Effect of sugars on the association between cowpea vicilin ( 7 S storage proteins ) and fungal cells

Vicilins (7S storage proteins) found in various legume seeds have been previously shown to interfere with the germination of spores or conidia of phytopathogenic fungi and inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells. In the present work vicilins from cowpea (Vigna unguiculata) seeds were added to the growth medium of Saccharomyces cerevisiae cells and Fusarium oxysporum conidia. Helix pomatia lectin, wheat germ agglutinin and Ulex europaeus lectin were used to identify differences in the binding of the vicilins to the surface of cells of S. cerevisiae and F. oxysporum treated with this protein. After the growth period, the material in suspension (yeast cells) was centrifuged and the final pellet was also treated with different sugar (glucose, sucrose, glucosamine, N-acetyl-glucosamine) concentrations and 0.1 M HCl for extraction of vicilins associated to chitinous structures present in yeast cells. Our results showed that vicilin sub-units were present in the different sugar extracts of yeast cells pretreated with the vicilins and these proteins were eluted by 0.5 M solutions of sugars in the following order of efficiency of elution: N-acetyl-glucosamine, sucrose/glucose and glucosamine. BIOCELL 2003, 27(2): 173-179 ISSN 0327 9545 PRINTED IN ARGENTINA


Introduction
Vicilins are seed storage proteins of the 7S globulin class, which are present in seeds of leguminous and other plants.These proteins are characterized as oligomers of 150-170 kDa formed by three similar subunits of about 40-70 kDa with no disulfide linkages.All vicilins purified from legume seeds are highly heterogeneous and consist of many different subunits.This heterogeneity is due to the expression of multigene families whose individual genes are very closely related and also to post-transcriptional processing and glycosylation (Derbyshire et al., 1976;Casey et al., 1986).
We have recently shown that vicilins from cowpea (Vigna unguiculata) and other legume seeds strongly associate with chitin, chitosan and fully acetylated chitin (Sales et al., 1996).Recent results have also shown that vicilins interfere with the germination of spores or conidia of phytopathogenic fungi and inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells (Gomes et al., 1997;Gomes et al., 1998a).Chung et al. (1997) also showed that vicilinrelated basic proteins isolated from cotton seeds inhibit the growth of various filamentous fungi.The same way others different types of antimicrobial proteins have been also purified from plants seeds (Wang and Bunkers, 2000).
A mechanism by which vicilins exert their effects on fungi growth was recently proposed by us.This mechanism is based on the ability of vicilins to bind to the cell walls and the plasma membranes of fungi (Gomes et al., 1998b).A similar mechanism of action is attributed to proteins containing the so called chitin binding domains (specific chitinases, hevein and heveinlike proteins and peptides from Amaranthus caudatus seeds) which are involved in defense mechanisms of plants against pathogens (Raikhel et al., 1993;Broekaert et al., 1992).The aim of the present work was to investigate the binding of vicilins to fungal cells and whether this binding could be reversed by different sugars like glucose, sucrose and N-acetyl-D-glucosamine.

Seeds
Cowpea (Vigna unguiculata) seeds of the cultivar pitiúba were supplied by the Centro de Ciências Agrárias, Universidade Federal do Ceará, Fortaleza, Brasil where they were developed.Seeds of the cowpea line IT81D-1045, which are resistant to the bruchid beetle Callosobruchus maculatus, were obtained from IITA, Ibadan, Nigeria through the Centro Nacional de Pesquisa Arroz-Feijão (EMBRAPA), Goiânia, Goiás, Brasil.This line was bred at IITA from the bruchid-resistant cultivar TVu 2027.

