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Concanavalin A binds to a mannose-containing ligand in the cell wall of some lichen phycobionts
Plant Physiology and Biochemistry 42 (2004) 773–779 www.elsevier.com/locate/plaphy
Original article
Concanavalin A binds to a mannose-containing ligand in the cell wall of some lichen phycobionts Blanca Fontaniella, Ana-María Millanes, Carlos Vicente *, María-Estrella Legaz Department of Plant Physiology, Faculty of Biology, José Antonio Novais st. s/n, Complutense University, 28040 Madrid, Spain Received 15 April 2004; accepted 9 September 2004 Available online 07 October 2004
Abstract Concanavalin A, the lectin from Canavalia ensiformis, develops arginase activity depending on Mn2+. The cation cannot be substituted by Ca2+ which, in addition, inhibits Mn2+-supported activity. Fluorescein-labeled Concanavalin A is able to bind to the cell wall of algal cells recently isolated from Evernia prunastri and Xanthoria parietina thalli. This binding involves a ligand, probably a glycoprotein containing mannose, which can be isolated by affinity chromatography. Analysis by SDS-PAGE reveals that the ligand is a dimeric protein composed by two monomers of 54 and 48 kDa. This ligand shows to be different from the receptor for natural lichen lectins, previously identified as a polygalactosylated urease. © 2004 Elsevier SAS. All rights reserved. Keywords: Cell wall; Concanavalin A; Evernia prunastri; Lectin ligand; Phycobiont; Xanthoria parietina
1. Introduction Lichens are symbiotic associations between a fungus and a cyanobacterium (cyanolichens) or a green alga (phycolichens), joined to form a new biological entity different from its individual components. Both bionts appear in nature among a mixture of millions of non-symbiotic microorganisms and they have to select each other for a compatible combination [15]. Thus, specificity seems to be required for the lichen association. According to Bubrick [7], specificity is referred to a biont which associates preferentially but not exclusively with another biont. Moreover, culture experiments performed to investigate the selectivity of the myco-
Abbreviations: ABP, algal-binding protein; ConA, Concanavalin A; FITC, fluorescein isothiocyanate; i.d., internal diameter; a-Me-Man, a-methyl-mannopyranoside; RCA, Ricinus communis agglutinin; SAX, secreted arginase from Xanthoria parietina; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Tris–Gly, tris (hydroxymethyl) aminomethane–glycine buffer; Tris–HCl, tris (hydroxymethyl) aminomethane hydrochloride. * Corresponding author. E-mail address: [email protected] (C. Vicente). 0981-9428/$ - see front matter © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.plaphy.2004.09.003
biont of Fulgensia bracteata, the dominant species in a community, towards a variety of potential photobionts provide evidence for the selectivity of the mycobiont and varying compatibility of the respective symbionts that can be interpreted as a cascade of interdependent processes of specific and non-specific reactions of the symbionts involved [36]. On the other hand, specificity imposes a continuous recognition in nature. When the algal cells multiply inside a growing thallus, daughter cells are enveloped by fungal hyphae that recognize these new cells as compatible [1]. Thus, a recognition mechanism is absolutely required, not only for de novo formation of new associations, but also for the maintenance of the symbiotic equilibrium in the lichen symbiosis. Ultrastructural studies about the relationship between lichen symbionts in the lichen thallus or those performed by using resynthesis procedures suggest that a such relationship may be due to cell surface recognition factors [2]. Several phytohemagglutinins have been isolated from a number of lichen species [3,5], but the fact that these hemagglutinins bind to the appropriate phycobiont was firstly found by Lockhart et al. [26] using Peltigera canina and P. polydactyla, two cyanolichens containing Nostoc, a cyanobacterium,
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as photobiont. Bubrick and Galun [9] found that a protein fraction isolated from the thallus of Xanthoria parietina and labeled with fluorescamine bound heavily to cultured phycobionts obtained from X. parietina, Caloplaca auriantia and C. citrina. However, this protein fraction bound neither to the same, freshly isolated phycobionts nor isolated or cultured algae from Cladonia convoluta, Ramalina duriaei and R. pollinaria, species from taxonomic families other than Teloschistaceae. Protein binding was localized at the cell wall surface of algal cells. This location was confirmed by the binding of labeled protein to algal cell wall ghosts. The algal-binding protein (ABP) was also found in Xanthoria mycobiont cultured in vitro [10]. The protein was uniformly distributed along the length of the hyphae and appeared to be restricted to the hyphal cell walls. This protein (ABP) was partially purified and characterized by Bubrick et al. [8] as a polypeptide of low molecular mass (12.5 kDa). A protein with similar binding properties was later obtained from the cyanolichen, Nephroma laevigatum [17] and purified as a dimeric protein composed by two monomers of 52 and 55 kDa of molecular mass [18]. Other lectins have been found to be produced by Peltigera membranacea [21] and by cephalodia of P. aphthosa [22], this last having a molecular mass of 20 kDa. An ABP partially purified from X. parietina thallus according to that described by Bubrick et al. [8] behaved as glycosylated arginase, which hydrolyzed arginine into ornithine and urea [30]. Fluorescein-coupled ABP (glycosylated arginase) bound to cell walls of isolated phycobionts of X. parietina, although the efficiency of this binding was strongly enhanced after culturing algal cells on 40 mM urea for 2 h. This treatment induced a glycosylated urease, which was located in the algal cell wall. E. prunastri also produces cell wall urease after the induction of this enzyme effected by urea. This particulate enzyme is almost identical to that secreted from thalli to media. It is a glycoprotein with a large polygalactose moiety bound to polypeptide [36]. Binding of glycosylated arginase to urease inhibited both enzymatic activities. On the other hand, lectins from Leguminosae family, favin, pea lectin, and Concanavalin A bind D-mannose and 2+ D-glucose residues, and also bind Ca and Mn2+ [4]. A partially purified ABP from the lichen X. parietina binds to the lectins Concanavalin A (ConA) from Canavalia ensiformis and ricin (RCA) from Ricinus communis [8]. On the other hand, Marx and Peveling [28] found that many cultured phycobionts isolated from several lichen species bind to commercial lectins, including Con A and RCA. The sugar specificity of these lectins is to a-D-glucose and a-Dmannose (ConA) and to a- or b-D-galactose (RCA). In this paper, we attempt to study the occurrence of specific receptors for ConA in two different lichen phycobionts in order to establish if they are identical or different from that defined as the ligand for natural lichen lectins, a glycourease with a polygalactoside as polysaccharide moiety, located in the cell wall of the algal cell.
