Full text of DOI:10.1093/glycob/10.12.1333

Glycobiology vol. 10 no. 12 pp. 1333–1340, 2000
Anticoagulant sulfated glycosaminoglycans in the tissues of the primitive chordate
Styela plicata (Tunicata)
Mário Gandra, Moisés C.M.Cavalcante and
Mauro S.G.Pavão1
Cassaro and Dietrich, 1977). They are present in the extra-
cellular matrix, at the cell surface, and also in cytoplasmic
granules (Yurt et al., 1977a,b; Ruoslahti, 1996), and can
interact with several factors such as, morphogens, growth
factors, cytokines, enzymes, and surface proteins of micro-
organisms (Kjellén and Lindahl, 1991; Rostand and Esko,
1997; Conrad, 1998; Wodarz and Nusse, 1998).
Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas and
Laboratório de Tecido Conjuntivo, Hospital Universitário Clementino Fraga
Filho, Universidade Federal do Rio de Janeiro, Caixa Postal 68041,
Rio de Janeiro, RJ 21941–590, Brazil
Received on June 1, 2000; revised on August 14, 2000; accepted on August 16,
2000
Sulfated glycosaminoglycans with unique structures have
been reported in the visceras of several species of ascidians. A
highly sulfated dermatan sulfate-like polymer, composed
by the unusual di-sulfated disaccharide α-L-IdoA (2SO4)-
1→3-GalNAc(6SO4) was isolated from the visceras of the
ascidian Ascidia nigra (Pavão et al., 1995). Similarly, an over-
sulfated dermatan sulfate composed by disaccharide repeating
units of α-L-IdoA(2SO4)-1→3-GalNAc(4SO4) was recently
purified from the visceras of the ascidian Halocynthia
pyriformis and Styela plicata (Pavão et al., 1998). In addition,
heparan sulfate-/heparin-like polymers have also been detected
in the visceras (Pavão et al., 1994) and recently isolated from
the test cells of S.plicata (Cavalcante et al., 1999). We now
expand the study of these molecules to the tissue level as an
approach to understand their biological roles. Here we report a
biochemical and histochemical analysis of the distribution of
these unique glycosaminoglycans among the tissues of the
ascidian S.plicata. Evidence of the occurrence of a heparin-like
polymer in this animal is also presented.
We performed a biochemical and histochemical study of
sulfated glycosaminoglycans in the tissues of the ascidian
Styela plicata. A highly sulfated dermatan sulfate and a
heparin-like polymer, identified by incubation with specific
lyases, occur at different concentrations in intestine, heart,
pharynx, and cloak. Dermatan sulfate prevails in the
pharynx, whereas the heparin-like polymer abounds in the
intestine. Staining of tissues sections with the cationic dye
1,9-dimethylmethylene blue before and after incubation
with specific lyases revealed that the dermatan sulfate
occurs in the extracellular matrix, while the heparin-like
polymer is located within cytoplasmic granules of cells in
the lumen of intestine and pharynx. The dermatan sulfate
has a similar disaccharide composition in all tissues
studied, whereas the heparin-like polymer differs in sulfate
content. A direct relationship between sulfate content of the
heparin-like polymer and antithrombin activity was
observed. Analysis of the repeating disaccharide units of
the heparin-like polymer indicates the presence of rela-
tively high amounts of the disulfated disaccharide namely
∆UA-1→4-GlcN(SO4)-(6SO4), which may suggest the occur-
rence in ascidians of regulatory biosynthetic mechanisms
different from those observed for heparin in mammals.
