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Purification and characterization of two endopolygalacturonases secreted during the early stages of the saprophytic growth of Sclerotinia sclerotiorum
FEMS Microbiology Letters 158 (1998) 133^138
Puri¢cation and characterization of two endopolygalacturonases secreted during the early stages of the saprophytic growth of Sclerotinia sclerotiorum Marie-Beèneèdicte Martel, Robert Leètoublon *, Michel Feévre Laboratoire de Biologie Cellulaire Fongique, Centre de Geèneètique Moleèculaire et Cellulaire, UMR-CNRS 5534, Universiteè Lyon I (Baêt. 405), 43 Bd du 11 Novembre 1918, 69622 Villeurbanne Cedex, France Received 5 November 1997 ; accepted 11 November 1997
Abstract Two endopolygalacturonases (endo-PGs) secreted at the early stage of cultures of Sclerotinia sclerotiorum grown on polygalacturonate medium, were purified to apparent homogeneity, using ion exchange chromatography. They are glycoproteins of an apparent weight of 42 and 41.5 kDa and a pI of 4.8. The two purified isoforms found in early cultures were not detected in late cultures. Purification of the isoforms secreted at different stages of growth revealed that the increase of polygalacturonase activity during the culture corresponds to a sequential production of enzymes and to the successive replacement of isoforms by new enzymes. ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords : Polygalacturonase; Sclerotinia sclerotiorum
1. Introduction Most plant pathogenic fungi produce a number of cell wall degrading enzymes when grown in the presence of pectin [1,2]. Among these enzymes, polygalacturonases (PGs) have been implicated in facilitating the invasion and colonization of host tissue particularly in diseases characterized by tissue maceration [3,4]. In Sclerotinia sclerotiorum, an ubiquitous and devastating plant pathogen, the involvement of polygalacturonases and other cell wall degrading enzyme activities in pathogenesis of host * Corresponding author. Tel.: +33 (4) 72 44 85 44; Fax: +33 (4) 72 43 11 81; E-mail: [email protected]
tissue have been established [5,6]. Biochemical and genetic studies undertaken to characterize the polygalacturonases and to understand their role during plant infection, have revealed the extent of complexity of this lytic system. A high number of polygalacturonase iso-components are produced in vitro; 16 isoforms have been separated [7]. Seven genes constituting two subfamilies of a multigene family encoding endopolygalacturonase have been identi¢ed and three endo-PG genes have been cloned and characterized [8,9]. Multiplicity of PG enzyme forms is widespread in fungi and it has been suggested that this enzyme multiplicity confers a greater adaptive capacity and £exibility of pathogens. However very little is known about the control
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 5 1 3 - 2
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mechanisms regulating the secretion of pectic enzymes. Mechanism by which an insoluble substrate such as cellulose or pectin, triggers the synthesis of enzymes for its degradation is still a matter of speculation. In Trichoderma, the most likely mechanism of cellulase induction is that cell surface associated cellulases are able to release small amounts of cellobiose which acts as the inducer [10]. If a similar model concerning the induction of pectinolytic enzymes exists, one can imagine that early secreted enzymes are of importance in initiating the inductive process. The aim of this study was to characterize the endopolygalacturonases produced by S. sclerotiorum during early growth on polygalacturonate medium and to investigate the range and time of appearance of the endo-PG isoforms.
2. Material and methods 2.1. Culture conditions S. sclerotiorum (strain s5) was maintained on potato dextrose agar. For enzyme production, the fungus was ¢rst grown for 2 days with constant stirring at 22³C in a liquid minimal medium [7] supplemented with 0.5% of glucose and 25 mM ammonium acetate then transferred to liquid minimal medium containing polygalacturonic acid (0.5%) as the carbon source and 25 mM ammonium acetate. Cultures were inoculated with mycelial disks cut from margins of 4 days old colonies. 2.2. Enzyme production At di¡erent times of cultivation, the mycelium was discarded after centrifugation and the culture medium collected, dialyzed against distilled water overnight at 4³C and freeze-dried. The lyophilized ¢ltrate containing secreted enzymes was solubilized in distilled water and brought to 50% ammonium sulfate saturation. The precipitate collected by centrifugation (30 min at 20 000Ug) was discarded. The resulting supernatant was brought to 85% ammonium sulfate saturation. The ¢nal pellet obtained after centrifugation at 20 000Ug for 30 min was dialyzed and served as the starting material for polygalacturonase puri¢cation.
