Glycobiology Advance Access originally published online on January 3, 2008
Glycobiology 2008 18(3):250-259; doi:10.1093/glycob/cwm139
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A preponderantly 4-sulfated, 3-linked galactan from the green alga Codium isthmocladum
3 Laboratório de Biotecnologia de Polímeros Naturais - BIOPOL, Departamento de Bioquímica, Universidade Federal do Rio Grande do Norte, Natal, RN, 59072-970
4 Laboratório de Tecido Conjuntivo, Hospital Universitário Clementino Fraga Filho
5 Instituto de Bioquímica Médica, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Caixa Postal 68041, Rio de Janeiro, RJ, 21941-590
6 Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, 21941-590
7 Departamento de Bioquímica, Universidade Federal de São Paulo, SP, 04044-020, Brazil
1 To whom correspondence should be addressed: e-mail: pmourao{at}hucff.ufrj.br
Received on October 9, 2007; revised on November 28, 2007; accepted on December 28, 2007
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Abstract |
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The green algae of the genus Codium have recently been demonstrated to be an important source of sulfated galactans from the marine environment. Here, a sulfated galactan was isolated from the species Codium isthmocladum and its structure was studied by a combination of chemical analyses and NMR spectroscopy. Two fractions (SG 1,
14 kDa, and SG 2, 20 kDa) were derived from this highly polydisperse and heterogeneous polysaccharide. Both exhibited similar structures in 1H 1D NMR spectra. The structural features of SG 2 and its desulfated derivative were analyzed by COSY, TOCSY, DEPT-HSQC, HSQC, and HMBC. This sulfated galactan is composed preponderantly of 4-sulfated, 3-linked β-D-galactopyranosyl units. In minor amounts, it is sulfated and glycosylated at C-6. Pyruvate groups are also found, forming five-membered cyclic ketals as 3,4-O-(1'carboxy)-ethylidene-β-D-galactose residues. A comparison of sulfated galactans from different marine taxonomic groups revealed similar backbones of 3-β-D-Galp-1. Key words: Codium isthmocladum / green alga / green seaweed / phylogeny / pyruvylated-sulfated galactan
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Introduction |
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TopAbstractIntroductionResults and discussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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The sulfated polysaccharides comprise a complex group of macromolecules with a large range of important biological activities (Verli 2007
). These anionic polymers can occur as glycosaminoglycans, widely distributed in vertebrate tissues (Mathews 1975). In marine organisms (invertebrates and algae), the sulfated polysaccharides are found predominantly as sulfated fucans or sulfated galactans. The sulfated fucans are constituted mainly of 
-L-fucose residues in which the species-specific molecular structures vary in the glycosidic position and in the pattern of sulfation (Berteau and Mulloy 2003; Mourão 2004, 2007). The most common source of sulfated fucans is the cell wall of brown algae (Phaeophyta) (Pereira et al. 1999; Rocha et al. 2005), but they can also be found in the egg jelly layer of sea urchins (Mulloy et al. 1994; Alves et al. 1997) and in the body wall of the sea cucumber (Echinodermata, Holothuroidea)(Ribeiro et al. 1994).
The most common source of sulfated galactans is the cell wall of red algae (Rhodophyta) (Lahaye 2001; van de Velde et al. 2004). Like the sulfated fucans, the sulfated galactans can also be isolated from the outer layer of the sea-urchin egg (Echinodermata, Echinoidea) (Alves et al. 1997), or extracted from other classes of invertebrates, such as tunicates (Urochordata, Ascidiacea) (Pavão et al. 1989; Albano et al. 1990; Santos et al. 1992) and clams (Mollusca, Bivalvia) (Amornrut et al. 1999). Recently, a novel sulfated galactan was isolated from sea grass (Aquino et al. 2005), leading for the first time to a description of sulfated polysaccharides in the marine angiosperms, a group of vascular plants. Thus, sulfated galactans with different structures are widely distributed among marine organisms from distant phyla.
