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Ultrastructure of acidic polysaccharides from the cell walls of brown algae
Journal of
Structural Biology Journal of Structural Biology 145 (2004) 216–225 www.elsevier.com/locate/yjsbi
Ultrastructure of acidic polysaccharides from the cell walls of brown algae Leonardo R. Andrade,a Leonardo T. Salgado,a Marcos Farina,a Mariana S. Pereira,b Paulo A.S. Mour~ ao,b and Gilberto M. Amado Filhoc,* a
Laborat orio de Biomineralizacßa~o, Departamento de Histologia e Embriologia, Instituto de Ci^ encias Biom edicas, CCS, Universidade Federal do Rio de Janeiro, Cidade Universit aria, Rio de Janeiro, RJ 21941-590, Brazil b Laborat orio de Tecido Conjuntivo, Hospital Universit ario Clementino Fraga Filho e Departamento de Bioquımica M edica, Instituto de Ci^ encias Biom edicas, CCS, Cidade Universit aria, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-590, Brazil c Programa Zona Costeira, Instituto de Pesquisas Jardim Bot^ anico/MMA, Rua Pacheco Le~ ao 915, Jardim Bot^ anico, Rio de Janeiro, RJ 22460-030, Brazil Received 10 June 2003, and in revised form 2 October 2003
Abstract We have studied the ultrastructure of acidic polysaccharides from the cell walls of brown algae using a variety of electron microscopy techniques. Polysaccharides from Padina gymnospora present self assembled structures, forming trabecular patterns. Purified fractions constituted by alginic acid and sulfated fucan also form well-organized ultrastructures, but the pattern of organization varies depending on the polysaccharide species. Alginic acid presents sponge-like structures. Sulfated fucan exhibits particles with polygonal forms with a polycrystalline structure. These particles are in fact constituted by sulfated fucan molecules since they are recognized by a lectin specific for a-L -fucosyl residues. X-ray microanalysis reveal that S is a constituent element, as expected for sulfated groups. Finally, an exhaustive purified sulfated fucan shows the same ultrastructure formed by polygonal forms. Furthermore, elemental analyses of acidic polysaccharides indicate that they retain Zn, when algae were collected from a contaminated area. This observation is supported by direct quantification of heavy metal in the biomass and also in the solubilized polysaccharides compared with the algae from a non-contaminated site. We conclude that these molecules have specific ultrastructure and elemental composition; and act as metal binder for the nucleation and precipitation of heavy metals when the algae are exposed to a metal contaminated environment. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Alginic acid; Biomineralization; Brown algae; EDXA; Field emission scanning electron microscopy; Fucoidan; Heavy metals; Sulfated fucan; Polysaccharides; TEM; Ultrastructure; Zinc
1. Introduction Cell walls of brown algae have a fibrillar compartment formed mainly of cellulose microfibrils, which is embedded in an amorphous matrix of acid polysaccharides linked each other by proteins (Kloareg et al., 1986). The acid polysaccharides are mainly composed of alginic acids and sulfated fucans (also known as fucoidan). The alginic acids are linear carboxylated copolymers constituted by different proportions of 1,4-linked
* Corresponding author. Fax: +55-21-22942295. E-mail address: gfi[email protected] (G.M. Amado Filho).
1047-8477/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2003.11.011
b-D -mannuronic acid (M-block) and a-L -guluronic acid (G-block) (Haug et al., 1966). Algal sulfated fucans are polymers mainly formed by sulfated a-L -fucose but also containing small proportions of other sugars such as mannose and galactose (Dietrich et al., 1995; Kloareg et al., 1986; Pereira et al., 1999, 2002). The chemical structure, physico-chemical properties, and biological activities of alginic acids and sulfated fucans have been widely studied, but the ultrastructure of these molecules still unknown, especially in the case of the sulfated fucans. The characterization of polysaccharide morphology can contribute for the understanding of the cell wall architecture in the marine algae. The few available data about the morphology of these
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polysaccharides are generally related to alginic acid beads as scaffold to cell culture in tissue engineering; carriers for immobilized proteins and polymers used for the controlled release of drugs (Chung et al., 2002; Magnani et al., 2000; Zhou and Zhang, 2001). Acidic polysaccharides found in the cell wall of brown algae have also been associated with the capacity of these organisms to accumulate heavy metals when exposed to an environment containing high concentrations of these elements (Amado Filho et al., 1999; Andrade et al., 2002; Lignell et al., 1982; Pellegrini et al., 1993). The negatively charged polysaccharides from the mucilaginous matrix can adsorb and partially exclude mobile anions, and consequently act as cation-exchanger and ionic barrier, selecting the elements that will be absorbed by the algal cells (Kloareg et al., 1986; Mariani et al., 1990; Percival, 1979). Different seaweeds from a heavy metal contaminated area were analyzed by atomic absorption spectrophotometry (AAS) and the results indicated that the brown alga Padina gymnospora presented the highest concentrations of Zn and Cd among the species tested (Amado Filho et al., 1999). Analytical electron microscopy showed that cell walls located in median regions of thallus presented electron-dense granules composed mainly by Zn and S. Cd was not detected because of its low concentration in the samples. According to this result, it was proposed that the main mechanism presented by P. gymnospora to accumulate high heavy metal concentrations from contaminated areas is related to the precipitation of these elements into the cell walls, diminishing the availability of metals to cell absorption (Amado Filho et al., 1996, 1999). In addition, adult individuals of P. gymnospora exposed to sub-lethal concentrations of Cd and/or Zn in vitro presented numerous crystalline granules along the cell walls, formed mainly by Cd and/or Zn, which colocalized with S and O (Andrade et al., 2002). This observation suggested that the acidic polysaccharides (mostly sulfated fucans) play an important role in nucleation of Cd and Zn. Perhaps the acid micro-environment assured by these polysaccharides in the cell walls propitiates an increase of crystalline granules like a biomineralization process, and consequently turning the algae more tolerant to metals (Andrade et al., 2002). In this work we have studied the ultrastructure of alginic acid and sulfated fucan from the cell wall of brown algae using different electron microscopy techniques. Both polysaccharides presented a self-assembled, well-organized ultrastructure but the pattern of organization varies depending on the polysaccharide species. Alginic acid showed a sponge-like structure whereas sulfated fucans presented particles with polygonal forms. Furthermore, elemental analysis of these ultrastructures indicates they retain heavy metals when the algae were exposed to a contaminated area.
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2. Materials and methods 2.1. Polysaccharides from P. gymnospora Adult individuals of P. gymnospora were collected in Sepetiba Bay, a heavy metal (mostly Zn and Cd) contaminated area located in Rio de Janeiro State. Algae were also collected in an adjacent non-contaminated area, Ribeira Bay, for comparative analysis. Fresh algae were cleaned from epiphytes with a thin brush under a stereoscopic microscope, briefly washed in distilled water and dried at 50 °C. The glasses used in polysaccharide extraction were previously washed in a detergent solution (1% Extran) and an acid solution (2% HNO3 ). The polysaccharides were extracted from 4 g (dry weight) of biomass in 250 ml of ultra-pure water, under agitation for 24 h at room temperature. The samples were centrifuged and the supernatants with soluble components were precipitated with 70% ethanol. The precipitates were removed by centrifugation, dissolved, and dialyzed against distilled water for 24 h and freezedried. The powder obtained is denominated as total polysaccharides and used for ultrastructural observation and elemental composition studies. Alternatively, the polysaccharides were also extracted from the algae with 5% KOH solution (w/v) under agitation for 24 h at room temperature. The solution was centrifuged, and the polysaccharides precipitated from the clear supernatant with 70% ethanol (v/v) and freezedried. The powder was then dissolved in distilled water and 4 M CaCl2 was added to the solution in order to precipitate the alginates. The sample was then centrifuged and both, the precipitated and supernatant containing alginic acid and sulfated fucan, respectively, were dialyzed against distilled water for 24 h and freezedried. The powders of fractioned polysaccharides (alginic acids and sulfated fucans) were used for electron microscopy studies. 2.2. Sulfated fucan from Laminaria brasiliensis Polysaccharides were extracted from the cell wall of the brown algae L. brasiliensis by papain digestion and the sulfated fucan was purified by anion exchange chromatography, as described (Pereira et al., 1999). The purity of the sulfated fucan preparation was checked by agarose gel electrophoresis and chemical analysis. 2.3. Transmission electron microscopy For transmission electron microscopy (TEM) purposes, the total and fractionated polysaccharides were dissolved (5 mg/ml) in ultra-pure water and 10 ll were transferred to a Formvar film coated copper grids. The polysaccharidic solutions were dried and the samples were observed in a Jeol 1200 EX, operated at 80 kV.
