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Studies on purification of a lectin from fruiting bodies of the edible shiitake mushroom Lentinus edodes
PERGAMON
The International Journal of Biochemistry & Cell Biology 31 (1999) 595±599
Studies on puri®cation of a lectin from fruiting bodies of the edible shiitake mushroom Lentinus edodes H.X. Wang a, T.B. Ng b, *, V.E.C. Ooi a a
Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, People's Republic of China Department of Biochemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, People's Republic of China
b
Received 17 August 1998; accepted 30 November 1998
Abstract A lectin, with a speci®city for N-acetylgalactosamine and N-acetylglucosamine and a molecular weight of 43 kDa, was isolated from fruiting bodies of the edible shiitake mushroom Lentinus edodes. The puri®cation procedure entailed extraction with aqueous buer, ammonium sulfate precipitation, gel ®ltration on Sephadex G-100 and anity chromatography on N-acetylgalactosamine-agarose. The lectin was unique in that it was tenaciously bound on anion exchangers including DEAE-cellulose, DEAE-Sepharose, DEAE-Sephadex, Q-Sepharose, Dowex and PEIcellulose and also on hydroxyapatite and phenyl Sepharose. It was largely unadsorbed on cation exchangers including CM-cellulose, CM-Sepharose, SP-Sepharose and Amberlite, and also on protein G-Sepharose, Red Sepharose and A-gel Blue gel, wheat germ lectin-Sepharose and p-aminophenyl-D-glucopyranoside agarose. # 1999 Published by Elsevier Science Ltd. All rights reserved.
1. Introduction Lectins are elaborated by a diversity of living organisms including animals, plants and microorganisms. They are proteins or glycoproteins which, because of their speci®c binding to sugars, have been exploited in structural studies on the cell surface, oligosaccharides and/or carbohydrate * Corresponding author. Tel.: +852-2609-63-59; fax: 8522603-72-06; e-mail: [email protected].
moieties of glycoproteins [1±4]. Mushrooms represent a rich source of lectins and the physicochemical and biochemical characteristics together with the biological activities of lectins from a variety of mushroom species have recently been reviewed [5, 6]. Jeune et al. [7] reported a procedure for preparing a glucosamine- and N-acetylgalactosamine-speci®c lectin from fruiting bodies of the edible shiitake mushroom L. edodes. The same group of researchers subsequently utilized the same protocol to isolate more L. edodes lectin for
1357-2725/99/$ - see front matter # 1999 Published by Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 9 9 ) 0 0 0 0 6 - 0
596
H.X. Wang et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 595±599
further studies [8]. The procedure involved extraction with 0.15 M NaCl, precipitation with 80% saturated (NH4)2SO4, dialysis, adsorption of lectin on DEAE Sephadex A-50 and elution with a salt gradient and ®nally adsorption on hydroxyapatite followed by elution with a phosphate gradient. The ®nal lectin preparation exhibited four sharp bands in polyacrylamide gel electrophoresis. The amino acid compositions of 3 fractions, namely, the sample ready for ion exchange chromatography on DEAE-Sephadex, the fraction eluted with 0.05 M NaCl from the DEAE-Sephadex column and the fraction eluted with 0.05 M phosphate from the hydroxyapatite column, were found to be disparate. The present investigation was undertaken with an aim to purify and characterize the L. edodes lectin in view of the apparent heterogeneity of the lectin prepared by the aforementioned group of investigators. The study was also prompted by the report about the mitogenic activity of the lectin [7, 8].
