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.     

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 electrophoretic heterogeneity of bovine nasal cartilage proteoglycans.

1. Proteoglycans were extracted from bovine nasal cartilage with 2.0M-CaC2 or with 0.15M-KCl followed by 2.0M-CaC2.. Proteoglycan fractions were prepared from the extracts by density-gradient centrifugation in CsCl under 'associative' and 'dissociative' conditions. 2. The heterogeneity of the proteoglycan fractions was investigated by large-pore-gel electrophoresis. It was concluded that extracts made with 2.0M-CaCl2 or sequential 2.0M-CaCl2 contain two major species of proteoglycan 'subunit' of different hydrodynamic size, together with proteoglycan aggregates. Both 'subunits' have mobilities that are greater than those of proteoglycans obtained from pig articular cartilage McDevitt & Muir (1971) Anal. Biochem. 44, 612-622] and are therefore probably smaller in size than the latter. 3. Proteoglycan fractions isolated from cartilage extracted lith 0.15M-KCl separated into two main components on large-pore-gel electrophoresis with mobilities greater than those of proteoglycans extracted with 2.0M-CaCl2. Proteoglycans extracted at low ionic strength from bovine nasal cartilage are of similar hydrodynamic size to those extracted from pig articular cartilage under the same conditions [McDevitt & Muir (1971) Anal. Biochem. 44, 612-622]. 4. The role of endogenous proteolytic enzymes in producing proteoglycan heterogeneity, particularly in low-ionic-strength cartilage extracts is discussed. 5. Hyaluronic acid and 'link proteins' were present in the proteoglycan fraction separated from KCl extracts as well as in the fraction separated from CaCl2 extracts. Hyaluronic acid can only be identified in proteoglycan fractions by large-pore-gel electrophoresis after proteolysis and further purification of the fraction. 6. Collagen was extracted by both salt solutions and was tentatively identified as type II. Small amounts of collagen appear to be associated with the proteoglycan-aggregate fraction from the high-ionic-strength extract but not with the corresponding fraction from the KCl extract.     

Effect of interleukin-1 on the size distribution of cartilage proteoglycans as determined by sedimentation field flow fractionation

The effect of interleukin-1 (IL-1) on the size distribution of cartilage proteoglycans was studied using sedimentation field flow fractionation (SdFFF), a rapid, high-resolution technique for the separation of proteoglycan monomers and aggregates. During incubation of cartilage in control media, 35S-prelabeled proteoglycan was lost primarily from proteoglycan present in the monomer form; aggregates were conserved. In the presence of IL-1, both 35S-proteoglycan monomers and aggregates were lost, suggesting that IL-1 increases the susceptibility of aggregates to loss from the cartilage matrix. Evaluation of uronic acid as a measure of net change in proteoglycan content indicated that IL-1 causes a net decrease in both monomers and aggregates. Kinetic studies suggested that aggregates are degraded to monomers which then diffuse out of the matrix. Incorporation of [35S]sulfate into cartilage proteoglycans following exposure to IL-1 showed that synthesis of monomers and aggregates is inhibited similarly. SdFFF is a valuable technique for studying proteoglycan metabolism. With its use, changes in proteoglycan monomer and aggregate populations can be detected in response to cytokines such as IL-1.     

Biosynthesis of proteoglycans in cartilage slices. Fractionation by gel chromatography and equilibrium density-gradient centrifugation

The kinetics of incorporation of [(35)S]sulphate into slices of pig laryngeal cartilage in vitro was linear with time up to 6h. The specific radioactivities of the extracted proteoglycans (containing about 80% of the uronic acid of the cartilage) and the glycosaminoglycans remaining in the tissue after extraction were measured after various times of continuous and ;pulse-chase' radioactivity incorporation. Radioactivity was present in the isolated chondroitin sulphate after 2 min, but there was a 35min delay in its appearance in the extractable proteoglycan fraction. Fractionation of the proteoglycans by gel chromatography showed that the smallest molecules had the highest specific radioactivity, but ;pulse-chase' experiments over 5h did not demonstrate any precursor-product relationships between fractions of different size. Equilibrium density-gradient centrifugation in 4m-guanidine hydrochloride showed that among the proteoglycan fractions the specific radioactivity increased as the chondroitin sulphate content decreased, but with preparations from ;pulse-chase' experiments there was again no evidence for precursor-product relationships between the different fractions. Differences in radioactive incorporation would seem to reflect metabolic heterogeneity within the proteoglycans extracted from cartilage. This may be due either to a partial separation of different types of proteoglycans or to differences in the rates of degradation of the molecules of different size and composition as a result of the nature and specificity of the normal degrading enzymes. The results suggest that molecules of all sizes were formed at the same time.     