2.3. Enzyme assays The polygalacturonase activity was determined by measuring the amount of reducing sugars released from polygalacturonic acid according to the 2-cyanoacetamide assay [11]. The standard reaction mixture (0.5 ml) containing 0.5 mg of polygalacturonic acid dissolved in 35 mM acetate bu¡er pH 3.5 and 1 to 10 Wl of the enzymatic fraction was incubated at 48³C for 20 min. The reaction was stopped by addition of 1.2 ml of TBC reagent (100 mM sodium tetraborate, 100 mM boric acid and 0.1% 2-cyanoacetamide). After boiling for 10 min and cooling, the optical density was determined spectrophotometrically at 270 nm. One unit of polygalacturonase activity was de¢ned as the amount of enzyme required to liberate 1 Wmol of reducing group per minute at 48³C. Exopolygalacturonase activity was assayed using digalacturonic acid as the substrate and measuring the released reducing sugars as described above. Protein content is estimated spectrophotometrically. 2.4. Chemical modi¢cation of the enzymes Acetylation with N-acetylimidazole was carried out according to Stratilova et al. [12]. Deglycosylation with tri£uoromethane sulfonic acid was performed according to Karp et al. [13]. 2.5. Chromatography Liquid chromatography was performed on a Gilson HPLC system. Gel ¢ltrations were carried out on a Sephadex G-25 column (1.5U15 cm) and on a TSK (TosoHaas) G-2000 SWXL column (0.78U30 cm). Ion exchange chromatographies were carried out on 5 ml Econo-Pac Q or Econo-Pac S cartridges (BioRad) equilibrated in 10 mM ammonium acetate bu¡er (pH 5) and eluted with a linear gradient of NaCl (0 to 1 M) in the same bu¡er. Hydroxyapatite chromatography was carried out on a 1.7 ml HAUltrogel (IBF) column equilibrated in 10 mM phosphate bu¡er (pH 6). Elution was performed with a linear gradient of phosphate bu¡er (10 to 300 mM), pH 6. Chromatography on 2.5 ml column of the immobilized reactive dye Brown 10 (Sigma) was performed in 10 mM ammonium acetate bu¡er ¢rst at
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pH 4, then at pH 5 and eluted with a linear gradient of NaCl (0 to 1 M) in the pH 5 bu¡er. 2.6. Analytical electrophoresis SDS-PAGE of proteins was carried out in 5% stacking and 10% resolving gels. Proteins were stained either with Coomassie brilliant blue R or silver stain. IEF was carried out on ready precoated gels (Serva) containing 5% ampholytes (pH range: 3 to 10). Gels were prefocused up to 500 V before application of the samples, then the proteins were focused at a constant power (4 W) for 2^3 h at a ¢nal voltage up to 1700 V. The pH gradient was veri¢ed with a micro-contact electrode. 2.7. Immunological methods Proteins separated on SDS-polyacrylamide gels were electroblotted to nitrocellulose. The nitrocellulose membranes were incubated overnight in 5% non-fat dry bovine milk in 0.15 M NaCl, 50 mM Tris-HCl bu¡er (TBS, pH 7.4) at 4³C, rinsed in TBS then incubated 1 h at 20³C, with the rabbit polyclonal antiserum (1:1000 dilution) raised against an acidic endopolygalacturonase [11]. Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:1000 dilution) was used as a second antibody and color was developed in TBS with 0.05% diaminobenzidine (DAB) and 0.05% H2 O2 .
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3. Results and discussion 3.1. Puri¢cation and biochemical characterization of two early secreted endo-PGs Fungal mycelium was grown in glucose medium for 2 days in order to obtain a large amount of biomass, then transferred to polygalacturonate medium to induce the synthesis of polygalacturonases. Puri¢cation of the polygalacturonases secreted after 2 days of culture in the presence of polygalacturonate was achieved by three separation steps (Fig. 1). The enzymes were ¢rst bound to an anionic resin, eluted by 0.5 M NaCl, dialyzed then chromatographed on a Brown 10 dye column. A single active fraction was eluted at pH 5 then subjected to HAUltrogel chromatography and separated in two fractions. The unbound enzyme (PG 8u) represented 70% of the enzymatic activity. After this puri¢cation step, SDS-PAGE was used as a criterion of purity. One single protein band indicating homogeneous protein was obtained in each fraction. The molecular masses of PG 8b and PG 8u were 42 kDa and 41.5 kDa, respectively (Fig. 2). The enzymes were glycosylated based on the positive response with the Glycotrack detection kit (Oxford Glyco Systems). Following deglycosylation a single band of about 39 kDa was detected in each fraction. Both enzymes have a pI of 4.8 as determined by analytical IEF. The temperature optimum for both PG 8u and PG 8b was
Fig. 1. Puri¢cation pro¢les of PG 8 isoforms secreted by S. sclerotiorum. a: Anion exchange chromatography on Econo-Pac S. b : Immobilized Brown 10 dye chromatography. c: Hydroxyapatite chromatography on HA-Ultrogel. Hatched : PG activity. Dashed line : gradient.
