Physicochemical properties of cartilage proteoglycans extracted by lanthanum chloride

Bovine nasal cartilage was extracted with 0.5 M LaCl₃ and the extract then diluted with nine volumes of water. The resulting precipitate (P_LaCl3) contained the proteoglycan subunits, together with minor protein components, but was essentially free from hyaluronic acid. The properties of P_LaCl3 were investigated by chemical analysis, electrophoresis, viscometry and analytical ultracentrifugation, and the results compared with those for proteoglycan obtained by caesium chloride density gradient centrifugation of 2 M CaCl₂ cartilage extracts. Proteoglycan subunits (A1D1) prepared from P_LaCl3 showed identical properties to those obtained from other high ionic strength cartilage extracts.     

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.     

The inability to prepare high-buoyant-density proteoglycan aggregates from extracts of normal adult human articular cartilage.

High-buoyant-density proteoglycan aggregates could not be prepared from extracts of adult human cartilage by associative CsCl-density-gradient centrifugation with a starting density of 1.68 g/ml, even though proteoglycan subunits, hyaluronic acid and link proteins were all present. In contrast, aggregates could be prepared when extracts of neonatal human cartilage or bovine nasal cartilage were subjected to the same procedure. This phenomenon did not appear to be due to a defect within the hyaluronic acid-binding region of the adult proteoglycan subunit, but rather to an interference in the stability of the interaction between the proteoglycan subunit and hyaluronic acid towards centrifugation. The factor responsible for this instability was shown to reside within the low-density cartilage protein preparation obtained by direct dissociative CsCl-density-gradient centrifugation of the adult cartilage extract.     

Comparison of proteoglycans from bovine articular cartilage.

Four bovine articular cartilages have been compared with regard to the chemical composition of the whole cartilages, the amount of proteoglycan selectively extracted with 3 M MGCl2 or with 3 M guanidine-HCl, and the compositions and physical properties of the isolated proteoglycans. The whole cartilages differ but slightly in composition. Occipital condylar cartilage, a thin cartilage from the smallest joint, contains 4% more collagen and proportionately less proteoglycan than proximal humeral, the thickest cartilage from the largest joint. Each cartilage contains a pool of proteoglycan that resists extraction with 3 M MgCl2 but is extracted with 3 M guanidine-HCl. The proteoglycan extracted from each cartilage with 3 M guanidine-HCl contains a high molecular weight proteoglycan-collagen complex demonstrated by analytical ultracentrifugation and by the turbidity of its visible and ultra-violet spectra. The four cartilages appear to differ most remarkably in the fraction of total proteoglycan extracted from each as proteoglycan-collagen complex.     

Treatment of cartilage proteoglycan aggregate with hydrogen peroxide. Relationship between observed degradation products and those that occur naturally during aging.

The effects of treatment of purified neonatal human articular-cartilage proteoglycan aggregate with H2O2 were studied. (1) Exposure of proteoglycan aggregate to H2O2 resulted in depolymerization of the aggregate and modification of the core protein of both the proteoglycan subunits and the link proteins. (2) Treatment of the proteoglycan aggregate with H2O2 rendered the proteoglycan subunits unable to interact with hyaluronic acid, with minimal change in their hydrodynamic size. (3) Specific cleavages of the neonatal link proteins occurred. The order in which the major products were generated and their electrophoretic mobilities resembled the pattern observed during human aging. (4) The proteolytic changes in the link proteins were inhibited in the presence of transition-metal-ion chelators, thiourea or tetramethylurea, suggesting that generation of hydroxyl radicals from H2O2 by trace transition-metal ions via a site-specific Fenton reaction may be responsible for the selective cleavages observed. (5) Cleavage of the link proteins in proteoglycan aggregates by H2O2 was shown to have a limited effect on the susceptibility of these proteins to cleavage by trypsin. (6) The relationship between these changes and those observed in cartilage during human aging suggests that some of the age-related changes in the structure of human cartilage proteoglycan aggregate may be the result of radical-mediated damage.     

