Biosynthesis of proteoglycans and their assembly into aggregates in cultures of chondrocytes from the Swarm rat chondrosarcoma.

Cultured chondrocytes from the Swarm rat chondrosarcoma incorporate [35S]sulfate into proteoglycans typical of hyaline cartilage. The movement of newly synthesized proteoglycans from inside the cells into the extracellular matrix and, finally, into the culture medium was examined by measuring the distribution of 35S-labeled proteoglycans in the medium, a 4 M guanidine HCl extract of the cell layer, and in the remaining residue for a number of chase times following a 5-min pulse with [35S]sulfate. When hyaluronate oligosaccharides containing greater than or equal to 10 monosaccharides were included in the chase media, a proportion of newly synthesized proteoglycans were displaced from the matrix (4 M extract) into the culture medium. This displacement was greatest when oligomers were in the chase media between 10 and 20 min after the pulse, approximately the time when the molecules are being secreted from the cells. The proportion of link-stabilized aggregate in the medium was examined by Sepharose 2B chromatography after adding an excess of unlabeled monomer which displaces labeled monomer from complexes with hyaluronate which are not link-stabilized. The proportion of link-stabilized aggregate increased from 12% to about 70% between 12 and 120 min of chase. The presence of 40 micron hyaluronate oligosaccharides of 16 monosaccharides in the chase media retarded but did not prevent aggregate formation. Oligomers of about 50 monosaccharides, which are large enough to bind both a monomer proteoglycan and a link protein, almost completely prevented the formation of the large link-stabilized aggregates. The results suggest: (a) newly synthesized proteoglycans are not bound into link-stabilized aggregates at the time of secretion; (b) hyaluronic acid oligomers which are long enough to interact only with the hyaluronic acid-binding site of proteoglycans will retard but not prevent link-stabilized aggregation; and (c) hyaluronic acid oligomers long enough to accommodate additionally a link protein form a link-stabilized ternary complex and prevent aggregation with larger hyaluronic acid molecules.     

Proteoglycans from bovine nasal and articular cartilages. Fractionation of the link proteins by wheat germ agglutinin affinity chromatography

Two forms of link protein, 46 and 51 kDa, are present in proteoglycan aggregates from both bovine nasal and bovine articular cartilages. Studies reported here show that the link proteins bind to concanavalin A, Lens culinaris agglutinin, Ricinus communis agglutinin, soybean agglutinin, and wheat germ agglutinin lectins. When the link proteins are eluted from these lectins with appropriate competing sugars, the 46- and the 51-kDa link proteins elute together and no separation is achieved. However, when the link proteins bound to wheat germ agglutinin are eluted with a 0 to 4 M guanidine hydrochloride linear gradient, a good separation of the 46- and 51-kDa link proteins is achieved. Wheat germ agglutinin affinity chromatography has been used on a preparative scale to isolate the 51-kDa link protein from mature bovine articular cartilage to homogeneity, in amounts sufficient to examine its effect on proteoglycan aggregate size and stability in sedimentation velocity studies. Proteoglycan aggregates were reassembled from proteoglycan monomers and hyaluronate in the absence of link protein, in the presence of both 46- and 51-kDa link proteins, and in the presence of the individual 51-kDa link protein. The sizes of the aggregates were compared in terms of sedimentation coefficients (s(0)20). The stability of the aggregates was compared in terms of the per cent aggregate present at pH 7 and 5. At pH 7, the sedimentation coefficients (s(0)20) of link-free aggregates, aggregates formed with both link proteins, and aggregates formed with 51-kDa link protein were 72, 93, and 112 S, respectively. Thus, the 51-kDa link protein has a pronounced effect on aggregate size. The link-free aggregate was grossly unstable, and only 36% aggregate was present at pH 5. The aggregate formed with both link proteins was effectively stabilized against dissociation and 79% aggregate was present at pH 5. The aggregate formed with 51-kDa link protein was not effectively stabilized against dissociation, and only 60% aggregate was present at pH 5. Thus, despite its pronounced effect on aggregate size, the 51-kDa link protein does not effectively stabilize the proteoglycan aggregate against dissociation. These results suggest that the 51-kDa link protein may selectively increase aggregate size, while the 46-kDa link protein may be required to effectively stabilize the proteoglycan aggregate against dissociation.     

