Purification and Characterization of a Novel Antimicrobial Peptide from ...

Vol. 267, No. 26, Issue of September 15, pp. 18814-18820,1992 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Purification and Characterizationof a Novel Antimicrobial Peptide from Maize (Zea mays L.) Kernels* (Received for publication, October 21, 1991)

Jonathan P. DuvickS, Tracy Rood,A. Gururaj Rao, and Daniel R. Marshakg From Pioneer Hi-Bred International.. Inc.., Johnston. Iowa 50131 and the $Cold Spring Harbor hboratou, Cold Spring Harbor, New York 11724

Several small, acid-soluble, basic peptides with antimicrobial properties have beenisolatedfrommaize (inbred B73) kernels. One of these peptides (MBP-1) has been purified to homogeneity and characterized. Thepeptidehasamolecular weight of 4127.08 as determined by plasma desorption mass spectroscopy, has no free cysteines, and is predominantly a-helical as determined by circular dichroism. The primarysequence of the peptide (33 residues) has been determined by Edman degradation and shows no homology to the thionins, a group cysteine-rich of peptides found in some cereals including wheat, barley, and sorghum, as well as several dicot species. Likethe thionins, however, MBP-1 has been found to have antimicrobial properties in vitro.MBP- 1inhibits spore germination or hyphal elongation of several plant pathogenicfungi, including two seed pathogensof maize (Fusarium moniliforme Sheld. and Fusarium graminearum (Gibberella zeae(Schw.) Petsch)), and several bacteria, including a bacterial pathogen of maize(Clauibacter michiganense ssp. nebraskense).A synthetic MBP- 1 peptide, air-oxidized and purified byreverse phase chromatography, was equally antifungal as compared with the naturally occurring peptide.

Seeds of maize and other monocots contain a variety of proteins whose primary function is storage of nitrogen for germination and growth (Wilson, 1987). Other proteins can also be found in seed whose function, although unknown, is thought to include protection from predators or pathogens. These include a varietyof enzyme inhibitors (Garcia-Olmedo et al., 1987) lectins (Raikhel and Wilkins, 1987), and hydrolytic enzymes including chitinases and glucanases (Huynh et al., 1992; Roberts et al., 1988). In addition, a family of small, basic peptides called thionins has been identified in seeds of certain monocots including wheat, barley, rye, and oat (Apel et al., 1990; Garcia-Olmeda et al., 1989) and, more recently, sorghum (Bloch and Richardson, 1991) . Thionins have also been isolated from barley leaves (Bohlmann and Apel, 1991), and thionin-like peptides have been reported from several dicots including mistletoe (Apel et al., 1990; Garcia-Olmeda et al., 1989) and Crambe abyssinica (Teeter et al., 1981). Thionins are cysteine- and lysine-rich polypeptides 43-46 amino acids in lengthand show considerable homology across species, including almost complete conservation of the position of the cysteine residues (Bloch and Richardson, 1991).

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed Pioneer Hi-Bred International, Inc., 7250 N. W. 62nd Ave., Johnston, IA 50131. Tel.: 515-270-3176; Fax: 515-253-2149.

Several thionins areinhibitory to plant pathogenic fungi and bacteria in vitro (Apel et al., 1990; Garcia-Olmeda et al., 1989; Hernandez-Lucas et al., 1974), and others are reported to inhibit a-amylases (Bloch and Richardson, 1991). The function of thioninsintheplant is unknown, although their inhibitory activity toward microbes and enzymes suggests a possible role in defense against microbial and/or insect predators (Garcia-Olmeda et al., 1989). Other small, basic peptides from mammalian and invertebrate sources have been implicated in defense against pathogens (Berkowitz et al., 1990; Diamond et al., 1991; Lehrer et al., 1990a; Steiner et al., 1988). Of the agronomically important monocot species, only maize and rice have not been shown to containthionins. Jones(Jonesand Cooper, 1980) isolated from maize seed several cysteine-rich peptides, which were similar to thionins in several chemical and physical properties but differed substantially in amino acid composition from the thionins and which were not toxic to insects when injected into the hemolymph. However, the peptides were not completely characterized, and no amino acid sequence information was obtained. We decided to re-examine maize seeds as a source of thionin-like peptides, withthe additional aim of finding novel antimicrobial factors of plant origin. In this paper we report the purification to homogeneity of a small, basic peptide with antimicrobial properties and anamino acid sequence distinct from thionins. EXPERIMENTALPROCEDURES

