Chondroitin SO, Catabolism in Chick Embryo

Vol. 254, No. 7, Issue of April Printed LIZ U.S.A.

Chondroitin

10, pp. 2316-2325,

1979

SO, Catabolism

in Chick

Embryo

Chondrocytes*

(Received for publication, March 3, 1978, and in revised form, September 29, 1978) Janet

H. Glaser

and H. Edward

Conrad

From

the Department

of Biochemistry,

University

of Illinois,

Urbana,

Illinois

61801

EXPERIMENTAL

Cultured chick embryo chondrocytes incorporate ““S04”- or labeled D-glucosamine or acetate supplied in the culture medium into chondroitin SO, at rates that are 1 to 2 orders of magnitude greater than those observed in cell cultures prepared from other embryonic tissues (1). During the labeling period, a portion of the newly synthesized chondroitin SO.$ is secreted into the culture medium while the remainder is recovered with the washed cells (2-4). When the culture medium is replaced with fresh medium, the labeled chondroitin SO, remaining with cells is not degraded (3,4). Similarly, the labeled chondroitin SO, secreted into the culture medium is not degraded when incubated further with the culture medium in the absence of cells (4). Thus, if these chondrocytes contain enzymes capable of degrading chondroitin S04, such enzymes do not appear to act in the cell cultures. Davidson and co-workers ($6) have shown that chondroitin SO, chains are degraded by a rat liver lysosomal hyaluronidase to form oligosaccharides, some of which can be attacked by a sulfatase to yield free Sod”-. These studies led to the suggestion that chondroitin SO., is degraded in rat liver by a reaction sequence involving an initial endoglycosidase cleavage of the polymer by hyaluronidase followed by the sequential action of ,&glucuronidase, sulfatase(s), and ,&N-acetylhexosaminidase, exoenzymes which attack from the nonreducing termi* This work was supported by Public Health Service Grant HD 8057 and by a fellowship award to J. H. G. by The Arthritis Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 IJ.S.C. Section 1734 solely to indicate this fact.

PROCEDURES

Analysis of Labeled Products-For analysis of labeled samples, aliquots of culture medium or enzyme incubation mixtures were spotted directly on strips (1 x 22 inches) of Whatman No. 3MM chromatography paper and the chromatograms were developed by descending chromatography in System 1 (l-butanol:glacial acetic acid: 1 M ammonia (2:3:3)) or System 2 (10) (l-butanol:glacial acetic acid:1 M ammonia (2:3:1)). For paper electrophoretic analysis, samples were spotted 3’% inches from one end of a strip (1 x 22 inches) of Whatman No. 3MM paper and the strips were electrophoresed toward the anode in pyridine:glacial acetic acid:water (1:5:400) at 30 V/cm for 2 h. After chromatographic or electrophoretic separations, strips were dried and cut into s-inch segments which were counted in a Beckman scintillation counter as previously described (11, 12). Gel filtration chromatography of the chondroitin SO1 chains and their digestion products was performed on columns of Sephadex G200 (1 X 115 cm) prepared in 1.0 M NaCl. Columns were eluted with 1.0 M NaCl and 1.5.ml fractions were collected. Aliquots (50 to 100 ~1) of each fraction were spotted on segments (% x 1 inch) of Whatman No. 3MM paper which were dried and counted in a scintillation counter as above. The compositions and degrees of polymerization of the substrates and digestion products were determined after first digesting the samples exhaustively with bacterial chondroitinase as follows. To a IOO-~1 aliquot containing 3 to 5 X 10” ,‘“SO, cpm (55 to 85 nmol of bound SO,, see below) of sample in water was added 0.1 unit of chondroitinase AC (Miles Laboratories) in 10 ~1 of 25 mM Tris/acetate buffer, pH 7.3 (enriched Tris buffer, see Ref. 10). After digestion for 2 h at 37”C, aliquots were analyzed for labeled mono- and disaccharides by radiochromatography (11) as described under “Results.” Preparation of Labeled Chondroitin Sod-Cartilage from the distal halves of 20 to 30 13-day-old chick embryo tibiotarsi was dissected free of adhering tissue and the intact cartilage pieces were sliced and placed in a IOO-ml plastic Falcon tissue culture dish and incubated with isotopic precursors for 24 h in 10 ml of Dulbecco’s modified Eagle’s medium containing 2 g of o-glucose/l liter and 10% fetal calf serum (DME(2) medium, see Ref. 3). Incubation media contained 50 pCi/ml of n-[“Hlgalactose (2.15 Ci/mmol), 20 pCi/ml of H$‘“S]SO,% (43 Ci/mg), or both 20 @/ml of the Hz[““S]SO, and 50 pCi/ml of sodium[,‘H]acetate (685 mCi/mmol). All isotopically labeled

2316

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nal. Although individual enzymes with appropriate substrate specificities for such a sequence have been demonstrated in several different tissues (see Ref. 7 for a review), the actual pathway of chondroitin SO4 breakdown has not been established for any single system, and Dorfman et al. (8) and Davidson (9) both have pointed out that the distribution of lysosomal hydrolyses may vary from tissue to tissue, resulting in different pathways of chondroitin SO, degradation in different tissues. The present study was undertaken to resolve the question of whether cultured chick embryo vertebral chondrocytes, shown in our earlier studies not to catabolize chondroitin SO, in cultures of intact cells, contained enzymes for chondroitin SO, breakdown and, if so, whether the degradation pathway is the same as that proposed for the process in rat liver. The data show that these chondrocytes contain the full complement of enzymes necessary for conversion of chondroitin SO, to oligosaccharides via endoglycosidase action and for conversion of oligosaccharides to monosaccharides and free Sod”- via exoenzyme attack.

An enzyme preparation from cultured chick embryo vertebral chondrocytes attacks chondroitin SO4 oligosaccharides from the nonreducing terminal in a recycling pathway involving the sequential action of a /?glucuronidase, a 4- or a 6-sulfatase, and a /3-N-acetylgalactosaminidase. The sequence is blocked by saccharo-1,4-lactone, an inhibitor of the P-glucuronidase, or by 2-acetamido-2-deoxy-n-galactonolactone, an inhibitor of the P-N-acetylgalactosaminidase. The level of 4-sulfatase activity is low relative to the other activities and limits the rate of catabolism of hybrid oligosaccharide structures containing both 6-sulfated galactosamine residues and 4-sulfated galactosamine residues. This results in the accumulation of shortened oligosaccharides, most of which have galactosamine-4SO1 residues at their nonreducing terminals. In the presence of the lactone inhibitors, polymeric chondroitin SO1 is broken down by the enzyme preparation to oligosaccharides which are 10 to 15 monosaccharides long, indicating that degradation of chondroitin SO1 chains is initiated by an endoglycosidase which generates oligosaccharide substrates for the recycling exoglycosidase system.

