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Chondroitin 4-sulfotransferase, which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of N-acetylgalactosamine in chondroitin, was purified 1900-fold to apparent homogeneity with 6.1% yield from the serum-free culture medium of rat chondrosarcoma cells by affinity chromatography on heparin-Sepharose CL-6B, Matrex gel red A-agarose, 3',5'-ADP-agarose, and the second heparin-Sepharose CL-6B. SDS-polyacrylamide gel electrophoresis of the purified enzyme showed two protein bands. Molecular masses of these protein were 60 and 64 kDa under reducing conditions and 50 and 54 kDa under nonreducing conditions. Both the protein bands coeluted with chondroitin 4-sulfotransferase activity from Toyopearl HW-55 around the position of 50 kDa, indicating that the active form of chondroitin 4-sulfotransferase is a monomer. Dithiothreitol activated the purified chondroitin 4-sulfotransferase. The purified enzyme transferred sulfate to chondroitin and desulfated dermatan sulfate. Chondroitin sulfate A and chondroitin sulfate C were poor acceptors. Chondroitin sulfate E from squid cartilage, dermatan sulfate, heparan sulfate, and completely desulfated N-resulfated heparin hardly served as acceptors of the sulfotransferase. The transfer of sulfate to the desulfated dermatan sulfate occurred preferentially at position 4 of the N-acetylgalactosamine residues flanked with glucuronic acid residues on both reducing and nonreducing sides.

Mechanical stress is an important regulator of chondrocyte functions but the mechanisms by which chondrocytes sense mechanical signals are unknown. Receptors for matrix molecules are likely involved in the mechanical signaling. In the first part of this study we identified integrins with affinity for the cartilage-specific collagen type II. We report that the collagen-binding integrins alpha 1 beta 1 and alpha 2 beta 1 isolated from bovine chondrocytes or human chondrosarcoma cells bound collagen type II as judged from affinity chromatography. The integrins alpha 3 beta 1 or alpha 9 beta 1 did not bind collagen type II-Sepharose. In the second part of the study we investigated the effect of mechanical stress on expression of matrix molecules and integrin subunits. Chondrocytes and chondrosarcoma cells, cultured on uncoated flexible silicone membranes in the presence of serum, were exposed to mechanical stress by the Flexercell system. Dynamic stimulation of chondrocytes for 3 h increased the mRNA expression of collagen type II and aggrecan as judged by Northern blotting, while the beta 1-integrin subunit was not changed. When chondrosarcoma cells were exposed to mechanical stimulation under the same conditions, mRNA expression of alpha 5 was found to increase while beta 1, alpha 2, and alpha v did not increase to significant levels. In another study the effect of mechanical stress on integrins was investigated when the cells were cultured on collagen type II-coated flex-dishes. Three hours of dynamic stress increased the mRNA expression of alpha 2-integrin subunit while the level of mRNA for integrin subunits beta 1, alpha 1, alpha 5, and alpha v showed no or small changes, indicating that matrix components may modulate the expression of integrins during mechanical stress.

Keratan sulphate is an integral component of the large aggregating proteoglycans of mature human articular cartilage. The keratan sulphate content of chondrocytic proteoglycans increases during maturation, and it is a useful marker of mature-type chondrocytic proteoglycans. Ordinarily, in cell culture, chondrocytes from non-neoplastic tissues dedifferentiate, diminish or cease to synthesize aggregating proteoglycans with the same amount of keratan sulphate as those formed in vivo, and do not maintain their in vivo phenotype. In tissue culture, this down-regulation of synthesis of keratan sulphate is irreversible. The study of the metabolism of mature human chondrocytes has been hampered by the absence of stable models. We report a cell-line, 105KC, derived from a human chondrosarcoma, that has maintained a stable proteoglycan phenotype during more than three years of culture. Analysis with immunofluorescence suggested that 105KC cells continued to synthesize keratan sulphate in long-term culture. Biochemical analysis demonstrated that 105KC cells maintained the production of chondrocytic large-aggregating proteoglycans and that keratan sulphate composed 13 per cent of their glycosaminoglycan content. To our knowledge, 105KC represents the first model to have maintained the post-fetal chondrocytic proteoglycan phenotype in stable culture. This study documents the feasibility of the development of mature chondrocytic cell-lines and sheds light on the biological characteristics of chondrosarcomas.

