INTRODUCTION

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Sulfated polysaccharides constitute a complex group of macromolecules known to possess a wide range of important biological properties. These anionic polymers are widespread in nature, occurring in a great variety of organisms. Marine invertebrates are a rich source of sulfated polysaccharides with novel structures (1-15).

The ascidians (Chordata-Tunicata) are marine invertebrates covered by an external supportive tissue called the tunic, surrounding a body. The tunic contains large amounts of a unique high molecular mass sulfated -L-galactan (1, 2, 5-7). Recently, we reported the occurrence of dermatan sulfate-like glycosaminoglycan in the body of this invertebrate (11, 13).

Mammalian dermatan sulfate is an anticoagulant due to selective inhibition of thrombin by potentiating heparin cofactor II activity (16, 17). Although of lower in vitro anticoagulant potency than heparin, it has efficacy in vivo with less hemorrhagic risk (18). Thus several authors have suggested using mammalian dermatan sulfate as an alternative antithrombotic polysaccharide (18-21).

In view of the increasing interest in the anticoagulant and antithrombotic actions of dermatan sulfate, we have characterized the fine chemical structure of dermatan sulfates extracted from the ascidian body and tested this compound in coagulation assays, including activation of heparin cofactor II. In addition, there is now more interest in therapeutics prepared from non-mammalian sources, thus reducing the risk of contamination with pathogenic agents.

In the present work, we report that dermatan sulfates isolated from different ascidian species have distinguishable patterns and proportions of sulfate substitution. These ascidian dermatan sulfates (together with native and oversulfated mammalian dermatan sulfate), where the extent and position of sulfate substitution have been fully characterized, are a valuable tool to trace the relationship between structure versus anticoagulant activity of this glycosaminoglycan. Dermatan sulfates with potent anticoagulant potency and high heparin cofactor II activity have been found.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results & Discussion References

Materials-- Heparan sulfate from human aorta was extracted and purified as described previously (22). Chondroitin 4-sulfate from whale cartilage, chondroitin 6-sulfate from shark cartilage, dermatan sulfate from bovine intestinal mucosa, twice-crystallized papain (15 units/mg protein), dextran sulfates (average mass 8 and 500 kDa), and the standard disaccharides -UA(2SO₄)¹-13-GalNAc(4SO₄), -UA(2SO₄)-13-GalNAc(6SO₄), -UA-13-GalNAc(4,6-diSO₄), and -UA(2SO₄)-13-GalNAc(4,6-diSO₄) were purchased from Sigma; chondroitin AC lyase (EC 4.2.2.5) from Arthrobacter aurescens and chondroitin ABC lyase (EC 4.2.2.4) from Proteus vulgaris were from Seikagaku American Inc. (Rockville, MD); agarose (standard low M_r) was from Bio-Rad, and 1,9-dimethylmethylene blue was from Aldrich. The disaccharides -UA-13-GalNAc(4SO₄) and -UA-13-GalNAc(6SO₄) were prepared by digestion of a mixture of standard chondroitin 4- and 6-sulfates with chondroitin AC lyase. Partially oversulfated mammalian dermatan sulfate was prepared by reaction with sulfur trioxide/pyridine (23). Human heparin cofactor II and thrombin were purified as described previously (24). The full-length cDNA for human heparin cofactor II was cloned in the expression vector pET-3d (Novagen, Madison, WI) and mutated to produce the Lys¹⁷³  Glc, Lys¹⁸⁵ Asn, and Arg¹⁸⁹ His variants as described previously (25, 26). The mutations and ligation sites were verified by dideoxynucleotide sequencing. The recombinant proteins were expressed in Escherichia coli and purified as described previously (25, 26). The thrombin chromogenic substrate S-2238 and factor Xa chromogenic substrate S-2222 were obtained from Chromogenix AB, Molndal, Sweden. The heparin for the APTT assay was the 4th International Standard (85/502), from NIBSC, Potters Bar, UK.