2. Results 2.1. ConA binds to the cell wall of lichen phycobionts ConA was labeled with fluorescein isothiocyanate (FITC) and added to a suspension of algal cells recently isolated from E. prunastri and X. parietina thalli. Incubation was achieved for 1 h at 30 °C. Then, cells were collected by centrifugation, washed with phosphate buffer and observed in a fluorescence microscope, lighting with blue light (kmax of excitation = 468 nm). As it is shown in Fig. 1, FITC-ConA efficiently bound to the cell wall of E. prunastri (Fig. 1A) and X. parietina (Fig. 1B) phycobionts. Algal cells incubated in the same conditions with FITC showed very low fluorescence, probably due to the inefficient binding of the fluorophore to superficial proteins in the cell wall (Fig. 1C, D). As it could be expected, the presence of both Ca2+ and Mn2+ during the incubation of cells with the labeled lectin significantly increased the binding of FITC-ConA to its cell wall receptor (Fig. 2). When labeled cells were treated for 1 h at room temperature with 100 mM a-methyl-mannopyranoside and then collected by centrifugation, the fluorescence retained by the cells was recovered in the supernatant (Fig. 2). However, fluorescence was not removed from the labeled cells when they were incubated with 100 mM galactose, the sugar recognized by lichen lectins. 2.2. ConA behaves as an arginine-hydrolysing enzyme Since Xanthoria arginases have been identified as proteins used for compatible cells recognition, arginase activity of ConA and secreted arginase from X. parietina have been
Fig. 1. Binding of FITC-ConA to lichen phycobionts. A, cells of E. prunastri phycobiont after 1 h of contact with a solution of FITC (1.0 µg ml–1), observed under fluorescence microscopy in blue light; B, the same cells treated with FITC-ConA observed during irradiation with blue light. Green fluorescence indicates the attachment of FITC-ConA on the cell wall of phycobionts; C, cells of X. parietina phycobiont after 2 h of contact with a solution of FITC (1.0 µg ml–1), observed under fluorescence microscopy in blue light; D, the same cells treated with FITC-ConA observed during irradiation with blue light. Green fluorescence indicates the attachment of FITC-ConA on the cell wall of phycobionts. Bar = 5.0 µm.
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Table 1 Arginase activity of ConA and a secreted, glycosylated arginase purified from X. parietina thallus, as depending on Ca2+ and Mn2+ Cation added a None Ca2+ Mn2+ Ca2+ + Mn2+ a b
Fig. 2. Binding of FITC-ConA to isolated phycobionts from (A) E. prunastri, and (B) X. parietina. Treatments 1, total fluorescence emission from ConA solution before incubation with algal cells; 2, fluorescence emission from a supernatant obtained by removing algal cells after incubation with ConA for 1 h at 30 °C; 3, fluorescence emission from a supernatant obtained by removing algal cells after incubation with ConA, 1.67 mM Ca2+ and 1.67 mM Mn2+ for 1 h at 30 °C; 4, fluorescence obtained after desorption with 100 mM a-methyl mannopyranoside of bound ConA to algal cells; 5, total fluorescence obtained as the sum of non-adsorbed FITC-ConA (treatment 3) plus a-Me-Man-desorbed FITC-ConA (treatment 4). Values are the mean of three replicates, bars giving the standard error.
studied in the same experimental conditions. SAX was previously purified at homogeneity as described in Section 4 and commercial ConA, after dialysis, produced only one band in SDS-PAGE of about 32 kDa (Fig. 3). This result was in agreement with the specifications supplied by Sigma Chem Co. for its product. As it is shown in Table 1, commercial ConA developed low but significant arginase activity by comparing it to that developed by purified, secreted arginase
Arginase activity (nkats) ConA SAX b 0.25 ± 0.04 1.86 ± 0.2 0 0 10.34 ± 0.74 105.63 ± 10.1 5.21 ± 0.48 16.12 ± 2.0
Concentration of both cations in reaction mixtures was 1.67 mM. Purified at homogeneity according to Molina et al. [30].
from Xanthoria. This activity showed to be dependent on Mn2+. This cation could not be substituted by Ca2+ and, in addition, Ca2+ significantly inhibited Mn2+-dependent activity. However, the inhibitory action of Ca2+ on Mn2+depending hydrolase activity was several times higher for a purified, secreted arginase from X. parietina thallus. This fact revealed structural differences between ConA and SAX in the conformation of the site for Mn2+ binding and probably of the active site. 2.3. The cell wall of lichen phycobionts contains a specific ligand for ConA To confirm the occurrence of a specific ligand in algal cell wall for mannose-recognizing lectins, protein extracted from the cell wall of both Evernia and Xanthoria phycobionts were chromatographed on beads of activated agarose containing bound ConA and eluted with 100 mM a-methylmannopyranoside. As it is shown in Fig. 4, only one peak of protein eluted from the bead in the first 6.0 ml eluate with a recovery higher than 85%. When this peak, proceeding from cell walls of Evernia (Fig. 4A) or Xanthoria (Fig. 4B) phycobionts, was assayed for urease activity, the protein showed to be unable to hydrolyze urea. Other peaks of protein did not be eluted using 100 mM a-D-galactose as mobile phase. After isolation, protein eluted with 100 mM a-methyl-mannopyranoside from ConA-agarose column was analyzed by SDS-PAGE. As it is shown in Fig. 4C, both Evernia and Xanthoria ligands of ConA were resolved in two identical subunits of 54 and 48 kDa, respectively.
3. Discussion 3.1. Binding of ConA to lichen phycobionts
Fig. 3. SDS-PAGE of commercial ConA previously dialyzed against 10 mM Tris–HCl buffer (pH 9.1) that revealed only one band of about 32 kDa (lane b). Molecular markers (in kDa) in lane a.
