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Purification and characterization of a lectin from the mushroom Mycoleptodonoides aitchisonii
Phytochemistry 56 (2001) 53±58
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Puri®cation and characterization of a lectin from the mushroom Mycoleptodonoides aitchisonii Hirokazu Kawagishi *, Jun-ichi Takagi, Tomoko Taira, Takeomi Murata, Taichi Usui Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan Received 14 March 2000; received in revised form 26 July 2000
Abstract A lectin was isolated from the mushroom Mycoleptodonoides aitchisonii by means of anity chromatography on bovine submaxillary mucin (BSM)-Toyopearl and gel ®ltration on Superose 12 HR10/30 using a FPLC system. This lectin is composed of four identical 16 kDa subunits and the molecular mass of the intact lectin was estimated to be 64 kDa by gel ®ltration. In a hemagglutination inhibition assay, it exhibited strong sugar-binding speci®city towards asialo-BSM among the mono- or oligo-saccharides and glycoproteins tested. The binding speci®city of the lectin was also examined by surface plasmon resonance analysis. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Mycoleptodonoides aitchisonii; Climacodontaceae; Mushrooms; Fruiting body; Lectin; Surface plasmon resonance
1. Introduction
2. Results and discussion
Mushrooms can be de®ned as macrofungi with distinctive fruiting bodies which are either epigenous or hypogenous and are suciently conspicuous in size to the naked eye to be hand-picked (Chang and Miles, 1992; Wang et al., 1998). Many lectins have been isolated from mushrooms (Kawagishi, 1995; Wang et al., 1998) and some macrofungi produce them not only at their fruiting body-stages but also at the myceliumstages (Kino et al., 1989; Tanaka et al., 1989; Guillot et al., 1991; Kawagishi et al., 1997). However, the roles of lectins in fungi are still unknown. In the course of our continuing screening for mushroom lectins with novel carbohydrate speci®city, we found strong lectin activity in the extract of the edible mushroom Mycoleptodonoides aitchisonii. This paper reports the isolation and characterization of this lectin from this source.
The isolation procedure employed is summarized in Table 1. Thus, initially a saline extract of the fruiting bodies of M. aitchisonii was adjusted to 80% saturation with ammonium sulfate. Since the resulting extract exhibited no remarkable binding speci®city to any monosaccharides but showed speci®city against asialoBSM and BSM in an hemagglutination inhibition assay, BSM-Toyopearl was considered as an anity support. Accordingly, after centrifugation, the precipitate obtained by ammonium sulfate precipitation was applied to a BSM-Toyopearl column. All the lectin activity was adsorbed onto the column but could be eluted with 1 M NaCl. The lectin-containing fraction was then further puri®ed by FPLC using Superose 12, to give the puri®ed M. aitchisonii lectin (MAL). MAL displayed a single band on SDS-PAGE regardless of the presence (lane 1) or absence (lane 2) of 2mercaptoethanol (Fig. 1). Gel ®ltration of the lectin on Superose 12 gave a symmetrical single peak and the molecular weight of the intact protein was estimated to be 64,000 by gel ®ltration (Fig. 2). These results suggested the lectin to be composed of four identical subunits without any S±S linkages. Amino acid analysis using acid hydrolysis revealed a high content of acidic amino acids and Gly, a low
Abbreviations: MAL, Mycoleptodonoides aitchisonii lectin; PBS, 10 mM phosphate-buered saline (pH 7.4); HBS, 10 mM HEPES buffered saline (pH 7.4); BSM, bovine submaxillary mucin; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; ABEE, paminobenzoic ethyl ether; TFA, tri¯uoroacetic acid; NP, p-nitrophenyl; All sugars are of d-con®guration unless otherwise stated. * Corresponding author. Tel. fax: +81-54-238-4885. E-mail address: [email protected] (H. Kawagishi).
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(00)00351-4
54
H. Kawagishi et al. / Phytochemistry 56 (2001) 53±58
content of Met and Phe, and no detectable Tyr (Table 2). Although as much as 3 nmol of the protein was applied to a protein sequencer, the N-terminal amino acid could not be determined, suggesting that its N-terminus was blocked. Isoelectric focusing gave a few bands in the pH
zone 4.3±4.5 (Fig. 3) and the carbohydrate content of the protein was circa 4.9%. These results showed that the lectin was an acidic glycoprotein. The components of the sugar in the protein were determined as GlcNAc, Gal, Man, and Xyl (1.56, 4.89, 3.68, 1.00 in molar ratio)
Table 1 Puri®cation of MAL (from 6 kg of the fruiting bodies) Step
Total protein (mg)
Total agglutination activity (titer)a
Speci®c agglutination activity (titer/mg)
Recovery of activity (%)
1. 80% Ammonium sulfate precipitation 2. Eluate from BSM-Toyopearl 3. Eluate from Superose 12
328.0 37.0 29.8
83,968 37,888 30,515
256 1024 1024
100 45.1 36.3
a
Measured with type A neuraminidase-treated blood cell. Table 2 Amino acid composition of MAL (see Section 3) Amino acid
Mol%
Amino acid
Mol%
Asx Thr Ser Glx Gly Ala Val Cys Met
12.1 5.9 7.3 10.2 17.7 7.5 6.2 3.8 0.3
Ile Leu Tyr Phe Lys His Arg Pro Trp
3.4 4.4 0.0 1.8 5.9 2.7 3.5 7.3 n.d.a
a
Not determined.
