Occurrence of sulfated galactans in marine angiosperms: evolutionary implications

Rafael S. Aquino2, Ana M. Landeira-Fernandez3,Ana Paula Valente3,4, Leonardo R. Andrade5, andPaulo A. S. Mour~aao1,2,3

2Laborat�oorio de Tecido Conjuntivo, Hospital Universit�aario ClementinoFraga Filho, Universidade Federal do Rio de Janeiro, Caixa Postal 68041,Rio de Janeiro, RJ, 21941-590, Brasil; 3Departamento de BioquımicaM�eedica, Instituto de Cieencias Biom�eedicas, Universidade Federal do Riode Janeiro, Caixa Postal 68041, Rio de Janeiro, RJ, 21941-590, Brasil;4Centro de Ressonancia Nuclear Magn�eetica de Macromol�eeculas,Universidade Federal do Rio de Janeiro; and 5Laborat�oorio deBiomineralizac~aao, Departamento de Histologia e Embriologia, Institutode Cieencias Biom�eedicas, Universidade Federal do Rio de Janeiro

Received on May 21, 2004; revised on July 19, 2004;accepted on August 14, 2004

We report for the first time that marine angiosperms(seagrasses) possess sulfated polysaccharides, which areabsent in terrestrial and freshwater plants. The structureof the sulfated polysaccharide from the seagrass Ruppiamaritima was determined. It is a sulfated D-galactan com-posed of the following regular tetrasaccharide repeatingunit: [3-b-D-Gal-2(OSO3)-1!4-a-D-Gal-1!4-a-D-Gal-1!3-b-D-Gal-4(OSO3)-1!]. Sulfated galactans havebeen described previously in red algae and in marine inver-tebrates (ascidians and sea urchins). The sulfated galactanfrom the marine angiosperm has an intermediate structurewhen compared with the polysaccharides from these twoother groups of organisms. Like marine invertebrate galac-tan, it expresses a regular repeating unit with a homo-genous sulfation pattern. However, seagrass galactancontains the D-enantiomer of galactose instead of theL-isomer found in marine invertebrates. Like red algae, themarine angiosperm polysaccharide contains both a and bunits of D-galactose; however, these units are not distrib-uted in an alternating order, as in algal galactan. Sulfatedgalactan is localized in the plant cell walls, mostly inrhizomes and roots, indicative of a relationship with theabsorption of nutrients and of a possible structural func-tion. The occurrence of sulfated galactans in marine organ-isms may be the result of physiological adaptations, whichare not correlated with phylogenetic proximity. We suggestthat convergent adaptation, due to environment pressure,may explain the occurrence of sulfated galactans in manymarine organisms.

Key words: evolution of marine organisms/marineangiosperms/seagrass/sulfated galactans/sulfatedpolysaccharides

Introduction

Sulfated polysaccharides comprise a complex group ofmacromolecules with a wide range of important biologicalproperties. These anionic polymers are widespread innature, occurring in a great variety of organisms. Highamounts of sulfated galactans and sulfated fucans arefound in marine algae (Painter, 1983; Pervical andMcDowell, 1967). In the animal kingdom, sulfated glycos-aminoglycans abound in vertebrate tissues (C�aassaro andDietrich, 1977; Mathews, 1975). Invertebrate species arealso a rich source of sulfated polysaccharides with novelstructures (Mour~aao and Perlin, 1987; Mour~aao et al., 1996;Pav~aao et al., 1998; Vieira et al., 1991; Vilela-Silva et al.,2002). However, sulfated polysaccharides have not beenfound in higher vascular plants (C�aassaro and Dietrich,1977; Yoon et al., 2002).A curious observation is that evolutionary distant organ-

isms that share the marine environment possess structurallyrelated sulfated polysaccharides, mostly composed ofsulfated fucose or sulfated galactose. Thus, the marineinvertebrate ascidians (Chordata-Tunicata) contain highamounts of sulfated galactans as red algae (Mour~aao andPerlin, 1987; Pav~aao et al., 1989; Santos et al., 1992). In thesame way, marine echinoderms possess sulfated fucans asbrown algae (Alves et al., 1997; Mulloy et al., 1994; Ribeiroet al., 1994; Vilela-Silva et al., 2002). This raises an interest-ing question: Is the occurrence of sulfated polysaccharidesan adaptation to marine life?A possible approach to this question is to investigate the