Results
Analysis of the glycans from the ascidian tissues
In order to determine the tissue distribution of sulfated
glycosaminoglycans in ascidian body, tissues from different
organs, intestine, heart, pharynx, and cloak were subjected to
papain extraction and the glycans were analyzed by agarose
gel electrophoresis (Figure 1). The glycans from intestine and
heart display the same electrophoretic pattern, migrating as
two main metachromatic bands. The low mobility band
migrates as standard heparan sulfate, while the second major
band migrates between standard heparan sulfate and dermatan
sulfate (Figure 1). The glycans from pharynx display an array
of metachromatic bands with a less defined migration, while
the glycans from the cloak possess a main metachromatic band
migrating between heparan sulfate and dermatan sulfate, and a
less intense band migrating as heparan sulfate (Figure 1).
A qualitative analysis of the sulfated glycans extracted from
each tissue was carried out by agarose gel electrophoresis
before and after degradation of the glycans with specific lyases
(Figure 1). The low mobility band of the glycans from
Key words: sulfated glycosaminoglycans/dermatan sulfate/
heparin/anticoagulant/ascidian tissues
Introduction
Glycosaminoglycans are complex polysaccharides based on
repeating disaccharide units of hexuronic acid (α-L-iduronic
acid or β-D-glucuronic acid) and hexosamine (α-D-glucosamine
or β-D-galactosamine). The heterogeneity of these polymers
results from variations in the degree of sulfation and occurrence of
two types of hexuronic acid (Conrad, 1998). The glycosamino-
glycans have a ubiquitous distribution in living organisms
occurring in virtually all organs and tissues (Mathews, 1975;
1To whom correspondence should be addressed
© 2000 Oxford University Press
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M.Gandra et al.
intestine, heart, pharynx, and cloak, which has the same migra-
tion of heparan sulfate, resists degradation with chondroitin
ABC lyase (Figure 1A) and heparan sulfate lyase (Figure 1C),
but is totally degraded after incubation with heparin lyase,
except for the glycans from the pharynx, which is only
partially degraded (Figure 1B). The band migrating between
standard heparan sulfate and dermatan sulfate of the glycans
from intestine, heart, pharynx, and cloak is resistant to heparin
lyase (Figure 1 B) and chondroitin AC lyase (Figure 1C), but is
completely degraded by chondroitin ABC lyase (Figure 1 A).
A minor metachromatic band, migrating as chondroitin sulfate
standard, which resists degradation with both chondroitinase
ABC and heparin-lyase, as well as DNase are also observed in
the glycans from the intestine, heart, and pharynx (Figure 1).
The chemical nature of this material remains to be determined.
A quantitative analysis of the distribution of sulfated
glycosaminoglycans in ascidian tissues is shown in Table I.
Uronic acid–containing glycans were detected in all tissues
studied. They occur in higher amounts in the intestine and
cloak, representing about 0.3% of the dry weight, when
compared to the heart and pharynx, where they account for
about 0.1% of the dry weight of the tissue. Dermatan sulfate
and a heparin-like polymer are present in all tissues. Dermatan
sulfate occurs mainly in the pharynx, whereas the heparin-like
polymer prevails in the intestine (Table I).
Disaccharide composition of the dermatan sulfate and
heparin-like polymer from the ascidian tissues
The glycans from the various tissues were exhaustively degraded
with chondroitin ABC lyase or a mixture of heparan sulfate and
heparin lyases, and the disaccharides formed were separated by
gel filtration HPLC and strong anion-exchange HPLC.
The disaccharides formed by the action of chondroitin ABC
lyase on the glycans from the different tissues are shown in
Figure 2 and in Table II. The method used allowed us to separate
the following standard disaccharides: ∆UA-1→3-GalNAc(6SO4),
∆UA-1→3-GalNAc(4SO4), ∆UA(2SO4)-1→3-GalNAc(6SO4),
∆UA-1→3-GalNAc(4SO4)(6SO4) and ∆UA(2SO4)-1→3-Gal-
NAc(4SO4) (Figure 2A). The disaccharide composition of the
dermatan sulfate from intestine, heart, pharynx, and cloak is
very similar, yielding about 75% of the unusual disaccharide
∆UA(2SO4)-1→3-GalNAc(4SO4) and 25% of the ∆UA-1→3-
GalNAc(4SO4) (Figure 2B and Table II).
