Proteoglycans of human gingival epithelium and connective tissue.

Proteoglycans extracted from separated specimens of healthy human gingival epithelium and from connective tissue have been purified. The epithelial proteoglycans fractionated as a single included peak on Sepharose 4B-CL and contained heparan sulphate and dermatan sulphate glycosaminoglycans. The connective-tissue proteoglycans separated into three major populations on Sepharose 4B-CL, one of which was excluded from this gel under associative conditions (0.5 M-sodium acetate, pH 7.4). Subsequent fractionation of the excluded material under dissociative conditions (4 M-guanidinium chloride/0.05 M-sodium acetate, pH 7.4) revealed an absence of any aggregate formation of molecules within this population. The connective-tissue proteoglycans contained heparan sulphate, dermatan sulphate and chondroitin 4-sulphate, the proportions of which varied with the molecular size of the proteoglycans. Amino acid analysis of the protein cores of gingival-epithelial and connective-tissue proteoglycans revealed differences that were similar to the differences described between other types of proteoglycans such as those from skin.     

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

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

Glycosaminoglycans and proteoglycans of human chondrosarcoma

The glycosaminoglycans and proteoglycans of a human chondrosarcoma have been studied. Glycosaminoglycans were fractionated and identified by cetylpyridium chloride (CPC) cellulose chromatography, ECTEOLA cellulose ion-exchange chromatography and electrophoresis on cellulose acetate. Proteoglycans were extracted by low ionic strength solutions and by 4 M guanidinium chloride and fractionated by equilibrium density-gradient centrifugation and gel chromatography on Sepharose 2B. The tumour matrix contained both the 4- and 6-sulphate isomers of chondroitin sulphate and a high concentration (12% of hexosamine) of hyaluronic acid. Proteoglycans were poor in carbohydrate moieties and a proportion were capable of aggregation. Amino acid analysis of the fractionated proteoglycans suggested the presence of a single protein core. A substance with the characteristic amino acid composition of glycoprotein link was recovered from the top of the dissociative density gradient.     

Extracellular matrix metabolism by chondrocytes 5. The proteoglycans and glycosaminoglycans synthesized by chondrocytes in high density cultures

Proteoglycans were extracted from the extracellular matrix of cultures of embryonic chick chondrocytes grown at high density and were purified by CsCl density gradient centrifugation. The chemical, physical and hyaluronate binding properties of the proteoglycans were similar to those observed in proteoglycans from other hyaline cartilages. Proteoglycans in the media were also purified and on analysis showed three populations of proteoglycans to be present. One population had the physical characteristics of a typical proteoglycan subunit and bound hyaluronate, the other two populations were unable to complex with hyaluronate but one had the physical characteristics of the proteoglycan subunit and the other was of smaller molecular weight. The small molecular weight appears to be a product of the enzymatic degradation of the larger molecular weight species.     

Glycosaminoglycans and proteoglycans of human chondrosarcoma.

The glycosaminoglycans and proteoglycans of a human chondrosarcoma have been studied. Glycosaminoglycans were fractionated and identified by cetylpirdium chloride (CPC) cellulose chromatography, ECTEOLA cellulose ion-exchange chromatography and electrophoresis on cellulose acetate. Proteoglycans were extracted by low ionic strength solutions and by 4 M guanidinium chloride and fractioned by equilibrium density-gradient centrifugation and gel chromatography on Sepharose 2B. The tumour matrix contained both the 4- and 6-sulphate isomers of chondroitin sulphate and a high concentration (12% of hexosamine) of hyaluronic acid. Proteoglycans were poor in carbohydrate moieties and proportion were capable of aggregation. Amino acid analysis of the fractionated proteoglycans suggested the presence of a single protein core. A substance with the characteristic amino acid composition of glycoprotein link was recovered from the top of the dissociative density gradient.     

Proteoglycans of guinea-pig coastal cartilage. Fractionation and characterization.