Recently, the green algae, particularly the genus Codium (Bryopsidales, Chlorophyta) (Love and Percival 1964; Matsubara et al. 2001; Bilan et al. 2007), have proved to be another important source of sulfated galactans in the marine environment. Fewer structures of sulfated polysaccharides from green seaweed are known than are those from red or brown algae. In the genus Codium, different species demonstrate structural heterogeneity and complexity in their sulfated galactans. Bilan et al. (2007) described a complex and highly pyruvylated and sulfated galactan from C. yezoense, composed essentially of linear backbone segments of 3-linked β-D-galactopyranosyl units, ramified by short oligosaccharides attached by linkages at C-6. Sulfate groups were localized mainly at C-4 and in lower amounts at C-6. A few pyruvate groups (about 25% of total residues) were present, forming 3,4-O-(1'-carboxy)-ethylidene-β-D-galactopyranose residues located at nonreducing terminals of the chains of galactose. The sulfated galactans from C. fragile and C. cylindricum were heterogeneous polymers. In addition to its galactose content, C. fragile also contained arabinose residues (sulfated arabinogalactan) (Love and Percival 1964) and C. cylindricum contained glucose residues, probably forming a sulfated glucogalactan (Matsubara et al. 2001).
Here, we describe the isolation and structural characterization of the sulfated galactan from Codium isthmocladum, a green alga that occurs abundantly along the coast of Natal, Rio Grande do Norte, Brazil. We analyzed this sulfated galactan by a combination of chemical methods (desulfation and methylation reactions and measurements of sulfate content) and nuclear magnetic resonance (NMR) spectroscopy (1H 1D and 2D correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), distortionless enhancement by polarization transfer-hetero- nuclear single quantum coherence (DEPT-HSQC), HSQC, and heteronuclear multiple bound correlation (HMBC)). The sulfated galactan from C. isthmocladum is composed preponderantly of 3-linked β-D-galactopyranose residues that are extensively 4-sulfated and occasionally 6-sulfated. In minor amounts, the β-D-galactopyranosyl units are 6-linked. Also, based on HMBC experiments, the sulfated galactans from C. isthmocladum contain pyruvate groups that form five-membered cyclic ketals located in 3,4-O-(1'carboxy)-ethylidene-β-D-galactose units.
A comparison among different sulfated polysaccharides indicates that sulfated galactans with glycosidic linkage (β1
3) occur in some taxonomic groups of marine eukaryotic organisms (rhodophytes, chlorophytes, angiosperms, echinoderms, and molluscs). The sulfated galactans found among these phyla differ in sulfation sites, with a marked tendency toward 4-sulfation in algae and 2-sulfation in invertebrate animals. In minor amounts, 6-sulfation is dispersed throughout the phylogenetic tree.
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Results and discussion |
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Purification of the sulfated galactans from the green alga C. isthmocladum
The polysaccharides extracted from the alga with maxatase digestion (see the Materials and methods section) were fractionated by precipitation with increasing volumes of acetone (1:0.3, 1:0.5, 1:0.7, 1:0.9, and 1:1.2, v/v of sample and acetone). All the precipitated fractions, except the one that was obtained with 1:1.2 acetone, revealed a heterogeneous content of sugars (Table I). Mannose was the major constituent in fractions precipitated with 1:0.3 and 1:0.5 acetone. Galactose and arabinose were equally predominant in fractions precipitated with 1:0.7 and 1:0.9 acetone. The precipitate obtained with 1:1.2 acetone contained primarily one type of sugar, arabinose.
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The mixture of polysaccharides obtained by precipitation with 1:0.9 acetone was purified additionally by ion-exchange chromatography (Bayer Lewatit) (Figure 1A). The fraction eluted with 0.3 M NaCl contained almost 60% of the total polysaccharides applied to the column. However, the low sulfate content indicated that these polysaccharides were composed predominantly of neutral residues. The fractions eluted with 2.0 M and 3.0 M NaCl contained
16% and 13% of the total polysaccharides applied to the Lewatit column. These fractions were highly sulfated with a molar ratio of 1.4 and 1.9 sulfate/residue, respectively (Table I). The sugar analysis of these two fractions revealed mainly galactopyranose residues (Table I) and they were denominated as SG 1 and SG 2.
