). Since chondroitin sulfate does not generally occur in isolated form in tissue, but rather is bound covalently in proteoglycan, we investigated the extent to which chondroitin sulfate which is bound in such a sterically hindered complex can be utilized by intact bacteria. Intact cells of B. thetaiotaomicron were able to digest chondroitin sulfate in proteoglycan, although at a slightly slower rate than free chondroitin sulfate. Prior digestion of proteoglycan with trypsin to produce small fragments of protein with several chondroitin sulfate chains attached did not increase the rate at which the bound chondroitin sulfate was digested. Accordingly, the slower rate of digestion was probably due to attachment of chondroitin sulfate chains to the protein backbone rather than to steric hindrance by other components of the proteoglycan. When proteoglycan which had been incubated with intact bacteria was treated with sodium borohydride to release the undigested fragments of chondroitin sulfate from the protein backbone, the size and composition of the fragments indicated that intact bacteria were able to digest all but three monosaccharides of the chondroitin sulfate chains. Thus, despite steric hindrance due to attachment of the chondroitin sulfate chains to the protein backbone, digestion of bound chondroitin sulfate by intact bacteria was nearly complete.     

Isolation and Immunohistochemical Localization of a Chondroitin Sulfate Proteoglycan from Adult Rat Brain

A chondroitin sulfate proteoglycan called PGM1 has been isolated from the particulate fraction of adult rat forebrain. Delipidation of the material, solubilization of proteoglycans in guanidinium chloride, precipitation at low ionic strength, and final extraction at pH 5.0 were used for its isolation. Proteoglycans were subjected to further purification by diethylaminoethyl-cellulose chromatography. Individual components were separated by gel filtration. PGM1 appeared to be a high-molecular-weight chondroitin sulfate proteoglycan, capable of strong interaction with hyaluronic acid. It was finally isolated by gel filtration on Ultrogel AcA 22 in the presence of 4 M guanidinium chloride. Monospecific antibodies obtained in rabbits against the purified molecule did not cross-react with other brain proteoglycans. Immunocytochemical techniques revealed an almost unique association of this compound with axons, particularly those known to contain neurofilaments. However, not all these axons and all parts of these axons contained PGM1. This component was not detectable in liver, intestine, spleen, kidney, lung, heart, skin, hair, lens, and muscle, a finding suggesting a specificity for the nervous tissue. This component is expressed in neural cell cultures. Despite the preservation of the neuronal specificity, it seems to lose its specific axonal localization in vitro.     

Isolation of a chondroitin sulfate--dermatan sulfate proteoglycan from bovine aorta.

Material containing proteoglycans was extracted from bovine aorta by the dissociative solvent 3.0 m MgCl². The proteoglycan that remained in solution at low ionic strength was purified by isopycnic CsCl centrifugation (?, 1.75 – 1.89 g/ml). From the lower third of the gradient a proteoglycan was isolated which behaved as a homogeneous material when analyzed by the ultracentrifuge and by electrophoresis on cellulose acetate. The proteoglycan contained 12% protein, 21% uronic acid, and 28% hexosamine. Analyses by hyaluronidase digestion and gas-liquid chromatography of the polysaccharide moieties of the proteoglycan showed a composition of 56% chondroitin 6-sulfate, 20% chondroitin 4-sulfate, and 7% dermatan sulfate. A copolymeric structure for the polysaccharide of the proteoglycan is proposed.     

Organization of glycosaminoglycan chains in a chondroitin sulfate-dermatan sulfate proteoglycan from bovine aorta

A chondroitin sulfate-dermatan sulfate proteoglycan was isolated from bovine aorta intima by extraction of the tissue by 4 M guanidine hydrochloride. The proteoglycan was purified by CsCl isopycnic centrifugation followed by gel filtration and ion-exchange chromatography. The proteoglycan had 21.9% protein, 22.1% uronate, 21.4% hexosamine and 10.8% sulfate. Glycosaminoglycan chains obtained from the proteoglycan by beta-elimination were resolved by gel filtration into two fractions, one containing chondroitin 6-sulfate with an approximate molecular weight of 49 000 and the other containing chondroitin 4-sulfate and dermatan sulfate in a proportion of 2:1 with an approximate molecular weight of 37 000. Digestion of the proteoglycan by chondroitinase ABC or AC yielded a protein core with similar composition and behavior in gel filtration and SDS-polyacrylamide gel electrophoresis. An approximate molecular weight of 180 000 was estimated for the core protein. Dermatan sulfate chains with an approximate molecular weight of 10 000 were observed only in the digest of chondroitinase AC. Limited trypsin hydrolysis of the proteoglycan yielded three peptide fragments containing chondroitin 6-sulfate, chondroitin 4-sulfate and dermatan sulfate in varied proportions. A tentative structure for the proteoglycan was suggested.     