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2.4. Analytical TEM The elemental composition of the extracted polysaccharides was determined by energy dispersive X-ray analysis (EDXA). The samples were prepared for TEM as described above, and analyzed in a Jeol 1200 EX equipped with a Noran–Voyager analytical system. A focused spot (d 100 nm) was used to analyze the areas of interest in the polysaccharide samples. The areas probed were representative of the whole sample (we obtained several spectra from the individual particles). Micrographs were obtained from untilted samples (before analysis). During EDXA the samples showed in this work were stable under the electron beam. The original specimen-retainer was changed for a graphite one. Typical acquisition data were: accelerating voltage ¼ 80 kV, live-time ¼ 300 s, dead-time ffi 20%, and sample tilt angle ¼ 30°. 2.5. Field emission scanning electron microscopy The extracted powder of the total polysaccharides and sulfated fucan and alginic acid were directly deposited on a carbon tape coated aluminum stubs, and sputtered with gold (Balzers/Union FL-9496) for 1 min. High resolution field emission scanning electron microscopy (FESEM) was performed in a Jeol 6340F, operated at 5 kV. A working distance of 8 mm and slow scan mode were used for imaging.
but without the incubation procedures described above. The grids were observed in a Zeiss 900 EM operated at 80 kV. 2.7. Heavy metals quantification Concentrations of Zn and Cd in the algae biomass collected from a heavy metal contaminated area and an adjacent non-contaminated area and of their freezedried polysaccharides were determined using 250 mg samples (triplicates) digested with 5 ml of 65% HNO3 (Merck PA) in closed teflon flasks maintained in a microwave oven (CEM-MDS-2000) for 30 min. The digested solutions were evaporated in a hot plate and thereafter re-dissolved in 10 ml of 0.1 N HCl. Metal concentrations were measured by flame atomic absorption spectrophotometry (Varian AA-1475). The results were expressed as lg g1 of dry weight. Analytical procedures were tested by comparative analysis of International Atomic Energy Agency (IAEA) certified reference material IAEA-140 (seaweed homogenate, Fucus). The results obtained were within the confidence limit (significance level a < 0:05).
3. Results 3.1. Polysaccharides extracted from the brown algae cell wall present self-assembled structures with trabecular pattern
2.6. Labeling with lectins The gold-labeled lectins (10 nm) from Ulex europaeus (UEA), which recognizes specifically a-L -fucosyl units, and Concanavalin A (Con A), specific to terminal residues of a-D -mannosyl and a-D -glucosyl were used for identification of carbohydrate structures in the ultrastructures of the molecules isolated from the brown algae cell wall. The powders of the isolated molecules were directly embedded in LRGold resin at )20 °C for 2 days and polymerized under UV radiation at )20 °C for 3 days. Ultra-thin sections were obtained in an ultramicrotome (Reichert Super-Nova) using a diamond knife (Polyscience) and then collected on nickel grids (300 mesh). The ultra-thin sections were sequentially immersed in 0.1 M phosphate buffer saline (PBS) for 10 min, 3 and 1% bovine serum albumin (BSA) in PBS for 10 min each, and finally incubated with UEA or Con A overnight at 4 °C. Different concentrations of the lectins were tested in order to determine the appropriated dilution. The grids were washed in BSA, PBS, and distilled water. The pH of solutions was previously adjusted to 6.3 and 8 for labeling with UEA and Con A, respectively. The sections were stained with 2% uranyl acetate for 20 min. As a control, sections were incubated with pure lectins
Approximately 12 and 23% of the dry biomass of the alga P. gymnospora were solubilized by extraction with distilled water and 5% KOH solution, respectively. The powder of solubilized polysaccharides varied from light brown (water extraction) to dark brown color (KOH extraction). TEM observations of the total polysaccharides showed numerous stick-like structures joined to each other (Fig. 1A). Electron diffraction images obtained from this sample presented diffraction patterns compatible with a polycrystalline material (inset in Fig. 1A). FESEM of these polysaccharides showed selfassembled structures, forming different lamellar or trabecular patterns (Figs. 1B–D). Ca and S were associated with these well-organized structures as indicated by the EDXA spectra (Figs. 1E and F). This confirm that alginic acid and sulfated fucan are found in these trabecular structures since calcium ions strongly bind to the carboxyl groups of alginic acid while S is a constituent of sulfate groups found in the algal fucans. 3.2. Alginic acid and sulfated fucan differ in the pattern of their ultrastructures Alginic acid was separated from sulfated fucan in the acidic polysaccharide mixture based on its property to
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Fig. 1. Electron microscopy and EDXA of the acidic polysaccharides from the brown alga P. gymnospora. (A) TEM observation of the total polysaccharides extracted from algae collected in SB presenting numerous stick-like structures joined to each other. The inset image is electron diffraction pattern obtained in an area similar to figure 1, formed by spots regularly distributed. (B) FESEM of total polysaccharides extracted from algae collected in SB showing self-assembled structures, forming different lamellar or trabecular patterns arranged transversally. (C) Low magnification image of an assembly of total polysaccharide fraction extracted from algae collected in SB, showing a coil-like appearance. (D) Detailed view of polysaccharides showing a plate-like arrangement. (E) and (F) EDXA spectra of total polysaccharides from Sepetiba Bay, showing Zn (E) and from an adjacent non-contaminated site, Ribeira Bay (image of the polysaccharides not shown), (F) C (Ka ¼ 0:28 keV), O (Ka ¼ 0:52 keV), S (Ka ¼ 2:31 keV), Cl (Ka ¼ 2:62 keV), and Ca (Ka ¼ 3:69 keV; Kb ¼ 4:01 keV). Inset in (E): Part of spectra showing the Zn peak (Ka ¼ 8:63 keV). Copper peaks (Ka ¼ 8:04 keV; Kb ¼ 8:91 keV) were originated from the grid. Arrows in (A) point to typical region that were analyzed by a focused beam (100 nm). (A) Bars in figures (A)–(D) correspond to 1 lm.
precipitate with CaCl2 (see Section 2). Macroscopically, the appearance of total polysaccharide sample was a fine powder, while sulfated fucan was granular and alginic acid presented flocculated forms. The two purified polysaccharides presented different ultrastructure and elemental composition. In the alginates fraction, TEM showed amorphous structures containing numerous pores (Fig. 2A). FE-
SEM revealed a sponge-like form (Fig. 2B) with their surfaces having a sheet appearance (Fig. 2C). The pores of alginate scaffold varied between 10 and 50 lm. Ca was determined as the characteristic element (Fig. 2D). In contrast, sulfated fucans showed numerous particles with polygonal projections, individualized or arranged in chains (Figs. 3A and B). Electron diffraction images revealed a polycrystalline structure of this fraction (inset
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Fig. 2. Electron microscopy and EDXA of alginate. (A) TEM of the alginates fraction presenting amorphous material with numerous pores. (B) FESEM of alginates fraction revealing a sponge-like structure. (C) FESEM of alginate fraction revealing a surface with a sheet appearance. (D) EDXA spectrum from alginate fraction. A focused beam of circa 100 nm was used (arrows point to typical analyzed regions). Ca was determined as the characteristic element. C, O, Cl, K, and Cu were also detected. (A) Bar ¼ 20 lm; (B) bar ¼ 10 lm; and (C) bar ¼ 50 lm.