occurrence of sulfated polysaccharides in marine angio-sperms or seagrasses. This is a polyphyletic assemblageof ~60 marine species (Les et al., 1997), concentrated inthe subclass Alismatidae (Cook, 1990; Cronquist, 1981;Tomlinson, 1982). They display several adaptations to themarine environment, such as a seawater submersed habitat,tolerance to salinity, hydrophily (water-pollinated), andan effective anchorage system. Marine angiosperms arecommonly found in the vicinity of mangroves, estuaries,hypo- or hypersaline coastal lagoons and fish ponds(Creed, 2000), recognized as ecologically important habitatsof the coast zone (Larkum and Hartog, 1989). These speciesprovide food and habitat for associated animals (Hartog,1970), enhancing near-shore productivity while bufferingwaves and currents; many species are uncovered in themeadows (Orth, 1992).We now reported for the first time that marine angio-

sperms contain high amounts of sulfated polysaccharidein contrast with the terrestrial and freshwater species. Theseagrass sulfated polysaccharide differs in its chemicalstructure from similar compounds found in red algae andmarine invertebrates. This observation raises interesting1To whom correspondence should be addressed; e-mail:

pmourao@hucff.ufrj.br

Glycobiology vol. 15 no. 1 # Oxford University Press 2005; all rights reserved. 11

Glycobiology vol. 15 no. 1 pp. 11–20, 2005doi:10.1093/glycob/cwh138Advance Access publication on August 18, 2004

questions, such as: Are the genes coding expression ofenzymes involved in the sulfation of polysaccharidespreserved during the evolution of vascular plants althoughnot expressed or repressed in terrestrial and freshwaterspecies? Did the seagrasses acquire the genes codingsulfotransferase expression by horizontal transfer? Is it plau-sible that the presence of sulfated galactan in these speciesis a convergent adaptation to the marine environment?

Results and discussion

Occurrence of sulfated galactans in the seagrasses

Species of marine angiosperms, namely, Ruppia maritima,Halodule wrightii, and Halophila decipiens, but not terres-trial angiosperms contain high amounts of sulfated poly-saccharides (Table I).The sulfated polysaccharides extracted from the species

R. maritimawere studied in more details. They were initiallypartial purified by gel chromatography on Sephacryl400HR, monitored for hexose, hexuronic acid, andmetachromasia (data not shown). The fraction sensitive tohexuronic acid, possibly due to the presence of pectin, wasdischarged. Anion exchange chromatography on MonoQ–fast performance liquid chromatography (FPLC) of thepectin-free polysaccharides yielded three major fractions,denoted as F1, F2, and F3, eluted from the column with1.0, 1.8, and 2.4 M NaCl, respectively (Figure 1A). Agarosegel electrophoresis of these fractions revealed F3 containsa single band (Figure 1B).Chemical analysis of fraction F3 revealed the occurrence

exclusively of galactose and sulfate. The trimethylsilylated(�)-2-butyl galactosides obtained from this polysaccharidehad the same retention times and peaks areas as the stan-dard D-galactose. Therefore, galactose occurs on theR. maritima galactan exclusively as the D-enantiomer.

Sulfated galactan from R. maritima has a regulartetrasaccharide repeating unit

We employed nuclear magnetic resonance (NMR) analysisto determine the structure of the sulfated galactan from

R. maritima. The 1H 1D spectra of the native and desulfatedgalactan are shown in Figure 2.The desulfated galactan shows two main anomeric reso-

nance, one at 5.2 ppm (a-unit) and another at 4.8 ppm(b-unit) (Figure 2B). Peak integration shows 0.4:0.6 ratio.The spectrum of the native galactan (Figure 2A) indicatesthat the b-unit is in fact composed by two anomeric signals,B1 and C1, whereas the a-unit has a single signal at5.21 ppm, A1. The integration of these peaks shows a0.45:0.26:0.29 ratio for A:B:C. Beyond the signals ascribedto anomeric protons, the spectrum of the native galactanshow two other signals with strong downfield shifts, asexpected for sulfation sites, and noted as C4 and B2.The assignment of peaks was achieved by analysis of 1H

correlations spectroscopy (COSY) (Figure 3A), 1H totalcorrelation spectroscopy (TOCSY) (Figure 3B and C),and 1H/13C heteronuclear multiple quantum coherence(HMQC) (Figure 4) spectra, giving the values presented inTable II. Both the b-H2 (denominated as B2) of residue Band b-H4 (C4) of residue C show strong downfield shifts

