raia

1. Structural composition and differential anticoagulant activities
of dermatan sulfates from the skin of four species of rays,
Dasyatis americana, Dasyatis gutatta, Aetobatus narinari
and 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 the
Brazilian seacoast, Dasyatis americana, Dasyatis gutatta, Aetobatus narinari and of Potamotrygon motoro, a fresh water species that habits
the Amazon River. DS obtained from the four species were composed of non-sulfated, mono-sulfated disaccharides bearing esterified sulfate
groups at positions C-4 or C-6 of N-acetyl galactosamine (GalNAc), and disulfated disaccharides bearing esterified sulfate groups at positions
C-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 the
disulfated disaccharides. The anticoagulant actions of ray skin DS, measured by bothAPTT clotting and HCII-mediated inhibition of thrombin
assays, were compared to that of mammalian DS. DS from D. americana had both highAPTT and HCII activities, whereas DS from D. gutatta
showed activity profiles similar to those of mammalian DS. In contrast, DS from both A. narinari and P. motoro had no measurable activity in
the APTT assay. Thus, the anticoagulant activity of ray skin DS is not merely a consequence of their charge density. We speculate that the
differences among the anticoagulant activities of these three DS may be related to both different composition and arrangements of the
disulfated 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) chains
are composed of linear polysaccharides assembled as disac-
charide units containing a hexosamine, N-acetyl galac-
tosamine (GalNAc) and L-iduronic acid (IdoA) joined by b
1,4 or 1,3 linkages respectively and usually sulfated at posi-
tion 4 of GalNAc. Disaccharides with a different number and
position 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 sulfate
groups 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 of
percentage of variously sulfated disaccharides and degree of
sulfation, depending on the tissue of origin, are responsible
for different and more specialized functions of these GAGs
(for a review see [1,3]).
* Corresponding author. Departamento de Bioquímica Médica, Centro
de 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
www.elsevier.com/locate/biochi
0300-9084/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.biochi.2004.09.002

2. DS has been implicated in cardiovascular disease, tu-
morogenesis, infection, wound repair, and fibrosis [1,3]. The
interactions 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/scatter
factor (HGF/SF) activate the HGF/SF signaling pathway [4].
One particularly well-studied DS binding interaction occurs
with heparin cofactor II (HCII). This serpin homolog of
antithrombin III acts by inhibiting the procoagulative effect
of thrombin. This effect is enhanced 1000-fold in the pres-
ence of DS. Oversulfated DS preparations purified from the
bodies of two kinds of ascidians, which are characterized by
the 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-promoting
activities [6].
DS is the predominant GAG expressed in the skin of
marine vertebrates. DS isolated from the skin of the eel,
Anguilla japonica, was shown to be composed mainly of
mono-sulfated disaccharides bearing esterified sulfate
groups at positions C-4 of GalNAc, and disulfated disaccha-
rides units of disaccharide B. The presence of 3-sulfated
and/or 2,3-sulfated uronic acid residues was also suggested
[7]. On the other hand, studies on DS from the skin of the ray
Raja clavata, which habits the Greek seacoast, showed the
presence of two DS with different degrees of sulfation: one
with low sulfated content and composed of non-sulfated, and
mono-sulfated disaccharides bearing esterified sulfate
groups at positions C-4 or C-6 of GalNAc and the other DS
was 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 sulfation
pattern. The presence of 2-sulfated and/or 2,3-sulfated uronic
acid residues in this ray skin DS was suggested [8–11]. Here,
we provide additional information on DS of ray skin by
analyzing DS from the ventral skin of three species that habit
the Brazilian seacoast, Dasyatis americana, Dasyatis gu-
tatta, Aetobatus narinari and of Potamotrygon motoro, a
species 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 standard
disaccharides 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) and
a-DUA(2SO4)-1→3-GalNAc(4,6-diSO4) (Ddi-triS) were
