Purification and properties of a lectin from ascomycete mushroom, Ciborinia camelliae

Phytochemistry 60 (2002) 103–107 www.elsevier.com/locate/phytochem

Purification and properties of a lectin from ascomycete mushroom, Ciborinia camelliae Yumi Otta, Koh Amano, Kano Nishiyama, Akikazu Ando, Shigeru Ogawa, Yoshiho Nagata* Department of Bioresources Chemistry, Faculty of Horticulture, Chiba University, Matsudo 271-8510 Japan Received 24 October 2001; received in revised form 11 February 2002

Abstract A lectin was isolated from an ascomycete mushroom, Ciborinia camelliae which was specific to N-acetyl-d-galactosamine. On SDS-polyacrylamide gel electrophoresis; this lectin gave a single band of  17-kDa in the presence of 2-mercaptoethanol, but formed dimers, trimers and tetramers in its absence. Amino acid analysis revealed the lectin contained two cysteines and no methionine. The N-terminal sequence was determined up to residue 21, and no homologous proteins including other ascomycete lectins were found. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Ascomycete mushroom; Ascomycete lectin; Ciborinia camelliae lectin

1. Introduction Lectins are carbohydrate-binding proteins found in a variety of organisms including fungi. Although lectins are useful tools in the study of molecular cell biology, their physiological roles in fungi are not well understood (Guillot and Konska, 1997; Wang et al., 1998; Nagata, 2000). Many lectins have been found in mushrooms, but only a few in ascomycete mushrooms. We have investigated ascomycete lectins obtained from the fruiting bodies of Aleuria aurantia (AAL) (Fukumori et al., 1990; Nagata et al., 1991) and Melastiza chateri (MCL) (Ogawa et al., 2001). AAL has been widely used as a specific probe for fucose-containing oligosaccharides on the cell surface, and as a tool for isolation of glycoproteins, and fractionation of oligosaccharides (Yamashita et al., 1985; Debray and Montreuil, 1989). We have also studied the possible roles of lectins in Abbreviations: AAL, Aleuria aurantia lectin; AOL, Arthrobotrys oligospora lectin; CCL, Ciborinia camelliae lectin; GalNAc, N-acetyld-galactosamine; MCL, Melastiza chateri lectin; MSH, 2-mercaptoethanol; PBS, phosphate-buffered saline; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SSL, Sclerotinia scleotiorum lectin. * Corresponding author. Tel./fax: +81-47-308-8869. E-mail address: [email protected] (Y. Nagata).

basidiomycete and ascomycete mushrooms (Oguri et al., 1996; Ogawa et al., 1996, 1998). In this report, we describe the isolation and characterization of a new Ciborinia camelliae lectin (CCL). The properties of CCL were compared with those of other ascomycete lectins.

2. Results and discussion 2.1. Purification A summary of the process used to purify CCL is given in Table 1. Dialyzed crude extract prepared from the fruiting bodies of C. camelliae was applied to a hydroxyapatite column. CCL was adsorbed weakly on the column, and eluted with 10 mM phosphate buffer, pH 6.8 (Fig. 1). From 2 g of the fruiting body (fr. wt), 160 mg of the lectin was obtained. 2.2. Properties The sugar-binding specificity of CCL is shown in Table 2. The lectin recognized N-acetyl-d-galactosamine (GalNAc) as the most effective monosaccharide. CCL was also specific to d-galactose, d-galactosamine as well

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Table 1 Purification of CCLa Step

Total proteinb (mg)

Total titer (104 units)

Specific activity (103 units/mg)

Yield (%)

Crude extract Hydroxyapatite

4.65 0.16

4.16 2.28

9.0 141

100 55

a b

Starting from 2.0 g fr. wt of fruiting bodies. Protein was measured by the method of Bradford (1976).

as galactose-containing disaccharides. Other sugars and glycoproteins were ineffective. As shown in Fig. 2A, purified CCL gave a single band of ca. 17-kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) in the presence of 2-mercaptoethanol (MSH), while in its absence, CCL formed dimers, trimers and tetramers, suggesting that inter-subunit disulfide-bridges were formed. It should be noted that the hemagglutinating activity of CCL was not inhibited by MSH. Immunoblotting showed that CCL cross-reacted, although weakly, with anti-AAL serum (Fig. 2B). CCL is not a glycoprotein, since staining with performic acid-Schiff reagent (Zacharius et al., 1969) was negative (data not shown). The lectin activity was stable below 40  C and completely inactivated at 60  C. CCL was most stable at pH 6.0, and relatively stable over a broad pH range (data not shown). The amino acid composition of CCL is shown in Table 3 in comparison with other ascomycete lectins. Since the molecular mass of CCL was estimated to be 17-kDa from SDS-PAGE, the total number of amino acid residues could be calculated to be 134, giving a Mr of 16,830. The N-terminal amino acid sequence of CCL (accession No. PC7176) is shown in Fig. 3. Although CCL showed immunochemical similarity with AAL (Fig. 2B), no similarity was found between the sequences up to the 21st N-terminal residue. A homology search with this sequence identified no protein in the FASTA Search.

