Glycobiology, 2001, Vol. 11, No. 6 433-440
© 2001 Oxford University Press
Embryos of the sea urchin Strongylocentrotus purpuratus synthesize a dermatan sulfate enriched in 4-O- and 6-O-disulfated galactosamine units
2Laboratório de Tecido Conjuntivo, Hospital Universitário Clementino Fraga Filho and Departamento de Bioquímica Médica, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Caixa Postal 68041, Rio de Janeiro, RJ, 21941-590, Brazil, 3Centro Nacional de Ressonância Nuclear Magnética, Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil, and 4Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, 92093-0202, USA
Received on November 3, 2000; revised on January 29, 2001; accepted on January 29, 2001.
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Abstract |
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Unfertilized eggs of the sea urchin Strongylocentrotus purpuratus are surrounded by a gelatinous layer rich in sulfated fucan. Shortly after fertilization this polysaccharide disappears, but 24 h later the embryos synthesize high amounts of dermatan sulfate concomitantly with the mesenchyme blastula–early gastrula stage when the larval gut is forming. This glycosaminoglycan has the same backbone structure [4-
-L-IdoA-1
3-ß-D-GalNAc-1]n as the mammalian counterpart but possesses a different sulfation pattern. It has a high content of 4-O- and 6-O-disulfated galactosamine units. In addition, chains of this dermatan sulfate are considerable longer than those of vertebrate tissues. Adult sea urchin tissues contain high concentrations of sulfated polysaccharides, but dermatan sulfate is restricted to the adult body wall where it accounts for 
20% of the total sulfated polysaccharides. In addition, sulfation at the 4-O-position decreases markedly in the dermatan sulfate from adult sea urchin when compared with the glycan from larvae. Overall, these results demonstrate the occurrence of dermatan sulfates with unique sulfation patterns in this marine invertebrate. The physiological implication of these oversulfated dermatan sulfates is unclear. One hypothesis is that interactions between components of the extracellular matrix in marine invertebrates occur at higher salt concentrations than in vertebrates and therefore require glycosaminoglycans with increased charge density. Key words: dermatan sulfate/glycosaminoglycan/sea urchin/echinoderm/larval development
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Introduction |
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TopAbstractIntroductionResults and discussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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Sea urchin eggs are surrounded by a transparent gelatinous layer rich in sulfated L-fucans or sulfated L-galactans that induces the exocytotic sperm acrosome reaction (Alves et al., 1997
, 1998; Vilela-Silva et al., 1999). This is an obligatory event for sperm binding to and fusing with the egg. Initial studies on the presence of sulfated polysaccharides in sea urchin embryos indicated that embryonic cells synthesize these macromolecules (Yamagata and Okazaki, 1974; Oguri and Yamagata, 1978; Solursh and Katow, 1982).
Several authors demonstrated that sea urchin embryos synthesize mainly a dermatan sulfate–like polysaccharide instead of the sulfated L-fucans or sulfated L-galactans reported in the eggs (Yamagata and Okazaki, 1974; Oguri and Yamagata, 1978; Solursh and Katow, 1982). Apparently, the dermatan sulfate from sea urchin embryos has a sulfation pattern distinct from similar glycosaminoglycans from mammalian tissues. Yamagata and Okazaki (1974) reported the occurrence of a dermatan sulfate in larvae of the sea urchin Pseudocentrotus depressus being mostly sulfated at O-4 and O-6 positions of galactosamine residues. In contrast, Oguri and Yamagata (1978) reported a dermatan sulfate mostly sulfated at O-4 position in larvae of the sea urchin Hemicentrotus pulcherrimus as being similar to dermatan sulfates from vertebrate tissues.
Electron microscopic studies indicated that the sulfated glycosaminoglycans occur as 30-nm-diameter granules within the blastocel of the sea urchin embryo (Katow and Solursh, 1979). On the blastocel surface these granules are attached to fine fibers, forming a structure probably important to mesenchyme cell migration (Lane and Solursh, 1991). When embryos are grown in sulfate-deficient sea water, these granules are reduced in number. The chemical structure of these sulfated polysaccharides was not determined in these earlier studies. Analysis of the blastula of the sea urchin Strongylocentrotus purpuratus suggest that both dermatan sulfate and chondroitin sulfate are major components. However, these glycosaminoglycans possibly account for only 30% of the total sulfated polysaccharide in the sea urchin Lytechinus pictus. The pattern of sulfation also differs between the two species, but disulfated disaccharides account for only minor amounts of the total polysaccharide present (Solursh and Katow, 1982).
