Structural composition and differential anticoagulant activitiesof dermatan sulfates from the skin of four species of rays,

Dasyatis americana, Dasyatis gutatta, Aetobatus narinariand Potamotrygon motoro

João M.M. Dellias a,b, Glaucia R. Onofre a,b, Cláudio C. Werneck a,b,Ana M. Landeira-Fernandez b, Fabio R. Melo a,b,c, Wladimir R.L. Farias c,

Luiz-Claudio F. Silva a,b,*a Laboratório de Tecido Conjuntivo, Hospital Universitário Clementino Fraga Filho, Brasil

b Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro,21941-590, Caixa Postal 68041, Rio de Janeiro, Brasil

c Departamento de Engenharia de Pesca, Universidade Federal do Ceará, Ceará, Brasil

Received 19 April 2004; accepted 10 September 2004

Available online 02 October 2004

Abstract

We compared the disaccharide composition of dermatan sulfate (DS) purified from the ventral skin of three species of rays from theBrazilian seacoast, Dasyatis americana, Dasyatis gutatta, Aetobatus narinari and of Potamotrygon motoro, a fresh water species that habitsthe Amazon River. DS obtained from the four species were composed of non-sulfated, mono-sulfated disaccharides bearing esterified sulfategroups at positions C-4 or C-6 of N-acetyl galactosamine (GalNAc), and disulfated disaccharides bearing esterified sulfate groups at positionsC-2 of the uronic acid and at position C-4 or C-6 of GalNAc. However, DS from the skin of P. motoro presented a very low content of thedisulfated disaccharides. The anticoagulant actions of ray skin DS, measured by both APTT clotting and HCII-mediated inhibition of thrombinassays, were compared to that of mammalian DS. DS from D. americana had both high APTT and HCII activities, whereas DS from D. gutattashowed activity profiles similar to those of mammalian DS. In contrast, DS from both A. narinari and P. motoro had no measurable activity inthe APTT assay. Thus, the anticoagulant activity of ray skin DS is not merely a consequence of their charge density. We speculate that thedifferences among the anticoagulant activities of these three DS may be related to both different composition and arrangements of thedisulfated disaccharide units within their polysaccharide chains.© 2004 Elsevier SAS. All rights reserved.

Keywords: Ray skin; Glycosaminoglycans; Dermatan sulfate, Disaccharides

1. Introduction

Dermatan sulfate (DS) glycosaminoglycan (GAG) chainsare composed of linear polysaccharides assembled as disac-charide units containing a hexosamine, N-acetyl galac-tosamine (GalNAc) and L-iduronic acid (IdoA) joined by b1,4 or 1,3 linkages respectively and usually sulfated at posi-

tion 4 of GalNAc. Disaccharides with a different number andposition of sulfate groups can be located, in different percent-ages, inside the polysaccharide chains, such as the non-sulfated or disulfated disaccharides in which two sulfategroups are O-linked in position 2 of IdoA and 4 of GalNAc(disaccharide B), in position 2 of IdoA and 6 of GalNAc(disaccharide D), or in positions 4 and 6 of GalNAc (disac-charide E) [1,2]. These heterogeneous structures, in terms ofpercentage of variously sulfated disaccharides and degree ofsulfation, depending on the tissue of origin, are responsiblefor different and more specialized functions of these GAGs(for a review see [1,3]).

* Corresponding author. Departamento de Bioquímica Médica, Centrode Ciências da Saúde, Universidade Federal do Rio de Janeiro, Caixa Pos-tal 68041, Rio de Janeiro, RJ, 21941-590, Brasil. Fax: +55-21-2562-2090.

E-mail address: lclaudio@hucff.ufrj.br (L.-C. Silva).

Biochimie 86 (2004) 677–683

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0300-9084/$ - see front matter © 2004 Elsevier SAS. All rights reserved.doi:10.1016/j.biochi.2004.09.002

DS has been implicated in cardiovascular disease, tu-morogenesis, infection, wound repair, and fibrosis [1,3]. Theinteractions of DS chains with fibroblast growth factor(FGF)-2 and FGF-7 have been studied with respect to cellu-lar proliferation and are implicated in wound repair [1],whereas interactions with hepatocyte growth factor/scatterfactor (HGF/SF) activate the HGF/SF signaling pathway [4].One particularly well-studied DS binding interaction occurswith heparin cofactor II (HCII). This serpin homolog ofantithrombin III acts by inhibiting the procoagulative effectof thrombin. This effect is enhanced 1000-fold in the pres-ence of DS. Oversulfated DS preparations purified from thebodies of two kinds of ascidians, which are characterized bythe predominant disulfated disaccharide units of disaccha-ride B, were shown to exert strong anticoagulant activity [5].It was recently demonstrated that these ascidian DS prepara-tions also exert marked in vitro neurite outgrowth-promotingactivities [6].