2.3. Comparison with other ascomycete lectins Only two ascomycete lectins, AAL (Kochibe and Furukawa, 1980) and MCL (Ogawa et al., 2001) have been isolated from fruiting bodies, though several have been purified from mycelia or sclerotia. One such lectin is AOL, a product of Arthrobotrys oligospora, a nematode-trapping fungus (Rosen et al., 1992, 1996). The sexual generation of this fungus was identified as Orbilia auricolor, an ascomycete mushroom of the order Heliotiales (Pfister, 1997). Ascomycete lectins were also isolated from mycelia of Neurospora crassa (Prick and Diekmann, 1979) and several species in Sclerotiniaceae (Kellens et al., 1992). Among ascomycete lectins, determination of the N-terminal amino acid sequence was done on three lectins, AAL, MCL and AOL. The sequence of CCL was compared with that of AAL and of AOL (Fig. 3), and the three sequences were found to be quite different. It was reported that the sequence of AAL was quite similar to that of MCL (Ogawa et al., 2001). The properties of the known ascomycete lectins are presented in Table 4. Although several ascomycete lectins have been isolated, all can be grouped into three types. The first group includes AAL and MCL, which are l-fucose-specific. The organisms producing these lectins belong to the same family, Pyronemataceae (Ogawa et al., 2001). The second group includes AOL

Table 2 Sugar-binding specificity of CCL Sugar

Minimum concentration for complete inhibition of hemagglutination (mM)a

N-Acetyl-d-galactosamine (GalNAc)4 Melibiose Lactose d-Galactose d-Galactosamine

12.5 12.5 12.5 25 50 100

a N-Acetyl-d-glucosamine, d-glucosamine, d-glucose, d-fructose, d-mannose, d- and l-arabinose, d- and l-fucose, d-xylose, maltose and sucrose exhibited no inhibition at concentrations up to 100 mM. Glycoproteins such as bovine submaxillary mucin, pocin stomach mucin, fetuin and asialofetuin exhibited no inhibition at concentrations up to 1 mg/ml.

Fig. 1. Purification of CCL. Crude extract of C. camelliae fruiting bodies was dialyzed against 10 mM phosphate buffer, pH 6.8, and applied to a hydroxyapatite column (18 cm). CCL was eluted with the same buffer, and absorbance at 280 nm and hemagglutinating activity were measured.

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Y. Otta et al. / Phytochemistry 60 (2002) 103–107 Table 3 Amino acid composition of CCL in comparison with other ascomycete lectins Amino acida

CCL

AAL

AOL

SSL

Asx Thr Ser Glx Pro Gly Ala Cysb Val Met Ile Leu Tyr Phe His Lys Arg Trpd

8.3 10.2 8.9 11.8 4.6 11.8 9.6 1.6 5.4 ndc 5.0 5.6 2.8 4.0 0.7 4.8 2.7 0.9

7.4 5.4 12.2 8.7 4.5 13.1 9.6 0.6 6.1 0 7.4 3.8 4.2 2.9 0.6 4.5 3.8 5.1

10.9 7.3 5.1 12.9 2.5 11.6 8.1 0 6.0 0 6.6 8.7 5.0 3.2 3.5 3.7 4.8 –e

15.9 7.1 7.7 8.3 6.8 6.3 9.7 0.8 4.3 0.2 2.6 3.1 8.8 3.1 1.1 10.2 4.0 –

References

This study

Fukumori et al. (1990)

Rosen et al. (1992)

Kellens et al. (1992)

a b c d e

Values are expressed in mol%. Determined as cysteic acid. Not detectable. Determined spectrophotometrically. Not determined.

