Isolation and Characterization of a Novel Class of Plant Antimicrobial ...

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Belgium, and SSICI Agrochemicals, Jealotts Hill Research Station, Bracknell, Berks RG126E Y, United Kingdom. We have ..... Leah et al. (7), using assay ...

JOURNAL OF BIOLOGICAL CHEMISTRY 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 4, Issue of February 5, pp. 2228-2233, 1992 Printed in U S.A.


Isolation and Characterization of a Novel Class of Plant Antimicrobial Peptides from Mirabilis jalapa L. Seeds* (Received for publication, June 27, 1991)

Bruno P. A. CammueSBfl, Miguel F. C. De BolleSIl, Franky R. G. TerrasSII, Paul Proost**, Jo Van Damme**,Sarah B. ReesSS, Jozef VanderleydenS, and Willem F. BroekaertSgg From the SF. A. Janssens Laboratory of Genetics, Catholic Universityof Leuven, Willem De Croylaan42, B-3001 Heverlee, Belgium, the **Rega Institute forMedical Research, Catholic University of Leuuen, Minderbroedersstraat 10, B-3000 Leuven, Belgium, and SSICI Agrochemicals, Jealotts Hill Research Station, Bracknell, BerksRG126E Y, United Kingdom

We have isolated from seeds of Mirabilis jalapa L. two antimicrobial peptides, designated Mj-AMP1 and Mj-AMPS, respectively. These peptides arehighly basic and consist of 37 and 36 residues for Mj-AMP1 and Mj-AMPB, respectively. Both peptides contain three disulfide bridges and differ from one another only by 4 amino acids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the reduced and unreduced peptides suggests that the peptides associate into dimers in their native form. The Mj-AMPs exhibit a broad spectrum of antifungal activity since they are active against all 13 tested plant pathogenic fungi. Concentrations required for 50%inhibition of fungal growth vary from 8 to 300 pg/ml for Mj-AMP1 and from 0.5 to 20 pg/ml for Mj-AMPB. These peptides were also active on two tested Gram-positive bacteria but were apparently nontoxic for Gram-negative bacteria and cultured human cells. Although the Mj-AMPs show sequence similarityto pagatoxins, a class of insecticidal neurotoxic peptides isolated from the venom of spiders, they do notaffect pulse transmission in insect nerves.

of aseries of genesencodingenzymes of the phytoalexin biosynthetic pathway (2). During the lastdecade, however, it has become increasingly clear that in some plants proteins can play a more direct role in the controlof microorganisms. Several classes of proteins with antifungal or antibacterial properties have now been identified, including chitinases (3, 4), P-1,3-glucanases ( 5 ) ,ribosome-inactivating proteins(6, 7), thionins (8), chitin-bindinglectins (9, lo), and permatins (11). In the present paper we describe the purification of a new class of potent antimicrobial peptides fromseeds of the fouro’clock plant (Mirabilis jalapa L). Their primary structure differs from allother known plant proteins, but instead, shows homology to insect neurotoxins found in venom the of spiders. EXPERIMENTAL PROCEDURES’ RESULTS