Identification of a hyaluronic acid-binding protein that interferes with the preparation of high-buoyant-density proteoglycan aggregates from adult human articular cartilage.

Adult human articular cartilage contains a hyaluronic acid-binding protein of Mr 60 000-75 000, which contains disulphide bonds essential for this interaction. The molecule can compete with proteoglycan subunits for binding sites on hyaluronic acid, and can also displace proteoglycan subunits from hyaluronic acid if their interaction is not stabilized by the presence of link proteins. The abundance of this protein in the adult accounts for the reported inability to prepare high-buoyant-density proteoglycan aggregates from extracts of adult human cartilage [Roughley, White, Poole & Mort (1984) Biochem. J. 221, 637-644], whereas the deficiency of the protein in newborn human cartilage allows the normal recovery of proteoglycan aggregates from this tissue. The protein shares many common features with a hyaluronic acid-binding region derived by proteolytic treatment of a proteoglycan aggregate preparation, and this may also represent its origin in the cartilage, with its production increasing during tissue maturation.     

Antigenic determinants of two components of proteoglycan complex from bovine cartilage

The immunological properties of a glycoprotein fraction and of proteoglycan subunits obtained from bovine nasal cartilage by nondisruptive methods of isolation have been studied. Using the techniques of hemagglutination and hemagglutination inhibition, we found that the glycoprotein contains most of the species-specific determinants, whereas the proteoglycan subunits contain most of the cross-reacting ones.     

Cartilage proteoglycan aggregates. Electronmicroscopic studies of native and fragmented molecules

1. Proteoglycan aggregates from bovine nasal cartilage were studied by using electron microscopy of proteoglycan/cytochrome c monolayers. 2. The aggregates contained a variably long central filament of hyaluronic acid with an average length of 1037nm. The proteoglycan monomers attached to the hyaluronic acid appeared as side chain filaments varying in length (averaging 249nm). They were distributed along the central filament at an average distance of about 36nm. 3. Chondroitin sulphate side chains were removed from the proteoglycan monomers of the aggregates by partial chondroitinase digestion. The molecules obtained had the same general appearance as intact aggregates. 4. Proteoglycan aggregates were treated with trypsin and the largest fragment, which contains the hyaluronic acid, link protein and hyaluronic acid-binding region, was recovered and studied with electron microscopy. Filaments that lacked the side chain extensions and had the same length as the central filament in the intact aggregate were observed. 5. Hyaluronic acid isolated after papain digestion of cartilage extracts gave filaments with similar length and size distribution as observed for the central filament both in the intact aggregate and in the trypsin digests. 6. Umbilical-cord hyaluronic acid was also studied and gave electron micrographs similar to those described for hyaluronic acid from cartilage. However, the length of the filament was somewhat shorter. 7. The electron micrographs of both intact and selectively degraded proteoglycans corroborate the current model of cartilage proteoglycan structure.     

The composition of cartilage proteoglycans. An investigation using high-and low-ionic-strength extraction procedures