Fig. 1. Purification of the sulfated galactan fromR. maritima on aMonoQ-FPLC column (A) and analysis of the purified fractions by agarose gelelectrophoresis (B). (A) Sulfated polysaccharides previously purified on aSephacryl column (10 mg) were applied to a Mono Q-FPLC and elutedas described under Materials and methods. Fractions were assayed formetachromasia (closed circles) and NaCl concentration (dashed line).The fractions indicated by the horizontal bar were pooled, dialyzedagainst distilled water, and lyophilized. (B) The three fractions of sulfatedpolysaccharides (15 mg each) were applied to a 0.5% agarose gel in 50 mm1,3-diaminopropane:acetate (pH 9.0) and run at 110 V for 1 h. Gel wasfixed with 0.1% N-cetyl-N,N,N-trimethylammonium bromide solutionovernight, dried, and stained with 0.1% toluidine blue in aceticacid:ethanol:water (0.1:5:5, v/v).

Table I. Concentration of sulfated polysaccharide in species of marineand terrestrial angiosperms

Angiosperm SpeciesSulfated polysaccharide(mg/mg dry tissue)

Marine R. maritima 10.3

H. wrightii 8.5

H. decipiens 7.7

Terrestrial Z. mays (maize) 50.001

P. vulgaris (bean) 50.001

Sulfated polysaccharides were extracted from the angiosperm species byprotease digestion and their concentrations were determined based on themetachromatic assay using 1,9-dimethylmethylene blue (Farndale et al.,1986) and a standard curve with the purified sulfated galactan fromR. maritima.

R.S. Aquino et al.

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(~0.8 ppm), indicative of sulfation sites (Figures 3A, 3B,and 4A; Table II).The position of the glycosidic linkage was not easily

deduced from the 1H chemical shifts, but the 13C chemical

shifts of the desulfated galactan displayed two signals, a-C4and b-C3 (Figure 4B, Table II), which are ~10 ppm down-field shifts. This indicates glycosilated positions (Falshawand Furneaux, 1994). Methylation analysis using the desul-fated galactan yielded equimolar proportions of 2,3,6-tri-O-methylgalactose (49%) and 2,4,6-tri-O-methylgalactose(51%), as expected. Therefore, a-units and b-units are4-linked and 3-linked, respectively.The distribution of the different residues in the native

sulfated galactan was deduced from the nuclear Overhauser

Fig. 2. 1H NMR spectra at 400 MHz of native (A) and desulfated (B)galactan from R. maritima (fraction P3). The spectra were recorded at60�C for samples in D2O solution. The residual water signal has beensuppressed by presaturation. Chemical shifts are relative to externaltrimethylsilylpropionic acid at 0 ppm. The anomeric protons assigned by1H/13C HMQC (see Figure 4A) are labeled in the native sample (A). Thesignal designated A1 refer to this produced by a(1!4) units, whereasthose produced by b(1!3) residues are labeled B1 and C1. B2 and C4 aresignals of protons bearing sulfation sites assigned to b-H2 and b-H4,respectively. The presence of a less intense C4 signal in the desulfatedgalactan (B) indicates that some 4-sulfate ester resists to the desulfationreaction. The integrals listed under the anomeric signals are normalizedto the sum of all the anomeric protons.

Fig. 3. COSY (A) and TOCSY (B, C) spectra of native (A, B) anddesulfated (C) galactan from R. maritima at 400 MHz, 60�C, in D2O.

Sulfated galactans in seagrass

13

enhancements spectroscopy (NOESY) spectrum (Figure 5).A strong NOE is seen between residue B and A and betweenresidue A and C. Because residue A forms a single typeof unit and peak integration shows 0.45:0.26:0.29 (A:B:C)ratio, we deduced that two residues A are linked to eachother. An interresidue NOE can be visualized between resi-dues C and B. Overall these results indicate the sequence-B-A-A-C-, as shown in Figure 6B.