The physiological importance of the sulfated polysaccharides for the development of sea urchin embryos was demonstrated in experiments where inhibitors of the glycosaminoglycan metabolism were added to the culture. After the addition of either ß-D-xyloside, sodium selenate, or 2-deoxy-D-glucose, the development of the sea urchin embryos ceased at the blastula stage. These compounds inhibit either the attachment of the glycosaminoglycan to the protein core, sulfation, or elongation of the saccharide chains (Kinoshita and Saiga, 1979; Akasaka et al., 1980; Solursh et al., 1986; Lane and Solursh, 1991).
We concentrated our studies on the characterization of the sulfated polysaccharide synthesized by the larvae of the sea urchin S. purpuratus. Shortly after fertilization, the sulfated fucan of egg jelly disappears, and by 24 h of development the embryos begin to synthesize large amounts of dermatan sulfate. In contrast with the same glycosaminoglycan from vertebrate tissue, the dermatan sulfate from sea urchin embryos has a longer saccharide chain and high amounts of O-4 and O-6 disulfated galactosamine units.
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Results and discussion |
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TopAbstractIntroductionResults and discussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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Sea urchin embryos synthesize a highly sulfated dermatan sulfate
Sulfated glycoconjugates extracted from unfertilized sea urchin eggs by 5% SDS showed a single component close to the top of the lane (SF in Figure 1). This polysaccharide was characterized as the sulfated fucan from the egg jelly coat (Alves et al., 1998
). It disappears shortly after fertilization. Thereafter, the embryos contain no detectable amounts of sulfated glycoconjugate on the polyacrylamide gel until 24 h after fertilization. At this moment, which corresponds to the blastula–early gastrula stage, they begin to synthesize another sulfated glycoconjugate (DS in Figure 1) which reaches a plateau at 52–70 h.
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The sulfated glycoconjugates were extracted from 72-h embryos by papain digestion and purified by anion exchange chromatography on a Mono Q-FPLC. The column (Figure 2B) yields a major peak, containing both metachromasia produced by sulfated polysaccharides with 1,9-dimethylmethylene blue (closed circles) and hexuronic acid (open circles). The sea urchin polysaccharide is eluted from the Mono Q column at a higher NaCl concentration (
2.4 M NaCl) than mammalian chondroitin-dermatan sulfate (1.0 NaCl) and even higher than heparin (1.3 M) (Figure 2A).
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Cleavage of the sea urchin polysaccharide by digestion with specific chondroitin lyases or by deamination with nitrous acid was followed by polyacrylamide gel electrophoresis (PAGE). The molecular mass of this polysaccharide was not reduced by digestion with chondroitin AC lyase or by deamination with nitrous acid, but it was totally digested by chondroitin ABC lyase (Figure 3).
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The products formed by exhaustive digestion by chondroitin ABC lyase were analyzed on a strong anion-exchange SAX–high-performance liquid chromatography column (Figure 4, Table I). This procedure separates the several disaccharides formed by chondroitin ABC lyase, including the three disulfated disaccharides
-
UA(2SO4)-13GalNAc(6SO4), -UA-13GalNAc(4,6diSO4), and -UA(2SO4)-13Gal-NAc(4SO4). Nearly 74% of the disaccharides obtained from the glycosaminoglycan of 72-h embryos are -UA-13Gal-NAc(4,6diSO4), whereas mammalian dermatan sulfate yields only minor amounts of the disulfated disaccharides (compare panels B and C in Figure 4).
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1H nuclear magnetic resonance (NMR) spectrum at 600 MHz of the sea urchin polysaccharide recorded at 60°C showed broader and poorly resolved signals (not shown). Possibly this is a consequence of the high molecular mass of the polysaccharide (Figure 3), giving viscous solutions at high concentrations. We tried to overcome this problem performing NMR analysis of the major disaccharide formed by digestion with chondroitin ABC lyase. 1H NMR spectrum of the disaccharide is shown in Figure 5.