purchased 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). The
abbreviations used were a-DUA(2SO4), a-D4,5
-unsaturated
hexuronic acid 2-sulfated; a-DUA, a-D4,5
-unsaturated hexu-
ronic acid; GalNAc, N-acetylated galactosamine;
GalNAc(4SO4) and GalNAc(6SO4) derivatives of
N-acetylated galactosamine bearing a sulfate ester at position
4 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, and
A. narinari, which habit the seacoast of the state of Ceará in
Brazil and from P. motoro, a fresh water species that habits
the 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 samples
were suspended in sodium acetate buffer, pH 5.5, containing
40 mg papain in the presence of 5 mM EDTA and 5 mM
cysteine at 60 °C for 24 h. The incubation mixtures were then
centrifuged (2000 × g for 10 min at room temperature) and
GAGs in the supernatants were precipitated with three vol-
umes of absolute ethanol and maintained at 4 °C for 24 h. The
precipitates formed were collected by centrifugation, freeze
dried and dissolved in 2 ml of distilled water. The crude GAG
preparations were applied to DEAE-cellulose columns (10 ×
1.5 cm) equilibrated with 0.5 M sodium acetate (pH 5.0). The
column was washed with 20 ml of 0.15 M NaCl in the same
buffer. Then, the column-bound skin GAGs were eluted
step-wise with 20 ml of 2.0 M NaCl in the same acetate
buffer. The GAGs eluted from the column were exhaustively
dialyzed against distilled water, lyophilised and dissolved in
1.0 ml of distilled water. The partially purified GAG fractions
were applied to a Mono Q-FPLC column (HR 5/5), equili-
brated with 20 mM Tris:HCl (pH 8.0). The column was
washed with 20 ml of the same buffer. The column-bound
skin GAGs were eluted with 30 ml of a salt linear gradient
ranging from 0 to 3 M NaCl in the same Tris buffer. Fractions
were monitored by their metachromatic property using 1,9-
dimethylmethylene blue [13], and by the carbazole reaction
for hexuronic acid [14]. The fractions corresponding to sul-
fated GAGs, composed of a metachromatic peak containing-
678 J.M. Dellias et al. / Biochimie 86 (2004) 677–683

3. uronic acid, were pooled, exhaustively dialyzed against dis-
tilled water, lyophilised and dissolved in 1.0 ml of distilled
water.
2.4. Identification of the skin GAGs
Skin GAGs were characterized by agarose gel electro-
phoresis, before and after digestion with chondroitin lyases
and deaminative cleavage with nitrous acid [15], as described
below.
2.4.1. Agarose gel electrophoresis
Approximately 0.01 mg of skin sulfated GAGs, before or
after enzymatic or chemical treatments, as well as a mixture
of standard C-4S, DS and HS (0.01 mg of each) were applied
to 0.5% agarose gels in 0.05 M 1,3-diaminopropane:acetate
(pH 9.0). After electrophoresis, GAGs were fixed in the gel
with 0.1% N-cetyl-N,N,N-trimethylammonium bromide in
water, 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 with
chondroitin AC and ABC lyases were carried out according
to Saito et al. [16].Approximately 0.1 mg of skin GAGs were
incubated with 0.3 units of chondroitinAC orABC lyases for
8 h at 37 °C in 0.1 ml of 50 mM Tris:HCl (pH 8.0) containing
5 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 by
Shively and Conrad [17]. Briefly, approximately 0.1 mg of
skin GAGs were incubated with 0.2 ml of fresh generated
HNO2 at room temperature for 90 min.
2.5. Analysis of the disaccharides from purified skin DS
formed by enzymatic depolymerization on a SAX-HPLC
Purified skin sulfated GAGs obtained on Mono Q-FPLC
were submitted to exhaustive digestion with chondroitin AC
lyase (see above). Disaccharides and chondroitin AC lyase-
resistant GAGs (composed of either intact DS or DS plus HS
chains) were recovered by a Superdex peptide-column (Am-
ersham Pharmacia Biotech) linked to a HPLC system from
Shimadzu (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 their
metachromatic 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 DS
plus HS chains), obtained after chondroitin AC lyase diges-
tion (void volume of Superdex peptide HPLC, see above)
were submitted to exhaustive digestion with chondroitin
ABC lyase. Unsaturated disaccharides, derived from DS
chains, were recovered by a Superdex peptide-column linked
to a HPLC system. The column was eluted with distilled
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 collected,
monitored for UV absorbance at 232 nm. Fractions corre-
sponding to unsaturated disaccharides were pooled, freeze
dried, and stored at –20 °C.