Fig. 1. SDS-PAGE of MAL. Lane 1 MAL (reduced with 2-mercaptoethanol); lane 2, MAL (non-reduced); lane M, marker.
Fig. 2. Gel ®ltration of MAL on a Superose 12 HR10/30 column.
Fig. 3. Isoelectric focusing of MAL; Lane 1, MAL, lane M, marker.
H. Kawagishi et al. / Phytochemistry 56 (2001) 53±58
by the analysis of their ABEE-derivatives. As far as we are aware, among mushroom lectins, there are only 2 lectins whose sugar compositions have been reported (Sueyoshi et al., 1985; Zhuang et al., 1996); ABA-I to IV from Agaricus bisporus had Fuc, Man, Gal and GlcNAc and APL from Amanita pantherina contained Glc, Man, Xyl and GlcNAc. Thus, the type of sugar chain observed for MAL has not yet been known as a sugar chain of mushroom lectins. The lectin activity was stable between pH 4.0 and 9.0, and below 45 C, but was rapidly deactivated over 50 C (not shown). EDTA treatment, or addition of metal cations such as CaCl2, MgCl2 and MnCl2, showed no eect on lectin activity, suggesting that MAL activity was not dependent on metal cations or that the lectin was already bound very tightly to metal cations. While MAL did not agglutinate any type of human erythrocytes, it was bound tightly to sialidase-treated erythrocytes regardless of blood type; the titer of the lectin (10 mg/ml) was 256 for all types of the enzymetreated erythrocytes. The sugar-binding speci®city of MAL was assayed by the hemagglutination inhibition method (Table 3), but it did not bind to any of the mono- or oligo-saccharides tested. Among the glycoproteins examined, asialo-BSM was the strongest inhibitor. BSM also inhibited hemagglutination by the lectin. Asialofetuin was bound to the lectin at a much higher concentration than asialo-BSM and BSM. Transferrin, a1-acid glycoprotein, and fetuin did not inhibit the hemagglutination by MAL. In addition, asialo-BSM was treated with trypsin, pronase, glycopeptidase A, and hydrazine. The resulting reaction mixtures showed a much weaker inhibitory activity
55
toward MAL-mediated hemagglutination at the same concentrations than intact asialo-BSM (data not shown). From this result, we could not therefore elucidate the detailed carbohydrate structure recognized by MAL. However both this and the hemagglutination assay indicated that MAL mainly recognized asialo-mucin type sugar chains, and that more than one sugar chain (close to each other in the glycoprotein) were necessary for the binding of MAL to the glycoprotein. This speculation was con®rmed by surface plasmon resonance analysis using BIAcore 2000. Four glycoproteins were immobilized on the sensor chip CM-5 by amine coupling. The sensorgrams and the kinetic data of the binding are summarized in Fig. 4 and Table 3, respectively. The binding of the lectin to all the glycoproteins ®tted best a heterogeneous ligand model among the various models displayed in the evaluation software. Therefore two kinetic parameters were given for each glycoproteins. The association rates (Kon) of the binding between four immobilized glycoproteins and the lectin were similar to each other. However, asialo-BSM and BSM showed slower dissociation rates (Ko) than asialo-fetuin and fetuin. The results of degradation of asialo-BSM, the hemagglutination inhibition assay and the BIAcore analysis may thus be explained in terms of either multivalent binding of MAL to glycoproteins, or an allosteric eect of the lctin; indeed, MAL caused a conformational change after binding of one sugar binding site to the lectin to the ®rst sugar chain of the glycoprotein, as well as being bound to the second dierent sugar chain. Accordingly, the binding was considered to be through a heterogeneous ligand. Interestingly, the degraded asialo-BSM did not bind to the lectin strongly.
Table 3 Comparison of kinetic parameters obtained from the interactions of immobilized glycoproteins with MAL by using BIAcore with the result of inhibition of MAL-mediated hemagglutination by the glycoproteins Ligands (inhibitors)a
Kon (Mÿ1 Sÿ1)
Ko (Sÿ1)
Rmax (RU)b
Kd (M)
Asialo-BSM
1.74 103 1.71 102
6.33 10ÿ4 3.00 10ÿ5
7.77 10 2.42 102
3.63 10ÿ7 1.76 10ÿ7
0.015
BSM
5.09 105 2.43 103
2.32 10ÿ4 2.57 10ÿ3
1.93 10 3.33 10
4.57 10ÿ10 1.06 10ÿ6
0.49
Asialo-fetuin
5.68 103 4.54 103
4.58 10ÿ2 2.93 10ÿ3
1.02 102 9.92 10
8.06 10ÿ6 6.45 10ÿ7
Fetuin
6.77 103 1.61 103
3.44 10ÿ3 2.10 10ÿ3
3.14 10 4.43 102
5.08 10ÿ7 1.31 10ÿ6
a
Hemagglutination inhibition activity (mg/ml)c
62.5 n.i.d
Glucosec, galactose, mannose, fucose, l-fucose, xylose, methyl a-glucoside, methyl b-glucoside, N-acetylglucosamine, N-acetylgalactosamine, methyl a-mannoside, methyl b-mannoside, l-arabinose, rhamnose, mannitol, N-acetylneuramic acid, lactose, N-acetyllactosamine, lactitol, lactulose, riburose, maltose, and sorbitol did not inhibit at all at concentrations up to 400 mM. Hyaluronic acid, a1-acid glycoprotein,transferrin, fetuin, Galb (1!3) GlcNAc, Galb(1!3)GalNAc, Galb (1!4) GlcNAc, GlcNAcb (1!3) Gal-bpNP, GlcNAcb (1!6) Gal-bpNP, Fuca (1!3) GlcNAc, Galb (1!3) Galb (1!4) GalNAcl-bpNP and Galb (1!4)Galb (1!4) GalNAcl-bpNP did not inhibit at all at concentrations up to 1 mg/ml. b Maxumum binding capacity of MAL to the surface. c Minimum inhibitor concentration required for inhibition of 4 hemagglutination doses of the lectin. d n.i.; not inhibited at all at concentrations up to 1.0 mg/ml.
