A hevein-like protein and a class I chitinasewith antifungal activity from leaves of thepaper mulberryMing Zhao,a Yan Ma,a Ying-Hong Pan,b* Chun-Hua Zhanga andWen-Xia Yuana

ABSTRACT: Paper mulberry (Broussonetia papyrifera, syn. Morus papyrifera L.) is a Chinese traditional medicine and its low-molecular-weight extracts are reported to have antifungal activity. In this study, two proteins (PMAPI and PMAPII) with activityagainst Trichoderma viride were obtained from paper mulberry leaves with a fast protein liquid chromatography (FPLC) unit.The purification protocol employed (NH4)2SO4 precipitation, ion-exchange chromatography and hydrophobic-interaction chro-matography on FPLC. Molecular masses were 18,798 Da for PMAPI, and 31,178 Da for PMAPII determined by Matrix-assistedlaser desorption ionization time-of-flight mass spectrometry. Peptide mapping fingerprint analysis showed that PMAPI has nopeptides similar to PMAPII. N-terminal amino acid sequencing revealed that PMAPI is a hevein-like protein, and PMAPII is aclass I chitinase. They both had a half-maximal inhibitory concentration (IC50) of 0.1 mg/mL against T. viride. This is the firstreport of high-molecular-weight extracts with antifungal activity from paper mulberry. Copyright © 2011 John Wiley & Sons,Ltd.

Keywords: antifungal protein; fast protein liquid chromatography; matrix-assisted laser desorption ionization time-of-flight massspectrometry; peptide mapping fingerprint analysis

IntroductionPaper mulberry (Broussonetia papyrifera, syn. Morus papyrifera L.)is a deciduous tree or shrub in the family Moraceaethat is native to eastern Asia, and widespread in China. Theroots, barks and fruits are all used in traditional Chinese medi-cine (Lee et al., 2001). The leaf juice is used to treat dysentery,and as a poultice for various skin disorders, and the leaves arereported to contain antibiotics (Duke and Ayensu, 1985).Wounded xylem tissue or fungal-infected shoot-corticaltissue of B. papyrifera produce a number of phytoalexinsincluding broussin, broussin-related derivatives, spirobrous-sonins A, spirobroussonins B, demethylbroussin, broussinol,broussonins A, broussonins B and broussonins A–F. Thesecompounds show antifungal activity against Bipolarisleersiae, Diaporthe nomurai or Fusarium solani (Gottstein andGross, 1992).

During a screening of antifungal compounds fromChinese medicinal herbs, we found that the growths of fungalmycelia were inhibited by the fractions with molecular massesmore than 3000 Da from leaves of the paper mulberry.This suggested that the high-molecular-weight extracts frompaper mulberry may have antifungal activity. To verify this, thepotential high-molecular-weight antifungal extracts from theleaves of wild paper mulberry were investigated on an FPLC.Two antifungal proteins, called PMAPI and PMAPII (papermulberry antifungal protein, PMAP) were purified. Homologysearches of the N-terminal sequences revealed that PMAPIis a hevein-like protein, and PMAPII belongs to classI chitinase.

Experimental

Materials

The wild paper mulberry grows around the Institute of Plant Protection,Chinese Academy of Agricultural Sciences, Beijing. The fresh leaves werecollected for further investigation. Fungi were obtained from the NationalKey Laboratory for Biology of Plant Diseases and Pests, China.

Antifungal activity assay

Antifungal activity against six plant fungal pathogens (Trichoderma viride,Botrytis cinerea, Phoma asparagi Sacc, Magnaporthe grissea, Fusariumoxysporum f.sp. vasinfectum and Sclerotinia sclerotiorum) was assessed

* Correspondence to: Ying H. Pan, Institute of Crop Sciences, ChineseAcademy of Agricultural Sciences, Beijing 100081, China. E-mail: yhpan@caas.net.cn

a College of Pu-erh Tea, Yunnan Agricultural University, Kunming 650201,China

b Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing100081, China

Abbreviations used: DTT, DL-dithiothreitol; EDTA, ethylenediaminetet-raacetic acid; FPLC, fast protein liquid chromatography; IC50, half- maximalinhibitory concentration; MALDI-TOF MS, matrix-assisted laser desorptionionization time-of-flight mass spectrometry; PMAP, paper mulberry antifun-gal protein; PVPP, polyvinylpyrrolidone; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis.

