Alveolarin, a novel antifungal polypeptide from the wild mushroom Polyporus alveolaris

Peptides 25 (2004) 693–696

Short communication

Alveolarin, a novel antifungal polypeptide from the wild mushroom Polyporus alveolaris Hexiang Wang a , T.B. Ng b,∗ , Qinghong Liu a a

State Key Laboratory of Agrobiotechnology, Beijing and Department of Microbiology, College of Biological Science, Chain Agricultural University, Beijing, China b Department of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China Received 17 September 2003; accepted 14 January 2004

Abstract An antifungal polypeptide, with a molecular mass of 28 kDa as judged by gel filtration and appearing as a single band with a molecular mass of 14 kDa in sodium dodecyl suflate-polyacrylamide gel electrophoresis, was isolated from fresh fruiting bodies of the mushroom Polyporus alveolaris. The antifungal polypeptide, designated as alveolarin, demonstrated an inhibitory action on mycelial growth in Botrytis cinerea, Fusarium oxysporum, Mycosphaerella arachidicola and Physalospora piricola. Alveolarin was isolated with a procedure that entailed ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, and gel filtration on Superdex 75 by fast protein liquid chromatography. © 2004 Elsevier Inc. All rights reserved. Keywords: Alveolarin; Antifungal polypeptide; Polyporus alveolaris

1. Introduction Antifungal proteins have been intensively studied because they have the potential of protecting organisms from the deleterious consequences of fungal attack. In crops the economic implications are substantial. Animals and plants produce a host of antifungal peptides and proteins with a spectacular diversity of structures, including thaumatin-like proteins [1,13], embryo-abundant proteins [9], allergen-like peptides [16], chitinases, glucanases [2,7], miraculin-like proteins [15], cyclophilin-like proteins [14], protease inhibitors [18], peroxidases [20], lectins [19], arginine- and glutamate-rich proteins [12], ribosome inactivating proteins and peptides [5,6], ribonucleases [4,8] and deoxyribonucleases [10]. In contrast to the voluminous literature on antifungal proteins originating from animals and flowering plants, knowledge about mushroom antifungal proteins is meager and restricted to only a few reports [2,5]. The objective of the present investigation was to isolate an antifungal ∗ Corresponding author. Tel.: +86-852-26096872; fax: +86-852-26035123. E-mail address: [email protected] (T.B. Ng).

0196-9781/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2004.01.026

polypeptide from the wild mushroom Polyporus alveolaris, and to compare its characteristics with known mushroom and angiosperm antifungal proteins.

2. Materials and methods 2.1. Isolation of antifungal polypeptide Fresh fruiting bodies (800 g) of the hexagonal-pored polypore mushroom P. alveolaris growing on the trunk of a Chinese banyan tree (Ficus microcarpa) were collected. The fruiting bodies were soaked and then homogenized in distilled water (3 ml/g mushroom) using a Waring blender. To the supernatant obtained following centrifugation (12,000 × g for 30 min at 4 ◦ C) of the homogenate was added Tris–HCl buffer (pH 7.3) until the concentration of Tris reached 10 mM. The supernatant was applied to a column of DEAE-cellulose (5 cm × 25 cm) (Sigma) in 10 mM Tris–HCl (pH 7.3). The unadsorbed fraction (D1) eluted with the same buffer was collected and applied to a column of Affi-gel blue gel (2.5 cm × 20 cm) (Bio-Rad) in 10 mM Tris–HCl buffer (pH 7.3). After elution of unadsorbed proteins (B1), the affinity column was eluted with a linear

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concentration (0–1 M) gradient of NaCl in the Tris–HCl buffer. The adsorbed fraction (B2) thus obtained was dialyzed, lyophilized and then subjected to gel filtration on a Superdex 75 HR 10/30 column (Amersham Biosciences) by fast protein liquid chromatography (FPLC) using an AKTA Purifier (Amersham Biosciences). The first peak obtained constituted the purified antifungal polypeptide designated as alveolarin. 2.2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis In order to estimate the molecular mass of the purified polypeptide, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) was performed as described by Laemmli and Favre [3]. FPLC on a Superdex 75 HR 10/30 column (Amersham Biosciences), which had been calibrated with molecular mass markers, was also conducted. The markers included bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa) and ␣-lactalbumin (14.4 kDa). The N-terminal sequence of the antifungal polypeptide was determined by using a Hewlett–Packard HP G1000A Edman degradation unit and an HP 1000 HPLC System [4].