Proteoglycans were extracted, in a yield of about 90%, from costal cartilage of young, growing guinea-pigs. Three solvents were used in sequence: 0.4 M guanidine - HCl, pH 5.8, 4 M guanidine - HCl, pH 5.8, and 4 M guanidine - HCl/0.1 M EDTA, pH 5.8. The proteoglycans were purified and fractionated by cesium chloride density gradient ultracentrifugation under associative and dissociative conditions. Gel chromatography on Sepharose 2 B of proteoglycan fractions from associative centrifugations showed the presence of both aggregated and monomer proteoglycans. The ratio of aggregates to monomers was higher in the second extract than in the other two extracts. Dissociative gradient centrifugation gave a similar distribution for proteoglycans from all three extracts. Thus, with decreasing buoyant density there were decreasing ratios of polysaccharide to protein, and of chondroitin sulfate to keratan sulfate. In addition, there was with decreasing density an increasing ratio of chondroitin 4-sulfate to chondroitin 6-sulfate. Amino acid analyses of dissociative fractions were inaccordance with previously published results. On comparing proteoglycan monomers of the three extracts, significant differences were found. Proteoglycans, extracted at low ionic strength, contained lower proportions of protein, keratan sulfate, chondroitin 6-sulfate and basic amino acids than those of the second extract. The proteoglycans of the third extract also differed from those of the other extracts. The results indicate that the proteoglycans of guinea-pig costal cartilage exist as a very polydisperse and heterogenous population of molecules, exhibiting variations in aggregation capacity, molecular size, composition of protein core, degree of substitution of the protein core, as well as variability in the type of polysaccharides substituted.     

Maturation-related changes in proteoglycans of fetal articular cartilage

Proteoglycans of the articulating and growing zones of maximum and minimum contact of bovine fetal articular cartilage were studied and compared to proteoglycans of immature calf and adult steer. During fetal maturation, localized changes were observed as early as the second trimester of fetal life but were restricted to the most superficial zones. Proteoglycans extracted from the growing zones were purified by density-gradient ultracentrifugation. The majority of proteoglycan monomers were able to interact with endogenous hyaluronate to form aggregates. Monomers had, at all fetal stages, similar elution profiles on Sepharose 2B and similar ratios of chondroitin sulfate chains/keratan sulfate chains/O-glycosidically linked oligosaccharides. Keratan sulfate chains were of similar size at all stages, but chondroitin sulfate chain size decreased markedly with fetal maturation. In the first and second trimesters of fetal life, the proteoglycans were poorly substituted with glycosaminoglycans. A major increase in the absolute number of glycosaminoglycans and oligosaccharides attached to core protein was detected during the third trimester of fetal life. No further changes in substitution occurred in early postnatal life. Enzymatic digestion of proteoglycan monomer demonstrated that the increase in substitution with keratan sulfate occurred to the same extent in the main polysaccharide attachment region and in the keratan sulfate-rich region.     

Isolation of proteoglycans from human articular cartilage.

Proteoglycans were extracted from normal human articular cartilage of various ages with 4M-guanidinium chloride and were purified and characterized by using preformed linear CsCl density gradients. With advancing age, there was a decrease in high-density proteoglycans of low protein/uronic acid weight ratio and an increase in the proportion of lower-density proteoglycans, richer in keratan sulphate and protein. Proteoglycans of each age were also shown to disaggregate in 4M-guanidinium chloride and at low pH and to reaggregate in the presence of hyaluronic acid and/or low-density fractions. Osteoarthrotic-cartilage extracts had an increased content of higher-density proteoglycans compared with normal cartilage of the same age, and results also suggested that these were not mechanical or enzymic degradation products, but were possibly proteoglycans of an immature nature.     

Turnover of proteoglycans in guinea pig costal cartilage

The turnover in vivo of proteoglycans of guinea pig costal cartilage was investigated using Na₂³⁵SO₄ as precursor. Proteoglycans were extracted with guanidine · HCl, at both low and high ionic strength, and purified and fractionated by ultracentrifugation in CsCl gradients under associative and dissociative conditions. The results suggest that the sulfate is incorporated into macromolecules of at least two major metabolic pools with half-lives of about 3 days and about 60–70 days, respectively. Molecules with a fast turnover were enriched in the low ionic strength extracts and in fractions containing small, nonaggregated proteoglycans. No substantial evidence was found for a precursor-product relationship between different fractions.     

Acquisition of hyaluronate-binding affinity in vivo by newly synthesized cartilage proteoglycans.