Sulfated galactan is located in the cell walls of theseagrass

To find out the tissue localization of the sulfated galactanin the marine seagrass R. maritima, we obtained semithinsections from three different regions of the plant, whichwere stained with toluidine blue and visualized by opticalmicroscopy (Figure 7A–C). The sulfated galactan is mostlylocalized in the plant cell wall, like the sulfated polysacchar-ides from marine algae. High amounts of the sulfatedgalactans were detected in the rhizomes of the seagrass,low proportion in the roots and practically absent in the

leaves as demonstrated by the different staining intensitiesat the microscopy experiments. Biochemical analysis(Figure 7D) indicates that although at lower proportion,leaves of R. maritima also possess sulfated polysaccharides.This observation suggests that the sulfated galactans havean osmotic function in the marine angiosperms, as alreadyreported for the sulfated polysaccharides in marine algae.In this way, sulfated galactan is more concentrated inthe rhizome and root and less in the leaves of the seagrass,which are compartments responsible for the nutrientabsorption from the sea water and, thus, more vulnerableto the salinity gradient. The histological analysis also indi-cates that the sulfated polysaccharides are more con-centrated in the external than in the internal regions of theplant. This is especially noted in the root (Figure 7C). Theseobservations suggest that the sulfated polysaccharidescould perform similar functions as in algae, selecting themolecules that enter into the cells and also maintaining theion balance between cytoplasm and environment (Kloaregand Quatrano, 1988). In addition, the roots and rhizomesare the regions that anchor the plant to the substrate, indi-cating that the presence of sulfated polysaccharide couldalso have a structural function, allowing the plant to survivein a high-kinetic-force environment like the sea coast.

Variety of chemical structures found in sulfated galactansfrom marine organisms

Sulfated galactans have been described in several organ-isms, which share the marine environment but belong tophylogenetic distant groups, such as red algae (Farias et al.,2000; Painter, 1983) and the invertebrate ascidians (Albanoand Mour~aao, 1986; Pav~aao et al., 1989; Santos et al., 1992)and sea urchins (Alves et al., 1997). We report for the firsttime that seagrasses, a group of angiosperms adapted to themarine environment, also contain high amounts of sulfatedgalactan. The presence of sulfated galactans in seagrassesthat evolved separately indicates that the occurrence ofsulfated polysaccharide is an important adaptation to themarine environment.The structure of the sulfated galactans found in these

marine organisms varies significantly. In the marine redalgae, the sulfated galactans (also known as carrageenans)have a backbone composed of [4-a-D-Gal-1!3-b-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 arealso reported in the polysaccharide from red algae (Painter,1983). Sulfated galactans from marine invertebrateshave regular and repetitive chemical structures. They are3-sulfated, 4-linked (Mour~aao and Perlin, 1987; Pav~aaoet al., 1989; Santos et al., 1992) (Figure 6C) and 2-sulfated,3-linked (Alves et al., 1997) (Figure 6D) in ascidian and seaurchin, respectively. In the invertebrate polysaccharides,galactose occurs exclusively as the unusual L-enantiomer(Mour~aao and Perlin, 1987; Pav~aao et al., 1989). Further-more, sulfated galactans from some species of ascidiansare highly branched polysaccharides (Mour~aao and Perlin,1987; Pav~aao et al., 1989). The sulfated galactan frommarineangiosperm has an intermediate structure. Like marineinvertebrate polysaccharide, it exhibits a regular repeating

Fig. 4. 1H/13C HMQC spectra of native (A) and desulfated (B) galactanfrom R. maritima at 400 MHz, 60�C, in D2O. The assignment was basedon TOCSY and COSY spectra. The values of chemical shifts reportedin Table II are relative to external trimethylsilylpropionic acid at 0 ppmfor 1H and methanol for 13C. The anomeric signals were identified bythe characteristic carbon chemical shifts.

R.S. Aquino et al.

14

sequence with a homogenous sulfation pattern. However,seagrass galactan contains the D-enantiomer of galactoseinstead of the L-isomer found in marine invertebrates. Likered algae galactan, the marine angiosperm polysaccharideis composed of a and b units of D-galactose. These units,

however, are not distributed in an alternating order, as inthe algal polysaccharide.

Evolutionarily distant species contain sulfated galactan:what’s the meaning?

The observation that in contrast with terrestrial and fresh-water plants the marine angiosperms contain high amountsof sulfated polysaccharides (like other marine species) raisespuzzling questions concerning the evolution of these organ-isms, such as described next.

Are the genes coding expression of enzymes involved in thesulfation of polysaccharides present but not expressed orrepressed in terrestrial and fresh water plants?. A possibleexplanation for the occurrence of sulfated galactans inmarinebut not in terrestrial and freshwater angiosperms could bethat the genes coding for the enzyme involved in the sulfationof these macromolecules remains preserved in plantsalthough not expressed or repressed in the terrestrial or fresh-water species. If this hypothesis held true, the marine envir-onment would induce the expression of these genes. Oursearch indicated that the known genomic sequences of twospecies of terrestrial plants have no homology to sulfotrans-ferase genes. Thus, genes coding enzymes involved insulfation of polysaccharides are not conserved during theevolution of terrestrial plants or have been extensivelymutated in higher plants and hence are no homologous tothe sulfotransferase genes.

