Isolation and Characterization of Proteoglycans from the Swarm Rat Chondrosarcoma*

Proteoglycan monomer (Dl) and aggregate (Al) preparations were isolated from 4 M guanidinium chloride extracts of the Swarm rat chondrosarcoma. When EDTA, 6-aminohexanoic acid, and benzamidine were present in the solutions, the Dl preparation contained a single component (so = 23 S), and the Al preparation contained 30% monomer (s,, = 23 S) and 70% aggregate (so = 111 S). In the absence of EDTA, 6-aminohexanoic acid, and benzamidine, the Al preparations contained only small proteoglycan fragments, indicating that extensive enzymatic degradation had occurred. The composition of the proteoglycan monomer was different from that of proteoglycan monomer preparations from normal hyaline cartilages in that it did not contain keratan sulfate and chondroitin 6-sulfate; only chondroitin 4-sulfate was found. The Al preparation from the chondrosarcoma contained only one link protein, which was like the smaller (molecular weight of 40,000) of the two link proteins present in Al preparations from bovine nasal cartilage. When the Al preparation from the chondrosarcoma was treated with chondroitinase ABC and trypsin and the digest was chromatographed on Sepharose

Proteoglycan monomer (Dl) and aggregate (Al) preparations were isolated from 4 M guanidinium chloride extracts of the Swarm rat chondrosarcoma.
When EDTA, 6-aminohexanoic acid, and benzamidine were present in the solutions, the Dl preparation contained a single component (so = 23 S), and the Al preparation contained 30% monomer (s,, = 23 S) and 70% aggregate (so = 111 S). In the absence of EDTA, 6-aminohexanoic acid, and benzamidine, the Al preparations contained only small proteoglycan fragments, indicating that extensive enzymatic degradation had occurred. The composition of the proteoglycan monomer was different from that of proteoglycan monomer preparations from normal hyaline cartilages in that it did not contain keratan sulfate and chondroitin 6-sulfate; only chondroitin 4-sulfate was found. The Al preparation from the chondrosarcoma contained only one link protein, which was like the smaller (molecular weight of 40,000) of the two link proteins present in Al preparations from bovine nasal cartilage. When the Al preparation from the chondrosarcoma was treated with chondroitinase ABC and trypsin and the digest was chromatographed on Sepharose 2B, a complex was isolated which contained the link protein and the segments of the protein core from the hyaluronic acid-binding region of the proteoglycan molecules.
Proteoglycans are present in the extracellular matrix of normal hyaline cartilages primarily as aggregates (1, 2). Solvents with high concentrations of electrolytes, such as 4 M guanidinium chloride, effectively dissociate the aggregates, minimize noncovalent interactions, and allow the proteoglycan molecules to be extracted from the tissue (3-6). Dialysis to lower electrolyte concentrations allows the molecules to reaggregate, and subsequently density gradient methods can be used to purify both aggregate and monomer proteoglycan preparations (7-g). The chemical and physical characteristics of such preparations isolated from a variety of cartilages have been described (S-21). The monomer preparation from bovine nasal cartilage contains a polydisperse population of macromolecules with molecular weights ranging from less than 1 x lo6 to greater than 4 x 106. The average molecule contains about 100 chondroitin sulfate chains with average molecular weights of about 2 x 10' and about 50 keratan sulfate chains with molecular weights of 4 x lo3 to 6 x lo3 (10,11,(22)(23)(24). The polysaccharide chains are attached to a core protein which has an average molecular weight of about 2 x lo5 (25). Recent investigations in a number of laboratories have advanced our understanding of the molecular interactions involved in proteoglycan aggregation (26)(27)(28)(29)(30)(31). Those proteoglycans which aggregate interact specifically with hyaluronic acid through a portion of their core protein (26,28,29,32). A number of proteoglycan molecules can bind to a single strand of hyaluranic acid. Small molecular weight link proteins are required to stabilize such aggregate structures (8,27,29,31). A model for the