A study of the interaction between cartilage proteoglycan and link protein.

The interaction between proteoglycan and link protein extracted from bovine articular cartilage (15-18-month-old animals) was investigated in 0.5 M-guanidinium chloride. The proteoglycans, radiolabelled as the aggregate (A1 fraction), were fractionated by two 'dissociative' density-gradient centrifugations (A1D1D1) followed by a rate-zonal centrifugation (S1) to yield an A1D1D1S1 preparation. At least 65% of these proteoglycans were able to bind to hyaluronate, but only 52% were able to bind to link protein as assessed by chromatography on Sepharose CL-2B. Over 80% of the [3H]link-protein preparation, radiolabelled as the aggregate, was able to interact with proteoglycan as assessed by chromatography on Sepharose CL-4B. Equilibrium-boundary-centrifugation studies performed at low link-protein concentrations (2.42 x 10(-9) M-5.93 x 10(-8) M) were analysed by Scatchard-type plots and indicated a Kd of 1.5 x 10(-8) M and a stoichiometry, n = 0.56, i.e. approx. 56% of those proteoglycans capable of binding to link protein had a strong site for link protein if a 1:1 stoichiometry were assumed. However, experiments performed at higher link-protein concentrations (3.5 x 10(-7) M and 8 x 10(-7) M) yielded stoichiometry values which were link-protein-concentration-dependent. Non-equilibrium binding studies using chromatography on Sepharose CL-2B and rate-zonal centrifugation yielded apparent stoichiometries between 0.6 and 7.5 link-protein molecules per proteoglycan monomer as a function of increasing link-protein concentration. It was concluded that a proportion of the proteoglycan molecules had a strong site for binding a single link protein (Kd 1.5 x 10(-8) M) and that at high link-protein concentrations a weaker, open-ended, process of link-protein self-association nucleated upon the strong link-protein-proteoglycan complex occurred. Hyaluronate oligosaccharides appeared to abolish a proportion of this self-association (as observed by Bonnet, Dunham & Hardingham [(1985) Biochem. J. 228, 77-85] in a study of link-protein-hyaluronate-oligosaccharide interactions) so as to leave a link protein:proteoglycan stoichiometry of 2. It is not clear whether this second link-protein molecule binds directly to the proteoglycan or to the first link protein.     

Assembly of proteoglycan aggregates in cultures of chondrocytes from bovine tracheal cartilage.

The assembly of proteoglycan aggregates in chondrocyte cell cultures was examined in pulse-chase experiments with the use of [35S]sulphate for labelling. Rate-zonal centrifugation in linear sucrose density gradients (10-50%, w/v) was used to separate the aggregated proteoglycans from monomers and to assess the size of the newly formed aggregates. The proportion of aggregates stabilized by link protein was assessed by competition with added exogenous aggregate components. The capacity of the proteoglycans synthesized in culture to compete with exogenous nasal-cartilage proteoglycans for binding was studied in dissociation-reassociation experiments. The results were as follows. (a) The proteoglycan monomers and the hyaluronic acid are exported separately and combined extracellularly. (b) The size of the aggregates increases gradually with time as the proportion of monomers bound to hyaluronic acid increases. (c) All of the aggregates present at a particular time appear to be link-stabilized and therefore not dissociated by added excess of nasal-cartilage proteoglycan monomer or hyaluronic acid oligomers. (d) The free monomer is apparently present as a complex with link protein. The monomer-link complexes are then aggregated to the hyaluronic acid. (e) The aggregates synthesized in vitro and the nasal-cartilage aggregates differ when tested for link-stabilization by incubation at low pH. The aggregates synthesized in vitro were completely dissociated whereas the cartilage proteoglycans remained aggregated. The results obtained from dissociation-reassociation experiments performed at low pH indicate that the proteoglycan monomer synthesized in vitro does not bind the hyaluronic acid or the link protein as strongly as does the nasal-cartilage monomer.     

Proteoglycan aggregate formation by articular chondrocytes. Decrease in link-protein synthesis during culture.