DS is the predominant GAG expressed in the skin ofmarine vertebrates. DS isolated from the skin of the eel,Anguilla japonica, was shown to be composed mainly ofmono-sulfated disaccharides bearing esterified sulfategroups at positions C-4 of GalNAc, and disulfated disaccha-rides units of disaccharide B. The presence of 3-sulfatedand/or 2,3-sulfated uronic acid residues was also suggested[7]. On the other hand, studies on DS from the skin of the rayRaja clavata, which habits the Greek seacoast, showed thepresence of two DS with different degrees of sulfation: onewith low sulfated content and composed of non-sulfated, andmono-sulfated disaccharides bearing esterified sulfategroups at positions C-4 or C-6 of GalNAc and the other DSwas composed mainly of mono-sulfated disaccharides bear-ing esterified sulfate groups at positions C-4 or C-6 of Gal-NAc, and disulfated disaccharides with a peculiar sulfationpattern. The presence of 2-sulfated and/or 2,3-sulfated uronicacid residues in this ray skin DS was suggested [8–11]. Here,we provide additional information on DS of ray skin byanalyzing DS from the ventral skin of three species that habitthe Brazilian seacoast, Dasyatis americana, Dasyatis gu-tatta, Aetobatus narinari and of Potamotrygon motoro, aspecies that habits the Amazon River.

2. Materials and methods

2.1. Materials

Chondroitin 4-sulfate (C-4S), DS, heparan sulfate (HS),twice-crystallized papain (15 U/mg protein), the standarddisaccharides a-DUA(2SO4)-1→3-GlcNAc(4SO4) (Ddi-diSB), a-DUA(2SO4)-1→3-GalNAc(6SO4) (Ddi-diSD),a-DUA-1→3-GalNAc(4,6-diSO4) (Ddi-diSE) anda-DUA(2SO4)-1→3-GalNAc(4,6-diSO4) (Ddi-triS) werepurchased from Sigma Chemical Co. (St. Louis, MO, USA).Chondroitin AC lyase (EC 4.2.2.5) from Arthrobacter aure-scens, chondroitin ABC lyase (EC 4.2.2.4) from Proteus

vulgaris were purchased from Seikagaku American Inc.(Rockville, MD, USA). The standard disaccharides a-DUA-1→3-GlcNAc (Ddi-0S), a-DUA-1→3-GalNAc(4SO4) (Ddi-4S) and a-DUA-1→3-GalNAc(6SO4) (Ddi-6S), were pur-chased from Seikagaku American Inc. (Rockville, MD). Theabbreviations used were a-DUA(2SO4), a-D4,5-unsaturatedhexuronic acid 2-sulfated; a-DUA, a-D4,5-unsaturated hexu-ronic acid; GalNAc, N-acetylated galactosamine;GalNAc(4SO4) and GalNAc(6SO4) derivatives ofN-acetylated galactosamine bearing a sulfate ester at position4 and at position 6, respectively; Ddi-0S, a-DUA-1→3-GlcNAc; Ddi-4S, a-DUA-1→3-GalNAc(4SO4); Ddi-6S,a-DUA-1→3-GalNAc(6SO4); Ddi-diSB, a-DUA(2SO4)-1→3-GlcNAc(4SO4); Ddi-diSD, a-DUA(2SO4)-1→3-GalNAc(6SO4); Ddi-diSE, a-DUA-1→3-GalNAc(4,6-diSO4) and Ddi-triS, a-DUA(2SO4)-1→3-GalNAc(4,6-diSO4). APTT, activated partial thromboplastin time.

2.2. Ray skin samples

Skin samples from the rays, D. americana, D. gutatta, andA. narinari, which habit the seacoast of the state of Ceará inBrazil and from P. motoro, a fresh water species that habitsthe Amazon river, were provided by the Department of Engi-neering of Fishes, Federal University of Ceará, Ceará state,Brazil.