Chondroitin

SO4 Catabolism

I The abbreviations fied Eagle’s medium calf serum; ADi-4S,

used are: DME (2) medium, Dulbecco’s modicontaining 2 g of o-glucose/liter and 10% fetal 2-acetamido-2-deoxy-3-O-(,f-o-gluco-4-enepyra-

is 1.04 pmol/ml; therefore, the final specific activity of the ,‘“SOr’ in the medium was 19.2 $Zi/pmol. Under the standard counting conditions used in this paper, 10 nmol of ,‘“SOI”- gave 57,300 cpm. The SO, ester groups in the newly synthesized chondroitin SO, have the same specific activity as the ““SO,’ in the culture medium, i.e. 5730 cpm/ nmol of ester SO,. Thus, the concentrations of substrates and products in all assays can be estimated from their ‘“SO, counts per min. In contrast, the molar specific activities of the “H-labeled precursors supplied in the culture medium cannot be used directly to calculate molar amounts of product since the specific activities of the [“HIacetate or the [,‘H]galactose supplied in the medium are diluted by the normal unlabeled cellular metabolites. Enzyme Preparation-Single cell suspensions were prepared from ventral halves of lo-day-old chick embryo vertebral cartilage and cultured in DME(2) medium as described previously (3). Cells were plated at an initial density of 5.6 x 10’ cells/lOO-mm plastic Falcon tissue culture dish containing 12.5 ml of culture medium. Medium was replaced on Days 3 and 6. On Day 8, when the culture was just subconfluent (approximately 5 x 10” cells), the medium was replaced with fresh DME(2) medium lacking fetal calf serum and the culture was incubated 48 h in the serum-free medium. Growth in serum-free medium approximately doubles the specific activity of the lysosomal enzymes described here. The medium was then removed and the cells (5.9 x 10” cells) were washed once with 5 ml of Dulbecco’s Tris/saline scraped from the dish with the aid of a rubber policeman, and transferred to a dialysis sac. The cell suspension was dialyzed overnight at 4°C against 10 IIIM sodium acetate buffer, pH 4.8, and stored frozen in small aliquots which were thawed as needed. The protein concentration in such preparations, determined by the Lowry procedure (15), was 2 to 3 mg/ml. Enzyme Inhibitors-n-Saccharic acid-1,4-lactone was obtained from Sigma. 2-Acetamido-2-deoxy-I>-galactonolactone was synthesized by hypoiodite oxidation of N-acetyl-I)-galactosamine and purified as described by Schaffer and Isbell (16). The product was crystallized from ethanol and characterized by KBr infrared spectroscopy. Rates ofchondroitin SOI and Collagen Synthesis-Cells prepared from the ventral halves of lo-day-old chick embryo vertebral cartilage as above were plated at 2 X lo4 cells/5 ml of DME(2) medium in 60. mm culture dishes and grown at 38°C. Medium changes were made on Days 3 and 5. On Day 7, one dish of cells was taken for measurement of cell density and two dishes each were taken for duplicate assays of chondroitin SO, and collagen synthesis rates. Cell counts were made on isotonically trypsinized cells using a Coulter counter. Chondroitin SO, synthesis was measured by incubation of the cells for 10 h in 5 ml of fresh DME(2) medium containing 20 KCi of ‘“SO,’ / ml. At the end of the incubation period, the labeling medium was removed from the culture dish and the cells were removed in 0.5 ml of a solution of 0.25% crystalline trypsin in water. An aliquot of the lysed cell suspension was streaked at the origin of a strip (1 x 22 inches) of Whatman No. 3MM chromatography paper. A second strip was streaked with an aliquot of the labeling medium. The chromatograms were developed for 15 h in System 2 to move the free ““SOI’~ away from the origins. The origin segments were then cut out of the dried chromatograms and counted in a scintillation counter as described above as a measure of the amount of chondroitin SOi synthesized. More than 95% of the material recovered in the origin segments was digestible with chondroitinase ABC. Collagen synthesis was estimated by incubation of the cultures fol 24 h with 5 ml of fresh DME(2) medium containing 5 PCi of [‘HIproline (42 Ci/mmol, Schwarz/Mann). The level of fetal calf serum was reduced to 0.5% in the incubation medium and 100 pg/ml each of sodium ascorbate and ,&aminopropionitrile fumarate were added. After the incubation, the medium was removed, the cells were washed with 1 ml of Tris/saline, and the wash was added to the medium. The cells were removed from the culture dish with 2 ml of O.l’+’ aqueous sodium dodecyl sulfate, the dish was washed with 1 ml of water, and the wash was added to the lysed cell suspension. The medium and .__ nosyluronic acid)-4-O-sulfo-u-galactose; ADi-6S, 2.acetamido-2deoxy-3-O-(P-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-n-galactose; ADi-OS, 2-acetamido-2-deoxy-3-0-(P-~)-gluco-4-enel)yranosyluronic acid)-o-galactose; Di-4S, 2-acetamido-2-deoxy-3-0-(jl-r,-glucopyranosyluronic acid)-4-O-sulfo-D-galactose; Di-GS, 2.acetamido-2deoxy-3-0-(/?-D-glucopyranosyluronic acid)-6.0-sulfo-D-galactose; Di-OS, 2-acetamido-2-deoxy-3-O-(P-D-glucopyranosyluronic acid)-ogalactose; DP, degree of polymerization in average number of monosaccharides per oligo- or polysaccharide; BrdUrd, bromodeoxyuridine; GlcUA, glucuronic acid.


compounds were obtained from New England Nuclear. Incubations were carried out at 38°C in a humidified incubator gassed with 7% COZ, 93% air. At the end of the labeling period, the tissues were washed in Dulbecco’s, pH 7.4, Tris/saline (0.8% NaCl solution) with Ca*+ and Mg” and blotted to remove excess fluid. Substrate Preparation: Chondroitin SOI Polysaccharide-Chondroitin SO, chains were prepared as described by Amado et al. (13). 35S04-labeled cartilage from 36 embryos was homogenized in 2.5 ml of 0.1 M sodium acetate, pH 5.5, containing 10 mM EDTA and 10 mM cysteine. Papain (10 mg, twice crystallized, Sigma) was added in 2.5 ml of the same buffer and the mixture was incubated at 60°C for 24 h. The papain-digested material was dialyzed against 50 mM NaySO, overnight at 4°C and then against distilled water for 7 h at room temperature. After centrifugation for 10 min at 12,000 X g, the supernatant was made 0.3 M in NaCl and the polysaccharide was precipitated by addition of 10% cetylpyridinium chloride (Sigma) in 0.3 M NaCl. The precipitate was dissolved in a mixture of 2 M NaCl and absolute ethanol (100:15, v/v) and the polysaccharide was reprecipitated by addition of 2 volumes of absolute ethanol. The precipitate was dissolved in water to give a solution containing 3.2 x IO” cpm/ml (558 nmol of bound Sod/ml, see below) from which aliquots were withdrawn for enzyme assays. Substrate Preparation: Chondroitin SO, Oligosaccharides-Cartilage slices labeled with n-[“Hlgalactose, or with sodium [ ‘HIacetate and ““SO*’ , were homogenized in a minimum volume of water. The homogenate was streaked along the origins of several l-inch strips of Whatman No. 3MM paper and chromatographed overnight in chromatography System 1 to move the residual isotopic precursor away from the origin. The dried origin segments were cut out and incubated for 24 h in 2 ml of 25 IIIM Tris/acetate buffer, pH 7.3 (10) containing thermolysin (Sigma type X, 2 units added at 0 and 8 h). The segments were removed from the solution and spun dry using a spin thimble (Reeve Angel), and the entire eluate, which contained 80 to 90% of the radioactivity on the segment, was evaporated to dryness in vacua. Oligosaccharides were prepared by digestion of the dried aliquots for 24 h at 37°C with Sigma type V ovine testicular hyaluronidase (1 mg/ ml of 0.1 M sodium acetate buffer, pH 5.0, contaming 0.15 M NaCl). The digested sample was paper chromatographed for 18 h in System 2 to remove the salt and leave the labeled oligosaccharides in the first 2% inches of the chromatogram. The labeled oligosaccharides were eluted with water using spin thimbles and the eluatc was concentrated to dryness and further purified by electrophoresis on Whatman No. 3MM paper as described above. More than 90% of the labeled oligosaccharides electrophoresed toward the anode as a single symmetrical peak. This peak was eluted with water, concentrated in vacua to dryness, and taken up in water to give a solution from which aliquots were withdrawn for enzyme assays. The oligosaccharide samples prepared from cartilage labeled with both ‘“SOi” and [.‘H]acetate gave 4.2 x 10” ““SO.I cpm (733 nmol of bound SOJml; see below) and 1.5 x 10” .‘H cpm. The sample prepared from I)-r’H]galactose-labeled cartilage gave 5.3 X 10” cpm/ml. Oligosaccharide preparations resulting from the hyaluronidase digestion have only D-GlcUA residues at the nonreducing terminals (see Ref. 14, and Table I). To prepare a substrate having primarily GalNAc-SO, residues at the nonreducing terminals, a second hyaluronidase-generated oligosaccharide preparation (2.5 x 10” ,‘“SO, cpm) was incubated at 37°C for 5 h with 0.5 mg of bovine liver ,&glucuronidase (Sigma, type B-3) in 1.0 ml of 50 mM sodium acetate, pH 4.8, containing 5 mM Na&‘O,. The product was purified by paper electrophoresis (see above) and dissolved in water to give a solution containing 5.3 x 10” ““SO, cpm/ml (925 nmol of bound SO,/ml; see below) for use in sulfatase assays. Calculation of Substrate Concentration.?-If the molar specific activities of the labeled metabolic precursors of chondroitin SOI are known, the molar amounts of the chondroitin SO, oligosaccharides, or their digestion products, can be calculated from the counts per min determined under standard counting conditions. In the present study, the standard counting conditions were those in which labeled metabolic products are counted on segments (I/L x 1 inch) of paper chromatograms or paper electrophoretograms following the separation process. The only direct metabolic precursor of chondroitin SO,, for which the specific activity can be readily determined is the ““S04”supplied in the culture medium. It was shown earlier (3) that the concentration of unlabeled Sod’- m the DME(2) medium’ used here