We have studied the hyaluronate-binding properties of aggregating cartilage proteoglycans synthesized in vivo by immature (6-week), mature (25-week) and aged (75-week) rabbits. Precursor isotope (35SO4) was given by intra-articular injection and articular cartilage was removed from rabbits after periods ranging from 1.5 h to 168 h. Proteoglycans were extracted with 4 M-guanidinium/HCl and monomers were isolated by CsCl gradient centrifugation under dissociative conditions. The percentages of both radiolabelled and total tissue monomers with a high affinity for hyaluronate [that is, capable of forming aggregates on Sepharose CL-2B in the presence of 0.8% (w/w) hyaluronate] were then determined. For all samples about 30% of the tissue monomers were high-affinity; however, less than 5% of the radiolabelled monomers were high-affinity at 1.5 h after injection, and this figure increased gradually with time in vivo. The increase was rapid in immature rabbits, such that after 24 h, about 30% of the radiolabelled monomers were high-affinity; on the other hand for mature and aged rabbits the increase was markedly slower such that 30% high-affinity was attained only after about 72 h. The results show that aggregating cartilage proteoglycans are secreted in vivo in a 'precursor' form with a low affinity for hyaluronate, and suggest that conversion of these monomers to a form with a higher binding affinity occurs with a half-time of about 12 h in immature cartilages but greater than 24 h in mature cartilages. The possible relationship of these findings to the process of proteoglycan aggregation in vivo is discussed.     

Structure of proteoglycans from different layers of human articular cartilage

Full-depth plugs of adult human articular cartilage were cut into serial slices from the articular surface and analysed for their glycosaminoglycan content. The amount of chondroitin sulphate was highest in the mid-zone, whereas keratan sulphate increased progressively through the depth. Proteoglycans were isolated from each layer by extraction with 4M-guanidinium chloride followed by centrifugation in 0.4M-guanidinium chloride/CsCl at a starting density of 1.5 g/ml. The efficiency with which proteoglycans were extracted depended on slice thickness, and extraction was complete only when cartilage from each zone was sectioned at 20 microns or less. When thick sections (250 microns) were extracted, hyaluronic acid was retained in the tissue. Most of the proteoglycans, extracted from each layer under optimum conditions, could interact with hyaluronic acid to form aggregates, although the extent of aggregation was less in the deeper layers. Two pools of proteoglycan were identified in all layers by gel chromatography (Kav. 0.33 and 0.58). The smaller of these was rich in keratan sulphate and protein, and gradually increased in proportion through the cartilage depth. Chondroitin sulphate chain size was constant in all regions. The changes in composition and structure observed were consistent with the current model for hyaline-cartilage proteoglycans and were similar to those observed with increasing age in human articular cartilage.     

The presence of a cartilage-like proteoglycan in the adult human meniscus.

Proteoglycans were extracted from the adult human meniscus under dissociative conditions and purified by CsCl-density-gradient centrifugation. The preparations of highest density contained proteoglycan that possessed the ability to interact with hyaluronic acid, was of large subunit size and was composed of chondroitin sulphate, keratan sulphate and sialic acid-containing oligosaccharides. This 'cartilage-like' proteoglycan also exhibited subunit and aggregate structures analogous to those of hyaline-cartilage proteoglycans when examined by electron microscopy. However, the composition of this proteoglycan was more comparable with proteoglycans from immature cartilage than from age-matched cartilage. The preparations from lower density, which were enriched in dermatan sulphate, contained smaller proteoglycan that was not able to interact with hyaluronic acid. This non-aggregating proteoglycan may be structurally distinct from the 'cartilage-like' proteoglycan, which does not contain dermatan sulphate.     

Differences in the rates of aggregation of proteoglycans from human articular cartilage and chondrosarcoma.

Pieces of adult human articular cartilage and chondrosarcoma were incubated in the presence of [35S]sulphate. After continuous or pulse-change incorporation of radioactivity, proteoglycans were extracted with 4.0 M-guanidinium chloride, purified by equilibrium density-gradient centrifugation and fractionated by gel chromatography. A comparison of the results suggests that the formation of stable aggregates occurs at a lower rate in articular cartilage than in chondrosarcoma.     