The lyase-derived DS unsaturated disaccharides and stan-
dard compounds were subjected to a SAX-HPLC analytical
column (250 × 4.6-mm, Sigma-Aldrich), as follows. After
equilibration in the mobile phase (distilled water adjusted to
pH 3.5 with HCl) at 0.5-ml/min, samples were injected and
unsaturated disaccharides eluted with a linear gradient of
NaCl from 0 to 1.5 M over 50 min in the same mobile phase.
The eluant was monitored for UV absorbance at 232 nm for
comparison with lyase derived disaccharide standards [18].
2.6. Anticoagulant action of the skin DS measured by
APTT clotting assay
Pure preparations of skin DS (free of contamination with
CS and/or HS) from the four species of ray were obtained by
submitting purified skin sulfated GAGs obtained on Mono
Q-FPLC to exhaustive digestion with chondroitin AC lyase
to degrade contaminant CS, in the case of all four ray species,
and sequentially treating only the A. narinari sample with
nitrous 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 agarose
gel electrophoresis to certify the absence of contamination by
other sulfated GAG species and used in the anticoagulant
assays described below.
APTT clotting assays were carried out as described [5].
Normal citrate-anticoagulated human plasma (0.09 ml) was
incubated with 0.01 ml of a solution of purified skin ray DS
or standard mammalian DS at different concentrations and
0.1 ml of kaolin + bovine brain phospholipid reagent (Re-
agent Celite, Biolab, Merieux). After 5 min of incubation at
37 °C, 0.1 ml of 0.25 M CaCl2 was added, and the clotting
time was recorded in a coagulometer (Amelung KC4A).
2.7. Inhibition of thrombin by HCII in the presence of skin
ray DS
Incubations were performed in disposable semimicrocu-
vettes. The final concentrations of reactants included 68 nm
HCII, 15 nm thrombin (both from Diagnostica Stago, As-
nières, France) and 0–1 mg/ml of DS samples in 0.1 ml of
0.02 Tris:HCl, 0.15 M NaCl, and 1.0 mg/ml polyethylene
glycol (p.H 7.4) (TS/PEG buffer). Thrombin was added last
to initiate the reaction. After a 60-s incubation at room
temperature, 0.5 ml of 0.1 mM chromogenic substrate
S-2238 (Chromogenix AB, Molndal, Sweden) in TS/PEG
679J.M. Dellias et al. / Biochimie 86 (2004) 677–683

4. buffer was added, and the absorbance at 405 nm was re-
corded for 120 s. The rate of change of absorbance was
proportional to the thrombin activity remaining in the incu-
bation. No inhibition occurred in control experiments in
which thrombin was incubated with HCII in absence of DS.
Nor did inhibition occur when thrombin was incubated with
DS 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 eluted
step-wise with 2 M NaCl. Subsequently, the crude GAG
fractions were applied into a Mono Q-FPLC column and the
GAGs were eluted by a linear salt gradient ranging from 0 to
3 M NaCl. The presence of sulfated GAGs in the chromato-
graphic fractions was monitored by their metachromatic
property and by the content of uronic acid (Fig. 1A–D). For
all four species of ray, sulfated GAGs eluted as nearly one
peak. The fractions containing the sulfated GAGs were
pooled, as indicated in Fig. 1, dialyzed, lyophilised and
characterized by agarose gel electrophoresis, as follows.
