©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Nonreducing End Structures of Chondroitin Sulfate Chains on Aggrecan Isolated from Swarm Rat Chondrosarcoma Cultures (*)

(Received for publication, December 14, 1994; and in revised form, February 8, 1995)

Ronald J. Midura (1)(§) Anthony Calabro (2) Masaki Yanagishita (2) Vincent C. Hascall (3)

From the  (1)Department of Orthopaedic Surgery, The University of Iowa, Iowa City, Iowa 52242, the (2)Proteoglycan Chemistry Section, NIDR, the National Institutes of Health, Bethesda, Maryland 20892, and the (3)Biomedical Engineering Department, The Cleveland Clinic Foundation, Cleveland, Ohio 44195

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Chondrocyte cultures derived from the Swarm rat chondrosarcoma were metabolically labeled with [S]sulfate or [6-^3H]GlcN. Radiolabeled aggrecan was purified from the cell layer and exhaustively digested with chondroitin ABC lyase. Digestion products were resolved into disaccharide and monosaccharide residues using Toyopearl HW40S chromatography. The separated saccharide pools were reduced with NaBH(4) and applied onto a CarboPac PA1 column to resolve all of the internal disaccharide alditols (unsaturated) from the nonreducing end disaccharide (saturated) and monosaccharide alditols. Mercuric acetate treatment was used prior to carbohydrate analysis to identify unambiguously the saturated from the unsaturated disaccharides. The chondroitin sulfate (CS) chains from these aggrecan preparations contained: (a) an internal disaccharide composition of unsulfated (3-4 per chain), 4-sulfated (32 per chain), 6-sulfated (1 per 14 chains), and 4,6-sulfated disaccharides (1 per 6 chains) and (b) a nonreducing terminal composition of 4-sulfated GalNAc (4 out of every 7 chains), 4,6-disulfated GalNAc (2 out of every 7 chains), and GlcUA adjacent to a 4-sulfated GalNAc residue (1 out of every 7 chains). Thus, the vast majority of these CS chains terminated with a sulfated GalNAc residue. The presence of 4,6-disulfated GalNAc at nonreducing termini is 60-fold more abundant than 4,6-disulfated GalNAc in interior disaccharides. This observation is consistent with the suggestion that disulfation of terminal GalNAc residues is involved in chain termination.


INTRODUCTION

Glycosaminoglycan chain termination is a long-standing issue in proteoglycan research(1, 2, 3, 4, 5, 6, 7, 8, 9, 10) . It has received renewed attention as a result of research suggesting that changes occur in the sulfation patterns of chondroitin sulfate (CS) (^1)chains on proteoglycans synthesized by chondrocytes in osteoarthritic cartilage (11, 12, 13) as well as during tissue development(11, 14, 15, 16) . CS chains can be terminated by either a GalNAc or a GlcUA, and each of these terminal residues can be sulfated in various positions. One approach to this problem has been to isolate and identify the nonreducing termini of glycosaminoglycans(5, 6) . The findings of these studies suggest that CS chains are often terminated with sulfated GalNAc residues. However, it is still not known precisely how many chains end with a GalNAc versus a GlcUA residue for a purified proteoglycan species. Furthermore, a quantitative assessment of the sulfation patterns at the nonreducing termini still needs to be rigorously addressed.

In this study, a strategy was devised to quantify the nonreducing termini on a model proteoglycan, aggrecan. Aggrecan was metabolically labeled with [S]sulfate and [^3H]GlcN precursors in chondrocyte cultures derived from rat chondrosarcoma tissue and purified by a series of chromatographic steps. A portion of the preparation was used to determine the number-average molecular weight of its CS chain population by monosaccharide analysis to compare the GalN to Gal (2 residues per chain) contents. Intact aggrecan was then digested exhaustively using highly purified chondroitin ABC lyase. This eliminase cleaves the beta1 4 bond between GalNAc and GlcUA residues in CS chains converting the GlcUA residue to a 4,5-unsaturated uronosyl and releasing a GalNAc with a free reducing group(17, 18) . In limit digests, internal GlcUA residues yield unsaturated disaccharides. A CS chain with a nonreducing terminal GlcUA residue will yield one disaccharide with a saturated GlcUA at its nonreducing end, while a chain with a substituted nonreducing GalNAc will yield this GalNAc monosaccharide and no saturated disaccharides(19) . The procedures used in this study resolves these nonreducing terminal monosaccharides and disaccharides from the interior unsaturated disaccharides. In addition, mercuric acetate treatment was employed to convert the unsaturated disaccharides to their respective substituted GalNAc derivatives by degrading the unsaturated uronosyl residue and cleaving the glycosidic bond(20, 21, 22) , while leaving the saturated disaccharides intact. This procedure unambiguously identifies a small number of saturated disaccharides in the presence of a large amount of unsaturated disaccharides. Altogether, this strategy provides an analysis of the spectrum of nonreducing termini and an estimate of the number-average molecular weight of the population of CS chains in the proteoglycan preparation.