2. Materials and methods 2.1. Isolation of lectin Fresh fruiting bodies of L. edodes purchased from a local supermarket were extracted with saline solution. After centrifugation, (NH4)2SO4 was added to the supernatant to 45% saturation and the mixture was allowed to stand overnight at 48C. It was found that only a little additional lectin activity could be precipitated by (NH4)2SO4 at a saturation higher than 45%. The precipitate obtained using (NH4)2SO4 at 45% saturation was subsequently dialyzed against distilled water and lyophilized to form a crude powder. The powder was dissolved in 0.05 M Tris±Cl buer (pH 7.8) and applied on a column of Sephadex G-100 (1.090 cm) eluted with the same buer. The peak with lectin activity was collected, dialyzed and chromatographed on a column of N-acetylgalactosamine-agarose equilibrated with 0.01 M phosphate buer (pH 7.1). Lectin activity was bound on the immobilized
sugar and eluted with 0.5 M N-acetylgalactosamine. 2.2. Hemagglutinating activity A serial two-fold dilution of the lectin solution in microtiter U-plates (50 ml) was mixed with 50 ml of a 2% suspension of rabbit erythrocytes in pH 7.2 phosphate buered saline at room temperature. The results were read after about 1 h when the blank had fully sedimented. The hemagglutination titer, de®ned as the reciprocal of the highest dilution exhibiting hemagglutination, was reckoned as one hemagglutination unit. Speci®c activity is the number of hemagglutination units per mg protein. 2.3. Test of hemagglutination inhibition by various carbohydrates The hemagglutination inhibition tests were performed in a manner analogous to the hemagglutination test. Serial twofold dilutions of sugar samples were prepared in phosphate buered saline. All of the dilutions were mixed with an equal volume (25 ml) of a solution of the lectin with 8 hemagglutination units. The mixture was allowed to stand for 30 min at room temperature and then mixed with 50 ml of a 2% rabbit erythrocyte suspension. The minimum concentration of the sugar in the ®nal reaction mixture which completely inhibited 8 hemagglutination units of the lectin preparation was calculated. 3. Results The results of chromatography of L. edodes crude powder on Sephadex G-100 are illustrated in Fig. 1. When the column fractions were monitored for hemagglutinating activity, it was found that the peaks of hemagglutinating activity did not coincide with the absorbance peaks. Two peaks of such bioactivity were detected, one eluted in the void volume and slightly ahead of the ®rst absorbance peak and a larger lectin peak with a molecular weight of 43 kDa lagging behind the second absorbance peak (Fig. 1). The
H.X. Wang et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 595±599
597
Fig. 1. Gel ®ltration of proteins derived from the (NH4)2SO4 precipitate of L. edodes fruiting body extract. Dimensions of Sephadex G-100 column: 190 cm. Buer = 0.05 M NH4HCO3, pH 8.5. The column had been calibrated with standard molecular weight markers; blue dextran (for determination of void volume), bovine serum (MW 67 kDa), ovalbumen (MW 43 kDa), chymotrypsinogen A (MW 25 kDa) and cytochrome C (MW 12.4 kDa).
peak with a molecular weight of 43 kDa was used for anity chromatography on N-acetylgalactosamine-agarose. The fraction adsorbed on the immobilized sugar contained a much more potent hemagglutinating activity compared with the large unadsorbed fraction (Fig. 2). The yields and speci®c hemagglutinating activities of the dierent chromatographic fractions are presented in Table 1. The fraction with molecular weight 43 kDa was subjected to cation exchange and anion exchange chromatography on a large number of chromatographic media including DEAE-cellulose, DEAE-Sepharose, DEAE-cellulose, Dowex, CM-cellulose and CM-Sepharose, SP-Sepharose and Amberlite. Hemagglutinating activity was largely unbound on the cation exchangers resulting in little puri®cation and so tightly bound on the anion exchangers that elution with concentrated salt solutions or buers with low pH was not useful in desorbing the activity. Chromatography on phenyl Sepharose and octyl agarose, adsorption chromatography on hydro-
xyapatite, anity chromatography on wheat germ lectin-Sepharose and p-aminophenyl-D-glucopyranoside agarose and chromatography on protein G-Sepharose, A-gel Blue gel and Red Sepharose were all of little use in purifying the lectin (data not shown). The activity was either largely unadsorbed or tightly adsorbed depending on the medium employed. N-acetylglucosamine, N-acetylgalactosamine and mannose inhibited 8 hemagglutinating titers of the L. edodes lectin preparation only at a dose of 0.5 M. Other sugars were devoid of inhibitory activity. 4. Discussion Previous studies conducted by Jeune et al. [7] and Moon et al. [8] resulted in a preparation of L. edodes lectin which was not homogeneous as witnessed in the presence of multiple bands in polyacrylamide gel electrophoresis. The absence of Asp, Thr and Gly residues from the amino acid composition of the ®nal lectin preparation
598
H.X. Wang et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 595±599
Fig. 2. Anity chromatography of peak 2 of lectin activity from Sephadex G-100 column on N-acetylgalactosamine-agarose column. Column dimensions: 15 cm. Starting buer: 0.01 M phosphate buer, pH 7.1. Arrow indicates the point at which 0.5 M Nacetylgalactosamine in the starting buer was applied.