Table II. 1H and 13C chemical shifts for residues of galactose in native and desulfated galactan from the seagrass R. maritima and standard compounda

1H chemical shifts (ppm)

Polysaccharide Unit H1 H2 H3 H4 H5 H6

Native galactan from R. maritima A 5.21 4.18 4.41 4.35 4.35 3.03

B 5.01 4.69 4.25 4.10 ND 3.93

C 4.91 3.73 3.97 4.97 4.08 3.93

Desulfated galactan from R. maritima a 5.21 ND 4.41 4.19 ND 3.93

b 4.84 3.85 4.36 4.19 ND 3.93

Desulfated galactan from a red algaa 4-a-D-Gal-1 5.29 3.85 3.95 4.22 4.16 3.78

3-bD-Gal-1 4.40 3.78 3.75 4.12 3.73 3.78

13C chemical shits (ppm)

C1 C2 C3 C4 C5 C6

Native galactan from R. maritima A 99.2 72.3 68.5 79.7 67.5 60.5

B 102.7 77.3 79.5 66.8 ND 60.5

C 103.5 71.8 72.8 77.4 68.7 60.5

Desulfated galactan from R. maritima a 99.0 ND 69.0 79.6 ND 60.5

b 104.4 72.3 78.3 68.4 ND 60.5

Desulfated galactan from a red algaa 4-a-D-Gal-1 102.9 71.5 70.9 80.9 73.9 63.0

3-b-D-Gal-1 105.3 72.3 82.7 70.4 77.5 63.0

Chemical shifts are relative to external trimethylsilylpropionic acid at 0 ppm for 1H and to methanol for 13C. Values in bold and italic indicate sulfatedand glycosilated positions, respectively.aSee Farias et al. (2000).

Fig. 5. NOESY spectrum of the sulfated galactan from R. maritima at400MHz, 60�C, in D2O. The spectrum shows signals connecting residuesA-C, C-B, and B-A. This indicates the following sequence of residues:-B-A-A-C-.

Sulfated galactans in seagrass

15

A horizontal gene transfer from another marineorganism?. The occurrence of sulfated galactan correlatesmore with physiological adaptation than with phylogeneticdistance, and hence fits a selective scenario. A possibleexplanation for this event is that the marine angiospermsacquire the genes for the enzymes involved in the sulfationof polysaccharides from other marine organism, such as redalgae. It is conceivable that marine angiosperms couldassimilate the genes for biosynthesis of the sulfated galactanfrom marine alga or invertebrate through microorganisminfection and use them as an adaptive device to themarine environment. In a similar way, Lidholt et al. (1994)reported that a bacterial glycosyltransferase involved inthe biosynthesis of a capsular polysaccharide mimics thehighly elaborate substrate specificity of the correspondingenzymes necessary for heparin biosynthesis. As a plausibleexplanation for this result, the authors speculated that themicroorganism had assimilated the gene for glycosyltrans-ferases from an infected mammalian host and used it togenerate a protective capsule. However, the structuraldifferences among the sulfated galactans from marine

angiosperms, invertebrates, and red algae (Figure 6) donot favor a horizontal transfer of genes coding enzymesfor the biosynthesis of the sulfated galactan, because inthat case we would expect higher homology between thegalactans. Furthermore, seagrasses make up a polyphyleticgroup, which makes the horizontal gene transfer even moreimprobable. Finally, the transfer of gene is an unusual eventduring the evolutionary process and has not been clearlydemonstrated during the evolution of animals, plants, andfungi (Cho et al., 1998; Goddard and Burt, 1999), althoughit has been reported in flowering plants (Bergthorsson et al.,2003).