Identification and characterization of a proteoglycan in embryonic chicken skin that can interact with hyaluronic acid

Sulfated proteoglycans of the dorsal skin of 8.5-day-old chick embryos have been characterized in terms of their extractability from the tissue, solubility, and sedimentation and chromatographic behavior. The proteoglycans described in this communication are those that remain soluble after dialysis against 0.5 m NaCl. Two chondroitin sulfate proteoglycans (PGCS-A and PGCS-C) and a heparan sulfate proteoglycan (PGHS) have been identified. PGCS-A is the only proteoglycan found in the medium in which the skins were cultured. Under associative conditions (0.4 M guanidine-HCl) PGCS-A and PGHS are extracted. The dissociative solvents (4 M guanidine-HCl) extract more PGCS-A and PGCS-C. PGCS-C has been shown to interact with hyaluronic acid to form aggregates. These proteoglycans have densities ranging from 1.49 to at least 1.59 g/ml. In contrast cartilage proteoglycans that can aggregate with hyaluronic acid have a density of at least 1.59 g/ml. It was not possible to determine if the PGCS-C aggregates exist in vivo.     

The ratio of protein and carbohydrate components of proteoglycans from normal and degenerative articular cartilage

The protein/uronic acid ratio in monomers and aggregates of proteoglycans in the human articular cartilage is investigated. It is shown that for the first two hours of cartilage extraction by isotonic solution proteoglycans with the low concentration of chondroitin sulphate are mainly removed; in the process of the subsequent extraction proteoglycans with a large amount of chondroitin sulphate; the quantity of chondroitin sulphate in the molecule does not effect the ability of proteoglycans to aggregation. The protein/uronic acid ratio increases in cartilage proteoglycans with aging and in the process of cartilage degeneration due to a decrease in the amount of the carbohydrate part of the molecule.     

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.     

Interaction of proteoglycans with proteins

The method has been developed for preparing complexes of lysozyme with chondroitin sulfate-4 and -6, non-aggregated (soluble) proteoglycans, cartilage proteoglycan aggregates, heparin fractions containing residues 3(H-3) and 4(H-4) of sulfuric acid per dimer of polymer. The studies of the chemical composition and IK-spectra of proteoglycan-lysozyme complexes have demonstrated the electrostatic nature of proteoglycan-lysozyme interaction.     

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.     

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 synthesis of dermatan sulphate proteoglycans by fetal and adult human articular cartilage.

Non-aggregating dermatan sulphate proteoglycans can be extracted from both fetal and adult human articular cartilage. The dermatan sulphate proteoglycans appear to be smaller in the adult, this presumably being due to shorter glycosaminoglycan chains, and these chains contain a greater proportion of their uronic acid residues as iduronate. Both the adult and fetal dermatan sulphate proteoglycans contain a greater amount of 4-sulphation than 6-sulphation of the N-acetylgalactosamine residues, in contrast with the aggregating proteoglycans, which always show more 6-sulphation on their chondroitin sulphate chains. In the fetus the major dermatan sulphate proteoglycan to be synthesized is DS-PGI, though DS-PGII is synthesized in reasonable amounts. In the adult, however, DS-PGI synthesis is barely detectable relative to DS-PGII, which is still synthesized in substantial amounts. Purification of the dermatan sulphate proteoglycans from adult cartilage is hampered by the presence of degradation products derived from the large aggregating proteoglycans, which possess similar charge, size and density properties, but which can be distinguished by their ability to interact with hyaluronic acid.     