in Fig. 3B). FESEM showed numerous cubic particles with sizes ranging of 4–20 lm (Figs. 3C and D). High magnification images of these particles showed a discrete undulated surface (Fig. 3E). Sulfur was detected as the characteristic element (Fig. 3F). One possible explanation for the unusual ultrastructure of the sulfated fucan extracted from brown algae is either the presence of covalent-linked proteins or others molecules which would bring together the polysaccharide chains. In order to clarify this aspect we extracted acidic polysaccharide from the brown algae using exhaustive protease digestion and the sulfated fucan was purified by anion exchange chromatography (Fig. 4A). For these experiments we used the species L. brasiliensis since the purification and analytical procedures for the sulfated fucan from this brown alga were already established (Pereira et al., 1999, 2002). The purity of the final product was assured by agarose gel electrophoresis (Fig. 4B). Again, TEM observations of the exhaustive purified sulfated fucan revealed the same ultrastructure, composed of numerous particles with polygonal projections (Fig. 4C), as already described for the polysac-
charide obtained from P. gymnospora. X-ray microanalysis detected the presence of S, besides C and O (Fig. 4D). The Na and Cl peaks originated from saline solution added to sulfated fucan during the purification procedures while Cu peaks are from the copper grids, as already mentioned. Further evidence that the observed ultrastructure is in fact constituted by the sulfated fucan from brown algae came from studies with specific lectin. The observations of ultra-thin sections of sulfated fucan ultrastructure revealed an intense labeled with UEA, a lectin specific for a-L -fucosyl (Fig. 5A). When ConA instead of UEA was used we observed a less intense label (Fig. 5B), as expected since mannose is only a minor component of the algal fucans (Dietrich et al., 1995; Pereira et al., 1999). The polygonal particles were labeled less intensively with both tested lectins (Figs. 5C and D). Ultra-thin sections of alginic acid incubated with the two lectins presented no significant label (data not shown). The grids previously incubated with pure lectins (control tests) did not exhibit any label.
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Fig. 3. Electron microscopy and EDXA of sulfated fucans. (A) TEM image of sulfated fucan fraction showing isolated particles with polygonal projections. (B) TEM of fucan fraction with particles arranged in chains. Inset in (B) electron diffraction image of sulfated fucans revealing the polycrystalline nature of the sample. (C) FESEM of sulfated fucans showing numerous particles with polyhedrical morphologies. (D) FESEM of a fucan particle. (E) High magnification of a fucan particle showing an undulated surface. (F) EDXA spectrum from sulfated fucans fraction obtained by TEM. The regions analyzed are the typical individual particles in (B) indicated by the arrows. S was detected as the characteristic element. The elements C, O, K (Ka ¼ 3:31 eV) (from KOH extraction), and Cu (from the grids) were also observed. (A) Bar ¼ 5 lm; (B) bar ¼ 18 lm; (C) bar ¼ 10 lm; (D) bar ¼ 1 lm; and (E) bar ¼ 0.1 lm.
3.3. Heavy metals are accumulated into the acidic polysaccharides ultrastructures EDXA spectra showed the presence of Zn in the ultrastructure of polysaccharides extracted from algae of
heavy metal contaminated area (Fig. 1E) while this element was absent from samples of control site (Fig. 1F). The elements C, O, S, K, and Ca were detected in samples obtained from the two sites and Cu peaks are provided from the copper grids. This observation is
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Fig. 4. Biochemical and microscopic analysis of sulfated fucans from L. brasiliensis. (A) Anion exchange chromatography on DEAE–cellulose column of sulfated fucan. Fractions were checked by the phenol–H2 SO4 (s) and carbazole (m) reactions, for metachromasia (d) and NaCl concentration (- - -) as described (Pereira et al., 1999). (B) Agarose gel electrophoresis of the crude polysaccharide (1) and the purified sulfated fucan (2) (15 lg of each) were applied to a 0.5% agarose gel and the electrophoresis was run and stained as described (Pereira et al., 1999). (C) TEM image showing particles presenting similar structures as observed in sulfated fucans fractions of P. gymnospora. (D) EDXA spectrum showing the elements C, O, and S. Na and Cl peaks were probably originated from saline solution added to fucans fraction during biochemical purification. Cu peaks are from the grids. (C) Bar ¼ 20 lm.
support by direct quantification of heavy metal in the biomass and also in solubilized polysaccharides. Zn and Cd contents were marked higher in algae from contaminated area comparing to the control algae (Table 1). Heavy metals were not detected in the samples of purified polysaccharides. Possibly, these elements were disrupted from the polysaccharides during the purification procedures.
4. Discussion 4.1. Acidic polysaccharides from the algal cell wall possess a well-organized ultrastructure The architecture of marine algae cell walls is maintained by the self-assembly of cellulose and alginic acid chains. Possibly, sulfated fucans play a key role crosslinking these two macromolecules (Kloareg and Quatrano, 1988). Studies about cell wall deposition during morphogenesis in Palvatia compressa reveal the wall strength in young zygotes depends on F-actin, whereas
cellulose and a sulfated polysaccharide (probably a sulfated fucan) are more important in tip growing zygotes (Bisgrove and Kropf, 2001). A microfibrillar arrangement of the brown algae cell walls is clearly observed by TEM at routine preparations. The cellulose microfibrils are embedded in an ‘‘amorphous’’ matrix, composed mostly by soluble polysaccharides. In some regions of the cell wall high amounts of this matrix masks the microfibrils, especially near the external region. The term ‘‘amorphous’’ is widely applied to the matrix polysaccharides because it is difficult to discriminate these compounds within the microfibrillar arrangement. The ‘‘amorphous’’ polysaccharides are easily extracted from the algal cell wall and purified as two major components, named alginic acid and sulfated fucan. The chemical structure and several biological properties of these polysaccharides have been widely studied. However no data is available concerning the ultrastructure and/or conformation of these soluble polysaccharides from the cell wall of brown algae, especially in the case of the sulfated fucans. It is generally assumed that the
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Fig. 5. TEM of ultra-thin sections (LRGold resin) of sulfated fucans fraction incubated with specific lectins labeled with colloidal gold. (A) Amorphous-like material intensively labeled with UEA. (B) Amorphous-like material intensively labeled with Con A. (C) Particle with a polygonal outline labeled in the border by UEA. (D) Particles with polygonal outlines also labeled in the border by Con A. Bar represents 200 lm in (A); 150 lm in (B); 100 lm in (C), and 100 lm in (D).
Table 1 Concentrations of Zn and Cd (determined by atomic absorption spectrophotometry) in samples of the brown algae P. gymnospora collected from a heavy metal contaminated area (Sepetiba Bay) and an adjacent non-contaminated area (Ribeira Bay) Sample
Collection site
Metals Zn
Cd
Total biomass
Sepetiba Bay Ribeira Bay
242 34 23 8
1.30 0.20 0.36 0.09
Solubilized polysaccharides
Sepetiba Bay
95 12
0.21 0.05
Ribeira Bay
9.3 0.4
ND
ND, not detected. Results in lg g1 of dry weight (mean SD).
physiological role of these molecules depends exclusively of the overall negative charged groups. Only very recent studies using synthetic oligosaccharides have suggested that sulfated fucans may assume specific conformation (Gerbst et al., 2001, 2002).