Fig. 2. SDS–PAGE and immunoblotting. (A) CCL was resolved by SDS–PAGE using 12.5% gel in the presence (1.3 mg; lane 1) or absence (5 mg; lane 2) of MSH. The gel was stained with Coomassie brilliant blue. (B) Purified CCL (1.3 mg; lanes 2 and 4) and AAL (5 mg; lanes 1 and 3) were resolved by SDS–PAGE, and stained with Coomassie Brilliant Blue (lanes 1 and 2). After being transferred to the membrane, they were reacted with anti-AAL serum (500 dilution), and visualized using alkaline phosphatase (lanes 3 and 4).

Table 4 Comparison of ascomycete lectins Lectin

CCL

AAL

MCL

AOL

Ciborinia camelliae fruiting body M.W. of subunit 17 Sugar-binding specificity GalNAc Cross-reactivity with anti-AAL Yes Expression in Fruiting body Yes Mycelium nd

Aleuria aurantia fruiting body 34 l-Fucose Yes

Melastiza chateri fruiting body 40 l-Fucose Yes

Arthrobotrys oligospora Sclerotinia sclerotiorum mycelium mycelium 15 17 Fetuin GalNAc nda nd

Yes Yes

Yes No

nd Yes

References

Ogawa et al. (1998) Ogawa et al. (2001) Rosen et al. (1996)

Source

a

This study

SSL

nd Yes Kellens et al. (1992)

Not determined.

Fig. 3. N-Terminal amino acid sequences of CCL, AOL and AAL. Amino acids identical in at least two lectins are indicated by black boxes. The amino acids were numbered taking the N-terminal residue as 1.

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which was a product of the nematode-trapping fungus. Although no similar lectin has been found in ascomycete fungi, a lectin having similar amino acid sequence was found in a basidiomycete mushroom, Agaricus bisporus (Rosen et al., 1996). The third group includes GalNAc-specific lectins produced by the members of Sclerotiniaceae. Lectins of this family are similar to CCL in terms of the molecular mass of the subunit and sugar-binding specificity. C. camelliae, the producer of CCL, belongs to the same family but another genus. To investigate the physiological roles of lectin in fungus, the expression of lectin genes in fruiting bodies and mycelia was studied, however, some fungus produced lectin in mycelia, but not all did, indicating that there is no general rule in the expression of lectin genes.

activity was assayed. For that of pH stability, CCL in the universal buffer (Johnson and Lindsey, 1939) was incubated for 2 h, and neutralized with 10-fold concentrated PBS, after which the residual activity was assayed. 3.5. Amino acid analysis and protein sequencing Amino acids were determined in a Hitachi Model 835 Analyzer. CCL (80 mg) was hydrolyzed in 6 M HCl containing 0.01% phenol in a sealed evacuated tube for 24 h at 105  C. Cysteine was measured as cysteic acid after performic acid oxidation (Hirs, 1967). Tryptophan was determined spectrophotometrically (Edelhoch, 1967). N-Terminal amino acid sequencing was done with an automated protein sequencer (model PPSQ-10, Shimadzu, Kyoto, Japan).

3. Experimental 3.1. Materials

Acknowledgements

Fruiting bodies of C. camelliae were collected at our campus in Chiba Prefecture, Japan, and stored at 80  C until use. The mushroom grows on the fallen petals of camellia, hence its name. The chemicals used were of the highest grade commercially available. (GalNAc)4 was a generous gift from Higeta Shoyu Co. Ltd.

A part of this work was supported financially by Higeta Shoyu Co., Ltd.

3.2. Hemagglutination and inhibition assay The lectin activity was titrated as reported (Fukumori et al., 1990) by serially diluting the sample (20 ml) with phosphate-buffered saline (PBS: 8 mM Na2HPO4, 1.5 mM KH2PO4, 137 mM NaCl, and 2.7 mM KCl, pH7.2), then mixing it with an equal volume of a 2% suspension of rabbit erythrocytes. After standing at room temperature for 1 h, hemagglutinating activity was determined. For inhibition assays, lectin solution (titer 8) was incubated with the test sugar, which was serially diluted with PBS. 3.3. SDS-PAGE and immunoblotting SDS-PAGE was performed as described before (Oguri et al., 1996) on 12.5% gel in the presence or absence of MSH. After each run, protein bands were stained with Coomassie brilliant blue R-250 (Merck). The preparation of anti-AAL serum and the immunoblotting were carried out as reported (Ogawa et al., 1998). 3.4. Temperature and pH stability For the determination of thermostability, CCL (titer 32) in PBS was heated for 10 min at designated temperatures before being cooled on ice, and the residual

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