1. A proteoglycan fraction (the proteoglycan subunit fraction) was prepared from extracts, with 0.15m-KCl (low-ionic-strength) and 0.5m-LaCl(3), 2.0m-CaCl(2) and 4.0m-guanidinium chloride (high-ionic-strength), of bovine nasal cartilage by equilibrium-density-gradient centrifugation, essentially as described by Hascall & Sajdera (1969). 2. The use of different centrifugation times showed that near-equilibrium conditions were reached by 48h for the fractions prepared from the high-ionic-strength extracts. The fraction isolated from the low-ionic-strength extract required a longer centrifugation time to reach equilibrium conditions. 3. The composition of the proteoglycan fractions from the various extracts was compared by analyses of their carbohydrate and amino acid contents. Difference indices were calculated from the amino acid analysis to compare the degree of compositional relationship between the protein components of the proteoglycans. 4. Small compositional differences were found between the proteoglycans isolated from the various high-ionic-strength extracts. The protein content of the fractions from the CaCl(2) extract and the guanidinium chloride extract showed the greatest difference in this respect, although their amino acid analysis was similar. 5. The proteoglycan fraction isolated from the low-ionic-strength extract shows marked differences in composition from the fractions isolated from the high-ionic-strength extracts. Its protein and glucosamine contents were lower whereas its hexuronic acid and galactosamine contents were higher than those of the latter. It also exhibits major differences in its amino acid composition. The glucosamine:galactosamine ratio of the fraction from the low-ionic-strength extract indicates that it may be an almost exclusively chondroitin sulphate-proteoglycan. Its analysis correlates closely with that of a low-molecular-weight proteoglycan isolated from pig laryngeal cartilage by Tsiganos & Muir (1969). 6. The proteoglycan fractions from both the low- and high-ionic-strength extracts migrate as a single band in zone electrophoresis carried out in a sucrose-density gradient at both pH3.0 and pH7.0, although each showed evidence of band widening during the electrophoresis. All the proteoglycan fractions migrated with the same electrophoretic mobility at pH3.0, irrespective of the differences in composition between them. 7. The differences between the proteoglycans from the low- and high-ionic-strength extracts are discussed and the view is advanced that they may be due to association between predominantly chondroitin sulphate-proteoglycans and a keratan sulphate-enriched proteoglycan species.     

Evidence for polymorphonuclear-leucocyte-derived proteinases in arthritic cartilage.

1. An enzyme that degrades proteoglycan at neutral pH was extracted with 4 M-guanidine hydrochloride from the articular cartilage of rabbits with antigen-induced arthritis. 2. The enzyme had an apparent molecular weight on Ultrogel AcA 54 of about 8000 and was optimally active at pH 7.5 in Tris/HCl buffer containing 0.2 M-NaCl. The partially purified preparation was totally inhibited by 0.01 mM-N-acetyldialanylprolylvalylchloromethane, severely inhibited by 2 mM-phenylmethanesulphonyl fluoride and soya-bean trypsin inhibitor (200 microgram/ml) and slightly inhibited by 10 mM-EDTA. Marked inhibition was also obtained with a cytosolic fraction prepared from rabbit polymorphonuclear leucocytes. 3. All properties of the enzyme were virtually identical with those of an 'elastase-like' proteinase that was isolated from rabbit polymorphonuclear-leucocyte granules. 4. The results are consistent with the idea that cartilage proteoglycan degradation in acute joint inflammation is due at least partly to the diffusion into the cartilage of proteinases derived from synovial-fluid polymorphonuclear leucocytes.     

Effects of ceramide on apoptosis, proteoglycan degradation, and matrix metalloproteinase expression in rabbit articular cartilage

Cartilage loss in osteoarthritis is characterized by matrix degradation and chondrocyte death. The lipid messenger ceramide is implicated in signal transduction of the catabolic cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1), as well as in apoptosis. The aim of this study was to examine the in vitro effects of ceramide on proteoglycan degradation, matrix-metalloproteinase (MMP) expression and activity, and chondrocyte apoptosis in rabbit articular cartilage. Cell-permeant ceramide C(2) stimulated proteoglycan degradation in cartilage explants starting from 3 x 10(-5) M, with 100% increase at the dose of 10(-4) M. This effect was probably due to MMPs since it was blocked by the MMP inhibitor batimastat. Furthermore, in isolated chondrocytes, C(2) stimulated the expression of MMP-1, 3, and 13 at the mRNA level, MMP activity, and MMP-3 production. Ceramide also caused chondrocyte apoptosis at doses ranging from 10(-5) to 10(-4) M. This study supports the hypothesis that ceramide might play a mediatory role in both matrix degradation and apoptosis in processes of cartilage loss such as those observed in osteoarthritis.     

Age-related changes in protein-related epitopes of human articular-cartilage proteoglycans.