and the presence of the same residues in large amounts in the lectin-containing fraction of a previous chromatographic step, infers that a substantial quantity of impurities was removed by chromatography on hydroxyapatite. By contrast, the results of our chromatographic experiments were not in accord with these early reports. Our data indicate that L. edodes lectin could not be puri®ed with the use of hydroxyapatite, DEAESephadex and a host of other anion exchangers owing to the fact that the lectin was very tightly bound on these chromatographic media and Table 1 Hemagglutinating activities of L. edodes chromatographic fractions (from 1 kg of fresh fruiting body) against rabbit erythrocytes Fraction
Yield (mg)
Speci®c hemagglutinating activity (titer/mg)
Crude powder Fraction A Fraction B Fraction B1 Fraction B2
5000 1320 1150 810 5
85 500 980 100 3700
could not be recovered even when concentrated salt solutions and pH changes were applied. The use of cation exchangers and hydrophobic interaction chromatographic media (phenyl Sepharose and octyl agarose) was to little avail in the puri®cation of the lectin. The lectin was mainly unadsorbed on the cation exchangers tested. Depending on the conditions of elution, the lectin was either largely unadsorbed or tenaciously bound on the hydrophobic interaction chromatographic column. Other chromatographic media tested including protein G Sepharose, A-gel Blue gel and Red Sepharose could not adsorb the lectin either. The protocol found to be satisfactory for the puri®cation of L. edodes lectin involved two main steps, gel ®ltration on Sephadex G-100 and anity chromatography on N-acetylgalactosamineagarose, neither of which had been utilized by Jeune et al. [7] and Moon et al. [8]. It can be deduced from the elution volume of the lectin from the Sephadex G-100 column and its mobility in SDS-PAGE that the lectin is a singlechained protein with a molecular weight of 43 kDa.
H.X. Wang et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 595±599
Another special feature of the L. edodes lectin is that it did not interact strongly with carbohydrates. All in all, L. edodes lectin is a unique lectin on account of its weak interaction with sugars. A procedure has been devised in this study to isolate a lectin from L. edodes fruiting bodies. It was found that, unlike other lectins, ion exchange chromatography was not useful for purifying this lectin. Acknowledgements The authors would like to express their thanks to the Research Grants Council, Government of Hong Kong for the award of an Earmarked Grant and to the Research Committee, Chinese University of Hong Kong, for the award of a postdoctoral fellowship to HXW. The excellent secretarial assistance of Ms. Christine Chung in the preparation of this manuscript is appreciated.
599
References [1] N. Sharon, H. Lis, Lectins: cell-agglutinating and sugarspeci®c proteins, Science 177 (1972) 949±959. [2] I.J. Goldstein, R.C. Hughes, M. Monsigny, T. Osawa, N. Sharon, What should be called a lectin?. Nature (London) 285 (1980) 66. [3] H. Franz, Mistletoe lectins and their A and B chains, Oncology 43 ((Suppl.)) (1986) 23±24. [4] N. Sharon, H. Lis, Lectins as cell recognition molecules, Science 246 (1989) 227±234. [5] J. Guillot, G. Konska, Lectins in higher fungi, Biochem. Syst. Ecol. 25 (1997) 203±230. [6] H.X. Wang, T.B. Ng, V.E.C. Ooi, Lectins from mushroom, Mycol. Res. 102 (1998) 897±906. [7] H.K. Jeune, I.J. Moon, M.K. Kim, S.R. Chung, Studies on lectins from Korean higher fungi: IV. A mitogenic lectin from the mushroom Lentinus edodes, Planta Med. 56 (1990) 592. [8] I.J. Moon, S.R. Chung, K.H. Jeune, Mitotic stimulation and cancer cell agglutination of the lectin from Lentinus edodes, Yakhak Hoeji 39 (1995) 260±267.