56 H. Kawagishi et al. / Phytochemistry 56 (2001) 53±58
Fig. 4. Sensorgrams showing the interaction between MAL and immobilized glycoproteins. [A] asialo-BSM, [B] BSM, [C] asialo-fetuin, [D] fetuin.
H. Kawagishi et al. / Phytochemistry 56 (2001) 53±58
To our knowledge, this is the ®rst lectin isolated from a family Climacodontaceae. 3. Experimental 3.1. Materials M. aitchisonii fruiting bodies were collected at Narusawa village, Yamanashi Prefecture, Japan, frozen upon collection and stored at ÿ30 C. The fungus was identi®ed by one of the authors (H.K.). Amino-Toyopearl 650M was purchased from Tosoh (Japan). FPLC system and Superose 12 HR10/30 column were products of Pharmacia (Sweden). BIAcore 2000 was a product of Biacore AB (Sweden). Galb1!3GlcNAc, Galb1!4GlcNAc, Galb1!6GlcNAc, Galb1!3GalNAc, Galb1! 4GalNAc, Galb1!6GalNAc, and Fucb1!3GlcNAc were synthesized by enzymatic methods (Hedbys, et al., 1989; Sakai et al., 1992; Usui et al., 1993). All other sugars for the hemagglutinating inhibition tests were products of Nacalai Tesque (Japan), Wako Pure Chemical (Japan) or Sigma (USA). All glycoproteins used were purchased from Sigma (USA). ABEE reagent and the Honenpak C18 reveresed-phase HPCC column were obtained from Honen Corporation (Japan). 3.2. Preparation of the anity adsorbent BSM was conjugated to Amino-Toyopearl 650M by following the instructions of manufacturer. 3.3. Isolation of MAL All procedures were carried out at 4 C except for defrosting of the fruiting bodies. After defrosting, fruiting bodies of M. aitchisonii were homogenized with saline (1.0 l per 100 g fruiting bodies) in a blender and extracted with stirring overnight. The resulting suspension was ®ltered through the ®ltrate was centrifuged (10,000 g) to remove insoluble residues. Solid ammonium sulfate was next added to the supernatant to 80% saturation. The resulting precipitate was collected by centrifugation (10,000 g, 20 min), resuspended in distilled water, dialyzed against distilled water and lyophilized. The lyophilized extract was redissolved in 10 mM PBS (pH 7.4) and applied to a BSM-Toyopearl column equilibrated in the same buer. After all unbound substances were removed by washing the column with buffer, the bound fraction was then desorbed by elution with 1 M NaCl. The eluates were concentrated by ultra®ltration, dialyzed against PBS, and further puri®ed by gel ®ltration on Superose 12 HR10/30 using a FPLC system in PBS. The hemagglutinating fraction was dialyzed against distilled water and lyophilized, to give the puri®ed (MAL) lectin.