Degradation of the glycans from the intestine, heart, and
pharynx with a mixture of heparan sulfate and heparin lyases
produces mainly (over 60%) of the trisulfated disaccharide
∆UA(2SO4)-1→4-GlcN(SO4)(6SO4), and smaller amounts of
the disulfated disaccharide ∆UA-1→4-GlcN(SO4)(6SO4)
(Figure 2D, Table III). On the contrary, the glycans from the
cloak yield mainly (about 60%) the disulfated disaccharide
∆UA1→4GlcN(SO4)(6SO4) and smaller amounts of the trisulfated
Table I. Distribution of the sulfated glycosaminoglycans among various tissues of the ascidian Styela plicata.
Distribution of glycosaminoglycans among various tissue (%)b
DS Heparin-like
Tissue
Concentration in each tissue
(µg of uronic acid/mg of dry weight)a
Intestine
Heart
2.6
1.1
1.6
3.2
7.3
46
10.3
63.0
19.4
23.3
10.5
20.2
Pharynx
Cloak
aThe content of uronic acid in the glycans extracted from each tissue was estimated by the carbazole reaction (Bitter and Muir,
1962).
bThe percentage of the glycosaminoglycans in the tissues was estimated by subtracting the densitometric units (OD/mm2) obtained
by densitometry of the metachromatic bands on agarose gel electrophoresis before and after degradation with specific glycosidases.
Table II. Disaccharide composition of the dermatan sulfate polymer from different ascidian tissues
Proportion of disaccharidesc
Position of elutiona
Disaccharide
TR
Intestine
<1
Heart
<1
Pharynx
<1
Cloak
<1
b
1
2
3
4
5
∆UA-1→3-GalNAc(6SO4)
∆UA-1→3-GalNAc(4SO4)
∆UA(2SO4)-1→3-GalNAc(6SO4)
∆UA-1→3-GalNAc(4SO4 )(6SO4)
∆UA(2SO4)-1→3-GalNAc(4SO4)
26.2
27.2
31.7
32.6
33.9
24.0
<1
25.0
<1
26.3
<1
28.4
<1
<1
<1
<1
<1
76.0
75.0
73.7
71.6
aStandard peak number in order of elution from a Spherisorb-SAX column.
bRetention time of the disaccharides on a Spherisorb-SAX column.
cThe areas under the peaks corresponding to the disaccharides from the Spherisorb-SAX column, were integrated to obtain the disaccharide proportions.
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Anticoagulant glycosaminoglycans in Tunicates
Table III. Disaccharide composition of the heparin-like polymer from different ascidian tissues
Proportion of disaccharidesc
Position of elutiona
Disaccharide
TR
Intestine
<1
Heart
<1
Pharynx
<1
Cloak
<1
b
1
2
3
4
5
6
∆UA-1→4-GlcNAc(6SO4)
∆UA(2SO4)-1→4-GlcNAc
∆UA-1→4-GlcN(SO4)(6SO4)
∆UA(2SO4)-1→4-GlcN(SO4)
∆UA(2SO4)-1→4-GlcNAc(6SO4)
∆UA(2SO4)-1→4-GlcN(SO4)( 6SO4)
21.7
22.8
23.7
24.9
27.5
29.3
<1
<1
<1
<1
39.3
<1
31.1
<1
25.5
<1
58.5
<1
<1
<1
<1
<1
60.7
68.9
74.5
41.5
aStandard peak number in order of elution from a Spherisorb-SAX column.
bRetention time of the disaccharides on a Spherisorb-SAX column.
cThe areas under the peaks corresponding to the disaccharides from the Spherisorb-SAX column, were integrated to obtain the disaccharide proportions.
disaccharide ∆UA(2SO4)-1→4-GlcN(SO4)(6SO4), after degra-
dation with the mixture of heparan sulfate and heparin lyases
(Table III).
pharynx stained with 1,9-dimethyl-methylene (asterisk in
Figure 4C). Treatment of the section with chondroitin ABC
lyase abolishes the metachromatic staining (asterisk in
Figure 4D). A layer of cells displaying a strong metachromasia
associated with intracellular granules is also present in the
sections from the pharynx treated with chondroitin ABC lyase
(Figure 4D, inset).