We now used a different approach to investigate whether the algal polysaccharides present specific ultrastructure. The polysaccharides were visualized by a variety of electron microscopy techniques. We observed that sulfated fucan and alginic acid present well-organized ultrastructure but the pattern of organization varies depending on the polysaccharide species. Alginic acid showed a sponge-like structure whereas sulfated fucan presented particles with polygonal forms. We have conclusive evidence that the ultrastructure composed of numerous particles with polygonal projection is in fact formed by sulfated fucan molecules. First, EDXA spectra reveal the presence of S in these structures, as expected for the sulfate groups found in the algal fucans. Second, these ultrastructures were intensively labeled with UEA, a specific lectin that recognize a-L -fucosyl residues. Finally, an exhaustive purified sulfated fucan showed the same morphology, composed of polyhedrical forms. A well-known example of polysaccharides assuming organized ultrastructure is the proteoglycans from
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vertebrate extracellular matrix (Rothenburger et al., 2002). However, the ultrastructure is observed exclusively on the intact molecules, which are glycosaminoglycan chains covalently attached to a protein core. Once the proteoglycan is digested with protease, the released glycosaminoglycan chains (molecular mass ranging from 10 to 40 kDa) no longer show ultrastructure organization under microscopic examination. The sulfated fucans still show the characteristic ultrastructure even after exhaustive protease digestion. These algal polysaccharides possess high molecular weight (>100 kDa, Pereira et al., 1999), well above the size of vertebrate glycosaminoglycan chains and similar to several intact proteoglycans. TEM images showed that the sulfated fucans of the two species studied in this work had similar morphologies (i.e., polyhedrical particles arranged in chains). It means that probably, the morphology of micron sized crystals of sulfated fucans from the two species was not influenced by the distinct proportions of the polysaccharides in these two algae, or the differences in polysaccharide composition is not significantly high to induce differences in macromolecular arrangement at the ultrastructural level. Our approach in the study of the algal polysaccharides may help the comprehension of some their physiological and biological role in terms of molecular structure. This is especially noteworthy for the sulfated fucans, which reveal an unusual polycrystalline structure. The chemical structure of these molecules may not reveal the spatial distribution of sulfate groups but this aspect may be clarified using structural analysis. It may clarify the drastic differences in biological activities between sulfated fucans with similar chemical structures (Alvez et al., 1997; Pereira et al., 2002).
In the present work, Zn was detected in the total biomass and in the solubilized polysaccharides from algae exposed to a heavy metal contaminated environment. In this sample, EDXA spectra showed that Zn was associated with Ca and S (besides C and O), indicating that alginic acid and sulfated fucan are the molecules responsible for the binding of heavy metal. Mariani et al. (1985) have studied the elemental composition of polysaccharides isolated from Fucus virsoides by X-ray microanalysis in SEM, and suggested that Ca2þ and Mg2þ were strongly complexed with alginic acid in algal cell walls, stabilizing the wall architecture. In contrast, the seawater cations were mainly bound to the sulfated polysaccharides, which regulate the passive ion disposed to the algal cells. Andrade et al. (2002) showed that when P. gymnospora was exposed to sub-lethal concentrations of Cd isolated and added with Zn in vitro, other cations such as Naþ , Kþ , Ca2þ found naturally in cell walls were substituted by Cd2þ and Zn2þ . In summary, the present work describes for the first time the ultrastructure of sulfated fucans and alginic acid extracted from the cell wall of brown algae. Alginic acid presented a sponge-like form with numerous pores while sulfated fucans showed polyhedrical forms with a unique crystalline nature. In addition, Zn was detected in these polysaccharides when the algae were collected from a heavy metal contaminated environment. This demonstrates the role of polyanionic polysaccharides as metal binders for the nucleation and precipitation of heavy metals in brown algae cell walls.
4.2. Acidic polysaccharides form the algal cell wall retain heavy metals
We thank Ms. Mair Machado for technical support, Ricardo Thomas for AAS measures and Dr. W. de Souza for electron microscopy facilities. This work was supported by PRONEX—CNPq, FAPERJ, JBRJ/ MMA. Paulo A.S. Mour~ao is a fellow of the John Simon Guggenheim Memorial Foundation.
Different species of brown algae have been used as biomonitor of heavy metal pollution due to their high capability of long-term uptake comparing with other seaweeds (Amado Filho et al., 1999; Engdahl et al., 1998; Karez et al., 1994; Murugadas et al., 1995). In a previous work, we have demonstrated that a brown algae collected from a heavy metal contaminated area accumulated Zn as electron-dense granules in the cell walls in median regions of thallus (Amado Filho et al., 1999). The polyanionic polysaccharides have been proposed as the molecules responsible for the binding of heavy metals when the algae are exposed to high concentrations of these elements (Amado Filho et al., 1996, 1999; Andrade et al., 2002; Ballan-Dufrancßais et al., 1991; Haug et al., 1974; Lignell et al., 1982; Pellegrini et al., 1991, 1993; Tretyn et al., 1996; Stengel and Dring, 2000).
Acknowledgments
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L.R. Andrade et al. / Journal of Structural Biology 145 (2004) 216–225 Andrade, L.R., Farina, M., Amado Filho, G.M., 2002. Role of cell walls of Padina gymnospora (Phaeophyta) in heavy metals (Zn and Cd) accumulation. Phycologia 41 (1), 39–48. Ballan-Dufrancßais, C., Marcaillou, C., Amiard-Triquet, C., 1991. Response of the phytoplancton alga Tetraselmis suecica to copper and silver exposure: vesicular metal bioaccumulation and lack of starch bodies. Biol. Cell. 72, 103–112. Bisgrove, S.R., Kropf, D.L., 2001. Cell wall deposition during morphogenesis in fucoid algae. Planta 212 (5–6), 648–658. Chung, T.W, Yang, J., Akaike, T., Cho, K.Y., Nah, J.W., Kim, S.I., Cho, C.S., 2002. Preparation of alginate/galactosylated chitosan scaffold for hepatocyte attachment. Biomaterials 23 (14), 2827–2834. Dietrich, C.P., Farias, G.G.M., De Abreu, L.R.D., Leite, E.L., Silva, L.F., Nader, H.B., 1995. A new approach for the characterization of polysaccharides from algae: presence of four main acidic polysaccharides in three species of the class Phaeophycea. Plant Sci. 108, 143–153. Engdahl, S., Mamboya, F., Mtolera, M., Semesi, A., Bjorg, M., 1998. The brown macroalgae Padina boergesenii as an indicator of heavy metal contamination in the Zanzibar Chanel. Ambio 27 (8), 694–700. Gerbst, A.G., Ustuzhanina, N.E., Grachev, A.A., Khatuntseva, E.A., Tsvetkov, D.E., Whitfield, D.M., Berces, A., Nifantiev, N.E., 2001. Synthesis, NMR, and conformational studies of fucoidan fragments. III. Effect of benzoyl group at O-3 on stereoselectivity of glycosylation by 3-O- and 3,4-di-O-benzoylated 2-O-benzyfucosyl bromides. J. Carbohydr. Chem. 20, 821–831. Gerbst, A.G., Ustuzhanina, N.E., Grachev, A.A., Zlotina, N.S., Khatuntseva, E.A., Tsvetkov, D.E., Shashkov, A.S., Uso, A.I., Nifantiev, N.E., 2002. Synthesis, NMR, and conformational studies of fucoidan fragments. IV. 4-mono- and 4,40 -disulfated (1! 3)-alpha-L -fucobioside and 4-sulfated fucoside fragments. J. Carbohydr. Chem. 21, 313–324. Haug, A., Larsen, B., Smidsrod, O., 1966. A study of the constitution of alginic acid by partial acid hydrolysis. Acta Chem. Scand. 20, 183–190. Haug, A., Larsen, B., Smidsrod, O., 1974. Uronic acid sequence in alginate from different sources. Carbohydr. Res. 32 (2), 217–225. Karez, C.S., Magalh~ aes, V.F., Pfeiffer, W.C., Amado Filho, G.M., 1994. Trace metal accumulation by algae in Sepetiba Bay, Brazil. Environ. Pollut. 83, 351–356. Kloareg, B., Quatrano, R.S., 1988. Structure of the cell walls of marine algae and ecophysiological functions of the matrix of polysaccharides. Oceanogr. Mar. Biol. Annu. Rev. 26, 259–315. Kloareg, B., Demarty, M., Mabeu, S., 1986. Polyanionic characteristics of purified sulfated homofucans from brown algae. Int. J. Biol. Macromol. 8, 380–386.