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The 1H spectrum of this disaccharide gave sharp lines compatible with a low molecular weight compound. The anomeric protons indicated in Figure 5 were identified using the 1H/13C heteronuclear multiple quantum coherence spectra (HMQC) (not shown) and gave characteristic chemical shifts for
(5.23–5.34 p.p.m.) and ß (4.79 p.p.m.) units. The spin systems for - and ß-N-acetyl-D-galactosamine and -unsaturated uronic acid can be traced in the total correlation spectroscopy (TOCSY) and correlation spectroscopy (COSY) spectra (not shown), giving the chemical shifts presented in Table II. All values are similar to those reported previously in the literature (Yamada et al., 1992), except for H4 of the -unsaturated uronic acid. Comparison of the 1H chemical shifts showed that H4 and H6 of the galactosamine are 0.6 p.p.m. downshifted from desulfated values, indicating these two positions are sulfated. We also reported the values of 13C chemical shifts for the disulfated disaccharide obtained from the sea urchin dermatan sulfate.
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These results indicate that the 72-h sea urchin embryos contain a dermatan sulfate with a distinctive structure due to a higher anionic charge density than mammalian dermatan sulfate. It contains alternative units of
-L-iduronic acid and 4-O-sulfated N-acetyl-ß-D-galactosamine, as does mammalian dermatan sulfate, but most of the galactosamine residues are also 6-O-sulfated. In addition, chains of the sea urchin dermatan sulfate are considerable longer than those of vertebrate tissue (Figure 3).
Dermatan sulfate in adult sea urchin tissue
We attempted to find dermatan sulfate in several tissues of adult sea urchin. Sulfated polysaccharides were detected in several tissues (Figure 6A), but only the body wall contains small amounts of chondroitin ABC lyase–sensitive and chondroitin AC lyase–resistant glycan (Figure 6B,C). The proportions of the disaccharide formed by exhaustive action of this enzyme on the dermatan sulfate from the body wall are given in Table I. Clearly, the proportion of 4-O-sulfation decreases from the embryo to adult tissue, whereas the 6-O-sulfation is almost unaffected.
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Summary of dermatan sulfate structures
Dermatan sulfates with the same backbone structure (4-
-L-Ido-13-ß-D-GalNAc-1)n, but with different patterns and proportions of sulfate substitutions, have been described in this and in previous studies (Figure 7). The repetitive disaccharide units of mammalian dermatan sulfate are sulfated at carbon 4 of the hexosamine moiety; small amounts of 2-O-sulfated -L- iduronic acid and 6-O-sulfated N-acetyl-ß-D-galactosamine units are also found in this glycosaminoglycan. We report that embryonic and adult sea urchin tissue contain dermatan sulfates with a distinctive sulfation pattern, both highly sulfated at the 6-position of the galactosamine, but with variable sulfation at the 4-O-position. Embryos contain dermatan sulfate highly sulfate at the 4-O-position, but sulfation at this position decreases sharply in the adult.
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In a previous study we have isolated dermatan sulfates from the body of ascidians (tunicates). All the ascidian dermatan sulfates have a high content of 2-O-sulfated
-L-iduronic acid residues but differ in the pattern of sulfation of the N-acetyl-ß-D-galactosamine units. For example, the species Styela plicata has 4-O-sulfated units, but Ascidian nigra has 6-O-sulfated units (Pavão et al., 1995, 1998). This collection of dermatan sulfates, in which the extension and position of sulfation vary greatly, raises interesting questions concerning the evolutionary history of these glycosaminoglycans and their biological roles.
Possible physiological role of dermatan sulfate in sea urchins
Dermatan sulfate–rich proteoglycans are an important component of vertebrate extracellular matrix. Thus, decorin, one of the dermatan sulfate–rich proteoglycans, is found mostly in cartilage and bone. It binds to different sites of collagen and is important in forming an ordered extracellular matrix. This proteoglycan is also involved in the regulation of several other cellular events, such as cell cycle and inflammatory process. Biglycan, another dermatan sulfate–rich proteoglycan, in addition to being found in cartilage and bone is found in endothelial cells and smooth muscle cells. It is frequently associated with the cell surface or with the pericellular matrix (for a review see Neame and Kay, 2000).
Dermatan sulfate was also found in invertebrates, specifically in ascidians (Pavão et al., 1995, 1998) and echinoderms (Yamagata and Okazaki, 1974; Oguri and Yamagata, 1978; Solursh and Katow, 1982; and this study), but its physiological role remains unknown. In the case of ascidian, a highly sulfated dermatan sulfate is found in the extracellular matrix of intestine, heart, cloak, and pharynx (Gandra et al., 2000). Dermatan sulfate is also a potent anticoagulant. It is likely, however, that the physiological function of this glycosaminoglycan has no relation to its anticoagulant activity, because the prevention of body fluid loss in the invertebrate does not involve coagulation of the hemolymph.