57
3.4. Hemagglutination test A 10% suspension of erythrocytes in PBS (10 ml) was treated with sialidase (10 ml, 1 U/ml) for 1 h at 37 C, then washed three times with PBS and suspended at a concentration of 3% in PBS. Agglutination of the erythrocytes by the lectin and inhibition of the agglutination by sugars and glycoproteins were carried out using microtiter U-plates. For the hemagglutination assay, erythrocyte-suspensions were added to lectin solutions of various concentrations in the wells, with reaction mixtures incubated for 1 h at room temperature. The titer was de®ned as the reciprocal of the end-point dilution causing hemagglutination. For the hemagglutination inhibition assay, lectin solutions of titer 4 were added to each sugar solutions of various concentrations, and the reaction mixtures were incubated for 1 h at room temperature. To the mixtures, erythrocyte-suspensions were added and further incubated for 1 h at room temperature. Inhibition was expressed as the minimum concentration of each sugar or glycoprotein required for inhibition of hemagglutination of titer 4 of the lectin. 3.5. SDS-PAGE SDS-PAGE was carried out by the method of Laemmli (Laemmli, 1970). Samples were heated in the presence or absence of 2-mercaptoethanol for 10 min at 100 C. Gels were stained with Coomassie Brilliant Blue. The molecular weight standards (Pharmacia, Sweden) used were phosphorylase b (Mr 94,000), albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), trypsin inhibitor (20,100), and a-lactalbumin (14,400). 3.6. Gel ®ltration for molecular weight estimations Gel ®ltration for measuring the molecular weights of native lectin was carried out on a Superose 12 HR10/30 column with a FPLC system. The molecular weight standards (Pharmacia, Sweden) used were ferritin (440,000), catalase (232,000), bovine serum albumin (67,000), ovalbumin (43,000), chymotrypsinogen A (25,000), and ribonuclease A (13,700). 3.7. Sugar analysis Sugar contents were measured by the phenol-sulfuric acid method with reference to glucose. Sugar compositions were determined as follows; puri®ed lectin (200 mmg) was dissolved in 20 ml distilled water in a test tube to which 4 M TFA (20 ml) was added. The test tube was incubated at 100 C in a hot block bath. After 4 h, the tube was cooled to room temperature and the acid was removed by using a centrifugal concentrator at 35 C. The dried sample was
58
H. Kawagishi et al. / Phytochemistry 56 (2001) 53±58
derivatized with ABEE in the presence of borane±pyridine complex at 80 C. After 1 h, the reaction mixture was cooled to room temperature. The distilled water (0.2 ml) and an equal volume of chloroform were added to the reaction mixture. After vigorous vortexing, it was centrifuged (6000 g, 1 min). The upper aqueous layer was analyzed by reversed±phase HPLC under the condition as follows; column, Honenpak C18 (75 mm 4.6 mm I.D.); solvent, A 0.02% TFA/CH3CN(90/10), B 0.02% TFA/CH3CN (50:50); program, 0±45 min (B conc. 0%), 45±55 min (B conc. 100%), 55±70 min (B conc. 0%); ¯ow rate, 1 ml/min; temp., 45 C; detection, absorbance at 305 nm. The monosaccharide and amino monosaccharide standards used were N-acetylglucosamine, N-acetylgalactosamine, galactose, glucose, mannose, xylose, and fucose. 3.8. Amino acid analysis The lectin was hydrolyzed with 6 M HCl at 110 C for 24 h in a sealed evacuated tube and analyzed on a Hitachi L-8500A amino acid analyzer. The cysteine and methionine contents were determined by oxidation of the lectin with performic acid followed by hydrolysis under the same condition to that of the intact protein and analysis on the analyzer. 3.9. N-terminal amino acid analysis Attempts to obtain the N-terminal amino acid of the protein used a PPSQ-10 Protein Peptide Sequencer (Shimadzu, Japan). 3.10. Thermostability Samples in PBS were heated for 30 min at the temperatures indicated, cooled on ice, and titrated. 3.11. pH stability The pH dependence of the lectin was measured by incubating the samples in the following buers for 24 h at room temperature, 0.02 M sodium acetate buer (pH 3.5±5.5), 0.02 M sodium phosphate buer (pH 6.0±7.5), 0.02 M Tris±HCl Buer (pH 8.0±9.0), and 0.02 M glycine±NaOH buer (9.5±11.0). 3.12. Eect of metal cations on lectin activity To examine metal cation requirements of the hemagglutination by the lectin, the sample (100 mg/ml) was incubated in 2 mM EDTA for 1 h at room temperature and titrated.
3.13. Surface plasmon resonance analysis Real time detection of MAL binding to glycoproteins was recorded by using a BIAcore 2000. Glycoproteins were immobilized covalently via their primary amines to a carboxyl group within a dextran layer on the sensor chip CM-5 according to the manufacture's speci®cations and our previous reported method (Zeng et al. 1998). MAL at various concentrations in HBS were injected over immobilized glycoproteins at 20 ml/min. After injection of the protein, HBS was introduced onto the sensor surface to initiate dissociation. The data were analyzed by BIAevaluation 3.0 software (Biacore AB, Sweden).