The uptake of [C]glucose into rabbit ear cartilage proteogylcans isolated by differential extraction and by collagenase digestion

Rabbit ear cartilage was incubated with [¹⁴C]glucose and proteoglycans were extracted from the crushed cartilage by differential extraction. The extraction was performed sequentially with 0.15, 0.45 and 1.0 M NaCl, followed by 0.1 M acetic acid. The tissue residue was digested with collagenase and finally with papain. Each extractant, with the exception of 0.1 M acetic acid, released uronic acid containing material into the solution. The extractable proteoglycans represented together about 20% of the whole tissue uronate, the proteoglycans released by collagenase treatment accounted for a further 30%. The rest was insoluble and was released by papain. The highest specific radioactivity was found in the 0.15 M NaCl extract, decreasing progressively in subsequent extracts. The lowest specific activity was found in the chondroitin sulfate released from the tissue residue by papain. Radioactivity in the collagen-associated proteoglycans was comparable to the radioactivity of 1.0 M NaCl extracts. All salt extractable proteoglycans were retarded by Sepharose 6B and were found to be heterogeneous in size and rate of precursor uptake. Chondroitin sulfate released from each proteoglycan fraction was also heterogeneous in size and metabolic activity.     

Degradation of proteoglycan in articular cartilage.

Adult rabbit articular cartilage was labelled in vivo over 48 h with [35S]sulphate and was then incubated in organ culture at pH 7.2. Approx. 65% of the tissue content of [35S]proteoglycan was released into the culture medium during the first 48 h of incubation. The average molecular size of the released proteoglycans, as assessed by fractionation on Sepharose 2B/CL and 4B/Cl, was only slightly smaller than that of the proteoglycans extracted from non-cultured cartilage with 4 M guanidine HCl. The percentage of released proteoglycans and extracted proteoglycans which formed aggregates with hyaluronic acid was approx. 25% and 75%, respectively. The results indicate that proteoglycan degradation in adult articular cartilage is initiated by a limited proteolysis of subunit core protein, with the production of non-aggregating species which diffuse readily from the tissue.     

The uptake of [14C]glucose into rabbit ear cartilage proteogylcans isolated by differential extraction and by collagenase digestion

Rabbit ear cartilage was incubated with [¹⁴C]glucose and proteoglycans were extracted from the crushed cartilage by differential extraction. The extraction was performed sequentially with 0.15, 0.45 and 1.0 M NaCl, followed by 0.1 M acetic acid. The tissue residue was digested with collagenase and finally with papain. Each extractant, with the exception of 0.1 M acetic acid, released uronic acid containing material into the solution. The extractable proteoglycans represented together about 20% of the whole tissue uronate, the proteoglycans released by collagenase treatment accounted for a further 30%. The rest was insoluble and was released by papain. The highest specific radioactivity was found in the 0.15 M NaCl extract, decreasing progressively in subsequent extracts. The lowest specific activity was found in the chondroitin sulfate released from the tissue residue by papain. Radioactivity in the collagen-associated proteoglycans was comparable to the radioactivity of 1.0 M NaCl extracts. All salt extractable proteoglycans were retarded by Sepharose 6B and were found to be heterogeneous in size and rate of precursor uptake. Chondroitin sulfate released from each proteoglycan fraction was also heterogeneous in size and metabolic activity.     

