2 Laboratório de Tecido Conjuntivo, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Caixa Postal 68041, Rio de Janeiro, RJ, 21941-590, Brasil; 3 Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Caixa Postal 68041, Rio de Janeiro, RJ, 21941-590, Brasil; 4 Centro de Ressonância Nuclear Magnética de Macromoléculas, Universidade Federal do Rio de Janeiro; and 5 Laboratório de Biomineralização, Departamento de Histologia e Embriologia, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro
Received on May 21, 2004; revised on July 19, 2004; accepted on August 14, 2004
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Abstract |
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Key words: evolution of marine organisms / marine angiosperms / seagrass / sulfated galactans / sulfated polysaccharides
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Introduction |
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A curious observation is that evolutionary distant organisms that share the marine environment possess structurally related sulfated polysaccharides, mostly composed of sulfated fucose or sulfated galactose. Thus, the marine invertebrate ascidians (Chordata-Tunicata) contain high amounts of sulfated galactans as red algae (Mourão and Perlin, 1987; Pavão et al., 1989
; Santos et al., 1992
). In the same way, marine echinoderms possess sulfated fucans as brown algae (Alves et al., 1997
; Mulloy et al., 1994
; Ribeiro et al., 1994
; Vilela-Silva et al., 2002
). This raises an interesting question: Is the occurrence of sulfated polysaccharides an adaptation to marine life?
A possible approach to this question is to investigate the occurrence of sulfated polysaccharides in marine angiosperms or seagrasses. This is a polyphyletic assemblage of 60 marine species (Les et al., 1997
), concentrated in the subclass Alismatidae (Cook, 1990
; Cronquist, 1981
; Tomlinson, 1982
). They display several adaptations to the marine environment, such as a seawater submersed habitat, tolerance to salinity, hydrophily (water-pollinated), and an effective anchorage system. Marine angiosperms are commonly found in the vicinity of mangroves, estuaries, hypo- or hypersaline coastal lagoons and fish ponds (Creed, 2000
), recognized as ecologically important habitats of the coast zone (Larkum and Hartog, 1989
). These species provide food and habitat for associated animals (Hartog, 1970
), enhancing near-shore productivity while buffering waves and currents; many species are uncovered in the meadows (Orth, 1992
).
We now reported for the first time that marine angiosperms contain high amounts of sulfated polysaccharide in contrast with the terrestrial and freshwater species. The seagrass sulfated polysaccharide differs in its chemical structure from similar compounds found in red algae and marine invertebrates. This observation raises interesting questions, such as: Are the genes coding expression of enzymes involved in the sulfation of polysaccharides preserved during the evolution of vascular plants although not expressed or repressed in terrestrial and freshwater species? Did the seagrasses acquire the genes coding sulfotransferase expression by horizontal transfer? Is it plausible that the presence of sulfated galactan in these species is a convergent adaptation to the marine environment?
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Results and discussion |
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Sulfated galactan from R. maritima has a regular tetrasaccharide repeating unit
We employed nuclear magnetic resonance (NMR) analysis to determine the structure of the sulfated galactan from R. maritima. The 1H 1D spectra of the native and desulfated galactan are shown in Figure 2.
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The assignment of peaks was achieved by analysis of 1H correlations spectroscopy (COSY) (Figure 3A), 1H total correlation spectroscopy (TOCSY) (Figure 3B and C), and 1H/13C heteronuclear multiple quantum coherence (HMQC) (Figure 4) spectra, giving the values presented in Table II. Both the ß-H2 (denominated as B2) of residue B and ß-H4 (C4) of residue C show strong downfield shifts (0.8 ppm), indicative of sulfation sites (Figures 3A, 3B, and 4A; Table II).
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The distribution of the different residues in the native sulfated galactan was deduced from the nuclear Overhauser enhancements spectroscopy (NOESY) spectrum (Figure 5). A strong NOE is seen between residue B and A and between residue A and C. Because residue A forms a single type of unit and peak integration shows 0.45:0.26:0.29 (A:B:C) ratio, we deduced that two residues A are linked to each other. An interresidue NOE can be visualized between residues C and B. Overall these results indicate the sequence -B-A-A-C-, as shown in Figure 6B.
