2 Department of Biochemistry, Karnatak University, Dharwad 580 003, India, and 3 Department of Plant Pathology, University of Agricultural Sciences, Dharwad 580 005, India
Received on February 22, 2004; revised on July 9, 2004; accepted on July 12, 2004
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Abstract |
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Key words: fungal lectins / glycosphingolipids / glycosylinositolphosphoceramides / lectin receptor / Sclerotium rolfsii lectin
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Introduction |
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Contrary to established roles of bacterial lectins in hostparasite interactions, functional roles assigned for fungal lectins are speculative. Although many believe that fungal lectins do mediate hostparasite interactions (Rudiger, 1998) similar to bacterial adhesins, several other roles are also put forth. Often physiological roles for fungal lectins were attributed based on the location and selection of the source for isolation (Barak and Chet, 1990
; Elad et al., 1983
; Inbar and Chet, 1994
; Kellens and Peumans, 1990
). Some of the roles assigned to fungal lectins are storage proteins (Kellens and Peumans, 1990
), fungalfungal interactions (mycoparasitism), and hostparasite interactions (Fukazawa and Kagaya, 1997
; Hostetter, 1994
; Rudiger, 1998
). Another argued function gaining greater attention is their involvement in morphogenesis and development of the fungi (Cooper et al., 1997
; Yatohgo et al., 1988
). However, none of these assigned roles are established.
Similar uncertainty also exists for the functional roles of membrane-bound fungal sphingolipids referred to as phytosphingosines. Inositolphosphorylceramides (IPCs), a family of membrane lipids with characteristic glycosyl moieties, occur exclusively in fungi (Dickson, 1998). These molecules, once believed to be simply structural components of membranes, are now implicated for their diversified roles as second messengers that activate intracellular signal transduction pathways leading to regulation of cell cycle and growth (Dickson, 1998
). Our findings with Sclerotium rolfsii lectin (SRL) and its putative endogenous receptor suggest that the cell wallassociated lectins and the sphingolipids in fungi with obscure functions collude to carryout important biological function in the development of the fungus.
Earlier we reported the purification and the fine sugar specificity of a Thomsen-Friedenreich antigenspecific lectin from S. rolfsii (Swamy et al., 2001; Wu et al., 2001
); its preliminary X-ray crystallographic data has been reported recently (Leonidas et al., 2003
). Interesting sugar specificity exhibited by SRL toward Galß1
3GalNAc-ser/thr, an oncofetal antigen, prompted us to study its functional role. The present article describes the localization and expression of the lectin and identification of its putative endogeneous glycosphingolipid receptor, to explain the functional role of the lectin and the receptor in the development and morphogenesis of S. rolfsii.
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Results |
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Inhibition of germination by neutralizing antibody
To further investigate the involvement of lectin in growth of the fungus, we examined the germination of sclerotial bodies after capping the lectin sites by anti-SRL. Interestingly, sclerotial bodies treated with anti-SRL did not germinate (Figure 3b) even after 7 days, but the sclerotial bodies treated with normal rabbit serum germinated normally and lavish growth was observed (Figure 3a). Similarly, the mycelia treated with anti-SRL also failed to grow. Not only anti-SRL but also SRL-binding glycoproteins fetuin and mucin inhibited the germination similarly (data not shown). Observations of inhibition of germination by extraneous lectin-binding molecules suggests that the lectin expressed in response to nutrient stress interacts with the endogenous receptor, apart from facilitating the mycelial aggregation, to form sclerotial bodies, which also play key role in germination process.
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Identification of free amino groups in the receptors on TLC by spraying with ninhydrin indicated positive reaction only for RI but not for RII (Figure 4, 4), even with excess amounts of lipid. However positive indication for the presence of phosphate moiety in both RI and RII was demonstrated by detection with ammonium molybdate (Figure 4, 5). From these analytical findings, we concluded that both the receptors (RI and RII) identified are ceramide-type glycosphingophospholipids. Also, orcinol and ninhydrin staining revealed that RI is different than RII by having higher content of carbohydrate and with free amino groups (ethanolamine or serine). These differences suggest that RII could probably be a modified form of RI. Heterogeneity associated with the receptor bands leading to such discrepancies could not be ruled out. Indeed both the receptors contain Galß13GalNAc, an essential criterion for binding by SRL and PNA.
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Discussion |
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Similar observations were made in Rhizoctonia solani, where in the lectin occurring in small amounts on young hyphae increased dramatically at the time of maturation and accumulated in mature sclerotina (Kellens and Peumans, 1990). Lectin accumulated in the sclerotina, represented as high as 40% of the total sclerotial protein, hence they concluded that the lectin serve as reserve storage protein as in plant seeds. In contrast Cooper et al. (1997)
suggested that the small, saline-soluble galactose-binding lectins (fungal galectins) secreted by many fungi are developmentally regulated with high expression in fruiting bodies. A fucose-specific lectin secreted by Rhizopus stolonifer only under spore-forming conditions reported recently (Oda et al., 2003
) supports this latter view.