The importance of dermatan sulfate for the development of sea urchin embryos was emphasized in several experiments where inhibitors of sulfation, chain elongation, or proteoglycan formation were added to the medium and inhibited embryonic development (Oguri and Yamagata, 1978; Kinoshita and Saiga, 1979; Heifetz and Lennarz, 1979; Akasaka et al., 1980; Solursh et al., 1986; Lane and Solursh, 1991). This glycosaminoglycan appears at the mesenchyme blastula–early gastrula stage when the embryonic gut is forming. However, dermatan sulfate is also present on the body wall of adult sea urchins, which indicates that it retains a biological role in the adult stage.
Especially noteworthy is the observation that dermatan sulfates from marine invertebrates are mostly highly sulfated, as shown in this and previous paper (Pavão et al., 1995, 1998). Perhaps interactions between components of the extracellular matrix in these organisms occur at higher salt concentrations than in vertebrates and therefore require glycosaminoglycans with higher charge density. Such a hypothesis will require future investigation of the adhesive properties of these molecules.
Finally, our results indicate that the 4-O-sulfotransferase involved on the sulfation of dermatan sulfate is a developmentally expressed enzyme in sea urchins. It appears 24 h after fertilization and remains active until 70 h postfertilization. However, its level is markedly attenuated in the adult invertebrate. We believe that cloning of this enzyme and determination of how its expression is regulated may constitute a significant contribution for understanding the control of glycosaminoglycan biosynthesis during the development of sea urchin.
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Materials and methods |
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TopAbstractIntroductionResults and discussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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Sea urchin embryos
Embryos of the sea urchin S. purpuratus were grown at 16°C in Millipore filtered sea water (50 mg/L penicillin G and 50 mg/L streptomycin sulfate) with constant gentle agitation. Embryos were collected at the indicated times by gentle hand centrifugation and dissolved in 5% SDS. In some experiments the embryos were immersed in 10 vol of acetone and kept for 24 h at 4°C. The sulfated polysaccharides were then extracted from the dried tissue by papain digestion and partially purified by ethanol precipitation (Alves et al., 1997
).
Purification of dermatan sulfate from sea urchin embryos
Sulfated polysaccharides extracted from sea urchin embryos by papain digestion were applied to a Mono Q-FPLC column (HR 5/5, Amersham Pharmacia Biotech) equilibrated with 20 mM Tris–HCl (pH 8.0). The column was developed by a linear gradient of 0–2.0 M NaCl in the same buffer. The flow rate of the column was 0.5 ml/min, and fractions of 0.5 ml were collected and assayed by metachromasia using 1,9-dimethylmethylene blue (Farndale et al., 1986) and by the carbazole reaction for hexuronic acid (Dische, 1947). The fractions containing the dermatan sulfate (as identified by the positive metachromatic and hexuronic acid assays) were pooled, dialyzed against distilled water, and lyophilized.
PAGE
Sulfated glycoconjugates and sulfated polysaccharides were analyzed by PAGE using two different systems. An initial approached was used for the materials from the embryos dissolved in 5% SDS. These samples (1 µg protein) were precipitated with 9 vol acetone, dissolved in Laemmli buffer with 5% fresh ß-mercaptoethanol, and boiled for 5 min. Five percent polyacrylamide gels in 0.1% SDS were used for electrophoresis. The electrophoresis chamber buffer (0.1% SDS) was diluted 50% (to 1.5 g Tris–OH and 7.2 g glycine per L). Gels were soaked 12 h in 10% acetic acid/7% methanol, and then the sulfated glycoconjugates were stained for 5 h with 0.1% toluidine blue in acetic acid/methanol, followed by destaining in tap water.
Sulfated polysaccharides extracted from the tissue by papain were analyzed by a different system of PAGE. Samples (5 µg, as hexuronic acid) were applied to a 1-mm-thick 6% polyacrylamide slab gel; after electrophoresis at 100 V for 1 h in 0.06 M sodium barbital (pH 8.6), the gel was stained with 0.1% toluidine blue in 1% acetic acid. After staining, the gel was washed overnight in 1% acetic acid. This methodology allow us to estimate the average molecular mass of sulfated glycosaminoglycans (Cardoso and Mourão, 1994; Tovar et al., 1998).