Molina et al. [30] were able to find that a glycosylated arginase from X. parietina, almost identical to the lectin ABP described by Bubrick et al. [8], actively binds to recently isolated phycobionts of the same lichen species. Even the possibility that this ABP recognizes with low efficiency phycobionts of E. prunastri was described. In addition, a secreted, glycosylated arginase produced by X. parietina thallus binds to Xanthoria phycobionts with an affinity higher than that found for ABP [31,32]. This imply that both phyco-
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3.2. ConA develops arginase activity depending on Mn2+
Fig. 4. Separation of a specific ligand for ConA. Separation of a specific ligand for ConA from a mixture of proteins isolated from cell walls of phycobionts from (A) E. prunastri and (B) X. parietina. Separation was performed by affinity chromatography on beads of ConA bound to activated agarose. Protein was desorbed with 100 mM a-methyl mannopyranoside and monitored by absorbance at 280 nm. (C), SDS-PAGE of the ConA ligand obtained from E. prunastri (lane b) and X. parietina (lane c) phycobionts. Molecular markers (in kDa) in lane a.
bionts contains the same ligand for the lectins, later described as a polygalactosylated urease [34]. Probably, this ligand for natural lichen lectins, containing a polygalactose moiety, is functional in many other lichen photobionts since Lehr et al. [21,22] found that lectins from Peltigera aphtosa and P. membranacea bound to the cyanobiont cell wall can be desorbed with lactose. On the other hand, the ability of ConA to bind to the cell wall of several lichen photobionts has been described by Marx and Peveling [28]. This ability has been here confirmed by using phycobionts recently isolated from E. prunastri and X. parietina thalli (Fig. 1). However, the fact that the desorption of FITC-ConA was achieved with a-MeMan (Fig. 2), an a-D-mannose derivative that shows a very high efficiency to bind to the lectin [13] indicates that the cell wall of these phycobionts contain a glycosylated protein (containing mannose), other than polygalactosylated urease, that does not act as ligand for the lectins produced by lichen mycobionts in natural conditions but as receptors for other external signals or developing another enzymatic activity.
Binding of Mn2+ to arginases from different sources has been extensively studied. According to Kanyo et al. [17], rat liver arginase has a binuclear cluster that is located at the base of a 15 Å-deep active site cleft in each monomer. His101, Asp124, Asp128, Asp 232 and a solvent molecule coordinate the metal ion more deeply situated. His126, Asp124, Asp232, Asp234 and a bridging solvent molecule coordinate the second metal ion. These positions are well conserved in yeast [38] and soybean [16] arginases. However, a similar, but not identical, situation to the first domain can be seen in ConA involving His51, Asp71, Asp78 and Glu171, according to the sequence established by Min et al. [29]. Moreover, Canales et al. [11] define His141 of rat liver arginase as the point of binding of the substrate molecule, arginine. This His141 may serve as a catalytic proton shuttle. A salt link between Glu277 and the substrate guanidinium group may orient the substrate for nucleophilic attack by metal-bridging solvent. The role of these amino acid residues in ConA molecule can be assumed by His24-Glu163, with a substantial loss of catalytic activity. Studies carried out by using a-Me-Man as a ligand suggest that the sugar forms seven hydrogen bonds with the peptide of ConA, four with –NH groups of Lys99, Tyr100, Arg228 and Lys229, and three with amino acids interacting with Ca2+, Asn 14 and Asp208 [13]. The comparison between crystalline structures of ConA-containing or not Ca2+ suggests that the cation pulls from Tyr12, Asp208 and Arg228 to conform the site to bind the specific sugar [37]. It is the probable that the binding of Ca2+ to the specific domain for the cation changes the tertiary structure of the domain defined as site for the sugar binding and, for the same reason, the structure of the catalytic site for arginine. As a consequence, a loss of arginase activity can be expected after Ca2+ to ConA, as it is shown in Table 1. 3.3. The cell wall ligand for ConA is different to the receptor for lichen lectins A first attempt to identify the ConA ligand has been performed by isolating protein from the cell wall of phycobionts of both E. prunastri and X. parietina, which were chromatographed on ConA-containing agarose. Only one peak of protein was eluted from the agarose beads in the first 6.0 ml of eluate (Fig. 4), achieved with 100 mM a-Me-Man. This indicates that the ligand for ConA must contain a biantennary polysaccharide chain, as demonstrated by Kobata and Yamashita [19]. Moreover, analysis by SDS-PAGE of Evernia and Xanthoria ligands reveals that both proteins are a dimer composed by two monomers of 54 and 48 kDa, respectively (Fig. 4C). However, this ligand does not developed urease activity and, then, it must be considered as a glycoprotein different from that which acts as ligand of natural lichen lectins [31].
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4. Methods 4.1. Plant material Evernia prunastri (L.) Ach, and X. parietina (L.) Th. Fr., growing on Quercus rotundifolia Willd, were collected in La Quinta (El Pardo, Madrid), dry in air flow and stored in the dark no more than 2 weeks. 4.2. Purification of secreted arginase from X. parietina (SAX) Samples of 15 g of X. parietina thalli were floated on 150 ml 10 mM Tris–HCl buffer (pH 9.1), for 1 h at 26 °C in the dark. Secreted arginase (SAX) was purified from the incubation media according to Molina and Vicente [33]. To test the homogeneity of the protein, 150 µl of the SAX solution, containing about 6.0 µg protein, were mixed with 75 µl of aqueous glycerol (v/v) and applied onto 12% (w/v) polyacrylamide gels. The running buffer was 50 mM Tris– glycine (pH 8.3) and, at this pH, the current generated at 180 mV was about 25 mA at 4 °C after equilibration. 