Articular cartilage chondrocytes maintain biosynthetic heterogeneity in cell culture, but undergo irreversible dedifferentiation of their proteoglycan phenotype, as defined by keratan sulfate content. A recently described cell line of malignant human chondrocytes, 105KC, was the first to maintain a differentiated keratan sulfate-proteoglycan phenotype in long-term culture. A clone of 105KC, labeled KC2H3, is currently described and represents a distinct and metabolically more homogeneous population of mature chondrocytes than 105KC. KC2H3 cells universally express keratan sulfate biosynthesis, as defined by indirect immunofluorescence. In addition, KC2H3 expresses a more mature proteoglycan phenotype than 105KC, as demonstrated by the keratan sulfate content: 24% of glycosaminoglycan content of the aggregating proteoglycans of KC2H3 versus 13% for 105KC. Further reported are the effects of long term monolayer culture on the proteoglycan phenotype expressed by KC2H3. After more than 16 months in continuous monolayer, KC2H3 cells remained morphologically indistinguishable from those maintained in suspension alternating with monolayer. In addition, the proteoglycan phenotype remained mature, without a tendency towards dedifferentiation. The flattened morphology adopted by chondrocytes while in monolayer has been considered a stimulus of dedifferentiation; the present study is the first to examine the direct effects of physical state on a homogeneous and stable population of chondrocytes.

Difficulty in maintaining phenotypic stability of the Swarm rat chondrosarcoma in long-term monolayer cultures has prompted investigation of alternative conditions that would enable extended maintenance of these cells, permitting use of the tumor as a model system for the long-term study of proteoglycan metabolism. Morphological analysis of the growth of the chondrosarcoma chondrocytes in agarose has shown stability of the culture over a 20 day period with respect to the ability of the cells to proliferate and synthesize an Alcian blue-positive extracellular matrix. The present study confirms these findings through analysis of the growth characteristics of the culture and the pattern of proteoglycan and collagen synthesis. The chondrocytes actively synthesize a proteoglycan-rich matrix at a rate dependent on the initial plating density and concentration of serum in the culture medium. These factors similarly affect the proliferative capabilities of the culture as demonstrated by the growth curves obtained at different culture conditions. During 20 days in culture, the cells synthesize an aggregating chondroitin sulfate proteoglycan and collagen type II, typical of cartilage and this chondrosarcoma. In addition, small molecular weight proteoglycans were found to be present at concentrations of up to 10% of the total proteoglycan population. Degradative rates are slow, the proteoglycan half-life is about 30 days, but can be enhanced with retinol, reducing the half-life to 2 days.

Two classes of oligosaccharides were identified in alkaline borohydride digests of monomer proteoglycans from the Swarm rat chondrosarcoma. The first class consists of three similar oligomers, each of which is linked to the proteoglycan core proteins by an O-glycosidic bond between N-acetylgalactosamine and hydroxyl groups on serine and threonine. The structures of the two smallest oligosaccharides are: (formula: see text). Proteoglycan monomers from the chondrosarcoma contain about 1.3 of these oligosaccharides for every chondroitin sulfate chain. Their structures and distribution on the core protein suggest that they are related to the linkage region between keratan sulfate chains and the core protein of cartilage proteoglycans. The second class of oligomers consists of oligosaccharide-peptides that contain mannose and are probably linked to the core protein by N-glycosylamine bonds to asparagine. There appear to be about 15 of these oligosaccharides/ proteoglycan molecule, and they are primarily located on the core protein nearer to the hyaluronic acid-binding site than both the oligosaccharides linked by O-glycoside bonds and the chondroitin sulfate chains. Chondrocytes isolated from the chondrosarcoma and grown in culture synthesize both classes of oligosaccharides.