The International Journal of Biochemistry & Cell Biology 31 (1999) 595±599
Studies on puri®cation of a lectin from fruiting bodies of the edible shiitake mushroom Lentinus edodes H.X. Wang a, T.B. Ng b, *, V.E.C. Ooi a a
Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, People's Republic of China Department of Biochemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, People's Republic of China
b
Received 17 August 1998; accepted 30 November 1998
Abstract A lectin, with a speci®city for N-acetylgalactosamine and N-acetylglucosamine and a molecular weight of 43 kDa, was isolated from fruiting bodies of the edible shiitake mushroom Lentinus edodes. The puri®cation procedure entailed extraction with aqueous buer, ammonium sulfate precipitation, gel ®ltration on Sephadex G-100 and anity chromatography on N-acetylgalactosamine-agarose. The lectin was unique in that it was tenaciously bound on anion exchangers including DEAE-cellulose, DEAE-Sepharose, DEAE-Sephadex, Q-Sepharose, Dowex and PEIcellulose and also on hydroxyapatite and phenyl Sepharose. It was largely unadsorbed on cation exchangers including CM-cellulose, CM-Sepharose, SP-Sepharose and Amberlite, and also on protein G-Sepharose, Red Sepharose and A-gel Blue gel, wheat germ lectin-Sepharose and p-aminophenyl-D-glucopyranoside agarose. # 1999 Published by Elsevier Science Ltd. All rights reserved.
1. Introduction Lectins are elaborated by a diversity of living organisms including animals, plants and microorganisms. They are proteins or glycoproteins which, because of their speci®c binding to sugars, have been exploited in structural studies on the cell surface, oligosaccharides and/or carbohydrate * Corresponding author. Tel.: +852-2609-63-59; fax: 8522603-72-06; e-mail: [email protected].
moieties of glycoproteins [1±4]. Mushrooms represent a rich source of lectins and the physicochemical and biochemical characteristics together with the biological activities of lectins from a variety of mushroom species have recently been reviewed [5, 6]. Jeune et al. [7] reported a procedure for preparing a glucosamine- and N-acetylgalactosamine-speci®c lectin from fruiting bodies of the edible shiitake mushroom L. edodes. The same group of researchers subsequently utilized the same protocol to isolate more L. edodes lectin for
1357-2725/99/$ - see front matter # 1999 Published by Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 9 9 ) 0 0 0 0 6 - 0
596
H.X. Wang et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 595±599
further studies [8]. The procedure involved extraction with 0.15 M NaCl, precipitation with 80% saturated (NH4)2SO4, dialysis, adsorption of lectin on DEAE Sephadex A-50 and elution with a salt gradient and ®nally adsorption on hydroxyapatite followed by elution with a phosphate gradient. The ®nal lectin preparation exhibited four sharp bands in polyacrylamide gel electrophoresis. The amino acid compositions of 3 fractions, namely, the sample ready for ion exchange chromatography on DEAE-Sephadex, the fraction eluted with 0.05 M NaCl from the DEAE-Sephadex column and the fraction eluted with 0.05 M phosphate from the hydroxyapatite column, were found to be disparate. The present investigation was undertaken with an aim to purify and characterize the L. edodes lectin in view of the apparent heterogeneity of the lectin prepared by the aforementioned group of investigators. The study was also prompted by the report about the mitogenic activity of the lectin [7, 8].