2.3. Isolation and purification of skin GAGs

GAGs were isolated from skin samples following a previ-ously described method [12]. Briefly, dried skin sampleswere suspended in sodium acetate buffer, pH 5.5, containing40 mg papain in the presence of 5 mM EDTA and 5 mMcysteine at 60 °C for 24 h. The incubation mixtures were thencentrifuged (2000 × g for 10 min at room temperature) andGAGs in the supernatants were precipitated with three vol-umes of absolute ethanol and maintained at 4 °C for 24 h. Theprecipitates formed were collected by centrifugation, freezedried and dissolved in 2 ml of distilled water. The crude GAGpreparations were applied to DEAE-cellulose columns (10 ×1.5 cm) equilibrated with 0.5 M sodium acetate (pH 5.0). Thecolumn was washed with 20 ml of 0.15 M NaCl in the samebuffer. Then, the column-bound skin GAGs were elutedstep-wise with 20 ml of 2.0 M NaCl in the same acetatebuffer. The GAGs eluted from the column were exhaustivelydialyzed against distilled water, lyophilised and dissolved in1.0 ml of distilled water. The partially purified GAG fractionswere applied to a Mono Q-FPLC column (HR 5/5), equili-brated with 20 mM Tris:HCl (pH 8.0). The column waswashed with 20 ml of the same buffer. The column-boundskin GAGs were eluted with 30 ml of a salt linear gradientranging from 0 to 3 M NaCl in the same Tris buffer. Fractionswere monitored by their metachromatic property using 1,9-dimethylmethylene blue [13], and by the carbazole reactionfor hexuronic acid [14]. The fractions corresponding to sul-fated GAGs, composed of a metachromatic peak containing-

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uronic acid, were pooled, exhaustively dialyzed against dis-tilled water, lyophilised and dissolved in 1.0 ml of distilledwater.

2.4. Identification of the skin GAGs

Skin GAGs were characterized by agarose gel electro-phoresis, before and after digestion with chondroitin lyasesand deaminative cleavage with nitrous acid [15], as describedbelow.

2.4.1. Agarose gel electrophoresisApproximately 0.01 mg of skin sulfated GAGs, before or

after enzymatic or chemical treatments, as well as a mixtureof standard C-4S, DS and HS (0.01 mg of each) were appliedto 0.5% agarose gels in 0.05 M 1,3-diaminopropane:acetate(pH 9.0). After electrophoresis, GAGs were fixed in the gelwith 0.1% N-cetyl-N,N,N-trimethylammonium bromide inwater, and stained with 0.1% toluidine blue in acetic acid:e-thanol:water (0.1:5:5, v/v).

2.4.2. Enzymatic depolymerization of the skin GAGs

2.4.2.1. Digestion with condroitin lyases. Digestions withchondroitin AC and ABC lyases were carried out accordingto Saito et al. [16]. Approximately 0.1 mg of skin GAGs wereincubated with 0.3 units of chondroitin AC or ABC lyases for8 h at 37 °C in 0.1 ml of 50 mM Tris:HCl (pH 8.0) containing5 mM EDTA and 15 mM sodium acetate.

2.4.2.2. Deaminative cleavage with nitrous acid. Deamina-tion by nitrous acid at pH 1.5, was performed as described byShively and Conrad [17]. Briefly, approximately 0.1 mg ofskin GAGs were incubated with 0.2 ml of fresh generatedHNO2 at room temperature for 90 min.

2.5. Analysis of the disaccharides from purified skin DSformed by enzymatic depolymerization on a SAX-HPLC

Purified skin sulfated GAGs obtained on Mono Q-FPLCwere submitted to exhaustive digestion with chondroitin AClyase (see above). Disaccharides and chondroitin AC lyase-resistant GAGs (composed of either intact DS or DS plus HSchains) were recovered by a Superdex peptide-column (Am-ersham Pharmacia Biotech) linked to a HPLC system fromShimadzu (Tokyo, Japan). The column was eluted with dis-tilled water:acetonitrile:trifluoroacetic acid (80:20:0.1,v/v)at a flow rate of 0.5 ml/min. Fractions of 0.5 ml were col-lected, monitored for UV absorbance at 232 nm and by theirmetachromatic property using 1,9-dimethylmethylene blue.Fractions corresponding to the chondroitin AC lyase-resistant GAGs (eluted at the void volume) were pooled,freeze dried, and stored at –20 °C.