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The purity of these fractions was demonstrated by agarose gel electrophoresis (Figure 1B). For both fractions, there was only a single band in the gel, indicating purified polysaccharides when compared with the polysaccharides precipitated with 1:0.9 acetone and with the crude polysaccharide before the serial precipitation with acetone. In conclusion, these two fractions contained exclusively sulfated galactan and were chosen for subsequent structural studies.
The molecular masses of SG1 and SG2 were determined by PAGE (Figure 1C). Their electrophoretic mobilities were compared with different molecular markers as indicated in the figure. Both fractions of sulfated galactan showed a dispersive migration due to a heterogeneous molecular weight as is the characteristic for the sulfated polysaccharides. However, the predominance of bands at 14 kDa and 20 kDa can be noted for SG1 and SG2, respectively. Obviously, the greater molecular weight of SG 2 together with its higher sulfate content (Table I) explains its elution from the Lewatit column with a higher saline concentration (3.0 M NaCl) than that observed for SG 1 (2.0 M NaCl) (Figure 1A).
Presence of 4- and 6-sulfation in the galactans
In an initial attempt to determine the structure of the sulfated galactan obtained from the green alga, we methylated the native polysaccharides and their chemically desulfated derivatives. In spite of limitations to this type of methodology, such as partial desulfation and methylation or preferential sites of methylation in the sugar ring, this analysis provides valuable information about the structure (Table II).
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In fact, comparing the methyl galactitols produced from the native sulfated galactan and desulfated derivative, we can observe the disappearance of the 2-O-methyl galactitol and a significant increase in 2,4-di-O-methyl galactitol (indicative of 4-sulfation) and of the 2,4,6-tri-O-methyl galactitol (indicative of 4- and 6-sulfation). The production of significant amounts of 2,4,6-tri-O-methyl galactitol from the methylation of the desulfated galactans suggests the predominance of 3-linked galactose units. In addition, the production of significant amounts of 2,4- and 2,6-di-O-methyl galactitols may indicate partial desulfation of the molecules, the presence of branching residues or the presence of other types of substituents, as discussed below.
Preponderance of 4-sulfated, 3-linked β-D-galactopyranose residues
For a more detailed structural analysis of the sulfated galactans from the green alga, we employed one-dimensional and two-dimensional NMR spectroscopy. The fractions of native sulfated galactans and their desulfated derivatives produced similar signals in the 1H 1D NMR spectra (Figure 2), which indicates similar structures for SG 1 and SG 2. Therefore, the 2D NMR spectra were recorded exclusively with fraction SG 2 and its desulfated derivative.
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The 1H-signals between 4.4 and 5.0 ppm in the spectra of the sulfated galactan contained a mixture of signals of H-1 from the β-anomers of galactopyranoses and of H-4 from the sugar rings; these exhibited a down-field shift (
0.6 ppm) due to sulfation (Pomin, Pereira, et al. 2005; Pomin, Valente, et al. 2005; Bilan et al. 2007). This conclusion is reinforced by the analysis of the 1H/13C DEPT-HSQC of the native polysaccharide (Figure 3A) and the disappearance of the signal at 
H/C 4.94/77.8 ppm, after the desulfation reaction (Figure 3B).
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The 1H 1D spectra (Figure 2C and D) and especially the 1H/13C DEPT-HSQC (Figure 3) showed two preponderantly anomeric signals, denominated as A and B, for the native sulfated galactan SG 2, as well as for its desulfated derivative. Through the 2D COSY spectra (Figure 4A and C) and TOCSY spectra (Figure 4B and D), it was possible to trace the spin systems of these two signals, especially for the desulfated SG 2 (Figure 4A and B). Thus, we obtained the 1H chemical shifts indicated in Table III. Only the chemical shifts of H-6 could not be determined. However, these values were easily deduced due to the negative-phase signals relative to CH2 (blue-contour peaks in Figure 3B) in the 1H/13C DEPT-HSQC spectrum. We identified two signals of H-6/C-6 associated with spin systems A and B (preponderant and minority blue signals, respectively). The analysis of the 13C chemical-shift values indicated unequivocally that the units designated A and B (structures 1 and 2 in Table III, respectively) were associated with 3- and 6-linked β-D-galactopyranose residues, respectively, as indicated by the typical low-field shift of carbons (
10 ppm) in sites of glycosylation (Table III). This 13C shift was also seen in reference compounds of 3- and 6-linked β-D-galactosyl units (structures 5 and 6 in Table III) and in galactose residues located at nonreducing terminals (structure 7 in Table III).