Purification of the M. jalapa Antimicrobial Peptides-The starting materialfor the isolationof the M. jalapa antimicrobial peptides was the basic heat-stable protein fraction extracted from the mature seeds. Purification was performed by chromatographic procedures,whereby the eluates were assayed for growth inhibition activity on the fungus Fusarium Although plants normally grow on substrates that are ex- culmorum. Upon fractionation by gel filtration on Superosetremely rich inmicroorganisms,infection remains a rare 12, the mixture resolved into three peaks, with the antifungal event. To keep out potential invaders, plants produce a wide activitycoeluting withthemostretarded peak(Fig. lA). array of antimicrobial compounds, either in a constitutive or Material from this peak was subjected to cation exchange a n inducible manner. The best studied of these are phytoalex- chromatography on S-Sepharose High Performance (Fig. 1B). ins, secondary metabolites with a broad antimicrobial activity No antifungal activity was found in the unbound fraction. spectrum, that are specifically synthesized upon perception of Application of a linear NaCl gradient allowed the separation appropriate defense-related signal molecules (1).The produc- of two factors with antifungalactivity. The first factor, heretion of phytoalexins depends on the transcriptional activation after called Mj-AMP1 (M. jalapa-antimicrobial peptide 1)’ eluted as amajorpeak around 50 mM NaC1, whereas the * Research was supported in part by the European Collaborative second factor, designatedanalogously as Mj-AMPB, eluted a t Linkage of Agriculture and Industry through Research programme 100 mM NaC1. The purityof the isolated antimicrobial factors (AGRE-0005) of the Commission of the European Communities. The costs of publication of this article were defrayed in part by the was verified by reversed-phase chromatography (Fig. 2). Both Mj-AMP1 and Mj-AMP2 yielded single well resolved major payment of page charges. Thisarticlemusttherefore be hereby activity. Moremarked “advertisement” in accordance with 18 U.S.C. Section 1734 peaks thatcoeluted exactlywith the antifungal solely to indicate this fact. 0 Postdoctoral fellowship from the Belgian Instituut ter Aanmoediging van het Wetenschappelijk Onderzoek in de Nijverheid en de Landbouw. T o whom correspondence should be addressed: F. A. ,Janssens Laboratory of Genetics,Catholic University of Leuven, Willem De Croylaan 42, B-3001 Heverlee, Belgium. Tel.: 0032-16-2209-21 (ext. 2403). !lContributed equally to this work. 11 Predoctoral fellowship from the Belgian Instituut ter Aanmoediging van het Wetenschappelijk Onderzoek in de Nijverheid en de Landbouw. $0 Research Associate of the National Fundfor Scientific Research (Belgium).

’ The “Experimental Procedures” and tables are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press. The abbreviations used are: Mj-AMP1, Mirabilis jalapa-antimicrobial peptide 1; DTT, dithiothreitol; HEPES, 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid; HPLC, high performance liquid chromatography; IC,,, concentration required for 50% inhibition of fungal growth; MES, 2-(N-morpholino)ethanesulfonicacid SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; UDA, Urtica dioica agglutinin.


M. jalapa L. Seeds

Antimicrobial Peptides from



100 80 60 40 20 0





1.C I 0 . !5 -

0 0























45 (ML)



1.5 0.2


0.1 0











FIG. 1. Purification of antimicrobial peptides from M. jalapa seeds. The basic heat stable protein fractionwas the starting material for further purification by gelfiltration chromatography( A ) . One-ml fractions were applied on a Superose-12 column (50 X 1.6 cm; Pharmacia LKBBiotechnology Inc.) previously equilibrated with PBS. Running buffer was PBS and the flow rate 1 ml/min. The eluate was monitored for absorbance a t 280 nm (lower panel) and collected in 2.5-ml fractions of which 2 p1 were used in the microspectrophotometric antifungal activity assay(upperpanel).The fractions containing antifungal activitywere pooled, dialyzed against 50 mM Na-MES (pH 5), and subjected to cation exchange chromatography ( B ) .Active fractions combined from three parallel gel filtration chromatographic runs were loaded on a S-Sepharose high performance column (10 X 1.6 cm; Pharmacia) in equilibrium with 50 mM Na-MES (pH 5). The column was eluted at 3 ml/min with a linear gradient of 450 ml from 0 to 150 mM NaCl in 50 mM Na-MES (pH 5). The eluate was monitored for protein by measurement of the absorbance a t 280 nm (lower panel) and collected in 15-ml fractions of which 2 pl were tested in the microspectrophotometric antifungal activity assay (upper panel).All columns were from Pharmacia and chromatography was done on a Waters 650 FPLC station.


10 N

A.000 NO. 20 > 1.000 > 1,000 NO. 200




20 0.5 15

wrrent pulses.



TABLE I l l Antibacterial dctrvlty

gels were cooled at'l0.C during e1ect;ophoreris.