Organization of glycosaminoglycan chains in a chondroitin sulfate - dermatan sulfate proteoglycan from bovine aorta

A chondroitin sulfate - dermatan sulfate proteoglycan was isolated from bovine aorta intima by extraction of the tissue by 4 M guanidine hydrochloride. The proteoglycan was purified by CsCl isopycnic centrifugation followed by gel filtration and ion-exchange chromatography. The proteoglycan had 21.9% protein, 22.1% uronate, 21.4% hexosamine and 10.8% sulfate. Glycosaminoglycan chains obtained from the proteoglycan by β-elimination were resolved by gel filtration into two fractions, one containing chondroitin 6-sulfate with an approximate molecular weight of 49 000 and the other containing chondroitin 4-sulfate and dermatan sulfate in a proportion of 2:1 with an approximate molecular weight of 37 000. Digestion of the proteoglycan by chondroitinase ABC or AC yielded a protein core with similar composition and behavior in gel filtration and SDS-polyacrylamide gel electrophoresis. An approximate molecular weight of 180 000 was estimated for the core protein. Dermatan sulfate chains with an approximate molecular weight of 10 000 were observed only in the digest of chondroitinase AC. Limited trypsin hydrolysis of the proteoglycan yielded three peptide fragments containing chondroitin 6-sulfate, chondroitin 4-sulfate and dermatan sulfate in varied proportions. A tentative structure for the proteoglycan was suggested.     

Proteochondroitin sulfate synthesized in cartilages induced in vivo and in vitro by bone matrix gelatin.

Implanted allogeneic demineralized bone matrix gelatin induced sequential development of cartilage and bone in the recipient rat muscle tissue. Proteoglycans of the implants labeled in vivo with [35S]sulfate at different stages of development were analyzed by sucrose density gradient centrifugation. The major proteoglycan synthesized in day-5 implant, just prior to onset of chondrogenesis, was a dermatan sulfate-containing proteoglycan with relatively slow sedimentation rate. Additionally, a small amount of a faster sedimenting component could be detected. The faster sedimenting proteoglycan, in which chondroitin 4-sulfate accounted for 85% of total radioactivity, became predominant in day-10 sample when cartilage formation was maximal. By day 30, when cartilage had been replaced by newly formed bone, the synthesis of this faster sedimenting component had ceased. A similar, if not identical, proteoglycan was found to be a major one synthesized by the in vitro-induced cartilage. This proteoglycan was smaller in overall size and shorter in length of its chondroitin sulfate chains than a major proteoglycan component obtained from neonatal rat epiphyseal cartilage. Concurrent with these changes in proteoglycan type, there appeared to be a change in collagen type, since type II collagen, in addition to type I collagen, was synthesized in day-10 implant. These results indicate that the proteoglycan can be used as a molecular marker for chondrogenesis by bone matrix gelatin.     

Occurrence and structural characterization of versican-like proteoglycan in human vitreous

Human vitreous gel is a special type of extracellular matrix, in which interpenetrating networks of collagen fibrils and hyaluronan are found. In this study, we report that apart from significant amounts of collagen, hyaluronan and sialylated glycoproteins, it was found that the human vitreous gel also contained low amounts of versican-like proteoglycan. The concentration of versican-like proteoglycan in the whole vitreous is 0.06 mg protein/ml of vitreous gel and represents a small percentage (about 5%) of the total protein content. The versican-like proteoglycan has a molecular mass of 380 kDa, as estimated by gel chromatography. Its core protein is substituted by chondroitin sulphate side chains (average molecular weight 37 kDa), in which 6-sulphated disaccharides predominated. According to the physicochemical data, the number of chondroitin sulphate chains is likely to be 5-7 per molecule. These proteoglycan monomers form large aggregates with endogenous hyaluronan. Versican, which is able to bind lectins via its C-terminal region, may bridge or interconnect various constituents of the extracellular matrix via its terminal domains in order to stabilize large supramolecular complexes at the vitreous, contributing towards the integrity and specific properties of the tissue.     

Quantitative measurements of the proton-motive force and its relation to steady state lactose accumulation in Escherichia coli.