Maize Seed-Untreated maize (Zea mays L.) seed, public inbred line B73,was obtained from the Research Samples Laboratory at Pioneer Hi-Bred, Johnston, IA. Fungal Isolates-Thefollowing isolates were obtained from the strain collection of Pioneer Hi-Bred Fusarium graminearum (Gibberella zeae (Schw.) Petsch), isolated from soil; Fusarium monilijorme Sheld., isolated from corn seed; Sclerotinia sclerotiorum (Lib.) de B a y , isolated from sunflower; and Sclerotinia trijoliorum Eriks., isolated from alfalfa. Aspergillus flauus (Schw.) Petsch, isolate CP22, was obtained from Don Sumner at theUniversity of Georgia (Tifton, GA). Alternaria longipes (Ellis et Everhart) Mason 15052was obtained from the American Type Culture Collection (Rockville, MD). Clavibacter michiganense ssp. nebraskense (Schuster, Hoff, Mandel, and Lazar) Carson and Davis was obtained from the University of Nebraska (Lincoln, NE). Escherichia coli strain DH5 was obtained from Bethesda Research Labs (Gaithersburg, MD). Other Reagents and Materials-CM-cellulose’ was obtained from Sigma. All chemicals were of highest purity available. Extraction Methods-Mature maize kernels were used to extract acid-soluble peptides. In some experiments, the embryo was separated

’ The abbreviations used are: CM-cellulose, carboxymethylcellulose; HPLC, high performance liquid chromatography; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; CAPS, 3(cyclohexy1amino)-1-propanesulfonic acid; PVDF, polyvinylidine difluoride; DTNB, 5,5’-dithiobis(2-nitrobenzoate);NBS, N-bromosuccinimide; FPLC, fast protein liquid chromatography; PDMS, plasma desorption mass spectrometry; Trt, trityl; NBY, nutrient broth/yeast extract; MBP-1, maize basic peptide 1; amu, atomic mass unit(s).