The synthesis and subsequent assembly of proteoglycan monomer and link protein into aggregates were examined using cultured chondrocytes from the Swarm rat chondrosarcoma. Proteoglycans purified directly from culture medium after a 6-h label with [14C]serine contained up to 80% of the radioactivity in aggregates, 8% of which was in link protein. The addition of hyaluronic acid oligosaccharides (averaging 50 monosaccharides) to the culture medium during labeling inhibited aggregate formation by 94%. More than 90% of the labeled link molecules (about 7% of the total radioactivity in the proteoglycan fraction) was associated in ternary complexes with monomeric proteoglycan and HA approximately 50. The stoichiometry of labeled link protein to labeled proteoglycan core protein aggregated with either macromolecular hyaluronic acid or hyaluronic acid oligosaccharides was calculated to be 1:1. The effect of exogenous proteoglycan on aggregate formation was examined by incubating cultures for 4 h with up to 5 mg of exogenous monomer. Over this time, about 85 micrograms of newly synthesized proteoglycan accumulated in the culture medium. With increasing amounts of exogenous monomer, up to 80 micrograms of 35S-labeled exogenous monomer was incorporated into link-stabilized aggregate, while the aggregation of endogenous proteoglycan decreased from 73 to 44%. The total endogenous plus exogenous monomer in link-stabilized aggregate increased to about 130 micrograms, approximately 1 1/2 times the total amount of endogenous monomer in the culture medium, indicating that at least 1 1/2 times more link molecules than endogenous proteoglycan molecules were present. Further, the molar ratio of labeled link to labeled monomer molecules associated with the nonaggregated proteoglycan fraction remained constant at approximately 1:1, indicating that the exogenous proteoglycans were ineffective in displacing link molecules from endogenous monomer.link complexes. The data suggest that the formation of proteoglycan aggregates may be a sequential process in which a link.monomer complex forms and this complex subsequently binds to hyaluronic acid.

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.

Rat chondrosarcoma chondrocytes were labeled with [3H]serine or [3H]mannose as a precursor. Intracellular proteoglycan core protein precursor was purified from cell lysates by immunoprecipitation with polyclonal antibodies against the hyaluronic acid-binding region, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The core precursor was eluted from the gels and treated with alkaline borohydride in order to convert serine residues substituted with xylose or N-acetylgalactosamine to alanine (or with alkaline sulfite to convert them to cysteic acid). After acid hydrolysis, the proportions of labeled serine and alanine (or cysteic acid) were determined by high performance liquid chromatography, and the results were compared with those obtained for the completed proteoglycan molecules isolated from the same cultures. In the completed proteoglycans, about 55% of the serine residues were substituted with xylose or N-acetylgalactosamine, while the corresponding figure for the intracellular precursor molecules was less than 5%. These results indicate, in agreement with our previous kinetic data, that the major part of the xylosyl transfer to the chondrosarcoma proteoglycan core protein precursor must occur late in the processing sequence, i.e. after about 85% of its intracellular lifetime and no more than 7 min before the addition of the rest of the chondroitin sulfate chain. The ratio of [3H]mannose to [3H]fucose in the core precursor was about 19, while that for the complete proteoglycan was about 2. This indicates the presence of high mannose, N-linked oligosaccharides on the core protein precursor which are converted to the complex forms on the completed proteoglycan. These data provide further support that the core precursor resides mainly in the pre-Golgi compartment and that xylosylation occurs mainly in a Golgi compartment.

Chondrocytes from the Swarm chondrosarcoma, a transplantable rat tumor, have been difficult to maintain in tissue culture for extended periods due to a time-dependent alteration of the culture to a more fibroblastic phenotype. This feature precluded the use of these cultures to examine chronic conditions that may affect cell metabolism, and the homogeneity or heterogeneity of the tumor cells within the culture population could not be examined. Use of suspension culture in agarose stabilized the chondrocyte phenotype, permitting long-term culture. Clones of tumor chondrocytes were established in agarose and were examined over 2-3 weeks for evidence that the cells were accumulating a proteoglycan-rich extracellular matrix, as determined by positive staining by Alcian blue, and were undergoing cell division. Nearly 90% of the cloned cells exhibited a prominent extracellular matrix by day 7 of culture and greater than 99% did so by day 14. Cell division did not occur to any great extent until days 6-7 of culture. After this lag, the cells appeared to undergo logarithmic growth, with a cell generation time of about 12 days. By 20 days of culture, between 80 and 90% of the initial clones contained multiple cells, indicating that nearly all the cells were in, or had entered, the cell cycle. These results suggest that the chondrocytes from the rat chondrosarcoma form a homogeneous cell population with respect to their ability to synthesize an extra-cellular matrix and divide.