A convergent adaptation?. A more plausible explanationfor the presence of related polysaccharides, the sulfatedgalactans, in evolutionary distant organisms that share themarine environment is a convergent adaptation due toenvironmental selective pressure. It has been reported pre-viously in seagrasses, related with the hydrophily of theseplants (Les et al., 1997). Convergent adaptation (orconvergent evolution) is a common biological event, when

Fig. 6. Phylogenetic relationships and divergence times of marine red algae, angiosperms and invertebrates and structure of their sulfated galactans. (A)The sulfated galactans from marine red algae have a repeating structure (-4-a-D-Gal-1!3-b-D-Gal-1!)n, with a variable sulfation pattern. In thespecies B. occidentalis, ~ one-third of the units are 2,3-disulfated and another third are 2-sulfated (Farias et al., 2000). (B) The sulfated galactan from themarine angiosperm R. maritima has the repeating structure (3-b-D-Gal-2(OSO3)-1!4-a-D-Gal-1!4-a-D-Gal-1!3-b-D-Gal-4(OSO3)-1!)n. (C) In thecase of tunicates, the sulfated galactans have 3-sulfated, 4-linked a-L-galactose units, and in several species nonsulfated L-galactose residues occur asbranched units (Mour~aao and Perlin, 1987; Pav~aao et al., 1989; Santos et al., 1992). (D) Sulfated galactan from sea urchin has a regular repeating structure,with a single sulfated monosaccharide unit, composed of 2-sulfated, 3-linked a-L-galactose (Alves et al., 1997). Land angiosperms and vertebratesdo not contain sulfated galactan.

R.S. Aquino et al.

16

similar-appearing structures evolved in entirely unrelatedgroups of organisms, such as the classical examples of thevertebrate eye and the cephalopod eye, the bivalve shellsof mollusks and of brachiopods, and so on (Brusca andBrusca, 1990). However, convergent evolution has not yetbeen suggested for molecules found in the extracellularmatrix of phylogenetic unrelated organisms, as in thepresent study.

Major conclusions

We reported the occurrence of sulfated galactan in themarine angiosperm. This polysaccharide has a unique struc-ture, with a regular repeating tetrasaccharide sequence,composed of [3-b-D-Gal-2(OSO3)-1! 4-a-D-Gal-1! 4-a-D-Gal-1! 3-b-D-Gal-4(OSO3)-1!]. The occurrence of sul-fated galactan in living organisms correlates more withphysiological adaptation than with phylogenetic distanceand hence fits a selective scenario. We suggest that a con-vergent adaptation due to environmental pressure mayexplain the occurrence of high concentration of sulfatedpolysaccharide in marine organisms.

Materials and methods

Sulfated galactans from seagrasses

Extraction. The seagrasses R. maritima and H. wriightwere collected at Ilha do Japonees, Cabo Frio, andH. decipiens at Urca, Rio de Janeiro, Brazil. They were

carefully separated from other organisms and sun-dried.The dried tissue (20 g) was crushed with a blender (Oster,Super Deluxe), suspended in 400 ml 0.1 M sodium acetate(pH 6.0), containing 2 g papain (Merck, Darmstadt,Germany), 5 mM ethylenediamine tetra-acetic acid, and5 mM cysteine. After incubation for 24 h at 60�C, themixture was filtrated and the supernatant saved. The sul-fated polysaccharides in the solution were precipitated with800 ml absolute ethanol. After 24 h at 4�C, the precipitateformed was collected by centrifugation (2560� g for 20 minat 5�C). The final precipitate was dried at 60�C for 12 h.Approximately 1.6 g (dry weight) of crude polysaccharidewas obtained after these procedures. The sulfated galactanwas purified by a combination of gel filtration and anionexchange chromatography, as described next.

Purification. The crude polysaccharide (100 mg) wasapplied to a Sephacryl 400 HR column (200� 3.5 cm),equilibrated with 0.2 M sodium bicarbonate (pH 6.0), andeluted with the same solution. The flow rate of the columnwas 5 ml/h and fractions of 4.0 ml were collected in regularintervals. Fractions were checked for hexose and hexuronicacid by the Dubois reaction (Dubois et al., 1956) andcarbazol reaction (Dische, 1947), respectively, and alsoby metachromatic assay using 1,9-dimethylmethyleneblue (Farndale et al., 1986). Fractions containing thesulfated galactan (indicated by the positive metachromaticreaction) and free of contaminants such as pectins(indicated by positive carbazole reaction) or a yellow

Fig. 7. Concentration of sulfated polysaccharide in different regions of the seagrass R. maritima, determined by histological (A–C) and biochemical (D)analysis. Optical microscopy images of the leaf (A), rhizome (B), and root (C) of the seagrass show differences in the metachromasia intensity oftoluidine blue. The metachromasia observed in these compartments were restricted to cell walls, whereas rhizomes and roots had presented more labelingintensity compared with leaves. Original magnification was 400� for all images. These observations were confirmed by biochemical determinationof the sulfated polysaccharide content in the different regions of the plant (D).