18814

Antimicrobial from Peptide from the endosperm and seed coat by first cutting the mature seed in two longitudinally and thenprying out the embryo halves with a fine scalpel blade. Tissues were ground to a fine powder in a Tecator rotary mill using a fine mesh screen or (for smaller tissue amounts) in a coffee grinder. The resulting flour was extracted in 0.1 M HZSO, ( 3 3 w/v) with stirring, for 1h a t room temperature. The extractwas squeezed through a muslin cloth and centrifuged for 10 min a t 9000 X g, 4 "C. The supernatant liquid was adjusted to pH 7.0 with ammonium hydroxide and the resulting precipitate removed by centrifugation. The resulting supernatant liquid was adjusted to pH 5.2 with glacial acetic acid and centrifuged to remove the precipitate. The final supernatant, designated as "crude acid extract," was filtersterilized and stored at -20 "C until further use. Chromatography-Crude acid extract (500 ml) was loaded onto a CM-cellulose column (2.5 X 75 cm), equilibrated with 0.3 M ammonium acetate, pH 5.2, and washed with 2 liters of the same buffer. A 2000-ml gradient of 0.4-1.2 M ammonium acetate, pH 5.2, wasapplied at a flow rate of 2 ml/min. Fractions (10 ml) were collected, and pooled fractions representing absorbance peaks at 280 nmwere pooled, resuspended in 0.01% acetic acid, lyophilizedagain, and stored at -20 "C. Alternatively, the crude acid extract was dialyzed into 0.2 M ammonium acetate, pH 5.2, using Spectra-mesh dialysis tubing with an exclusion limit of 1000 Da (Spectrum, Los Angeles, CA), and 10-ml aliquots loaded onto a cation exchange HPLC column (Mono-S HR 5/5; Pharmacia LKB Biotechnology Inc.) that was previously equilibrated with the same buffer. A gradient of0.2-1.2 M ammonium acetate, pH 5.2, was applied at a flow rate of 2 ml/min (650 ml total). Fractions (5 ml) were collected and lyophilized, resuspended in 0.01% acetic acid, re-lyophilized, and stored a t -20 "C. Fractions purified by cation exchange chromatography were analyzed by reverse phase HPLC using an Altex ODS 2.5 X 150-mm Cls column (Beckman Instruments, Fullerton, CA) equilibrated in 10% acetonitrile, 0.1% trifluoroacetic acid. An 8-ml gradient of 10-40% acetonitrile in the presence of 0.1% trifluoroacetic acid was applied at a flow rate of 1ml/min, and 0.5-ml fractions were collected, pooled, lyophilized, and stored at -20 "C. Polyacrylamide Gel Electrophoresis-Two SDS-PAGE methods suitable for resolution of small peptides were used one employing 50% urea in the separating gel to slow down migration (Anderson et al., 1983) or one in which Tricine was substituted for glycine in the gel buffer leading to better separationof small proteinsin the stacking layer (Schagger and von Jagow, 1987). Samples (1-5 pg) were dissolved in sample buffer containing 0.1% SDS and8-mercaptoethanol, heated 5 min at 95 "C, and electrophoresed in 0.5-mm slab gels at 50 mA. Gels were fixed in 30% MeOH, 10%acetic acid and stained with Coomassie Blue. Protein Blotting-SDS-PAGE gels run with Tris-Tricine as described above were fixed in CAPS-MeOH, pH 10.5 (LeGendre and Matsudaira, 1988), and proteins transferred onto PVDF membranes (Immobilon P, Millipore, Bedford, MA), using a semidry electroblotting apparatus, at 30mA for 30-60 min. The membrane was stained with Coomassie Blue in 50% methanol, and Coomassiestained bands were excised for analysis. Amino Acid Analysis-The sample was hydrolyzed at 150 "C for 1 h, and themembrane was placed directly on an Applied Biosystems 420A derivatizer to convert free amino acids to phenylthiocarbamyl derivatives. The derivatized residues were separated on a C18reverse phase column and detected a t 254 nm. Determination of Free Sulfhydryl Groups-The sulfhydryl content of MBP-1 was determined by reaction with DTNB in the presence of 6 M guanidinium hydrochloride as described by Riddles et al. (1983). Tryptophan Estimation-Tryptophan was estimated by the NBS oxidation method of Spande and Witkop (1967). One ml of protein, having an absorbance of -0.7 a t 280 nm in 0.1 M ammonium acetate containing 4M urea at pH4.0, was titrated with 5-pl aliquots of NBS (aqueous stock solution of 5 mM) until the decreasing absorbance a t 280 nm reached a constant value. Tryptophan content was then calculated using the appropriate equation. Peptide Sequencing-Peptide sequencing was carried out on an Applied Biosystems 477A protein sequenator/l20A analyzer, by the Edman degradation method. The PVDF-blotted sample was loaded directly into the instrument. Fluorescence Measurement-Fluorescence measurement was made in a 1-cm cuvette at 25 'C with a Perkin-Elmer LS50 spectrofluorometer, equipped with a xenon flash tube and excitation and emission reflection grating monochromators. The excitation and emission slit widths were set at 5 nm, and the excitation wavelength was 280 nm.