4.3. Assay of arginase activity Commercial ConA (from Sigma Chemical Co., St. Louis, MO) was firstly dialyzed against 10 mM Tris–HCl buffer (pH 9.1) for 24 h at 2 °C, and then tested by SDS-PAGE according to Laemmli [20] as described later. Dialyzed ConA was assayed for arginase activity according to Legaz and Vicente [23], including crystalline urease in the reaction mixtures. These contained, in a final volume of 3.0 ml, 10 µmol Tris– HCl buffer (pH 9.1), 7.5 µmol maleic acid, 5.0 µmol Mn2+ (as manganese sulfate), 0.4 µmol arginine, 8.1 mg crystalline urease, and 10.0 µg ConA [25]. When indicated, Mn2+ was substituted by Ca2+ at the same concentration or added together. Reaction was carried out at 37 °C for 30 min and was stopped by adding 0.5 ml of a saturated potassium carbonate solution. The amount of ammonia produced was measured by the Conway microdiffusion method [13]. 4.4. Isolation of lichen phycobionts Phycobionts were isolated from thalli of E. prunastri and X. parietina according to Fontaniella et al. [14]. Samples of dry thalli (0.5 g) were rinsed in distilled water to remove superficial contaminations. Samples were then macerated in a mortar with 10 ml distilled water. Homogenates were filtered through six layers of cheese-cloth and both mycoand phycobionts were separated by centrifugation in sucrose:potassium iodide and phosphate:sucrose gradients, as described. 4.5. Labeling of ConA with FITC and its binding to algal cells ConA was labeled with FITC according to Molina et al. [30]. A buffered solution of ConA-containing 10 µg
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protein·ml–1 was mixed with 100 µg FITC and maintained in the dark, at 30 °C for 2 h with continuous shaking. After this, mixture was dialyzed against 10 mM Tris–HCl buffer (pH 9.15) to remove free fluorophore. Algal cells recently isolated from E. prunastri and X. parietina thalli were incubated with fluorescent ConA (about 30 µg protein) or only with FITC (1.0 µg ml–1) in a final volume of 3.0 ml for 1 h at 30 °C in the dark. Cells were then collected by centrifugation at 12,000 × g for 15 min at 2 °C and examined under a fluorescence microscope Olympus DP50 equipped with a CCD camera and using a Viewfinder Lite program captured images. Samples of collected cells were immobilized on a slide previously covered with a layer of polylysine (50 µg ml–1 MilliQ water) for 12 h and fixed with MowiolDABCO to avoid the decay of fluorescence. Supernatants were used to measure fluorescence of the protein which was not retained by algal cells using a spectrofluorimeter Kontron SF25. Samples were excited using light of 468 nm wavelength and the fluorescence emission was measured at 512 nm. After microscopic observation, cells were incubated for 1 h at 30 °C in the dark with 3.0 ml of 100 mM a-methylD-mannose in 10 mM Tris–HCl buffer (pH 9.15). After this last incubation, cells were removed by centrifugation as described and fluorescence emission of supernatant was measured. 4.6. Extraction of protein from phycobiont cell walls Recently isolated phycobionts were mechanically disrupted and dispersed in 3.0 ml 75 mM phosphate buffer (pH 6.9). Homogenates were centrifuged at 3200 × g for 10 min at 2 °C [6]. Supernatants were discarded and pellets were resuspended in the same volume of buffer containing 0.1% Triton X-100 (w/v). Mixtures were continuously shaken at 4 °C for 2 h, and then dialyzed against 75 mM phosphate buffer (pH 6.9) to remove completely the detergent. After this, mixtures were newly centrifuged at 3200 × g for 15 min at 2 °C and pellets were resuspended in 3.0 ml of aqueous 0.5 M NaCl. Mixtures were stored at 4 °C for 12 h, including 2 min periods of 15 s each hour of sonication at 20 kcycles·s–1 with ice-cold protection. Mixtures were then centrifuged at 12,000 × g for 20 min at 2 °C and supernatants were dialyzed against 75 mM phosphate buffer (pH 6.9) for 4 h at 4 °C [24]. Dialysates were used as solutions of protein extracted from the phycobiont cell walls. 4.7. Isolation of ConA ligand by affınity chromatography Cell wall ligands were isolated from affinity chromatography using agarose activated with cyanogen bromide from Sigma Chemical Co. About 2.0 g of this agarose were fully hydrated with MilliQ water and then mixed with 20 ml of a solution of ConA-containing 2.0 mg protein·ml–1 in 0.1 M phosphate buffer (pH 7.4), for 16 h at 4 °C. After this, 0.6 g glycine were added to the mixture and stored at room temperature for 8 h [39]. Beads (4.0 cm × 1.0 cm i.d.) of activated
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agarose containing linked ConA were packed in columns and washed with 300 ml 0.1 M phosphate buffer (pH 7.4) at a flux rate of 1.0 ml min–1. Samples of 2.0 ml of soluble cell wall protein extracted from phycobionts of E. prunastri and X. parietina were loaded onto the bead and maintained in contact for 2 h at room temperature. Protein was eluted with 100 mM a-methyl-D-mannopyranoside in buffer and monitored by the method of Lowry et al. [27]. Eluted protein from agarose beads was assayed for urease activity in reaction mixtures containing 20 µg protein, 40 µmol urea and 150 µmol potassium phosphate (pH 6.9), in a final volume of 3.0 ml [36]. Incubation was carried out for 30 min at 37 °C and the ammonia produced was estimated according to Conway [12]. Activity was expressed as nkats.
[7]
[8]
[9]
[10]
[11]
[12]
4.8. SDS-PAGE of ConA ligands [13]
Peaks of protein eluted from agarose columns were analyzed by SDS-PAGE according to Laemmli [20]. Eluates were lyophilized and residues were redissolved in Laemmli buffer containing 50 µM 2-mercaptoethanol. Samples were clarified by centrifugation at 20,000 × g for 15 min at 2 °C. Supernatants were boiled for 5 min and loaded onto 15% (w/v) polyacrylamide gel and developed by SDS/PAGE. Fifty milimols Tris/0.1 M Gly containing 0.1% (w/v) SDS was used as running buffer. Protein was revealed with colloidal Coomassie blue (GelCode Blue stain reagent, Pierce, Rockford, IL, USA), the gel was dried in vacuum and scanned. Colloidal dye was selected since its sensitivity in revealing protein bands is similar to that obtained with silver stain [35], about 6.0 ng protein (2.5 ng for the silver method using bovine serum albumin). The mixture SeeBlue 2 plus, from Novex Innogenetics was used as molecular marker.
[14] [15] [16]
[17]
[18]
[19]
[20]
Acknowledgements This work was supported by a grant from the Ministerio de Ciencia y Tecnología (Spain), BFI2000-0610. We wish to thank Miss Raquel Alonso for her excellent technical assistance.