Sulfated galactans in seagrass

17

pigment were pooled, dialyzed against distilled water, andlyophilized.The sulfated galactan obtained from the Sephacryl col-

umn (10 mg) was applied to a Mono Q-FPLC column (HR5/5, Amersham Pharmacia Biotech, Little Chalfont, UK),equilibrated with 20 mM Tris–HCl (pH 8.0), containing10 mM ethylenediamine tetra-acetic acid. The column wasdeveloped by a linear gradient of 1–4 M NaCl in the samesolution. The flow rate of the column was 0.5 ml/min andfractions of 0.5 ml were collected and assayed by metachro-masia. The fractions containing the sulfated galactan werepooled, dialyzed against distilled water, and lyophilized.

Agarose gel electrophoresis

Sulfated galactans were analyzed by agarose gel electro-phoresis as described (Alves et al., 1997; Vieira et al.,1991). Samples (~15 mg) were applied to a 0.5% agarosegel and run for 1 h at 110 V in 50 mM 1,3-diaminopropa-ne:acetate buffer (pH 9.0). The sulfated polysaccharides inthe gel were fixed with 0.1% N-cetyl-N,N,N-trimethylam-monium bromide solution. After 12 h, the gel was dried andstained 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 for5 h) of the sulfated polysaccharide, the hexose was identi-fied by paper chromatography in n-butanol:pyridine:water(3:2:1, v/v) for 48 h on Whatman No. 1 paper, followed bystaining with silver nitrate and by gas-liquid chromatogra-phy (GLC) of derived alditol acetates (Kircher, 1960).Sulfate was measured by the BaCl2/gelatin method (Saitoet al., 1968).

Determination of the D or L configuration of galactose

The enantiomeric form of the galactose was assigned basedon analysis of the trimethylsilylated (�)-2-butyl glycoside,as described (Gerwig et al., 1978). The polysaccharide fromR. maritima (1 mg) was mixed with 0.5 ml (�)-2-butanol,containing 1 M HCl (Aldrich, Milwaukee, WI). After buta-nolysis for 18 h at 80�C, the solution was neutralized withAg2CO3, and the supernatant was concentrated and dis-solved in 50 ml. Thereafter, we added 50 ml bis(trimethylsi-lyl)trifluoro acetamide (Sigma, St. Louis, MO) and kept thesolution for 30 min at room temperature. The butanolyzedand trimethylsilylated derivatives were analyzed on a DB-5GLC column. The temperature was programmed from120�C to 240�C at 2�C/min. The injector and detector tem-peratures were 220�C and 260�C, respectively. Appropriatecontrols of trimethylsilylated (�)-2-butyl-D- and L-galacto-sides were analyzed under the same conditions.

Desulfation and methylation of the sulfated galactan

Desulfation of the sulfated galactan was performed asdescribed (Mour~aao and Perlin, 1987; Vieira et al., 1991).The sulfated polysaccharide (10 mg) was dissolved in 5 mldistilled water and mixed with 1 g (dry weight) of Dowex50-W (H1, 200–400 mesh). After neutralization with pyri-dine, the solution was lyophilized. The resulting pyridinium

salt of the sulfated galactan was dissolved in 2.5 ml dimethylsulfoxide:methanol (9:1,v/v). The mixture was heated at80�C for 4 h, and the desulfated product was exhaustivelydialyzed against distilled water and lyophilized.The desulfated galactan (5 mg) was subjected to three

rounds of methylation as described previously (Ciucanuand Kerek, 1984) with the modifications suggested byPatankar et al. (1993). The methylated polysaccharide washydrolyzed in 6 M trifluoroacetic acid for 5 h at 100�C andreduced with borohydride. The alditols were acetylated withacetic anhydride:pyridine (1:1, v/v) (Kircher, 1960). Thealditol acetates of the methylated sugar was dissolved inchloroform and analyzed in a gas chromatography–massspectrometer (DB-1 capillary column).