structure and aggregation of proteoglycans from cartilages that is consistent with the available data has been summarized elsewhere (33). This report describes attempts to use the methodology developed with normal cartilages to isolate and purify proteoglycan fractions from a neoplastic tissue, the Swarm rat chondrosarcoma, a tumor first described by Choi et al. (34). A preliminary report of some aspects of this investigation has been presented (35). was free of necrosis was used in the experiments described herein. The tumors lose 93% of their weight when dried at loo", and proteoglycans, estimated on the basis of hexuronic acid content, account for about 27% of the dry weight. These values are similar to those reported by Choi et al. (34).
fore, these were the basic conditions for the extraction step in subsequent experiments.
The proportion of the extracting solvent to tissue was lower than that used for extracting normal cartilages because of the higher water content of the tumor. Further, aqueous extracts of the tumor degraded proteoglycans at pH 4, in a manner analogous to that described for cathepsins isolated from cartilages (46-50).
The presence of proteases in the extracts impaired the recovery of intact proteoglycan preparations. Therefore, the effects of several protease inhibitors were determined. Al preparations were isolated with the use of Solution A, Solution B, or Solution C as described in "Experimental Procedure." A tracing of a schlieren pattern observed in the ultracentrifuge for the Al preparation isolated with no inhibitors present (Solution A) is shown in Fig. 2~. The solute concentration was about 2 mg/ml. No aggregate component was observed, but several small proteoglycan fragments were found with sedimentation coefficients of 3.5, 7, 10.4, and 12.3 S. The inclusion of 6-aminohexanoic acid and EDTA (Solution B) in the isolation and purification steps yielded an Al preparation with significant amounts of aggregate. Integral G(s) curves (51) were constructed from ultracentrifugal analyses of the sample for solute concentrations between 2 and 0.5 mg/ml, Fig. 3 sample using Solution C, Curves C, through C, of Fig. 5.3 The G(s) curve at zero concentration for this preparation, which showed a single, symmetrical peak in the ultracentrifuge, Fig. 26, is shown in Fig. 5  Al preparation (see Fig. 3) is indicated by the dashed line in Fig. 5. The latter curve has a pronounced skewed distribution toward lower sedimentation coefficients, indicating that the monomer in this Al preparation contained a larger fraction of molecules with lower sedimentation coefficients than for the molecules in the Dl preparation.
When benzamidine, an inhibitor of trypsin-like activity (53), was used in addition to EDTA and 6-aminohexanoic acid, Solution C, proteolysis was inhibited more effectively. The G(s) curves, Fig. 6, and the chromatographic analysis, Fig. 4c, indicate that about 70% of the proteoglycan molecules in this Al preparation were present as aggregates. The value of s, for the aggregate component increased to 111 S. The G(s) curves for the monomer component yielded an extrapolated curve at zero concentration identical with that for the Dl preparation shown in Fig. 5. A tracing of the schlieren pattern for the Al preparation isolated with Solution C is shown in Fig. 2c. Centrifugal and Chromatographic Analyses of Bovine Nasal Cartilage AI-For comparison, experiments were done with bovine nasal cartilage. The Al preparation was isolated with the use of Solution B. Integral G(s) curves, Fig. 7, indicated that the monomer and aggregate components in this preparation had extrapolated weight average sedimentation coefficients of 27.7 and 126 S, respectively.
The centrifugal analyses also suggested that the aggregate fraction accounted for about 85% of the sample. The Sepharose 2B chromatogram, Fig. 4a, showed that about 84% of the sample was excluded.

DI-The
Dl core preparation showed a single peak in the ultracentrifuge, Fig. 2d, with a s, of 7.3 S. A symmetrical, included peak was eluted from Sepharose 2B and Sepharose 4B with K,, values of 0.74 and 0.60, respectively. These values are similar to those observed in comparable experiments with preparations from the proteoglycan monomer of bovine nasal septa (25, 29, 30).