Zea mays Kernels

18815

A solution of the protein in 10 mM phosphate buffer, pH 5.8, having an absorbance < 0.1 at 280 nm was used. CD Measurements-The CD spectrum (190-250 nm) of MBP-1 was recorded a t 25 "C with a Jasco 600 spectropolarimeter using 0.1cm cells.The protein concentrationwas 0.137mg/ml in 10 mM sodium phosphate, pH 5.8. Mean residue ellipticity was calculated using a mean residue weight of 110. Calculations of the secondary structure combinations which provide the best fit to the CD data were made with the Jasco secondary structure estimation software using the CD spectra of proteins with known secondary structure content. Mass Spectrometry-PDMS was performed on a Bio-Ion BIN2OK plasma desorption mass spectrometer. An aliquot of 200 pmol of MBP-1 in 20 pl of 0.1% (w/v) trifluoroacetic acid was applied to a target of aluminized Mylar that had been electrosprayed previously with 50 pg of nitrocellulose. The sample was allowed to adsorb for 1 min, spun dry, and washed once with 50 p1 of trifluoroacetic acid. Spectra were recorded at an accelerating voltage of 16 kV at 16,000ns intervals. Peptide Fragments-Peptide fragments suitable for sequencing were obtained by enzymatic or chemical digestion of the reducedcarboxymethylated MBP-1. Typically, about 350 pg of protein in 400 pl of 0.1 M ammonium bicarbonate, pH 7.8, was incubated at 37 "C with either trypsin (for 15 min), endoproteinase Glu-C (for 50 min), or thermolysin (for 75 min), at an enzyme to substrate ratio of 1:25. Peptide fragments were also obtained by cyanogen bromide (CNBr) digestion of MBP-1 as follows. To 280 pg of MBP-1 was added 1 ml of70 mg/ml CNBr in 70% (w/v) formic acid, and the mixture was incubated in the dark at room temperature for 24 h. Subsequently, the mixture was diluted 10-fold with water and lyophilized. The lyophilized powder was then solubilized in an appropriate volume of 0.1% trifluoroacetic acid in water. Enzymatic or CNBr digests were injected directly onto anFPLC reverse phase column (C2/C18,HR 5/ 5 Pep-RPC; Pharmacia) and thepeptides separated using a 120-min linear gradient of 0-50% eluting buffer at a flow rate of 0.5 ml/min. The starting buffer was 0.1% trifluoroacetic acid, while the eluting buffer was 100% acetonitrile containing 0.1% trifluoroacetic acid. The elution was monitored a t 214 nm, and 0.5-ml fractions were collected. Peak fractions were appropriately pooled and lyophilized, and the amino acid sequence was determined on an Applied Biosystems 477A sequenator. For N-terminal sequence, the intact peptide was electrophoresed on a Tris-Tricine SDS-PAGEgel in the presence of dithiothreitol, and was blotted onto PVDF for sequencing. C-terminal Sequencing-C-terminal sequencing of the intact protein (1 nmol) was performed with carboxypeptidase Y at an enzyme to substrate ratio of approximately 1:lO. Norleucine was used as an internalstandard. The reaction wasallowed to proceed at room temperature for 60 min in 50 mM sodium acetate buffer, pH 5.5. Aliquots were removed at 10-min intervals and analyzed for amino acid composition. Sequence Analysis-Peptide database searches were performed using the FASTA search algorithm (Pearson and Lipman, 1988) with GCG software from the Genetics Computer Group (Madison, WI). Solid Phase Synthesis of MBP-1-MBP-1 was synthesized on an Applied Biosystems 431 A peptide synthesizer by the FastMocTM strategy (Applied Biosystems, Foster City, CA) involving the use of 2-(1H-benzotriazol-l-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate for coupling amino acids (Fields, 1991). Preloaded glycine on 4-hydroxymethylphenoxymethyl, copolystyrene, 1%divinylbenzene resin (0.69 mmol/g) served as the C-terminal amino acid. Stepwise synthesis proceeded from the C-terminal as single couplings of each amino acid. All amino acids were purchased as 1 mmol cartridges from Applied Biosystems. The following amino acids were used as their side chain-protected derivatives: Arg(2,2,5,7&pentamethylchroman-6-sulfonyl), Cys(Trt), Glu(tert-butyl ester), Gln(Trt), and His(Trt). Upon the completion of synthesis, the peptide-resin was dried under vacuum for 1 h. Cleavage of the peptide from the resin and simultaneous deprotection of the side chains was performed by treating a 0.1-g portion of the peptide-resin with 10 ml of cleavage mixture (90% trifluoroacetic acid, 7.5% phenol, 5% thioanisole, and 2.5% ethanedithiol) for 3 h at room temperature. Free peptide was precipitated by direct filtration into ice-cold methyl-t-butyl ether. The crude peptide was recovered by centrifugation and dried under vacuum. Folding and oxidation of MBP-1 toform disulfide bonds was achieved by solubilizing 40 mg of the crude peptide in 400 ml of 100 mM ammonium bicarbonate adjusted to pH 8.5 (0.1 mg/ml) and stirring at room temperature for 64 h. Following lyophilization, the peptide was purified as described elsewhere on a reverse phase column. The purified peptide was analyzed by PDMS and the antifungal activity compared with that of the naturally occurring peptide.