Antithrombin activity of the heparin-like polymers from the
ascidian tissues
The most well known characteristic of heparin is its anti-
coagulant activity. This activity is related mainly to the ability
of heparin to catalyze inhibition of thrombin by antithrombin
(Bjork and Lindahl, 1982). In order to verify whether the
heparin-like polymers from the ascidian tissues posses anti-
coagulant activity we assayed the thrombin inhibitory activity
of the chondroitin ABC lyase-resistant glycans from the
various tissues in the presence of antithrombin.
The dermatan sulfate-free glycans from all tissues studied
were able to catalyze thrombin inhibition by antithrombin
(Figure 3), however with a concentration at least 20 times
higher, when compared to mammalian heparin. Among the
glycans from the ascidian tissues, those from the pharynx
possess the higher activity (IC50 = 0.03 µg/ml), whereas the
glycans from the cloak are the less active (IC50 = 0.3 µg/ml).
The antithrombin activity of the material obtained from the
tissues disappears after degradation with heparin-lyase, excluding
the presence of active contaminants in our preparations.
Discussion
Sulfated glycosaminoglycans have been previously detected in
the ascidian Styela plicata (Pavão et al., 1994, 1998; Cavalcante
et al., 1999). A nitrous acid-sensitive glycan (Pavão et al.,
1994) and an oversulfated dermatan sulfate with high heparin
cofactor II activity, composed mainly by disaccharide units of
∆UA(2SO4)-1→3GalNAc(4SO4) (Pavão et al., 1998) occur in
the visceras of this ascidian. In the present study, we investigated
the distribution of these glycosaminoglycans in the various
tissues of S.plicata.
Initially we performed a biochemical analysis of the glycans
extracted from different tissues: intestine, pharynx, heart, and
cloak. We found that dermatan sulfate (identified by its sensitivity
to chondroitin ABC lyase and resistance to chondroitin AC
lyase) and a heparin-like polymer (identified by its sensitivity
to heparin lyase and resistance to heparan sulfate lyase) are
present at different concentrations in the tissues studied (see
Figure 1 and Table I). The dermatan sulfate prevails in the
pharynx, while the heparin-like polymer occurs in higher
amounts in the intestine (Table I). The former polymer shows
a retarded electrophoretic migration on agarose gel, when
compared to the vertebrate standard (Figure 1A). This pattern
is probably due to the higher charge density of the invertebrate
glycan, allowing a stronger interaction between the sulfate
groups on the polysaccharide and the diamine groups of the gel
buffer.
Histochemical analysis of the ascidian tissues
We used histochemical analysis of the intestine and pharynx
with the cationic dye 1,9-dimethyl-methylene blue to determine
the location of sulfated glycosaminoglycans in these tissues
(Figure 4). Staining of intestine sections reveals a layer of cells
along the intestinal lumen displaying a strong metachromasia
associated with intracellular granules (arrow in Figure 4A). A
diffuse extracellular metachromatic material is also observed
(asterisk in Figure 4A). The metachromatic staining of the
granules resists treatment with chondroitin ABC lyase (arrow
in Figure 4B), while the metachromatic extracellular material
completely disappears after treatment with the enzyme (asterisk in
Figure 4B). The basal membrane is also metachromatic, but in
this case, treatment with chondroitin ABC lyase has no effect
on the staining (arrow in the inset of Figure 4B).