Interaction of Chondrosarcoma
Proteoglycan with Hyaluronic Acid-One of the requisites for proteoglycan aggregation is the ability of proteoglycan monomers to interact with hyaluronic acid (26-31, 60). Chromatography of proteoglycans with and without hyaluronic acid on Sepharose 2B can be used to monitor this interaction (28). The elution profile for the chondrosarcoma Dl without added hyaluronic acid is shown in Fig. 8a; an included peak (K,, = 0.30) was found. The addition of 12 ~g of hyaluronic acid to an identical Dl sample resulted in a shift of material into the excluded volume of the column, Fig. 8b. The elution profiles of Dl admixed with different concentrations of hyaluronic acid showed that the percentage of Dl molecules excluded increased with hyaluronic acid concentration up to 20 wg of hyaluronic acid/3 mg of Dl, at which ratio 55% of the Dl molecules were excluded. Reduction and alkylation of the Dl preparation completely destroyed the ability of the proteoglycans to bind to hyaluronic acid. Previously it was shown that such treatment also prevents aggregation of bovine nasal proteoglycan preparations (3,8). Properties of Aggregate from Rat Chondrosarcoma-At least one of the two small molecular weight link proteins (61) present in Al-D4 preparations from bovine nasal cartilage is necessary for aggregation (27,29,31). Therefore, the Al-D4 fraction was isolated from the chondrosarcoma Al and compared with the same fraction isolated from bovine nasal cartilage. Samples were subjected to electrophoresis on 7% polyacrylamide gels in the presence of Na dodecyl-SO,.
Al-D4 from bovine nasal cartilage showed two predominant protein-staining bands, Fig. 9b, as previously observed (29, 60). Their molecular weights were about 45,000 and 40,000, as estimated from graphs of logarithm of molecular weight against mobility of proteins with known molecular weights on identical gels. In contrast, the chondrosarcoma Al-D4 fraction contained only a single major protein component, with an estimated molecular weight of 40,000, Fig. 9a were mixed, the protein in the chondrosarcoma Al-D4 underwent co-electrophoresis with the smaller of the two proteins in the nasal cartilage Al-D4, Fig. 9c.

Reaggregation
Experiments-Because of the difference in the Al-D4 fractions, attempts were made to determine if they could be interchanged in reaggregation experiments. Mixtures of the Al-D1 fractions with the Al-D2 through Al-D4 fractions were prepared and treated as described under "Experimental Procedure." The Al-D2 and Al-D3 fractions were included because it has been shown that these fractions contain the hyaluronic acid molecules required for the formation of aggregates with normal cartilages (28, 60). Fig. 2e shows a tracing of a schlieren pattern for a mixture in which chondrosarcoma Al-D2 through Al-D4 was added to bovine nasal Al-Dl, while Fig. 2f shows a tracing of the converse experiment, i.e. a mixture of bovine nasal Al-D2 through Al-D4 with chondrosarcoma Al-Dl. The mixture which contained only chondrosarcoma fractions gave a pattern identical with that in Fig. 2e, while the mixture which contained only bovine nasal fractions gave a pattern identical with that in Fig. 2f A brief treatment of bovine nasal Al with chondroitinase followed by digestion with trypsin yielded a large molecular weight complex that contained hyaluronic acid, the smaller link protein, and a large segment of protein, referred to as the hyaluronic acid-binding region, from the proteoglycan molecules (31). The chondrosarcoma Al fraction was treated in a similar manner. Chromatography of the chondroitinase ABCtrypsin incubation mixture on Sepharose 2B gave the pattern shown in Fig. 10a. The protein-hyaluronic acid complex eluted as a broad peak shortly after the excluded volume; it was isolated and subjected to electrophoresis on a Na dodecyl-SO, polyacrylamide gel, Fig and 65,000 would migrate. These proteins correspond to the hyaluronic acid-binding polypeptide fraction derived from the core protein of proteoglycan molecules in the analogous experiment with bovine nasal Al (31). However, in the latter case only one broad, polydisperse band, with an average molecular weight of approximately 90,000 was observed; the polydispersity was attributed to the presence of some keratan sulfate in the preparation (31). The fact that two closely spaced, narrow bands were observed in the experiment with the chondrosarcoma Al, which does not contain keratan sulfate, suggests that there may be two different, though related polypeptides in this fraction.
Alternatively, a peptide bond in the hyaluronic acid-binding region of the proteoglycan molecules may be partially resistant to treatment with trypsin, and limit digestion may not have been reached. It is of interest, however, that two proteins were present in small concentrations in the chondrosarcoma Al-D4 preparation which had mobilities similar to those for the two closely spaced bands in the chondroitinase-trypsin experiment, Fig. 9a. This is consistent with the suggestion by Heinegdrd and Hascall (33,62) that proteoglycans in Al preparations which are substituted with few or no polysaccharide chains contain little or no polypeptide beyond the hyaluronic acid-binding region polypeptide (see also the discussion by Rosenberg (63)). The ratio of the area under the link protein peak to that under the two hyaluronic acidbinding region polypeptides, Fig. 106, was about 1.3. If the amounts of dye bound per unit weight of protein were equiva-1 t 2 3 4 5 6 7 8 9 10 lent, these results would suggest that the original hyaluronic acid-protein complex contained two link proteins for each hyaluronic acid-binding region polypeptide. 4 In contrast, it was suggested that there was only one link a protein for each hyaluronic acid-binding region protein in Al preparations from bovine nasal cartilage (29).