The synthesis of hyaluronic acid by cultured chondrocytes from the Swarm rat chondrosarcoma was examined using [3H]glucosamine as a precursor. [3H]Hyaluronate in samples was estimated as the specific unsaturated disaccharide released by incubation with chondroitinase ABC under conditions where all [3H]hyaluronate was converted to disaccharide even in the presence of excess chondroitin sulfate. The products of digestion of chondroitin sulfate and hyaluronate were well separated by cellulose thin layer chromatography. A second method involving quantitation of the 3H-oligosaccharides released by digestion with Streptomyces hyaluronidase gave nearly identical results. [3H]Hyaluronate formed about 12% of the [3H]glycosaminoglycans synthesized by the chondrocytes, although levels as high as 20% were found in one set of cultures. The incorporation of [3H]glucosamine into chondroitin sulfate and hyaluronate was linear and at a constant ratio over a 24-h period. The distributions of [35S]proteoglycans, [3H]chondroitin sulfate, and [3H]hyaluronate between the medium, a 4.0 M guanidine HCl extract of the cell layer, and the cell residue, were investigated over 24 h. Both hyaluronate and proteoglycan accumulated in the medium with little retention in the cell layer. Pulse-chase experiments indicated that a greater proportion of the [3H]hyaluronate than the [3H]chondroitin sulfate was initially retained in the cells, but that subsequently [3H]hyaluronate accumulated more rapidly in the medium, suggesting differences in the transit time through the extracellular matrix for the two molecules once secreted from the cell. In cultures labeled for 6 h, 4.0 M guanidine HCl extracted 66% of the hyaluronate and 58% of the chondroitin sulfate associated with the cell layer, while 1% Zwittergent, a zwitterionic detergent, extracted 59% and 29%, respectively. The selective extraction with detergent suggests that there is a pool of hyaluronate in the cell layer which is not associated with proteoglycan aggregates. Equilibrium density gradient centrifugation of cell layer extracts and culture media, under dissociative conditions, showed that 89% of the [3H]hyaluronate banded at densities between 1.43-1.55 g/ml with a peak at 1.47 g/ml. Disaccharide analyses of the gradient fractions showed that the main proteoglycan component contained primarily chondroitin 4-sulfate but also revealed a minor proteoglycan at lower densities which was considerably enriched in chondroitin 6-sulfate.

After incubating cultured chondrocytes from the Swarm rat chondrosarcoma for 30 min with [3H]serine, a labeled macromolecule was found predominantly as Mr = approximately 370,000 species which was subsequently identified as a core protein precursor to cartilage proteoglycan from the following properties: (a) it was immunoprecipitated along with completed proteoglycan from cell extracts by an antiserum to the complex of hyaluronic acid-binding region, link protein, and hyaluronic acid. Its immunoprecipitation could be inhibited completely by the addition of purified hyaluronic acid-binding region to the extracts, indicating the presence of common antigenic determinants with this region of the proteoglycan core protein. (b) the core protein precursor was able to interact with the hyaluronic acid and link protein in proteoglycan aggregates added as carrier to extracts to form mixed aggregates of high buoyant density in associative CsCl density gradients. Labeled core protein precursor and link protein were subsequently isolated from the mixed aggregates from the top of dissociative CsCl density gradients. (c) radioactivity in core protein precursor after a 30-min pulse of [3H]serine disappeared after inhibiting further protein synthesis with cycloheximide concurrent with the appearance of label in completed proteoglycan molecules.

Tunicamycin, an inhibitor of dolichol-diphospho-N-acetylglucosamine formation and hence an inhibitor of N-linked oligosaccharide biosynthesis, suppressed total proteoglycan synthesis by Swarm rat chondrosarcoma chondrocytes without affecting the size of the proteoglycan molecule, its secretion from the cell, or its ability to be retained in the extracellular matrix. In addition, tunicamycin did not substantially alter the ability of the chondrocytes to polymerize glycosaminoglycan onto an exogenous beta-D-xyloside acceptor. A secondary effect of tunicamycin suppression of proteoglycan synthesis was that a lesser amount of newly synthesized proteoglycan diffused from the extracellular matrix into the culture medium. The ability of exogenous hyaluronic acid and proteoglycan to increase the percentage of newly synthesized 35S-proteoglycan in the medium in tunicamycin-treated cultures indicates that matrix retention of 35S-proteoglycan is related to the total extracellular uronic acid content rather than to the presence or absence of mannose oligosaccharides bound to the proteoglycan molecule. These noncytotoxic concentrations of tunicamycin (33-333 ng/ml) decreased [3H]mannose incorporation to the same extent that they decreased total [35S]sulfate and [3H]serine incorporation, and caused the chondrocyte to synthesize and secrete a species of beta-hexosaminidase that was mannose-deficient as assessed by its failure to bind to concanavalin A. The additional finding of decreased insulin binding to tunicamycin-treated chondrosarcoma chondrocytes suggested that the inhibition of proteoglycan synthesis was due to diminution of receptors which respond to stimulatory hormones.