The synthesis of link-stabilized proteoglycan aggregates by rabbit articular chondrocytes was investigated by [35S]sulphate labelling of primary monolayer cultures maintained for up to 21 days. (1) At all culture times the cells secreted a high-molecular-weight cartilage-type proteoglycan monomer of which 75%-80% formed aggregates with hyaluronic acid. (2) At 2 days of culture all of the aggregates were in link-stabilized form, but by 21 days only 5% were link-stabilized, as shown by displacement of monomers from the aggregate by hyaluronic acid oligosaccharides. (3) The addition of purified link protein to 21-day culture medium increased the proportion of link-stable aggregate from 5% to 70%. (4) Analysis of [3H]serine-labelled proteoglycan aggregates in the medium showed a marked decrease with culture time in the ratio of 3H-labelled link protein to 3H-labelled core protein present. The results suggest that the secretion of proteoglycan monomers and link protein by articular chondrocytes changes independently during prolonged monolayer culture.     

The influence of link protein stabilization on the viscometric properties of proteoglycan aggregate solutions

The dynamic, steady-shear and transient shear flow properties of precisely prepared link-stable (s0 136, 66% aggregate) and link-free (s0 93, 59% aggregate) proteoglycan aggregate solutions at concentrations ranging from 10 to 50 mg/ml were determined using a cone-on-plate viscometer in a mechanical spectrometer. All proteoglycan solutions tested possessed: (1) linear viscoelastic properties - as measured by the dynamic complex modulus under small amplitude steady oscillatory conditions (1 less than or equal to omega less than or equal to 100 rad/s) - and (2) nonlinear shear-rate dependent apparent viscosities and primary normal stress difference under steady shearing conditions (0.25 less than or equal to gamma less than or equal to 250 s-1). Our transient flow data show that all proteoglycan aggregate solutions exhibited transient stress overshoot effects in shear stress and normal stress. From these steady and transient flow data, we conclude that link protein stabilized aggregates have significant effects on their dynamic and steady-shear properties as well as transient flow properties. The transient stress overshoot data provide a measure of the energy per unit volume of fluid required to overcome the proteoglycan networks in solution from a resting state. Thus we found that link-stable aggregates form much stronger networks than link-free aggregates. This is corroborated by the fact that link-stable aggregates form more elastic (lower than delta) and stiffer (higher [G*]) networks than link-free aggregates. The complete spectrum of viscometric flow data is entirely compatible with the proposed role of link protein in adding structural stability to the proteoglycan-hyaluronate bond. In cartilage, the enhanced strength of the networks formed by link-stable aggregates may play an important role in determining the material properties of the tissue and thereby contribute to the functional capacity of cartilage in diarthrodial joints.     

Superficial and deeper layers of dog normal articular cartilage. Role of hyaluronate and link protein in determining the sedimentation coefficients distribution of the nondissociatively extracted proteoglycans

Proteoglycans were extracted under nondissociative conditions from superficial and deeper layers of dog normal articular cartilage. The purified a-A1 preparations were characterized by velocity gradient centrifugation. Superficial specimens exhibited an abundant population of slow sedimenting aggregates whereas the aggregates of deeper preparations sedimented as two well-defined families of molecules. These dissimilarities in the size distribution of the aggregates observed between superficial and deeper a-A1 preparations derived most of all from differences in their content of hyaluronate and link proteins: (a) superficial preparations contained twice as much hyaluronate as deeper specimens; (b) superficial aggregates were link-free and unstable at pH 5.0 whereas deeper preparations contained link-proteins and their faster sedimenting aggregates were stabilized against dissociation at pH 5.0. In these proteoglycan preparations from different cartilage layers, the monomers exhibited an identical capacity for aggregation and the hyaluronate molecules displayed quite similar molecular weight (Mr = 5 x 10(5] and aggregating capacity. These observations as well as aggregating studies conducted with highly purified link protein and purified hyaluronate specimens of different molecular weights support the following conclusions: (a) link protein not only stabilizes proteoglycan aggregates but also enhances the aggregating capacity of hyaluronate; (b) for all practical purposes, the slow sedimenting aggregates represent a secondary complex of hyaluronate and proteoglycan monomers whereas the fast sedimenting aggregates may be considered as a ternary complex wherein link protein stabilizes the hyaluronate-proteoglycans interaction; (c) the distinctive heterogeneity of articular cartilage can be related to structurally different proteoglycan aggregates. The structural dissimilarities observed between superficial and deeper aggregates could reflect the different macromolecular organization of the proteoglycan molecules in the territorial and interterritorial matrices, respectively.     

