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.     

Age-related changes in the composition and structure of human articular-cartilage proteoglycans.

1. Analysis of the purified proteoglycans extracted from normal human articular cartilage with 4M-guanidinium chloride showed that there was an age-related increase in their content of protein and keratan sulphate. 2. The hydrodynamic size of the dissociated proteoglycans also decreased with advancing age, but there was little change in the proportion that could aggregate. 3. Results suggested that some extracts of aged-human cartilage had an increased content of hyaluronic acid compared with specimens from younger patients. 4. Dissociated proteoglycans, from cartilage of all age groups, bind to hyaluronic acid and form aggregates in direct proportion to the hyaluronic acid concentration. 5. Electrophoretic heterogeneity of the dissociated proteoglycans was demonstrated on polyacrylamide/agarose gels. The number of proteoglycan species observed was also dependent on the age of the patient.     

Articular-cartilage proteoglycans in aging and osteoarthritis.

The composition of macroscopically normal hip articular cartilage obtained from dogs of various ages was studied. Pieces of cartilage with signs of degeneration were studied separately. In normal aging, the extraction yield of proteoglycans decreased; the keratan sulphate content of extracted proteoglycans increased and the chondroitin sulphate content decreased. The extracted proteoglycans were smaller in the older cartilage, mainly owing to a decrease in the chondroitin sulphate-rich region of the proteoglycan monomers. The hyaluronic acid-binding region and the keratan sulphaterich region were increased and the molar concentration of proteoglycan probably increase with increasing age. The degenerated cartilage had higher water content and the proteoglycans, as well as other tissue components, gave higher yields. The proteoglycan monomers from the degenerated cartilage were smaller than those from normal cartilage of the same age, and hence had a smaller chondroitin sulphate-rich region and some of the molecules also appeared to lack the hyaluronic acid-binding region. Increased proteolytic activity may be involved in the process of cartilage degeneration.     

Variations in the composition of bovine hip articular cartilage with distance from the articular surface.

Punch biopsies of bovine hip articular cartilage was sectioned according to depth and the proteoglycans were isolated. The mid-sections of the cartilage contained more proteoglycans than did either the superficial or the deepest portions of the cartilage proteoglycans than did either the superficial or the deepest portions of the cartilage. The most superficial 40 micrometer of the cartilage contained relatively more glucosaminoglycans compared with the remainder of the cartilage. The proteoglycans recovered from the surface 200 micrometer layer contained less chondroitin sulphate, were smaller and almost all of these molecules were able to interact with hyaluronic acid to form aggregates. From about 200 micrometer and down to 1040 micrometer from the surface, the proteoglycans became gradually somewhat smaller, probably owing to decreasing size of the chondroitin sulphate-rich region. The proportion of molecules that were able to interact with the hyaluronic acid was about 90% and remained constant with depth. The proteoglycans from the deepest layer near the cartilage-bone junction contained a large proportion of non-aggregating molecules, and the average size of the proteoglycans was somewhat larger. The alterations of proteoglycan structure observed with increasing depth of the articular cartilage beneath the surface layer (to 200 micrometer) are of the same nature as those observed with increasing age in full-thickness articular cartilage. The articular-cartilage proteoglycans were smaller and had much higher keratan sulphate and protein contents that did molecules isolated from bovine nasal or tracheal cartilage.     