Purified skin sulfated GAGs (containing either DS or DSplus HS chains), obtained after chondroitin AC lyase diges-tion (void volume of Superdex peptide HPLC, see above)

were submitted to exhaustive digestion with chondroitinABC lyase. Unsaturated disaccharides, derived from DSchains, were recovered by a Superdex peptide-column linkedto a HPLC system. The column was eluted with distilledwater:acetonitrile:trifluoroacetic acid (80:20:0.1,v/v) at aflow rate of 0.5 ml/min. Fractions of 0.5 ml were collected,monitored for UV absorbance at 232 nm. Fractions corre-sponding to unsaturated disaccharides were pooled, freezedried, and stored at –20 °C.

The lyase-derived DS unsaturated disaccharides and stan-dard compounds were subjected to a SAX-HPLC analyticalcolumn (250 × 4.6-mm, Sigma-Aldrich), as follows. Afterequilibration in the mobile phase (distilled water adjusted topH 3.5 with HCl) at 0.5-ml/min, samples were injected andunsaturated disaccharides eluted with a linear gradient ofNaCl from 0 to 1.5 M over 50 min in the same mobile phase.The eluant was monitored for UV absorbance at 232 nm forcomparison with lyase derived disaccharide standards [18].

2.6. Anticoagulant action of the skin DS measured byAPTT clotting assay

Pure preparations of skin DS (free of contamination withCS and/or HS) from the four species of ray were obtained bysubmitting purified skin sulfated GAGs obtained on MonoQ-FPLC to exhaustive digestion with chondroitin AC lyaseto degrade contaminant CS, in the case of all four ray species,and sequentially treating only the A. narinari sample withnitrous acid to degrade HS contaminant present in this frac-tion. Degradative products and intact DS chains were sepa-rately recovered by a Superose 12-FPLC gel filtration col-umn. These purified DS fractions were analyzed by agarosegel electrophoresis to certify the absence of contamination byother sulfated GAG species and used in the anticoagulantassays described below.

APTT clotting assays were carried out as described [5].Normal citrate-anticoagulated human plasma (0.09 ml) wasincubated with 0.01 ml of a solution of purified skin ray DSor standard mammalian DS at different concentrations and0.1 ml of kaolin + bovine brain phospholipid reagent (Re-agent Celite, Biolab, Merieux). After 5 min of incubation at37 °C, 0.1 ml of 0.25 M CaCl2 was added, and the clottingtime was recorded in a coagulometer (Amelung KC4A).

2.7. Inhibition of thrombin by HCII in the presence of skinray DS

Incubations were performed in disposable semimicrocu-vettes. The final concentrations of reactants included 68 nmHCII, 15 nm thrombin (both from Diagnostica Stago, As-nières, France) and 0–1 mg/ml of DS samples in 0.1 ml of0.02 Tris:HCl, 0.15 M NaCl, and 1.0 mg/ml polyethyleneglycol (p.H 7.4) (TS/PEG buffer). Thrombin was added lastto initiate the reaction. After a 60-s incubation at roomtemperature, 0.5 ml of 0.1 mM chromogenic substrateS-2238 (Chromogenix AB, Molndal, Sweden) in TS/PEG

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buffer was added, and the absorbance at 405 nm was re-corded for 120 s. The rate of change of absorbance wasproportional to the thrombin activity remaining in the incu-bation. No inhibition occurred in control experiments inwhich thrombin was incubated with HCII in absence of DS.Nor did inhibition occur when thrombin was incubated withDS alone over the range of concentrations tested [5].

3. Results

3.1. Isolation and purification of skin ray sulfated GAGs

GAGs were extracted from skin samples by papain diges-tion and partially purified on an anion-exchange chromatog-raphy column, where the column-bound GAGs were elutedstep-wise with 2 M NaCl. Subsequently, the crude GAGfractions were applied into a Mono Q-FPLC column and theGAGs were eluted by a linear salt gradient ranging from 0 to3 M NaCl. The presence of sulfated GAGs in the chromato-graphic fractions was monitored by their metachromaticproperty and by the content of uronic acid (Fig. 1A–D). Forall four species of ray, sulfated GAGs eluted as nearly onepeak. The fractions containing the sulfated GAGs werepooled, as indicated in Fig. 1, dialyzed, lyophilised andcharacterized by agarose gel electrophoresis, as follows.