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Lignell, A., Roomans, G.M., Pedersen, M., 1982. Localization of adsorbed cadmium in Fucus vesiculosus L. by X-ray microanalysis. Zeitschrift fur Pflanzenphysiologie 105, 103–109. Magnani, A., Rappuoli, R., Lamponi, S., Barbucci, R., 2000. Novel polysaccharides hydrogels: characterization and properties. Polym. Advan. Technol. 11 (8-12), 488–495. Mariani, P., Tolomio, C., Braghetta, P., 1985. An ultrastructural approach to the adaptive role of the cell wall in the intertidal alga Fucus virsoides. Protoplasma 128, 208–217. Mariani, P., Tolomio, C., Baldan, B., Braghetta, P., 1990. Cell wall ultrastructure and cation localization in some benthic marine algae. Phycologia 29 (2), 253–262. Murugadas, T.L., Phang, S.M., Tong, S.L., 1995. Heavy metal accumulation patterns in selected seaweed species of Malaysia. Asia Pac. J. Mol. Biol. Biothechnol. 3 (4), 209–310. Pellegrini, L., Pellegrini, M., Delivopoulos, S., Berail, G., 1991. The effects of cadmium on the fine structure of the brown alga Cystoseira barbata forma repens Zinova et Kalugina. Br. Phycol. J. 26, 1–8. Pellegrini, M., Laugie, A., Sergent, M., Phan-Tan-Lun, R., Valls, R., Pellegrini, L., 1993. Interactions between the toxicity of heavy metals cadmium, copper, zinc in combinations and detoxifying role of calcium in the brown alga Cystoseira barbata. J. Appl. Phycol. 5, 351–361. Percival, E., 1979. The polysaccharides of green, red and brown seaweeds: their basic structure, biosynthesis and function. Br. Phycol. J. 14, 103–117. Pereira, M.S., Mulloy, B., Mour~ao, P.A.S., 1999. Structure and anticoagulant activity of sulfated fucans. J. Biol. Chem. 274 (12), 7656–7667. Pereira, M.S., Melo, F.R., Mour~ao, P.A.S., 2002. Is there a correlation betwen structure and anticoagulant action of sulfated galactans and sulfated fucans? Glycobiology 12, 573–580. Rothenburger, M., Volker, W., Vischer, P., Glasmacher, B., Scheld, H.H., Deiwick, M., 2002. Ultrastructure of proteoglycans in tissueengineered cardiovascular structures. Tissue Eng. 8 (6), 1049–1056. Stengel, D.B., Dring, M.J., 2000. Copper and iron concentrations in Ascophyllum nodosum (Fucales, Phaeophyta) from different sites in Ireland and after culture experiments in relation to thallus age and epiphytism. J. Exp. Mar. Biol. Ecol. 246, 145–161. Tretyn, A., Grolig, F., Magdowski, G., Wagner, G., 1996. Selective binding of Ca, Zn, Cu and K by the physods of the green alga Mougeotia scalaris. Folia Histochemica et Cytobiologica 34 (2), 103–108. Zhou, J.P., Zhang, L.N., 2001. Structure and properties of blend membranes prepared from cellulose and alginate in NaOH/urea aqueous solution. J. Polym. Sci. B 39 (4), 451–458.
Structural Biology Journal of Structural Biology 145 (2004) 216–225 www.elsevier.com/locate/yjsbi
Ultrastructure of acidic polysaccharides from the cell walls of brown algae Leonardo R. Andrade,a Leonardo T. Salgado,a Marcos Farina,a Mariana S. Pereira,b Paulo A.S. Mour~ ao,b and Gilberto M. Amado Filhoc,* a
Laborat orio de Biomineralizacßa~o, Departamento de Histologia e Embriologia, Instituto de Ci^ encias Biom edicas, CCS, Universidade Federal do Rio de Janeiro, Cidade Universit aria, Rio de Janeiro, RJ 21941-590, Brazil b Laborat orio de Tecido Conjuntivo, Hospital Universit ario Clementino Fraga Filho e Departamento de Bioquımica M edica, Instituto de Ci^ encias Biom edicas, CCS, Cidade Universit aria, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-590, Brazil c Programa Zona Costeira, Instituto de Pesquisas Jardim Bot^ anico/MMA, Rua Pacheco Le~ ao 915, Jardim Bot^ anico, Rio de Janeiro, RJ 22460-030, Brazil Received 10 June 2003, and in revised form 2 October 2003
Abstract We have studied the ultrastructure of acidic polysaccharides from the cell walls of brown algae using a variety of electron microscopy techniques. Polysaccharides from Padina gymnospora present self assembled structures, forming trabecular patterns. Purified fractions constituted by alginic acid and sulfated fucan also form well-organized ultrastructures, but the pattern of organization varies depending on the polysaccharide species. Alginic acid presents sponge-like structures. Sulfated fucan exhibits particles with polygonal forms with a polycrystalline structure. These particles are in fact constituted by sulfated fucan molecules since they are recognized by a lectin specific for a-L -fucosyl residues. X-ray microanalysis reveal that S is a constituent element, as expected for sulfated groups. Finally, an exhaustive purified sulfated fucan shows the same ultrastructure formed by polygonal forms. Furthermore, elemental analyses of acidic polysaccharides indicate that they retain Zn, when algae were collected from a contaminated area. This observation is supported by direct quantification of heavy metal in the biomass and also in the solubilized polysaccharides compared with the algae from a non-contaminated site. We conclude that these molecules have specific ultrastructure and elemental composition; and act as metal binder for the nucleation and precipitation of heavy metals when the algae are exposed to a metal contaminated environment. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Alginic acid; Biomineralization; Brown algae; EDXA; Field emission scanning electron microscopy; Fucoidan; Heavy metals; Sulfated fucan; Polysaccharides; TEM; Ultrastructure; Zinc
1. Introduction Cell walls of brown algae have a fibrillar compartment formed mainly of cellulose microfibrils, which is embedded in an amorphous matrix of acid polysaccharides linked each other by proteins (Kloareg et al., 1986). The acid polysaccharides are mainly composed of alginic acids and sulfated fucans (also known as fucoidan). The alginic acids are linear carboxylated copolymers constituted by different proportions of 1,4-linked
* Corresponding author. Fax: +55-21-22942295. E-mail address: gfi[email protected] (G.M. Amado Filho).