2317

Chondroitin SO, Catabolism

2318

L

-I

was transfer,red to the dish containing the cells. The cell suspension was then stirred gently on a magnet stirrer for 20 h at 4°C to extract the chondromucoprotein, and the cell residue was pelleted at 12,000 x R. The supernatant was stored at 0°C prior to density gradient centrifugation. Sucrose gradients were prepared and run as described by Kimata et al. (22). A 0.5.ml cushion of 40% sucrose was layered on the bottom of each tube and the tubes were filled with 13 ml of a linear gradient

generated from 5 and 20% sucrose solutions prepared in the dissociative solvent above. To the top of each gradient was added 0.1 to 0.5 ml of the sample to be analyzed, and the sedimentation was run at 85,000 X g (26,000 rpm) for 26 h at 20°C in a Beckman SW-41 rotor. Tubes were then punctured at the bottom and 0.5.ml fractions were collected. An aliquot (0.1 ml) of each fraction was mixed with 1.5 ml of water and counted in a scintillation vial containing 15 ml of a xylene/Triton X114 scintillation fluid containing 3 g of diphenyloxazole/liter (23). RESULTS

Characterization of Standard Mono- and DisaccharidesIn the studies described below, a number of the products formed in the enzymatic breakdown of labeled chondroitin SO, are identified by their relative migration rates on paper chromatograms. Fig. 1 shows the chromatographic behaviors of standard mono- and disaccharides encountered in this work. The chromatographic migrations of the unsaturated disaccharides (ADi-6S and ADi-4S), the major products formed when polymeric chondroitin ‘?S04 is digested exhaustively with chondroitinase AC, have been noted in previous reports (3, 10, 12). Brief hydrolysis (0.1 N HzS04, lOO”C, 60 min) of these unsaturated disaccharides releases the corresponding sulfated forms of GalNAc (24) which serve as standards for their identification in Fig. 1. The saturated disaccharide standards, Di-6S and DidS, were prepared by treating a mixture of chondroitin SO, tetra- and hexasaccharides with chondroitinase AC. These saturated disaccharides are released from the nonreducing terminals of the oligosaccharides (10, 14). Fig. 1

shows that saturated disaccharides are readily separated from the unsaturated disaccharides. The saturated disaccharides were identified by their specific susceptibilities to desulfation by the bacterial chondro-6-sulfatase and chondro-4-sulfatase (10). Complete separation of Di-4S and GalNAc-6-SO4 requires elution of the combined peak and paper electrophoretic separation as described under “Experimental Procedures.” Degree of Polymerization of Oligo- and PolysaccharidesThe oligo- and polysaccharide substrates used here were uniformly labeled with “5S042m, [3H]acetate, and/or D-[“HIgalactose. Exhaustive digestion of these substrates with chondroitinase AC yields unsaturated disaccharides from all internal disaccharide units in the chain, saturated disaccharides from the nonreducing terminals of chains which terminate with D-&CurOniC acid, and GaINAc, GalNAc-4-S04, or GaINAc-6-SO4 from chains which have GalNAc residues at their nonreducing ends. Thus, in a sample labeled with a single isotopic precursor, the number of nonreducing terminals in the sample is represented by the sum of the counts per min in Di-GS, DG4S, Di-OS, GalNAc-4-S04, and GalNAc-6-SO+ The total number of monosaccharide residues in the sample is represented by twice the total counts per min in disaccharides plus the total counts per min in monosaccharides. Thus, the degree of polymerization (DP) is given by [ADi-6S

DP= Di-6S

cpm + ADi-4S cpm + AD&OS cpm + Di-6$ cpm + Di-4S cpm + Di-OS cpm] x 2 + GalNAc-6-SO., cpm + GAlNAc-4-SO4 cpm + GalNAc cpm + Di-4S cpm + Di-OS cpm + GalNAc-6-SO4 cpm + GalNAc-4-SOr cpm

cpm

+ GalNAc cpm This treatment of the data assumes uniform distribution of label in the substrate or product samples. In samples labeled with two isotopic precursors, e.g. [3H]acetate and 35S042-, the counts per min from only one of the labels is used in the calculation. In samples labeled with 35S042m alone, unsulfated products of chondroitinase digestion (ADi-OS, Di-OS, and GalNAc) cannot be measured. This may introduce a small error into the calculated DP since unsulfated GalNAc residues represent about 10% of the total GalNAc residues in the polymeric chondroitin SO4 (12,25). However, in the oligosaccharide substrates isolated for use in this study, no unsulfated mono- or disaccharides were found. Characterization of Oligosaccharide Substrate Used for Exoenzyme Studies-The studies on oligosaccharide diges-

0

4 8 12 16 20 24 28 Relative Distance along Chromatogram

32

FIG. 1. Chromatographic separation of standards. Descending chromatograms run on Whatman No. 3MM chromatography paper were developed with 1-butanol:glacial acetic acid:1 M ammonia (2:3:1) at room temperature. Development for 18 h separates GalNAc but 40- to 48-h development is required for disaccharide separation.