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The structure of the sulfated galactans found in these marine organisms varies significantly. In the marine red algae, the sulfated galactans (also known as carrageenans) have a backbone composed of [4--D-Gal-1
3-ß-D-Gal-1] but with a heterogeneous sulfation pattern (Farias et al., 2000
; Painter, 1983
) (Figure 6A). Small amounts of L-enantiomer of galactose and 3,6-anhydro-galactose are also reported in the polysaccharide from red algae (Painter, 1983
). Sulfated galactans from marine invertebrates have regular and repetitive chemical structures. They are 3-sulfated, 4-linked (Mourão and Perlin, 1987
; Pavão et al., 1989
; Santos et al., 1992
) (Figure 6C) and 2-sulfated, 3-linked (Alves et al., 1997
) (Figure 6D) in ascidian and sea urchin, respectively. In the invertebrate polysaccharides, galactose occurs exclusively as the unusual L-enantiomer (Mourão and Perlin, 1987
; Pavão et al., 1989
). Furthermore, sulfated galactans from some species of ascidians are highly branched polysaccharides (Mourão and Perlin, 1987
; Pavão et al., 1989
). The sulfated galactan from marine angiosperm has an intermediate structure. Like marine invertebrate polysaccharide, it exhibits a regular repeating sequence with a homogenous sulfation pattern. However, seagrass galactan contains the D-enantiomer of galactose instead of the L-isomer found in marine invertebrates. Like red algae galactan, the marine angiosperm polysaccharide is composed of
and ß units of D-galactose. These units, however, are not distributed in an alternating order, as in the algal polysaccharide.
Evolutionarily distant species contain sulfated galactan: what's the meaning?
The observation that in contrast with terrestrial and freshwater plants the marine angiosperms contain high amounts of sulfated polysaccharides (like other marine species) raises puzzling questions concerning the evolution of these organisms, such as described next.
Are the genes coding expression of enzymes involved in the sulfation of polysaccharides present but not expressed or repressed in terrestrial and fresh water plants?
A possible explanation for the occurrence of sulfated galactans in marine but not in terrestrial and freshwater angiosperms could be that the genes coding for the enzyme involved in the sulfation of these macromolecules remains preserved in plants although not expressed or repressed in the terrestrial or freshwater species. If this hypothesis held true, the marine environment would induce the expression of these genes. Our search indicated that the known genomic sequences of two species of terrestrial plants have no homology to sulfotransferase genes. Thus, genes coding enzymes involved in sulfation of polysaccharides are not conserved during the evolution of terrestrial plants or have been extensively mutated in higher plants and hence are no homologous to the sulfotransferase genes.
A horizontal gene transfer from another marine organism?
The occurrence of sulfated galactan correlates more with physiological adaptation than with phylogenetic distance, and hence fits a selective scenario. A possible explanation for this event is that the marine angiosperms acquire the genes for the enzymes involved in the sulfation of polysaccharides from other marine organism, such as red algae. It is conceivable that marine angiosperms could assimilate the genes for biosynthesis of the sulfated galactan from marine alga or invertebrate through microorganism infection and use them as an adaptive device to the marine environment. In a similar way, Lidholt et al. (1994) reported that a bacterial glycosyltransferase involved in the biosynthesis of a capsular polysaccharide mimics the highly elaborate substrate specificity of the corresponding enzymes necessary for heparin biosynthesis. As a plausible explanation for this result, the authors speculated that the microorganism had assimilated the gene for glycosyltransferases from an infected mammalian host and used it to generate a protective capsule. However, the structural differences among the sulfated galactans from marine angiosperms, invertebrates, and red algae (Figure 6) do not favor a horizontal transfer of genes coding enzymes for the biosynthesis of the sulfated galactan, because in that case we would expect higher homology between the galactans. Furthermore, seagrasses make up a polyphyletic group, which makes the horizontal gene transfer even more improbable. Finally, the transfer of gene is an unusual event during the evolutionary process and has not been clearly demonstrated during the evolution of animals, plants, and fungi (Cho et al., 1998
; Goddard and Burt, 1999), although it has been reported in flowering plants (Bergthorsson et al., 2003
).
A convergent adaptation?
A more plausible explanation for the presence of related polysaccharides, the sulfated galactans, in evolutionary distant organisms that share the marine environment is a convergent adaptation due to environmental selective pressure. It has been reported previously in seagrasses, related with the hydrophily of these plants (Les et al., 1997). Convergent adaptation (or convergent evolution) is a common biological event, when similar-appearing structures evolved in entirely unrelated groups of organisms, such as the classical examples of the vertebrate eye and the cephalopod eye, the bivalve shells of mollusks and of brachiopods, and so on (Brusca and Brusca, 1990
). However, convergent evolution has not yet been suggested for molecules found in the extracellular matrix of phylogenetic unrelated organisms, as in the present study.