18816

Antimicrobial Peptide from Zea mays Kernels

Antifungal Assays-F. graminearum was grown in half-strength CM-cellulose-yeastextract broth(7.5 g of CM-cellulose,0.5 g of yeast extract, 0.25 g of MgS04.7H20,0.5 g of NH4NOI,and 0.5 g of KHzP04 per liter of distilled water). Cultures were shaken a t 200 rpm at 28 "C in thelight. After 7 days, cultures were filtered through two layers of sterile cheesecloth and theresulting filtrate passed through aNalgene 0.45-pm disposable filter unit. Conidia (spores) were collected on the membrane, washed with sterile distilled water, and resuspended in a small volume of sterile water. Conidia were counted with a hemocytometer and stored a t 4 "C for not longer than 1 month. A. longipes cultures were grown on carrot agar at 28 "C under continuous fluorescent light, and F. moniliforme and A. flavus weregrown on oatmeal agar at 28 "C under ambient light. For these three fungi, conidia were collected by scraping a sterile inoculating loop across the surface of the plate. Concentrated suspensions were made in sterile water with 0.1% Tween 20. Conidia were counted with a hemocytometer and used immediately. For an assay, fungal spore suspensions were diluted to give a concentration of 250 spores/gO pl of dilute culture medium (0.037g of NaCl, 0.0625 g of MgSO,. 7H20, 0.25 g of calcium nitrate, 2.5 g of glucose, 0.25 g of yeast extract, 0.125 g of casein hydrolysate (enzyme), and 0.125 g of casein hydrolysate (acid) in 7.5 mM sodium phosphate buffer, pH 5.8). For Sclerotinia cultures, mycelia were grown on cellophane discs (52 mm) overlain on V8 agar. When hyphal growth reached the margin of the disc, the cellophane was removed and the mycelium dislodged by vortexing in 10 ml of diluted culture medium, followed by filtration through two layers of cheesecloth. Hyphal pieces were washed by centrifugation at 2000 rpm for 5 min and resuspended in diluted growth medium to give a concentration of approximately 50 hyphal pieces/90 pl. To perform antifungal assays, 10 pl of test material in water or 0.01% acetic acid were added to wells of a microtiter plate. Ninety microliters of spores or hyphal pieces were added and mixed. Plates were covered and incubated at 28" in the dark for 24-48 h. Growth was evaluated visually using an inverted microscope, and a scale of 0-4 was used to rate the effect of added peptide (0 = no observable inhibition relative to water control; 1 = slight inhibition; 2 = substantial inhibition; 3 = almost complete inhibition; 4 = complete inhibition). (See Fig. 6 for photomicrographs of F. graminearum conidia showing levels of germination/growth inhibition ranging from 0 to 4.) Antibacterial Assays-Cultures were grown to midlog phase (E. coli in LB broth and C. nebruskense in NBY) and then harvested by centrifugation (2000 X g for 10 min). Cells were washed with 10 mM sodium phosphate buffer, pH 5.8 (C. nebruskense) or pH 6.5 (E. coli) by centrifugation and then colony forming units estimated spectrophotometrically a t 600 nm, with previously established colony forming unit-opticaldensity relationships used as a reference. Assays for bactericidal activity were performed by incubating lo6 bacterial colony forming units in90 p1 with 10 ml of peptide (or water for control). After 60 min at 37 "C (E. coli) or 25 "C (C. nebruskense), four serial, 10-fold dilutions were made in sterile phosphate buffer. Aliquots of 100 p1 were plated on LB or NBY plates, using 1 or 2 plates/dilution. Resulting colonies were counted, and theeffect of the peptide was expressed as percent of initial colony count (Selsted et al., 1984). Assays forbacteriostatic activity were performed by incubating lo6 bacteria with MBP-1 in 200 p1 of dilute medium (1 part NBY broth t o 4 parts 10 mM sodium phosphate, pH 5.8) in microtiter plate wells. Plates werecovered, sealed, and incubated at 28 "C. Growth was monitored spectrophotometrically a t 600 nm. After 41 h controls had grown sufficiently (optical density > 0.20) to measure effect of peptide as percent of control.