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Fig. 2. Electrophoretic analysis of the puri¢ed PG 8u and PG 8b from S. sclerotiorum. a: Silver staining of the proteins separated by SDS-PAGE. b: SDS-PAGE immunoblot of the enzymes probed with antibodies to an acidic endo-PG. 1: PG 8u; 2: PG 8b.
50³C. They did however show di¡erent optimal pH values, pH 3.5 and 4.0 for PG 8u and PG 8b, respectively. The two enzymes were identi¢ed as endopolygalacturonases based on the rapid reduction of substrate viscosity and by the higher release of reducing groups from polygalacturonate (99 Wmol min31 ) than from digalacturonate (1 Wmol min31 ). Thin layer chromatography revealed that di- and trigalacturonic acid oligosaccharides were the degradation products of polygalacturonate. The enzymes also cross-reacted with the antibodies raised against a puri¢ed acidic endo-PG (Fig. 2). The Km and Vm of PG 8b were 0.8 mg/ml and 8.6 Wmoles galacturonic acid min31 mg31 . Km and Vm of PG 8u were 0.5
mg/ml and 8.7 Wmoles galacturonic acid min31 mg31 . Both puri¢ed endo-PGs were treated with N-acetylimidazole which is known to acetylate tyrosine residues and to inhibit polygalacturonases from Aspergillus sp. [12]. Acetylation inhibited PG 8u completely indicating the presence of an accessible important tyrosine residue while PG 8b was insensitive to the inhibitor. This con¢rms that PG 8u and PG 8b are di¡erent enzymes. Several endo-PGs secreted by S. sclerotiorum at di¡erent times of cultures have been puri¢ed. They have a similar mode of action by causing a rapid reduction in substrate viscosity accompanied by the production of small oligomers of galacturonic acid units. Comparisons of their enzymatic parameters revealed that their Km are similar, varying from 0.5 to 1 mg/ml while their Vmax are highly di¡erent. The early secreted enzymes that we have characterized exhibit a Vmax about one hundred times lower than the enzymes produced in late cultures (12 days of culture) i.e. 8.5 Wmol min31 mg31 versus 620 to 1100 Wmol min31 mg31 [14]. 3.2. Separation of the PG isoforms secreted during cultivation on polygalacturonate medium In order to know if these enzymes are continuously produced in inducing cultures, enzymes secreted during cultivation on polygalacturonate medium were collected at three stages corresponding to di¡erent phases of growth: during the exponential phase of growth and polygalacturonate consumption (2 and 4 days after transfer) and during the stationary phase of growth when the pectic polymer had been exhausted (10 days after transfer). The time course production of hydrolytic enzymes revealed
Table 1 Enzymatic activity of the puri¢ed polygalacturonase isoforms secreted by Sclerotinia sclerotiorum grown in the presence of polygalacturonate Polygalacturonase isoforms Days of culture
PG 9
PG 8
PG 7
PG 6
PG 5
PG 4
PG 3
PG 2
PG 1
2 4 10
nd 57 3
10 27 nd
nd 3 nd
nd nd 1.4
nd nd 2
nd 40 8
nd nd 30
nd nd nd
nd 0.6 90
Puri¢ed fractions were obtained according to the £ow scheme described Fig. 3. PG activity is expressed as the amount of enzyme required to liberate 1 Wmol of reducing sugar per minute. nd, not detected.