the cell suspension were heated at 100°C for 10 min to destroy collagenase and other proteases, and both samples were dialyzed against four changes of 1 mM EDTA solution. The volumes of the dialyzed samples were measured and IOO-yl aliquots were taken for scintillation counting. Radioautography of polyacrylamide electrophoretograms of thelabeled material(i7-20)shbwed that more than 80% of the nondialvzable l”Hlm-oline-labeled material was found in collagen chains or their procollagen precursors. In experiments in which chondroitin SO4 and collagen synthesis were assayed in serum-deprived cells, the DME(2) medium in which the cells had been growing was replaced on Day 7 with DME(2) medium from which fetal calf serum had been deleted. After 48 h, the serum-free medium was replaced with normal DME(2) medium and 10 h later the rates of chondroitin SO4 and collagen synthesis were determined as above. For measurement of polymer synthesis in bromodeoxyuridine-treated cells, the cells were initially plated in DME(2) medium as above. At Days 3 and 5, the medium was replaced with DME(2) medium containing 10 pg of BrdUrd/ml. At Day 7, chondroitin SO, and collagen synthesis were measured as above in fresh medium containing 10 pg of BrdUrd/ml. Sucrose Density Gradient Analysis of ““S-labeled Chondromucoproteins-Five dishes each of normal, serum-deprived, and BrdUrdtreated cells, grown as above, were labeled for 10 h in 2 ml of fresh DME(2) medium containing 400 PCi of ““SO,” ~/ml. The medium was removed and the cells were washed twice with 1 ml of phosphatebuffered saline. The washes were combined with the culture medium and the solution was dialyzed overnight at 4°C against 200 volumes of 50 mM Na2S04 containing 10 mM Na2EDTA, 100 ItIM 6-aminopropionic acid (Aldrich), and 5 IIIM benzamidine hydrochloride (Aldrich). The sample was then lyophilized and taken up in 2.5 ml of the dissociative solvent described by Oegema et al. (21). This solvent contained 4 M guanidine hydrochloride in 50 ITIM sodium acetate, pH 5.8, plus 10 mM Na2EDTA, 100 mM 6-aminopropionic acid, and 5 mM benzamidine hydrochloride. The solution was stored at 0°C prior to centrifugation. The washed cell monolayers were scraped from the dishes with a rubber policeman and transferred to a single 35.mm glass dish. Each dish was rinsed with 0.5 ml of the dissociative solvent and the wash

Chondroitin

SO4 Catabolism

of substrates

Characterization Oligosaccharide

analyzed

Total ““SO, on chromatogram

and products

Di-GS

I

ADi-GS 60.3

20

Segment

30

1

Number

FIG. 2. Paper electrophoretic separation of products formed by incubation of 3H/3”S04-labeled chondroitin SO4 oligosaccharide (Line 1, Table I) with vertebral chondrocyte extracts. Substrate (352,000 ““SO, cpm, 101,000 ‘H cpm) was incubated for 18 h at 37°C with 488 pg of enzyme protein in a final volume of 300 ~1 of 0.05 M sodium formate, pH 4.0, containing 0.05 M NaCl. The incubation mixture was analyzed by paper electrophoresis as described under “Experimental Procedures.”

of their chondroitinase digestion products”

Di-4S ADi-4S % total ~‘“SO4 cpm 15.2 16.4

49.4

0

10.7

4.4 4.9

14.8 11.4

as described

I c

10

34.2 34.9

under “Experimental Procedures” and the total counts per min in each chondroitinase product was measured after chromatography as shown in Fig. 1. Only %04 counts per min were used in these analyses.

I

H.=

TABLE I by analysis Chondroitinase

CPm

1. Exoenzyme sub89,000 8.2 strate 2. Peak II, Fig. 2 133,690 0 3. Sulfatase substrate 160,040 2.8 4. Sulfatase product, 160,690” 3.3 Fig. 7, 6 h n Oligosaccharides were digested with chondroitinase

ratio of 6-sulfated to 4-sulfated residues of 1 to 2. The only nonreducing terminals in the products were sulfated GalNAc residues, and, of these, 87% were sulfated in position 4. Paper chromatographic examination of Peak II showed that it contained a mixture of penta- and trisaccharides. Fig. 3 shows the time course of release of 3”S04’m and [“H]GalNAc under the incubation conditions described above. The data show that “5S042m and [“H]GalNAc are released in equimolar amounts throughout the incubation period. The initial rate of release of SOP- and GalNAc was 8 nmol of each/h/mg of protein. After 24 h, approximately 60% of the starting 3”S04 and 3H are recovered in these two products. The effects of pH and ionic strength on release of ““Sod2 and 3H are shown in Fig. 4. Maximum rates of release of both products are observed at pH 4.0 in 50 mM sodium formate buffer containing 50 mM NaCl in the incubation mixture. The


tion were carried out using as substrate an oligosaccharide preparation prepared by hyaluronidase digestion of a chondroitin SO, sample labeled with [“HIacetate in the GalNAc residues and with 3”S04. The composition of the oligosaccharide substrate, as determined from the products formed by chondroitinase digestion, is shown in Line 1 of Table I. Since no products containing 3H but not ““SO, were found in the chondroitinase products (i.e. no unsulfated mono- or disaccharides), only the data for the ““SO, counts per min are presented for characterization of the substrate. The average degree of polymerization of the substrate, calculated from these data, is 8.6 monosaccharides/chain. The ratio of 6- to 4sulfated GalNAc residues is 2.1:1. Since no sulfated monosaccharides were formed, the only nonreducing terminal residue in the sample was D-glUCUrOniC acid, consistent with the known specificity of testicular hyaluronidase used to prepare the oligosaccharide (26). Two-thirds of the nonreducing terminals were Di-4s; one-third were Di-6s. From the substrate characterization data in Table I, it appears that the substrate preparation contains hybrid structures with both 6- and 4-sulfated disaccharides in the same chain. This conclusion is indicated by the following. If the preparation were a mixture of oligosaccharides containing only B-sulfated disaccharides and oligosaccharides containing only 4sulfated disaccharides, the DP of the former would be 16.7 monosaccharides/chain while the DP of the latter would be 4.2 monosaccharides/chain. The extensive hyaluronidase digestion used for preparation of the substrate (see “Experimental Procedures”) would not be expected to give 4- and 6sulfated oligosaccharides of such different sizes, nor would fragments with DP values as high as 17 remain after the exhaustive digestion. The hybrid nature of the substrate is further indicated by an analysis of the products formed when it is incubated with chondrocyte extracts as described below. It is possible that the hybrid oligosaccharides are formed by transglycosidase action of the hyaluronidase (26) and are not a reflection of hybrid sequences in the chondroitin SO4 starting material. Enzymatic Cleavage of Chondroitin SO, Oligosaccharides-Incubation of the oligosaccharide substrate for 18 h at 37°C with an unfractionated extract of chick embryo chondrocytes at pH 4.0 released free ““SO,“- and [“H]GalNAc. Paper electrophoresis of the products, shown in Fig. 2, gave three peaks, only one of which (Peak II) migrated in approximately the same position as the starting material. Peak I, which contained 60% of the original “H but no ““Sod, was identified as [“H]GalNAc by its co-migration with a standard on a paper chromatogram in System 2. Peak III contained 62% of the original ““SO, but not 3H and was identified by its electrophoretic migration as inorganic ,?SOq2-. Analysis of Peak II (Line 2, Table I) showed an average DP of 4.0 and a

GalNAc-6-S

digestion

woducts

GalNAc-4-S

Aver%ge DP

C6S/C4S’

0

0

8.6

2.1

5.2 30.1 11.1

34.8 13.8 12.5

4.0 3.1 (3.1)

1.2 2.0 1.7

b Calculated as described under “Results.” ’ Ratio of total GalNAc-6-SO, residues in the oligosaccharide total GalNAc-4-SO4 residues. ’ Includes 35,400 cpm of free %O,‘-

(22% of the total).

to

Chondroitin

2320

SO4 Catabolism

pH optimum suggests a lysosomal origin for these activities. The enzyme system is 1.5 times more active in formate than in acetate buffer, presumably because of inhibition of the Nacetylhexosaminidase by acetate (27). Exo Activity of P-Glucuronidase-The parallel release of “SOq2- and [“H]GalNAc in all of the incubations described above suggests that the products are released from the oligosaccharide substrate by reactions which attack the substrate from the nonreducing terminal in an ordered and recycling sequence. Such a sequence would require a /?-glucuronidase, one or more sulfatases, and a ,8-N-acetylgalactosaminidase, with the rate of the reaction catalyzed by one of these enzymes limiting the overall rate of ““S04’- and [“H]GalNAc release. Since the only nonreducing terminal residues in the product

1

2

Hours

Hours

of Incubation

FIG. 3. Time course of inorganic ““SO,“(0) and free [ ‘H]GalNAc (0) release when “H/%0,-labeled chondroitin SO, oligosaccharide (Line 1, Table I) was incubated with vertebral chondrocyte extract. For each time point, an incubation mixture containing substrate (25,300 ““SO, cpm, 10,300 :‘H cpm) and 163 pg of enzyme protein in a final volume of 100 ~1 of 0.05 M sodium formate buffer, pH 4.0, containing 0.05 M NaCl was analyzed by paper electrophoresis as in Fig. 2.