1. Developing tail tendons from rats (19-day foetal to 126 days post partum) were examined by electron microscopy after staining for proteoglycan with a cationic copper phthalocyanin dye. Cuprolinic Blue, in a "critical electrolyte concentration" method. Hydroxyproline was measured on papain digests of tendons, from which glycosaminoglycuronans were isolated, characterized and quantified. 2. Mean collagen fibril diameters increased more than 10-fold with age according to a sigmoid curve, the rapid growth phase 2 being during 30-90 days after conception. Fibril periodicities were considerably smaller (50-55 nm) in phases 1 and 2 than in phase 3 (greater than 62 nm). 3. Dermatan sulphate is the main glycosaminoglycuronan in mature tendon. Chondroitin sulphate and hyaluronate preponderate in foetal tissue. 4. Proteoglycan was seen around but not inside collagen fibrils. Proteoglycan and collagen were quantified from electron micrographs. Their ratios behaved similarly to uronic acid/hydroxyproline and hyaluronate/hydroxyproline ratios, which decreased rapidly around birth, and then levelled off to a low plateau coincident with the onset of rapid growth in collagen fibril diameter. 5. Dermatan sulphate/hydroxyproline ratios suggest that the proteoglycan orthogonal array around the fibril is largely dermatan sulphate. In the foetus hyaluronate and chondroitin sulphate exceed that expected to be bound to collagen. 6. An inhibiting action of chondroitin sulphate-rich proteoglycan on fibril diameter growth is suggested. 7. The distributions of hyaluronate, chondroitin sulphate and dermatan sulphate are discussed in the light of secondary structures suggested to be present in hyaluronate and chondroitin sulphate, but not in dermatan sulphate.     

The biosynthesis of proteoglycans and interstitial collagens by bovine pericardial fibroblasts

The biosynthesis of interstitial collagens (types I and III) and proteoglycans was studied in fibroblasts isolated from the parietal layer of bovine pericardium. Confluent cultures were labeled with Na2 35SO4 for proteoglycans or 14C-proline for collagens. The proteoglycans synthesized by pericardial fibroblasts were purified by DEAE-Sephacel chromatography and further fractionated into three components by gelfilitration. Two minor high molecular weight proteoglycans were shown by SDS-PAGE to be resistant to chondroitinase ABC and AC, and partially degraded by nitrous acid. The major, low molecular weight proteoglycan had a core protein of 45 kDa and is considered to be a dermatan sulfate/chondroitin sulfate proteoglycan since it was resistant to nitrous acid, but digested partially by chondroitinase AC and completely by ABC. The pericardial fibroblasts synthesized predominantly type I collagen and low amounts (about 10%) of type III collagen which was detected by delayed reduction on SDS-PAGE. The data show that pericardial fibroblasts synthesize the same macromolecules that can be extracted from the intact tissue and suggest that the proteoglycan may play a structural as well as physiological role.     

Purification and characterization of human platelet proteoglycan.

Freshly prepared platelets were shown to contain glycosaminoglycans equivalent to 530 micrograms of hexuronate/10(11) platelets. When the platelets were extracted with 4 M-guanidinium chloride containing proteinase inhibitors, and the extract was dialysed extensively against 7 M-urea solution, almost all of proteoglycan was recovered in the urea-soluble fraction. The proteoglycan was purified from the urea-soluble fraction with a yield of 47% by DEAE-Sephacel chromatography, CsCl-density-gradient centrifugation, Bio-Gel A-15m gel filtration and then rechromatography on DEAE-Sephacel. The purified proteoglycan contained 30% glucuronic acid, 32% N-acetylgalactosamine, 14% sulphate and 15% protein. Serine, glutamic acid, glycine, aspartic acid and leucine accounted for 64% of the total amino acids. The Mr of the proteoglycan was assessed to be approx. 136000 by sedimentation-equilibrium methods. The galactosaminoglycan released by alkaline-borohydride treatment of the proteoglycan was converted stoichiometrically into 4-sulphated unsaturated disaccharide by digestion with chondroitinase AC-II, indicating that the galactosaminoglycan was fully sulphated chondroitin 4-sulphate. The apparent Mr of the chondroitin sulphate was assessed to be 28000 by gel filtration on Bio-Gel A-0.5m (KD 0.18). On two-dimensional electrophoresis on a cellulose acetate membrane, the chondroitin sulphate gave a single compact spot co-migrating with a reference chondroitin sulphate, indicating that the chondroitin sulphate chains were homogeneous in both length and charge density. On the basis of these results, the proteoglycan in human platelets was concluded to be a macromolecule of Mr 136000 containing four chondroitin 4-sulphate chains each with the apparent Mr of 28000.     