Monoclonal antibodies were prepared that recognize different age-related epitopes on proteoglycan subunits of high buoyant density isolated from human epiphysial and articular cartilages. Antibody EFG-4 (IgG1) recognizes a proteinase-sensitive segment associated with the core protein. Antibody BCD-4 (IgG1) reacts with keratan sulphate bound to core protein. Both epitopes are minimally expressed in foetal cartilage and increase with age after birth to become maximally expressed in adult cartilage by about 30 years of age. In contrast, monoclonal antibody alpha HFPG-846 (IgM) recognizes a core-protein-related epitope that is maximally expressed in young foetal cartilage, declines up to birth and thereafter and is almost absent after about 30 years of age. Antibody alpha HFPG-846 was used to isolate by immuno-affinity chromatography two subpopulations of proteoglycan subunits from a 16-year-old-human cartilage proteoglycan subunit preparation. Only the antibody-unbound population showed a significant reaction with antibodies EGF-4 and BCD-4. The amino acid and carbohydrate compositions of these proteoglycan fractions were different, and one (antibody-bound) resembled those of foetal and the other (antibody-unbound) resembled those of adult proteoglycans isolated from 24-27-week-old-foetal and 52-56-year-old-adult cartilage respectively. These observations demonstrate that human cartilages contain at least two chemically and immunochemically distinct populations of proteoglycans, the proportions and content of which are age-dependent. It is likely that these populations represent the products of different genes, though their heterogeneity may be compounded by the result of different post-translation modifications.     

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.     

Hyaluronate-Binding Proteins of Murine Brain

The distribution of hyaluronate-binding activity was determined in the soluble and membrane fractions derived from adult mouse brain by sonication in low-ionic-strength buffer. Approximately 60% of the total activity was recovered in the soluble fraction and 33% in membrane fractions. In both cases, the hyaluronate-binding activities were found to be of high affinity (KD = 10(-9) M), specific for hyaluronate, and glycoprotein in nature. Most of the hyaluronate-binding activity from the soluble fraction chromatographed in the void volume of Sepharose CL-4B and CL-6B. Approximately 50% of this activity was highly negatively charged, eluting from diethylaminoethyl (DEAE)-cellulose in 0.5 M NaCl, and contained chondroitin sulfate chains. This latter material also reacted with antibodies raised against cartilage link protein and the core protein of cartilage proteoglycan. Thus, the binding and physical characteristics of this hyaluronate-binding activity are consistent with those of a chondroitin sulfate proteoglycan aggregate similar to that found in cartilage. A 500-fold purification of this proteoglycan-like, hyaluronate-binding material was achieved by wheat germ agglutinin affinity chromatography, molecular sieve chromatography on Sepharose CL-6B, and ion exchange chromatography on DEAE-cellulose. Another class of hyaluronate-binding material (25-50% of that recovered) eluted from DEAE with 0.24 M NaCl; this material had the properties of a complex glycoprotein, did not contain chondroitin sulfate, and did not react with the antibodies against cartilage link protein and proteoglycan. Thus, adult mouse brain contains at least three different forms of hyaluronate-binding macromolecules. Two of these have properties similar to the link protein and proteoglycan of cartilage proteoglycan aggregates; the third is distinguishable from these entities.     

Interaction of proteoglycans with the pericellular (1 alpha, 2 alpha, 3 alpha) collagens of cartilage