References Chang, S.T., Miles, P.G., 1992. Mushroom biology Ð a new discipline. The Mycologist 6, 64±65. Guillot, J., Giollant, M., Damez, M., Dusser, M., 1991. Isolation and characterization of a lectin from the mushroom Lactarius deliciosus. J. Biochem. (Tokyo) 109, 840±845. Hedbys, L., Johansson, E., Mosbach, K., Larsson, P.-O., Gunnarsson, A., Svensson, S., LoÈnn, H., 1989. Synthesis of Galb1-3GlcNAc and GAlb1-3GlcNAcb-SEt by an enzymatic method comprising the sequential use of b-galactosidases from bovine testes and Escherichia coli. Glycoconjugate J., 161±168. Kawagishi, H., 1995. Mushroom lectins. Food Rev. Int 11, 63±68. Kawagishi, H., Mitsunaga, S., Yamawaki, M., Ido, M., Shimada, A., Kinoshita, T., Murata, T., Usui, T., Kimura, A., Chiba, S., 1997. A lectin from the mycelia of a fungus Ganoderma lucidum. Phytochemistry 44, 7±10. Kino, K., Yamashita, A., Yamaoka, K., Watanabe, J., Tanaka, S., Ko, K., Shimizu, K., Tsunoo, H., 1989. Isolation and characterization of a new immunomodulatory protein, Ling Zhi-8 (LZ-8), from Ganoderma lucidum. J. Biol. Chem. 264, 472±478. Laemmli, U., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680±685. Sakai, K., Katsumi, K., Ohi, H., Usui, T., Ishido, Y., 1992. Enzymatic synthesis of N-acetyllactosamine and N-acetylallolactosamine by the use b-d-galactosidases. J. Carbohydr. Chem. 11, 553±565. Sueyoshi, S., Tsuji, T., Osawa, T., 1985. Puri®cation and characterization of four isolectins of mushroom (Agaricus bisporus). Biol. Chem. Hoppe-Seyler 366, 213±221. Tanaka, S., Ko, K., Kino, K., Tsuchiya, K., Yamashita, A., Murasugi, A., Sakuma, S., Tsunoo, H., 1989. Complete amino acid sequence of an immunomodulatory protein, Ling Zhi-8 (LZ-8). J. Biol. Chem. 264, 6372±16777. Usui, T., Kubota, S., Ohi, H., 1993. A convenient synthesis of b-dgalactosyl disaccharide derivatives using the b-d-galactosidase from Bacillus circulans. Carbohydr. Res. 244, 315±323. Wang, H., Ng, T.B., Ooi, V.E., 1998. Lectins from mushrooms. Mycol. Res. 102, 897±906. Zeng, X., Murata, T., Kawagishi, H., Usui, T., Kobayashi, K., 1998. Analysis of speci®c interactions of synthetic glycopolypeptides carrying N-acetyllactosamine and related compounds with lectins. Carbohydr. Res. 312, 209±217. Zhuang, C., Murata, T., Usui, T., Kawagishi, H., Kobayashi, K., 1996. Puri®cation and characterization of a lectin from the toxic mushroom Amanita pantherina. Biochim. Biophys. Acta 1291, 40±44.
www.elsevier.com/locate/phytochem
Puri®cation and characterization of a lectin from the mushroom Mycoleptodonoides aitchisonii Hirokazu Kawagishi *, Jun-ichi Takagi, Tomoko Taira, Takeomi Murata, Taichi Usui Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan Received 14 March 2000; received in revised form 26 July 2000
Abstract A lectin was isolated from the mushroom Mycoleptodonoides aitchisonii by means of anity chromatography on bovine submaxillary mucin (BSM)-Toyopearl and gel ®ltration on Superose 12 HR10/30 using a FPLC system. This lectin is composed of four identical 16 kDa subunits and the molecular mass of the intact lectin was estimated to be 64 kDa by gel ®ltration. In a hemagglutination inhibition assay, it exhibited strong sugar-binding speci®city towards asialo-BSM among the mono- or oligo-saccharides and glycoproteins tested. The binding speci®city of the lectin was also examined by surface plasmon resonance analysis. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Mycoleptodonoides aitchisonii; Climacodontaceae; Mushrooms; Fruiting body; Lectin; Surface plasmon resonance
1. Introduction
2. Results and discussion
Mushrooms can be de®ned as macrofungi with distinctive fruiting bodies which are either epigenous or hypogenous and are suciently conspicuous in size to the naked eye to be hand-picked (Chang and Miles, 1992; Wang et al., 1998). Many lectins have been isolated from mushrooms (Kawagishi, 1995; Wang et al., 1998) and some macrofungi produce them not only at their fruiting body-stages but also at the myceliumstages (Kino et al., 1989; Tanaka et al., 1989; Guillot et al., 1991; Kawagishi et al., 1997). However, the roles of lectins in fungi are still unknown. In the course of our continuing screening for mushroom lectins with novel carbohydrate speci®city, we found strong lectin activity in the extract of the edible mushroom Mycoleptodonoides aitchisonii. This paper reports the isolation and characterization of this lectin from this source.
The isolation procedure employed is summarized in Table 1. Thus, initially a saline extract of the fruiting bodies of M. aitchisonii was adjusted to 80% saturation with ammonium sulfate. Since the resulting extract exhibited no remarkable binding speci®city to any monosaccharides but showed speci®city against asialoBSM and BSM in an hemagglutination inhibition assay, BSM-Toyopearl was considered as an anity support. Accordingly, after centrifugation, the precipitate obtained by ammonium sulfate precipitation was applied to a BSM-Toyopearl column. All the lectin activity was adsorbed onto the column but could be eluted with 1 M NaCl. The lectin-containing fraction was then further puri®ed by FPLC using Superose 12, to give the puri®ed M. aitchisonii lectin (MAL). MAL displayed a single band on SDS-PAGE regardless of the presence (lane 1) or absence (lane 2) of 2mercaptoethanol (Fig. 1). Gel ®ltration of the lectin on Superose 12 gave a symmetrical single peak and the molecular weight of the intact protein was estimated to be 64,000 by gel ®ltration (Fig. 2). These results suggested the lectin to be composed of four identical subunits without any S±S linkages. Amino acid analysis using acid hydrolysis revealed a high content of acidic amino acids and Gly, a low
Abbreviations: MAL, Mycoleptodonoides aitchisonii lectin; PBS, 10 mM phosphate-buered saline (pH 7.4); HBS, 10 mM HEPES buffered saline (pH 7.4); BSM, bovine submaxillary mucin; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; ABEE, paminobenzoic ethyl ether; TFA, tri¯uoroacetic acid; NP, p-nitrophenyl; All sugars are of d-con®guration unless otherwise stated. * Corresponding author. Tel. fax: +81-54-238-4885. E-mail address: [email protected] (H. Kawagishi).