Proteoglycan synthesis by cultured chondrocytes from the Swarm rat chondrosarcoma was examined after treatment with 0.1 mg/ml of cycloheximide which inhibited [3H]serine incorporation into total protein by greater than 90%. Incorporation of [35S]sulfate into proteoglycans decreased with nearly first order kinetics (t 1/2 = 96 +/- 6 min) with an accompanying increase in the size of the proteoglycan molecules, primary due to an increase in chondroitin sulfate chain sizes. After 5 h of cycloheximide treatment, when [35S]sulfate incorporation was inhibited by about 90%, addition of 1 mM beta-D-xyloside restored 76% of the incorporation into chondroitin sulfate observed in cultures treated only with xyloside. This suggests that the biochemical pathways for the affected by cycloheximide treatment. Cultures were prelabeled for 15 min with either [3H]serine or [35S]-methionine, and then cycloheximide was added to block further protein synthesis. Both precursors appeared in completed proteoglycan molecules with nearly first order kinetics with t 1/2 values of 92 +/- 8 and 101 +/- 11 min for [3H]serine and [35S]methionine, respectively, values in close agreement with the t 1/2 from the [35S]sulfate data. These results suggest that after cycloheximide treatment, the rate of [35S]sulfate incorporation into proteoglycan, after a correction for increases in chondroitin sulfate chain size, was directly proportional to the size of the intracellular pool of core protein. From the steady state rate of proteoglycan synthesis (estimated to be about 80 ng/min/10(6) cells in separate experiments) and a corrected t 1/2 value of 60 min, the amount of precursor core protein can be calculated to be about 500 ng/10(6) cells in these experiments.

The core protein of proteoglycans from cartilage is substituted with glycosaminoglycans as well as N- and O-glycosidically linked oligosaccharides. We have taken advantage of the long intracellular half-life of the core protein precursor to the rat chondrosarcoma proteoglycan to study the temporal relationship between the addition of the chondroitin sulfate chains and the O-linked oligosaccharides onto the core protein during the formation of the completed proteoglycan molecule. Chondrocyte cultures were pulsed on day 2 with [6-3H]glucosamine for times ranging from 30-420 min. Media and corresponding 4% zwittergent, 4 M guanidine HCl extracts were then pooled and subjected to dissociative density gradient ultracentrifugation to yield purified proteoglycan monomers which were then subjected to alkaline borohydride treatment. The released chondroitin sulfate chains were then purified by precipitation with 50% (v/v) ethanol. The O-linked oligosaccharide-alditols in the supernatant fractions were purified by molecular sieve chromatography on Bio-Gel P-6, and analyzed after digestion with alpha-neuraminidase and subsequent chromatography on Bio-Gel P-2. The different O-linked oligosaccharide-alditols were identified from their hexosamine and hexosaminitol contents. The kinetics of entry of 3H label into N-acetylgalactosamine of chondroitin sulfate was indistinguishable from that into either N-acetylglucosamine or N-acetylgalactosaminitol residues of the oligosaccharide-alditols, with half-times to linear incorporation of 10-17 min. These results show that initiation as well as completion of the O-linked oligosaccharides on the core protein occurs essentially at the same time that chondroitin sulfate chains are added. The results suggest that these biosynthetic processes occur in the Golgi apparatus during the last few minutes of the total intracellular dwell time (half-time of about 90 min) of the core protein acceptor.