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B. Fontaniella et al. / Plant Physiology and Biochemistry 42 (2004) 773–779 [30] M.C. Molina, E. Muñiz, C. Vicente, Enzymatic activities of algalbinding protein and its algal cell wall receptor in the lichen Xanthoria parietina. An approach to the parasitic basis of mutualism, Plant Physiol. Biochem. 31 (1993) 131–142. [31] M.C. Molina, E. Stocker-Wörgötter, R. Turk, C. Bajon, C. Vicente, Secreted, glycosylated arginase from Xanthoria parietina thallus induces loss of cytoplasmic material from Xanthoria photobionts, Cell Adhes. Commun. 6 (1999) 481–490. [32] M.C. Molina, C. Vicente, Correlationships between enzymatic activity of lectins, putrescine content and chloroplast damage in Xanthoria parietina phycobionts, Cell Adhes. Commun. 3 (1995) 1–12. [33] M.C. Molina, C. Vicente, Purification and characterization of two isolectins with arginase activity from the lichen Xanthoria parietina, J. Biochem. Mol. Biol. 33 (2000) 300–307. [34] M.C. Molina, C. Vicente, M.M. Pedrosa, M.E. Legaz, M.E. Isoforms of arginase in the lichens Evernia prunastri and Xanthoria parietina: physiological roles and their implication in the controlled parasitism of the mycobiont, in: H.E.A. Schenk, R.G. Herrmann, K.W. Jeon, N.E. Müller, W. Schwemmler (Eds.), Eukaryotism and Symbiosis, Springer Verlag, Berlin, 1997, pp. 477–483.
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Original article
Concanavalin A binds to a mannose-containing ligand in the cell wall of some lichen phycobionts Blanca Fontaniella, Ana-María Millanes, Carlos Vicente *, María-Estrella Legaz Department of Plant Physiology, Faculty of Biology, José Antonio Novais st. s/n, Complutense University, 28040 Madrid, Spain Received 15 April 2004; accepted 9 September 2004 Available online 07 October 2004
Abstract Concanavalin A, the lectin from Canavalia ensiformis, develops arginase activity depending on Mn2+. The cation cannot be substituted by Ca2+ which, in addition, inhibits Mn2+-supported activity. Fluorescein-labeled Concanavalin A is able to bind to the cell wall of algal cells recently isolated from Evernia prunastri and Xanthoria parietina thalli. This binding involves a ligand, probably a glycoprotein containing mannose, which can be isolated by affinity chromatography. Analysis by SDS-PAGE reveals that the ligand is a dimeric protein composed by two monomers of 54 and 48 kDa. This ligand shows to be different from the receptor for natural lichen lectins, previously identified as a polygalactosylated urease. © 2004 Elsevier SAS. All rights reserved. Keywords: Cell wall; Concanavalin A; Evernia prunastri; Lectin ligand; Phycobiont; Xanthoria parietina
1. Introduction Lichens are symbiotic associations between a fungus and a cyanobacterium (cyanolichens) or a green alga (phycolichens), joined to form a new biological entity different from its individual components. Both bionts appear in nature among a mixture of millions of non-symbiotic microorganisms and they have to select each other for a compatible combination [15]. Thus, specificity seems to be required for the lichen association. According to Bubrick [7], specificity is referred to a biont which associates preferentially but not exclusively with another biont. Moreover, culture experiments performed to investigate the selectivity of the myco-
Abbreviations: ABP, algal-binding protein; ConA, Concanavalin A; FITC, fluorescein isothiocyanate; i.d., internal diameter; a-Me-Man, a-methyl-mannopyranoside; RCA, Ricinus communis agglutinin; SAX, secreted arginase from Xanthoria parietina; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Tris–Gly, tris (hydroxymethyl) aminomethane–glycine buffer; Tris–HCl, tris (hydroxymethyl) aminomethane hydrochloride. * Corresponding author. E-mail address: [email protected] (C. Vicente). 0981-9428/$ - see front matter © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.plaphy.2004.09.003
biont of Fulgensia bracteata, the dominant species in a community, towards a variety of potential photobionts provide evidence for the selectivity of the mycobiont and varying compatibility of the respective symbionts that can be interpreted as a cascade of interdependent processes of specific and non-specific reactions of the symbionts involved [36]. On the other hand, specificity imposes a continuous recognition in nature. When the algal cells multiply inside a growing thallus, daughter cells are enveloped by fungal hyphae that recognize these new cells as compatible [1]. Thus, a recognition mechanism is absolutely required, not only for de novo formation of new associations, but also for the maintenance of the symbiotic equilibrium in the lichen symbiosis. Ultrastructural studies about the relationship between lichen symbionts in the lichen thallus or those performed by using resynthesis procedures suggest that a such relationship may be due to cell surface recognition factors [2]. Several phytohemagglutinins have been isolated from a number of lichen species [3,5], but the fact that these hemagglutinins bind to the appropriate phycobiont was firstly found by Lockhart et al. [26] using Peltigera canina and P. polydactyla, two cyanolichens containing Nostoc, a cyanobacterium,
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as photobiont. Bubrick and Galun [9] found that a protein fraction isolated from the thallus of Xanthoria parietina and labeled with fluorescamine bound heavily to cultured phycobionts obtained from X. parietina, Caloplaca auriantia and C. citrina. However, this protein fraction bound neither to the same, freshly isolated phycobionts nor isolated or cultured algae from Cladonia convoluta, Ramalina duriaei and R. pollinaria, species from taxonomic families other than Teloschistaceae. Protein binding was localized at the cell wall surface of algal cells. This location was confirmed by the binding of labeled protein to algal cell wall ghosts. The algal-binding protein (ABP) was also found in Xanthoria mycobiont cultured in vitro [10]. The protein was uniformly distributed along the length of the hyphae and appeared to be restricted to the hyphal cell walls. This protein (ABP) was partially purified and characterized by Bubrick et al. [8] as a polypeptide of low molecular mass (12.5 kDa). A protein with similar binding properties was later obtained from the cyanolichen, Nephroma laevigatum [17] and purified as a dimeric protein composed by two monomers of 52 and 55 kDa of molecular mass [18]. Other lectins have been found to be produced by Peltigera membranacea [21] and by cephalodia of P. aphthosa [22], this last having a molecular mass of 20 kDa. An ABP partially purified from X. parietina thallus according to that described by Bubrick et al. [8] behaved as glycosylated arginase, which hydrolyzed arginine into ornithine and urea [30]. Fluorescein-coupled ABP (glycosylated arginase) bound to cell walls of isolated phycobionts of X. parietina, although the efficiency of this binding was strongly enhanced after culturing algal cells on 40 mM urea for 2 h. This treatment induced a glycosylated urease, which was located in the algal cell wall. E. prunastri also produces cell wall urease after the induction of this enzyme effected by urea. This particulate enzyme is almost identical to that secreted from thalli to media. It is a glycoprotein with a large polygalactose moiety bound to polypeptide [36]. Binding of glycosylated arginase to urease inhibited both enzymatic activities. On the other hand, lectins from Leguminosae family, favin, pea lectin, and Concanavalin A bind D-mannose and 2+ D-glucose residues, and also bind Ca and Mn2+ [4]. A partially purified ABP from the lichen X. parietina binds to the lectins Concanavalin A (ConA) from Canavalia ensiformis and ricin (RCA) from Ricinus communis [8]. On the other hand, Marx and Peveling [28] found that many cultured phycobionts isolated from several lichen species bind to commercial lectins, including Con A and RCA. The sugar specificity of these lectins is to a-D-glucose and a-Dmannose (ConA) and to a- or b-D-galactose (RCA). In this paper, we attempt to study the occurrence of specific receptors for ConA in two different lichen phycobionts in order to establish if they are identical or different from that defined as the ligand for natural lichen lectins, a glycourease with a polygalactoside as polysaccharide moiety, located in the cell wall of the algal cell.