EXPERIMENTAL PROCEDURES

Materials

Protease-free chondroitin ABC lyase (Proteus vulgaris) and all chondroitin sulfate, dermatan sulfate, and hyaluronan unsaturated disaccharide standards (high performance liquid chromatography grade) were obtained from Seikagaku America; hyaluronidase (bovine testes) was obtained from Calbiochem; [S]sulfate (40 Ci/mg) was from ICN Radiochemicals; D-[6-^3H]glucosamine (35 Ci/mmol) was from DuPont NEN; all culture media and HEPES were purchased from Life Technologies Inc.; highly purified bovine serum albumin was from Calbiochem; a CarboPac PA1 column (4.6 times 250 mm), high purity monosaccharide standards, and a Dionex DX-300 chromatography system with a pulsed electrochemical detector and a flow-cell spectrophotometer were from Dionex Corp.; an Aminex A-9 column (4.6 times 250 mm) was from Bio-Rad; Toyopearl HW-40S was obtained from Toso Haas; Sephacryl S-1000 (superfine), Sephadex G-50 (fine), Q-Sepharose (fast flow), prepacked Superose 6 (HR 10/30) columns, and empty HR columns (10/30 and 16/50) were purchased from Pharmacia Biotech Inc.; Dowex AG 50W-X8 (200-400 mesh) was obtained from Bio-Rad; Microcon 3 (3-kDa cut-off) and Centricon 30 (30-kDa cut-off) microconcentrators were from Amicon; Ultrafree MC filter units (0.45 µm porosity) were from Millipore; high purity sodium acetate and NaCl were from Mallinckrodt; trifluoroacetic acid (>99% purity) was from Pierce; high purity carbazole was from J. T. Baker; NaBH(4) (>99% purity) and mercuric acetate were from Aldrich Chemical; 50% NaOH (19.2 M) was from Fisher Scientific; HighSafe 3 scintillation mixture was from Wallac. A Speed Vac concentrator was from Savant Instruments, a division of Forma Scientific. All other chemicals and reagents were of the highest commercially available grade of purity. All aqueous solutions were made with ultrapure water obtained from a Nanopure water purification system (Barnstead-Thermolyne, Dubuque, IA). Swarm rat chondrosarcoma tissue was kindly provided by Dr. James H. Kimura (Henry Ford Hospital, Detroit, MI).

Cell Culture and Metabolic Labeling

A monodisperse cell suspension was prepared from Swarm rat chondrosarcoma tissue using established procedures(23) . These chondrocytes were seeded at a confluent density (300,000 cells/cm^2) and cultured for 3 days in RPMI 1640 (4.5 g/liter glucose) containing 10% fetal bovine serum at 37 °C in a humidified 5% CO(2) atmosphere with daily media replacements(24) . At 72 h of incubation, the cultures were metabolically labeled with 100 µCi/ml [S]sulfate or 400 µCi/ml [^3H]GlcN for 8 h. After labeling, the medium was collected and made 4 M with solid guanidine HCl and 2% (v/v) with Triton X-100. The cell layer was extracted with 4 M guanidine HCl, 2% Triton X-100, 50 mM sodium acetate, pH 6.0, containing protease inhibitors (25) for 24 h at 4 °C.

Purification of Aggrecan

Labeled macromolecules from medium and tissue extracts were isolated from unincorporated radioactive precursors using 4-ml Sephadex G-50 columns eluted with 10 M formamide, 0.5% Triton X-100, 0.3 M NaCl, and 50 mM sodium acetate, pH 6.0, as described previously (25) . Cell layer samples contained 68% of the S (8.6 times 10^7 cpm) and 62% of the ^3H (6.4 times 10^6 cpm) radioactivity. Labeled macromolecules, recovered in the excluded volume fractions from the Sephadex G-50 columns, were added to 10 ml of a 50% (v/v) Q-Sepharose slurry equilibrated in the same formamide buffer. Q-Sepharose columns (1 times 6 cm) were packed and then washed with 5 bed volumes of 10 M formamide, 0.5% Triton X-100, 0.3 M NaCl, 50 mM sodium acetate, pH 6.0, to collect the unbound material (glycoproteins). Proteoglycans were then batch-eluted by resuspending the Q-Sepharose beads with 1 bed volume of 4 M guanidine HCl, 0.5% CHAPS, 0.1% Triton X-100, 50 mM sodium acetate, pH 6.0, followed by washing the column with 3 more bed volumes of this solution. Cell layer samples contained >99% of the S and 79% of the ^3H in proteoglycans, while medium samples contained 99% and 65% of the S and ^3H, respectively, in proteoglycans (overall recovery was 85-90%). Proteoglycan samples were subsequently concentrated by ultrafiltration using Centricon 30 microconcentrators pretreated with 100 µg/ml serum albumin as described previously(26) . Radiolabeled aggrecan was isolated from smaller proteoglycans and hyaluronan using chromatography on Sephacryl S-1000 (1 times 30 cm, Pharmacia HR 10/30) eluted with 4 M guanidine HCl, 0.5% CHAPS, 0.1% Triton X-100, and 50 mM sodium acetate, pH 6.0, at a flow rate of 0.4 ml/min; 1-min fractions were collected and analyzed for radioactivity. Aggrecan (K(d) from 0.13 to 0.56; peak at 0.34) was recovered, concentrated in Centricon 30 devices (albumin-blocked), and stored at 4 °C.