as

1.2 MBP-1

Ob

B

OR

80.2 0.1

0.0

w-

B @CI "

o w 0.01

-

Elution volume (ml)

FIG. 1. Purification of MBP-1 by column chromatography. a, CM-cellulose cation exchange chromatography of crude maize seed extract. The column (2.5 X 75 cm) was equilibrated with 0.3 M ammonium acetate, pH 5.2, and washed with 2 liters of the same buffer. A 2000-ml gradient of 0.4-1.2 M ammonium acetate, pH 5.2, was applied at a flow rate of 2 ml/min. Fractions (10 ml) were collected. b, Mono-S cation exchange FPLC of crude maizeseed extract. c, reverse phase chromatography of MBP-1 from CM-cellulose column (see Fig. la) on CISreverse phase HPLC column.

fractions showed activity. The fraction that eluted at 0.850.88 M ammonium acetate (Fig. 1, a and b ) was chosen for additional analysis, as itwas well resolved from adjacent peaks and was the most active against F. graminearum. The total yield of this fraction averaged approximately 12 mg/kg of dry seed. This represents approximately 0.014% of the total protein, based on an average protein content of 9% by weight for mature maize seed (Wall and Paulis, 1987)). This fraction, designated as Maize BasicPeptide 1 or MBP1, was re-chromatographed on a CIS reverse phase column, resulting in a single major peak eluting at -25% B (Fig. IC). This peak contained essentially all the antifungal activity of the starting material when assayed against F. graminearurn (not shown). In addition, there was a minor peak (-10% of total) eluting at -24% acetonitrile. This peak was not tested RESULTS for antifungal activity. Ion exchange fractionation of crude, acid-extracted maize The ion-exchange-purified material, when reduced, denakernels on CM-cellulose resulted in threemajor UV-absorbing tured and electrophoresed on a Tris-Tricine SDS-polyacrylpeaks (Fig. la). When the same material was chromato- amide gel, migrated as a single Coomassie staining band (Fig. graphed on a high resolution Mono-S cation column, 16 peaks 2, lane B ) . The estimated molecular weight was 3,740.Similar were observed (Fig. Ib). Analysis by SDS-PAGE of pooled results were obtained with SDS-polyacrylamide gels in the peak fractions eluting during application of the gradient on presence of 4 M urea (not shown). either CM-cellulose or Mono-S showed that most of the Amino acid analysis of MBP-1 blotted onto PVDF indifractions contained one or more low molecular weight, COO- cated ahigh content of Glx and Arg, with smaller amounts of massie-staining bands (not shown). Each fraction was tested Ser, His, Thr, Ala, Pro, Leu, Lys, and Asx (Table I). Cys was for its ability to inhibit spore germination/hyphal elongation detected, but could not be quantified. No free sulfhydryl of F. graminearum macroconidia (not shown). Several of the groups were detected by titration with DTNB.

Kernels mays Antimicrobial Zeafrom Peptide A

B

C

D

18817 4 1

10

m A

1

4

7

46.5 1

29.1

23 t

20

Nl

4

KDa

N2

1

E

5

Ti

0 7l 7

10

t

14

R

c

31 CP 33

24 2 5 6 5 27 b -

l6

15

27

20

25

* " , 30

R-S-G-R-G-E-C-R-R-Q-C-L-R-R-H-E-G-Q-P-W-E-T-Q-E-C-M-R-R-C-R-R-R-G 4 1

19.1

Cl

b

26 27

M

X3

FIG.3. Deduced sequence of MBP-1. Lines with arrows indicate peptide sequence obtained from N-terminal sequencing of the native peptide ( N I , N 2 ) ; proteolytic fragments from digestion with endo-Glu-C (G5, G7, G8),trypsin ( T I , T2),and thermolysin (2%-A); and fragments derived from cyanogen bromide treatment ( C I , C2). Carboxypeptidase cleavage products ( C P ) were analyzed to confirm the last 3 amino acids of MBP-1.