Our results of localization and expression of SRL revealed that the SRL is developmental-stage specific lectin secreted in response to stress, playing key role in the formation of sclerotial bodies rather than simply serving as reserve storage protein. Germination of sclerotial bodies is another event in the development of the fungus, which also involves the role of the lectin as shown by lectin capping studies. Capping of the lectin sites by anti-SRL strongly inhibits germination of the sclerotial bodies. Similar inhibition was also found by treating these bodies with mucin or fetuin, with which SRL strongly binds (Swamy et al., 2001). These observations suggest that the lectin in a bound form with any extraneous lectin-binding molecules, such as antibodies, fetuin, or mucin will result in inhibition of sclerotial body germination. For the onset of sclerotial body germination, lectinreceptor complex could be mediating as critical signaling molecule, probably this signaling event is interrupted when the lectin is complexed with anti-SRL or a hapten.
Recently it was shown that the disruption of the glucosyl ceramide synthesis using inhibitors of UDP-Glc, ceramide glucosyltransferase, leads to inhibition of spore germination, cell cycle, and hyphal growth in Aspergillus nidulans and Aspergillus fumigatus (Levery et al., 2002). Membrane-bound glycosyl ceramides are reported to be widely distributed in many fungi, and during their syntheses, sugar moieties are added directly to ceramides, which are referred to as glycosylinositol phosphorylceramides (GIPCs). This class of glycosyl ceramides occurring in fungi (Lester and Dickson, 1993
; Dickson, 1998
) are mostly glucosylceramides. However there are also reports of galactosylceramides occurring in some fungi (Levery et al., 2000
; Toledo et al., 1999
, 2000
). GIPCs are believed to act as signaling molecules and are implicated for their diversified roles in viability (Zhong et al., 2000
) and cell growth (Chung et al., 2001
). We believe that the inhibition of spore germination by disrupting the ceramide synthesis using inhibitors of glycosyl transferases in A. nidulans and A. fumigatus (Levery et al., 2002
) is analogous to our observed inhibition of sclerotial body germination by capping the lectin sites on sclerotial bodies.
Identification of lectin binding receptors in the lipid extracts of sclerotial bodies by specific lectin labeling method substantiates the significance of the lectinreceptor interaction in the development of the fungus. Considering the sugar specificity of SRL, which binds specifically to Galß13GalNAc- residues of glycoproteins (Swamy et al., 2001
), it becomes evident that lectin-binding receptor could essentially contain this moiety. Preliminary characterization studies of the lectin-binding receptors after TLC using specific detection reagents indicate that the receptor RI and RII resemble phytoceramides reported in fungi (Dickson, 1998
), in particular IPCs. The lectin receptor identified in S. rolfsii appears to be galactosyl ceramide. Based on our findings, we propose that the lectin expressed abundantly on the mycelia at the time of sclerotial body formation facilitates the aggregation of the mycelium by interacting with endogenous glycosyl ceramide receptor(s) having specific carbohydrate moiety. Furthermore, it appears that the lectinreceptor complex formed at the time of sclerotial body formation will remain intact during the dormant stage, and the complex would undergo changes to facilitate germination. In the near future these findings would shed light on the importance of fungal lectins as well the IPC receptors.
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Materials and methods |
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Immunolocalization of lectin
Localization of lectin in vegetative mycelia and immature and mature sclerotial bodies of S. rolfsii during development was carried out by immunolabeling the lectin sites with FITC anti-SRL. Vegetative mycelial mass from the S. rolfsii culture broth of 10 days was washed repeatedly by centrifugation at 8000 x g. Washed mycelial filaments were suspended in phosphate buffered saline (PBS) containing p-BSA (3%) and incubated for 30 min at 37°C to block nonspecific binding by lectin antibodies, followed by extensive washing with PBS. Subsequently the filaments were incubated with FITC anti-SRL in PBS (50 µg/ml) for 30 min with gentle shaking. Excess unbound antibodies were removed by washing with PBS on centrifugation. Essentially the same procedure was adopted for lectin localization on sclerotial bodies. Interaction of FITC anti-SRL on vegetative mycelia and sclerotial bodies was observed and photographed under fluorescence microscope (Carl Zeiss Jenalumar, model Fluoval 2) using excitation filter G-247 in the path of excitation light and barrier filter B 450 in the path of emitted light.