Isolation of CS Chains

Portions of the S- and ^3H-labeled aggrecan preparations were mixed to achieve a 2:1 ratio of S:^3H. Guanidine HCl was removed from the sample by repeated dilution in water and concentration using an albumin-blocked, Centricon 30 device (overall recovery was 92%). This desalted sample (48 µg of uronic acid as determined by carbazole assay) was treated with alkaline borohydride (1 M NaBH(4) in 50 mM NaOH) for 24 h at 45 °C(27) . After neutralization, methylation of the residual borate, and vacuum drying(26) , the resultant glycoconjugates were applied to a Superose 6 column eluted at 0.4 ml/min with 0.5 M ammonium acetate, pH 7.0; 1-min fractions were collected and analyzed for radioactivity. CS chains were recovered (K(d) from 0.35 to 0.78, peak at 0.56), vacuum-dried, and then reapplied to the Superose 6 column to remove trace amounts of smaller oligosaccharides. These highly purified CS chains were vacuum-dried and then analyzed for GalN and Gal contents as described below.

GalN and Gal Compositional Analyses

A portion of the CS chains recovered from Superose 6 (10 µg of uronic acid) was acid-hydrolyzed (4 M trifluoroacetic acid for 3 h at 100 °C), vacuum-evaporated, reconstituted in water, and then applied to a CarboPac PA1 column eluted with 16 mM NaOH at a flow rate of 1 ml/min. Monosaccharides were detected by integrated pulsed amperometric detection (IPAD program 1) using a gold working electrode and a Ag/AgCl(2) reference electrode(28) . Data were collected digitally using a Dionex Advanced Computer Interface and Dionex AI-450 software. High purity monosaccharide standards were used to formulate PAD response curves for each saccharide. The amounts of GalN and Gal in the samples were determined via these standard curves. [^3H]GlcN was used as an internal chromatography standard for each run; 1-min fractions were collected and analyzed for radioactivity.

Analysis of CS Tetrasaccharides

A portion of the desalted CS chains described above (15 µg of uronic acid) was taken up to a final reaction volume of 200 µl in 50 mM sodium acetate, pH 7.0, containing 200 turbidity-reducing units of testicular hyaluronidase. Samples were digested for 18 h at 37 °C and then applied to Microcon 3 microconcentrators to isolate the digestion products (97% of the S and 93% of the ^3H in the ultrafiltrate) from the enzyme (retentate). These digestion products were injected into a Toyopearl HW40S column (1.6 times 50 cm, Pharmacia HR 16/50) eluted with 0.5 M ammonium acetate, pH 7.0, at a flow rate of 0.8 ml/min. A total of 75 fractions (0.53 ml each) were collected starting at 36 min after injection of the sample. Typically, V(o) was at fraction 10 and V(t) was at fraction 63.

CS tetrasaccharides recovered from the Toyopearl HW40S column were vacuum-dried, reconstituted in 100 µl of 0.1 M sodium acetate, pH 7.0, and then digested with 0.01 unit of chondroitin ABC lyase (1 h at 38 °C). Digestion products (ultrafiltrate) were isolated from the enzyme (retentate) using Microcon 3 microconcentrators. These digestion products were reduced using a modified borohydride reduction procedure(29) . Ultrafiltrates (diluted to leq1 mM uronic acid) were adjusted to 50 mM NaBH(4) in 50 mM sodium acetate, pH 7.0, by adding neutral-pH Nanopure water and an aliquot of 500 mM NaBH(4) in 10 mM sodium acetate, pH 7.0 (prepared immediately before adding to the sample). Reduction of the reducing-end, aldehyde group was achieved by a 30-min incubation at 38 °C; the unsaturated bond within the uronosyl residue was not reduced by this procedure(29) . Following this reaction, the sample was placed on ice, and ice-cold 0.1 M acetic acid was added until all excess borohydride was exhausted. The reduced disaccharide products were isolated (and simultaneously desalted) by Toyopearl HW40S chromatography followed by Speed Vac drying. A portion of this dried sample was treated with 35 mM mercuric acetate, pH 5.0, for 30 min at room temperature to degrade unsaturated disaccharides (20, 21, 22) . As described previously(22) , residual mercury was removed from the samples by Dowex AG 50W-X8 cation exchange before any additional analyses were performed.