3.2. Characterization of skin sulfated GAGs

The purified sulfated GAGs isolated from the skin of thefour ray species were characterized by agarose gel electro-phoresis, before and after enzymatic digestions with chon-droitin AC and ABC lyases and by deaminative cleavage bynitrous acid (Fig. 2A–D). In D. americana, D. gutatta and

P. motoro, nearly only one electrophoretic band with migra-tion similar to that of DS standard, which was resistant totreatment with chondroitin AC lyase (that specifically de-grades CS standard) and completely removed by treatmentwith chondroitin ABC lyase (that specifically degrades bothCS and DS standards), was observed, demonstrating that DSwas the main sulfated GAG species in these fractions. Thepresence of a faint band, with electrophoretic migration simi-lar to that of CS standard and that was removed after treat-ment with chondroitin AC lyase was also detected, showing asmall contamination of the DS fractions with CS(Fig. 2A,B,D, respectively). The sulfated GAG fraction ofA. narinari showed a more heterogeneous composition. Twoelectrophoretic bands were distinguished, where the majorone was partially degraded by treatment with chondroitinaseAC lyase and completely removed by treatment with chon-droitinase ABC, showing the presence of both CS and DSchains. A faint electrophoretic band showing migration simi-lar to that of HS standard and that was resistant to treatmentwith both condroitin lyases, but disappeared after treatmentwith nitrous acid (that degrades HS standard) was also de-tected (Fig. 2C). These results showed that CS, DS and asmall amount of HS were present in the sulfated GAG frac-tion of A. narinari.

3.3. Disaccharide composition of skin ray DS chains

In order to remove the presence of CS chains in thepurified ray skin sulfated GAG fractions, which will affectthe disaccharide analysis of DS, these fractions were exhaus-tively treated with chondroitin AC lyase and the chondroitinAC lyase-resistant GAGs (composed of either intact DS orDS plus HS chains) were recovered by gel filtration chroma-tography (not shown). The CS-depleted fractions were ex-

Fig. 1. Purification of sulfated GAGs from ray skin of D. americana (A), D. gutatta (B), A. narinari (C) and P. motoro (D) on a Mono Q-FPLC column. TheDEAE-cellulose-purified GAGs were applied into a Mono Q-FPLC and purified as described. Fractions were monitored by their metachromatic property (+)and by the content of uronic acid (O). The fractions corresponding to sulfated GAGs, composed of a metachromatic peak containing-uronic acid (as indicatedby horizontal bars) were pooled, dialyzed against distilled water and lyophilized.

680 J.M. Dellias et al. / Biochimie 86 (2004) 677–683

haustively treated with chondroitin ABC lyase and the unsat-urated disaccharides formed by the depolimerization of DSchains were analysed by HPLC-anion exchange chromatog-raphy. The disaccharide composition of ray skin DS is shownin Table 1. The main unsaturated disaccharide found in allfour species of rays was Ddi-4S. Variable small amounts ofDdi-0S and Ddi-6S were also observed (Table 1a–d). Signifi-cant amounts of Ddi-diSB, accompanied by small content ofDdi-diSD were found in the ray species, D. americana, D. gu-tatta and A. narinari (Table 1a–c). Small amount of Ddi-diSB

was also detected in DS from the fresh water ray species,P. motoro (Table 1d).

3.4. DS from D. americana, D. gutatta and A. narinari,which show similar disaccharide compositions, presenteddifferences in their anticoagulant activities

Differences were observed in the anticoagulant action ofthe three ray skin DS, measured by APTT clotting assay,when compared to mammalian DS. Among the three ray skinDS, the most effective in prolonging the APTT was that fromD. Americana (Fig. 3, closed circles), even when comparedto mammalian DS (Fig. 3, open circles), whereas DS fromD. gutatta (Fig. 3, closed squares) exhibited similar activity

as that of mammalian DS. In contrast, DS from A. narinaripresented no measurable activity in the APTT assay (Fig. 3,open squares). As expected, DS from P. motoro also showedno activity in this assay (Fig. 3, closed triangles). Fig. 4shows direct measurement of inhibition of thrombin by hep-arin cofactor II in the presence ray skin and mammalian DS.The IC50 for thrombin inhibition is 12.0 mg/ml for mamma-lian DS (Fig. 4, open circles). Interestingly, the IC50 forthrombin inhibition in the presence of heparin cofactor II forskin DS from rays D. americana (IC50 = 1.0 mg/ml) andD. guttata (IC50 = 3.0 mg/ml) are 12 and 4 times lower whencompared with the IC50 for mammalian DS (Fig. 4, closed

Fig. 2. Agarose gel electrophoresis of purified sulfated GAGs from the skin of D. americana (A), D. gutatta (B), A. narinari (C) and P. motoro (D) (for detailssee Fig. 1), before (–) and after (+) chondroitin AC and ABC lyase digestions or deaminative cleavage by nitrous acid. After enzymatic or chemical incubation,the sulfated GAGs were applied to 0.5% agarose gel and electrophoresis was carried out as described. CS, chondroitin 4-sulfate; DS, dermatan sulfate; HS,heparan sulfate.