2. Results 2.1. ConA binds to the cell wall of lichen phycobionts ConA was labeled with fluorescein isothiocyanate (FITC) and added to a suspension of algal cells recently isolated from E. prunastri and X. parietina thalli. Incubation was achieved for 1 h at 30 °C. Then, cells were collected by centrifugation, washed with phosphate buffer and observed in a fluorescence microscope, lighting with blue light (kmax of excitation = 468 nm). As it is shown in Fig. 1, FITC-ConA efficiently bound to the cell wall of E. prunastri (Fig. 1A) and X. parietina (Fig. 1B) phycobionts. Algal cells incubated in the same conditions with FITC showed very low fluorescence, probably due to the inefficient binding of the fluorophore to superficial proteins in the cell wall (Fig. 1C, D). As it could be expected, the presence of both Ca2+ and Mn2+ during the incubation of cells with the labeled lectin significantly increased the binding of FITC-ConA to its cell wall receptor (Fig. 2). When labeled cells were treated for 1 h at room temperature with 100 mM a-methyl-mannopyranoside and then collected by centrifugation, the fluorescence retained by the cells was recovered in the supernatant (Fig. 2). However, fluorescence was not removed from the labeled cells when they were incubated with 100 mM galactose, the sugar recognized by lichen lectins. 2.2. ConA behaves as an arginine-hydrolysing enzyme Since Xanthoria arginases have been identified as proteins used for compatible cells recognition, arginase activity of ConA and secreted arginase from X. parietina have been
Fig. 1. Binding of FITC-ConA to lichen phycobionts. A, cells of E. prunastri phycobiont after 1 h of contact with a solution of FITC (1.0 µg ml–1), observed under fluorescence microscopy in blue light; B, the same cells treated with FITC-ConA observed during irradiation with blue light. Green fluorescence indicates the attachment of FITC-ConA on the cell wall of phycobionts; C, cells of X. parietina phycobiont after 2 h of contact with a solution of FITC (1.0 µg ml–1), observed under fluorescence microscopy in blue light; D, the same cells treated with FITC-ConA observed during irradiation with blue light. Green fluorescence indicates the attachment of FITC-ConA on the cell wall of phycobionts. Bar = 5.0 µm.
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Table 1 Arginase activity of ConA and a secreted, glycosylated arginase purified from X. parietina thallus, as depending on Ca2+ and Mn2+ Cation added a None Ca2+ Mn2+ Ca2+ + Mn2+ a b
Fig. 2. Binding of FITC-ConA to isolated phycobionts from (A) E. prunastri, and (B) X. parietina. Treatments 1, total fluorescence emission from ConA solution before incubation with algal cells; 2, fluorescence emission from a supernatant obtained by removing algal cells after incubation with ConA for 1 h at 30 °C; 3, fluorescence emission from a supernatant obtained by removing algal cells after incubation with ConA, 1.67 mM Ca2+ and 1.67 mM Mn2+ for 1 h at 30 °C; 4, fluorescence obtained after desorption with 100 mM a-methyl mannopyranoside of bound ConA to algal cells; 5, total fluorescence obtained as the sum of non-adsorbed FITC-ConA (treatment 3) plus a-Me-Man-desorbed FITC-ConA (treatment 4). Values are the mean of three replicates, bars giving the standard error.
studied in the same experimental conditions. SAX was previously purified at homogeneity as described in Section 4 and commercial ConA, after dialysis, produced only one band in SDS-PAGE of about 32 kDa (Fig. 3). This result was in agreement with the specifications supplied by Sigma Chem Co. for its product. As it is shown in Table 1, commercial ConA developed low but significant arginase activity by comparing it to that developed by purified, secreted arginase
Arginase activity (nkats) ConA SAX b 0.25 ± 0.04 1.86 ± 0.2 0 0 10.34 ± 0.74 105.63 ± 10.1 5.21 ± 0.48 16.12 ± 2.0
Concentration of both cations in reaction mixtures was 1.67 mM. Purified at homogeneity according to Molina et al. [30].
from Xanthoria. This activity showed to be dependent on Mn2+. This cation could not be substituted by Ca2+ and, in addition, Ca2+ significantly inhibited Mn2+-dependent activity. However, the inhibitory action of Ca2+ on Mn2+depending hydrolase activity was several times higher for a purified, secreted arginase from X. parietina thallus. This fact revealed structural differences between ConA and SAX in the conformation of the site for Mn2+ binding and probably of the active site. 2.3. The cell wall of lichen phycobionts contains a specific ligand for ConA To confirm the occurrence of a specific ligand in algal cell wall for mannose-recognizing lectins, protein extracted from the cell wall of both Evernia and Xanthoria phycobionts were chromatographed on beads of activated agarose containing bound ConA and eluted with 100 mM a-methylmannopyranoside. As it is shown in Fig. 4, only one peak of protein eluted from the bead in the first 6.0 ml eluate with a recovery higher than 85%. When this peak, proceeding from cell walls of Evernia (Fig. 4A) or Xanthoria (Fig. 4B) phycobionts, was assayed for urease activity, the protein showed to be unable to hydrolyze urea. Other peaks of protein did not be eluted using 100 mM a-D-galactose as mobile phase. After isolation, protein eluted with 100 mM a-methyl-mannopyranoside from ConA-agarose column was analyzed by SDS-PAGE. As it is shown in Fig. 4C, both Evernia and Xanthoria ligands of ConA were resolved in two identical subunits of 54 and 48 kDa, respectively.