CarboPac PA1 Chromatography

Desalted, reduced samples were applied to a CarboPac PA1 column (with guard column) that was eluted at 1 ml/min with a sodium trifluoroacetate gradient in 0.1 M NaOH(29) . Fractions were of 1-min duration. Trifluoroacetic acid was prepared as a 1 M stock (pH 7.0) as follows. Concentrated trifluoroacetic acid (13.4 M) was diluted into Nanopure water and carefully titrated to pH 7.0 with freshly prepared NaOH solutions made from a high purity 50% NaOH stock. This trifluoroacetic acid solution was brought up to final volume, filtered (0.2 µm), and stored at room temperature. The working solutions for CarboPac PA1 chromatography consisted of two solutions: solution 1 (0.1 M NaOH; prepared fresh from a 50% NaOH stock) and solution 2 (0.5 M trifluoroacetic acid in 0.1 M NaOH). The programmed trifluoroacetic acid gradient was (% solution 1:% solution 2): 0 min (97:3), 12 min (97:3), 32 min (74:26), 42 min (74:26), 62 min (40:60), 72 min (40:60), 82 min (0:100), 90 min (0:100); linear elution between time points.

Analysis of Disaccharides and Monosaccharides Resulting from Chondroitin ABC Lyase Digestion of Aggrecan

Portions of the S- and ^3H-labeled aggrecan samples recovered from Sephacryl S-1000 were mixed to achieve a 2:1 ratio of S:^3H, and the solvent was exchanged to 0.1 M sodium acetate, pH 7.0, by repeated concentration and resuspension using albumin-blocked Centricon 30 devices. The final guanidine HCl concentration was calculated to be 1 µM, and sample recovery in the retentate was 94%. This aggrecan sample (450 µg of uronic acid) was then digested for 10 h at 38 °C with chondroitin ABC lyase (0.2 unit) in a final reaction volume of 100 µl. Microcon 3 devices, pretreated with 100 µg/ml serum albumin, were used to isolate the digestion products (ultrafiltrate) from the core protein and enzyme (retentate). The ultrafiltrate sample (96% of the S and 84% of the ^3H) was eluted on a Toyopearl HW40S column to separate mono-, di-, and trisaccharide digestion products. This chromatography step was sufficient to desalt the disaccharides, but not the monosaccharide products. Monosaccharides were desalted by a procedure involving cation removal using Dowex AG 50W-X8 and vacuum drying(29) . Dowex resin, stored as a 50% slurry in Nanopure water, was washed three times with ice-cold, neutral-pH Nanopure water by centrifugation through an Ultrafree MC filter unit (3000 times g, 2-3 min). Typically, a 25-µl packed bed of washed Dowex was used for every 100 µl of sample. Samples (precooled on ice) were exposed to the Dowex resin for 3-5 min at 4 °C. Filtrates were collected by centrifugation and then dried on a Speed Vac.

Desalted monosaccharide and disaccharide samples were reduced with 50 mM NaBH(4) in 50 mM sodium acetate, pH 7.0, and subsequently retreated with Dowex as described above. After Speed Vac drying, samples were resuspended with 1 ml of ice-cold, absolute methanol and dried again to remove residual borate. Generally, a second cycle of methanol drying was needed to remove all traces of borate. Samples were then applied to and eluted from a CarboPac PA1 column, before and after mercuric acetate treatment, as described above.

Other Procedures

Hexuronic acid concentrations of appropriate samples were determined using a carbazole assay (30) that has been modified for automation (31) and adapted for manual operation. Select samples were acid-hydrolyzed, vacuum-evaporated, and analyzed for [^3H]galactosaminitol content using an Aminex A-9 column as described previously(32, 33) . Sample radioactivity was determined by liquid scintillation spectrometry with a Beckman LS-5801 beta-counter using HighSafe 3 at a 5:1 volume ratio of scintillant to sample. S and ^3H activities were discriminated using an external standard of S and calculating its spillover into the ^3H channel.


RESULTS

Preparation of Reduced, Saturated Chondroitin 4-Sulfate Disaccharide from Labeled Aggrecan

S- and ^3H-labeled aggrecan was purified from the cell layer of rat chondrosarcoma cultures as described under ``Experimental Procedures.'' A portion of this aggrecan preparation was treated with alkaline borohydride to release their glycoconjugates. The CS chains (K(d) = 0.56) were separated from N-linked glycopeptides and O-linked oligosaccharides (K(d) = 0.98) using Superose 6 chromatography (data not shown). The CS peak (98% of the S and 89% of the ^3H) was reapplied to Superose 6 to remove all traces of smaller glycoconjugates. Monosaccharide analyses detected 21.33 nmol of GalN and 1.14 nmol of Gal in this CS sample. Given 2 mol of Gal per mol of CS chain, this sample contained 0.57 nmol of CS chains (=1.14/2). Therefore, the molar ratio of GalN within these CS chains is 37.4 (=21.33/0.57). This ratio yields a number-averaged molecular weight for these CS chains of 18 kDa. (^2)