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Fig. 3. Flow scheme of the puri¢cation of polygalacturonase fractions (PG) from cultures of S. sclerotiorum. Thin line : unbound fraction and thick line: bound fraction to each stated matrix.
that the crude PG activity (170 units) detected 2 days after the transfer increased to 580 units 4 days after transfer, then stabilized reaching 650 units after 10 days of culture. We observed that the crude enzymatic activity at each stage of the culture was much higher than the sum of the puri¢ed enzyme activities (see Table 1). These di¡erences indicated synergetic action of the enzymes in the crude extract rather than their instability during puri¢cation because puri¢ed enzymes remained stable for several weeks at room temperature. Separation of extracellular polygalacturonases was achieved by using several chromatographic steps according to the puri¢cation protocol shown in Fig. 3. Using the enzyme preparation from the 4 days old culture, the puri¢cation scheme described above allows to separate the anionic matrix bound enzymes into three active fractions (PG 7, PG 8, PG 9). The Econo-Pac S unbound fraction was chromatographed on a Econo-Pac Q column which allowed the separation of two other PGs (PG 1 and PG 4). Except for PG 9, all the enzymes found in the medium after 10 days of culture were not bound to the anionic matrix. They were divided in two unequal active fractions on the cationic matrix at pH 5. From the bound material, three active fractions
(PG 4, PG 5 and PG 6) were separated after Brown 10; the two most active fractions (PG 1 and PG 3) were issued from the unbound material on the anion exchange column. The isoforms characterizing the polygalacturonase activity collected after 2 days of culture PG 8u and PG 8b were not detected after 10 days and PG 9, the most active isoform (corresponding to 45% of the total activity of the puri¢ed isoforms) of the 4 days old culture represented only 2% of the activity of the puri¢ed fractions after 10 days. In contrast, PG 1 which was not detected after 2 days and which corresponded to 0.5% of the activity of the separated enzymes after 4 days, was the main fraction after 10 days of culture representing 67% of the activity of the puri¢ed enzymes. All the enzymes tested were glycoproteins, exhibiting a similar molecular mass (around 42 kDa) but di¡ering in their pI and charge as attested by their separation using ion exchange chromatographies. These results are consistent with the expression of a large gene family of endo-PGs [9]. The sequence in the secretion of endo-PG isoforms observed, does not correspond to the appearance and accumulation of isoforms during growth but is characterized by the successive replacement of iso-
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forms by new enzymes. If the enzymes that we have characterized in the early stages have an essential role for the initial degradation of the polymer, they will become ine¤cient in late cultures when the polymer has been degraded to oligomers and they will be replaced by enzyme exhibiting di¡erent biochemical properties. This sequential pattern of endo-PG secretion could be interpreted as successive induction and repression of enzyme synthesis for the complete degradation of the pectic polymer. The early secreted endo-PGs could release products which would repress their synthesis and induced the secretion of other enzymes with di¡erent biochemical properties. In this respect it will be of interest to know if each step of the sequential endo-PG production is constituted by the secretion of isoforms with a higher Vmax and if this sequence is related to the extent of degradation of the inducing polymer or to the availability of assimilable products.
Acknowledgments This work was supported by grants from the CNRS (UMR 5534) and the Universiteè Claude Bernard- LYON (FRANCE). The authors thank Annie Verrier for typing the manuscript.
References [1] Bateman, D. and Basham, H. (1976) Degradation of plant cell walls and membranes by microbial enzymes. In: Physiological Plant Pathology (Heitefuss, R. and Williams, P., Eds.), pp. 316^355. Springer-Verlag, Berlin. [2] Cooper, R.M. (1984) The role of cell wall-degrading enzymes in infection and damage. In: Plant Diseases : Infection, Damage and Loss (Wood, R.K.S. and Jelis, G.J., Eds.), pp. 13^28. Blackwell Scienti¢c Publications, Oxford.