I O2Q

I 30

I

I 40

PH

I

I 50

I 6.0

I 0

I 0.1

I 02

I 0.3

O

M NaCl

FIG. 4. Effect of pH and ionic strength on rate of release of “‘SO,‘(Cl, W) and ]‘H]GalNAc (0, 0) from “H/““SOJabeled chondroitin SO, oligosaccharide (Line 1, Table I). Each incubation was carried out for 6% h at 37°C in a mixture containing substrate (42,000 %O, cpm, 15,000 “H cpm) and 163 pg of enzyme protein in a final volume of 100 ~1 of 0.05 M buffer. a, effect of pH. Products were assayed after incubation in sodium formate (open symbols) or sodium acetate (closed symbols) buffer without addition of NaCl. b, effect of ionic strength. Products were assayed after incubation in sodium formate buffer, pH 4.0, with varying levels of NaCl.

3

4

5

6

7

of Incubation

FIG. 5. Release of ‘“SOr2~ (0, W) and [“H]GalNAc (0,O) from “H/ ““SO4-labeled chondroitin SO, oligosaccharide (Table I) at 37’C in the absence (open symbols) and presence (closed symbols) of lactone inhibitors. a, inhibition by saccharo-1,4-lactone. For each time point, the incubation mixture contained substrate (30,000 ““SO4 cpm, 16,800 “H cpm), 0.2 mM lactone, and 163 pg of enzyme protein in 100 ~1 of 0.05 M sodium formate, pH 4.0, containing 0.05 M NaCl. b, inhibition by 2-acetamido-2-deoxy-n-galactonolactone. For each time point, the incubation mixture contained substrate (80,000 ““SO4 cpm, 29,300 “H cpm), 0.4 mM lactone, and 105 pg of enzyme of protein in 100 ~1 of 0.05 M sodium formate, pH 4.0, containing 0.05 M NaCl.

are GalNAc-4-SO4 and GalNAc-6-SO., (Line 2, Table I), it appears that the removal of SOb2- must precede GalNAc release and that the rate-limiting reactions are catalyzed by the sulfatases. If such a recycling sequence operates, inhibitors which block the action of one of the proposed enzymes would prevent release of all subsequent products normally generated in the sequence. This hypothesis was tested by examining the effects of saccharo-1,4-lactone and 2-acetamido-2-deoxy-n-galactonolactone, inhibitors of P-glucuronidase (28) and P-Nacetylgalactosaminidase (29), respectively, on oligosaccharide degradation. Since the substrate has n-glucuronic acid at its nonreducing terminal (Line 1, Table I), the initial step in the cycle would be blocked by the saccharolactone and release of both “!S04”- and [“H]GalNAc would be prevented. The 2acetamido-2-deoxy-n-galactonolactone, on the other hand, would permit release of the nonreducing terminal D-ghCurOnk acid and only 1 mol of ““S042m/mol of oligosaccharide. The effect of 0.2 mM 1,4-saccharolactone on product formation is shown in Fig. 5a. In the absence of inhibitor, the initial rates of appearance of both ““SO,‘- and [“H]GalNAc were rapid. The inhibitor reduced both of these initial rates to 10 to 15% of the uninhibited rates. Furthermore, no disaccharides were released from the substrate when the inhibitor was present. These data indicate ,&glucuronidase must act on the substrate before the sulfatase or the galactosaminidase can act. A direct demonstration of the ,&glucuronidase action is shown in Fig. 6. For this experiment, a second oligosaccharide substrate prepared from chondroitin SO, labeled by addition of n-[3H]galactose to cartilage tissue cultures were used. This isotopic precursor gives relatively specific labeling in the GlcUA residues of the polymer (30). The oligosaccharide substrate used here was labeled both in the GlcUA residues


‘0

Chondroitin

SO, Catabolism

(75% of total “H in substrate) and the GalNAc residues. The data presented in Fig. 6 show that free GlcUA is released when this substrate is incubated with the chondrocyte enzyme and that the saccharolactone inhibits GlcUA release. Since these assays were run in the presence of 2-acetamido-2-deoxyD-galactonolactone, release of internal GlcUA residues was blocked. Exo Activity of Galactosaminidase and Sulfatases-The effect of 0.4 mM 2-acetamido-2-deoxy-D-galactonolactone on the catabolism of the “H/“5S04-labeled oligosaccharide is shown in Fig. 56. In this case, the initial rate of [“H]GalNAc release is reduced in the presence of the lactone to approximately 10% of the control rate. Initially, the rate of ““SO4’release is only slightly decreased, but after the first 3 h of the incubation in the presence of the inhibitor, the rate of ““Sod”release is sharply reduced. According to the hypothesis that the exoenzyme attack on these oligosaccharides must release Sob”- before GalNAc can be released, one would expect that,

8T 2 x

1

in the presence of the 2-acetamido-2-deoxy-n-galactonolactone, the enzyme would release 1 mol of ““S04”-/mol of substrate before further SO,“- release is blocked. The data in Fig. 56 show that SO.,- release has slowed well before 1 mol of sod2- is obtained. A further examination of this stoichiometry showed that the failure to obtain 1 mol of S04”-/mol of substrate is due to the extremely slow release of Sod”- from the 4-sulfated GalNAc residues. Low 4-sulfatase activity, implied by the high percentage of nonreducing terminal GalNAc4-SO4 residues in the products from the uninhibited reaction (Line 2, Table I), would explain the low yield of free SO*“- in the inhibited reaction since 65% of the nonreducing terminals on the substrate are Di-4S disaccharides (Line 1, Table I). A direct comparison of the relative rates of removal of 6- and 4SO, groups from GalNAc terminals was made using an oligosaccharide substrate prepared by isolation of a second hyaluronidase-generated chondroitin SO, oligosaccharide and treating it with bovine liver P-glucuronidase to expose the sulfated GalNAc residues at the nonreducing terminals of the oligosaccharide preparation. The composition of the substrate, as determined from the products of chondroitinase digestion, is shown in Line 3 of Table I. Fifty-nine per cent of the nonreducing terminals were GalNAc-6-SO4 residues; 27% were GalNAc-4-SO4 residues. When this substrate was incubated with enzyme in the presence of 0.5 mM 2-acetamido-2-deoxyD-galaCtOnOkiCtOne, there was a rapid release of free Sod’from the nonreducing terminals. The desulfated oligosaccharide product was analyzed for its chondroitinase AC digestion products at intervals during the incubation. These data, presented in Fig. 7, showed that the free “5SOs2m was released primarily from the GalNAc-6-SO4 nonreducing terminals while the GalNAc-4-SO4 nonreducing terminals were desulfated very slowly. The analysis of the product remaining after a 6-h digestion period is shown in Line 4 of Table I. As expected, the yield of unsaturated disaccharides formed by

I

r-

I

,

I

I

I

I

50
-

sidered, since serum hyaluronidase has been reported (31). Direct assay of the fetal calf serum using the pH 4 assay conditions described above showed a very low level of hyalu-

ofproducts

I

I-

(Line 3, Table II) and from Fig. 8c (Line 4, Table II). In each case, the DP of the starting material was decreased while the overall ratio of g-sulfated to 4-sulfated GalNAc residues was unchanged. These data indicate that the polymer chains were cleaved by endoglycosidase activity in the cell extracts. The possibility that the endoglycosidase activity found in these cell extracts may have come from the serum in the culture medium in which the cells were grown must be con-

Characterization

I

,-

The data presented in Fig. 8, b and c, show that both of the lactone inhibitors prevent the release of inorganic ““S04’-. However, the retardation of the radioactive peak indicates that

I

i-

or 2-acetamido-2-deoxy-D-galactonolactone.

firmed by the analyses of the combined

I

a

with enzyme as described oligosaccharide. The latter described in the text. I.