Formation of proteoglycan aggregates in rat chondrosarcoma chondrocyte cultures treated with tunicamycin

Proteoglycan monomer and link protein isolated from the Swarm rat chondrosarcoma both contain glycosylamine-linked oligosaccharides. In monomer, these N-linked oligosaccharides are concentrated in a region of the protein core which interacts specifically with both hyaluronate and link protein to form proteoglycan aggregates present in cartilage matrix. Chondrocyte cultures were treated with tunicamycin to inhibit synthesis of the N-linked oligosaccharides, and the ability of the deficient proteoglycan and link protein to form aggregates was studied. Cultures were pretreated with tunicamycin for 3 h and then labeled with either [3H]mannose, [3H]glucosamine, [3H]serine, or with [35S]sulfate for 6 h in the presence of tunicamycin. Formation of link protein-stabilized proteoglycan aggregates in the culture medium was inhibited by up to 40% when the cells were treated with 3 micrograms of tunicamycin/ml, a concentration which inhibited 3H incorporation with mannose as a precursor by about 90%, but by only 15% with glucosamine as a precursor. When exogenous proteoglycan aggregate was added to the culture medium, however, it was found that both endogenous monomer and link protein synthesized in the presence of tunicamycin were fully able to form link-stabilized aggregates. This suggests that glycosylamine-linked oligosaccharides on monomer and on link protein are not necessary for their specific interactions with hyaluronate and with each other. Further, although tunicamycin did not inhibit net synthesis of hyaluronate, transfer of hyaluronate from the cell layer to the culture medium was retarded. This phenomenon accounted for most if not all of the decrease in the amount of proteoglycan which formed aggregates in the medium of cultures treated with tunicamycin.     

Age-related decrease in the link-stability of proteoglycan aggregates formed by articular chondrocytes

Chondrocytes were isolated from the articular cartilage of rabbits aged between 6 and 50 weeks and labelled with [35S]sulphate after 48 h in monolayer culture. The percentage of the total proteoglycan monomers synthesized by each culture that were present as link-stabilized aggregates was shown to be about 83% at 6, 9 and 12 weeks, 73% at 15 weeks, 48% at 30 weeks and 32% at 50 weeks. The proliferative activity of the cells in culture also decreased markedly with the age of the donor. The results suggest that aging of chondrocytes in vivo is accompanied by a decrease in their capacity for link-protein synthesis.     

Effects of tissue compression on the hyaluronate-binding properties of newly synthesized proteoglycans in cartilage explants.

The effects of tissue compression on the hyaluronate-binding properties of newly synthesized proteoglycans in calf cartilage explants were examined. Pulse-chase experiments showed that conversion of low-affinity monomers to the high-affinity form (that is, to a form capable of forming aggregates with 1.6% hyaluronate on Sephacryl S-1000) occurred with a t1/2 of about 5.7 h in free-swelling discs at pH 7.45. Static compression during chase (in pH 7.45 medium) slowed the conversion, as did incubation in acidic medium (without compression). Both effects were dose-dependent. For example, the t1/2 for conversion was increased to about 11 h by either (1) compression from a thickness of 1.25 mm to 0.5 mm or (2) medium acidification from pH 7.45 to 6.99. Oscillatory compression of 2% amplitude at 0.001, 0.01, or 0.1 cycles/s during chase did not, however, affect the conversion. Changes in the hyaluronate-binding affinity of [35S]proteoglycans in these experiments were accompanied by no marked change in the high percentage (approximately 80%) of monomers which could form aggregates with excess hyaluronate and link protein. Since static tissue compression would result in an increased matrix proteoglycan concentration and thereby a lower intra-tissue pH [Gray, Pizzanelli, Grodzinsky & Lee (1988) J. Orthop. Res. 6, 777-792], it seems likely that matrix pH may influence proteoglycan aggregate assembly by an effect on the hyaluronate-binding affinity of proteoglycan monomer. Such a pH mechanism might have a physiological role, promoting proteoglycan deposition in regions of low proteoglycan concentration.     