[3] Alghisi, P. and Favaron, F. (1995) Pectin-degrading enzymes and plant parasite interactions. Eur. J. Plant. Pathol. 101, 365^375. [4] Cervone, F., de Lorenzo, G., Aracri, B., Bellincampi, D., Caprari, C., Clark, A.J., Desiderio, A., Devoto, A., Leckie, F., Mattei, B., Nuss, L. and Salvi, G. (1996) The role of polygalacturonase, PGIP and pectin oligomers in fungal infection. In: Pectins and Pectinases (Visser, J. and Voragen, A.G.J., Eds.), pp. 191^205. Elsevier, Amsterdam. [5] Hancock, J.C. (1966) Degradation of pectic substances associated with pathogenesis by Sclerotinia sclerotiorum in Sun£ower and Tomato stems. Phytopathology 56, 975^979. [6] Lumsden, R.D. (1969) Sclerotinia sclerotiorum infection of bean and the production of cellulase. Phytopathology 59, 653^657. [7] Martel, M.B., Leètoublon, R. and Feévre, M. (1996) Puri¢cation of endo-polygalacturonases from Sclerotinia sclerotiorum : multiplicity of the complex enzyme system. Curr. Microbiol. 33, 243^248. [8] Reymond, P., Deleèage, G., Rascle, C. and Feévre, M. (1994) Cloning and sequence analysis of a polygalacturonase-encoding gene from the phytopathogenic fungus S. sclerotiorum. Gene 146, 233^237. [9] Fraissinet-Tachet, L., Reymond-Cotton, P. and Feévre, M. (1995) Characterization of a multigene family encoding an endopolygalacturonase in Sclerotinia sclerotiorum. Curr. Genet. 29, 96^100. [10] Kubicek, C.P., Messner, R., Gruber, F., Mach, R.L. and Kubicek-Pranz, M. (1993) The Trichoderma cellulase regulatory puzzle : From the interior life of a secretory fungus. Enzyme Microb. Technol. 15, 90^99. [11] Honda, S., Nishimura, Y., Takahashi, M., Chiba, H. and Kakehi, K. (1982) A manual method for the spectrophotometric determination of reducing carbohydrates with 2-cyanoacetamide. Anal. Biochem. 119, 194^199. [12] Stratilova, E., Dzurova, M., Markovic, O. and Joërnvall, H. (1996) An essential tyrosine residue of Aspergillus polygalacturonase. FEBS Lett. 382, 164^166. [13] Karp, D.R., Atkinson, J.P. and Shre¡er, D.C. (1982) Genetic variation in glycosylation of the fourth component of murine complement. J. Biol. Chem. 257, 7330^7335. [14] Waksman, G., Keon, J.P. and Turner, G. (1991) Puri¢cation and characterization of two endopolygalacturonases from Sclerotinia sclerotiorum. Biochim. Biophys. Acta 1073, 43^48.
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Puri¢cation and characterization of two endopolygalacturonases secreted during the early stages of the saprophytic growth of Sclerotinia sclerotiorum Marie-Beèneèdicte Martel, Robert Leètoublon *, Michel Feévre Laboratoire de Biologie Cellulaire Fongique, Centre de Geèneètique Moleèculaire et Cellulaire, UMR-CNRS 5534, Universiteè Lyon I (Baêt. 405), 43 Bd du 11 Novembre 1918, 69622 Villeurbanne Cedex, France Received 5 November 1997 ; accepted 11 November 1997
Abstract Two endopolygalacturonases (endo-PGs) secreted at the early stage of cultures of Sclerotinia sclerotiorum grown on polygalacturonate medium, were purified to apparent homogeneity, using ion exchange chromatography. They are glycoproteins of an apparent weight of 42 and 41.5 kDa and a pI of 4.8. The two purified isoforms found in early cultures were not detected in late cultures. Purification of the isoforms secreted at different stages of growth revealed that the increase of polygalacturonase activity during the culture corresponds to a sequential production of enzymes and to the successive replacement of isoforms by new enzymes. ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords : Polygalacturonase; Sclerotinia sclerotiorum
1. Introduction Most plant pathogenic fungi produce a number of cell wall degrading enzymes when grown in the presence of pectin [1,2]. Among these enzymes, polygalacturonases (PGs) have been implicated in facilitating the invasion and colonization of host tissue particularly in diseases characterized by tissue maceration [3,4]. In Sclerotinia sclerotiorum, an ubiquitous and devastating plant pathogen, the involvement of polygalacturonases and other cell wall degrading enzyme activities in pathogenesis of host * Corresponding author. Tel.: +33 (4) 72 44 85 44; Fax: +33 (4) 72 43 11 81; E-mail: [email protected]
tissue have been established [5,6]. Biochemical and genetic studies undertaken to characterize the polygalacturonases and to understand their role during plant infection, have revealed the extent of complexity of this lytic system. A high number of polygalacturonase iso-components are produced in vitro; 16 isoforms have been separated [7]. Seven genes constituting two subfamilies of a multigene family encoding endopolygalacturonase have been identi¢ed and three endo-PG genes have been cloned and characterized [8,9]. Multiplicity of PG enzyme forms is widespread in fungi and it has been suggested that this enzyme multiplicity confers a greater adaptive capacity and £exibility of pathogens. However very little is known about the control
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 5 1 3 - 2
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mechanisms regulating the secretion of pectic enzymes. Mechanism by which an insoluble substrate such as cellulose or pectin, triggers the synthesis of enzymes for its degradation is still a matter of speculation. In Trichoderma, the most likely mechanism of cellulase induction is that cell surface associated cellulases are able to release small amounts of cellobiose which acts as the inducer [10]. If a similar model concerning the induction of pectinolytic enzymes exists, one can imagine that early secreted enzymes are of importance in initiating the inductive process. The aim of this study was to characterize the endopolygalacturonases produced by S. sclerotiorum during early growth on polygalacturonate medium and to investigate the range and time of appearance of the endo-PG isoforms.