Ascidian Dermatan Sulfates

Isolation-- The ascidian Styela plicata was collected in Rio de Janeiro, Brazil, and Halocynthia pyriformis was collected in Stonington, ME. The body was separated from the tunic, cut in small pieces, and dried. The sulfated polysaccharides were extracted from the dried tissues (20 g) by papain digestion and partially purified by cetylpyridinium and ethanol precipitations, using the same methodology described for other tissues (8, 22), yielding ~200 mg (as dry weight).

Purification-- The sulfated polysaccharides extracted from the body of the ascidians (200 mg) were then applied to a DEAE-cellulose column (10 × 1.5 cm) equilibrated with 0.5 M sodium acetate (pH 5.0). The column was eluted stepwise with 50 ml each of 0.5 M and 1.0 M NaCl in the same buffer. The flow rate of the column was 8.0 ml/h. The fractions eluted with 1.0 M NaCl were pooled, dialyzed against distilled water, and lyophilized.

Electrophoresis

Agarose Gel Electrophoresis-- Glycosaminoglycans were analyzed by agarose gel electrophoresis, as described (13). Briefly, glycosaminoglycans (15 µg) were applied to an agarose gel (0.5%, w/v) and run in 0.05 M 1,3-diaminopropane/acetate (pH 9.0) for 1 h at 120 V. The glycosaminoglycans in the gel were fixed with 0.1% N-cetyl-N,N,N-trimethylammonium bromide in water and stained with 0.1% toluidine blue in acetic acid/ethanol/water (0.1:5:5, v/v). After staining, the gel was washed for about 15 min in acetic acid/ethanol/water (0.1:5:5, v/v).

Polyacrylamide Gel Electrophoresis-- The molecular masses of the glycosaminoglycans were estimated by polyacrylamide gel electrophoresis. Samples (10 µg) were applied to a 1-mm-thick 6% polyacrylamide slab gel, and after electrophoresis at 100 V for ~1 h in 0.06 M sodium barbital (pH 8.6), the gel was stained with 0.1% toluidine blue in 1% acetic acid. After staining, the gel was washed overnight in 1% acetic acid. The molecular mass markers were the same as those used previously (14).

Analysis of the Products Formed by Digestion of the Dermatan Sulfates with Chondroitin AC and ABC Lyases-- Mammalian and ascidian dermatan sulfates (200 µg of each) were incubated with 0.1 unit of chondroitin AC lyase or chondroitin ABC lyase in 300 µl of 50 mM Tris/HCl (pH 8.0), containing 5 mM EDTA and 15 mM sodium acetate. After incubation at 37 °C for 12 h, aliquots containing the enzyme-resistant glycosaminoglycans in the reaction mixtures were analyzed by agarose and polyacrylamide gel electrophoresis, as described above. Thereafter, the reaction mixtures were mixed with 3 volumes of absolute ethanol. The precipitate formed after standing at 10 °C for 24 h, containing the enzyme-resistant glycosaminoglycans, was removed by centrifugation (2,000 × g for 15 min at room temperature). The clear supernatant, containing the released disaccharides, was dried on a rotary evaporator and dissolved in 100 µl of distilled water. This disaccharide solution (20 µl) and standard compounds were analyzed by strong anion-exchange chromatography on a 25-cm × 4.6-mm Spherisorb-SAX column (Sigma/Aldrich), linked to a HPLC system from Shimadzu (Tokyo, Japan). After sample injection, the column was washed with 5 ml of acidified water (pH 3.5), followed by elution with a 40-ml gradient of 0-1.0 M NaCl, (pH 3.5), at a flow rate of 1 ml/min. The eluant was monitored for UV absorbance at 232 nm. Disaccharides were identified by comparison with the elution positions of known disaccharide standards.

NMR Experiments-- ¹H and ¹³C spectra were recorded using a Bruker DRX 600 with a triple resonance 5-mm probe. About 5 mg of each dermatan sulfate was dissolved in ~0.7 ml of 99.8% D₂O (NMR grade from Sigma), and the pH was adjusted to ~7.0. All spectra were recorded at 60 °C, with HOD suppression by presaturation or WATERGATE method (29). COSY, TOCSY, and ¹H/¹³C heteronuclear correlation (HMQC) spectra were recorded using states time proportional phase incrementation for quadrature detection in the indirect dimension. TOCSY spectra were run with 4,096 × 400 points with a spin-lock field of about 10 kHz and a mixing time of 80 ms, which was previously determined to give optimum results for these samples. HMQC spectra were run with 1,024 × 256 points, and globally optimized alternating phase rectangular pulses was used during acquisition for ¹³C decoupling. All chemical shifts are relative to internal or external trimethylsilylpropionic acid and methanol.