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(00)00351-4
54
H. Kawagishi et al. / Phytochemistry 56 (2001) 53±58
content of Met and Phe, and no detectable Tyr (Table 2). Although as much as 3 nmol of the protein was applied to a protein sequencer, the N-terminal amino acid could not be determined, suggesting that its N-terminus was blocked. Isoelectric focusing gave a few bands in the pH
zone 4.3±4.5 (Fig. 3) and the carbohydrate content of the protein was circa 4.9%. These results showed that the lectin was an acidic glycoprotein. The components of the sugar in the protein were determined as GlcNAc, Gal, Man, and Xyl (1.56, 4.89, 3.68, 1.00 in molar ratio)
Table 1 Puri®cation of MAL (from 6 kg of the fruiting bodies) Step
Total protein (mg)
Total agglutination activity (titer)a
Speci®c agglutination activity (titer/mg)
Recovery of activity (%)
1. 80% Ammonium sulfate precipitation 2. Eluate from BSM-Toyopearl 3. Eluate from Superose 12
328.0 37.0 29.8
83,968 37,888 30,515
256 1024 1024
100 45.1 36.3
a
Measured with type A neuraminidase-treated blood cell. Table 2 Amino acid composition of MAL (see Section 3) Amino acid
Mol%
Amino acid
Mol%
Asx Thr Ser Glx Gly Ala Val Cys Met
12.1 5.9 7.3 10.2 17.7 7.5 6.2 3.8 0.3
Ile Leu Tyr Phe Lys His Arg Pro Trp
3.4 4.4 0.0 1.8 5.9 2.7 3.5 7.3 n.d.a
a
Not determined.
Fig. 1. SDS-PAGE of MAL. Lane 1 MAL (reduced with 2-mercaptoethanol); lane 2, MAL (non-reduced); lane M, marker.
Fig. 2. Gel ®ltration of MAL on a Superose 12 HR10/30 column.
Fig. 3. Isoelectric focusing of MAL; Lane 1, MAL, lane M, marker.
H. Kawagishi et al. / Phytochemistry 56 (2001) 53±58
by the analysis of their ABEE-derivatives. As far as we are aware, among mushroom lectins, there are only 2 lectins whose sugar compositions have been reported (Sueyoshi et al., 1985; Zhuang et al., 1996); ABA-I to IV from Agaricus bisporus had Fuc, Man, Gal and GlcNAc and APL from Amanita pantherina contained Glc, Man, Xyl and GlcNAc. Thus, the type of sugar chain observed for MAL has not yet been known as a sugar chain of mushroom lectins. The lectin activity was stable between pH 4.0 and 9.0, and below 45 C, but was rapidly deactivated over 50 C (not shown). EDTA treatment, or addition of metal cations such as CaCl2, MgCl2 and MnCl2, showed no eect on lectin activity, suggesting that MAL activity was not dependent on metal cations or that the lectin was already bound very tightly to metal cations. While MAL did not agglutinate any type of human erythrocytes, it was bound tightly to sialidase-treated erythrocytes regardless of blood type; the titer of the lectin (10 mg/ml) was 256 for all types of the enzymetreated erythrocytes. The sugar-binding speci®city of MAL was assayed by the hemagglutination inhibition method (Table 3), but it did not bind to any of the mono- or oligo-saccharides tested. Among the glycoproteins examined, asialo-BSM was the strongest inhibitor. BSM also inhibited hemagglutination by the lectin. Asialofetuin was bound to the lectin at a much higher concentration than asialo-BSM and BSM. Transferrin, a1-acid glycoprotein, and fetuin did not inhibit the hemagglutination by MAL. In addition, asialo-BSM was treated with trypsin, pronase, glycopeptidase A, and hydrazine. The resulting reaction mixtures showed a much weaker inhibitory activity
55
toward MAL-mediated hemagglutination at the same concentrations than intact asialo-BSM (data not shown). From this result, we could not therefore elucidate the detailed carbohydrate structure recognized by MAL. However both this and the hemagglutination assay indicated that MAL mainly recognized asialo-mucin type sugar chains, and that more than one sugar chain (close to each other in the glycoprotein) were necessary for the binding of MAL to the glycoprotein. This speculation was con®rmed by surface plasmon resonance analysis using BIAcore 2000. Four glycoproteins were immobilized on the sensor chip CM-5 by amine coupling. The sensorgrams and the kinetic data of the binding are summarized in Fig. 4 and Table 3, respectively. The binding of the lectin to all the glycoproteins ®tted best a heterogeneous ligand model among the various models displayed in the evaluation software. Therefore two kinetic parameters were given for each glycoproteins. The association rates (Kon) of the binding between four immobilized glycoproteins and the lectin were similar to each other. However, asialo-BSM and BSM showed slower dissociation rates (Ko) than asialo-fetuin and fetuin. The results of degradation of asialo-BSM, the hemagglutination inhibition assay and the BIAcore analysis may thus be explained in terms of either multivalent binding of MAL to glycoproteins, or an allosteric eect of the lctin; indeed, MAL caused a conformational change after binding of one sugar binding site to the lectin to the ®rst sugar chain of the glycoprotein, as well as being bound to the second dierent sugar chain. Accordingly, the binding was considered to be through a heterogeneous ligand. Interestingly, the degraded asialo-BSM did not bind to the lectin strongly.