Hyaluronic acid in cartilage and proteoglycan aggregation

1. Dissociation of purified proteoglycan aggregates was shown to release an interacting component of buoyant density higher than that of the glycoprotein-link fraction of Hascall & Sajdera (1969). 2. This component, which produced an increase in hydrodynamic size of proteoglycans on gel chromatography, was isolated by ECTEOLA-cellulose ion-exchange chromatography and identified as hyaluronic acid. 3. The effect of pH of extraction showed that the proportion of proteoglycan aggregates isolated from cartilage was greatest at pH4.5. 4. The proportion of proteoglycans able to interact with hyaluronic acid decreased when extracted above or below pH4.5, whereas the amount of hyaluronic acid extracted appeared constant from pH3.0 to 8.5. 5. Sequential extraction of cartilage with 0.15m-NaCl at neutral pH followed by 4m-guanidinium chloride at pH4.5 was shown to yield predominantly non-aggregated and aggregated proteoglycans respectively. 6. Most of the hyaluronic acid in cartilage, representing about 0.7% of the total uronic acid, was associated with proteoglycan aggregates. 7. The non-aggregated proteoglycans were unable to interact with hyaluronic acid and were of smaller size, lower protein content and lower keratan sulphate content than the disaggregated proteoglycans. Together with differences in amino acid composition this suggested that each type of proteoglycan contained different protein cores.     

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 the intervertebral disc. Absence of degradation during the isolation of proteoglycans from the intervertebral disc.

Proteoglycans extracted with 4M-guanidinium chloride from pig intervetebral discs, and purified by equilibrium density-gradient centrifugation in CsCl, were of smaller hydrodynamic size than those extracted and purified in the same way from the laryngeal cartilage of the same animal. Whether this difference in size arose from degradation during the extraction and purification of the proteoglycans of the disc was investigated. Purified proteoglycans labelled either in the chondroitin sulphate chains or in the core protein were obtained from laryngeal cartilage by short-term organ culture. These labelled proteoglycans were added at the beginning of the extraction of the disc proteoglycans, and labelled cartilage and unlabelled disc proteoglycans were isolated and purified together. There was no appreciable loss of radioactivity after density-gradient centrifugation nor decrease in hydrodynamic size of the labelled cartilage proteoglycans on chromatography on Sepharose 2B, when these were present during the extraction of disc proteoglycans. It is concluded that disc proteoglycans are intrinsically of smaller size than cartilage proteoglycans and this difference in size does not arise from degradation during the extraction.     

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.     

Proteoglycans of the intervertebral disc. Homology of structure with laryngeal proteoglycans.

The structure of the proteoglycans from normal pig nucleus pulposus and relatively normal human annulus fibrosus and nucleus pulposus was investigated in detail and the results were compared with the current structural model of proteoglycans of hyaline cartilage. Like proteoglycans of cartilage, those of intervertebral disc contain keratan sulphate and chondroitin sulphate attached to a protein core; they are able to aggregate to hyaluronic acid; the protein core likewise has three regions, one lacking glycosaminoglycans, another rich in keratan sulphate and a third region rich in chondroitin sulphate. However, disc proteoglycans contain more keratan sulphate and protein and less chondroitin sulphate and are also considerably smaller than cartilage proteoglycans. In proteoglycans of human discs, these differences appeared to be due principally to a shorter region of the core protein bearing the chondroitin sulphate chains, whereas in proteoglycans of pig discs their smaller size and relatively low uronic acid content were due to shorter chondroitin sulphate chains. There were subtle differences between proteoglycans from the nucleus and annulus of human discs. In the latter a higher proportion of proteoglycans was capable of binding to hyaluronate.     

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.     

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.     

Biosynthesis and metabolism in vivo of intervertebral-disc proteoglycans in the mouse.

The synthesis and turnover in vivo of 35S-labelled proteoglycans in mouse cervical, thoracic and lumbar intervertebral discs, and in costal cartilage, was investigated after intraperitoneal injection of [35S]sulphate. Intervertebral discs and costal cartilage synthesize similar amounts of 35S-labelled proteoglycans per microgram of DNA. Discs and cartilage all synthesize a major proteoglycan species (approx. 85%) of large hydrodynamic size and a minor species (approx. 15%) of small size. Both proteoglycans carry chondroitin sulphate chains. Keratan sulphate was not found associated with either species. The total 35S-labelled proteoglycan pool had a metabolic half-life (t1/2) of 10-12 days in discs, and 17 days in cartilage. The extractable major and minor species turned over at similar rates. Those proteoglycans left in the tissue after 29 days turn over very slowly. Approx. 50% of the major 35S-labelled proteoglycan species formed mixed aggregates with hyaluronic acid and rat chondrosarcoma proteoglycan. The ability to form aggregates did not decrease up to 45 days after synthesis. Of the heterogeneous population of proteoglycans comprising the major species, those remaining in the tissue 9 days after synthesis were of smaller average hydrodynamic size and had shorter chondroitin sulphate side chains than the average size at the time of synthesis. With increasing time after synthesis, proteoglycans were less readily extracted from the tissue by 4.0 M-guanidinium chloride than at the time of synthesis.     