The dermatan sulfate possesses the same chemical composition
in all tissues: ∆UA(2SO4)-1→3GalNAc(4SO4) (∼75%) and
∆UA-1→3GalNAc(4SO4) (∼25%), as indicated by the analysis
of the disaccharide units shown in Table II. In vertebrate
dermatan sulfate only about 5% of the disaccharide units are
O-sulfated at carbon 2 of iduronic acid moieties. This result is
in accordance to our previous data on the chemical composition of
Similarly to the intestine, a diffuse extracellular meta-
chromatic material is also present in the sections from the
1335

M.Gandra et al.
Fig. 2. Strong anion-exchange HPLC analysis of the disaccharides formed by
specific lyases on the ascidian glycosaminoglycans. A mixture of chondroitin
(A) and heparin (C) standard disaccharides and the disaccharides formed by
exhaustive action of chondroitinase ABC (B) and heparin- plus heparan
sulfate-lyases (D) on the glycans from intestine (see Materials and methods)
were applied to a 25 cm × 4.6 mm Spherisorb-SAX column, linked to an
HPLC system. The column was eluted with a linear gradient of NaCl, as
described in Materials and methods. The eluent was monitored for UV
absorbance at 232 nm. The chondroitin sulfate disaccharide standards used
were: 6S, ∆UA-1→3-GalNAc(6SO4); 4S, ∆UA-1→3-GalNAc(4SO4); 2,6,
∆UA(2SO4)-1→3-GalNAc(6SO4); 4,6, ∆UA-1→3-GalNAc(4SO4 )(6SO4);
2,4, ∆UA(2SO4)-1→3-GalNAc(4SO4). The heparin disaccharide standards used
were: 6S, ∆UA-1→4-GlcNAc(6SO4); N,6, ∆UA-1→4-GlcN(SO4)(6SO4); tri,
∆UA(2SO4)-1→4-GlcN(SO4)( 6SO4). X, Unidentified peak.
Fig. 1. Agarose gel electrophoresis of the sulfated glycans from the various
tissues of S.plicata before and after incubation with chondroitin ABC lyase or
heparin lyase. The glycans from intestine, heart, pharynx and cloak (15 µg as
dry weight of each), before (–) and after (+) incubation with chondroitin ABC
lyase (Chase ABC) (A), heparin lyase (B), and chondroitin AC lyase (Chase
AC) and heparan sulfate lyase (C) (see Materials and methods), as well as a
mixture of standard glycosaminoglycans, containing chondroitin sulfate (CS),
dermatan sulfate (DS) and heparan sulfate (HS) (1.5 µg as uronic acid of each)
were applied to a 0.5% agarose gel in 0.05 M 1,3-diaminopropane/acetate
(pH 9.0), and run for 1 h at 110 mV. After electrophoresis the glycans were
fixed with aqueous 0.1% cetylmethylammonium bromide solution and stained
with 0.1% toluidine blue in acetic acid/ethanol/water (0.1:5:5, v/v).
Fig. 3. Antithrombin activity of the glycans from the tissues of S.plicata.
Antithrombin (100 nM) was incubated with thrombin (20 nM) in the presence
of various concentrations of the chondroitin ABC lyase-treated glycans (see
Materials and methods) from intestine (squares), heart (solid circles), pharynx
(diamonds), and cloak (asterisks) of S.plicata, and mammalian heparin (open
circles). After 60 s, the remaining thrombin activity was determined with a
chromogenic substrate (A405nm/min).
the S.plicata oversulfated dermatan (Pavão et al., 1998) and
suggests the presence of only one type of dermatan sulfate in
this ascidian.
The ascidian dermatan sulfate occurs in the extracellular
matrix surrounding the cells (asterisk in Figure 4A,C), as
indicated by the sensitivity of a metachromatic extracellular
material to chondroitin ABC lyase in the sections from
intestine and pharynx (asterisk in Figures 4B,D).
more sulfated polymer, composed mainly by the tri-sulfated disac-
charide ∆UA(2SO4)-1→4-GlcN(SO4)(6SO4) is present in the intes-
tine, heart and pharynx, whereas a less sulfated molecule, composed
mainly of the disaccharide ∆UA-1→4-GlcN(SO4)(6SO4) occurs in
the cloak (Table III).