Cartilage proteoglycan aggregate is degraded more extensively by cathepsin L than by cathepsin B.

The degradative actions of cathepsins L and B on human articular-cartilage proteoglycan aggregates were examined. Cathepsin L was found to be much more extensive than cathepsin B in degrading proteoglycan aggregates. It released products with size similar to that of single chondroitin sulphate chains, and a series of degraded link-protein fragments in the digestion mixtures. These proteolytically modified link-protein components (Mr 25,000 and 33,000) have similar Mr values to those of fragments observed in adult human cartilage. In contrast, cathepsin B exhibited a much more limited degradation on both proteoglycan subunits and link-protein components. Both cathepsins L and B generate multiple but distinct cleavage sites on human link proteins, and the hydrolysed bonds have been identified in the region between residues 18 and 29. Protein sequencing analysis of these modified link-protein components also provided evidence for the location of a second N-linked glycosylation site at residue 41 in human link proteins, in addition to that previously described at residue 6 on a proportion of the link proteins. Furthermore, it allows us to report the sequence of human link protein up to residue 65.     

Turnover of proteoglycans in cultures of bovine articular cartilage

Proteoglycans in cultures of adult bovine articular cartilage labeled with [35S]sulfate after 5 days in culture and maintained in medium containing 20% fetal calf X serum had longer half-lives (average 11 days) compared with those of the same tissue maintained in medium alone (average 6 days). The half-lives of proteoglycans in cultures of calf cartilage labeled after 5 days in culture and maintained in medium with serum were considerably longer (average 21 days) compared to adult cartilage. If 0.5 mM cycloheximide was added to the medium of cultures of adult cartilage, or the tissue was maintained at 4 degrees C after labeling, the half-lives of the proteoglycans were greater, 24 and greater than 300 days, respectively. Analyses of the radiolabeled proteoglycans remaining in the matrix of the tissue immediately after labeling the tissue and at various times in culture revealed two main populations of proteoglycans; a large species eluting with Kav of 0.21-0.24 on Sepharose CL-2B, of high bouyant density and able to form aggregates with hyaluronate, and a small species eluting with a Kav of 0.63-0.70 on Sepharose CL-2B, of low buoyant density, containing only chondroitin sulfate chains, and unable to form aggregates with hyaluronate. The larger proteoglycan had shorter half-lives than the smaller proteoglycan; in cartilage maintained with serum, the half-lives were 9.8 and 14.5 days, respectively. Labeling cartilage with both [3H]leucine and [35S]sulfate showed the small proteoglycan to be a separate synthetic product. The size distribution of 35S-labeled proteoglycans lost into the medium was shown to be polydisperse on Sepharose CL-2B, the majority eluting with a Kav of 0.27 to 0.35, of high buoyant density, and unable to aggregate with hyaluronate. The size distribution of glycosaminoglycans from 35S-labeled proteoglycans appearing in the medium did not differ from that associated with labeled proteoglycans remaining in the matrix.     

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.     

Isolation and characterization of a link protein from bovine aorta proteoglycan aggregate

Proteoglycan aggregates were isolated from bovine aorta by extraction with 0.5 M guanidine hydrochloride in the presence of proteinase inhibitors and purified by isopycnic CsCl centrifugation. The bottom two-fifths (A1) of the gradient contained 30% of proteoglycans in the aggregated form. The aggregate had 14.8% protein and 20.4% hexuronic acid with hyaluronic acid, dermatan sulfate and chondroitin sulfates in a proportion of 18:18:69. A link protein-containing fraction was isolated from the bottom two-fifths by dissociative CsCl isopycnic centrifugation. The link protein that floated to the top one-fifth of the gradient was purified by chromatography on Sephadex G-200 in the presence of 4 M guanidine hydrochloride. It moved as a single band in SDS-polyacrylamide gel electrophoresis with a molecular weight of 49 000. The amino acid composition of link protein resembled that of link protein from cartilage, but was strikingly different from that of the protein core of the proteoglycan monomer. The neutral sugar content of link protein was 3.5% of dry weight. Galactose, mannose and fucose constituted 21, 62 and 16%, respectively of the total neutral sugars. In aggregation studies the link protein was found to interact with both proteoglycan monomer and hyaluronic acid. Oligosaccharides derived from hyaluronic acid decreased the viscosity of link protein-free aggregates of proteoglycan and hyaluronic acid but not of link-stabilized aggregates, demonstrating that the link protein increases the stability of proteoglycan aggregates.     