Interaction between cartilage proteoglycan and the collagen(s) composed of 1 alpha, 2 alpha, and 3 alpha chains was studied in vitro. Most of the collagen was insoluble under the conditions of assay (0.15 M NaCl, 0.008 M phosphate buffer, pH 7.4; 4 degrees C) and was in the form of fibrils 20 nm in diameter or thinner. The larger fibrils had 60-70 nm periodicity, characteristic of native collagens. Proteoglycan monomers which had been labeled by incubating cartilage slices in vitro with Na2 35SO4 were used to assay the interaction. The insoluble collagen fraction bound proteoglycan from solution. At proteoglycan:collagen ratios lower than 1:2, binding was rapid and linear, and the dissociation constant was 1.7 X 10(-9) M. At higher proteoglycan:collagen ratios, more proteoglycan was bound, but at a slower rate. Binding of proteoglycan to collagen did not require fibrils, since soluble 1 alpha, 2 alpha, and 3 alpha containing collagen also bound to proteoglycan and formed an insoluble complex. Denatured collagens did not bind proteoglycan or compete for binding with normal collagen. Optimum binding occurred with intact proteoglycan, but proteoglycan which had been treated with protease was also bound at low levels. Both protease-treated proteoglycan and free chondroitin sulfate competed with intact proteoglycan in the binding assays, but neither chondroitinase ABC-treated proteoglycan nor the oligosaccharides produced by digestion of chondroitin sulfate with testicular hyaluronidase altered the binding of proteoglycan to collagen. Hyaluronic acid did not compete with radioactive proteoglycan, but heparin and dextran sulfate were extremely effective inhibitors of binding. These data suggest a relatively nonspecific interaction between sulfated polyanions and 1 alpha, 2 alpha, and 3 alpha containing collagens. However, given the location of these collagens near the chondrocyte surface, the interaction of fibrillar 1 alpha, 2 alpha, 3 alpha collagen with proteoglycan is likely to occur and to be of biological importance.     

The action of human articular-cartilage metalloproteinase on proteoglycan and link protein. Similarities between products of degradation in situ and in vitro.

Interleukin 1 stimulation of human articular cartilage in organ culture produced the concomitant release of proteoglycan fragments and latent metalloproteinase. The released fragments ranged in size from that of almost intact proteoglycan subunits to the product of limiting digestion generated by the activated metalloproteinase. None of the fragments possessed the ability to interact with hyaluronic acid. Analysis of proteoglycan aggregate digested with the activated metalloproteinase showed that isolated hyaluronic acid-binding regions were produced from the proteoglycan subunits, and that the two higher-Mr link-protein components (Mr 48,000 and 44,000) were converted into the lowest-Mr component (Mr 41,000). Link protein extracted from cartilage under stimulation with interleukin 1 showed a similar conversion. These results suggest that interleukin 1 stimulates the release of latent metalloproteinase from chondrocytes and that a proportion of the enzyme is activated in situ in the cartilage matrix. The mode of action of the activated enzyme is compatible with a role in the changes in proteoglycan structure seen in aging.     

Ultrastructural characterization of embryonic chick cartilage proteoglycan core protein and the mapping of a monoclonal antibody epitope.

The ultrastructure of embryonic chick cartilage proteoglycan core protein was investigated by electron microscopy of specimens prepared by low angle shadowing. The molecular images demonstrated a morphological substructural arrangement of three globular and two linear regions within each core protein. The internal globular region (G2) was separated from two terminally located globular regions (G1 and G3) by two elongated strands with lengths of 21 +/- 3 nm (E1) and 105 +/- 22 nm (E2). The two N-terminal globular regions, separated by the 21-nm segment, were consistently visualized in well spread molecules and showed little variation in the length of the linear segment connecting them. The E2 segment, however, was quite variable in length, and the C-terminal globular region (G3) was detected in only 53% of the molecules. The G1, G2, and G3 regions in chick core protein were 10.1 +/- 1.7 nm, 9.7 +/- 1.3 nm, and 8.3 +/- 1.3 nm in diameter, respectively. These results are similar to those described previously for proteoglycan core proteins isolated from rat chondrosarcoma, bovine nasal cartilage, and pig laryngeal cartilage (Paulsson, M., Morgelin, M., Wiedemann, H., Beardmore-Gray, M., Dunham, D., Hardingham, T., Heinegard, D., Timpl, R., and Engel, J. (1987) Biochem. J. 245, 763-772). However, a significant difference was detected between the length of the elongated strand (E2) of core proteins isolated from chick cartilage, E2 length = 105 +/- 22 nm, compared to bovine nasal cartilage, E2 length = 260 +/- 39 nm. The epitope of the proteoglycan core protein-specific monoclonal antibody, S103L, was visualized by electron microscopy, and the distance from the core protein N terminus to the S103L binding site was measured. The S103L binding site was localized to the E2 region, 111 +/- 20 nm from the G1 (N terminus) domain and 34 nm from the G3 (C terminus) domain. cDNA clones selected from an expression vector library of chicken cartilage mRNA also show this epitope to be located near the C-terminal region (R. C. Krueger, T. A. Fields, J. Mensch, and B. Schwartz (1990) J. Biol. Chem. 265, 12088-12097).     