Anticoagulant Action of the Ascidian Dermatan Sulfate Measured by Activated Partial Thromboplastin Time (APTT)-- APTT clotting assays were carried out as described (30, 31). Normal citrate-anticoagulated human plasma (90 µl) was incubated with 10 µl of a solution of glycosaminoglycan (0-100 µg) and 100 µl of kaolin + bovine brain phospholipid reagent (Reagent Celite, Biolab, Mérieux). After 5 min of incubation at 37 °C, 100 µl of 0.25 M CaCl₂ was added, and the clotting time was recorded. The activity was expressed as units/mg using a parallel standard curve based on the International Heparin Standard (193 units/mg).

Inhibition of Thrombin by Heparin Cofactor II in the Presence of Glycosaminoglycans-- Incubations were performed in disposable UV semi-microcuvettes. The final concentrations of reactants included 68 nM plasma-derived or recombinant heparin cofactor II, 15 nM thrombin, and 0-100 µg/ml glycosaminoglycan in 100 µl of 0.02 M Tris/HCl, 0.15 M NaCl, and 1.0 mg/ml polyethylene glycol (pH 7.4) (TS/PEG buffer). Thrombin was added last to initiate the reaction. After a 60-s incubation at room temperature, 500 µl of 100 µM chromogenic substrate S-2238 (Chromogenix AB, Molndal, Sweden) in TS/PEG buffer was added, and the absorbance at 405 nm was recorded by a computer at 5-s intervals for 100 s. The rate of change of absorbance was proportional to the thrombin activity remaining in the incubation. No inhibition occurred in control experiments in which thrombin was incubated with heparin cofactor II in the absence of glycosaminoglycan. Nor did inhibition occur when thrombin was incubated with glycosaminoglycan alone over the range of concentrations tested.

	RESULTS AND DISCUSSION

Top Abstract Introduction Procedures Results & Discussion References

Isolation of Highly Anionic Dermatan Sulfates from Ascidians-- The sulfated polysaccharides extracted from the body of the ascidians were partially purified on a DEAE-cellulose column (not shown). On Mono Q-FPLC, these DEAE-cellulose-purified polysaccharides elute primarily at ~1.5 M NaCl (Fig. 1, A and C). Rechromatography on another Mono Q-FPLC column yields a symmetric peak for H. pyriformis (Fig. 1D), containing both strong metachromasia produced by the sulfated glycosaminoglycans with 1,9-dimethylmethylene blue (open circles) and hexuronic acid (closed circles). In the case of S. plicata (Fig. 1B), a contaminant polysaccharide is eluted at the right portion of the major peak. This contaminant has strong metachromasia but is devoid of hexuronic acid. Nevertheless, the fractions pooled as indicated by the horizontal bar in Fig. 1B contain an electrophoretically homogeneous sulfated polysaccharide (Fig. 2).

View larger version (44K): [in this window] [in a new window]   Fig. 1.   Purification of the dermatan sulfate (DS) from S. plicata (A and B) and from H. pyriformis (C and D) on a Mono Q-FPLC column. A and C, the DEAE-cellulose-purified sulfated polysaccharides from the ascidian body (60 mg) were applied to a Mono Q-FPLC column and purified as described under "Experimental Procedures." Fractions were assayed by the carbazole reaction (), for metachromasia (), and NaCl concentration (- - - -). The fractions indicated by horizontal bars were pooled, dialyzed against distilled water, and lyophilized. B and D, the Mono Q-purified sample (10 mg) was reapplied to the same column, eluted, and recovered as described above. The vertical arrows in the panels indicate the elution of standard mammalian dermatan sulfate (DS) and heparin on the Mono Q-FPLC column.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Agarose gel electrophoresis of the ascidian and mammalian dermatan sulfates and of standard glycosaminoglycans, before and after incubation with chondroitin AC and ABC lyases. Purified ascidian dermatan sulfates (see Fig. 1), native and oversulfated mammalian dermatan sulfates, and a mixture of standard glycosaminoglycans (GAGs) containing 10 µg each of chondroitin 4-sulfate (CS), dermatan sulfate (DS), and heparan sulfate (HS) before (None) and after incubation with chondroitin AC (Chase AC) and ABC (Chase ABC) lyases (see "Experimental Procedures") were applied to a 0.5% agarose gel and run for 1 h at 110 V in 1,3-diaminopropane/acetate (pH 9.0). The glycosaminoglycans in the gel were fixed with 0.1% N-cetyl-N,N,N-trimethylammonium bromide solution. After 12 h, the gel was dried and stained with 0.1% toluidine blue in acetic acid/ethanol/water (0.1:5:5, v/v).