The influence of environmental agents on prostaglandin biosynthesis and metabolism in the lung. Inhibition of lung 15-hydroxyprostaglandin dehydrogenase by exposure of guinea pigs to 100 per cent oxygen at atmospheric pressure.

The methods of Hascall & Sajdera (1969) were used to compare the proteoglycans of human intervertebral disc with those of bovine nasal cartilage. In contrast with cartilage, most of the hexuronate of disc could be extracted at low shear with water or dilute salt solutions. Extracts of disc with 4M-guanidinium chloride were centrifugated in 0.4M-guanidinium chloride in a CsCl gradient. Analytical ultracentrifugation of the hexuronate-containing heavy component revealed two fractions. both more polydisperse than those of cartilage. Also the more rapidly sediminting component was a much smaller fraction of the total. After prior extraction with 0.4M-guanidinium chloride, 4M-guanidinium chloride extracts of disc were found, by ultracentrifugal analysis, to be enriched in components resembling the proteoglycan monomer and aggregating factors of cartilage.     

Cartilage proteoglycans. Structure and heterogeneity of the protein core and the effects of specific protein modifications on the binding to hyaluronate.

Purified proteoglycans extracted from pig laryngeal cartilage in 0.15 M-NaCl and 4 M-guanidinium chloride were analysed and their amino acid compositions determined. Selective modification of amino acid residues on the protein core confirmed that binding to hyaluronate was a function of the protein core, and was dependent on disulphide bridges, intact arginine and tryptophan residues, and epsilon-amino groups of lysine. Fluorescence measurement suggested that tryptophan was not involved in direct subsite interactions with the hyaluronate. The polydispersity in size and heterogeneity in composition of the aggregating proteoglycan was compatible with a structure based on a protein core containing a globular hyaluronate-binding region and an extended region of variable length also containing a variable degree of substitution with chondroitin sulphate chains. The non-aggregated proteoglycan extracted preferentially in 0.15 M-NaCl, which was unable to bind to hyaluronate, contained less cysteine and tryptophan than did other aggregating proteoglycans and may be deficient in the hyaluronate-binding region. Its small average size and low protein and keratan sulphate contents suggest that it may be a fragment of the chondroitin sulphate-bearing region of aggregating proteoglycan produced by proteolytic cleavage of newly synthesized molecules before their secretion from the cell.     

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

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

Enhanced extractability of articular cartilage proteoglycans in osteoarthrosis (Short Communication)

Tissue contents of small, easily extracted, proteoglycans, relatively poor in keratan sulphate, were compared in normal and osteoarthrotic cartilage. Although the amounts of small proteoglycans were similar in each tissue, as were the collagen contents, some proteoglycans in the diseased cartilage were much more readily extracted than those in the normal tissue.     

Separation and characterization of two populations of aggregating proteoglycans from cartilage.