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The spin system traced for the native SG 2 was more complex due to the greater heterogeneity of the polymer. However, we identified two spin systems, denoted by A and B (Figure 4C and D) (Table III). Again, it was difficult to identify the signals that correspond to H-6, but the DEPT-HSQC spectrum (Figure 3A) was especially useful for this assignment (blue-contour peaks). Signals from glycosylated 6-position (
H,H'/C 4.36, 4.01/69.9 ppm of units denominated as B) and unsubstituted 6-position (units A) were identified by comparison with the spectra of the desulfated SG 2 (Figure 3B). Moreover, we identified another signal (H,H'/C 4.42, 4.32/66.8 ppm) with a typical 1H low-field shift (0.6 ppm) that indicates 6-sulfation (Figure 3A).
The 6-sulfation is necessarily associated with the system A, while the system B is glycosylated at this site. The system A is mainly sulfated at C-4, as indicated by the typical low-field shift (0.65 ppm) of the H-4 (structure 3 in Table III). But, there are also minor amounts of nonsulfated units, as indicated by the methylation analysis. The system B is mainly 4-sulfated (as indicated by the down-field shift of H-4 and structure 4 in Table III), but nonsulfated units also co-exist with these units.
In synthesis, these results indicate, for the sulfated galactan from the green alga, a preponderant constitution of 3-β-D-Galp-1, about 80% of the total residues, as indicated by the integrals of the NMR signals (Figure 2B and D). Most of these units are 4-sulfated, but, in minor amounts, there are also units sulfated at the 6-position, as well as nonsulfated units. The sulfated galactan also contains 6-linked units of β-D-Galp-4(SO4), and, in minor amounts, 6-linked nonsulfated galactopyranose residues. The nuclear overhauser enhancement spectroscopy (NOESY) spectra (data not shown) did not reveal a clear distribution pattern for these minority residues in the structure of the polymer.
Occurrence of 3,4-O-(1'carboxy)-ethylidene residues in the sulfated galactans
Curiously, in addition to the 1H- and 13C-signals from the galactose ring and its anomeric protons and carbons, the native SG 1 and SG 2 and their desulfated derivatives showed an intense high-field 1H-signal at 1.7 ppm (Figure 2), which strongly indicates methyl groups. Furthermore, in the methylation analysis the desulfated galactan was still highly substituted at C-3 and C-4, as observed from the production of 2,6-di-O-methyl galactitols (Table II). These data together suggest the presence of an extra-ring methyl group bound to C-3 and/or C-4 of the galactose ring.
We used 1H/13C heteronuclear NMR experiments to prove that this methyl group comes from pyruvate groups. The HSQC spectrum (Figure 5A) revealed only a singlet at H/C 1.77/23.4 ppm that may correspond to methyl signals from pyruvic acid. The HMBC spectrum (Figure 5B) showed a doublet signal at H/C 1.87/109.3 ppm and H/C 1.67/109.3 ppm due to nondecoupling during the acquisition of the pulse sequence. Moreover, due to identified signals of proton nuclei bound to carbon nuclei that are separated by more than one bond, we assigned two other signals coupled to the H 1.77 ppm frequency. These peaks resonate at C 109.1 ppm and C 176.6 ppm and correspond respectively to the groups O–C–O and COOH of the pyruvate (Figure 5B and Table IV). The low-field proton chemical shift of this system (1.77 ppm) clearly reveals a typical pyruvate involved in a five-membered cyclic ketal including O-3 and O-4 of the nonreducing-terminal galactoses, instead of a six-membered cyclic ketal including O-4 and O-6 positions (Shashkov et al. 2000; Bilan et al. 2007). These data together indicate the presence of galactose residues with 3,4-O-(1'carboxy)-ethylidene cyclic ketals.