Research article

Received 23 July 2010, Accepted 8 September 2010 Published online in Wiley Online Library: 26 January 2011

(wileyonlinelibrary.com) DOI 10.1002/bmc.1543

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using sterile Petri plates (9 ¥ 15 cm) containing 15 mL potato dextroseagar. After mycelial colonies developed, an Oxford cup was laid 1 cm fromthe mycelia colony rim. Protein aliquots in 10 mm Tris–HCl buffer (pH 7.2)were applied to the cup, and the plates incubated at 28°C for 48 h untilmycelia growth enveloped the periphery of the cup containing thecontrol, and had produced crescents of inhibition around the cup withthe antifungal samples.

To determine the half maximal inhibitory concentration (IC50) value forantifungal activity, the inhibition of fungal growth was determined for5 mL of six different doses (10, 1, 1 ¥ 10-1, 1 ¥ 10-2, 1 ¥ 10-3 and 1 ¥10-4 mg/mL) of protein.

Protein isolation

Wild paper mulberry leaves (5000 g) were ground to a powder in liquidnitrogen. Proteins were extracted from the powder after 3 h incubationwith 10.0 L of extraction buffer (30 mM Na2HPO4, 15 mM NaH2PO4, 150 mM

NaCl, 25 mM EDTA, 1.30 mM DTT and 0.05 mM PVPP), pH 7.2, at 4°C. Aftercentrifugation (12,000g, 20 min, 4°C), proteins in the supernatant wereprecipitated by adding ammonium sulfate to 40% saturation, and centri-fuging (12,000g, 20 min, 4°C). The precipitate was dissolved in, and dia-lyzed against, 25 mM sodium acetate buffer (pH 5.0), and subjected toion-exchange chromatography by fast protein liquid chromatography ona SP-Sepharose Fast-Flow (2.6 ¥ 30 cm) column (GE Healthcare) equili-brated with the sodium acetate buffer. After eluting unadsorbed proteins,

the column was eluted with 130 mM NaCl in sodium acetate buffer toyield adsorbed fractions. Fractions with antifungal activity were dilutedwith sodium acetate buffer with 1 M (NH4)2SO4, and subjected tohydrophobic-interaction chromatography on a Phenyl Sepharose 6 FastFlow (1.6 ¥ 34 cm; GE Healthcare) column equilibrated with 2 M (NH4)2SO4

in sodium acetate buffer (pH 5.0). The column was eluted with a linearconcentration (2–0 M) gradient of (NH4)2SO4 in sodium acetate buffer. Allfractions were analyzed for antifungal activity and active fractions weresubjected to hydrophobic-interaction chromatography on a Resource ISOcolumn (1 mL; GE Healthcare). The column was eluted with a linear con-centration (2–0 M) gradient of (NH4)2SO4 in sodium acetate buffer. Frac-tions with antifungal activity were dialyzed against sodium acetate bufferand subjected to ion-exchange chromatography on a Mono S column(1 mL; GE Healthcare) equilibrated with sodium acetate buffer, and elutedwith a linear concentration (0–0.12 M) gradient of NaCl in the sodiumacetate buffer. Fractions with antifungal activity were resubjected to ion-exchange chromatography on the Mono S column to yield a single peak.