Intermediary gel immunoelectrophoresis was used to show that purified aggregating cartilage proteoglycans from 2-year-old steers contain two distinct populations of molecules and that only one of these is immunologically related to non-aggregating cartilage proteoglycans. The two types of aggregating proteoglycans were purified by density-gradient centrifugation in 3.5M-CsCl/4M-guanidinium chloride and separated by zonal rate centrifugation in sucrose gradients. The higher-buoyant-density faster-sedimenting proteoglycan represented 43% of the proteoglycans in the extract. It had a weight-average Mr of 3.5 X 10(6), did not contain a well-defined keratan sulphate-rich region, had a quantitatively dominant chondroitin sulphate-rich region and contained 5.9% protein and 23% hexosamine. The lower-buoyant-density, more slowly sedimenting, proteoglycan represented 15% of the proteoglycans in the extract. It had a weight-average Mr of 1.3 X 10(6), contained both the keratan sulphate-rich and the chondroitin sulphate-rich regions and contained 7.3% protein and 23% hexosamine. Each of the proteoglycan preparations showed only one band on agarose/polyacrylamide-gel electrophoresis. The larger proteoglycan had a lower mobility than the smaller. The distribution of chondroitin sulphate chains along the chondroitin sulphate-rich region was similar for the two types of proteoglycans. The somewhat larger chondroitin sulphate chains of the larger proteoglycan could not alone account for the larger size of the proteoglycan. Peptide patterns after trypsin digestion of the proteoglycans showed great similarities, although the presence of a few peptides not shared by both populations indicates that the core proteins are partially different.     

Studies on the extraction of different proteoglycan populations in bovine articular cartilage

Large proteoglycan monomers and small dermatan sulfate proteoglycans were extracted from explants of bovine articular cartilage with increasing (0-4 M) concentrations of guanidinium chloride (GuHCl). The first extractions were followed by a second extraction with 4 M GuHCl. The amount of proteoglycans extracted in the first buffer depended on the GuHCl concentration. At low concentrations of GuHCl, a relatively high amount of small proteoglycans was obtained. Fifty percent of the small proteoglycans was extracted in buffer with 0.85 M GuHCl, while 2.0-2.2 M GuHCl was needed to extract half of the large proteoglycans. Immediately after synthesis, 35S-labeled large proteoglycans were extracted much easier (50% at 1.4 M GuHCl), and those extracted at low concentrations of GuHCl were less capable of aggregation with hyaluronic acid. After 7 days of 'chase' these differences between endogenous and 35S-labeled proteoglycans had disappeared.     

Comparisons between extracted and residual proteoglycans on the glycosaminoglycan level and changes with ageing

Sheep nasal cartilage from animals of five different ages were studied. Significant variations in the composition and on the extractability of the tissue occur with ageing. The ratio chondroitin sulphate to keratan sulphate in extracted and residual proteoglycans does not change in the same manner with ageing. The relative distribution of molecular sizes of keratan sulphate differs between extracted and residual proteoglycans. Hyaluronic acid and chondroitin sulphate appear homogeneous on the gel chromatography for all ages both in extracted and residual proteoglycans.     

Isolation and partial characterization of proteoglycans from rat incisors.

Newly synthesized proteoglycans of rat incisors were labelled in vivo for 6h with [35S]-sulphate in order to facilitate their detection during purification and characterization. Proteoglycans were extracted from non-mineralized portions (predentine) of rat incisors with 4M-guanidinium chloride and subsequently from dentine by demineralization with a 0.4M-EDTA solution containing 4M-guanidinium chloride. Both extractions were performed at 4 degrees C in the presence of proteinase inhibitors. Purification of proteoglycans was achieved with a procedure involving gel-filtration chromatography, selective precipitation of phosphoproteins, affinity chromatography and ion-exchange chromatography. Two proteoglycan populations were found in the initial extract (Pd-PG I and Pd-PG II), whereas only one fraction (D-PG) was obtained after demineralization. The minor proteoglycan fraction from the first extract, Pd-PG I, although not totally characterized, differed sharply from the other proteoglycans in that it had a larger molecular size with larger glycosaminoglycan chains composed of chondroitin 4- and 6-sulphate isomers. In contrast, the major proteoglycans Pd-PG II and D-PG had smaller hydrodynamic sizes with smaller glycosaminoglycan chains (but larger than those from bovine nasal cartilage proteoglycans) composed exclusively of chondroitin 4-sulphate. The major proteoglycans were incapable of interacting with hyaluronic acid. In general, the amino acid compositions of the major proteoglycans of rat incisors resembled that of bovine nasal cartilage proteoglycans, but the former had lower proline, valine, isoleucine, leucine, and higher aspartic acid, contents.