Effects of ceramide on aggrecanase activity in rabbit articular cartilage

Ceramide participates in signal transduction of IL-1 and TNF, two cytokines likely involved in cartilage degradation in osteoarthritis. We previously showed that ceramide stimulates proteoglycan degradation, mRNA expression of matrix metalloproteinase (MMP)-1, -3, and -13, and pro-MMP-3 production in rabbit cartilage. Since aggrecan, the main cartilage proteoglycan, can be cleaved by metalloproteinases both of MMP and aggrecanase type, the aim of this study was to determine if ceramide stimulates aggrecanase action and, if that is the case, in which measure aggrecanase mediates the degradative effect of ceramide. To this end, antibodies were used against the C terminal aggrecan neoepitopes generated by aggrecanases (NITEGE(373)) and MMPs (DIPEN(341)). Ceramide C(2) at 10(-5) to 10(-4) M dose-dependently increased NITEGE signal, without changing that of DIPEN, in cultured explants of rabbit cartilage. The effects of 10(-4) M C(2) on NITEGE signal and proteoglycan degradation were similarly antagonized by the metalloproteinase inhibitor batimastat, with return to the basal level at 10(-6) M. These results show that, similarly to IL-1 and TNF, ceramide-induced aggrecan degradation is mainly due to aggrecanases. That no increase of MMP activity was detected, despite stimulation of MMP expression, was probably due to lack of proenzyme conversion to mature form, since addition of a MMP activator to C(2)-treated cartilage increased both DIPEN signal and proteoglycan degradation. These findings support the hypothesis that cytokine-induced ceramide could play a mediatory role in situations of increased degradation of cartilage matrix.     

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.     

Viscoelastic properties of proteoglycan subunits and aggregates in varying solution concentrations

Using a cone-on-plate mechanical spectrometer, we have measured the linear and non-linear rheological properties of cartilage proteoglycan solutions at concentrations similar to those found in situ. Solutions of bovine nasal cartilage proteoglycan subunits (22S) and aggregates (79S) were studied at concentrations ranging from 10 to 50 mg ml-1. We determined: (1) the complex viscoelastic shear modulus G (omega) under small amplitude (0.02 radians) oscillatory excitation at frequencies (omega) ranging from 1.0 to 20.0 Hz, (2) the non-linear shear rate (gamma) dependent apparent viscosity napp (gamma) in continuous shear, and (3) the non-linear shear rate dependent primary normal stress difference sigma 1 (gamma) in continuous shear. Both the apparent viscosity and normal stress difference were measured over four decades of shear rates ranging from 0.25 to 250 s-1. Analysis of the experimental results were performed using a variety of materially objective non-linear viscoelastic constitutive laws. We found that the non-linear, four-coefficient Oldroyd rate-type model was most effective for describing the measured flow characteristics of proteoglycan subunit and aggregate solutions. Values of the relaxation time lambda 1, retardation time lambda 2, zero shear viscosity no, and nonlinear viscosity parameter muo were computed for the aggregate and subunit solutions at all of the solute concentrations used. The four independent material coefficients showed marked dependence on the two different molecular conformations, i.e. aggregate or subunit, of proteoglycans in solution.