When chondrocytes from the Swarm rat chondrosarcoma are isolated by trypsin and collagenase digestion and cultured in Petri dishes, they form a new extracellular matrix within 24 hours, with proteoglycan matrix granules and collagen fibrils. This rapid synthesis of new matrix, together with the biochemical and morphological characterization of the proteoglycans as typical of cartilage, demonstrates the value of these cultures as a model system for studies of synthesis, secretion, and organization of extracellular matrix.

Biosynthesis of cartilage proteoglycan was examined in a model system of cultured chondrocytes from a transplantable rat chondrosarcoma. Extensive modification with the addition of chondroitin sulfate glycosaminoglycan, N-linked oligosaccharide, and O-linked oligosaccharide is required to convert a newly synthesized core protein precursor into a proteoglycan. Kinetic analyses revealed the presence of a large pool of core protein precursor (t 1/2 approximately 90 min) awaiting completion into proteoglycan. The large t 1/2 of this pool allowed kinetic labeling experiments with a variety of radioactive precursors to distinguish between early biosynthetic events associated primarily with the rough endoplasmic reticulum from late events associated primarily with the Golgi apparatus. The results of a series of experiments indicated that the addition of N-linked oligosaccharide chains occurs early in the biosynthetic process in association with the rough endoplasmic reticulum, whereas the initiation and completion of O-linked oligosaccharides occurs much later, at about the same time as chondroitin sulfate synthesis. This also indicated that keratan sulfate chains, when present in the completed molecule, are added in the Golgi apparatus, as they are probably built on oligosaccharide primers closely related to the O-oligosaccharide chains. Furthermore, when 3H-glucose was used as the precursor, the entry of label into xylose, the linkage sugar between the core protein and the chondroitin sulfate chain, was found to occur within 5 min of the entry of label into galactose and galactosamine in the remainder of the chondroitin sulfate chain. This indicated that the initiation and completion of the chondroitin sulfate chain occurs late in the pathway probably entirely in the Golgi apparatus. Thus, proteoglycan synthesis can be described as occurring in two stages in this system, translation and N-glycosylation of a core protein precursor which has a long half-life in the rough endoplasmic reticulum, followed by extensive rapid modification in the Golgi complex in which the majority of glycosaminoglycan and oligosaccharide chains are added to the core protein precursor with subsequent rapid secretion into the extracellular matrix.

The population of proteoglycan monomers in aggregates was purified from chondrocyte cultures after labeling with either [3H]serine and [35S]methionine or [3H]serine and [35S]sulfate. Digestion of the monomers labeled with [3H]serine and [35S]methionine with trypsin indicated that serine was enriched (approximately 80%) in the chondroitin sulfate attachment region of the core protein, while methionine was enriched (approximately 60%) in the hyaluronic acid-binding region. Sepharose CL-2B chromatography and velocity gradients were used to isolate monomer subfractions which differed in molecular size. The 3H/35S ratios for the subfractions from monomers labeled with [3H]serine and [35S]methionine were nearly constant, indicating that the core protein lengths were constant regardless of the size of the monomer. Conversely, the 3H/35S ratios for subfractions of monomers labeled with [3H]serine and [35S]sulfate increased significantly with decreasing sizes. Chondroitin sulfate chains in subfractions were released by alkaline borohydride treatment. The mean molecular weights of the chondroitin sulfate chains decreased from approximately 18,500 in the largest subclass of monomers to approximately 12,500 in the smallest. [3H]Serine-labeled monomer subfractions were digested with papain to determine the proportion of serine residues substituted with chondroitin sulfate. The distribution of label was constant for greater than 90% of the monomers from the largest to the smallest. The results indicate that, for at least 90% of the newly synthesized monomers that are able to aggregate, variation in chondroitin sulfate chain size is the only contributing factor to polydispersity in their molecular size.

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.