Proteoglycans of fetal bovine tendon

The proteoglycans (PG) of bovine fetal tendon (4-8 months in utero) were extracted with 4 M guanidine HCl and partially purified by ion exchange chromatography. Proteoglycans from fetal tendon were virtually entirely small molecules (Kav approximately equal to 0.55 by Sepharose CL-4B chromatography). These small proteoglycans had dermatan sulfate glycosaminoglycan chains and a core protein (after chondroitinase ABC digestion) with Mr approximately equal to 45,000 on sodium dodecyl sulfate-polyacrylamide gels. By electrophoretic mobility, immunocross-reactivity, and V8 protease sensitivity, these proteoglycans were determined to be of both PG I and PG II types. In contrast, adult tendon contains only the PG II type of small proteoglycan. Proteoglycans synthesized by fetal tendon explant cultures were, by both chromatographic and electrophoretic mobilities, somewhat larger than those extracted from the same tissue. There was no difference in the spectrum of proteoglycans observed between those regions of fetal tendon destined to receive only tensional forces (proximal) and those regions that will be subjected as well to compressive forces (distal) in the adult. These observations indicate that the proteoglycan content and synthetic capability of all regions of fetal tendon are constant and significantly different from those of both the tensional and fibrocartilaginous regions of adult tendon.     

Immunochemical analysis of cartilage proteoglycans. Cross-reactivity of molecules isolated from different species.

Antibodies directed against whole bovine nasal-cartilage proteoglycan and against the hyaluronic acid-binding region and chondroitin sulphate peptides from the same molecule were used in immunodiffusion and immunoelectromigration experiments. Proteoglycans from bovine nasal and tracheal cartilage showed immunological identity, with all three antisera. Proteoglycans from pig hip articular cartilage, dog hip articular cartilage, human tarsal articular cartilage and rat chondrosarcoma reacted with all the antisera and showed immunological identity with the corresponding structures isolated from bovine nasal-cartilage proteoglycans. In contrast, proteoglycans from rabbit articular cartilage, rabbit nasal cartilage and cultured chick limb buds did not react with the antibodies directed against the hyaluronic acid-binding region, though reacting with antibodies raised against whole proteoglycan monomer and against chondroitin sulphate peptides. All the proteoglycans gave two precipitation lines with the anti-(chondroitin sulphate peptide) antibodies. Similarly, the proteoglycans reacting with the anti-(hyaluronic acid-binding region) antibodies gave two precipitation lines. The results indicate the presence of at least two populations of aggregating proteoglycan monomers in cartilage. The relative affinity of the antibodies for cartilage proteoglycans and proteoglycan substructures from various species was determined by radioimmunoassay. The affinity of the anti-(hyaluronic acid-binding region) antibodies for the proteoglycans decreased in the order bovine, dog, human and pig cartilage. Rat sternal-cartilage and rabbit articular-cartilage proteoglycans reacted weakly, whereas chick limb-bud and chick sternal-cartilage proteoglycans did not react. In contrast, the affinity of antibodies to chondroitin sulphate peptides for proteoglycans increased in the order bovine cartilage, chick limb bud and chick sternal cartilage, dog cartilage, rat chondrosarcoma, human cartilage, pig cartilage, rat sternal cartilage and rabbit cartilage.     

Proteoglycans of the human intervertebral disc. Electrophoretic heterogeneity of the aggregating proteoglycans of the nucleus pulposus.