On the contrary, the heparin-like polymer possesses a more
heterogeneous sulfation pattern in the tissues of this ascidian. A
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Anticoagulant glycosaminoglycans in Tunicates
Fig. 4. Histological sections from the intestine and pharynx of S.plicata stained with 1,9-dimethylmethylene blue before and after incubation with chondroitin
ABC lyase. Sections from intestine (A and B) and pharynx (C and D) were stained with the cationic dye 1,9-dimethylmethylene blue before (A and C) and after
(B and D) incubation with chondroitin ABC lyase (see Materials and methods). Arrows indicate the metachromatic granules inside cells, and basal membrane
(inset in B). Asterisks indicate the extracellular metachromatic material.
The occurrence of high percentage (about 25%) of the disulfated
disaccharide ∆UA-1→4-GlcN(SO4)(6SO4) in the ascidian
heparin is noteworthy (Table III). In the biosynthetic pathway
of heparin, described by Lindahl and co-workers for mammalian
systems (Lindahl, 1989; Lindahl et al., 1989; Salmivirta et al.,
1996), the 6-O-sulfation of GlcNSO3 occurs preferentially after
iduronic acid formation and 2-O-sulfation. During the biosyn-
thesis of the ascidian heparin, this mechanism seems to be less
relevant.
In addition to the susceptibility to heparin lyase and the
occurrence of high amounts of trisulfated disaccharide units in
the ascidian heparin-like polymers, two other features of these
glycans are present in mammalian heparin, namely, antithrombin
activity and localization in cytoplasmic granules. Although
less potent than heparin, the heparin-like polymers from all
ascidian tissues are able to catalyze thrombin inhibition by
antithrombin (Figure 3). Preliminary data of our laboratory
indicated that the ascidian heparin is eluted from an anti-
thrombin affinity column with approximately the same salt
concentration (not shown), suggesting the presence of the anti-
thrombin-high-affinity pentasaccharide in the ascidian
heparin. However, at the moment, we do not have any
chemical evidence that confirm this evidence. More detailed
experiments are required to properly address this question.
It is important to note the relationship between the sulfate
content of the ascidian glycans with antithrombin activity. The
heparin-like polymer from pharynx, which has the higher
content of trisulfated disaccharide (see Table III), possesses the
higher antithrombin activity (0.03 µg/ml), as measured by the
IC50 for thrombin inhibition (see Table IV). The less sulfated
glycans from the cloak, enriched in disulfated disaccharides
(see Table III) is 10 times less potent (0.3 µg/ml) (Table IV).
It is known that heparin occurs exclusively within cyto-
plasmic granules of mast cells (Straus et al., 1982; Stevens,
1987). In fact, some authors (Kjellén and Lindahl, 1991)
suggest that the term heparin should be reserved for those
glycosaminoglycans that are synthesized by mast cells and
stored in cytoplasmic granules. In Figure 4, we provide
1337

M.Gandra et al.
evidence that suggests the occurrence of the heparin-like
polymer in cytoplasmic granules of cells that are located at the
lumen of the intestine (arrow in Figure 4A) and pharynx (inset
in Figure 4C). The evidence comes from the fact that the meta-
chromatic materials in the cytoplasmic granules are resistant to
treatment with chondroitin ABC lyase (arrows in Figure 4B
and inset in Figure 4D). Recent data from our laboratory
reinforces this evidence. We detected the presence of an over-
sulfated heparan sulfate, enriched in trisulfated disaccharide
units, in cytoplasmic granules of the test cells that surround the
oocytes of S.plicata (Cavalcante et al., 1999).
containing 100 mg papain, 5 mM EDTA, and 5 mM cysteine
and incubated at 60°C for 24 h. The mixtures were centrifuged
(2000 × g for 10 min at room temperature), another 100 mg of
papain in 20 ml of the same buffer, containing 5 mM EDTA
and 5 mM cysteine was added to the precipitate, and the
mixture was incubated for another 24 h. The clear supernatants
from the two extractions were combined and the polysaccharides
precipitated with 2 vol. of 95% ethanol and maintained at 4°C
for 24 h. The precipitates formed were collected by centrifugation
(2000 × g for 10 min at room temperature), freeze-dried, and
dissolved in 2 ml of distilled water.