3. Discussion 3.1. Binding of ConA to lichen phycobionts
Fig. 3. SDS-PAGE of commercial ConA previously dialyzed against 10 mM Tris–HCl buffer (pH 9.1) that revealed only one band of about 32 kDa (lane b). Molecular markers (in kDa) in lane a.
Molina et al. [30] were able to find that a glycosylated arginase from X. parietina, almost identical to the lectin ABP described by Bubrick et al. [8], actively binds to recently isolated phycobionts of the same lichen species. Even the possibility that this ABP recognizes with low efficiency phycobionts of E. prunastri was described. In addition, a secreted, glycosylated arginase produced by X. parietina thallus binds to Xanthoria phycobionts with an affinity higher than that found for ABP [31,32]. This imply that both phyco-
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3.2. ConA develops arginase activity depending on Mn2+
Fig. 4. Separation of a specific ligand for ConA. Separation of a specific ligand for ConA from a mixture of proteins isolated from cell walls of phycobionts from (A) E. prunastri and (B) X. parietina. Separation was performed by affinity chromatography on beads of ConA bound to activated agarose. Protein was desorbed with 100 mM a-methyl mannopyranoside and monitored by absorbance at 280 nm. (C), SDS-PAGE of the ConA ligand obtained from E. prunastri (lane b) and X. parietina (lane c) phycobionts. Molecular markers (in kDa) in lane a.
bionts contains the same ligand for the lectins, later described as a polygalactosylated urease [34]. Probably, this ligand for natural lichen lectins, containing a polygalactose moiety, is functional in many other lichen photobionts since Lehr et al. [21,22] found that lectins from Peltigera aphtosa and P. membranacea bound to the cyanobiont cell wall can be desorbed with lactose. On the other hand, the ability of ConA to bind to the cell wall of several lichen photobionts has been described by Marx and Peveling [28]. This ability has been here confirmed by using phycobionts recently isolated from E. prunastri and X. parietina thalli (Fig. 1). However, the fact that the desorption of FITC-ConA was achieved with a-MeMan (Fig. 2), an a-D-mannose derivative that shows a very high efficiency to bind to the lectin [13] indicates that the cell wall of these phycobionts contain a glycosylated protein (containing mannose), other than polygalactosylated urease, that does not act as ligand for the lectins produced by lichen mycobionts in natural conditions but as receptors for other external signals or developing another enzymatic activity.
Binding of Mn2+ to arginases from different sources has been extensively studied. According to Kanyo et al. [17], rat liver arginase has a binuclear cluster that is located at the base of a 15 Å-deep active site cleft in each monomer. His101, Asp124, Asp128, Asp 232 and a solvent molecule coordinate the metal ion more deeply situated. His126, Asp124, Asp232, Asp234 and a bridging solvent molecule coordinate the second metal ion. These positions are well conserved in yeast [38] and soybean [16] arginases. However, a similar, but not identical, situation to the first domain can be seen in ConA involving His51, Asp71, Asp78 and Glu171, according to the sequence established by Min et al. [29]. Moreover, Canales et al. [11] define His141 of rat liver arginase as the point of binding of the substrate molecule, arginine. This His141 may serve as a catalytic proton shuttle. A salt link between Glu277 and the substrate guanidinium group may orient the substrate for nucleophilic attack by metal-bridging solvent. The role of these amino acid residues in ConA molecule can be assumed by His24-Glu163, with a substantial loss of catalytic activity. Studies carried out by using a-Me-Man as a ligand suggest that the sugar forms seven hydrogen bonds with the peptide of ConA, four with –NH groups of Lys99, Tyr100, Arg228 and Lys229, and three with amino acids interacting with Ca2+, Asn 14 and Asp208 [13]. The comparison between crystalline structures of ConA-containing or not Ca2+ suggests that the cation pulls from Tyr12, Asp208 and Arg228 to conform the site to bind the specific sugar [37]. It is the probable that the binding of Ca2+ to the specific domain for the cation changes the tertiary structure of the domain defined as site for the sugar binding and, for the same reason, the structure of the catalytic site for arginine. As a consequence, a loss of arginase activity can be expected after Ca2+ to ConA, as it is shown in Table 1. 3.3. The cell wall ligand for ConA is different to the receptor for lichen lectins A first attempt to identify the ConA ligand has been performed by isolating protein from the cell wall of phycobionts of both E. prunastri and X. parietina, which were chromatographed on ConA-containing agarose. Only one peak of protein was eluted from the agarose beads in the first 6.0 ml of eluate (Fig. 4), achieved with 100 mM a-Me-Man. This indicates that the ligand for ConA must contain a biantennary polysaccharide chain, as demonstrated by Kobata and Yamashita [19]. Moreover, analysis by SDS-PAGE of Evernia and Xanthoria ligands reveals that both proteins are a dimer composed by two monomers of 54 and 48 kDa, respectively (Fig. 4C). However, this ligand does not developed urease activity and, then, it must be considered as a glycoprotein different from that which acts as ligand of natural lichen lectins [31].