sorbance increase at 260 nm of 1 min−1 in the acid-soluble fraction per ml of reaction mixture under the specified conditions. 2.5. Assay for deoxyribonuclease activity The reaction mixture consisted of 0.2 ml of 0.1 M NH4 OAc buffer (pH 5.5), 0.2 ml of a herring sperm DNA solution (Gibco BRL) (5 mg/ml) and 10 ␮l of the solution of the antifungal polypeptide (5 mg/ml). Incubation of the reaction mixture was carried out at 25 ◦ C for 15 min. The reaction was terminated by addition of 0.3 ml of ice-cold 20 mM lanthanum nitrate in 1.2% (v/v) perchloric acid. After 20 min at 0 ◦ C the reaction mixture was centrifuged at 3000 × g for 5 min. The supernatant was diluted three-fold with water and the optical density was read at 260 nm against a blank reaction mixture without the antifungal polypeptide. One unit of enzymatic activity is defined as the amount of enzyme which produces an absorbance increase at 260 nm of 0.001 min−1 ml−1 at pH 5.5 and 37 ◦ C using herring sperm DNA. It is slightly different from 1 Kunitz unit of DNase activity which is based on an assay conducted at a pH of 5, a temperature of 25 ◦ C and DNA type 1 as substrate [11].

2.3. Assay for antifungal activity 2.6. Assay for lectin activity In the assay for antifungal activity, sterile petri plates (100 mm × 15 mm) containing 10 ml potato dextrose agar were used. After the mycelial colony had developed, sterile blank paper disks (0.625 cm in diameter) were placed at a distance of 1 cm from the rim of the mycelial colonly. A 15 ␮l aliquot of the test sample in 0.05 M MES buffer (pH 6.0) was applied to a disk. Incubation of the petri plate was carried out at 23 ◦ C for 72 h until mycelial growth had surrounded peripheral disks containing the control and had generated crescents of inhibition around disks with antifungal samples. Four fungal species, Botrytis cinerea, Fusarium oxysporum, Mycosphaerella arachidicola and Physalospora piricola were examined in the assay [13]. 2.4. Assay for ribonuclease activity The activity toward tRNA was estimated by measuring the production of acid-soluble, UV-absorbing species as previously described [4]. Yeast tRNA (200 ␮g) was incubated with the isolated antifungal polypeptide in 150 ␮l 100 mM MES buffer (pH 6.0) at 37 ◦ C for 15 min. The reaction was terminated by introducing 350 ␮l of ice-cold 3.4% (v/v) perchloric acid. After standing on ice for 15 min, the mixture was centrifuged at 15,000 × g for 15 min at 4 ◦ C. The absorbance of the supernatant, after suitable dilution, was measured at 260 nm. One unit of ribonuclease activity is defined as the amount of ribonuclease that produces an ab-

In the assay for lectin (hemagglutinating) activity, a serial two-fold dilution of the solution of the test sample (antifungal polypeptide) in microtiter U-plates (50 ␮l) was mixed with 50 ␮l of a 2% suspension of rabbit red blood cells in phosphate buffered saline (pH 7.2) at 20 ◦ C. The results were recorded after about 1 h when the blank had fully sedimented [11]. 2.7. Assay of protease activity A solution of casein (Sigma), which was used as substrate in the protease assay, was freshly prepared as follows. To 2 g casein 10 ml distilled water and 10 ml 0.2 M NaOH were added. After addition of another 60 ml distilled water, the mixture was stirred to make a solution. The pH of the solution was adjusted to 7.5 and the solution was heated at 90 ◦ C for 15 min before cooling down and dilution with 100 ml 10 mM Tris–HCl (pH 8) containing 40 mM CaCl2 . The precipitate was removed and the resulting solution could be used. The test sample (antifungal polypeptide) or trypsin solutin (50 ␮l) was mixed with 350 ␮l of the above casein solution. After 25 min 1 ml 5% (w/v) trichloroacetic acid was added. The reaction mixture was allowed to stand at room temperature for 30 min before centrifugation for 15 min. The absorbance of the supernatant was read at 280 nm against water as blank [11].

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Fig. 2. Antifungal activity of alveolarin toward Physalospora piricola. (A) Control (15 ␮l 0.05 M MES buffer pH 6.0), (B) 120 ␮g alveolarin in 15 ␮l MES buffer, (C) 24 ␮g alveolarin in 15 ␮l Mes buffer.

4. Discussion

Fig. 1. (a) Affinity chromatography of fraction D1 on an Affi-gel blue gel column. Starting buffer: 10 mM Tris–HCl buffer (pH 7.3). Dotted line across the chromatogram represented the 0–1 M NaCl gradient used to elute fraction B2. (b) Gel filtration of fraction B2 on a Superdex 75 HR 10/30 column. Buffer: 0.2 M NH4 HCO3 (pH 8.5). Flow rate: 0.4 ml/min. Fraction size: 0.8 ml. Peak SU1 represented purified antifungal protein designated alveolarin.