A comparison of isometallothionein synthesis in rat liver after partial hepatectomy and parenteral zinc injection.

Proteoglycan aggregates free of non-aggregating proteoglycan have been prepared from the annuli fibrosi and nuclei pulposi of intervertebral discs of three human lumbar spines by extraction with 4M-guanidinium chloride, associative density gradient centrifugation, and chromatography on Sepharose CL-2B. The aggregate (A1-2B.V0) was subjected to dissociative density-gradient ultracentrifugation. Three proteins of Mr 38 900, 44 200 and 50 100 found in the fraction of low buoyant density (A1-2B.V0-D4) reacted with antibodies to link protein from newborn human articular cartilage. After reduction with mercaptoethanol, two proteins of Mr 43 000 and two of Mr 20 000 and 14 000 were seen. The A1-2B.V0-D4 fraction, labelled with 125I, coeluted with both hyaluronate and a hyaluronate oligosaccharide (HA14) on a Sepharose CL-2B column. HA10 and HA14 reduced the viscosity of A1 fractions; HA4, HA6 and HA8 did not. HA14 decreased the viscosity of disc proteoglycans less than it did that of bovine cartilage proteoglycans. Thus, although a link protein was present in human intervertebral disc, it stabilized proteoglycan aggregates less well than did the link protein from bovine nasal cartilage.     

The density and strength of proteoglycan-proteoglycan interaction sites in concentrated solutions.

Rheological flow properties of link-stable and link-free proteoglycan (PG) aggregates in concentrated solutions were measured using a cone-on-plate viscometer. A second-order constitutive model, based upon the statistical-network theories of Lodge, [Rheol. Acta 7, 379-392 (1968)] and De Kee and Carreau [J. Non-Newtonian Fluid Mech. 6, 127-143 (1979)], was developed to describe the measured steady and transient flow responses exhibited by the PG solutions. Our measurements confirmed previous experimental findings that the complex shear modulus of PG solutions depends on the frequency of the imposed small-amplitude oscillatory shear, and the apparent viscosity and primary normal-stress difference depend nonlinearly on the shear rate under steady-shear flow conditions [Mow et al., J. Biomechanics 17, 325-338 (1984b); Hardingham et al., J. orthop. Res. 5, 36-46 (1987)]. In the present study, we found that PG solutions exhibit pronounced stress overshoot responses and large hysteresis loop effects. These transient responses were shown to be sensitive to acceleration strain (i.e. the second rate of strain) as well as PG structure (i.e. link-protein stabilization). The model parameters were determined by curvefitting of the second-order constitutive model and experimental data from steady, oscillatory and transient shear flow measurements. Using this network model, we calculated the density of the idealized interaction sites existing in the PG network, and the average strength of these interaction sites. The results indicate that link-protein stabilization of PG aggregates does not change the density of interaction sites formed in the PG network, rather, it increases the average strength of these interaction sites.     

Characterization of link protein(s) from human intervertebral-disc tissues.