Table 1Disaccharide composition of DS from ray skin

Unsaturated disaccharide (%)Ray species Ddi-0S Ddi-4S Ddi-6S Ddi-diSB Ddi-diSD

a) D. americana 12 50 5 30 3b) D. gutatta 3 51 11 33 2c) A. narinari 10 47 11 24 8d) P. motoro 3 75 16 6 n.d.

n.d.: not detected.

Fig. 3. Analysis of the anticoagulant activity of skin ray DS as measured bythe APTT test. Mammalian DS is included for comparison. The panel showsa typical result representative of two different experiments.

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circles and closed squares, respectively). In contrast, the IC50

for skin DS from rays A. narinari (Fig. 4, open squares) andP. motoro (Fig. 4, closed triangles) are 40.0 mg/ml and>100.0 mg/ml, respectively.

4. Discussion

DS is the predominant GAG expressed in the skin ofmarine vertebrates. DS isolated from the skin of the eel,Anguilla japonica, was shown to be composed mainly ofmono-sulfated disaccharides bearing esterified sulfategroups at positions C-4 of GalNAc (81%), and disulfateddisaccharides units of disaccharide B (12%). Based on 1Hnuclear magnetic resonance spectroscopy, the presence of3-sulfated and/or 2,3-sulfated IdoA residues was suggested[7]. Interestingly, Sakai et al. [7] showed that although thecontent of the disaccharide B sequence (which is required forthe binding of DS to HCII) in eel skin DS was twofold higherthan that of DS from porcine skin, the eel skin DS showed alesser anticoagulant activity when compared to that of theporcine DS sample [7].

DS with distinguished disaccharide composition and de-gree of sulfation has been reported in ray skin [8,10,11]. DSfrom the skin of the ray R. clavata, which habits the Greekseacoast, was shown to contain two DS with different de-grees of sulfation: DSI, which was the major DS species inthe ray skin, and a low-sulfated DS (LSDS) as the minor one[8]. LSDS was shown to be composed of non-sulfated (44%),and mono-sulfated disaccharides bearing esterified sulfatedgroups at positions C-4 or C-6 of GalNAc (53% and 3%,respectively) [11]. DSI was composed mainly of mono-sulfated disaccharides bearing esterified sulfate groups atpositions C-4 or C-6 of GalNAc (61% and 4%, respectively),

and disulfated disaccharides with a peculiar sulfation pattern(32%) and a minor portion of non-sulfated disaccharides(3%). Using disaccharide analysis by HPLC methods, theauthors suggested that the disulfated disaccharides werecomposed of: units bearing esterified sulfate groups at posi-tions C-2 and C-3 of the uronic acid residues linked toGalNAc (64%), units bearing esterified sulfate groups atpositions C-3 of the uronic acid and at position C-6 ofGalNAc (disaccharide K) (20%) and units bearing esterifiedsulfate groups at positions C-2 of the uronic acid and atposition C-6 of GalNAc (disaccharide D) (13%) [8,10].

In the present work, we provide additional information onDS of ray skin by analysing DS from the ventral skin of threespecies that habit the Brazilian seacoast, D. americana,D. gutatta and A. narinari, and of P. motoro, a fresh waterspecies that habits theAmazon River. Our results showed thatDS from the four ray species were composed mainly ofmono-sulfated disaccharides bearing esterified sulfategroups at positions C-4 of GalNAc (50%, 51% and 47% forD. americana, D. gutatta and A. narinari, respectively, and75% for P. motoro). Variable small amounts of non-sulfated(12%, 3%, 10% and 12%) and of mono-sulfated disaccha-rides bearing esterified sulfate groups at positions C-6 ofGalNAc (5%, 11%, 11% and 16%) were also observed forD. americana, D. gutatta, A. narinari, and P. motoro, respec-tively. Significant amounts of disulfated disaccharides bear-ing esterified sulfate groups at positions C-2 of the uronicacid and at position C-4 of GalNAc (disaccharide B) (30%,33% and 24%), accompanied by small content of disulfateddisaccharides bearing esterified sulfate groups at positionsC-2 of the uronic acid and at position C-6 of GalNAc (disac-charide D) (3%, 2% and 8%) were found in the marine rayspecies, D. americana, D. gutatta and A narinari, respec-tively. Small amount of disaccharide B (6%) was also de-tected in P. motoro. We found no evidences to the presence of2-sulfated or 3-sulfated and/or 2,3-sulfated uronic acid resi-dues among the ray skin DS analysed here.