1047-8477/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2003.11.011
b-D -mannuronic acid (M-block) and a-L -guluronic acid (G-block) (Haug et al., 1966). Algal sulfated fucans are polymers mainly formed by sulfated a-L -fucose but also containing small proportions of other sugars such as mannose and galactose (Dietrich et al., 1995; Kloareg et al., 1986; Pereira et al., 1999, 2002). The chemical structure, physico-chemical properties, and biological activities of alginic acids and sulfated fucans have been widely studied, but the ultrastructure of these molecules still unknown, especially in the case of the sulfated fucans. The characterization of polysaccharide morphology can contribute for the understanding of the cell wall architecture in the marine algae. The few available data about the morphology of these
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polysaccharides are generally related to alginic acid beads as scaffold to cell culture in tissue engineering; carriers for immobilized proteins and polymers used for the controlled release of drugs (Chung et al., 2002; Magnani et al., 2000; Zhou and Zhang, 2001). Acidic polysaccharides found in the cell wall of brown algae have also been associated with the capacity of these organisms to accumulate heavy metals when exposed to an environment containing high concentrations of these elements (Amado Filho et al., 1999; Andrade et al., 2002; Lignell et al., 1982; Pellegrini et al., 1993). The negatively charged polysaccharides from the mucilaginous matrix can adsorb and partially exclude mobile anions, and consequently act as cation-exchanger and ionic barrier, selecting the elements that will be absorbed by the algal cells (Kloareg et al., 1986; Mariani et al., 1990; Percival, 1979). Different seaweeds from a heavy metal contaminated area were analyzed by atomic absorption spectrophotometry (AAS) and the results indicated that the brown alga Padina gymnospora presented the highest concentrations of Zn and Cd among the species tested (Amado Filho et al., 1999). Analytical electron microscopy showed that cell walls located in median regions of thallus presented electron-dense granules composed mainly by Zn and S. Cd was not detected because of its low concentration in the samples. According to this result, it was proposed that the main mechanism presented by P. gymnospora to accumulate high heavy metal concentrations from contaminated areas is related to the precipitation of these elements into the cell walls, diminishing the availability of metals to cell absorption (Amado Filho et al., 1996, 1999). In addition, adult individuals of P. gymnospora exposed to sub-lethal concentrations of Cd and/or Zn in vitro presented numerous crystalline granules along the cell walls, formed mainly by Cd and/or Zn, which colocalized with S and O (Andrade et al., 2002). This observation suggested that the acidic polysaccharides (mostly sulfated fucans) play an important role in nucleation of Cd and Zn. Perhaps the acid micro-environment assured by these polysaccharides in the cell walls propitiates an increase of crystalline granules like a biomineralization process, and consequently turning the algae more tolerant to metals (Andrade et al., 2002). In this work we have studied the ultrastructure of alginic acid and sulfated fucan from the cell wall of brown algae using different electron microscopy techniques. Both polysaccharides presented a self-assembled, well-organized ultrastructure but the pattern of organization varies depending on the polysaccharide species. Alginic acid showed a sponge-like structure whereas sulfated fucans presented particles with polygonal forms. Furthermore, elemental analysis of these ultrastructures indicates they retain heavy metals when the algae were exposed to a contaminated area.
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2. Materials and methods 2.1. Polysaccharides from P. gymnospora Adult individuals of P. gymnospora were collected in Sepetiba Bay, a heavy metal (mostly Zn and Cd) contaminated area located in Rio de Janeiro State. Algae were also collected in an adjacent non-contaminated area, Ribeira Bay, for comparative analysis. Fresh algae were cleaned from epiphytes with a thin brush under a stereoscopic microscope, briefly washed in distilled water and dried at 50 °C. The glasses used in polysaccharide extraction were previously washed in a detergent solution (1% Extran) and an acid solution (2% HNO3 ). The polysaccharides were extracted from 4 g (dry weight) of biomass in 250 ml of ultra-pure water, under agitation for 24 h at room temperature. The samples were centrifuged and the supernatants with soluble components were precipitated with 70% ethanol. The precipitates were removed by centrifugation, dissolved, and dialyzed against distilled water for 24 h and freezedried. The powder obtained is denominated as total polysaccharides and used for ultrastructural observation and elemental composition studies. Alternatively, the polysaccharides were also extracted from the algae with 5% KOH solution (w/v) under agitation for 24 h at room temperature. The solution was centrifuged, and the polysaccharides precipitated from the clear supernatant with 70% ethanol (v/v) and freezedried. The powder was then dissolved in distilled water and 4 M CaCl2 was added to the solution in order to precipitate the alginates. The sample was then centrifuged and both, the precipitated and supernatant containing alginic acid and sulfated fucan, respectively, were dialyzed against distilled water for 24 h and freezedried. The powders of fractioned polysaccharides (alginic acids and sulfated fucans) were used for electron microscopy studies. 2.2. Sulfated fucan from Laminaria brasiliensis Polysaccharides were extracted from the cell wall of the brown algae L. brasiliensis by papain digestion and the sulfated fucan was purified by anion exchange chromatography, as described (Pereira et al., 1999). The purity of the sulfated fucan preparation was checked by agarose gel electrophoresis and chemical analysis. 2.3. Transmission electron microscopy For transmission electron microscopy (TEM) purposes, the total and fractionated polysaccharides were dissolved (5 mg/ml) in ultra-pure water and 10 ll were transferred to a Formvar film coated copper grids. The polysaccharidic solutions were dried and the samples were observed in a Jeol 1200 EX, operated at 80 kV.
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2.4. Analytical TEM The elemental composition of the extracted polysaccharides was determined by energy dispersive X-ray analysis (EDXA). The samples were prepared for TEM as described above, and analyzed in a Jeol 1200 EX equipped with a Noran–Voyager analytical system. A focused spot (d 100 nm) was used to analyze the areas of interest in the polysaccharide samples. The areas probed were representative of the whole sample (we obtained several spectra from the individual particles). Micrographs were obtained from untilted samples (before analysis). During EDXA the samples showed in this work were stable under the electron beam. The original specimen-retainer was changed for a graphite one. Typical acquisition data were: accelerating voltage ¼ 80 kV, live-time ¼ 300 s, dead-time ffi 20%, and sample tilt angle ¼ 30°. 2.5. Field emission scanning electron microscopy The extracted powder of the total polysaccharides and sulfated fucan and alginic acid were directly deposited on a carbon tape coated aluminum stubs, and sputtered with gold (Balzers/Union FL-9496) for 1 min. High resolution field emission scanning electron microscopy (FESEM) was performed in a Jeol 6340F, operated at 5 kV. A working distance of 8 mm and slow scan mode were used for imaging.
but without the incubation procedures described above. The grids were observed in a Zeiss 900 EM operated at 80 kV. 2.7. Heavy metals quantification Concentrations of Zn and Cd in the algae biomass collected from a heavy metal contaminated area and an adjacent non-contaminated area and of their freezedried polysaccharides were determined using 250 mg samples (triplicates) digested with 5 ml of 65% HNO3 (Merck PA) in closed teflon flasks maintained in a microwave oven (CEM-MDS-2000) for 30 min. The digested solutions were evaporated in a hot plate and thereafter re-dissolved in 10 ml of 0.1 N HCl. Metal concentrations were measured by flame atomic absorption spectrophotometry (Varian AA-1475). The results were expressed as lg g1 of dry weight. Analytical procedures were tested by comparative analysis of International Atomic Energy Agency (IAEA) certified reference material IAEA-140 (seaweed homogenate, Fucus). The results obtained were within the confidence limit (significance level a < 0:05).