Agarose gel electrophoresis
Glycosaminoglycans were analyzed by agarose gel electrophoresis, as described elsewhere (Vieira et al., 1991; Alves et al., 1997). Briefly, glycosaminoglycans (15 µg) were applied to an agarose gel (0.5%, w/v) and run in 0.05 M 1,3-diaminopropane/acetate (pH 9.0) for 1 h at 120 V. The glycosaminoglycan in the gel were fixed with 0.1% N-cetyl-N,N,N-trimethylammonium bromide in water and stained with 0.1% toluidine blue in acetic acid/ethanol/water (0.1:5:5, v/v/v). After staining, the gel was washed for about 15 min in acetic acid/ethanol/water (0.1:5:5, v/v/v).
Analysis of products formed by digestion of dermatan sulfate with chondroitin AC and ABC lyases
Mammalian and sea urchin dermatan sulfates (200 µg of each) were incubated with 0.1 unit of chondroitin AC lyase or chondroitin ABC lyase in 300 µl of 50 mM Tris–HCl (pH 8.0), containing 5 mM EDTA and 15 mM sodium acetate. After incubation at 37°C for 12 h, aliquots containing the enzyme-resistant glycosaminoglycans in the reaction mixture were analyzed by agarose and/or polyacrylamide gel electrophoresis, as described above. Thereafter, the reaction mixtures were mixed with 3 vol of absolute ethanol. The precipitate formed after standing at –10°C for 24 h, containing the enzyme-resistant glycosaminoglycans, was removed by centrifugation (2000 x g for 15 min at 23°C). The clear supernatant, containing the released disaccharides, was dried on a rotary evaporator and dissolved in 100 µl distilled water. This disaccharide solution (20 µl) and standard compounds were analyzed by strong anion-exchange chromatography on a 250 x 4.6-mm Spherisorb-SAX column (Sigma/Aldrich), linked to an HPLC system from Shimadzu (Tokyo). After sample injection, the column was washed with 5 ml of acidified water (pH 3.5), followed by elution with 40-ml gradient of 0–1.0 M NaCl (pH 3.5) at a flow rate of 1 ml/min. The eluant was monitored for UV absorbance at 233 nm. Disaccharides were identified by comparison with the elution positions of known disaccharide standards (Pavão et al., 1998).
Deaminative cleavage by nitrous acid
Deaminative cleavage by nitrous acid at pH 1.5 was performed as described (Shively and Conrad, 1976). This method cleaves glycosidic linkages occurring on N-sulfate glycosamine residues. PAGE of control and nitrous acid–incubated polysaccharides was used to assess the deaminative cleavage.
NMR experiments
Spectra were recorded using a Bruker DRX 600 triple resonance 5 mm probe. About 1.5 mg of the disaccharide obtained from the sea urchin dermatan sulfate was dissolved in 0.5 ml of 99.9% D2O (NMR grade from Cambridge Isotope Laboratories), and the spectra were recorded at 60°C, with HOD suppression by presaturation. TOCSY, COSY, and 1H/13C heteronuclear correlation (HMQC) spectra were recorded using states time proportional phase incrementation for quadrature detection in the indirect dimension. The COSY spectrum was run with 2048 and 200 points. The TOCSY spectrum was run with 4096 x 200 points with a spin-lock field of about 10 kHz and a mixing time of 80 ms, which was previously determined to give optimum results with these samples. HMQC spectra were run with 1024 x 128 points, and globally optimized alternating phase rectangular pulses were used during acquisition for 13C decoupling. All chemical shifts are relative to external trimethylsilylpropionic acid and methanol.
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Acknowledgments |
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This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq: FNDCT, PADCT, and PRONEX), Financiadora de Estudos e Projetos, Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERS), and NIH Grant HD 12986 (to V.D.V.). We thank Adriana A. Piquet for technical assistance.
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Abbreviations |
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TopAbstractIntroductionResults and discussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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COSY, correlation spectroscopy; HMQC, heteronuclear multiple quantum coherence spectra; HPLC, high-performance liquid chromatography; NMR, nuclear magnetic resonance; PAGE, polyacrylamide gel electrophoresis; TOCSY, total correlation spectroscopy.
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1 To whom correspondence should be addressed
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References |
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