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 21941590, Brazil
Received on June 1, 2000; revised on August 14, 2000; accepted on August 16, 2000.
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Abstract |
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Key words: sulfated glycosaminoglycans/dermatan sulfate/heparin/anticoagulant/ascidian tissues
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Introduction |
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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 -L-IdoA (2SO4)-1
3-GalNAc(6SO4) was isolated from the visceras of the ascidian Ascidia nigra (Pavão et al., 1995
). Similarly, an oversulfated dermatan sulfate composed by disaccharide repeating units of
-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.
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Results |
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A quantitative analysis of the distribution of sulfated glycosaminoglycans in ascidian tissues is shown in Table I. Uronic acidcontaining 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).
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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).
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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.
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Discussion |
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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.
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 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).
On the contrary, the heparin-like polymer possesses a more heterogeneous sulfation pattern in the tissues of this ascidian. A more sulfated polymer, composed mainly by the tri-sulfated disaccharide UA(2SO4)-1
4-GlcN(SO4)(6SO4) is present in the intestine, 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).
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 biosynthesis 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 antithrombin affinity column with approximately the same salt concentration (not shown), suggesting the presence of the antithrombin-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 cytoplasmic 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 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 metachromatic 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 oversulfated 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
).
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.
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Materials and methods |
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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), containing 100 mg papain, 5 mM EDTA, and 5 mM cysteine and incubated at 60°C for 24 h. The mixtures were centrifuged (2000 x 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 x g for 10 min at room temperature), freeze-dried, and dissolved in 2 ml of distilled water.
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-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).
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 TrisHCl 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.
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.
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 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.
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 x 25 cm Spherisorb SAX column, using a linear gradient of 01.0 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.
Inhibition of thrombin by antithrombin in the presence of the glycans from the various ascidian tissues
The glycans extracted from intestine, heart, pharynx, and cloak were incubated separately with chondroitin ABC lyase, as described above. After the incubation period, 2 volumes of 95% ethanol was added. The precipitate formed after incubating the sample at 10°C for 12 h, containing the enzyme-resistant glycans, was separated by centrifugation (2000 x g for 15 min at room temperature) and used to estimate the antithrombin activity of the chondroitin ABC lyase-resistant glycans from different tissues. Incubations were performed in disposable polystyrene cuvettes. The reactants included 100 nM antithrombin, 20 nM thrombin and 010 µg/ml (as uronic acid) chondroitin lyase ABC-resistant glycans from different tissues in 100 µl 0.02 M TrisHCl, 0.15 M NaCl, and 1.0 mg/ml polyethylene glycol, pH 7.4 (TS/PEG buffer). Thrombin was added last to initiate the reaction. After a 60 s incubation at room temperature, 500 µl of 100 µl Chromosym TH in TS/PEG buffer was added, and the absorbance at 405 nm was recorded continuously for 100 s. The rate of change of absorbance was proportional to the thrombin activity remaining in the incubation. No inhibition occurred in control experiments in which thrombin was incubated with antithrombin in the absence of glycan, nor did inhibition occur when thrombin was incubated with glycan alone over the range of concentrations tested.
Histochemistry of the ascidian tissues
For histochemical preparations the intestine and pharynx were carefully isolated from other tissues and fixed in 5% formaldehyde in seawater for 2 h at room temperature. After fixation, the organs were washed with water, dehydrated in graded ethanol, cleared in xylol, and embedded in Para-plast (m.p. 55.6°C). Approximately 7 µm sections from intestine and pharynx were cut longitudinally on a Spencer microtome. Sections were stained with 0.05 M 1,9-dimethyl-methylene blue in 0.1 M HCl, before or after incubation with chondroitin ABC lyase in 50mM TrisHCl buffer (pH 8.0), containing 5 mM EDTA and 15 mM sodium acetate, for 12 h at room temperature.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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