2. Materials and methods 2.1. Isolation of lectin Fresh fruiting bodies of L. edodes purchased from a local supermarket were extracted with saline solution. After centrifugation, (NH4)2SO4 was added to the supernatant to 45% saturation and the mixture was allowed to stand overnight at 48C. It was found that only a little additional lectin activity could be precipitated by (NH4)2SO4 at a saturation higher than 45%. The precipitate obtained using (NH4)2SO4 at 45% saturation was subsequently dialyzed against distilled water and lyophilized to form a crude powder. The powder was dissolved in 0.05 M Tris±Cl buer (pH 7.8) and applied on a column of Sephadex G-100 (1.090 cm) eluted with the same buer. The peak with lectin activity was collected, dialyzed and chromatographed on a column of N-acetylgalactosamine-agarose equilibrated with 0.01 M phosphate buer (pH 7.1). Lectin activity was bound on the immobilized
sugar and eluted with 0.5 M N-acetylgalactosamine. 2.2. Hemagglutinating activity A serial two-fold dilution of the lectin solution in microtiter U-plates (50 ml) was mixed with 50 ml of a 2% suspension of rabbit erythrocytes in pH 7.2 phosphate buered saline at room temperature. The results were read after about 1 h when the blank had fully sedimented. The hemagglutination titer, de®ned as the reciprocal of the highest dilution exhibiting hemagglutination, was reckoned as one hemagglutination unit. Speci®c activity is the number of hemagglutination units per mg protein. 2.3. Test of hemagglutination inhibition by various carbohydrates The hemagglutination inhibition tests were performed in a manner analogous to the hemagglutination test. Serial twofold dilutions of sugar samples were prepared in phosphate buered saline. All of the dilutions were mixed with an equal volume (25 ml) of a solution of the lectin with 8 hemagglutination units. The mixture was allowed to stand for 30 min at room temperature and then mixed with 50 ml of a 2% rabbit erythrocyte suspension. The minimum concentration of the sugar in the ®nal reaction mixture which completely inhibited 8 hemagglutination units of the lectin preparation was calculated. 3. Results The results of chromatography of L. edodes crude powder on Sephadex G-100 are illustrated in Fig. 1. When the column fractions were monitored for hemagglutinating activity, it was found that the peaks of hemagglutinating activity did not coincide with the absorbance peaks. Two peaks of such bioactivity were detected, one eluted in the void volume and slightly ahead of the ®rst absorbance peak and a larger lectin peak with a molecular weight of 43 kDa lagging behind the second absorbance peak (Fig. 1). The
H.X. Wang et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 595±599
597
Fig. 1. Gel ®ltration of proteins derived from the (NH4)2SO4 precipitate of L. edodes fruiting body extract. Dimensions of Sephadex G-100 column: 190 cm. Buer = 0.05 M NH4HCO3, pH 8.5. The column had been calibrated with standard molecular weight markers; blue dextran (for determination of void volume), bovine serum (MW 67 kDa), ovalbumen (MW 43 kDa), chymotrypsinogen A (MW 25 kDa) and cytochrome C (MW 12.4 kDa).
peak with a molecular weight of 43 kDa was used for anity chromatography on N-acetylgalactosamine-agarose. The fraction adsorbed on the immobilized sugar contained a much more potent hemagglutinating activity compared with the large unadsorbed fraction (Fig. 2). The yields and speci®c hemagglutinating activities of the dierent chromatographic fractions are presented in Table 1. The fraction with molecular weight 43 kDa was subjected to cation exchange and anion exchange chromatography on a large number of chromatographic media including DEAE-cellulose, DEAE-Sepharose, DEAE-cellulose, Dowex, CM-cellulose and CM-Sepharose, SP-Sepharose and Amberlite. Hemagglutinating activity was largely unbound on the cation exchangers resulting in little puri®cation and so tightly bound on the anion exchangers that elution with concentrated salt solutions or buers with low pH was not useful in desorbing the activity. Chromatography on phenyl Sepharose and octyl agarose, adsorption chromatography on hydro-
xyapatite, anity chromatography on wheat germ lectin-Sepharose and p-aminophenyl-D-glucopyranoside agarose and chromatography on protein G-Sepharose, A-gel Blue gel and Red Sepharose were all of little use in purifying the lectin (data not shown). The activity was either largely unadsorbed or tightly adsorbed depending on the medium employed. N-acetylglucosamine, N-acetylgalactosamine and mannose inhibited 8 hemagglutinating titers of the L. edodes lectin preparation only at a dose of 0.5 M. Other sugars were devoid of inhibitory activity. 4. Discussion Previous studies conducted by Jeune et al. [7] and Moon et al. [8] resulted in a preparation of L. edodes lectin which was not homogeneous as witnessed in the presence of multiple bands in polyacrylamide gel electrophoresis. The absence of Asp, Thr and Gly residues from the amino acid composition of the ®nal lectin preparation
598
H.X. Wang et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 595±599
Fig. 2. Anity chromatography of peak 2 of lectin activity from Sephadex G-100 column on N-acetylgalactosamine-agarose column. Column dimensions: 15 cm. Starting buer: 0.01 M phosphate buer, pH 7.1. Arrow indicates the point at which 0.5 M Nacetylgalactosamine in the starting buer was applied.