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The occurrence of 3,4-O-(1'carboxy)-ethylidene-galactose residues in the sulfated galactans from the green alga allows us to reinterpret the methylation analysis of these polysaccharides. The observation that significant amounts of 2,6-di-O-methyl derivative obtained from the desulfated galactans (Table II) may be explained by the presence of pyruvylated groups substituted at 3- and 4-positions of the galactoses that are located at nonreducing ends of the polysaccharide. In addition, a re-analysis of the NMR spectra (TOCSY and DEPT-HSQC) of the spin system A reveals an additional heterogeneity compatible with 3,4-O-(1'carboxy)-ethylidene-β-D-galactopyranosyl units from nonreducing terminals, especially coincident signals of H-3 and H-4 of pyruvated units and the low-field shift of the adjacent H-5 (denoted by A5*) (Figures 3 and 4).
Major conclusions about the structure of the sulfated galactans from the green alga C. isthmocladum
The sulfated galactan from the green alga C. isthmocladum is a complex polysaccharide with different structural components. The main variations come from different positions of glycosidic linkages (3- and 6-linked units), from different sulfation sites (positions 4 and/or 6), and from the presence of pyruvate groups involved in cyclic ketals with the positions O-3 and O-4 of the β-D-galactoses located at nonreducing ends. The studies of methylation and the NMR spectra ensure that the 3-β-Galp-4(SO4)-1 is the preponderant unit of these polysaccharides (Figure 6A). However, galactopyranosyl units linked by β13 linkages and disulfated at 4- and 6-positions are also found (Figure 6B), as are 4-sulfated, 6-linked residues (Figure 6C). Finally, units at nonreducing terminals contain 3,4-O-(1'carboxy)-ethylidene (Figure 6D). The structural complexity of this polysaccharide prevented us from determining the sites of substitution of the nonreducing terminals of the main backbone. Another possible source of heterogeneity in these molecules is the presence of minor amounts of nonsulfated units. The attempt to determine a repetitive pattern of distribution in the polymer using NMR (especially the NOESY spectra) proved to be unsuccessful.
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Apparently, the sulfated galactans from C. isthmocladum and C. yezoense are similar polysaccharides. But a more in-depth analysis of the structure of these two polysaccharides reveals some differences. In particular, the sulfated galactan from C. isthmocladum has a more simple structure than the polysaccharide from C. yezoense, as revealed by the HMQC spectra (compare Figure 3 and the results of Bilan et al. 2007
) and the methylation analysis. Clearly, the sulfated galactan from C. isthmocladum has no 3,6-linked units and no six-membered cyclic ketals including O-4 and O-6 positions. In addition, the sulfated galactan from C. isthmocladum is less branched than the polymer from C. yezoense.
The preponderance of the 3-β-D-Galp-4(SO4)-1 unit in these sulfated galactans from the green alga stimulated us to review the distribution of this structure in the animal and vegetal kingdoms (Whittaker 1969). The sulfated 3-β-D-Galp-1 units are preserved among species of phyla that cohabit the marine environment. These species appear among brown algae (Phaephyta), green algae (Chlorophyta), red seaweeds (Rhodophyta), marine seagrass (Angiospermae, Spermatophyta), invertebrates [sea urchins (Echinodermata, Echinoidea), clams (Mollusca, Bivalvia), tunicates (Urochordata, Ascidiacea)], and vertebrates such as fishes (Teleostei, Chordata) that express keratan sulfate, although 6-sulfation is less evident in this glycosaminoglycan (Scudder et al. 1986). Although the 3-β-D-Galp-1 units are preserved in these phyla, the preferential sulfation site varies: 4-sulfation in the algae and marine angiosperm and 2- or 6-sulfation in the galactans from invertebrates and in the keratan sulfate (and the marine angiosperm).