3.2. Characterization of skin sulfated GAGs
The purified sulfated GAGs isolated from the skin of the
four 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 by
nitrous 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 to
treatment with chondroitin AC lyase (that specifically de-
grades CS standard) and completely removed by treatment
with chondroitin ABC lyase (that specifically degrades both
CS and DS standards), was observed, demonstrating that DS
was the main sulfated GAG species in these fractions. The
presence of a faint band, with electrophoretic migration simi-
lar to that of CS standard and that was removed after treat-
ment with chondroitinAC lyase was also detected, showing a
small contamination of the DS fractions with CS
(Fig. 2A,B,D, respectively). The sulfated GAG fraction of
A. narinari showed a more heterogeneous composition. Two
electrophoretic bands were distinguished, where the major
one was partially degraded by treatment with chondroitinase
AC lyase and completely removed by treatment with chon-
droitinase ABC, showing the presence of both CS and DS
chains. A faint electrophoretic band showing migration simi-
lar to that of HS standard and that was resistant to treatment
with both condroitin lyases, but disappeared after treatment
with nitrous acid (that degrades HS standard) was also de-
tected (Fig. 2C). These results showed that CS, DS and a
small 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 the
purified ray skin sulfated GAG fractions, which will affect
the disaccharide analysis of DS, these fractions were exhaus-
tively treated with chondroitin AC lyase and the chondroitin
AC lyase-resistant GAGs (composed of either intact DS or
DS 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. The
DEAE-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 indicated
by horizontal bars) were pooled, dialyzed against distilled water and lyophilized.
680 J.M. Dellias et al. / Biochimie 86 (2004) 677–683

5. haustively treated with chondroitin ABC lyase and the unsat-
urated disaccharides formed by the depolimerization of DS
chains were analysed by HPLC-anion exchange chromatog-
raphy. The disaccharide composition of ray skin DS is shown
in Table 1. The main unsaturated disaccharide found in all
four species of rays was Ddi-4S. Variable small amounts of
Ddi-0S and Ddi-6S were also observed (Table 1a–d). Signifi-
cant amounts of Ddi-diSB, accompanied by small content of
Ddi-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, presented
differences in their anticoagulant activities
Differences were observed in the anticoagulant action of
the three ray skin DS, measured by APTT clotting assay,
when compared to mammalian DS.Among the three ray skin
DS, the most effective in prolonging the APTT was that from
D. Americana (Fig. 3, closed circles), even when compared
to mammalian DS (Fig. 3, open circles), whereas DS from
D. gutatta (Fig. 3, closed squares) exhibited similar activity
as that of mammalian DS. In contrast, DS from A. narinari
presented no measurable activity in the APTT assay (Fig. 3,
open squares). As expected, DS from P. motoro also showed
no activity in this assay (Fig. 3, closed triangles). Fig. 4
shows 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 for
thrombin inhibition in the presence of heparin cofactor II for
skin DS from rays D. americana (IC50 = 1.0 mg/ml) and
D. guttata (IC50 = 3.0 mg/ml) are 12 and 4 times lower when
compared 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 details
see 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 1
Disaccharide 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 3
b) D. gutatta 3 51 11 33 2
c) A. narinari 10 47 11 24 8
d) 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 by
theAPTT test. Mammalian DS is included for comparison. The panel shows
a typical result representative of two different experiments.
681J.M. Dellias et al. / Biochimie 86 (2004) 677–683

6. circles and closed squares, respectively). In contrast, the IC50
for skin DS from rays A. narinari (Fig. 4, open squares) and
P. 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 of
marine vertebrates. DS isolated from the skin of the eel,
Anguilla japonica, was shown to be composed mainly of
mono-sulfated disaccharides bearing esterified sulfate
groups at positions C-4 of GalNAc (81%), and disulfated
disaccharides units of disaccharide B (12%). Based on 1
H
nuclear magnetic resonance spectroscopy, the presence of
3-sulfated and/or 2,3-sulfated IdoA residues was suggested
[7]. Interestingly, Sakai et al. [7] showed that although the
content of the disaccharide B sequence (which is required for
the binding of DS to HCII) in eel skin DS was twofold higher
than that of DS from porcine skin, the eel skin DS showed a
lesser anticoagulant activity when compared to that of the
porcine DS sample [7].
DS with distinguished disaccharide composition and de-
gree of sulfation has been reported in ray skin [8,10,11]. DS
from the skin of the ray R. clavata, which habits the Greek
seacoast, was shown to contain two DS with different de-
grees of sulfation: DSI, which was the major DS species in
the 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 sulfated
groups 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 at
positions 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, the
authors suggested that the disulfated disaccharides were
composed of: units bearing esterified sulfate groups at posi-
tions C-2 and C-3 of the uronic acid residues linked to
GalNAc (64%), units bearing esterified sulfate groups at
positions C-3 of the uronic acid and at position C-6 of
GalNAc (disaccharide K) (20%) and units bearing esterified
sulfate groups at positions C-2 of the uronic acid and at
position C-6 of GalNAc (disaccharide D) (13%) [8,10].