Determination of molecular mass

Homogeneity and molecular mass of the purified antifungal proteins wasestimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). Protein bands were stained with 0.1% Coomassie BrilliantBlue R250. Molecular mass marker proteins were from GE Healthcare.Matrix-assisted laser desorption ionization time-of-flight mass spectrom-

Figure 1. (A) Ion-exchange chromatography with a SP-Sepharose Fast-Flow column. (B) Hydrophobic-interaction chromatography of fraction S1 onPhenyl Sepharose 6 Fast-Flow. (C) Hydrophobic-interaction chromatography of fraction S1 on a Resrouce ISO column. (D) Ion-exchange chromatographyof fraction R1 on a Mono S column. (E) Ion-exchange chromatography of PMAPI on a Mono S column. (F) Ion-exchange chromatography of PMAPII ona Mono S column.

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etry (MALDI-TOF MS) was used determine the molecular mass of thehomogeneous proteins using an Applied Biosystems 4700 ProteomicsAnalyzer.

Peptide mapping fingerprint analysis

Lyophilized dried proteins were dissolved in 10 mM ammonium bicarbon-ate (pH 8.0), 10 mL of trypsin (12.5 mg/mL; Trypsin Gold, Mass Spectrom-etry grade, Promega Corp., USA) was added, and the mixtures agitated at37°C overnight (16 h). Tryptic peptides were analyzed by MALDI-TOF asabove.

N-terminal amino acid sequence analysis

Amino acid sequence analysis used an HP G1000A Edman degradationunit and an HP 1000 HPLC system from Hewlett/Packard (Palo Alto, CA,USA). Sequence homologies were determined with the BLASTp program(Altschul et al., 1997).

Results and discussion

Isolation of antifungal proteins

Two antifungal proteins named PMAPI and PMAPII were obtainedfrom the extracts from wild paper mulberry leaves, using a pro-cedure of (NH4)2SO4 precipitation, ion-exchange chromatographyon SP-Sepharose Fast-Flow (Fig. 1A), hydrophobic-interactionchromatography on Phenyl Sepharose 6 Fast-Flow (Fig. 1B) orResource ISO (Fig. 1C), and ion-exchange chromatography onMono S (Fig. 1D–F).

To compare the chromatographic features of PMAPI andPMAPII, each fraction was subjected to ion-exchange chromatog-raphy on Mono S columns, and eluted with a linear concentration(0–0.12 M) gradient of NaCl in sodium acetate. The retentionvolumes were 4.889 mL for PMAPI, and 5.725 mL for PMAP II, at aflow rate of 1 mL/min, and elution volume of 8 mL.

Molecular mass of antifungal proteins

SDS–PAGE profiles showed molecular masses of approximately20 kDa for PMAPI and 30 kDa for PMAPII (Fig. 2). MALDI-TOF MSrevealed a molecular mass of 18,798 Da for PMAPI, and 31,178 Dafor PMAPII (Fig. 3).

Antifungal activities

Antifungal activities against six plant fungal pathogens (T. viride,B. cinerea, P. asparagi Sacc, M. grissea, F. oxysporum f.sp. vasinfec-tum and S. sclerotiorum) were tested. The paper mulberry leafextracts showed activity against only T. viride. We assume this wasbecause T. viride is widespread in the environment, and may havebeen on the paper mulberry leaves that we collected, stimulatingthe tree to produce the antifungal compounds. The IC50 for theantifungal activity of PMAPI and PMAPII were both 0.1 mg/mL.

Peptide mapping and fingerprinting

Of the two antifungal proteins purified from paper mulberry, thechromatographic features suggested that they were similar.Therefore, the tryptic peptides of PMAPI and PMAPII were ana-

Figure 2. SDS-PAGE of PMAPI and PMAPII.

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lyzed by MALDI-TOF mass spectrometry (Supporting Table 1). Nosimilarity was observed between the tryptic peptides of PMAPIand PMAPII. This demonstrated that PMAPI and PMAPII are twodifferent proteins.