3.1 2.8 10.0

5.5

1.8

1.1

32.8 6.7 10.6

3.2

15.5

1.7

1.1

1.8

in Fig. 8 and 10% of each assay volume was electrophoresed on paper to were eluted from the paper strip and digested with chondroitinase AC. The


chondroitinase action on the internal residues of the products did not change during the incubation period since the 2acetamido-2-deoxy-n-galactonolactone prevented action of the N-acetyl-n-galactosaminidase. These data show that the sulfatases act directly on the nonreducing terminal GalNAcSO, residues and that the 4-sulfatase activity in the extracts is much lower than the 6-sulfatase activity under these assay conditions. Degradation of Polymeric Chondroitin Sod--To determine whether the chondrocyte enzyme preparation could act on chondroitin SO, polymers, monomeric chondroitin SO4 chains, prepared by papain digestion of chondroitin 3”S04, were used as substrate. As shown in Table II (Line l), this substrate had an average DP of 33 monosaccharides/chain and a ratio of 6sulfated to 4-sulfated GalNAc residues of 1.8. Incubation of this substrate with the chondrocyte extract under the same conditions found to be optimal for oligosaccharide digestion (see Fig. 4) resulted in extensive degradation of the polysaccharide with release of 53% of the labeled SO4 as inorganic ““S04’- in an 18-h incubation period. Sephadex G-200 chromatography of the digestion mixture, shown in Fig. Ba, gave a large peak of radioactivity emerging in the fully included volume which was identified by paper electrophoresis as free ““S04”-, and a very broad peak of retarded oligosaccharides. Analysis of the combined oligosaccharide fraction (Line 2, Table II) showed an average DP of 6.7. More than 70% of the nonreducing terminals were 4-sulfated mono- or disaccharides and the ratio of 6- to 4-sulfated GalNAc residues in the product was lowered to 1.1, indicating once again that more free ‘5S042- was released from the 6-sulfated GalNAc residues than from the 4-sulfated residues. To determine whether the observed degradation was due solely to the sequential action of the exoenzymes, shown above to act on the oligosaccharide substrates, the incubation was carried out in the presence of

Chondroitin

SO4 Catabolism

calf serum, which was heated at 60°C for 1 h, conditions shown previously to inactivate a number of lysosomalenzymes (32), lost all hyaluronidase activity but retained its capacity to support chondrocyte growth. Enzyme preparations from cells grown in medium containing the heat-inactivated fetal calf serum were shown to have the same specific activities for the endoglycosidase, the /3-glucuronidase, and the overall chondroitin SO, depolymerizing activity as enzyme prepared from cells grown in the presence of normal fetal calf serum. Thus, the endoglycosidase activity measured here must be derived from the chondrocytes. Characterization

of

Cells

Used

for

Enzyme

Source

as

* Very few authors have converted data for incorporation of isotopic precursors, expressed in counts per min of product, into absolute rates. In those cases where sufficient information is given for estimation of the absolute rates from the counts per min data (3,4,45-53), the values reported for chondroitin SO, synthesis by chondrocytes range from 0.02 to 2.9 nmol of Sod*- incorporated/h/IO6 cells, with the most common values being approximately 1 to 2 nmol/h/106 cells.

of chondroitin deprived,

TABLE III SO, and collagen synthesis by normal, and BrdUrd-treated chondrocytes

See “Experimental Procedures” activitv measurements.

for details of cell growth and

Cell density”

Cells

cells/dish

serum-

Chondroitin SO4 synthesis

x 10m” nmol

SO,/h/lO”

Collagen synthesis cells

cpn/h/lO”

cells

2.2 3.1 2200 Normal Serum-deprived 3.0 2.5 2090 BrdUrd-treated 1.3 0.25 1800 “Cell density at time when biosynthetic activity measurements were initiated.

s 3

41’A

* ‘2x

2

2 1

w

Ir2

0.3

1.2

g a

0.2

0.8

p

0.1

0.4

3 s

6

FIG. 9. Sucrose density gradient analysis of chondromucoproteins prepared from normal (Panels A and B), BrdUrd-treated (Panels C and D), and serum-deprived (Panels E and fl chondrocytes. In each pair the left panel shows the profile of the chondromucoprotein prepared from the cells while the rightpanel shows the profile of the material

recovered

in the culture

medium.

the labeling period was secreted into the culture medium by both the serum-grown and the serum-deprived cells. The BrdUrd-treated cells secreted 70% of their newly synthesized chondroitin SO, into the medium. Finally, the morphological appearance of the serum-grown and the serum-deprived chondrocytes were identical. The serum-deprived cells retained their polygonal shape and accumulated large quantities of extracellular matrix. The BrdUrd-treated cultures contained large, flattened, irregularly shaped cells identical with those seen in cultures of dedifferentiated chondrocytes (41). DISCUSSION

It has been proposed that the complete catabolism of chondroitin SO, in animal tissues involves the initial action of a hyaluronidase followed by a sequential exoenzyme attack by a ,&glucuronidase, a 6- or a 4sulfatase, and a /3-N-acetylgalactosaminidase (8). Each of these types of enzymes has been extensively characterized in terms of their activities on artificial substrates. However, the activities of these catabolic enzymes has been measured on chondroitin SO4 substrates in only a few instances and in quite different tissues. Buddecke and Hoefele (54) reported that a ,&glucuronidase purified