Altogether, our results on DS from the skin of the four rayspecies studied and the data reported on DS structure fromeel skin (Anguilla japonica) and from ray skin of R. clavatashowed structural heterogeneities among DS from skin ofdifferent marine vertebrates. The nature of such structuraldiversity among these skin DS is not yet known, in particular,among skin DS from different ray species, as commentedabove.

DS from eel skin, which was shown to present a highcontent of the disaccharide B sequence, showed a lesseranticoagulant activity when compared to that of the porcineDS skin, which was shown to present twofold lesser disac-charide B sequences than the DS from eel skin [7]. It hasbeen reported that the presence in the structure of DS ofcontiguous sequences of disaccharide B, of at least fourrepeating sequences, is required for anticoagulant activitythrough HCII [19]. Therefore, Sakai et al. [7] suggested, asone possible explanation why eel skin DS has a very lowanticoagulant activity, that the disaccharide B sequences in

Fig. 4. Dependence on the concentration of skin ray DS for inactivation ofthrombin by HCII. Mammalian DS is included for comparison. The assayswere performed with a chromogenic substrate for thrombin as referred to inSection 2. After 120 s, the remaining thrombin activity was determined(A405 nm/min). The panel shows a typical result representative of twodifferent experiments.

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DS from eel skin might be delocalised, and there was nocontiguous sequences of these disaccharide units. Whether ornot ray skin DS from R. clavata presents appreciable antico-agulant activity is not yet known.

Differences were observed in the anticoagulant action ofskin DS from D. americana, D. gutatta and A. narinari,measured by both APTT clotting and HCII-mediated inhibi-tion of thrombin assays, when compared to mammalian DS.DS from D. americana had both high APTT and HCII activi-ties, whereas DS from D. gutatta showed activity profilessimilar to those of mammalian DS. In contrast, DS fromA. narinari had no measurable activity in the APTT assay. Aswe used pure preparations of ray skin DS, free of CS and/orHS contaminations (see Section 2), these differences wereexclusively due to the ray skin DS. We speculate that apossible explanation for these different anticoagulant activi-ties would be related to differences in the disaccharide com-position and/or in the arrangement of the disulfated disaccha-ride B units within the chains of these three ray skin DS. DSfrom both D. americana and D. gutatta presented a verysimilar disaccharide composition, but very different antico-agulant activities. DS from D. americana might present con-tiguous disulfated disaccharide B units, whereas in DS ofD. gutatta these units might be delocalised resulting in thelack of contiguous sequences of this unit, likely to what waspreviously suggested by Sakai et al. [7] in the case of eel skinDS. DS from A. narinari presented a content of disaccharideD units higher than those in the DS from the other two rayspecies. This DS had no measurable activity in the APTTassay. In this case, beside the possible delocalisation of thedisaccharide B units, the presence of the disaccharide D unitsin this DS might be also negatively affecting its anticoagulantactivity. Ascidian DS with a high content of disaccharide Dunits has been reported to has no discernible anticoagulantactivity [5]. A detailed study of this point on the DS chainsfrom the skin of these rays is necessary to clarify these issues.

Acknowledgments

This work was supported by Conselho Nacional de De-senvolvimento Científico e Tecnológico (CNPq: PADCT andPRONEX), Fundação de Amparo à Pesquisa do Estado doRio de Janeiro (FAPERJ) and Coordenação de Aperfeiçoa-mento de Pessoal de Ensino Superior (CAPES: PROCAD).

References

[1] J.M. Trowbridge, R.L. Gallo, Dermatan sulfate: new functions froman old glycosaminoglycan, Glycobiology 12 (2002) 117R–125R.

[2] N. Volpi, Disaccharide mapping of chondroitin sulfate of differentorigins by high-performance capillary electrophoresis and high-performance liquid chromatography, Carbohydrate Polymers 55(2004) 273–281.

[3] K. Sugahara, T. Mikami, T. Uyama, S. Mizuguchi, K. Nomura,H. Kitagawa, Recent advances in the structural biology of chondroitinsulfate and dermatan sulfated, Current Opinion in Structural Biology13 (2003) 612–620.