This is the first report on the distribution and localization of
sulfated glycosaminoglycans in the tissues of ascidians. The
results described here may bring new insights about the
function of this class of polysaccharides in animals. It is likely,
however, that the physiological function of the sulfated
glycosaminoglycans described here has no relation to their
anticoagulant activity, since the prevention of body fluid loss
in this invertebrate does not involve coagulation of the
hemolymph.
Chemical analysis
The hexuronic acid content of the glycans from the various
tissues was estimated by the carbazole reaction (Bitter and
Muir, 1962).
Agarose gel electrophoresis
The intact or enzyme-degraded glycans from the ascidian
tissues were analyzed by agarose gel electrophoresis, as
described previously (Dietrich and Dietrich, 1976). About
1.5 µg (as uronic acid) of the glycans, and a mixture of
standard glycosaminoglycans, containing chondroitin sulfate,
dermatan sulfate, and heparan sulfate (1.5 µg as uronic acid of
each) were applied to a 0.5% agarose gel in 0.05 M 1,3-diamino-
propane/acetate (pH 9.0), and run for 1 h at 110 mV. After
electrophoresis the glycans were fixed with aqueous 0.1%
cetylmethylammonium bromide solution and stained with
0.1% toluidine blue in acetic acid/ethanol/water (0.1:5:5, v/v).
Materials and methods
Materials
Heparan sulfate from human aorta was extracted and purified
as described previously (Cardoso and Mourão, 1994). Chon-
droitin 4-sulfate from whale cartilage, dermatan sulfate from
bovine intestinal mucosa, twice-crystallized papain (15 units/mg
protein) and the standard disaccharides α-∆UA-1→3-Gal-
NAc(6SO4), α-∆UA-1→3-GalNAc(4SO4), α-∆UA(2SO4)-
1→3-GalNAc(6SO4), α-∆UA-1→3-GalNAc(4SO4)(6SO4), α-
∆UA(2SO4)-1→3-GalNAc(4SO4), α-∆UA-1→4-GlcN(SO4),
α-∆UA-1→4-GlcNAc(6SO4), α-∆UA(2SO4)-1→4-GlcNAc,
Enzymatic treatments
Chondroitin lyases. the glycans extracted from the ascidian
tissues (~100 µg) were incubated with 0.01 U of chondroitin
AC or ABC lyase in 0.1 ml 50 mM Tris–HCl buffer (pH 8.0),
containing 5 mM EDTA and 15 mM sodium acetate. After
incubation at 37°C for 12 h, another 0.01 U of enzyme was
added to the mixture, and the reaction continued for an
additional 12 h period. The percentage of the sulfated glycans
degraded by the enzymes was estimated by densitometry of the
metachromatic bands on a Bio-Rad densitometer, following
agarose gel electrophoresis of intact and enzyme-degraded
glycans.