Preparation of vicilins
Vicilin type proteins from V. unguiculata were prepared following the protocol developed by Macedo et al. (1995) with some modifications.We extracted the finely ground seed meal (1:10 meal to buffer ratio) with 100 mM Tris-HCl buffer, pH 8.0, for 30 min at room temperature.After centrifugation for 30 min at 8,000 x g and 4 o C, the proteins in the supernatant were fractionated by ammonium sulphate precipitation and the 70 to 90% saturation fraction was dialyzed against water and freeze dried.The vicilins from cowpea seeds were further purified by chromatography on a Sephacryl S-200 column (3 x 40 cm), equilibrated and eluted with the same buffer used for extraction.The vicilin-rich fractions were recovered and submitted to an ion-exchange chromatography step on a DEAE-Sepharose column (2 x 12 cm) equilibrated with 50 mM Tris-HCl, pH 8.0, and eluted with a NaCl gradient (0 to 1 M) in the same buffer.Vicilin-rich fractions were recovered and submitted to chromatography on a Sephacryl S-400 column (2.5 x 60 cm) in 100 mM Tris-HCl, 0.25 M NaCl, pH 8.0.Vicilins were recovered by dialysis and freeze drying.The degree of purification of the vicilins obtained by the above procedure was ascertained during the purification process itself since only the most quantitatively significant and symmetrical protein peaks were collected.Purified vicilins showed similar patterns to those obtained previously by us (Macedo et al., 1995) and by others (Derbyshire et al., 1976;Sammour et al., 1984).

Fungi
Fungal isolates utilised were: Fusarium oxysporum, kindly supplied by CNPAF/EMBRAPA, Goiania, Goiás, Brazil, and Saccharomyces cerevisiae (1038) obtained from Departamento de Biologia, Universidade Federal do Ceará, Fortaleza, Brazil.The fungi were maintained on Agar Sabouraud.For the preparation of yeast cell cultures, inoculum was transferred to Petri dishes containing Agar Sabouraud and allowed to grow at 28 o C for 2 days; after this period cells were transferred to sterile 0.15 M saline solution (10 ml).Yeast cells were quantified in a Neubauer chamber for further calculation of appropriate dilutions.For the preparation of conidia of F. oxysporum, fungal cultures were transferred to Petri dishes containing Agar Sabouraud for 12 days (Leah et al., 1991); after this period, sterile distilled water (10 ml) was added to the dishes and these were gently agitated for 1 min for spore liberation with the help of a Drigalski spatula.Spores were quantified in a Neubauer chamber for appropriate dilutions.

Incubation of fungi in the presence of vicilins
For the growth of fungi in the presence of vicilins, yeast cells and fungal conidia (2 x 10 4 ml -1 in Sabouraud broth) were incubated in 10 ml Kitasato flasks followed by the addition of cowpea (pitiúba and IT81D-1045) vicilins (0.8 mg/ml).The flasks were then maintained at room temperature (25 o C).The fungi were grown for the following 72 h.A general control without addition of protein was also utilized.
Lectin-binding assay S. cerevisiae cells and F. oxysporum conidia, grown for 72 h in Sabouraud broth in the presence of vicilins, were washed with 0.1 M phosphate, pH 7.3 and submitted to fixation for 2 h at room temperature in a solution containing 0.1% glutaraldehyde, 2.0% formaldehyde in the same buffer.The materials were rinsed three times with 0.1 M phosphate buffer, pH 7.3, dehydrated in solutions of increasing concentrations of methanol (30% -90%) and processed for LR Gold embedding.For labelling binding assay of the Helix pomatia, wheat germ (WGA) and Ulex europaeus (UEA I) lectins (Sigma Chemical Co.), ultrathin sections were treated following the protocol described by Benhemau and Quellete (1986) with some modifications.PBS/BSA (10 mM phosphate and 0.15 M NaCl pH 7.5, containing 1% bovine serum albumin) was used for all rinsing steps and for dilution of the reagents.Sections were labelled by immersing grids in drops (30 µl) of solutions in the following sequence: (1) 50 mM ammonium chloride in PBS, 30 min; (2) PBS/BSA, 30 min; (3) lectin solution (1:10) in PBS/BSA, over night, 4°C; (4) five changes of PBS/BSA, 10 min each; (5) five changes of buffer, 10 min each; (6) five changes of deionized water, 10 min each.Sections were then air-dried onto a Formvar film, stained with uranyl acetate and lead citrate and observed in a ZEISS 900 transmission electron microscope.Control sections were made by labelling of yeast cells grown for 72 h in Sabouraud broth not treated with vicilins.

Immunogold labelling of vicilins in fungi structures
For immunocytochemical analysis, fungi were first submitted to fixation for 2 h at room temperature in a solution containing 0.1% glutaraldehyde, 2.0% formaldehyde in 0.1 M phosphate buffer, pH 7.3, rinsed three times with 0.1 M phosphate buffer, pH 7.3, dehydrated in solutions of increasing concentrations of methanol (30% -90%) and processed for LR Gold embedding.For immunogold labelling, ultrathin sections were treated following the protocol described by Gomes et al. (1998b).