The Products Formed by Digestion with Chondroitin ABC Lyase Indicate That the Ascidian Dermatan Sulfate Has a Preponderance of 2-O-Sulfated -L-Iduronic Acid and 4-O-Sulfated N-Acetyl--D-galactosamine Residues-- The digestion of ascidian and mammalian dermatan sulfate by chondroitin AC and ABC lyases was followed by polyacrylamide gel electrophoresis. The molecular masses of these dermatan sulfates were not reduced by digestion with chondroitin AC lyase, but they were totally digested by chondroitin ABC lyase (Fig. 3).

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Polyacrylamide gel electrophoresis of the ascidian and mammalian dermatan sulfates, before and after incubation with chondroitin AC and ABC lyases. Purified ascidian and mammalian dermatan sulfates, before () and after (+) incubation with chondroitin AC (Chase AC) and ABC (Chase ABC) lyases (10 µg of each glycosaminoglycan) were applied to 6% 1-mm-thick polyacrylamide gel slab in 0.02 M sodium barbital (pH 8.6) and run for 30 min at 100 V. After electrophoresis the dermatan sulfates were stained with 0.1% toluidine blue in 1% acetic acid and then washed for about 4 h in 1% acetic acid. The molecular mass (MM) markers were high molecular mass dextran sulfate (S1, ~500 kDa), chondroitin 6-sulfate from shark cartilage (S2, 60 kDa), chondroitin 4-sulfate from whale cartilage (S3, 40 kDa), and low molecular mass dextran sulfate (S4, 8 kDa).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Strong anion-exchange HPLC analysis of the disaccharides formed by chondroitin ABC lyase digestion of ascidian and mammalian dermatan sulfates. A mixture of disaccharide standards (A) and the disaccharides formed by exhaustive action of chondroitin ABC lyase on the dermatan sulfate from S. plicata (B), from H. pyriformis (C), and from bovine mucosa (D) were applied to a 25-cm × 4.6-mm Spherisorb-SAX column, linked to an HPLC system. The column was eluted with a gradient of NaCl as described under "Experimental Procedures." The eluant was monitored for UV absorbance at 232 nm. The numbered peaks correspond to the elution positions of known disaccharide standards as follows. Peak 1, -UA-13-GalNAc(6SO4); peak 2, -UA-13-GalNAc(4SO4); peak 3, -UA(2SO4)-13-GalNAc(6SO4); peak 4, -UA-13-GalNAc(4,6-diSO4); peak 5, -UA(2SO4)-13-GalNAc(4SO4); peak 6, -UA(2SO4)-13-GalNAc(4,6-diSO4).

NMR Spectra Confirm the Preponderance of [4--L-IdceA(2SO₄)-13--D-GalNAc(4SO₄)-1]_n Units in the Ascidian Dermatan Sulfates-- The ¹H NMR spectra of the ascidian and mammalian dermatan sulfates are shown in Fig. 5. The chemical shifts reported in Table II are based on interpretations of the COSY (not shown), TOCSY (Fig. 6), and HMQC (Fig. 7) spectra.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   1H NMR spectra at 600 MHz of the dermatan sulfate from S. plicata (A), H. pyriformis (B), and from mammalian tissue (C). All spectra were recorded at 60 °C for samples in D2O solution. Chemical shifts are relative to internal or external trimethylsilylpropionic acid at 0 ppm. The HOD signal has been partially suppressed by presaturation. Signals designated A refer to those produced by N-acetyl--D-galactosamine, whereas those produced by unsulfated and 2-O-sulfated -L-iduronic acid residues are labeled I and Ia, respectively. X are signals from non-carbohydrate contaminants. Expansions of the 5.5 to 4.7 ppm region of the spectra of the ascidian dermatan sulfates are shown in the insets in A and B. The integrals listed under the proton regions of the spectra are normalized to a total of 100 =protons.