Qualitative diseisment o f f r e e cysteine thlol groups Hundred p g amou#ntr of reduced or unledUCed peptides Wele dissolved in 6 M guanidlniun-C1 containlnq 100 mll sodium phosphate buffer (pH 7) and 1 mM EOTA. The mIXtUI.eB w e ~ e allowed to r w c t with 5, 5'dithionitlobenmlc acid and monitored for ele ease of nitrobenmate 31 described by Creighton (30). ReductIan o f the peptldes vas done by addltlon of Trli-HCl (pH 8.6) to 100 mM End dlthiotPeltO1 to 30 mM. followed by incubatIan at 45'C for 1 h. The peptlder Y e w separated from the excess reagents by revel'red-phase ChPOmdtography con L CzjC18 111icd c01umn (see legend to Flg. 2). S - c d l - b o x y a i o l d o m e t h y l d f l o n and a m n o acid sequence dndiyiis---CYSteine residues Of peptides were modified by S-c~rboxy..ldo.ethyl.ti~" as follows: 100 pg amounts o f peptides W ~ P B dlsrolved in 150 pl 0.3 M Trir-HCI (pH 8.6) containing 30 mM DTT and r#!acted far I h at 45'c. lodoacetanide was added to a final concentration O f 100 mi! and the mixture was kept in the dark at 37'C for I h. The reaction was finally quenched by addition of OTT to a final Concentration o f 100 mM and allowedto react for an additimll hour at 37.C. Desalting vas done by High Performance Liquid Chromatography (HPLC) or, a P e p 4 (porous silica C2/C18) (Pharmacia) column (25 x 0.4 cm). The carboxyamidomethyllited peptides were

recovered by eluting the column with 8 linear gradient from 0.1 I tPiflUOrOaCetic acid (TFA) to 2-propanol containing 0.1% TFA. The resulting peptjde fractions Yere Subjected to amlno acid sequence analysis in a 477A Protein Sequencer (Applled Bioryriemr) with on-line detrctlanofphenylthiohydlntoinamino acid derivatives in a 1201InalYler (Applled with pyroglutanate aminopeptidase (Boehrinqer. Bloryrtemr). Tmatment o f pepttdel Mannheim, FRG) V I S done according to thesupplier's inrtructionr.

Ipectlo.etTy---P0litive ma$* spectra were obtained by fast atom bombardment mars spectrometry on a VG 70 SEQ double-focuring instrument. lmilation "3% performed w t h xenon atoms at 8 Lev and 1 ml emission current. The matrix consisted of 1.4-dithiothreitol : 1.4-dithiaerythritol (5:l v/v). One pl of the underlvatized protein ranple (2.5 pg/pl in "ate?) YIP nixedHith 2 ul O f thematrix. Hass calibration was done urinr IDectra generited by cesium iodide. Mass

Extractran of basic heat-stable proteinsfrom PIirabrlir ~ a l a p d reedr---One kg o f PI. jalapa seeds was ground in a coffee m l l and the resulting m a l was extracted fol 2 h at 4'C with 2litres O f an ice-coldextractionbuffercontaining10 nM ! NsH2POq. 15 WI Na2HPO loo d KCl, 2 lnll EDTA. 2 mM thiourea. 1 Iw PRSF and 1 mgll leupeptin. The homogenate w% q u e e r e d through cheesecloth and clarified by centrifugation ( 5 min at 7,000 x 9). Solid ammiurn sulphate was added to the supematant to obtain 35 X relative saturation and the precipitate formed a f t e r standing f o v 1 h at room temperature was removed by centrifugation (10 min at 7.000 x 9). The supernatant was adjusted to 65 X relative amaniun sulphate saturation and the precipitate formed overnight at loom temperstore collected by centrifugation (30 min at 7,000 x g). After redissolving the pellet in 3110 ml 10 mM sodium phosphate buffer (pH 61. the solution was heated at 75'C for 10 nin. The coagulated insoluble material was discarded after centrifugation (20 nin at 7.000 x 9) and the using benroylated cellulose supernatant was dialyzed extensively against distilled water tubing (Sigma) with a noleculsr weight Cut off o f 2.000 Oa. After dialysis the solution was adjusted to 50 rA Trir-HC1 (pH 9) by addition of the ten-fold concentrated buffer. and subsequently parsed over a Q-Sepharore Fast Flow(Pharnacia)column(12 x 5 cm) in equilibriumwith 50 mi Tlir-HC1. pH 9. Theproteins in theunboundfraction were precipitated by addition Of amonium sulphate t o 7 5X relative saturation. The precipitate was collected by centPifugation (20 mi" at 7.000 x 9) and the pellet redirrelved in 15 m1 PBS. This materia1 rePrelentl the baric heat-stable DrOtein fraction o f PI. lalam seeds. containing approximately 16 g/nl of total protein.


Final concentration l"O/rnl


X viability' O f

fibroblasts endothelial cells 98 94

97 98




22 67

70 91 0

5 UDA P-purothionin 500 5 50 8-purothionin 95 8-purothionin 5 'Oetermined a$ the percentage o f cells that remained unstained by trypan

3 12 NO. 15 1 0.5

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