NMR spectroscopy1H and 13C spectra of the native and desulfated galactanwere recorded using a Bruker DRX 400 apparatus with atriple-resonance probe. About 4 mg of each sample wasdissolved in 0.5 ml 99.9% D2O (Cambridge Isotope Labora-tory, Andover, MA). All spectra were recorded at 60�Cwith HOD suppression by presaturation. COSY, TOCSY,NOESY, and 1H/13C HMQC spectra were recorded usingstates-time proportion phase incrementation for quadraturedetection in the indirect dimension. TOCSY spectra wererun with 4046� 400 points with a spin-lock field of ~10 kHzand a mixed time of 80 ms. HMQC spectra were run with1024� 256 points and globally optimized alternating phaserectangular pulses for decoupling. NOESY spectra were runwith a mixing time of 100 ms. Chemical shifts are relative toexternal trimethylsilylpropionic acid at 0 ppm for 1H and tomethanol for 13C.

Sulfated polysaccharides localization byoptical microscopy

Samples of the seagrass R. maritima were collected in thesame area described, transported to the laboratory in plasticbags 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% glutar-aldehyde (Merck EM grade) buffered with 0.2 M sodiumcacodylate (pH 7.2) in sea water for 2 h at room tempera-ture. The fragments were rinsed in the same buffer, dehy-drated with acetone graded series, and embedded in Spurrresin. Semithin sections (2 mm) were obtained in a micro-tome (Sorvall, Asheville, NC) and stained with 1% toluidineblue (Sigma), pH 4.4, for 3 min at 40�C. This stain indicatesthe presence of sulfated polysaccharides by metachromasia.The slides of the three compartments of the seagrass wereobserved in a Nikon Eclipse, and digital images wereacquired using the software Image Pro Plus using thesame conditions for all slides.

GenBank database analysis

To look for homologous sequences of the sulfotransferasegene, we conducted a search on GenBank database of twoterrestrial plants with known genomic sequences. Becausevarious enzymes act on the sulfation of diverse compounds,such as alcohols, phenols, and steroids, we had to comparethe sequences of genes for an enzyme specific for sulfation

R.S. Aquino et al.

18

of polysaccharide between several species. Thus, we tested aconsensus sequence of eight 2-O-sulfotransferases (the sea-grass polysaccharide has a sulfate at the position 2). Thesulfotransferase sequences were identified using an in silicoapproach. The screening for 2-O-sulfotransferase sequenceswas performed on the National Center for BiotechnologyInformation GenBank sequence database (accessionnumbers: 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 theExpert Protein Analysis System translate tool. A highlyconserved consensus sequence (MFRKMGLLRIMMPP-KHWLQLLAVVAFAVAMLFLENQIQKLEESRAKLE-RAIARHEVREIEQRHTMDGPRQDATLDEEEDIIIIY-NRVPKTASTSFTNIAYDLCAKNRYHVLHINTTKN-NPVMSLQDQVRFVKNITTWNEMKPGFYHGHISYL-DFAKFGVKKKPIYINVIRDPIERLVSYYYFLRFGD-DYRPGLRRRKQGDKKTFDECVAEGGSDCAPEKL-WLQIPFFCGHSSECWNVGSRWAMDQAKSNLINEY-FLVGVTEELEDFIMLLEAALPRFFRGATDLYRTG-KKSHLRKTTEKKLPTKQTIAKLQQSDIWKMENEF-YEFALEQFQFIRAHAVREKDGDLYILAQNFFYEKI-YPKSN) was obtained after alignment using CLUSTALW(Thompson et al., 1994). tBLASTn searches against theplant genome data base of Arabidopsis thaliana and Oryzasativa using the consensus sequence of 2-O-sulfotranferaseas a query to identify a plant homologous sequence topolysaccharide sulfotranferase.

Acknowledgments

This work was supported by grants from Conselho Nacio-nal de Desenvolvimento Cientıfico e Tecnol�oogico andFundac~aao de Amparo a Pesquisa do Estado do Rio deJaneiro. We thank Adriana A. Piquet for technicalassistance, Dr. Orlando B. Martins for help on the searchof homology sequences of sulfotransferase genes in plantspecies, and Hailton G. Aquino and Antonia G. S. Aquinofor their help in the collection of seagrasses. This work wassubmitted as part of a Ph.D. thesis by R.S. Aquino to theDepartamento de Bioquımica, Instituto de Quımica,Universidade Federal do Rio de Janeiro.

Abbreviations

COSY, correlation spectroscopy; FPLC, fast performanceliquid chromatography; GLC, gas-liquid chromatography;HMQC, heteronuclear multiple quantum coherence;NOESY, nuclear Overhauser enhancements spectro-scopy; NMR, nuclear magnetic resonance; TOCSY, totalcorrelation spectroscopy.

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