View this table: [in this window] [in a new window]   Table II Proton chemical shifts for residues of -L-iduronate and of N-acetyl--D-galactosamine in ascidian and mammalian dermatan sulfates

View larger version (45K): [in this window] [in a new window]   Fig. 6.   Part of the TOCSY spectrum of the dermatan sulfate from the ascidian H. pyriformis, at 600 MHz, 60oC, in D2O. The spin systems for both 2-O-sulfated (Ia) and unsulfated (I) -L-iduronic acid residues are traced through the spectrum. A similar TOCSY spectrum was obtained for the dermatan sulfate from the ascidian S. plicata (not shown).

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Part of the HMQC spectra of the mammalian dermatan sulfate (A) and of the dermatan sulfate from the ascidian H. pyriformis (B) at 60oC, in D2O. Starting from the proton chemical shifts, it was possible to obtain the values of carbon chemical shifts for mammalian and ascidian dermatan sulfates. Signals designated by A refer to those produced by N-acetyl--D-galactosamine, whereas those of unsulfated and 2-O-sulfated -L-iduronic acid units are labeled I and Ia, respectively.

View this table: [in this window] [in a new window]   Table III Carbon chemical shifts for residues of -L-iduronate and of N-acetyl--D-galactosamine in ascidian and mammalian dermatan sulfates

Summary of Dermatan Sulfate Structures-- Dermatan sulfates with the same backbone structure [4--L-IdceA-13--D-GalNAc-1]_n but with different patterns and proportions of sulfate substitutions have been described in this and in previous studies (Fig. 8). The repetitive disaccharide units of native mammalian dermatan sulfate are sulfated at carbon 4 of the hexosamine moiety; small amounts (~5%) of 2-O-sulfated -L-iduronic acid residues are also found in this mammalian glycosaminoglycan.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Major repetitive disaccharide units of ascidian and mammalian dermatan sulfates. These glycosaminoglycans have the same backbone structure [4--L-IdceA-13--D-GalNAc-1]n but have different patterns of sulfate substitutions. The ascidian dermatan sulfates are highly sulfated at the 2-position of -L-iduronic acid units but differ in the pattern of sulfation of the N-acetyl--D-galactosamine residues. In the species S. plicata and H. pyriformis the hexosamine moieties are 4-O-sulfated, whereas in A. nigra they are 6-O-sulfated. On the partially oversulfated mammalian dermatan sulfate most of the galactosamine residues are sulfated at both 4- and 6-positions; the nonsulfated, 2-sulfated, and 3-sulfated -L-iduronic acid residues are in the proportion of 55:25:20. The repetitive disaccharide units of mammalian dermatan sulfate are sulfated at carbon 4 of the hexosamine moiety, and small amounts of 2-O-sulfated -L-iduronic acid (~5%) residues are also found in this mammalian glycosaminoglycan.

4-O-Sulfation of the N-Acetyl--D-galactosamine Residues Is Essential for the Anticoagulant Activity of Dermatan Sulfate-- Anticoagulant activities determined by the APTT assay are listed in Table IV for ascidian dermatan sulfates and for mammalian dermatan sulfates, before and after chemical oversulfation. Mammalian dermatan sulfate has a specific activity of ~2 units/mg. Oversulfation of this glycosaminoglycan, with the introduction of extra 2-O- and 3-O-sulfation of the -L-iduronic acid residues and extra 6-O-sulfation of the N-acetyl--D-galactosamine residues increases the specific activity in the APTT assay to 13 units/mg.