In the present work, we provide additional information on
DS of ray skin by analysing DS from the ventral skin of three
species that habit the Brazilian seacoast, D. americana,
D. gutatta and A. narinari, and of P. motoro, a fresh water
species that habits theAmazon River. Our results showed that
DS from the four ray species were composed mainly of
mono-sulfated disaccharides bearing esterified sulfate
groups at positions C-4 of GalNAc (50%, 51% and 47% for
D. americana, D. gutatta and A. narinari, respectively, and
75% 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 of
GalNAc (5%, 11%, 11% and 16%) were also observed for
D. 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 uronic
acid and at position C-4 of GalNAc (disaccharide B) (30%,
33% and 24%), accompanied by small content of disulfated
disaccharides bearing esterified sulfate groups at positions
C-2 of the uronic acid and at position C-6 of GalNAc (disac-
charide D) (3%, 2% and 8%) were found in the marine ray
species, 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 of
2-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 ray
species studied and the data reported on DS structure from
eel skin (Anguilla japonica) and from ray skin of R. clavata
showed structural heterogeneities among DS from skin of
different marine vertebrates. The nature of such structural
diversity among these skin DS is not yet known, in particular,
among skin DS from different ray species, as commented
above.
DS from eel skin, which was shown to present a high
content of the disaccharide B sequence, showed a lesser
anticoagulant activity when compared to that of the porcine
DS skin, which was shown to present twofold lesser disac-
charide B sequences than the DS from eel skin [7]. It has
been reported that the presence in the structure of DS of
contiguous sequences of disaccharide B, of at least four
repeating sequences, is required for anticoagulant activity
through HCII [19]. Therefore, Sakai et al. [7] suggested, as
one possible explanation why eel skin DS has a very low
anticoagulant activity, that the disaccharide B sequences in
Fig. 4. Dependence on the concentration of skin ray DS for inactivation of
thrombin by HCII. Mammalian DS is included for comparison. The assays
were performed with a chromogenic substrate for thrombin as referred to in
Section 2. After 120 s, the remaining thrombin activity was determined
(A405 nm/min). The panel shows a typical result representative of two
different experiments.
682 J.M. Dellias et al. / Biochimie 86 (2004) 677–683

7. DS from eel skin might be delocalised, and there was no
contiguous sequences of these disaccharide units.Whether or
not ray skin DS from R. clavata presents appreciable antico-
agulant activity is not yet known.
Differences were observed in the anticoagulant action of
skin 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 profiles
similar to those of mammalian DS. In contrast, DS from
A. narinari had no measurable activity in theAPTT assay.As
we used pure preparations of ray skin DS, free of CS and/or
HS contaminations (see Section 2), these differences were
exclusively due to the ray skin DS. We speculate that a
possible 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. DS
from both D. americana and D. gutatta presented a very
similar disaccharide composition, but very different antico-
agulant activities. DS from D. americana might present con-
tiguous disulfated disaccharide B units, whereas in DS of
D. gutatta these units might be delocalised resulting in the
lack of contiguous sequences of this unit, likely to what was
previously suggested by Sakai et al. [7] in the case of eel skin
DS. DS from A. narinari presented a content of disaccharide
D units higher than those in the DS from the other two ray
species. This DS had no measurable activity in the APTT
assay. In this case, beside the possible delocalisation of the
disaccharide B units, the presence of the disaccharide D units
in this DS might be also negatively affecting its anticoagulant
activity. Ascidian DS with a high content of disaccharide D
units has been reported to has no discernible anticoagulant
activity [5]. A detailed study of this point on the DS chains
from 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 and
PRONEX), Fundação de Amparo à Pesquisa do Estado do
Rio de Janeiro (FAPERJ) and Coordenação de Aperfeiçoa-
mento de Pessoal de Ensino Superior (CAPES: PROCAD).
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