Extraction of vicilins associated to yeast cells by different sugars
After a 72 h growth period, the yeast cells in suspension and corresponding to each treatment with vicilins were centrifuged at 3,000 x g in an Eppendorf microcentrifuge for 2 min.The supernatant was discarded and the pellet was exhaustively washed with 0.1 M Tris-HCl, pH 8.0, until no more protein could be measured by the Bradford (1976) method.Each pellet was treated with 1 ml of different sugars (N-acetyl-Dglucosamine, glucose, sucrose and glucosamine) at different concentrations (0.05, 0.1, 0.2, 0.4, 0.5 M).The final precipitates were then treated with 1 ml of 0.1 N HCl.All resulting supernatants were concentrated in a speed vac apparatus.The dried samples were redissolved in 0.1 ml of 0.1M Tris-HCl pH 8.0 and utilized for SDS-PAGE and western blotting.

Quantitative analysis of proteins associated to yeast cells and extracted by different sugars
The quantitative analysis of the proteins extracted by different sugars was carried out by the method of Bradford employing bovine serum albumin as standard (Bradford, 1976).After extraction of the pellets with 1 ml of the different sugars (N-acetyl-D-glucosamine, glucose, sucrose and glucosamine) in different concentrations (0.1, 0.5 M) and 0.1 N HCl, the resulting supernatants were collected and utilized for the Bradford assay.The reaction was performed by incubating 0.04 ml of supernatant with 1 ml of the Bradford reagent and the absorbance was measured at 595 nm.

Gel electrophoresis and immunoblotting
Sodium dodecyl sulfate electrophoresis (SDS-PAGE) was carried out according to the denaturing method of Laemmli (1970).Protein markers employed were bovine serum albumin (66 kDa), ovalbumin (45 kDa), glyceraldehyde-3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa) and lysozyme (14 kDa).The transfer of proteins to nitrocellulose membranes was carried out as described by Towbin et al. (1979).Blots were developed using a chemiluminescence detection Kit (Amersham ECL reagent) accord-ing to the manufacturer's instructions.Antisera against purified vicilins (from cowpea seeds) were prepared by immunization of white rabbits (Macedo et al., 1995).Purified antisera were obtained by affinity chromatography of the crude immune sera in a column of protein A covalently bound to Sepharose CL-4B.