Chondrocytes-The enzyme system in the present study was prepared from monolayer cultures of chondrocytes which were cultured in serum-free DME(2) medium for 48 h prior to harvest. Consequently, it was necessary to demonstrate that these cells, after temporary deprivation of serum, retained the chondrocyte phenotype (33) which is characterized by (a) high rates of chondroitin SO, and type II collagen synthesis, (b) the capacity to synthesize the high molecular weight chondromucoprotein found in definitive chondrocytes (34,35), and (c) a typical polygonal cell morphology. Loss of phenotype has been observed when monolayers of chondrocytes are subcultured (4, 36) or treated with bromodeoxyuridine or chick embryo extract (35, 37-41). The loss of phenotype in cultured chondrocytes is not readily reversible (42). In order to determine the phenotypic behavior of the serumdeprived cells, the activities of these cells were compared with those of the chondrocytes grown continuously in the presence of 10% fetal calf serum. The data in Table III compare the rates of chondroitin SO, and collagen synthesis by normal and serum-deprived cells with those of BrdUrd-treated cells. The serum-deprived cells synthesized chondroitin SO4 and collagen at rates greater than 80 and 95%, respectively, of the rates found for the serum-grown cells. Slab gel electrophoresis (43, 44) gave identical banding patterns for the collagens synthesized by normal and serum-deprived cells with only traces of a band in the position of the a2 chain (44). Although serum depletion for 48 h results in some decrease in the rate of chondroitin SO4 synthesis, these cells still exhibit activity greater than most of the activities reported previously for monolayer chondrocyte cultures.2 By comparison, the BrdUrd-treated cells synthesized chondroitin SO1 and collagen at 8 and 82%, respectively, of the rates observed in the normal chondrocytes. The synthesis of the high molecular weight chondromucoprotein that is typical of definitive chondrocytes is shown in Fig. 9. Panels A and B show that most of the chondromucoprotein recovered from both the culture medium and the washed monolayer of serum-grown cells sediments rapidly in sucrose gradients as observed earlier for the product synthesized by chondrocytes (34). In contrast, the major peak of labeled chondroitin SO, recovered from BrdUrd-treated cells sediments much more slowly (Panels C and D) as reported for the products from non-chondrogenic cells and for BrdUrdsuppressed chondrocytes (34). Panels E and F show that the sedimentation behavior of the products synthesized by the serum-deprived cells is similar to that shown in Panels A and B for the products obtained from the definitive chondrocytes. Approximately 45% of the chondroitin SO, synthesized during

Rates

2323

2324

Chondroitin

SO, Catabolism incorporated/h/lo” cells. Since 10” chondrocytes contain approximately 0.2 mg of protein (4), the biosynthetic rate is 15.5 nmol of S04”-/h/mg of protein. Thus, the capacity for chondroitin SO, catabolism by cell extracts is approximately half of the capacity for chondroitin SO, synthesis by the monolayer cultures. In spite of this high catabolic capacity, very little chondroitin SO, breakdown is observed when prelabeled cells are cultured in fresh medium (3, 4). Hyaluronic acid catabolism by lysosomal hyaluronidase (5, 67-69), P-glucuronidase, and fi-N-acetyl-D-glucosaminidase (58, 59) has also been described in previous work, but once again, there is no single case in which all three of these activities have been measured in a single tissue using hyaluronic acid substrates. It is generally assumed that the lysosoma1 enzymes which act on hyaluronic acid substrates are the same enzymes that participate in chondroitin SO, catabolism, but this has not been rigorously established. If this assumption is correct, there remains the question of whether the cell can regulate the separate catabolism of the two polymers. Achnowledgments-The excellent technical assistance of WenNan Wang is aratefullv acknowledged. We also wish to thank Warren Knudson for- the sucrose gradient centrifugation runs and Tom Schmid for performing the gel electrophoresis on the collagen samples. REFERENCES 1. Dorfman, A. (1972) in The Comparative Molecular Biology of Extracellular Matrices (Slavkin, H. C., ed) pp. 27-28, Academic Press, New York 2. Schwartz, N. B., and Dorfman, A. (1975) Connect. Tissue Res. 3, 115-122 3. Kim, J. J., and Conrad, H. E. (1976) J. Biol. Chem. 251, 62106217 4. Kim, J. J., and Conrad, H. E. (1977) J. Biol. Chem. 252, 82928299 5. Aronson, N. N., Jr., and Davidson, E. A. (1967) J. Biol. Chem. 242,441-444 6. Tudball, M., and Davidson, E. A. (1969) Biochim. Biophys. Actu 171, 113-120 7. Buddecke, E., and Kresse, H. (1974) in Connective Tissues (Fricke, R., and Hartmann, F., eds) pp. 131-145, SpringerVerlag, New York 8. Dorfman, A., Matalon, R., Cifonelli, J. A., Thompson, J., and Dawson, G. (1972) in Sphingolipids, Sphingolipidoses, and Allied Disorders (Volk, B. W., and Aronson, S. M., eds) pp. 195-210, Plenum Press, New York 9. Davidson, E. A. (1970) in Metabolic Conjugation and Metabolic Hydrolysis (Fishman, W. H., ed) Vol. 2, pp. 327-353, Academic Press, New York 10. Yamagata, Y., Saito, H., Habuchi, O., and Suzuki, S. (1968) J. Biol. Chem. 243, 1523-1535 11. Conrad, H. E., Varboncoeur, E., and James, M. E. (1973) Anal. Biochem. 51,486-500 12. Kim, J. J., and Conrad, H. E. (1974) J. Biol. Chem. 249, 30913097 13. Amado, R., Ingmar, B., Lindahl, U., and Wasteson, A. (1974) FEBS Lett. 39,49-52 14. Linker, A., Meyer, K., and Weissmann, B. (1955) J. Biol. Chem. 213, 237-248 15. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 16. Schaffer, R., and Isbell, H. S. (1963) Methods Curbohydr. Chem. 2,11-12 LJ. K. (1970) Nature (Land.) 227, 680-685 17. Laemmli, 18. Bonner, W. M., and Laskey, R. A. (1974) Eur. J. Biochem. 46, 83-88 19. Laskey, R. A., and Mills, A. D. (1975) Eur. J. Biochem. 56, 335341 20 Burke, J. M., Balian, G., Ross, R., and Bornstein, P. (1977) Biochemistry 16, 3243-3249 21. Oegema, T. R., Jr., Hascall, V. C., and Dziewiatkowski, D. D. (1975) J. Biol. Chem. 250, 6151-6159 22. Kimata, K., Okayama, M., Oohira, A., and Suzuki, S. (1974) J. Biol. Chem. 249, 1646-1653


from bovine aorta would release free GlcUA from a chondroitin-4-SO4 tetrasaccharide. P-N-Acetylgalactosaminidase action on a chondroitin SO, substrate has been reported by Thompson et al. (55), who showed that an unfractionated extract of human skin fibroblasts would release GalNAc from a chondroitin-4-SO4 heptasaccharide. Sulfatases from a chick embryo lysosomal fraction (13), a rat liver lysosomal fraction (6), and an unfractionated human skin fibroblast extract (56, 57) have been shown to release Sod’- from chondroitin SO, oligosaccharides. The proposed pathways for chondroitin SO, catabolism (7-9) have been based largely upon these few reports and upon studies of the actions of hyaluronidase, pglucuronidase, and /?-N-acetylglucosaminidase on hyaluronic acid (14, 26, 58, 59) and hyaluronodextrins, substrates which are structurally similar to the chondroitin SO, substrates. It may be noted, however, that there are no previous literature reports in which all of the activities required for chondroitin SO, catabolism have been demonstrated in a single enzyme source. Furthermore, while the release of free GlcUA, GalNAc, and SOS” were demonstrated in the assays with chondroitin SO, substrates referenced above, the other products formed in each of the incubations were not characterized. Consequently, the proposed pathway has remained hypothetical. In fact, while lysosomal hyaluronidase action on chondroitin SO, has been observed in a variety of tissues (5, 13, 26, 60-64), it is not ubiquitous (65), and Dorfman et al. (8) and Davidson (9) both point out that the distribution of all these lysosomal hydrolases may vary from one tissue to another, resulting in different pathways of degradation in different tissues. For example, Arbogast et al. (65) suggest that human skin fibroblasts, which lack hyaluronidase, degrade chondroitin SO, solely by exoenzyme attack. In the present report, the individual steps in the release of free GlcUA, SO1’-, and GalNAc from chondroitin SO, substrates are demonstrated in extracts of chick embryo chondrocytes, and the obligatory sequence of exoenzyme attack is shown. In the absence of lactone inhibitors, multiple residues of free GalNAc and SOd2- are released per mol of oligosaccharide substrate, and the shortened oligosaccharide products have sulfated GalNAc residues at their nonreducing terminals. In spite of the fact that the sulfatase activity seems to limit the overall rate of free SO,“- release, no free GalNAc-SO4 is found among the reaction products. This suggests that the sulfatase must act before the N-acetyl-D-galactosaminyl bond is enzymatically cleaved. The data obtained with the lactone inhibitors show the further features of the obligatory sequence of the exoenzyme attack on chondroitin SO, oligosaccharides. Saccharo-1,4-lactone, which blocks release of free GlcUA (Fig. 6), also inhibits Sob’- and GalNAc release (Fig. 5). Thus, ,& glucuronidase action must precede the sulfatase and galactosaminidase action. The 2-acetamido-2-deoxy-n-galactonolactone, on the other hand, inhibits GalNAc release but not the release of the nonreducing terminal GlcUA (Fig. 6) or SO,“(Fig. 7). In the presence of either lactone, polymeric chondroitin SO, is broken down into oligosaccharides but only limited exoenzyme action can occur. These data confirm the sequence of exoenzyme action on chondroitin SO, previously proposed. Evidence consistent with some of the present findings has been described in a note by Wasteson et al. (66). The previous studies have measured products formed by exoenzyme action, but the assays used were not appropriate for measuring rates of chondroitin SO, catabolism. In the present study, the initial rates of S04’- and GalNAc release were found to be approximately 8 nmol/h/mg of protein. It is interesting to compare this rate with the rate at which monoSO,. In this layer cultures incorporate ““SO4 ‘- into chondroitin study (Table III), the biosynthetic rate was 3.1 nmol of SO,“-