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Materials and methods |
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TopAbstractIntroductionResults and discussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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Extraction of polysaccharides
The green alga C. isthmocladum was collected from the sublittoral coast of Natal, Rio Grande do Norte, Brazil. Immediately after collection, the alga was air-dried at 50°C (under ventilation) and ground in a blender. The seaweed was then treated with ethanol and acetone to remove pigments and lipids, respectively. One hundred grams of delipidated, dry powdered alga was suspended in 500 mL of an aqueous solution of 0.25 M NaCl and adjusted to pH 8.0 with NaOH. About 20 mg of maxatase, an alkaline protease from Esporobacillus (Biobrás, Montes Claros, Minas Gerais, Brazil) was added to the solution for proteolytic digestion. After incubation for 18 h at 60°C under agitation, the mixture was filtered through cheesecloth. The filtrate was fractionated through a serial precipitation with increasing volumes of acetone (1:0.3, 1:0.5, 1:0.7, 1:0.9, and 1:1.2, v/v). For each precipitation, the volume of ice-cold acetone was added under gentle agitation and maintained at 4°C for 24 h. The pellets formed in each suspension were collected by centrifugation separately (10,000 x g for 20 min), dried under vacuum, resuspended in distilled water, and analyzed for chemical composition (content of sugars and sulfate molar ratio were estimated as described below). The measurements are indicated in Table I.
Chemical analyses of the precipitates obtained with different volumes of acetone
Total sugar content of each pellet was measured by the phenol–H2SO4 reaction (Dubois et al. 1956), using D-galactose as standard. After acid hydrolysis of the polysaccharides (6 M HCl for 4 h at 100°C), the sulfate contents were measured by the toluidine method as previously described (Nader and Dietrich 1977). Again, the polysaccharides of each pellet were hydrolyzed (2 M HCl for 2 h at 100°C) and the type of hexose was determined by gas–liquid chromatography of alditol acetate derivatives (Kircher 1960), as described below. As standards, fucose, glucose, rhamnose, arabinose, mannose, galactose, and xylose were converted to their respective alditol acetates.
Purification of the sulfated galactan
To obtain the sulfated galactan, the precipitate formed with 1:0.9 acetone was fractionated by ion-exchange chromatography. After centrifugation and drying, 12 mg was dissolved in 5 mL of distilled water and applied to a column (19.0 x 4.5 cm) of MP500 Lewatit resin (Bayer Chemicals, São Paulo, Brazil). First, the column was washed with 300 mL of distilled water and developed by a stepwise gradient system using 300 mL of aqueous solutions with different concentrations of NaCl (0.3 M, 0.5 M, 0.7 M, 1.0 M, 1.5 M, 2.0 M, 3.0 M, and 4.0 M) at 1 mL/min. The first 300 mL at each step was collected in a single recipient; then, a second recipient was used to collect an additional 300 mL of the same saline molarity to check for the complete elution of the polysaccharides. To precipitate the polysaccharides, two volumes (600 mL) of methanol were added to both recipients for each step. However, only the first 300 mL of the steps at 0.3 M, 2.0 M, and 3.0 M precipitated polysaccharides when methanol was added. These suspensions were centrifuged separately (10,000 x g for 20 min), dried under vacuum, dissolved in distilled water to a final concentration of 10 mg/mL, and analyzed for hexose (Dubois et al. 1956) and sulfate molar ratio (Nader and Dietrich 1977).
Agarose and polyacrylamide gel electrophoresis
The precipitate with 1:0.9 acetone and the fractions of sulfated galactan (SG 1 and SG 2) were analyzed by agarose gel electrophoresis as described previously (Dietrich CP and Dietrich SMC 1977). The samples (20 µg) were applied to a 5-mm-thick 0.5% agarose gel and run for 1 h at 110 V in 0.05 M 1,3-diaminopropane–acetate (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).
The molecular masses of the sulfated galactan (SG 1 and SG 2) were estimated by PAGE in comparison with the electrophoretic mobility of standard compounds (Pomin, Pereira, et al. 2005). The sulfated polysaccharides (10 µg of each) were applied to a 1-mm-thick 10% polyacrylamide slab gel in 0.02 M sodium barbital (pH 8.6). After electrophoresis (100 V for 30 min), the sulfated polysaccharides were stained with 0.1% toluidine blue in 1% acetic acid and washed for about 1 h in 1% acetic acid. The molecular-mass markers used were high-molecular-weight dextran sulfate (500 kDa), chondroitin 6-sulfate from shark cartilage (60 kDa), chondroitin 4-sulfate from whale cartilage (40 kDa), unfractionated heparin from porcine intestinal mucosa (15 kDa), low-molecular-weight-heparin (7.5 kDa), and low-molecular-weight dextran sulfate (8 kDa).