2. Material and methods 2.1. Culture conditions S. sclerotiorum (strain s5) was maintained on potato dextrose agar. For enzyme production, the fungus was ¢rst grown for 2 days with constant stirring at 22³C in a liquid minimal medium [7] supplemented with 0.5% of glucose and 25 mM ammonium acetate then transferred to liquid minimal medium containing polygalacturonic acid (0.5%) as the carbon source and 25 mM ammonium acetate. Cultures were inoculated with mycelial disks cut from margins of 4 days old colonies. 2.2. Enzyme production At di¡erent times of cultivation, the mycelium was discarded after centrifugation and the culture medium collected, dialyzed against distilled water overnight at 4³C and freeze-dried. The lyophilized ¢ltrate containing secreted enzymes was solubilized in distilled water and brought to 50% ammonium sulfate saturation. The precipitate collected by centrifugation (30 min at 20 000Ug) was discarded. The resulting supernatant was brought to 85% ammonium sulfate saturation. The ¢nal pellet obtained after centrifugation at 20 000Ug for 30 min was dialyzed and served as the starting material for polygalacturonase puri¢cation.
2.3. Enzyme assays The polygalacturonase activity was determined by measuring the amount of reducing sugars released from polygalacturonic acid according to the 2-cyanoacetamide assay [11]. The standard reaction mixture (0.5 ml) containing 0.5 mg of polygalacturonic acid dissolved in 35 mM acetate bu¡er pH 3.5 and 1 to 10 Wl of the enzymatic fraction was incubated at 48³C for 20 min. The reaction was stopped by addition of 1.2 ml of TBC reagent (100 mM sodium tetraborate, 100 mM boric acid and 0.1% 2-cyanoacetamide). After boiling for 10 min and cooling, the optical density was determined spectrophotometrically at 270 nm. One unit of polygalacturonase activity was de¢ned as the amount of enzyme required to liberate 1 Wmol of reducing group per minute at 48³C. Exopolygalacturonase activity was assayed using digalacturonic acid as the substrate and measuring the released reducing sugars as described above. Protein content is estimated spectrophotometrically. 2.4. Chemical modi¢cation of the enzymes Acetylation with N-acetylimidazole was carried out according to Stratilova et al. [12]. Deglycosylation with tri£uoromethane sulfonic acid was performed according to Karp et al. [13]. 2.5. Chromatography Liquid chromatography was performed on a Gilson HPLC system. Gel ¢ltrations were carried out on a Sephadex G-25 column (1.5U15 cm) and on a TSK (TosoHaas) G-2000 SWXL column (0.78U30 cm). Ion exchange chromatographies were carried out on 5 ml Econo-Pac Q or Econo-Pac S cartridges (BioRad) equilibrated in 10 mM ammonium acetate bu¡er (pH 5) and eluted with a linear gradient of NaCl (0 to 1 M) in the same bu¡er. Hydroxyapatite chromatography was carried out on a 1.7 ml HAUltrogel (IBF) column equilibrated in 10 mM phosphate bu¡er (pH 6). Elution was performed with a linear gradient of phosphate bu¡er (10 to 300 mM), pH 6. Chromatography on 2.5 ml column of the immobilized reactive dye Brown 10 (Sigma) was performed in 10 mM ammonium acetate bu¡er ¢rst at
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pH 4, then at pH 5 and eluted with a linear gradient of NaCl (0 to 1 M) in the pH 5 bu¡er. 2.6. Analytical electrophoresis SDS-PAGE of proteins was carried out in 5% stacking and 10% resolving gels. Proteins were stained either with Coomassie brilliant blue R or silver stain. IEF was carried out on ready precoated gels (Serva) containing 5% ampholytes (pH range: 3 to 10). Gels were prefocused up to 500 V before application of the samples, then the proteins were focused at a constant power (4 W) for 2^3 h at a ¢nal voltage up to 1700 V. The pH gradient was veri¢ed with a micro-contact electrode. 2.7. Immunological methods Proteins separated on SDS-polyacrylamide gels were electroblotted to nitrocellulose. The nitrocellulose membranes were incubated overnight in 5% non-fat dry bovine milk in 0.15 M NaCl, 50 mM Tris-HCl bu¡er (TBS, pH 7.4) at 4³C, rinsed in TBS then incubated 1 h at 20³C, with the rabbit polyclonal antiserum (1:1000 dilution) raised against an acidic endopolygalacturonase [11]. Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:1000 dilution) was used as a second antibody and color was developed in TBS with 0.05% diaminobenzidine (DAB) and 0.05% H2 O2 .