A portion of the CS chains was exhaustively digested with testicular hyaluronidase. The digestion products resolved into one major and three minor peaks on Toyopearl HW40S (Fig. 1A). The major peak (89% of the S and 82% of the ^3H) contains CS tetrasaccharides that eluted in a position between HA tetra- and hexasaccharides (Fig. 1A, bar). A portion of the tetrasaccharide peak was digested with chondroitin ABC lyase, reduced with NaBH(4), and reapplied to the HW40S column (Fig. 1B). Two major and three minor peaks were observed. The first of the two major peaks (49% of the S and 45% of the ^3H) eluted slightly earlier than the CS disaccharide peak generated by testicular hyaluronidase (A), while the second (48% of the S and 44% of the ^3H) eluted slightly earlier than the saturated HA disaccharide standard (arrowhead 2, A). These two major peaks were recovered together as one sample (bar, B). A portion of this sample was reapplied to the HW40S column without further treatment (Fig. 1C, peaks Iand IIa), while another aliquot was treated with mercuric acetate as described under ``Experimental Procedures'' and then reapplied to the HW40S column (Fig. 1D). Peak I was unaltered by mercuric ion treatment, while peak IIa was cleaved into monosaccharides by the treatment (peak IIb). These data indicate that peak I consists of saturated CS disaccharide alditols, while peak IIa consists of unsaturated CS disaccharide alditols.


Figure 1: Isolation of saturated chondroitin 4-sulfate disaccharide. A, CS chains digested with testicular hyaluronidase were applied to a Toyopearl HW40S column. The bar indicates the CS tetrasaccharides recovered for further analysis. Numbered arrowheads (8, 6, 4, 2, and 1) denote the elution positions for authentic standards of HA(8), HA(6), HA(4), HA disaccharide, and GlcN, respectively. B, CS tetrasaccharides from A were digested with chondroitin ABC lyase, reduced with NaBH(4), and eluted on the HW40S column. The bar indicates the disaccharides recovered for further analysis. The arrow denotes the elution position of the CS tetrasaccharide. These disaccharides were reapplied to the HW40S column in C (designated I and IIa). D, the disaccharides recovered in B were treated with mercuric acetate prior to elution on the HW40S column (products are designated I and IIb).



Equal aliquots of the samples represented in Fig. 1, C and D, were analyzed on a CarboPac PA1 column (29) in order to identify these disaccharides. The untreated sample (Fig. 2A) resolved into two peaks (Iand IIa) of nearly equal proportions. Peak I eluted in a position (32 min) that was unique from all known unsaturated disaccharide standards, while peak IIa eluted in the position of the DeltaDi-4S(r) standard. Analysis of the mercuric acetate-treated sample (Fig. 2B) indicated that peak I was resistant to mercuric ion cleavage, while peak IIa (DeltaDi-4S(r)) was converted to a new structure (peak IIb) with a unique elution position (24 min) from all of the disaccharide standards. Peak IIb is deduced to be N-acetylgalactosaminitol-4-sulfate (4SGalNAc(r)) since it is a sulfated monosaccharide derived from DeltaDi-4S(r) and contains only [^3H]galactosaminitol after acid hydrolysis and hexosamine/hexosaminitol analysis (data not shown).


Figure 2: Elution position of saturated chondroitin 4-sulfate disaccharide alditol (Di-4S(r)) and 4-sulfated N-acetylgalactosaminitol (4SGalNAc(r)) on CarboPac PA1. The column was eluted with a gradient of sodium trifluoroacetate (TFA) in 0.1 M NaOH (indicated by a dashed line in A). A depicts the elution profile for the intact disaccharides (I and IIa) recovered from HW40S (Fig. 1B). Numbers indicate the elution positions of the following disaccharide standards: 1, DeltaDi-0S(r); 2, DeltaDi-HA(r); 3, DeltaDi-4S(r); 4, DeltaDi-2S(r); 5, DeltaDi-6S(r); 6, DeltaDi-2,4S(r); 7, DeltaDi-4,6S(r); 8, DeltaDi-2,6S(r). B shows the elution positions of the mercuric acetate-treated products (designated I and IIb) derived from disaccharides I and IIa.



Peak I is deduced to be reduced, saturated chondroitin 4-sulfate disaccharide (Di-4S(r)) because it (a) is derived from a chondroitin 4-sulfate tetrasaccharide generated by the hydrolase testicular hyaluronidase, which leaves a GlcUA residue at the nonreducing end of all digestion products(34) , (b) elutes in a disaccharide size range on HW40S after chondroitin lyase digestion, (c) is resistant to mercuric acetate treatment, and (d) contains only [^3H]galactosaminitol after acid hydrolysis and hexosamine/hexosaminitol analysis (data not shown). Altogether, these analyses establish the unique elution positions of Di-4S(r) and 4SGalNAc(r) (two potential nonreducing termini of CS chains) on the CarboPac PA1 column.

Separation of Di- and Monosaccharide Products Resulting from Chondroitin ABC Lyase Digestion of Aggrecan

A portion of the labeled aggrecan preparation was exhaustively digested with chondroitin ABC lyase, and the resultant digestion products were applied onto a HW40S column before borohydride reduction (Fig. 3). Three peaks, one major and two minor, were detected. The major peak eluted in the disaccharide size range (peak 2; 96.8% of the S and 97.1% of the ^3H). One minor peak eluted in the monosaccharide size range (peak 1; 3.1% of the S and 2.8% of the ^3H), while the second minor peak eluted in the trisaccharide range (indicated by an asterisk in Fig. 3; 0.1% of both S and ^3H). (^3)Peaks 1 and 2 were recovered for further analysis.