Major conclusions
We reported the occurrence of sulfated galactan in the marine angiosperm. This polysaccharide has a unique structure, with a regular repeating tetrasaccharide sequence, composed of [3-ß-D-Gal-2(OSO3)-14-
-D-Gal-1
4-
-D-Gal-1
3-ß-D-Gal-4(OSO3)-1
]. The occurrence of sulfated galactan in living organisms correlates more with physiological adaptation than with phylogenetic distance and hence fits a selective scenario. We suggest that a convergent adaptation due to environmental pressure may explain the occurrence of high concentration of sulfated polysaccharide in marine organisms.
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Materials and methods |
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Purification
The crude polysaccharide (100 mg) was applied to a Sephacryl 400 HR column (200 x 3.5 cm), equilibrated with 0.2 M sodium bicarbonate (pH 6.0), and eluted with the same solution. The flow rate of the column was 5 ml/h and fractions of 4.0 ml were collected in regular intervals. Fractions were checked for hexose and hexuronic acid by the Dubois reaction (Dubois et al., 1956) and carbazol reaction (Dische, 1947
), respectively, and also by metachromatic assay using 1,9-dimethylmethylene blue (Farndale et al., 1986
). Fractions containing the sulfated galactan (indicated by the positive metachromatic reaction) and free of contaminants such as pectins (indicated by positive carbazole reaction) or a yellow pigment were pooled, dialyzed against distilled water, and lyophilized.
The sulfated galactan obtained from the Sephacryl column (10 mg) was applied to a Mono Q-FPLC column (HR 5/5, Amersham Pharmacia Biotech, Little Chalfont, UK), equilibrated with 20 mM TrisHCl (pH 8.0), containing 10 mM ethylenediamine tetra-acetic acid. The column was developed by a linear gradient of 14 M NaCl in the same solution. The flow rate of the column was 0.5 ml/min and fractions of 0.5 ml were collected and assayed by metachromasia. The fractions containing the sulfated galactan were pooled, dialyzed against distilled water, and lyophilized.
Agarose gel electrophoresis
Sulfated galactans were analyzed by agarose gel electrophoresis as described (Alves et al., 1997; Vieira et al., 1991
). Samples (
15 µg) were applied to a 0.5% agarose gel and run for 1 h at 110 V in 50 mM 1,3-diaminopropane:acetate buffer (pH 9.0). The sulfated polysaccharides in the gel were fixed with 0.1% N-cetyl-N,N,N-trimethylammonium bromide solution. After 12 h, the gel was dried and stained with 0.1% toluidine blue in acetic acid:ethanol:water (0.1:5:5, v/v).
Chemical analysis
After acid hydrolysis (6.0 M trifluoroacetic acid 100°C for 5 h) of the sulfated polysaccharide, the hexose was identified by paper chromatography in n-butanol:pyridine:water (3:2:1, v/v) for 48 h on Whatman No. 1 paper, followed by staining with silver nitrate and by gas-liquid chromatography (GLC) of derived alditol acetates (Kircher, 1960). Sulfate was measured by the BaCl2/gelatin method (Saito et al., 1968
).
Determination of the D or L configuration of galactose
The enantiomeric form of the galactose was assigned based on analysis of the trimethylsilylated ()-2-butyl glycoside, as described (Gerwig et al., 1978). The polysaccharide from R. maritima (1 mg) was mixed with 0.5 ml ()-2-butanol, containing 1 M HCl (Aldrich, Milwaukee, WI). After butanolysis for 18 h at 80°C, the solution was neutralized with Ag2CO3, and the supernatant was concentrated and dissolved in 50 ml. Thereafter, we added 50 ml bis(trimethylsilyl)trifluoro acetamide (Sigma, St. Louis, MO) and kept the solution for 30 min at room temperature. The butanolyzed and trimethylsilylated derivatives were analyzed on a DB-5 GLC column. The temperature was programmed from 120°C to 240°C at 2°C/min. The injector and detector temperatures were 220°C and 260°C, respectively. Appropriate controls of trimethylsilylated ()-2-butyl-D- and L-galactosides were analyzed under the same conditions.
Desulfation and methylation of the sulfated galactan
Desulfation of the sulfated galactan was performed as described (Mourão and Perlin, 1987; Vieira et al., 1991
). The sulfated polysaccharide (10 mg) was dissolved in 5 ml distilled water and mixed with 1 g (dry weight) of Dowex 50-W (H1, 200400 mesh). After neutralization with pyridine, the solution was lyophilized. The resulting pyridinium salt of the sulfated galactan was dissolved in 2.5 ml dimethyl sulfoxide:methanol (9:1,v/v). The mixture was heated at 80°C for 4 h, and the desulfated product was exhaustively dialyzed against distilled water and lyophilized.