Determination of SRL activity during growth and development of S. rolfsii
In mycelia. For the determination of lectin activity in developing mycelia, S. rolfsii cultures were grown in Byrde's mineral medium (100 ml) in different flasks by innoculating one sclerotial body for each flask. On different days from the date of inoculation, the mycelial mass formed was collected from three flasks separately (triplicate) after filtration on a sintered funnel. Individual mycelial lots of each day were separately washed with distilled water and freeze-dried. Dried mycelium (10 mg/ml) was homogenized with PBS (50 mM, pH 7.2) using pestle and mortar, briefly sonicated, and kept for extraction on a rotary shaker (150 rpm) for 1 h. The homogenate was subjected to centrifugation at 10,000 x g for 30 min, at 4°C (Kubota refrigerated centrifuge, RA 300F angle rotor). Clear supernatant after ultrafiltration on 0.2 µ membrane was used for the determination of lectin activity.
In sclerotial bodies. Sclerotial bodies formed during the course of development were harvested from each flask (in triplicate) by picking up manually from the culture broths on different days, washed thoroughly with distilled water, and powdered after freeze drying. Dry powder of the sclerotial bodies was suspended in PBS (5 mg/ml), sonicated to obtain homogeneous suspension and extracted for 1 h on a rotary shaker. Clear supernatant obtained on centrifugation at 8000 x g for 30 min was used for the determination of lectin activity. Lectin activity in the extracts of mycelia and sclerotial bodies harvested on different days was determined by hemagglutination assay. Total carbohydrate, protein content, and dry weight were also determined. The results of the lectin activity, total sugar, protein content, and dry weight of mycelium/sclerotial bodies were plotted against the age of culture.
Hemagglutination assay
The hemagglutinating activity of the lectin was routinely assayed by serial twofold dilution technique of Liener and Hill (1953) in microtiter assay plates using trypsinized human (O group) erythrocytes. The highest dilution of the extract causing visible hemagglutination was regarded as the titer and the protein content in the highest dilution causing visible agglutination as 1 unit of hemagglutination activity (minimum concentration of protein required for agglutination). Total activity in the extracts was determined from the total protein content.
Estimation of protein and sugar
Protein concentration was estimated by the method of Lowry et al. (1951) using BSA as standard. The total sugar content was estimated by the phenol-sulfuric acid method of Dubois et al. (1956)
using glucose as standard sugar.
Inhibition of sclerotial body germination by anti-SRL
Sclerotial bodies from a fresh culture were incubated for 10 min at 37°C with anti-SRL in a screw-capped vial. Treated sclerotial bodies were allowed to germinate in a petri dish containing Byrde's media with 1.5% agar at 28°C. As control, a sclerotial body that was treated with normal rabbit serum was inserted in another well of the plate. Germination of the sclerotial body in the plate was photographed after fourth day of inoculation.
Identification of putative endogenous lectin receptor
Extraction and thin layer chromatography of membrane lipids. Total lipids were extracted from the powdered mature sclerotial bodies using 20 volumes (w/v) of chloroform: methanol (2:1). The extract was dried by rotary evaporation and partitioned between chloroform and water. The resulting organic layer was washed twice with water and finally dried by flushing nitrogen and stored at 20°C for further fractionation by TLC. Dried lipid extracted from sclerotial bodies in chloroform (2 µl) was applied by streaking onto high-performance TLC plates (1 x 10 cm), and developed with chloroform:methanol:water (13:5:0.9) and dried at 60°C for 1 h.
Biotinylated lectin labeling assay
Lectin-binding lipid components on TLC plates were identified by enzyme-linked lectinosorbent detection using biotinylated SRL. In princple, the method we adopted is similar to solid-phase methods, used to demonstrate binding of viruses and bacteria and to characterize receptor carbohydrates based on their specific binding to glycolipids separated on thin-layer chromatograms (Karlsson, 1989). In this detection assay, the TLC plates with fractionated lipid components were immersed in PBS containing polyvinyl pyrrolidone (1%) for 5 min. Subsequently the plates were blocked with p-BSA (3%) in PBS for 1 h to prevent nonspecific binding and washed gently three times with PBS and incubated overnight at 4°C in screw-capped tubes containing biotinylated SRL or biotinylated PNA in PBS (20 µg/ml). The plates were gently washed with PBS to remove unbound lectin and treated with streptavidin horseradish peroxidase. The plates were finally washed with distilled water and incubated in tetramethyl benzidine/H2O2 for 5 min; the blue-colored bands developed were photographed immediately.
Analytical characterization of lectin binding receptors
Analytical characterization of lectin-binding receptors resolved on TLC plates was carried out using specific spraying reagents. The chromatograms were dried and sprayed separately for detection of glycolipids by orcinol, free amino groups by ninhydrin, and phospholipids by ammonium molybdate reagents. Reagents and the methods used were essentially as described by Kates (1972). After the development of the color the plates were photographed.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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