Figure 3: Isolation of disaccharides and monosaccharides generated by chondroitin ABC lyase digestion of aggrecan. Labeled aggrecan was exhaustively digested with chondroitin ABC lyase, and its digestion products were applied onto a Toyopearl HW40S column. The monosaccharide peak (1) was well separated from the disaccharide peak (2). The asterisk denotes a trisaccharide peak mentioned in the text. The elution positions of the CS tetrasaccharide (CS tetra) isolated in Fig. 1A and glucosamine (GlcN) are provided as reference standards. The inset depicts the full scale profile.



CarboPac PA1 Analyses of the Monosaccharides Released from Aggrecan by Chondroitin ABC Lyase

After reduction, the monosaccharide alditols in the peak 1 sample were analyzed on CarboPac PA1 before (Fig. 4A) and after mercuric acetate treatment (Fig. 4B). Two major and two minor peaks were observed in the untreated sample. One minor peak eluted in the position of DeltaDi-0S(r) (1.5% of the ^3H in the sample), and the other eluted in the position of DeltaDi-4S(r) (3% of the S and 4% of the ^3H). As expected, both of these minor peaks were susceptible to mercuric ion degradation. However, the major peaks were resistant to mercuric acetate treatment. The first major peak eluted in the position of 4SGalNAc(r) and contained 47% of the S and 63% of the ^3H with a S/^3H ratio of 2.4 (similar to the S/^3H ratio of 2.3 for the disaccharide peak 2 in Fig. 3). The second, later eluting peak contained 50% of the S and 31.5% of the ^3H with a S/^3H ratio of 5.0. This peak is assigned as N-acetylgalactosaminitol-4,6-disulfate (4,6SGalNAc(r)) since it elutes in the position of authentic 4,6SGalNAc(r) generated by mercuric acetate treatment of DeltaDi-4,6S(r) (described below). The production of these sulfated GalNAc residues, as a result of chondroitin ABC lyase digestion of aggrecan, provides strong evidence that they are at the nonreducing ends of CS chains.


Figure 4: Identification of the monosaccharides generated by chondroitin ABC lyase digestion of aggrecan. Monosaccharides recovered in Fig. 3(peak 1) were reduced with NaBH(4) and applied to a CarboPac PA1 column. Samples were applied either before (A) or after (B) mercuric acetate treatment. The presence of minor amounts of DeltaDi-0S(r) and DeltaDi-4S(r) is the result of cross-contamination of peak 1 with disaccharides from peak 2.



CarboPac PA1 Analyses of the Disaccharides Released from Aggrecan by Chondroitin ABC Lyase

After reduction, the disaccharide alditols in the peak 2 sample from Fig. 3were analyzed on CarboPac PA1 either before (Fig. 5, A and C) or after mercuric acetate treatment (Fig. 5, B and D). The untreated sample eluted as one major peak and five minor, identifiable peaks. The major peak (97.9% of the S and 89.2% of the ^3H in the sample with a S/^3H ratio of 2.5) eluted in the position of DeltaDi-4S(r). Analysis of the minor peaks indicated that: (a) one (9.7% of the ^3H) eluted in the position of DeltaDi-0S(r); (b) a second (0.4% of the S) eluted in the position of free sulfate; (c) a third (1% of the S and 0.5% of the ^3H with a S/^3H ratio of 4.9) eluted in the position of DeltaDi-4,6S(r); (d) a fourth (0.5% of the S and 0.4% of the ^3H with a S/^3H ratio of 2.5) eluted in the position of Di-4S(r); and (e) a fifth (0.2% of the S and 0.2% of the ^3H with a S/^3H ratio of 2.1) eluted in the position of DeltaDi-6S(r) (Fig. 5C, inset). The presence of free sulfate detected in these analyses is most likely the result of a small amount of the disaccharides undergoing desulfation during the processing of the sample.


Figure 5: Identification of the disaccharides generated by chondroitin ABC lyase digestion of aggrecan. Disaccharides recovered in Fig. 3(peak 2) were reduced with NaBH(4) and applied to a CarboPac PA1 column. Samples were applied either before (A and C) or after (B and D) mercuric acetate treatment. C and D provide expanded views of the baselines in A and B, respectively. The asterisk in D denotes a minor peak that might possibly represent a small amount (0.1%) of Di-6S(r) (Anna H. K. Plaas, personal communication). Insets in C and D depict a greatly magnified view of the baseline between fractions 41 and 56. SO(4), inorganic sulfate.



After mercuric acetate treatment, one major and three minor identifiable species were detected during the CarboPac PA1 run. The major peak eluted in the position of 4SGalNAc(r) and is the degradation product derived from DeltaDi-4S(r). An expanded scale in Fig. 5D revealed three minor peaks: (a) GalNAc(r) which is derived from DeltaDi-0S(r), (b) 4,6SGalNAc(r) which is derived from DeltaDi-4,6S(r), and (c) Di-4S(r). Additionally, the disaccharide peak that eluted in the position of DeltaDi-6S(r) was susceptible to mercuric acetate treatment (Fig. 5D, inset). Thus, mercuric acetate treatment converted quantitatively all of the unsaturated disaccharide alditols into their respective, earlier eluting monosaccharide alditols, thereby exposing the intact Di-4S(r) peak.