Dermatan Sulfates from the Ascidians H. pyriformis and S. plicata Are Potent Activators of Thrombin Inhibition by Heparin Cofactor II-- Fig. 9 shows direct measurement of inhibition of thrombin by heparin cofactor II in the presence of ascidian and mammalian dermatan sulfates. The IC₅₀ for thrombin inhibition is 3.0 and 2.0 µg/ml for native and oversulfated mammalian dermatan sulfates, respectively. More interestingly, however, the IC₅₀ for inhibition of thrombin in the presence of heparin cofactor II for the dermatan sulfates from the ascidians H. pyriformis and S. plicata are ~8.6 and ~9.7 times lower when compared with the IC₅₀ for native mammalian dermatan sulfate (Fig. 9 and Table V). In contrast, the IC₅₀ for the dermatan sulfate from the ascidian A. nigra is 320 µg/ml (13).

View larger version (29K): [in this window] [in a new window]   Fig. 9.   Dependence on the dermatan sulfate and heparin concentration for inactivation of thrombin by heparin cofactor II. Heparin cofactor II (68 nM) was incubated with thrombin (15 nM) in the presence of various concentrations of native mammalian dermatan sulfate (), oversulfated mammalian dermatan sulfate (), dermatan sulfate from the ascidians S. plicata () and H. pyriformis (), and of heparin (), before and after incubation with chondroitin ABC lyase (chase ABC). After 60 s, the remaining thrombin activity was determined with a chromogenic substrate (A405 nm/min).

View larger version (24K): [in this window] [in a new window]   Fig. 10.   Dependence on the dermatan sulfate and heparin concentration for inactivation of thrombin (A) or factor Xa (B) by antithrombin. Antithrombin (50 nM) was incubated with thrombin (15 nM) or factor Xa (15 nM) in the presence of various concentrations of native () and oversulfated mammalian dermatan sulfate (), ascidian dermatan sulfate from S. plicata () and heparin (). After 60 s, the remaining thrombin or factor Xa activity was determined with a chromogenic substrate (A405 nm/min).

Activity of the Ascidian Dermatan Sulfates with Mutants of Heparin Cofactor II-- The binding sites in heparin cofactor II for mammalian dermatan sulfate and heparin overlap but are not identical (24, 36, 37). Arg¹⁸⁹ and Lys¹⁷³ residues are specific for dermatan sulfate and heparin binding, respectively, whereas Lys¹⁸⁵ is involved in the binding of both glycosaminoglycans. As a consequence, the Arg¹⁸⁹  His variant requires >50-fold higher than normal concentrations of mammalian dermatan sulfate to accelerate inhibition of thrombin when compared with native recombinant heparin cofactor II. However, mutation of Lys¹⁷³  Gln has no effect on the cofactor activity of mammalian dermatan sulfate (Fig. 11A). By contrast, the Lys¹⁷³  Gln and Lys¹⁸⁵  Asn variants require higher concentrations of heparin for thrombin inhibition than native recombinant heparin cofactor II (Fig. 11B).

View larger version (37K): [in this window] [in a new window]   Fig. 11.   Dependence on the dermatan sulfate and heparin concentration for inactivation of thrombin by native recombinant heparin cofactor II and mutants of Lys173  Gln, Lys185  Asn, and Arg189  His. Native recombinant heparin cofactor II () and mutants of Lys173  Gln (), Lys185  Asn (), and Arg189  His () (68 nM) were incubated with thrombin (15 nM) in the presence of various concentrations of native mammalian dermatan sulfate (A), heparin (B), dermatan sulfate from the ascidians S. plicata (C) and H. pyriformis (D), and chemically oversulfated mammalian dermatan sulfate (E), as described in the legend of Fig. 9. DS, dermatan sulfate.

Conclusions-- Following the report that mammalian dermatan sulfate has anticoagulant activity, although significantly weaker than that of a comparable dose of heparin, several authors attempted to obtain derivatives with higher activity. Among these studies are chemical oversulfation of mammalian dermatan sulfate (23, 38-40), isolation of natural highly sulfated dermatan sulfates from hagfish (41) or from mammalian tissues (42), and preparation of naturally oversulfated sequences from mammalian dermatan sulfate (35, 43).