Chondroitin

46. Nevo, Z., and Dorfman, A. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 2069-2072 47. Schwartz, N. B., Galligani, L., Ho, P.-L., and Dorfman, A. (1974) Proc. Natl. Acad. Sci. U. S. A. 71,4047-4051 48. Schwartz, N. B., and Dorfman, A. (1975) Connect. Tissue Res. 3, 115-122 49. Schwartz, N. B., Ho, P.-L., and Dorfman, A. (1976) Biochem. Biophys. Res. Commun. 71,851-856 50. Schwartz, N. B. (1977) J. Biol. Chem. 252, 6316-6321 51. Palmoski, M. J., and Goetinck, P. J. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 3385-3388 52. Huang, D. (1974) J. Cell Biol. 62, 881-886 53. Muto, M., Yoshimura, M., Okayama, M., and Kaji, A. (1977) Proc. Natl. Acad. Sci. U. S. A. 74,4173-4177 54. Buddecke, E., and Hoefele, 0. (1966) Hoppe-Seyler’s Z. Physiol. Chem. 347, 173-191 55. Thompson, J. N., Stoolmiller, A. C., Matalon, R., and Dorfman, A. (1973) Science 181,866-867 56. Matalon, R., Arbogast, B., and Dorfman, A. (1974) Biochem. Biophys. Res. Commun.. 61, 1450-1457 57. Shapira, E., DeGregorio, R. R., Matalon, R., and Nader, H. G. (1975) Biochem. Biophys. Res. Commun. 62,448-455 58. Weissmann, B., Hadjiioannou, S., and Tornheim, J. (1964) J. Biol. Chem. 239, 59-63 59. Weissmann, B., Cashman, D. C., and Santiago, R. (1975) Connect. Tissue Res. 3, 7-15 60. Hutterer, F. (1966) Biochim. Biophys. Acta 115, 312-319 61. Tan, Y. H., and Bowness, J. M. (1968) Biochem. J. 110,9-17 62. Filipovic, I., and Buddecke, E. (1968) Hoppe-Seyler’s Z. Physiol. Chem. 349,533-543 63. Cashman, D. C., Laryea, J. U., and Weissmann, B. (1969) Arch. Biochem. Biophys. 135, 387-395 64. Margolis, R. U.; Margolis, R. K., Santella, R., and Atherton, D. M. (1972) J. Neurochem. 19, 2325-2332 65. Arbogast, B., Hopwood, J. T., and Dorfman, A. (1975) Biochem. Biophys. Res. Commun. 67,376-382 66. Wasteson, A., Amado, R., Ingmar, B., and Heldin, C.-H. (1975) Protides Biol. Fluids Proc. Colloq. Bruges 431-435 67. Aronson, N. N., Jr., and de Duve, C. (1968) J. Biol. Chem. 243, 4564-4573 68. desalegui, M., and Pigman, W. (1967) Arch. Biochem. Biophys.

120,60-67 69. Orkin, R. W., Jackson, G., and Toole, Biophys. Res. Commun. 77, 132-138

B. P. (1977)

Biochem.


23. Anderson, L. E., and McClure, W. 0. (1973) Anal. Biochem. 51, 173-179 24. Blake, D. A. (1977) Ph.D. thesis, University of Illinois, Urbana 25. Saito, H., Yamagata, T., and Suzuki, S. (1968) J. Biol. Chem. 243, 1536-1542 26. Weissmann, B. (1955) J. Biol. Chem. 216, 783-794 27. Pugh, D., Leaback, D. H., and Walker, P. G. (1957) Biocham. J. 65, 464-469 28. Levvy, G. A. (1952) Biochem. J. 52,464-472 29. Findlay, J., Levvy, G. A., and Marsh, C. A. (1958) Biochem. J. 69, 467-476 30. Bach, G., Eisenberg, F., Jr., Cantz, M., and Neufeld, E. F. (1973) Proc. Natl. Acad. Sci. U. S. A. 70,2134-2138 31. desalegui, M., Plonska, H., and Pigman, W. (1967) Arch. Biochem. Biophys. 121,548-554 32. Glaser, J. H., and Sly, W. S. (1973) J. Lab. Clin. Med. 82, 969977 33. Levitt, D., and Dorfman, A. (1974) in Current Topics in Deuelopmental Biology (Moscona, A. A., and Monroy, A., eds) Vol. 8, pp. 103-149, Academic Press, New York 34. Okayama, M., Pacifici, M., and Holtzer, H. (1976) Proc. Natl. Acad. Ski. U. S. A. 73,3224-3228 35. Holtzer, H., Rubenstein, N., Fellini, S., Yeah, G., Chi, J., Birnbaum. J.. and Okavama. M. (1975) Q. Reu. Bioahvs. 8. 523-557 36. Chacko, S., Abbott, J,, Hohzer, S., and Holtzer, H. (1969) J. Exp. Med. 130,417-442 37. Abbott, J., and Holtzer, H. (1968) Proc. Natl. Acad. Sci. U. S. A. 59,1144-1151 38. Schiltz, J. R., Mayne, R., and Holtzer, H. (1973) Differentiation 1, 97-108 39. Mayne, R., Schiltz, J. R., and Holtzer, H. (1973) in Biology of the Fibroblast (Kulonen, E., and Pikkarainen, J., eds) pp. 61-78, Academic Press, New York 40. Mayne, R., Vail, M. S., and Miller, E. J. (1976) Dev. Biol. 54,230240 41. Holtzer, H., and Abbott, J. (1968) in The Stability of the Differentiated State (Ursprung, H., ed) pp. 1-16, Springer-Verlag, New York 42. Maym, R., Abbott, J., and Holtzer, H. (1973) Exp. Cell Res. 77, 255-263 43. Uitto, J. (1977) Biochemistry 16, 3421-3429 44. Burke, J. M., Balian, G., Ross, R., and Bornstein, I’. (1977) Biochemistry 16. 3243-3249 45. Nevo, Z., Horwitz, L., and Dorfman, A. (1972) Dev. Biol. 28,219228

SO, Catabolism