These data demonstrate that the CS chains from rat chondrosarcoma aggrecan contain a small amount of Di-4S(r) after chondroitin lyase digestion. The presence of saturated disaccharides after chondroitin lyase digestion indicates that they are at the nonreducing ends of CS chains. Table 1summarizes the proportion of each internal disaccharide and nonreducing terminal unit accounted for in these analyses as a percentage of the ^3H (from GlcN) released from aggrecan by chondroitin ABC lyase. The nonreducing termini identified in this study account for 3.1% of this ^3H activity.




DISCUSSION

This study has devised a strategy to analyze the nonreducing termini of CS chains. An underlying premise of this strategy is that, when completely degraded by chondroitin lyase, CS chains terminating with a GalNAc residue will yield free, variably sulfated GalNAc monosaccharides, while those terminating with a GlcUA will yield saturated disaccharides of various degrees of sulfation(19) . The strategy involves: (a) exhaustive degradation of the CS chains using highly purified chondroitin ABC lyase which releases nonreducing terminal residues from the internal, repeating disaccharide units; (b) separation of resultant disaccharide and monosaccharide products using high resolution, low molecular weight gel permeation chromatography; and (c) identification of these digestion products using high performance anion exchange chromatography on CarboPac PA1. Additionally, mercuric acetate treatment was used to identify unambiguously any saturated (mercuric ion resistant) from unsaturated (mercuric ion sensitive) disaccharides. Exhaustive chondroitin ABC lyase digestion was critical for this approach since limited digestions revealed trisaccharides that contained a nonreducing terminal GalNAc residue and the adjacent internal disaccharide, thus complicating subsequent structural analyses (see asterisk in Fig. 3).

To achieve validation of this strategy, this study focused on analyzing the saccharide composition of CS chains on aggrecan synthesized by chondrocytes isolated from the Swarm rat chondrosarcoma. This is a well established chondrocyte model system capable of producing a large quantity of aggrecan. Aggrecan synthesized by these cells is relatively easy to purify, and the overall structure of its CS chains is well characterized, with nearly uniform sulfation on C4 of the GalNAc residues(22) . A metabolic labeling approach was chosen because it provides the sensitivity and quantitation required for this analysis and emphasizes a structural analysis of the stable, nonreducing end structures on newly synthesized aggrecan. This strategy is optimal for identifying and quantifying the nonreducing termini of CS chains from an intact proteoglycan.

Table 1and Fig. 6summarize our findings. A novel observation was made concerning the internal disaccharide composition of the CS chains from rat chondrosarcoma aggrecan. Minute amounts of both DeltaDi-6S(r) and DeltaDi-4,6S(r) were detected as a result of the high resolution capacity of the CarboPac PA1 column. Based on the molar ratio of GalN per 2 Gal residues (37), the number of internal and terminal GalNAc residues released from one CS chain by chondroitin ABC lyase would be 36. The enzyme leaves one disaccharide, mostly unsulfated(22) , bound on the linkage oligosaccharide(35, 36) . Excluding this first disaccharide, the proportions of the internal disaccharides are 32 4-sulfated and 3-4 unsulfated internal disaccharide units per CS chain, about one 4,6-sulfated disaccharide per 6 CS chains, and approximately one 6-sulfated disaccharide per 14 CS chains. (^4)


Figure 6: Data summary for nonreducing terminal structures on CS chains from rat chondrosarcoma aggrecan. Ratios for nonreducing termini were calculated from data in Table 1as follows: 1.76/3.03 4/7 for those chains ending with a 4SGalNAc residue, 0.88/3.03 2/7 for those chains ending with a 4,6SGalNAc residue, and 0.39/3.03 1/7 for those chains ending with a GlcUA residue. Arrows indicate the cleavage positions of chondroitin ABC lyase. Trisaccharides, obtained in small yields, are products of cleavage of chains terminating in GalNAc at the internal sites indicated by asterisks. These trisaccharides are digested only very slowly by chondroitin ABC lyase (R. J. Midura, A. Calabro, M. Yanagishita, and V. C. Hascall, unpublished data).



The expected proportion of terminal residues compared to the total combined internal and terminal units per chain is calculated to be 1 out of 36 or 2.8%. In close agreement with this value, the actual proportion of nonreducing termini in these chains is calculated to be 3.1% of the total chondroitin ABC lyase-digestible products (Table 1). Three nonreducing termini were identified: 4 out of 7 CS chains terminated with a 4SGalNAc residue, 2 out of 7 with a 4,6SGalNAc residue, and 1 out of 7 with a GlcUA residue adjacent to a 4SGalNAc residue (Fig. 6). Lacking appropriate authentic standards, this study cannot exclude the possibility that extremely small amounts of 6-sulfated or 4,6-disulfated saturated disaccharides, or 6SGalNAc, were also present in these aggrecan preparations. These data are consistent with: (i) studies reporting that a majority of CS chains from embryonic cartilage terminate with either 4SGalNAc or 4,6SGalNAc residues (5, 6) and (ii) studies suggesting that 4-sulfation of GalNAc termini on CS chains may stop chain elongation because CS chains terminating with a 4SGalNAc residue are poor acceptors for subsequent GlcUA addition in vitro(1, 2, 3, 4) .