Desulfation and methylation reactions
Desulfation of the sulfated galactan was performed as detailed previously (Mourão and Perlin 1987; Vieira et al. 1991). About 20 mg of each sulfated galactan (SG 1 and SG 2) was dissolved in 5 mL of distilled water and mixed with 1 g (dry weight) of Dowex 50-W (H+, 200–400 mesh). After neutralization with pyridine, solutions were lyophilized. The resulting pyridinium salts were dissolved in 2.5 mL of dimethyl sulfoxide:methanol (9:1, v/v). The mixtures were heated at 80°C for 4 h, and the desulfated products were exhaustively dialyzed against distilled water and lyophilized. The extent of desulfation was estimated by the molar ratio of sulfate/total sugars. This method allows us to detect desulfation down to a molar ratio of 
0.1 sulfate/total sugar. About 5 mg of each desulfated fraction was obtained. The native and desulfated galactans were subjected to three rounds of methylation, as described (Ciucanu and Kerek 1984) and with the modifications suggested by Patankar et al. (1993). The methylated galactans were hydrolyzed with 6.0 M trifluoroacetic acid for 5 h at 100°C, reduced with borohydride, and the alditols were acetylated with 1:1 acetic anhydride/pyridine (Kircher 1960). The alditol acetates from the methylated sugars were dissolved in chloroform and analyzed in a Hewlett-Packard gas chromatography/mass spectrometry unit, model 5987-A. Injection was made in the splitless mode in a DB-1 capillary column (25 m x 0.3 mm). The column was programmed to run at 120°C for 2 min, then raised to 230°C at 2°C/min, and held for 5 min.
NMR experiments
1H and 13C, one-dimensional and two-dimensional spectra of the native sulfated galactan and the desulfated galactan were recorded using a Bruker DRX 400 MHz apparatus with a triple resonance probe as detailed previously (Pomin, Valente, et al. 2005). About 5 mg of each sample was dissolved in 0.5 mL 99.9% deuterium oxide (Cambridge Isotope Laboratory, Cambridge, MA). All spectra were recorded at 50°C with HOD suppression by presaturation. The 1D 1H NMR spectra were recorded with 16 scans. The 2D 1H/1H COSY, TOCSY, NOESY, and 1H/13C HSQC spectra were recorded using states-time proportion phase incrementation (states-TPPI) for quadrature detection in the indirect dimension. The TOCSY spectra were run with 4046 x 400 points with a spin-lock field of 10 kHz and a mixed time of 80 ms. The NOESY spectra were recorded with a mixing time of 100 ms. The 1H/13C DEPT-HSQC and 1H/13C HSQC spectra were run with 1024 x 256 points and globally optimized alternating phase rectangular pulses (GARP) for decoupling. The 1H/13C HMBC spectrum was recorded with 1024 x 256 points, with a 60-ms delay for evolution of long-range couplings and was set with no decoupling during acquisition time. Chemical shifts are displayed relative to external trimethylsilylpropionic acid at 0 ppm for 1H and relative to methanol for 13C.
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Supplementary Data |
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TopAbstractIntroductionResults and discussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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Supplementary data for this article is available online at http://glycob.oxfordjournals.org.
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Funding |
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TopAbstractIntroductionResults and discussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
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Conflict of interest statement |
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TopAbstractIntroductionResults and discussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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None declared.
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Acknowledgements |
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We are grateful to Anderson Pinheiro and Fabiana Albernaz from the Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, CCS, Brazil for help with the NMR experiments and figures and especially to Martha Sorenson for editing the manuscript.
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Footnotes |
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2 These two authors contributed equally to the work.
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Abbreviations |
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COSY, correlation spectroscopy; DEPT, distortionless enhancement by polarization transfer; HMBC, heteronuclear multiple bound coherence; HSQC, heteronuclear single quantum coherence; NMR, nuclear magnetic resonance; NOESY, nuclear overhauser enhancement spectroscopy; PAGE, polyacrylamide gel electrophoresis; SG, sulfated galactan; TOCSY, total correlation spectroscopy.
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References |
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TopAbstractIntroductionResults and discussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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