α-∆UA(2SO4)-1→4-GlcN(SO4),
α-∆UA(2SO4)-1→4-Glc-
NAc(6SO4), α-∆UA-1→4-GlcN(SO4)-(6SO4) and α-∆UA-
(2SO4)-1→4-GlcN(SO4)-(6SO4) were purchased from Sigma
(St. Louis, MO); chondroitin AC lyase (EC 4.2.2.5) from
Arthrobacter aurenses, chondroitin ABC lyase (EC 4.2.2.4)
from Proteus vulgaris, heparan sulfate lyase (EC 4.2.2.8) and
heparin lyase (EC4.2.2.7) from Flavobacterium heparinum
were from Seikagaku America Inc. (Rockville, MD); agarose
(standard low Mr) was from Bio-Rad (Richmond, CA);
toluidine blue was from Fisher Scientific (USA) and 1,9-
dimethylmethylene blue from Serva Feinbiochimica (Heidelberg,
Germany); cetyltrimethylammonium bromide from Merck
(Darmstadt, Germany) and DEAE-cellulose (DE-52) from
Whatman International (Maidstone UK); human antithrombin
and thrombin were from Hematologica Technologies Inc.
(USA); thrombin chromogenic substrate tosyl-Gly-Pro-Arg-p-
nitroanilide acetate (Chromozyn TH) was from Boehringer
Mannheim (Germany).
Heparan sulfate and heparin lyases. About 50 µg (as dry
weight of each) of the glycans extracted from the ascidian
tissues were incubated with 0.005 U of either heparan sulfate
lyase or heparin lyase in 100 µl of 100 mM sodium acetate
buffer (pH 7.0), containing 10 mM calcium acetate for 17 h at
37°C. At the end of the incubation period the mixtures were
analyzed by agarose gel electrophoresis, as described earlier.
The percent of the sulfated glycans degraded by the enzymes
was estimated by densitometry of the metachromatic bands, as
described in the previous paragraph.
Isolation of the glycans
Adult specimens of S.plicata were collected at Guanabara Bay,
Rio de Janeiro. Animals were maintained in an aerated
aquarium until use. Tissues from different organs (intestine,
heart, pharynx, and cloak) of several ascidians were carefully
isolated from other tissues, under magnifying lenses, cut in
small pieces and dried. The dried tissues (∼1 g) were individually
suspended in 20 ml of 0.1 M sodium acetate buffer (pH 5.5),
In order to determine the disaccharide composition of the
glycosaminoglycans present in the various ascidian tissues, the
glycans obtained from each tissue were incubated with
chondroitin ABC lyase, or with a mixture of heparin and
heparan sulfate lyase, as described above. After the incubation
period, the reaction mixture was applied to a Superdex-Peptide
10/30 column (Amersham Pharmacia Biotech), connected to
1338

Anticoagulant glycosaminoglycans in Tunicates
an HPLC system. The column was equilibrated with an
aqueous solution of 20% acetonitrile (pH 3.5) and developed at
a flow rate of 0.25 ml/min. Fractions of 0.5 ml were collected
and checked for absorbance at 232 nm. The fractions
containing the disaccharides, identified by their position of
elution from the column, were pooled, freeze-dried, and
analyzed by HPLC.
Acknowledgments
We express their appreciation to Dr. Silvana Alodi (Departa-
mento de Histologia e Embriologia–ICB –UFRJ) for assistance
with the histochemistry. This work was supported by grants
from the Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq: FNDCT, PADCT and PRONEX),
Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro
(FAPERJ), Fundação Universitária José Bonifácio (FUJB) and
PEW-Latin American Fellow Program.
Analysis of the disaccharides formed by digestion of the
glycans with specific glycosidases
The disaccharides formed by digestion of the glycans from
different tissues with chondroitin ABC lyase, and/or with
heparin and heparan sulfate lyases were analyzed by HPLC on
a Supelco 4.5 mm × 25 cm Spherisorb SAX column, using a
linear gradient of 0–1.0 M aqueous NaCl (pH 3.5) at a flow rate
of 0.5 ml/min. The elution of the disaccharides was followed
by absorbance at 232 nm, and they were identified by comparison
with elution positions of known disaccharide standards.
Abbreviations
α-∆UA(2SO4), α-∆4,5 unsaturated hexuronic acid 2-sulfate;
HPLC, high-performance liquid chromatography; FPLC, fast
protein liquid chromatography.
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