3. Results 3.1. Polysaccharides extracted from the brown algae cell wall present self-assembled structures with trabecular pattern
2.6. Labeling with lectins The gold-labeled lectins (10 nm) from Ulex europaeus (UEA), which recognizes specifically a-L -fucosyl units, and Concanavalin A (Con A), specific to terminal residues of a-D -mannosyl and a-D -glucosyl were used for identification of carbohydrate structures in the ultrastructures of the molecules isolated from the brown algae cell wall. The powders of the isolated molecules were directly embedded in LRGold resin at )20 °C for 2 days and polymerized under UV radiation at )20 °C for 3 days. Ultra-thin sections were obtained in an ultramicrotome (Reichert Super-Nova) using a diamond knife (Polyscience) and then collected on nickel grids (300 mesh). The ultra-thin sections were sequentially immersed in 0.1 M phosphate buffer saline (PBS) for 10 min, 3 and 1% bovine serum albumin (BSA) in PBS for 10 min each, and finally incubated with UEA or Con A overnight at 4 °C. Different concentrations of the lectins were tested in order to determine the appropriated dilution. The grids were washed in BSA, PBS, and distilled water. The pH of solutions was previously adjusted to 6.3 and 8 for labeling with UEA and Con A, respectively. The sections were stained with 2% uranyl acetate for 20 min. As a control, sections were incubated with pure lectins
Approximately 12 and 23% of the dry biomass of the alga P. gymnospora were solubilized by extraction with distilled water and 5% KOH solution, respectively. The powder of solubilized polysaccharides varied from light brown (water extraction) to dark brown color (KOH extraction). TEM observations of the total polysaccharides showed numerous stick-like structures joined to each other (Fig. 1A). Electron diffraction images obtained from this sample presented diffraction patterns compatible with a polycrystalline material (inset in Fig. 1A). FESEM of these polysaccharides showed selfassembled structures, forming different lamellar or trabecular patterns (Figs. 1B–D). Ca and S were associated with these well-organized structures as indicated by the EDXA spectra (Figs. 1E and F). This confirm that alginic acid and sulfated fucan are found in these trabecular structures since calcium ions strongly bind to the carboxyl groups of alginic acid while S is a constituent of sulfate groups found in the algal fucans. 3.2. Alginic acid and sulfated fucan differ in the pattern of their ultrastructures Alginic acid was separated from sulfated fucan in the acidic polysaccharide mixture based on its property to
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Fig. 1. Electron microscopy and EDXA of the acidic polysaccharides from the brown alga P. gymnospora. (A) TEM observation of the total polysaccharides extracted from algae collected in SB presenting numerous stick-like structures joined to each other. The inset image is electron diffraction pattern obtained in an area similar to figure 1, formed by spots regularly distributed. (B) FESEM of total polysaccharides extracted from algae collected in SB showing self-assembled structures, forming different lamellar or trabecular patterns arranged transversally. (C) Low magnification image of an assembly of total polysaccharide fraction extracted from algae collected in SB, showing a coil-like appearance. (D) Detailed view of polysaccharides showing a plate-like arrangement. (E) and (F) EDXA spectra of total polysaccharides from Sepetiba Bay, showing Zn (E) and from an adjacent non-contaminated site, Ribeira Bay (image of the polysaccharides not shown), (F) C (Ka ¼ 0:28 keV), O (Ka ¼ 0:52 keV), S (Ka ¼ 2:31 keV), Cl (Ka ¼ 2:62 keV), and Ca (Ka ¼ 3:69 keV; Kb ¼ 4:01 keV). Inset in (E): Part of spectra showing the Zn peak (Ka ¼ 8:63 keV). Copper peaks (Ka ¼ 8:04 keV; Kb ¼ 8:91 keV) were originated from the grid. Arrows in (A) point to typical region that were analyzed by a focused beam (100 nm). (A) Bars in figures (A)–(D) correspond to 1 lm.
precipitate with CaCl2 (see Section 2). Macroscopically, the appearance of total polysaccharide sample was a fine powder, while sulfated fucan was granular and alginic acid presented flocculated forms. The two purified polysaccharides presented different ultrastructure and elemental composition. In the alginates fraction, TEM showed amorphous structures containing numerous pores (Fig. 2A). FE-
SEM revealed a sponge-like form (Fig. 2B) with their surfaces having a sheet appearance (Fig. 2C). The pores of alginate scaffold varied between 10 and 50 lm. Ca was determined as the characteristic element (Fig. 2D). In contrast, sulfated fucans showed numerous particles with polygonal projections, individualized or arranged in chains (Figs. 3A and B). Electron diffraction images revealed a polycrystalline structure of this fraction (inset
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Fig. 2. Electron microscopy and EDXA of alginate. (A) TEM of the alginates fraction presenting amorphous material with numerous pores. (B) FESEM of alginates fraction revealing a sponge-like structure. (C) FESEM of alginate fraction revealing a surface with a sheet appearance. (D) EDXA spectrum from alginate fraction. A focused beam of circa 100 nm was used (arrows point to typical analyzed regions). Ca was determined as the characteristic element. C, O, Cl, K, and Cu were also detected. (A) Bar ¼ 20 lm; (B) bar ¼ 10 lm; and (C) bar ¼ 50 lm.
in Fig. 3B). FESEM showed numerous cubic particles with sizes ranging of 4–20 lm (Figs. 3C and D). High magnification images of these particles showed a discrete undulated surface (Fig. 3E). Sulfur was detected as the characteristic element (Fig. 3F). One possible explanation for the unusual ultrastructure of the sulfated fucan extracted from brown algae is either the presence of covalent-linked proteins or others molecules which would bring together the polysaccharide chains. In order to clarify this aspect we extracted acidic polysaccharide from the brown algae using exhaustive protease digestion and the sulfated fucan was purified by anion exchange chromatography (Fig. 4A). For these experiments we used the species L. brasiliensis since the purification and analytical procedures for the sulfated fucan from this brown alga were already established (Pereira et al., 1999, 2002). The purity of the final product was assured by agarose gel electrophoresis (Fig. 4B). Again, TEM observations of the exhaustive purified sulfated fucan revealed the same ultrastructure, composed of numerous particles with polygonal projections (Fig. 4C), as already described for the polysac-
charide obtained from P. gymnospora. X-ray microanalysis detected the presence of S, besides C and O (Fig. 4D). The Na and Cl peaks originated from saline solution added to sulfated fucan during the purification procedures while Cu peaks are from the copper grids, as already mentioned. Further evidence that the observed ultrastructure is in fact constituted by the sulfated fucan from brown algae came from studies with specific lectin. The observations of ultra-thin sections of sulfated fucan ultrastructure revealed an intense labeled with UEA, a lectin specific for a-L -fucosyl (Fig. 5A). When ConA instead of UEA was used we observed a less intense label (Fig. 5B), as expected since mannose is only a minor component of the algal fucans (Dietrich et al., 1995; Pereira et al., 1999). The polygonal particles were labeled less intensively with both tested lectins (Figs. 5C and D). Ultra-thin sections of alginic acid incubated with the two lectins presented no significant label (data not shown). The grids previously incubated with pure lectins (control tests) did not exhibit any label.
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Fig. 3. Electron microscopy and EDXA of sulfated fucans. (A) TEM image of sulfated fucan fraction showing isolated particles with polygonal projections. (B) TEM of fucan fraction with particles arranged in chains. Inset in (B) electron diffraction image of sulfated fucans revealing the polycrystalline nature of the sample. (C) FESEM of sulfated fucans showing numerous particles with polyhedrical morphologies. (D) FESEM of a fucan particle. (E) High magnification of a fucan particle showing an undulated surface. (F) EDXA spectrum from sulfated fucans fraction obtained by TEM. The regions analyzed are the typical individual particles in (B) indicated by the arrows. S was detected as the characteristic element. The elements C, O, K (Ka ¼ 3:31 eV) (from KOH extraction), and Cu (from the grids) were also observed. (A) Bar ¼ 5 lm; (B) bar ¼ 18 lm; (C) bar ¼ 10 lm; (D) bar ¼ 1 lm; and (E) bar ¼ 0.1 lm.
3.3. Heavy metals are accumulated into the acidic polysaccharides ultrastructures EDXA spectra showed the presence of Zn in the ultrastructure of polysaccharides extracted from algae of
heavy metal contaminated area (Fig. 1E) while this element was absent from samples of control site (Fig. 1F). The elements C, O, S, K, and Ca were detected in samples obtained from the two sites and Cu peaks are provided from the copper grids. This observation is
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Fig. 4. Biochemical and microscopic analysis of sulfated fucans from L. brasiliensis. (A) Anion exchange chromatography on DEAE–cellulose column of sulfated fucan. Fractions were checked by the phenol–H2 SO4 (s) and carbazole (m) reactions, for metachromasia (d) and NaCl concentration (- - -) as described (Pereira et al., 1999). (B) Agarose gel electrophoresis of the crude polysaccharide (1) and the purified sulfated fucan (2) (15 lg of each) were applied to a 0.5% agarose gel and the electrophoresis was run and stained as described (Pereira et al., 1999). (C) TEM image showing particles presenting similar structures as observed in sulfated fucans fractions of P. gymnospora. (D) EDXA spectrum showing the elements C, O, and S. Na and Cl peaks were probably originated from saline solution added to fucans fraction during biochemical purification. Cu peaks are from the grids. (C) Bar ¼ 20 lm.
support by direct quantification of heavy metal in the biomass and also in solubilized polysaccharides. Zn and Cd contents were marked higher in algae from contaminated area comparing to the control algae (Table 1). Heavy metals were not detected in the samples of purified polysaccharides. Possibly, these elements were disrupted from the polysaccharides during the purification procedures.