and the presence of the same residues in large amounts in the lectin-containing fraction of a previous chromatographic step, infers that a substantial quantity of impurities was removed by chromatography on hydroxyapatite. By contrast, the results of our chromatographic experiments were not in accord with these early reports. Our data indicate that L. edodes lectin could not be puri®ed with the use of hydroxyapatite, DEAESephadex and a host of other anion exchangers owing to the fact that the lectin was very tightly bound on these chromatographic media and Table 1 Hemagglutinating activities of L. edodes chromatographic fractions (from 1 kg of fresh fruiting body) against rabbit erythrocytes Fraction
Yield (mg)
Speci®c hemagglutinating activity (titer/mg)
Crude powder Fraction A Fraction B Fraction B1 Fraction B2
5000 1320 1150 810 5
85 500 980 100 3700
could not be recovered even when concentrated salt solutions and pH changes were applied. The use of cation exchangers and hydrophobic interaction chromatographic media (phenyl Sepharose and octyl agarose) was to little avail in the puri®cation of the lectin. The lectin was mainly unadsorbed on the cation exchangers tested. Depending on the conditions of elution, the lectin was either largely unadsorbed or tenaciously bound on the hydrophobic interaction chromatographic column. Other chromatographic media tested including protein G Sepharose, A-gel Blue gel and Red Sepharose could not adsorb the lectin either. The protocol found to be satisfactory for the puri®cation of L. edodes lectin involved two main steps, gel ®ltration on Sephadex G-100 and anity chromatography on N-acetylgalactosamineagarose, neither of which had been utilized by Jeune et al. [7] and Moon et al. [8]. It can be deduced from the elution volume of the lectin from the Sephadex G-100 column and its mobility in SDS-PAGE that the lectin is a singlechained protein with a molecular weight of 43 kDa.
H.X. Wang et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 595±599
Another special feature of the L. edodes lectin is that it did not interact strongly with carbohydrates. All in all, L. edodes lectin is a unique lectin on account of its weak interaction with sugars. A procedure has been devised in this study to isolate a lectin from L. edodes fruiting bodies. It was found that, unlike other lectins, ion exchange chromatography was not useful for purifying this lectin. Acknowledgements The authors would like to express their thanks to the Research Grants Council, Government of Hong Kong for the award of an Earmarked Grant and to the Research Committee, Chinese University of Hong Kong, for the award of a postdoctoral fellowship to HXW. The excellent secretarial assistance of Ms. Christine Chung in the preparation of this manuscript is appreciated.
599
References [1] N. Sharon, H. Lis, Lectins: cell-agglutinating and sugarspeci®c proteins, Science 177 (1972) 949±959. [2] I.J. Goldstein, R.C. Hughes, M. Monsigny, T. Osawa, N. Sharon, What should be called a lectin?. Nature (London) 285 (1980) 66. [3] H. Franz, Mistletoe lectins and their A and B chains, Oncology 43 ((Suppl.)) (1986) 23±24. [4] N. Sharon, H. Lis, Lectins as cell recognition molecules, Science 246 (1989) 227±234. [5] J. Guillot, G. Konska, Lectins in higher fungi, Biochem. Syst. Ecol. 25 (1997) 203±230. [6] H.X. Wang, T.B. Ng, V.E.C. Ooi, Lectins from mushroom, Mycol. Res. 102 (1998) 897±906. [7] H.K. Jeune, I.J. Moon, M.K. Kim, S.R. Chung, Studies on lectins from Korean higher fungi: IV. A mitogenic lectin from the mushroom Lentinus edodes, Planta Med. 56 (1990) 592. [8] I.J. Moon, S.R. Chung, K.H. Jeune, Mitotic stimulation and cancer cell agglutination of the lectin from Lentinus edodes, Yakhak Hoeji 39 (1995) 260±267.