DISCUSSION
In general, the data in this paper indicate that the chondrosarcoma contains proteoglycan aggregates with structures consistent with the emerging model for aggregates from normal cartilages (33). The Al preparation obtained when EDTA, 6-aminohexanoic acid, and benzamidine were used in the isolation steps represented about 90% of the total tissue hexuronic acid. It had properties very similar to those reported for bovine nasal and tracheal Al preparations (29) with respect to the following parameters: (a) monomer and aggregate size; (b) the presence of a large proportion of aggregate; (c) the ability of the monomer preparation to interact with hyaluronic acid; and (d) the presence of a hyaluronic acid-protein complex which resists digestion with chondroitinase and trypsin.

The chondrosarcoma
Al differs from Al preparations from 'For an equimolar complex the ratio of the areas of the peaks should be proportional to the ratio of the molecular weights, which in this case is 4 x lo':6 x 10' or 0.67. Since the actual ratio is 1.2, a complex of two link proteins and one hyaluronic acid-binding region polypeptide of the proteoglycan monomer (Dl) is suggested.
normal hyaline cartilages with respect to the following parameters: (a) the lack of keratan sulfate in the proteoglycan molecules; (b) the absence of chondroitin 6-sulfate; and (c) the presence of only one of the two link proteins. Further, two polypeptides were derived from the hyaluronic acid-binding region of the proteoglycans when chondrosarcoma Al was treated with chondroitinase and trypsin, while only one was observed in the comparable experiment with bovine nasal cartilage Al (31). The presence of keratan sulfate in the latter preparation, however, may introduce enough polydispersity such that two populations of polypeptides, if present, would not be observed. The absence of keratan sulfate and chondroitin-6-sulfate suggests that the chondrosarcoma proteoglycans are more nearly like those that are present in immature or embryonic cartilages (34,64).
Choi et al. (34) examined the characteristics of the polysaccharides isolated from the Swarm rat chondrosarcoma after papain digestion.
They found hyaluronic acid (1.2%), chondroitin 4-sulfate (98%), and a separate fraction which contained glucosamine but was not keratan sulfate. The presence of glucosamine, sialic acid, and alkali-labile galactosamine in the chondrosarcoma Dl preparation suggests that this latter fraction may well be an oligosaccharide fraction related to that normally involved in the attachment of keratan sulfate chains to the core protein.
The isolation of proteoglycans from the chondrosarcoma by procedures previously applied to hyaline cartilages yielded preparations with proteoglycans that were partially degraded. The neoplastic tissue or its vascular network (or both) contained enzymes, presumably proteases, which retained some activity even after exposure to 4 M GnHCl. Inclusion of the protease inhibitors 6-aminohexanoic acid, EDTA, and benzamidine hydrochloride in the solutions used for extraction and dialysis, and expeditious manipulation at low temperature, greatly reduced or completely abolished the effects of these endogenous enzymes. Because hyaline cartilages contain small amounts of proteases active at both acidic and neutral pH (46~50), it is advisable to utilize protease inhibitors and low temperatures in preparing the Al fractions of proteoglycans. For example, the use of 6-aminohexanoic acid and EDTA in the isolation of bovine nasal Al yielded a preparation in which about 85% of the material