4. Discussion 4.1. Acidic polysaccharides from the algal cell wall possess a well-organized ultrastructure The architecture of marine algae cell walls is maintained by the self-assembly of cellulose and alginic acid chains. Possibly, sulfated fucans play a key role crosslinking these two macromolecules (Kloareg and Quatrano, 1988). Studies about cell wall deposition during morphogenesis in Palvatia compressa reveal the wall strength in young zygotes depends on F-actin, whereas
cellulose and a sulfated polysaccharide (probably a sulfated fucan) are more important in tip growing zygotes (Bisgrove and Kropf, 2001). A microfibrillar arrangement of the brown algae cell walls is clearly observed by TEM at routine preparations. The cellulose microfibrils are embedded in an ‘‘amorphous’’ matrix, composed mostly by soluble polysaccharides. In some regions of the cell wall high amounts of this matrix masks the microfibrils, especially near the external region. The term ‘‘amorphous’’ is widely applied to the matrix polysaccharides because it is difficult to discriminate these compounds within the microfibrillar arrangement. The ‘‘amorphous’’ polysaccharides are easily extracted from the algal cell wall and purified as two major components, named alginic acid and sulfated fucan. The chemical structure and several biological properties of these polysaccharides have been widely studied. However no data is available concerning the ultrastructure and/or conformation of these soluble polysaccharides from the cell wall of brown algae, especially in the case of the sulfated fucans. It is generally assumed that the
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Fig. 5. TEM of ultra-thin sections (LRGold resin) of sulfated fucans fraction incubated with specific lectins labeled with colloidal gold. (A) Amorphous-like material intensively labeled with UEA. (B) Amorphous-like material intensively labeled with Con A. (C) Particle with a polygonal outline labeled in the border by UEA. (D) Particles with polygonal outlines also labeled in the border by Con A. Bar represents 200 lm in (A); 150 lm in (B); 100 lm in (C), and 100 lm in (D).
Table 1 Concentrations of Zn and Cd (determined by atomic absorption spectrophotometry) in samples of the brown algae P. gymnospora collected from a heavy metal contaminated area (Sepetiba Bay) and an adjacent non-contaminated area (Ribeira Bay) Sample
Collection site
Metals Zn
Cd
Total biomass
Sepetiba Bay Ribeira Bay
242 34 23 8
1.30 0.20 0.36 0.09
Solubilized polysaccharides
Sepetiba Bay
95 12
0.21 0.05
Ribeira Bay
9.3 0.4
ND
ND, not detected. Results in lg g1 of dry weight (mean SD).
physiological role of these molecules depends exclusively of the overall negative charged groups. Only very recent studies using synthetic oligosaccharides have suggested that sulfated fucans may assume specific conformation (Gerbst et al., 2001, 2002).
We now used a different approach to investigate whether the algal polysaccharides present specific ultrastructure. The polysaccharides were visualized by a variety of electron microscopy techniques. We observed that sulfated fucan and alginic acid present well-organized ultrastructure but the pattern of organization varies depending on the polysaccharide species. Alginic acid showed a sponge-like structure whereas sulfated fucan presented particles with polygonal forms. We have conclusive evidence that the ultrastructure composed of numerous particles with polygonal projection is in fact formed by sulfated fucan molecules. First, EDXA spectra reveal the presence of S in these structures, as expected for the sulfate groups found in the algal fucans. Second, these ultrastructures were intensively labeled with UEA, a specific lectin that recognize a-L -fucosyl residues. Finally, an exhaustive purified sulfated fucan showed the same morphology, composed of polyhedrical forms. A well-known example of polysaccharides assuming organized ultrastructure is the proteoglycans from
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vertebrate extracellular matrix (Rothenburger et al., 2002). However, the ultrastructure is observed exclusively on the intact molecules, which are glycosaminoglycan chains covalently attached to a protein core. Once the proteoglycan is digested with protease, the released glycosaminoglycan chains (molecular mass ranging from 10 to 40 kDa) no longer show ultrastructure organization under microscopic examination. The sulfated fucans still show the characteristic ultrastructure even after exhaustive protease digestion. These algal polysaccharides possess high molecular weight (>100 kDa, Pereira et al., 1999), well above the size of vertebrate glycosaminoglycan chains and similar to several intact proteoglycans. TEM images showed that the sulfated fucans of the two species studied in this work had similar morphologies (i.e., polyhedrical particles arranged in chains). It means that probably, the morphology of micron sized crystals of sulfated fucans from the two species was not influenced by the distinct proportions of the polysaccharides in these two algae, or the differences in polysaccharide composition is not significantly high to induce differences in macromolecular arrangement at the ultrastructural level. Our approach in the study of the algal polysaccharides may help the comprehension of some their physiological and biological role in terms of molecular structure. This is especially noteworthy for the sulfated fucans, which reveal an unusual polycrystalline structure. The chemical structure of these molecules may not reveal the spatial distribution of sulfate groups but this aspect may be clarified using structural analysis. It may clarify the drastic differences in biological activities between sulfated fucans with similar chemical structures (Alvez et al., 1997; Pereira et al., 2002).
In the present work, Zn was detected in the total biomass and in the solubilized polysaccharides from algae exposed to a heavy metal contaminated environment. In this sample, EDXA spectra showed that Zn was associated with Ca and S (besides C and O), indicating that alginic acid and sulfated fucan are the molecules responsible for the binding of heavy metal. Mariani et al. (1985) have studied the elemental composition of polysaccharides isolated from Fucus virsoides by X-ray microanalysis in SEM, and suggested that Ca2þ and Mg2þ were strongly complexed with alginic acid in algal cell walls, stabilizing the wall architecture. In contrast, the seawater cations were mainly bound to the sulfated polysaccharides, which regulate the passive ion disposed to the algal cells. Andrade et al. (2002) showed that when P. gymnospora was exposed to sub-lethal concentrations of Cd isolated and added with Zn in vitro, other cations such as Naþ , Kþ , Ca2þ found naturally in cell walls were substituted by Cd2þ and Zn2þ . In summary, the present work describes for the first time the ultrastructure of sulfated fucans and alginic acid extracted from the cell wall of brown algae. Alginic acid presented a sponge-like form with numerous pores while sulfated fucans showed polyhedrical forms with a unique crystalline nature. In addition, Zn was detected in these polysaccharides when the algae were collected from a heavy metal contaminated environment. This demonstrates the role of polyanionic polysaccharides as metal binders for the nucleation and precipitation of heavy metals in brown algae cell walls.
4.2. Acidic polysaccharides form the algal cell wall retain heavy metals
We thank Ms. Mair Machado for technical support, Ricardo Thomas for AAS measures and Dr. W. de Souza for electron microscopy facilities. This work was supported by PRONEX—CNPq, FAPERJ, JBRJ/ MMA. Paulo A.S. Mour~ao is a fellow of the John Simon Guggenheim Memorial Foundation.
Different species of brown algae have been used as biomonitor of heavy metal pollution due to their high capability of long-term uptake comparing with other seaweeds (Amado Filho et al., 1999; Engdahl et al., 1998; Karez et al., 1994; Murugadas et al., 1995). In a previous work, we have demonstrated that a brown algae collected from a heavy metal contaminated area accumulated Zn as electron-dense granules in the cell walls in median regions of thallus (Amado Filho et al., 1999). The polyanionic polysaccharides have been proposed as the molecules responsible for the binding of heavy metals when the algae are exposed to high concentrations of these elements (Amado Filho et al., 1996, 1999; Andrade et al., 2002; Ballan-Dufrancßais et al., 1991; Haug et al., 1974; Lignell et al., 1982; Pellegrini et al., 1991, 1993; Tretyn et al., 1996; Stengel and Dring, 2000).
Acknowledgments
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