Proteochondroitin sulfate synthesized in cartilages induced in vivo and in vitro by bone matrix gelatin

Implanted allogeneic demineralized bone matrix gelatin induced sequential development of cartilage and bone in the recipient rat muscle tissue. Proteoglycans of the implants labeled in vivo with [³⁵S]sulfate at different stages of development were analyzed by sucrose density gradient centrifugation. The major proteoglycan synthesized in day-5 implant, just prior to onset of chondrogenesis, was a dermatan sulfate-containing proteoglycan with relatively slow sedimentation rate. Additionally, a small amount of a faster sedimenting component could be detected. The faster sedimenting proteoglycan, in which chondroitin 4-sulfate accounted for 85% of total radioactivity, became predominant in day-10 sample when cartilage formation was maximal. By day 30, when cartilage had been replaced by newly formed bone, the synthesis of this faster sedimenting component had ceased. A similar, if not identical, proteoglycan was found to be a major one synthesized by the in vitro-induced cartilage. This proteoglycan was smaller in overall size and shorter in length of its chondroitin sulfate chains than a major proteoglycan component obtained from neonatal rat epiphyseal cartilage. Concurrent with these changes in proteoglycan type, there appeared to be a change in collagen type, since type II collagen, in addition to type I collagen, was synthesized in day-10 implant. These results indicate that the proteoglycan can be used as a molecular marker for chondrogenesis by bone matrix gelatin.     

Isolation and characterization of proteoglycans from the swarm rat chondrosarcoma.

Proteoglycan monomer (D1) and aggregate (A1) preparations were isolated from 4 M guanidinium chloride extracts of the Swarm rat chondrosarcoma. When EDTA, 6-aminohexanoic acid, and benzamidine were present in the solutions, the D1 preparation contained a single component (SO = 23 S), and the A1 preparation contained 30% monomer (SO = 23 S) and 70 percent aggregate (SO = 111 S). In the absence of EDTA, 6-aminohexanoic acid, and benzamidine, the A1 preparations contained only small proteoglycan fragments, indicating that extensive enzymatic degradation had occurred. The composition of the proteoglycan monomer was different from that of proteoglycan monomer preparations from normal hyaline cartilages in that it did not contain keratan sulfate and chondroitin 6-sulfate; only chondroitin 4-sulfate was found. The A1 preparation from the chondrosarcoma contained only one link protein, which was like the smaller (molecular weight of 40,000) of the two link proteins present in A1 preparations from bovine nasal cartilage. When the A1 preparation from the chondrosarcoma was treated with chondroitinase ABC and trypsin and the digest was chromatographed on Sepharose 2B, a complex was isolated which contained the link protein and the segments of the protein core from the hyaluronic acid-binding region of the proteoglycan molecules.     

Identification of chondroitin sulfate E in human lung mast cells

Human lung mast cells (HLMC) enriched up to 99% purity by counter current elutriation and density gradient centrifugation were labeled with 35S-sulfate to determine cell-associated proteoglycans. The 35S-labeled proteoglycans were extracted by the addition of detergent and 4 M guanidine-HCl, and separated from unincorporated precursor by Sephadex G-50 chromatography. 35S-Proteoglycans chromatographed over Sepharose 4B with a Kav of 0.48. 35S-Glycosaminoglycans separated from the parent 35S-proteoglycans by beta-elimination and chromatographed over Sepharose 4B with a Kav of 0.63. Characterization of 35S-proteoglycans by chondroitin ABC lyase treatment revealed approximately 36% of the proteoglycan to be composed of chondroitin sulfates. Analysis by HPLC of component disaccharides liberated by chondroitin ABC lyase using an amino-cyano-substituted silica column indicated that the chondroitin sulfates consisted of the monosulfated A disaccharide (GlcUA----GaINAc4SO4) (75%) and the over-sulfated E disaccharide (GlcUA----GaINAc4,6-diSO4) (25%). Nitrous acid/heparinase-susceptible heparin proteoglycans accounted for approximately 62% of the total 35S-proteoglycans present in the HLMC. Proteoglycans remaining after exposure of the original proteoglycan extract to either heparinase or chondroitin ABC lyase were of similar size, suggesting that the majority of heparin and chondroitin sulfate glycosaminoglycans were on separate protein cores. Proteoglycans extracted from HLMC were protease insensitive. Hence, in addition to heparin proteoglycans, HLMC synthesize a hitherto unrecognized quantity of chondroitin sulfate E proteoglycans.