[4] M. Lyon, J.A. Deakin, J.T. Gallagher, The mode of action of heparanand dermatan sulfates in the regulation of hepatocyte growthfactor/scatter factor, The Journal of Biological Chemistry 277 (2002)1040–1046.

[5] M.S.G. Pavão, K.R.M. Aiello, C.C. Werneck, L.C.F. Silva,A.P. Valente, B. Mulloy, et al., Highly sulfated dermatan sulfates fromascidians. Structure versus anticoagulant activity of these glycosami-noglycans, J. Biol. Chem. 273 (1998) 27848–27857.

[6] M. Hikino, T. Mikami, A. Faissner, A.C.E.S. Vilela-Silva,M.S.G. Pavão, K. Sugahara, Oversulfated dermatan sulfate exhibitisneurite outgrowth-promoting activity toward embryonic mouse hip-pocampal neurons, The Journal of Biological Chemistry 278 (2003)43744–43754.

[7] S. Sakai, W.S. Kim, I.S. Lee, Y.S. Kim, A. Nakamura, T. Toida,T. Imanari, Purification and characterization of dermatan sulfate fromthe skin of the eel, Anguilla japonica, Carbohydrate Research 338(2003) 263–269.

[8] N.K. Karamanos, A. Manouras, D. Politou, P. Gritsoni, T. Tsegenidis,Isolation and high-performance liquid chromatographic analysis ofRay (Raja clavata) skin glycosaminoglycans, Comp. Biochem.Physiol. B 100 (1991) 827–832.

[9] T. Tsegenidis, Influence of oversulphation and neutral sugar presenceon the chondroitinases AC and ABC actions towards glycosaminogly-cans from ray (Raja clavata) and squid (Illex illecebrosus coidentii)skin, Comp. Biochem. Physiol. B 103 (1992) 275–279.

[10] C.C. Chatziioannidis, N.K. Karamanos, T. Tsegenidis, Isolation andcharacterisation of a small dermatan sulphate proteoglycan from rayskin: Raja clavata, Comp. Biochem. Physiol. B 124 (1999) 15–24.

[11] C.C. Chatziioannidis, N.K. Karamanos, S.T. Anagnostides, T. Tseg-enidis, Purification and characterization of a minor low-sulphateddermatan sulphate-proteoglycan from ray skin, Biochimie 81 (1999)187–196.

[12] C.O. Passos, C.C. Werneck, G.R. Onofre, E.A. Pagani, A.L. Filgueira,L.C.F. Silva, Comparative biochemistry of human skin: glycosami-noglycans from different body sites in normal subjects and in patientswith localized scleroderma, Journal of the European Academy ofDermatology and Venereology 17 (2003) 14–19.

[13] R.W. Farndale, D.J. Buttle, A.J. Barret, Improved quantitation anddiscrimination of sulfated glycosaminoglycans by use of dimethylm-ethylene blue, Biochim. Biophys. Acta 883 (1986) 173–177.

[14] Z.A. Dische, A new specific color reaction of hexuronic acids, J. Biol.Chem. 167 (1947) 189–198.

[15] L.C.F. Silva, Isolation and purification of glycosaminoglycans, in:N. Volpi (Ed.), Analytical Techniques to Evaluate the Structure andFunctions of Natural Polysaccharides, Glycosaminoglycans,Research Signpost, Trivandrum, 2002, pp. 1–14.

[16] N. Saito, T.Yamagata, S. Suzuki, Enzymatic methods for the determi-nation of small quantities of isomeric chondroitin sulfates, J. Biol.Chem. 243 (1968) 1536–1544.

[17] J.E. Shively, H.E. Conrad, Formation of anhydrosugars in the chemi-cal depolymerization of heparin, Biochemistry 15 (1976) 3932–3942.

[18] K. Arcanjo, G. Belo, C. Folco, C.C. Werneck, R. Borojevic,L.C.F. Silva, Biochemical characterization of heparan sulfate derivedfrom murine hemopoietic stromal cell lines: a bone marrow-derivedcell line S17 and a fetal liver-derived cell line AFT024, Journal ofCellular Biochemistry 87 (2002) 160–172.

[19] G. Mascellani, L. Liverani, B. Parma, G. Bergonzini, P. Bianchini,Active site for heparin cofactor II in low molecular mass dermatansulfate. Contribution to the antithrombotic activity of fractions withhigh affinity for heparin cofactor II, Thromb. Res. 84 (1996) 21–32.

683J.M. Dellias et al. / Biochimie 86 (2004) 677–683