Passive loss of proteoglycan from articular cartilage explants

The addition of proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, 0.25 mM benzamidine hydrochloride, 6.25 mM EDTA, 12.5 mM 6-aminohexanoic acid and 2 mM iodoacetic acid) to explant cultures of adult bovine articular cartilage inhibits proteoglycan synthesis as well as the loss of the macromolecule from the tissue. Those proteoglycans lost to the medium of explant cultures treated with proteinase inhibitors were either aggregates or monomers with functional hyaluronic acid-binding regions, whereas proteoglycans lost from metabolically active tissue also included a population of monomers that were unable to aggregate with hyaluronate. Analysis of the core protein from proteoglycans lost into the medium of inhibitor-treated cultures showed the same size distribution as the core proteins of proteoglycans present in the extracellular matrix of metabolically active cultures. The core proteins of proteoglycans appearing in the medium of metabolically active cultures showed that proteolytic cleavage of these macromolecules occurred as a result of their loss from the tissue. Explant cultures of articular cartilage maintained in medium with proteinase inhibitors were used to investigate the passive loss of proteoglycan from the tissue. The rate of passive loss of proteoglycan from the tissue was dependent on surface area, but no difference in the proportion of proteoglycan aggregate to monomer appearing in the medium was observed. Furthermore, proteoglycans were lost at the same rate from the articular and cut surfaces of cartilage. Proteoglycan aggregates and monomer were lost from articular cartilage over a period of time, which indicates that proteoglycans are free to move through the extracellular matrix of cartilage. The movement of proteoglycans out of the tissue was shown to be temperature dependent, but was different from the change of the viscosity of water with temperature, which indicates that the loss of proteoglycan was not solely due to diffusion. The activation energy for the loss of proteoglycans from articular cartilage was found to be similar to the binding energies for electrostatic and hydrogen bonds.     

Influence of the cells on the pericellular environment. The effect of hyaluronic acid on proteoglycan synthesis and secretion by chondrocytes of adult cartilage.

The chondrocyte is a specialized cell that synthesizes proteoglycans of a type found only in cartilage and nucleus pulposus. These proteoglycans are distinct in forming multiple aggregates of unique structure in which hyaluronic acid provides a central chain to which many proteoglycan molecules are bound at one end only. Chondrocytes were isolated from adult cartilage and used in suspension culture to test the effect of compounds in the medium on the synthesis of proteoglycans. Hyaluronic acid alone, among a number of compounds extracted from or analogous to those in cartilage, reduced the incorporation of [35S] sulphate into macromolecular material.Oligosaccharides of hyaluronic acid of the size of decasaccharides and above also had this effect but hyaluronic acid already bound to proteoglycan did not. The proportion of total labelled material associated with the cells increased at the expense of that in the medium. Treatment of the cells with trypsin abolished the effect of hyaluronic acid but treatment with chondroitinase did not. It is suggested that hyaluronic acid interacts with proteoglycans at the cell surface by a specific mechanism similar to that involved in proteoglycan aggregation, as a result of which the secretion and synthesis of proteoglycans is reduced.     

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.     

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.     

The extractability of extracellular matrix components as a marker of cartilage remodeling in laryngeal squamous cell carcinoma

Sequential extraction was applied to investigate the proteoglycan (PG) organization in healthy laryngeal cartilage (HLC) and laryngeal cartilage squamous cell carcinoma (LCSCC). Highly stable aggrecan aggregates, extracted from both HLC and LCSCC with strong dissociative reagents, i.e., 4 M guanidine HCl (GdnHCl), represented 53% and 7%, respectively, of total extracted macromolecules. Less stable complexes/aggregates, extracted with mild dissociative reagents (1 and 2 M GdnHCl), represented 40% and 61% of total extracted PGs from healthy and cancerous cartilage, respectively. Interestingly, a relative high proportion (32%) of uronic acid (UA)-containing macromolecules were removed from the cancerous cartilage using associative extracting solutions (PBS and 0.5 M GdnHCl), which obviously represented molecules freely extractable from the tissue. In contrast, the corresponding proportion in HLC was impressively low (about 7%). The major proportion of these molecules was chondroitin sulfate-containing PGs (CSPGs), which identified mainly as aggrecan. Differential digestion of the sequential extracts with chondroitinase ABC and chondroitinase AC II demonstrated the presence of dermatan sulfate-containing PGs (DSPGs) in both HLC and LCSCC, being mainly present in the 1 M GdnHCl extract, and identified as decorin. All cancerous extracts were found to be rich in 4-sulfated disaccharides, mostly participating in DS structures. In conclusion, the applied procedure permitted the elucidation of the changes in the cartilage status, regarding the stability and identity of its proteoglycan aggregates/complexes, in both HLC and LCSCC.