Proteoglycan aggregates (A1) were prepared from the anulus fibrosus, nucleus pulposus and cartilage-endplate tissues of postnatal (0-6-month-old)-and young-adult (20-30-year-old)-human intervertebral discs. The A1 fractions from young-adult disc contained a greater proportion of non-aggregating proteoglycans than did postnatal tissues. After dissociative CsCl-density-gradient fractionation of the A1, more than 90% of the uronic acid was found in the postnatal A1D1, whereas only 60-80% of the hexuronate was present in the A1D1 isolated from young-adult disc tissues. These results indicated that more lower-buoyant-density proteoglycans occur in the young-adult disc. Link-protein-rich fractions (A1D3) were subjected to SDS/polyacrylamide-gel electrophoresis and immunolocation analyses using monoclonal antibodies specific for epitopes on link protein or proteoglycan. Under non-reducing conditions, the major link protein present in postnatal disc tissues was link protein 1. By contrast, all three link proteins (1, 2 and 3) were detected in young-adult tissues, with the smaller link protein 3 predominating. Analyses of the A1D3 fractions under reducing conditions also indicated the presence of link-protein-degradation peptides (Mr approx. 26,000) from young-adult disc tissues, but not from postnatal tissues. Sequential Sepharose CL-6B and Sephacryl S-300 chromatography in 4 M-guanidinium chloride was employed to separate the link proteins of the A1D3 fraction from protein-rich proteoglycan. Immunolocation analyses indicated that postnatal samples contained no detectable contaminating proteoglycan fragments. However, young-adult link-protein preparations could not be separated from hyaluronic acid-binding region and other proteoglycan fragments by means of these chromatographic procedures. The studies indicate that, compared with hyaline articular cartilage, degraded link protein and proteoglycan accumulate at an early age in young-adult disc tissues. These partially degraded proteoglycan aggregate components may significantly alter the biomechanical properties of disc tissues.     

Cartilage proteoglycan aggregate formation. Role of link protein.

Cartilage proteoglycan aggregate formation was studied by zonal rate centrifugation in sucrose gradients. Proteoglycan aggregates, monomers and proteins could be resolved. It was shown that the optimal proportion of hyaluronic acid for proteoglycan aggregate formation was about 1% of proteoglycan dry weight. The reaggregation of dissociated proteoglycan aggregate A1 fraction was markedly concentration-dependent and even at 9 mg/ml only about 90% of the aggregates were reformed. The lowest proportion of link protein required for maximal formation of link-stabilized proteoglycan aggregates was 1.5% of proteoglycan dry weight. It was separately shown that link protein co-sedimented with the proteoglycan monomer. By competition with isolated hyaluronic acid-binding-region fragments, a proportion of the link proteins was removed from the proteoglycan monomers, indicating that the link protein binds to the hyaluronic acid-binding region of the proteoglycan monomer.     

Aortic endothelial cells synthesize a large chondroitin sulphate proteoglycan capable of binding to hyaluronate.

Confluent cultures of mouse aortic endothelial (END-D) were incubated with either [35S]methionine or 35SO4 2-, and the radiolabelled proteoglycans in media and cell layers were analysed for their hyaluronate-binding activity. The proteoglycan subfraction which bound to hyaluronate accounted for about 18% (media) and 10% (cell layers) of the total 35S radioactivity of each proteoglycan fraction. The bound proteoglycan molecules could be dissociated from the aggregates either by digestion with hyaluronate lyase or by treatment with hyaluronate decasaccharides. Digestion of [methionine-35S]proteoglycans with chondroitinase and/or heparitinase, followed by SDS/polyacrylamide-gel electrophoresis, indicated that the medium and cell layer contain at least three chondroitin sulphate proteoglycans, one dermatan sulphate proteoglycan, and two heparan sulphate proteoglycans which differ from one another in the size of core molecules. Among these, only the hydrodynamically large chondroitin sulphate species with an Mr 550,000 core molecule was shown to bind to hyaluronate. A very similar chondroitin sulphate proteoglycan capable of binding to hyaluronate was also found in cultures of calf pulmonary arterial endothelial cells (A.T.C.C. CCL 209). These observations, together with the known effects of hyaluronate on various cellular activities, suggest the existence of possible specialized functions of this proteoglycan subspecies in cellular processes characteristic of vascular development and diseases.     

Lack of effect of lysozymes on cartilage proteoglycans.

Certain similarities between lysozyme and testicular hyaluronidase suggested that the putative action of the former in disaggregation of cartilage proteoglycans might be explained by a selective effect of the enzyme on the hyaluronic acid portion of the aggregate. Human lysozyme did not reduce the viscosity of either hyaluronate or of aggregated proteoglycans, it did not reduce the sedimentation velocity of hyaluronate, and it did not alter the chromatographic profile of the aggregate in a system sensitive to the difference between aggregate and its subunit. The role of human cartilage lysozyme in disaggregation of cartilage proteoglycans remains uncertain.