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3. Results and discussion 3.1. Puri¢cation and biochemical characterization of two early secreted endo-PGs Fungal mycelium was grown in glucose medium for 2 days in order to obtain a large amount of biomass, then transferred to polygalacturonate medium to induce the synthesis of polygalacturonases. Puri¢cation of the polygalacturonases secreted after 2 days of culture in the presence of polygalacturonate was achieved by three separation steps (Fig. 1). The enzymes were ¢rst bound to an anionic resin, eluted by 0.5 M NaCl, dialyzed then chromatographed on a Brown 10 dye column. A single active fraction was eluted at pH 5 then subjected to HAUltrogel chromatography and separated in two fractions. The unbound enzyme (PG 8u) represented 70% of the enzymatic activity. After this puri¢cation step, SDS-PAGE was used as a criterion of purity. One single protein band indicating homogeneous protein was obtained in each fraction. The molecular masses of PG 8b and PG 8u were 42 kDa and 41.5 kDa, respectively (Fig. 2). The enzymes were glycosylated based on the positive response with the Glycotrack detection kit (Oxford Glyco Systems). Following deglycosylation a single band of about 39 kDa was detected in each fraction. Both enzymes have a pI of 4.8 as determined by analytical IEF. The temperature optimum for both PG 8u and PG 8b was
Fig. 1. Puri¢cation pro¢les of PG 8 isoforms secreted by S. sclerotiorum. a: Anion exchange chromatography on Econo-Pac S. b : Immobilized Brown 10 dye chromatography. c: Hydroxyapatite chromatography on HA-Ultrogel. Hatched : PG activity. Dashed line : gradient.
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Fig. 2. Electrophoretic analysis of the puri¢ed PG 8u and PG 8b from S. sclerotiorum. a: Silver staining of the proteins separated by SDS-PAGE. b: SDS-PAGE immunoblot of the enzymes probed with antibodies to an acidic endo-PG. 1: PG 8u; 2: PG 8b.
50³C. They did however show di¡erent optimal pH values, pH 3.5 and 4.0 for PG 8u and PG 8b, respectively. The two enzymes were identi¢ed as endopolygalacturonases based on the rapid reduction of substrate viscosity and by the higher release of reducing groups from polygalacturonate (99 Wmol min31 ) than from digalacturonate (1 Wmol min31 ). Thin layer chromatography revealed that di- and trigalacturonic acid oligosaccharides were the degradation products of polygalacturonate. The enzymes also cross-reacted with the antibodies raised against a puri¢ed acidic endo-PG (Fig. 2). The Km and Vm of PG 8b were 0.8 mg/ml and 8.6 Wmoles galacturonic acid min31 mg31 . Km and Vm of PG 8u were 0.5
mg/ml and 8.7 Wmoles galacturonic acid min31 mg31 . Both puri¢ed endo-PGs were treated with N-acetylimidazole which is known to acetylate tyrosine residues and to inhibit polygalacturonases from Aspergillus sp. [12]. Acetylation inhibited PG 8u completely indicating the presence of an accessible important tyrosine residue while PG 8b was insensitive to the inhibitor. This con¢rms that PG 8u and PG 8b are di¡erent enzymes. Several endo-PGs secreted by S. sclerotiorum at di¡erent times of cultures have been puri¢ed. They have a similar mode of action by causing a rapid reduction in substrate viscosity accompanied by the production of small oligomers of galacturonic acid units. Comparisons of their enzymatic parameters revealed that their Km are similar, varying from 0.5 to 1 mg/ml while their Vmax are highly di¡erent. The early secreted enzymes that we have characterized exhibit a Vmax about one hundred times lower than the enzymes produced in late cultures (12 days of culture) i.e. 8.5 Wmol min31 mg31 versus 620 to 1100 Wmol min31 mg31 [14]. 3.2. Separation of the PG isoforms secreted during cultivation on polygalacturonate medium In order to know if these enzymes are continuously produced in inducing cultures, enzymes secreted during cultivation on polygalacturonate medium were collected at three stages corresponding to di¡erent phases of growth: during the exponential phase of growth and polygalacturonate consumption (2 and 4 days after transfer) and during the stationary phase of growth when the pectic polymer had been exhausted (10 days after transfer). The time course production of hydrolytic enzymes revealed
Table 1 Enzymatic activity of the puri¢ed polygalacturonase isoforms secreted by Sclerotinia sclerotiorum grown in the presence of polygalacturonate Polygalacturonase isoforms Days of culture
PG 9
PG 8
PG 7
PG 6
PG 5
PG 4
PG 3
PG 2
PG 1
2 4 10
nd 57 3
10 27 nd
nd 3 nd
nd nd 1.4
nd nd 2
nd 40 8
nd nd 30
nd nd nd
nd 0.6 90
Puri¢ed fractions were obtained according to the £ow scheme described Fig. 3. PG activity is expressed as the amount of enzyme required to liberate 1 Wmol of reducing sugar per minute. nd, not detected.