Table 3 Comparison of kinetic parameters obtained from the interactions of immobilized glycoproteins with MAL by using BIAcore with the result of inhibition of MAL-mediated hemagglutination by the glycoproteins Ligands (inhibitors)a
Kon (Mÿ1 Sÿ1)
Ko (Sÿ1)
Rmax (RU)b
Kd (M)
Asialo-BSM
1.74 103 1.71 102
6.33 10ÿ4 3.00 10ÿ5
7.77 10 2.42 102
3.63 10ÿ7 1.76 10ÿ7
0.015
BSM
5.09 105 2.43 103
2.32 10ÿ4 2.57 10ÿ3
1.93 10 3.33 10
4.57 10ÿ10 1.06 10ÿ6
0.49
Asialo-fetuin
5.68 103 4.54 103
4.58 10ÿ2 2.93 10ÿ3
1.02 102 9.92 10
8.06 10ÿ6 6.45 10ÿ7
Fetuin
6.77 103 1.61 103
3.44 10ÿ3 2.10 10ÿ3
3.14 10 4.43 102
5.08 10ÿ7 1.31 10ÿ6
a
Hemagglutination inhibition activity (mg/ml)c
62.5 n.i.d
Glucosec, galactose, mannose, fucose, l-fucose, xylose, methyl a-glucoside, methyl b-glucoside, N-acetylglucosamine, N-acetylgalactosamine, methyl a-mannoside, methyl b-mannoside, l-arabinose, rhamnose, mannitol, N-acetylneuramic acid, lactose, N-acetyllactosamine, lactitol, lactulose, riburose, maltose, and sorbitol did not inhibit at all at concentrations up to 400 mM. Hyaluronic acid, a1-acid glycoprotein,transferrin, fetuin, Galb (1!3) GlcNAc, Galb(1!3)GalNAc, Galb (1!4) GlcNAc, GlcNAcb (1!3) Gal-bpNP, GlcNAcb (1!6) Gal-bpNP, Fuca (1!3) GlcNAc, Galb (1!3) Galb (1!4) GalNAcl-bpNP and Galb (1!4)Galb (1!4) GalNAcl-bpNP did not inhibit at all at concentrations up to 1 mg/ml. b Maxumum binding capacity of MAL to the surface. c Minimum inhibitor concentration required for inhibition of 4 hemagglutination doses of the lectin. d n.i.; not inhibited at all at concentrations up to 1.0 mg/ml.
56 H. Kawagishi et al. / Phytochemistry 56 (2001) 53±58
Fig. 4. Sensorgrams showing the interaction between MAL and immobilized glycoproteins. [A] asialo-BSM, [B] BSM, [C] asialo-fetuin, [D] fetuin.
H. Kawagishi et al. / Phytochemistry 56 (2001) 53±58
To our knowledge, this is the ®rst lectin isolated from a family Climacodontaceae. 3. Experimental 3.1. Materials M. aitchisonii fruiting bodies were collected at Narusawa village, Yamanashi Prefecture, Japan, frozen upon collection and stored at ÿ30 C. The fungus was identi®ed by one of the authors (H.K.). Amino-Toyopearl 650M was purchased from Tosoh (Japan). FPLC system and Superose 12 HR10/30 column were products of Pharmacia (Sweden). BIAcore 2000 was a product of Biacore AB (Sweden). Galb1!3GlcNAc, Galb1!4GlcNAc, Galb1!6GlcNAc, Galb1!3GalNAc, Galb1! 4GalNAc, Galb1!6GalNAc, and Fucb1!3GlcNAc were synthesized by enzymatic methods (Hedbys, et al., 1989; Sakai et al., 1992; Usui et al., 1993). All other sugars for the hemagglutinating inhibition tests were products of Nacalai Tesque (Japan), Wako Pure Chemical (Japan) or Sigma (USA). All glycoproteins used were purchased from Sigma (USA). ABEE reagent and the Honenpak C18 reveresed-phase HPCC column were obtained from Honen Corporation (Japan). 3.2. Preparation of the anity adsorbent BSM was conjugated to Amino-Toyopearl 650M by following the instructions of manufacturer. 3.3. Isolation of MAL All procedures were carried out at 4 C except for defrosting of the fruiting bodies. After defrosting, fruiting bodies of M. aitchisonii were homogenized with saline (1.0 l per 100 g fruiting bodies) in a blender and extracted with stirring overnight. The resulting suspension was ®ltered through the ®ltrate was centrifuged (10,000 g) to remove insoluble residues. Solid ammonium sulfate was next added to the supernatant to 80% saturation. The resulting precipitate was collected by centrifugation (10,000 g, 20 min), resuspended in distilled water, dialyzed against distilled water and lyophilized. The lyophilized extract was redissolved in 10 mM PBS (pH 7.4) and applied to a BSM-Toyopearl column equilibrated in the same buer. After all unbound substances were removed by washing the column with buffer, the bound fraction was then desorbed by elution with 1 M NaCl. The eluates were concentrated by ultra®ltration, dialyzed against PBS, and further puri®ed by gel ®ltration on Superose 12 HR10/30 using a FPLC system in PBS. The hemagglutinating fraction was dialyzed against distilled water and lyophilized, to give the puri®ed (MAL) lectin.