N-terminal amino acid sequence analysis andprotein identification

N-terminal amino acid sequences are in Table 1. The N-terminalamino acid sequence of PMAPI had 66.6% identity to PMAPII.Using the N-terminal amino acid sequence of PMAPI as a query,Blastp search revealed that PMAPI had 65–82% homology tohevein-like proteins from several plants (Supporting Table 2),indicating that PMAPI is a hevein-like protein. Heveins arecysteine-rich proteins in rubber tree (Hevea brasiliensis) latex.Originally described by Archer, they have strong in vitro antifun-gal activity against several fungi (Parijs et al., 1991). Hevein andhevein-like antimicrobial proteins have been reported fromrubber tree latex (Parijs et al., 1991), amaranth seeds, Pharbitis nil,

elderberry fruit, sugar beet leaves, Arabidopsis and Eucommiaulmoides bark (Lee et al., 2003). Here, we report the isolation of ahevein-like protein (PMAPI) from paper mulberry.

Using the N-terminal amino acid sequence of PMAPII as query,Blastp search revealed that PMAPII had 80–90% homology toclass I chitinases from several plants (Supporting Table 3), sug-gesting that PMAPII belongs to class I chitinase. Chitin is a struc-tural component in a diverse array of organisms, including fungi,insects, crustaceans and nematode eggs. Chitinase (EG 3.2.1.14)hydrolyzes the chitin polymer (Punja and Zhang, 1993) and isthought to have a crucial function in plant defense against fungalpathogens (Ghosh et al., 2004; Huynh et al., 1992). Chitinases aredivided into three classes. Class I enzymes are basic isoforms withan amino-terminal cysteine-rich domain and a highly conservedcatalytic domain. Class II enzymes lack a cysteine-rich domain,but have a catalytic domain that is homologous to class I chiti-nases. Class III chitinases share no homology with the class I orclass II enzymes, but are homologous to the acidic chitinases ofcucumber and Arabidopsis (Huynh et al., 1992; Ye and Ng, 2005).Chitinases have been isolated from tobacco, cucumber, beans,peas, grains and other organisms (Selitrennikoff, 2001). Here, weisolated a novel class I chitinase (PMAPII) from paper mulberry.

Paper mulberry is used in traditional Chinese medicine (Leeet al., 2001). The low-molecular-weight extracts are reported tocontain antibiotics (Duke and Ayensu, 1985; Gottstein and Gross,1992). However, to our knowledge, the antifungal activity ofhigh-molecular-weight extracts were not tested. During thetesting of antifungal activity in Chinese medicinal herbs, wefound that the fractions with molecular masses more than3000 Da inhibit the growths of fungal mycelia. We then presumedthat paper mulberry may contain antifungal protein. In this work,the antifungal activity of high-molecular-weight extracts ofpaper mulberry leaves was analyzed, and a hevein-like proteinand a class I chitinase were first identified.

In conclusion, two proteins (PMAPI and PMAPII) with antifun-gal activity against T. viride were isolated from paper mulberryleaves. N-terminal amino acid sequence analysis revealed thatPMAPI is a hevein-like protein, and PMAP II ia a class I chitinase.This is the first report of high-molecular-weight extracts frompaper mulberry with antifungal activity.

Supporting informationSupporting information can be found in the online version of thisarticle.

AcknowledgementsThis work was supported by grants from the National NaturalSciences Foundation of China (no. 30471060) and the SpecialProgram for Key Basic Research of the Ministry of Science andTechnology of China (no. 2001CCA01100).

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Figure 3. MALDI-TOF mass spectra for PMAPI (A) and PMAPII (B).

Table 1. N-terminal amino acid sequences of PMAPI andPMAPII

Protein N-terminal amino acid sequence

PMAPI NH2-EQCGSQVGGKTCPNNLCCXKYGWCGDTDDHPMAPII NH2-EQCGRQAGGALCPGGLCCXKYGWCGNTPEY

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