Results and Discussion
Several carbohydrate-binding proteins with antifungal activity have been isolated from many plant species, especially from their seeds (Leah et al., 1991;Chrispeels and Raikhel, 1991).The present study was initiated in order to show whether vicilins (7S storage proteins), with chitin-binding properties, isolated from cowpea seeds can bind to different sugars like glucose, sucrose, N-acetylglucosamine, glucosamine and triacetyl-chitotriose.The association of proteins of the chitin-binding family is not restricted to chitin, since they also bind to various complex glycoconjugates containing N-acetylglucosamine (GlcNAc) or N-acetyl-Dneuraminic acid (NeuNAc) (Raikhel et al., 1993).Vicilins probably show affinity towards other glyconjugates as well.In order to reinforce this idea we utilized lectins (Helix pomatia lectin, wheat germ agglutinin and Ulex europaeus lectin) with different sugar specificities to identify N-acetylgalactosamine, Nacetylglucosamine and L-fucose sugar residues on the surface of S. cerevisiae cells and F. oxysporum conidia after treatment with vicilins.Lectins have prooved to be useful tools for identifying and locating glyconjugate components of microbiall cells (Recobet et al., 1996).Through immunogold labelling performed on sections of fungi (F.oxysporum and S. cerevisiae) structures, we have previously suggested the association of vicilins with fungi cell walls and/or plasma membranes (Gomes et al., 1998b).Figure 1 shows the results of lectin binding assays with cells of S. cerevisiae and F. oxysporum conidia after localization of glycoconjugated reaction.N-acetylgalactosamine and N-acetylglucosamine binding lectins are able to bind to cell wall and/or cell plasma membrane of F. oxysporum and no qualitative differences between the samples (treated or not treated with vicilins) were seen (Fig. 1A, 1C and 1E).These results suggest that not only the chitin monomer N-acetyl glucosamine is involved in the binding of vicilins to the cell wall of fungi.Glycoconjugates containing Nacetylgalactosamine residues may also be involved in the binding.However only N-acetylglucosamine binding lectin is able to bind to cell wall and/or plasma mem-brane of S. cerevisiae (Fig. 1B, 1D and 1F).Control fungi sections treated with Helix pomatia lectin without previous treatment with vicilins (data not shown) showed identical labbeling to fungi sections with previous treatment with vicilins (Fig. 1E and 1F).Cell walls of F. oxysporum conidia submitted to vicilin treatments were labelled by anti-vicilin serum as show in figure 1G.Apparently bound vicilins do not interfere with lectins association; furthermore, no binding occurs with the L-fucose binding lectin in both fungi (Data not shown).After a 72 h growth period, control cells or cells grown in the presence of cowpea vicilins from pitiúba and IT81D-1045 seeds were washed with buffer (Tris-HCl 0.1 M, pH 8.0) and also submitted to a final washing with different sugars and subsequently by 0.1 N HCl.Proteins extracted by all treatments were analyzed by western blotting using an antiserum raised against cowpea vicilins.All vicilins showed the same pattern of sequential sub-unit elution by 0.5 M sugars (glucose, Nacetylglucosamine, glucosamine and sucrose) and 0.1 M HCl indicating that some vicilin subunits are extracted by all sugars and others are more strongly bound to yeast cells since they are extracted only after subsequent acidic treatment as was already shown by Gomes et al. (1998a) (Fig. 2).Western blotting of vicilins eluted by increasing  sugar concentrations (Fig. 3) shows the same banding pattern of vicilins eluted by 0.5 M sugar solutions shown in figure 2. Glucose (Fig. 3A) and N-acetylglucosamine (Fig. 3B) as well as sucrose and glucosamine (data not shown) are able to extract vicilins in concentrations as low as 0.1 M. Based on previous findings that vicilins associate with chitin and chitin containing structures (Firmino et al., 1996;Yunes et al., 1998), we conclude that all sugars tested (N-acetylglucosamine, glucose, sucrose and glucosamine) are able to extract vicilin subunits when employed at appropriate concentrations.
In order to compare the ability of different sugars in extracting vicilins from yeast cells we measured the protein content of the resulting supernatants (see Methods).As seen in figure 4A, vicilins from pitiúba seeds were better eluted by 0.5 M solutions of sugars in the following elution efficiency order: N-acetylglucosamine, sucrose/glucose and glucosamine.In the case of vicilins from IT81D-1045 (Fig. 4B) a more subtle difference among sugars was observed even though the same order of elution efficiency was noted.However, a distinct behavior was shown when comparing the two cowpea vicilins.Similar measurements of extracted proteins were also made by employing 0.1 M sugar solutions with no detectable differences in elution efficiencies (data not shown).
We are systematically investigating the association of vicilins with chitin components of fungi (Gomes et al., 1997), chitin derivatives (Yunes et al., 1998) and chitinous structures of the midgut of insects (Firmino et al., 1996).The findings reported in this paper and other results on the association of vicilins with chitin structures suggest that vicilins associate with organisms containing chitin or various others complex glycoconjugates, and that different sugars are able to reverse this association.

FIGURE 1 .
FIGURE 1.Localization of glycoconjugates using lectin-gold complexes in cells of F. oxysporum conidia (A, C and E) and S. cerevisiae (B, D and F) after lectin binding assays (gold particles.10 nm).A and B -control fungi sections treated with WGA lectin without previous treatment with vicilins.C and D -Fungi sections treated with WGA lectin with previous treatment with vicilins.E and F-Fungi sections treated with Helix pomatia lectin with previous treatment with vicilins.G, Cell walls of F. oxysporum conidia submitted to vicilin treatments labelled by anti-vicilin serum.Cell wall (stars); cell plasma membrane (arrows); Bar represents 0.3 µm for all figures.

FIGURE 4 .
FIGURE 4. Vicilins extracted from yeast structures with different sugars (0.5 M). (A) Pitiúba vicilin, (B) IT81D-1045 vicilin.Experiments were run in triplicate and the standard errors (coefficient of variation not more than 20%) are omitted for clarity.