Of particular interest, the present study argues that there is a 60-fold greater incidence of 4,6SGalNAc residues at the nonreducing end position as compared to internal positions. (^5)Other groups (5, 6) have reported a higher frequency of 4,6-disulfation of GalNAc residues at the nonreducing terminal position when compared to those in internal positions of CS chains. This suggests the intriguing possibility that the disulfation of GalNAc is differentially regulated when this hexosamine resides at the terminus versus in an internal position within the chain. Indeed, some investigators have identified a 6-O-sulfotransferase activity which readily adds a sulfate group to the C6 position of 4SGalNAc at the chain terminus, but not when the 4SGalNAc is in an internal position within CS chains(7, 8, 37) . Perhaps, as previously suggested(6, 8, 37) , disulfation of GalNAc residues at the nonreducing end is involved in CS chain termination.

It is not known whether these nonreducing termini represent the actual residues that signal CS chain termination. It is possible that rapid trimming events might occur immediately after chain termination which, in effect, result in the stable, nonreducing end groups reported above. Additionally, the results of this study do not exclude the possibility that other classes of proteoglycans (or aggrecan from other tissue sources, including pathological or developmental conditions) may have a higher proportion of their CS chains terminating with a GlcUA residue or with 6-sulfated residues. Application of the methods described in this paper should help resolve these questions.


FOOTNOTES

*
This study was funded, in part, by a grant to the Dept. of Orthopaedic Surgery (University of Iowa) from the Roy J. Carver Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom reprint requests should be addressed: Biomedical Engineering Dept., Wb3, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195.

(^1)
The abbreviations used are: CS, chondroitin sulfate; HA, hyaluronan; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; mono- and disaccharide species (a subscripted ``r'' designates that it is in a reduced form): 4SGalNAc, 4-sulfated N-acetylgalactosamine; 6SGalNAc, 6-sulfated N-acetylgalactosamine; 4,6SGalNAc, 4,6-disulfated N-acetylgalactosamine; Di-4S, saturated chondroitin 4-sulfate disaccharide; DeltaDi-0S, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose; DeltaDi-HA, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-glucose; DeltaDi-4S, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose; DeltaDi-6S, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose; DeltaDi-4,6S, 2-acetamido-2-deoxy3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-D-galactose; DeltaDi-2S, 2-acetamido-2-deoxy-3-O-(2-O-sulfo-beta-D-gluco-4-enepyranosyluronic acid)-D-galactose; DeltaDi-2,4S, 2-acetamido-2-deoxy-3-O-(2-Osulfo-beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose; DeltaDi-2,6S, 2-acetamido-2-deoxy-3-O-(2-O-sulfo-beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose.

(^2)
Each CS chain contains 37 total GalN residues with 1 residue as a nonreducing end group (primarily a sulfated GalNAc, see ``Discussion''). Therefore, (36 internal disaccharides times 475 Da/disaccharide) + (1 nonreducing end unit times 300 Da/sulfated GalNAc) + (800 Da/linkage oligosaccharide) = 17,100 Da + 300 Da + 800 Da = 18,200 Da/CS chain.

(^3)
Preliminary analyses indicated the presence of two trisaccharides. Each could be converted into equal amounts of ^3H in unsaturated disaccharides and monosaccharides by additional chondroitin ABC lyase digestion. One appeared to contain a 4SGalNAc(r) and the other a 4,6SGalNAc(r) at their nonreducing ends (2:1 ratio, respectively). Together, these data suggest that these trisaccharides originally resided at the nonreducing end of the CS chains (see Fig. 6).

(^4)
Calculations are as follows: (86.73%/96.87%) times 36 = 32 DeltaDi-4S(r) per CS chain; (9.46%/96.87%) times 36 = 3.5 DeltaDi-0S(r) per CS chain; (0.49%/96.87%) times 36 = 0.18 DeltaDi-4,6S(r) per CS chain, or 1 out of 6; (0.19%/96.87%) times 36 = 0.07 DeltaDi-6S(r) per CS chain, or 1 out of 14.

(^5)
Calculations are as follows: for 4,6SGalNAc at terminus, 2/7 = 0.29; for 4,6SGalNAc in internal position, 1/210 = 0.0048 (6 chains having 35 internal disaccharides yields 210 total internal disaccharide residues); 0.29/0.0048 = 60.4.


ACKNOWLEDGEMENTS

We thank Dr. Anna H. K. Plaas for critically reviewing this manuscript prior to its submission.


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