57
3.4. Hemagglutination test A 10% suspension of erythrocytes in PBS (10 ml) was treated with sialidase (10 ml, 1 U/ml) for 1 h at 37 C, then washed three times with PBS and suspended at a concentration of 3% in PBS. Agglutination of the erythrocytes by the lectin and inhibition of the agglutination by sugars and glycoproteins were carried out using microtiter U-plates. For the hemagglutination assay, erythrocyte-suspensions were added to lectin solutions of various concentrations in the wells, with reaction mixtures incubated for 1 h at room temperature. The titer was de®ned as the reciprocal of the end-point dilution causing hemagglutination. For the hemagglutination inhibition assay, lectin solutions of titer 4 were added to each sugar solutions of various concentrations, and the reaction mixtures were incubated for 1 h at room temperature. To the mixtures, erythrocyte-suspensions were added and further incubated for 1 h at room temperature. Inhibition was expressed as the minimum concentration of each sugar or glycoprotein required for inhibition of hemagglutination of titer 4 of the lectin. 3.5. SDS-PAGE SDS-PAGE was carried out by the method of Laemmli (Laemmli, 1970). Samples were heated in the presence or absence of 2-mercaptoethanol for 10 min at 100 C. Gels were stained with Coomassie Brilliant Blue. The molecular weight standards (Pharmacia, Sweden) used were phosphorylase b (Mr 94,000), albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), trypsin inhibitor (20,100), and a-lactalbumin (14,400). 3.6. Gel ®ltration for molecular weight estimations Gel ®ltration for measuring the molecular weights of native lectin was carried out on a Superose 12 HR10/30 column with a FPLC system. The molecular weight standards (Pharmacia, Sweden) used were ferritin (440,000), catalase (232,000), bovine serum albumin (67,000), ovalbumin (43,000), chymotrypsinogen A (25,000), and ribonuclease A (13,700). 3.7. Sugar analysis Sugar contents were measured by the phenol-sulfuric acid method with reference to glucose. Sugar compositions were determined as follows; puri®ed lectin (200 mmg) was dissolved in 20 ml distilled water in a test tube to which 4 M TFA (20 ml) was added. The test tube was incubated at 100 C in a hot block bath. After 4 h, the tube was cooled to room temperature and the acid was removed by using a centrifugal concentrator at 35 C. The dried sample was
58
H. Kawagishi et al. / Phytochemistry 56 (2001) 53±58
derivatized with ABEE in the presence of borane±pyridine complex at 80 C. After 1 h, the reaction mixture was cooled to room temperature. The distilled water (0.2 ml) and an equal volume of chloroform were added to the reaction mixture. After vigorous vortexing, it was centrifuged (6000 g, 1 min). The upper aqueous layer was analyzed by reversed±phase HPLC under the condition as follows; column, Honenpak C18 (75 mm 4.6 mm I.D.); solvent, A 0.02% TFA/CH3CN(90/10), B 0.02% TFA/CH3CN (50:50); program, 0±45 min (B conc. 0%), 45±55 min (B conc. 100%), 55±70 min (B conc. 0%); ¯ow rate, 1 ml/min; temp., 45 C; detection, absorbance at 305 nm. The monosaccharide and amino monosaccharide standards used were N-acetylglucosamine, N-acetylgalactosamine, galactose, glucose, mannose, xylose, and fucose. 3.8. Amino acid analysis The lectin was hydrolyzed with 6 M HCl at 110 C for 24 h in a sealed evacuated tube and analyzed on a Hitachi L-8500A amino acid analyzer. The cysteine and methionine contents were determined by oxidation of the lectin with performic acid followed by hydrolysis under the same condition to that of the intact protein and analysis on the analyzer. 3.9. N-terminal amino acid analysis Attempts to obtain the N-terminal amino acid of the protein used a PPSQ-10 Protein Peptide Sequencer (Shimadzu, Japan). 3.10. Thermostability Samples in PBS were heated for 30 min at the temperatures indicated, cooled on ice, and titrated. 3.11. pH stability The pH dependence of the lectin was measured by incubating the samples in the following buers for 24 h at room temperature, 0.02 M sodium acetate buer (pH 3.5±5.5), 0.02 M sodium phosphate buer (pH 6.0±7.5), 0.02 M Tris±HCl Buer (pH 8.0±9.0), and 0.02 M glycine±NaOH buer (9.5±11.0). 3.12. Eect of metal cations on lectin activity To examine metal cation requirements of the hemagglutination by the lectin, the sample (100 mg/ml) was incubated in 2 mM EDTA for 1 h at room temperature and titrated.
3.13. Surface plasmon resonance analysis Real time detection of MAL binding to glycoproteins was recorded by using a BIAcore 2000. Glycoproteins were immobilized covalently via their primary amines to a carboxyl group within a dextran layer on the sensor chip CM-5 according to the manufacture's speci®cations and our previous reported method (Zeng et al. 1998). MAL at various concentrations in HBS were injected over immobilized glycoproteins at 20 ml/min. After injection of the protein, HBS was introduced onto the sensor surface to initiate dissociation. The data were analyzed by BIAevaluation 3.0 software (Biacore AB, Sweden).
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