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4. Methods 4.1. Plant material Evernia prunastri (L.) Ach, and X. parietina (L.) Th. Fr., growing on Quercus rotundifolia Willd, were collected in La Quinta (El Pardo, Madrid), dry in air flow and stored in the dark no more than 2 weeks. 4.2. Purification of secreted arginase from X. parietina (SAX) Samples of 15 g of X. parietina thalli were floated on 150 ml 10 mM Tris–HCl buffer (pH 9.1), for 1 h at 26 °C in the dark. Secreted arginase (SAX) was purified from the incubation media according to Molina and Vicente [33]. To test the homogeneity of the protein, 150 µl of the SAX solution, containing about 6.0 µg protein, were mixed with 75 µl of aqueous glycerol (v/v) and applied onto 12% (w/v) polyacrylamide gels. The running buffer was 50 mM Tris– glycine (pH 8.3) and, at this pH, the current generated at 180 mV was about 25 mA at 4 °C after equilibration. 4.3. Assay of arginase activity Commercial ConA (from Sigma Chemical Co., St. Louis, MO) was firstly dialyzed against 10 mM Tris–HCl buffer (pH 9.1) for 24 h at 2 °C, and then tested by SDS-PAGE according to Laemmli [20] as described later. Dialyzed ConA was assayed for arginase activity according to Legaz and Vicente [23], including crystalline urease in the reaction mixtures. These contained, in a final volume of 3.0 ml, 10 µmol Tris– HCl buffer (pH 9.1), 7.5 µmol maleic acid, 5.0 µmol Mn2+ (as manganese sulfate), 0.4 µmol arginine, 8.1 mg crystalline urease, and 10.0 µg ConA [25]. When indicated, Mn2+ was substituted by Ca2+ at the same concentration or added together. Reaction was carried out at 37 °C for 30 min and was stopped by adding 0.5 ml of a saturated potassium carbonate solution. The amount of ammonia produced was measured by the Conway microdiffusion method [13]. 4.4. Isolation of lichen phycobionts Phycobionts were isolated from thalli of E. prunastri and X. parietina according to Fontaniella et al. [14]. Samples of dry thalli (0.5 g) were rinsed in distilled water to remove superficial contaminations. Samples were then macerated in a mortar with 10 ml distilled water. Homogenates were filtered through six layers of cheese-cloth and both mycoand phycobionts were separated by centrifugation in sucrose:potassium iodide and phosphate:sucrose gradients, as described. 4.5. Labeling of ConA with FITC and its binding to algal cells ConA was labeled with FITC according to Molina et al. [30]. A buffered solution of ConA-containing 10 µg
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protein·ml–1 was mixed with 100 µg FITC and maintained in the dark, at 30 °C for 2 h with continuous shaking. After this, mixture was dialyzed against 10 mM Tris–HCl buffer (pH 9.15) to remove free fluorophore. Algal cells recently isolated from E. prunastri and X. parietina thalli were incubated with fluorescent ConA (about 30 µg protein) or only with FITC (1.0 µg ml–1) in a final volume of 3.0 ml for 1 h at 30 °C in the dark. Cells were then collected by centrifugation at 12,000 × g for 15 min at 2 °C and examined under a fluorescence microscope Olympus DP50 equipped with a CCD camera and using a Viewfinder Lite program captured images. Samples of collected cells were immobilized on a slide previously covered with a layer of polylysine (50 µg ml–1 MilliQ water) for 12 h and fixed with MowiolDABCO to avoid the decay of fluorescence. Supernatants were used to measure fluorescence of the protein which was not retained by algal cells using a spectrofluorimeter Kontron SF25. Samples were excited using light of 468 nm wavelength and the fluorescence emission was measured at 512 nm. After microscopic observation, cells were incubated for 1 h at 30 °C in the dark with 3.0 ml of 100 mM a-methylD-mannose in 10 mM Tris–HCl buffer (pH 9.15). After this last incubation, cells were removed by centrifugation as described and fluorescence emission of supernatant was measured. 4.6. Extraction of protein from phycobiont cell walls Recently isolated phycobionts were mechanically disrupted and dispersed in 3.0 ml 75 mM phosphate buffer (pH 6.9). Homogenates were centrifuged at 3200 × g for 10 min at 2 °C [6]. Supernatants were discarded and pellets were resuspended in the same volume of buffer containing 0.1% Triton X-100 (w/v). Mixtures were continuously shaken at 4 °C for 2 h, and then dialyzed against 75 mM phosphate buffer (pH 6.9) to remove completely the detergent. After this, mixtures were newly centrifuged at 3200 × g for 15 min at 2 °C and pellets were resuspended in 3.0 ml of aqueous 0.5 M NaCl. Mixtures were stored at 4 °C for 12 h, including 2 min periods of 15 s each hour of sonication at 20 kcycles·s–1 with ice-cold protection. Mixtures were then centrifuged at 12,000 × g for 20 min at 2 °C and supernatants were dialyzed against 75 mM phosphate buffer (pH 6.9) for 4 h at 4 °C [24]. Dialysates were used as solutions of protein extracted from the phycobiont cell walls. 4.7. Isolation of ConA ligand by affınity chromatography Cell wall ligands were isolated from affinity chromatography using agarose activated with cyanogen bromide from Sigma Chemical Co. About 2.0 g of this agarose were fully hydrated with MilliQ water and then mixed with 20 ml of a solution of ConA-containing 2.0 mg protein·ml–1 in 0.1 M phosphate buffer (pH 7.4), for 16 h at 4 °C. After this, 0.6 g glycine were added to the mixture and stored at room temperature for 8 h [39]. Beads (4.0 cm × 1.0 cm i.d.) of activated
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agarose containing linked ConA were packed in columns and washed with 300 ml 0.1 M phosphate buffer (pH 7.4) at a flux rate of 1.0 ml min–1. Samples of 2.0 ml of soluble cell wall protein extracted from phycobionts of E. prunastri and X. parietina were loaded onto the bead and maintained in contact for 2 h at room temperature. Protein was eluted with 100 mM a-methyl-D-mannopyranoside in buffer and monitored by the method of Lowry et al. [27]. Eluted protein from agarose beads was assayed for urease activity in reaction mixtures containing 20 µg protein, 40 µmol urea and 150 µmol potassium phosphate (pH 6.9), in a final volume of 3.0 ml [36]. Incubation was carried out for 30 min at 37 °C and the ammonia produced was estimated according to Conway [12]. Activity was expressed as nkats.
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[9]
[10]
[11]
[12]
4.8. SDS-PAGE of ConA ligands [13]
Peaks of protein eluted from agarose columns were analyzed by SDS-PAGE according to Laemmli [20]. Eluates were lyophilized and residues were redissolved in Laemmli buffer containing 50 µM 2-mercaptoethanol. Samples were clarified by centrifugation at 20,000 × g for 15 min at 2 °C. Supernatants were boiled for 5 min and loaded onto 15% (w/v) polyacrylamide gel and developed by SDS/PAGE. Fifty milimols Tris/0.1 M Gly containing 0.1% (w/v) SDS was used as running buffer. Protein was revealed with colloidal Coomassie blue (GelCode Blue stain reagent, Pierce, Rockford, IL, USA), the gel was dried in vacuum and scanned. Colloidal dye was selected since its sensitivity in revealing protein bands is similar to that obtained with silver stain [35], about 6.0 ng protein (2.5 ng for the silver method using bovine serum albumin). The mixture SeeBlue 2 plus, from Novex Innogenetics was used as molecular marker.
[14] [15] [16]
[17]
[18]
[19]
[20]
Acknowledgements This work was supported by a grant from the Ministerio de Ciencia y Tecnología (Spain), BFI2000-0610. We wish to thank Miss Raquel Alonso for her excellent technical assistance.
[21]
[22]
[23] [24]
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