The desulfated galactan (5 mg) was subjected to three rounds of methylation as described previously (Ciucanu and Kerek, 1984) with the modifications suggested by Patankar et al. (1993)
. The methylated polysaccharide was hydrolyzed in 6 M trifluoroacetic acid for 5 h at 100°C and reduced with borohydride. The alditols were acetylated with acetic anhydride:pyridine (1:1, v/v) (Kircher, 1960
). The alditol acetates of the methylated sugar was dissolved in chloroform and analyzed in a gas chromatographymass spectrometer (DB-1 capillary column).
NMR spectroscopy
1H and 13C spectra of the native and desulfated galactan were recorded using a Bruker DRX 400 apparatus with a triple-resonance probe. About 4 mg of each sample was dissolved in 0.5 ml 99.9% D2O (Cambridge Isotope Laboratory, Andover, MA). All spectra were recorded at 60°C with HOD suppression by presaturation. COSY, TOCSY, NOESY, and 1H/13C HMQC spectra were recorded using states-time proportion phase incrementation for quadrature detection in the indirect dimension. TOCSY spectra were run with 4046 x 400 points with a spin-lock field of 10 kHz and a mixed time of 80 ms. HMQC spectra were run with 1024 x 256 points and globally optimized alternating phase rectangular pulses for decoupling. NOESY spectra were run with a mixing time of 100 ms. Chemical shifts are relative to external trimethylsilylpropionic acid at 0 ppm for 1H and to methanol for 13C.
Sulfated polysaccharides localization by optical microscopy
Samples of the seagrass R. maritima were collected in the same area described, transported to the laboratory in plastic bags with sea water, and cleaned from other organisms. Fragments of 5 mm diameter were cut from the leaves, roots, and rhizomes and immediately fixed in 2.5% glutaraldehyde (Merck EM grade) buffered with 0.2 M sodium cacodylate (pH 7.2) in sea water for 2 h at room temperature. The fragments were rinsed in the same buffer, dehydrated with acetone graded series, and embedded in Spurr resin. Semithin sections (2 µm) were obtained in a microtome (Sorvall, Asheville, NC) and stained with 1% toluidine blue (Sigma), pH 4.4, for 3 min at 40°C. This stain indicates the presence of sulfated polysaccharides by metachromasia. The slides of the three compartments of the seagrass were observed in a Nikon Eclipse, and digital images were acquired using the software Image Pro Plus using the same conditions for all slides.
GenBank database analysis
To look for homologous sequences of the sulfotransferase gene, we conducted a search on GenBank database of two terrestrial plants with known genomic sequences. Because various enzymes act on the sulfation of diverse compounds, such as alcohols, phenols, and steroids, we had to compare the sequences of genes for an enzyme specific for sulfation of polysaccharide between several species. Thus, we tested a consensus sequence of eight 2-O-sulfotransferases (the seagrass polysaccharide has a sulfate at the position 2). The sulfotransferase sequences were identified using an in silico approach. The screening for 2-O-sulfotransferase sequences was performed on the National Center for Biotechnology Information GenBank sequence database (accession numbers: BC059008.1, BC025443.1, NM_204481.1, NM_012262.2, AB093516.1, NM_011828.2, AF169243, AB024568.1) and translated in all six frames using the Expert Protein Analysis System translate tool. A highly conserved consensus sequence (MFRKMGLLRIMMPPKHWLQLLAVVAFAVAMLFLENQIQKLEESRAKLERAIARHEVREIEQRHTMDGPRQDATLDEEEDIIIIYNRVPKTASTSFTNIAYDLCAKNRYHVLHINTTKNNPVMSLQDQVRFVKNITTWNEMKPGFYHGHISYLDFAKFGVKKKPIYINVIRDPIERLVSYYYFLRFGDDYRPGLRRRKQGDKKTFDECVAEGGSDCAPEKLWLQIPFFCGHSSECWNVGSRWAMDQAKSNLINEYFLVGVTEELEDFIMLLEAALPRFFRGATDLYRTGKKSHLRKTTEKKLPTKQTIAKLQQSDIWKMENEFYEFALEQFQFIRAHAVREKDGDLYILAQNFFYEKIYPKSN) was obtained after alignment using CLUSTAL W (Thompson et al., 1994). tBLASTn searches against the plant genome data base of Arabidopsis thaliana and Oryza sativa using the consensus sequence of 2-O-sulfotranferase as a query to identify a plant homologous sequence to polysaccharide sulfotranferase.
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Acknowledgements |
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Abbreviations |
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References |
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