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137
Fig. 3. Flow scheme of the puri¢cation of polygalacturonase fractions (PG) from cultures of S. sclerotiorum. Thin line : unbound fraction and thick line: bound fraction to each stated matrix.
that the crude PG activity (170 units) detected 2 days after the transfer increased to 580 units 4 days after transfer, then stabilized reaching 650 units after 10 days of culture. We observed that the crude enzymatic activity at each stage of the culture was much higher than the sum of the puri¢ed enzyme activities (see Table 1). These di¡erences indicated synergetic action of the enzymes in the crude extract rather than their instability during puri¢cation because puri¢ed enzymes remained stable for several weeks at room temperature. Separation of extracellular polygalacturonases was achieved by using several chromatographic steps according to the puri¢cation protocol shown in Fig. 3. Using the enzyme preparation from the 4 days old culture, the puri¢cation scheme described above allows to separate the anionic matrix bound enzymes into three active fractions (PG 7, PG 8, PG 9). The Econo-Pac S unbound fraction was chromatographed on a Econo-Pac Q column which allowed the separation of two other PGs (PG 1 and PG 4). Except for PG 9, all the enzymes found in the medium after 10 days of culture were not bound to the anionic matrix. They were divided in two unequal active fractions on the cationic matrix at pH 5. From the bound material, three active fractions
(PG 4, PG 5 and PG 6) were separated after Brown 10; the two most active fractions (PG 1 and PG 3) were issued from the unbound material on the anion exchange column. The isoforms characterizing the polygalacturonase activity collected after 2 days of culture PG 8u and PG 8b were not detected after 10 days and PG 9, the most active isoform (corresponding to 45% of the total activity of the puri¢ed isoforms) of the 4 days old culture represented only 2% of the activity of the puri¢ed fractions after 10 days. In contrast, PG 1 which was not detected after 2 days and which corresponded to 0.5% of the activity of the separated enzymes after 4 days, was the main fraction after 10 days of culture representing 67% of the activity of the puri¢ed enzymes. All the enzymes tested were glycoproteins, exhibiting a similar molecular mass (around 42 kDa) but di¡ering in their pI and charge as attested by their separation using ion exchange chromatographies. These results are consistent with the expression of a large gene family of endo-PGs [9]. The sequence in the secretion of endo-PG isoforms observed, does not correspond to the appearance and accumulation of isoforms during growth but is characterized by the successive replacement of iso-
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forms by new enzymes. If the enzymes that we have characterized in the early stages have an essential role for the initial degradation of the polymer, they will become ine¤cient in late cultures when the polymer has been degraded to oligomers and they will be replaced by enzyme exhibiting di¡erent biochemical properties. This sequential pattern of endo-PG secretion could be interpreted as successive induction and repression of enzyme synthesis for the complete degradation of the pectic polymer. The early secreted endo-PGs could release products which would repress their synthesis and induced the secretion of other enzymes with di¡erent biochemical properties. In this respect it will be of interest to know if each step of the sequential endo-PG production is constituted by the secretion of isoforms with a higher Vmax and if this sequence is related to the extent of degradation of the inducing polymer or to the availability of assimilable products.
Acknowledgments This work was supported by grants from the CNRS (UMR 5534) and the Universiteè Claude Bernard- LYON (FRANCE). The authors thank Annie Verrier for typing the manuscript.
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