Nuclei pulposi were dissected from lumbar discs of radiologically normal human spines of cadavers aged 17, 20 and 21 years. Proteoglycans were extracted with 4 M guanidine hydrochloride (dissociative conditions) with proteinase inhibitors and isolated as A1 fractions by associative density-gradient centrifugation. Aggregating and non-aggregating proteoglycans were separated by Sepharose 2B chromatography. Both aggregating and non-aggregating proteoglycans contained a keratan sulphate-rich region as isolated by chondroitinase/trypsin/chymotrypsin digestion and Sepharose CL-6B chromatography. Agarose/acrylamide-gel electrophoresis of individual fractions of a Bio-Gel A-50m dissociative-column separation of the aggregating proteoglycans revealed two, well-separated bands: S and F, the slower and faster migrating bands respectively. The non-aggregating proteoglycan fractions were eluted under associative conditions (0.5 M-sodium acetate, pH 6.8) and migrated as a single band in the electrophoretic system. The gel-electrophoretic heterogeneity of the aggregating proteoglycans was still evident after hydroxylamine fragmentation and removal of the hyaluronate-binding portion of the molecule. Dissociative density-gradient centrifugation of the aggregating proteoglycans partially separated the Band-S proteoglycans from the Band-F population. Subsequent dissociative chromatography of the high-buoyant-density Band F proteoglycans permitted discrimination of this band into two gel-electrophoresis-distinguishable populations (Bands F-1 and F-2). Enzyme-linked immunosorbent assays with a monoclonal antibody that recognized keratan sulphate demonstrated that the D1 fraction containing the Band F-1 proteoglycans was enriched in keratan sulphate compared with the total aggregating or non-aggregating pool of proteoglycans. The proteoglycans of young adult nucleus pulposus could then be ascribed to one of four structurally and/or electrophoretically distinct populations: (1) the non-aggregating population, which comprised about 70% of the total extractable proteoglycans; (2) the aggregating pool, comprising: (a) Band F-1 proteoglycans, which had a relatively large hydrodynamic size, uronate/protein weight ratio, were enriched in keratan sulphate and had a high buoyant density; (b) Band S proteoglycans, which migrated slower in agarose/acrylamide gels, had a smaller hydrodynamic size, lower buoyant density and a lower uronate/protein ratio than the Band F-1 population; (c) Band F-2 proteoglycans, which were lower in buoyant density, smaller in hydrodynamic size and slightly faster in electrophoretic mobility than the Band F-1 proteoglycans.     

Changes in the chondroitin sulfate-rich region of articular cartilage proteoglycans in experimental osteoarthritis

The chondroitin sulfate-rich region was cleaved from cartilage proteoglycans of experimental osteoarthritic canine joints to establish whether changes in this region of the molecule contribute to the well-documented increase in the chondroitin sulfate to keratan sulfate ratio in osteoarthritis. Experimental osteoarthritis was induced in eight dogs by severance of the right anterior cruciate ligament, the left joint serving as a control. Proteoglycans were extracted from the femoral cartilage of both joints, isolated as A1 fractions by associative density gradient centrifugation and cleaved with hydroxylamine. The chondroitin sulfate-rich region was isolated by either gel chromatography or dissociative density gradient centrifugation. The chondroitin sulfate-rich region from the proteoglycans of the experimental osteoarthritic joints was slightly larger in hydrodynamic size and had both a higher uronate/protein weight ratio and galactosamine/glucosamine molar ratio than the corresponding control. We conclude that the chondroitin sulfate-rich region of proteoglycans in articular cartilage of experimental osteoarthritic joints is larger and has more chondroitin sulfate than that of proteoglycans of normal cartilage.     

Fractionation of proteoglycans from bovine corneal stroma.

Proteoglycans were extracted from bovine corneal stroma with 4M-guanidinum chloride, purified by DEAE-dellulose chromatography (Antonopoulos et al., 1974) and fractionated by precipitation with ethanol into three fractions of approximately equal weight. One of these fractions consisted of a proteoglycan that contained keratan sulphate as the only glycosaminoglycan. In the othertwo fractions proteoglycans that contained chondroitin sulphate, dermatan sulphate and keratan sulphate were present. Proteoglycans which had a more than tenfold excess of galactosaminoglycans over keratan sulphate could be obtianed by further subfractionation. The gel-chromatographic patterns of the glucosaminoglycans before and after digestion with chondroitinase AC differed for the fractions. The individual chondroitin sulphate chains seemed to be larger in cornea than in cartilage. Oligosaccharides, possibly covalently linked to the protein core of the proteoglycans, could be isolated from all fractions. The corneal proteoglycans were shown to have higher protein contents and to be of smaller molecular size than cartilage proteoglycans.     

Matrix proteins bound to associatively prepared proteoglycans from bovine cartilage.

Proteoglycans were extracted from bovine tracheal cartilage by high-speed homogenization, the use of dissociative solvents being avoided. The homogenate was fractionated by gel chromatography, sucrose-density-gradient centrifugation and ion-exchange chromatography. A previously unrecognized protein, cartilage matrix protein, was identified by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It cofractionated with the proteoglycans in all systems, indicating an interaction. The cartilage matrix protein-proteoglycan complex was dissociated by treatment with 4M-guanidinium chloride. The complex again formed when the guanidine was removed. The cartilage matrix protein has a mol.wt. of more than 200000. On reduction it yields subunits with a mol.wt. of approx. 60000.