3. Results Antifungal activity resided in fraction D1 of the water extract of fruiting bodies, and in fraction B2 derived by affinity chromatography of D1 on Affi-gel blue gel. Fraction D2 adsorbed on DEAE-cellulose and fraction B1 unadsorbed on Affi-gel blue gel were devoid of antifungal activity. Gel filtration of B2 on Superdex 75 yielded two peaks of approximately equal size, SU1 and SU2 (Fig. 1). Only SU1 possessed antifungal activity. SU1 appeared as a single band with a molecular mass of 14 kDa (data not shown), in contrast to an estimated molecular mass of 28 kDa obtained by gel filtration. SU1, designated as alveolarin, demonstrated the single N-terminal sequence GVCDMADLA. The data indicate that alveolarin is probably homodimeric. The antifungal activity of alveolarin toward P. piricola is presented in Fig. 2. The activity toward B. cinerea, F. oxysporum and M. arachidicola was similar. The yields of fractions D1, D2, B1, B2, SU1 (purified antifungal polypeptide) and SU2 were 198.6, 427.5, 73.2, 55.7, 19.0 and 18.2 mg, respectively, from 800 g fruiting bodies. Alveolarin was devoid of ribonuclease, deoxyribonuclease, lectin and protease activities when tested at 10, 25, 50 and 100 ␮g, respectively.

The antifungal polypeptide isolated in this investigation is novel in several aspects. Alveolarin is active against a number of fungal species such as B. cinerea, F. oxysporum, M. arachidicola and P. piricola. In contradistinction, Lyophyllum antifungal protein from the mushroom Lyophyllum shimeiji inhibits P. piricola and M. arachidicola but is inactive toward the fungi Coprinus comatus, Colleotrichum gossypii and Rhizoctonia solani [5]. Alveolarin probably consists of two identical subunits because only one N-terminal sequence is discernible and the molecular mass determined by gel filtration is double that obtained from SDS–PAGE. Red kidney bean lectin [19], and ribonucleases from American ginseng and Chinese ginseng [8], which exhibit antifungal activity, are homodimeric. The vast majority of antifungal proteins such as thaumatin-like proteins [1,13], miraculin-like proteins [15], embryo-abundant proteins [9], chitinases [7], glucanases [2,8], cyclophilin-like proteins [14], allergen-like peptides [14] and protease inhibitors [18] are monomeric. The subunit molecular mass of alveolarin is only about half of those of red kidney bean lectin [19] and ginseng ribonucleases [4,8]. Their spectra of biological activities also differ although they have antifungal activity in common. The N-terminal sequence of alveolarin is different from that of Lyophyllum antifungal protein from the mushroom L. shimeiji [5]. It is noteworthy that alveolarin resembles sativin, an antifungal protein from sugar snap seeds [15] to some extent in N-terminal sequence. This structural feature, together with the dimeric nature and the broad spectrum of the antifungal action of alveolarin, suggest that alveolarin and Lyophyllum antifungal protein are very different proteins although they are both of mushroom origin.

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Antifungal proteins exhibit a wide range of molecular masses, from a few kilodaltons in case of antifungal peptides [16] to 67 kDa in case of red kidney bean lectin [19]. The molecular mass of alveolarin, 28 kDa, lies in the middle of the range, and is similar to those of some of the chitinases [17]. Alveolarin can be isolated with a simple chromatographic procedure involving ion exchange chromatography on DEAE-cellulose as the first step, followed by affinity chromatography on Affi-gel blue gel, and finally by gel filtration on Superdex 75. The fraction unadsorbed on DEAE-cellulose can be directly chromatographed on Affi-gel blue gel without the need for dialysis, making the isolation procedure simple and easy. The chromatographic behavior of alveolarin on DEAE-cellulose and Affi-gel blue gel is similar to that exhibited by previously isolated antifungal proteins and peptides [8,10,12–20]. The observation that alveolarin is devoid of hemagglutinating, deoxyribonuclease and ribonuclease activities rules out the possibility that alveolarin is a lectin or a ribonuclease. It is known that some lectins [19], deoxyribonucleases [10], and ribonucleases [4] may exhibit antifungal activity. Alveolarin is also lacking in protease activity. In summary, an antifungal polypeptide with characteristics different from mushroom antifungal proteins isolated earlier [2,5] has been isolated from the wild mushroom P. alveolaris.

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