Chondrocytes from the Swarm rat chondrosarcoma were pulse-labeled with [3H]serine for 30 min and chased, in the presence of cycloheximide, for times up to 300 min. The movement of newly synthesized core protein precursor of the proteoglycan through elements of the endoplasmic reticulum and Golgi complex was examined. Rough and smooth microsome fractions were obtained by centrifuging postmitochondrial supernatants from cell homogenates on discontinuous sucrose gradients. The core protein precursor was identified in subcellular fractions by (a) immunoprecipitation with an antiserum directed against the hyaluronate binding region of the core protein and the link protein and (b) its size on polyacrylamide gels. Labeled core protein precursor decreased from the microsomes with a t1/2 of 60 +/- 8 min, nearly the same as for the appearance of label in completed proteoglycan monomer (t1/2 = 58 +/- 13 min), consistent with a precursor-product relationship. After correcting for incomplete recovery of the core protein precursor in the microsomal fractions and for cross-contamination of the smooth microsomes by elements of rough endoplasmic reticulum, the redistribution of core protein precursor and completed proteoglycan in the intracellular compartments and of labeled extracellular proteoglycan were fit to a three-compartment model. A t1/2 of 98 +/- 7 min for the loss of core protein precursor from the rough microsomes and a t1/2 = 10 +/- 4 min for the completed proteoglycan in the intracellular compartment (Golgi and secretory vesicles) was obtained. The data indicate that at least 70% of the intracellular transit time for the core protein precursor is spent in the rough endoplasmic reticulum. The addition of glycosaminoglycan chains followed by secretion from the cell occurs relatively rapidly, occupying less than 30% of the total intracellular dwell time.

Preformed Cs2SO4 zonal gradients were used to purify aggregates from proteoglycan preparations derived from associative extracts of the Swarm rat chondrosarcoma. Zonal gradients were used in solvents with different concentrations of guanidine HCl and different solvent pH values to study the mechanisms for dissociating the aggregates. Aggregates are stable in concentrations of guanidine HCl up to 1.5 M at pH 6.6. At 2 M guanidine HCl, partial dissociation occurs over 20 h in which a link protein is completely dissociated for every monomer proteoglycan dissociated from the aggregate structure. This suggests that in this solvent disaggregation occurs concurrent with complete separation of link protein from monomer. At solvent pH 2.7 to 3.3 in ionic conditions which normally promote aggregation, dissociation occurs by a mechanism in which the link protein remains associated with monomer. Thus, link protein-monomer complexes dissociate as bimolecular units from hyaluronic acid; such complexes then exhibit physical properties indistinguishable from pure monomers. The link protein-monomer complexes reassociate with hyaluronic acid to form link-stabilized aggregates when the solvent pH is raised to pH 7, i.e. to associative conditions. The study provides additional evidence for the role that link protein-monomer interactions have in proteoglycan aggregate structures.

1. Primary cultures of chondrocytes from the Swarm rat chondrosarcoma were labelled with either [3H]glucosamine or [14C]glucosamine, and hyaluronate synthesized by the cells was isolated from the cell layer. Parallel cultures were labelled with either [3H]serine or [3H]lysine, and identical fractions were isolated from the cell layer. Some cultures were dual-labelled. 2. In cultures labelled with [3H]serine for between 30 min and 24 h and extracted with 4.0 M-guanidine, a procedure that solubilizes predominantly extracellular macromolecules, small amounts of [3H]serine-labelled molecules were found associated with the hyaluronate fraction purified from the extract by dissociative CsCl-density-gradient centrifugation and dissociative Sepharose CL-2B chromatography. About 75% of the [3H]serine-labelled molecules in the fraction were specifically associated with hyaluronate, since they could be removed by prior treatment with proteinase-free Streptomyces hyaluronidase. The association of the [3H]serine-labelled molecules with hyaluronate was non-covalent, since they could be separated from it by further centrifugation in CsCl density gradients containing 4 M-guanidinium chloride and a zwitterionic detergent. 3. In other experiments the cultures were extracted with a sequential zwitterionic-detergent/guanidinium chloride procedure that completely solubilized the cell layer and enabled fractions containing newly synthesized cell-associated hyaluronate to be isolated. Zwitterionic detergent was present throughout. No [3H]lysine was incorporated into these fractions, irrespective of whether the cultures were pulsed concurrently with [3H]lysine and [14C]glucosamine or sequentially with [3H]lysine to prelabel the protein pool (24 h) followed by [14C]-glucosamine to label hyaluronate (1 h). 4. The results show that newly synthesized hyaluronate is not associated with covalently bound protein, and suggest that chain synthesis is initiated by a mechanism other than on to a core protein. Small amounts of [3H]serine-labelled molecules are, however, non-covalently associated with extracellular hyaluronate. Their identity is at present unknown, but they are probably of low molecular weight.