15.1

6.2

3.4

FIG.2. SDS-PAGEof maize seedproteins, usingthe method of Schagger and co-workers (Schagger von and Jagow, 1987). Gel was stained withCoomassieBlue. Lane A , molecularweight markers; lane B, MBP-1 (2.5 pg); lanes C-E, crude acid extracts of whole kernel, endosperm + seed coat, and embryo, respectively (10 pl of crude extract/lane). TABLE I Amino acid composition of MBP-I peptide from maize seed Amino acid

Mol/mol of protein (rounded to the nearest whole number) Calculated"

Aspartic acid/asparagine Glutamic acid/glutamine Serine Glycine Histidine Arginine Threonine Alanine Proline Tyrosine Tryptophan Methionine Cysteine Leucine Phenylalanine Lysine Isoleucine

Total

0 8

Actualb

0

1

I 1

4

4

1 11 1 0 1 0 lC 1 >2 1 0 0

1 11 1 0 1 0 1 1

0

32

4

1 0 0 0

-

33 Calculations based on an apparentM , of 4,350g/mol. As determined by amino acid sequence analysis. Determinedindependently by oxidationwithN-bromosuccinimide. a

The Edman sequencing method was applied to the whole protein and to the peptide fragments in the determination of the amino acidsequence of MBP-1. Fig. 3 illustrates the sequencing strategy used and shows the positions and sequences of the peptides. In two separate runs (N1 and N2), N-terminal sequencing of the undigested proteingave 23 and 28 residues, respectively. Sequence N2 was obtainedwith PVDF-blottedMBP-1 whereassequence N1 was obtained with the HPLC-purified protein. Amino acid sequences of the endo-Glu-C fragments G7 and G8 matched residues 7-27 of the whole protein. The tryptic peptides T1 and T2 yielded sequence data that overlapped that of the endo-Glu-C fragments. Additionally, the thermolysin-generated peptide, Thl, confirmed the first11 residues at the N terminus of MBP-1. Final confirmation was obtained from twocyanogen bromide

FIG.4. PDMS of MBP-1. An aliquot of 200 pmol of MBP-1 in 20 p1 of 0.1% (w/v) trifluoroacetic acid was applied to a target of aluminized Mylarthat had been previously electrosprayed with50 pg of nitrocellulose. The sample was allowed to adsorb for 1 min, spun dry, and washed once with 50 pl of trifluoroacetic acid. Spectra were recorded using a Bio-Ion BIN2OK plasma desorption mass spectrometer at an acceleratingvoltage of 16 kV a t 16,000-ns intervals. fragments,C1and C2, which yielded, respectively, an Nterminal peptide of 26 residues ending in methionine and a C-terminal peptidewith glycine at theC terminus. Thesingle tryptophan residue obtainedthrough sequencing was also independently confirmed by oxidation with NBS (see "Experimental Procedures"). The three aminoacids at theC terminus of MBP-1 were determined by digesting the intactprotein with carboxypeptidaseY for various times. At 2 hthe released amino acid was glycine (32-89 pmol). At 19 h the released amino acids were glycine (17.2 pmol)andarginine (21.76 pmol). At 24 h the yield of glycine was reduced to 7.5 pmol and that of arginine increased to 45.5 pmol. These results confirmed the C-terminalsequence to be Arg-Arg-Gly. Based on these resultswe propose the complete sequence of MBP-1 to consist of 33 residues as depicted in Fig. 3. Analysis by PDMS indicated a measured mass of 4129.08, based on the average of singly, doubly, and triply charged species(average of 4127.47,4127.12, and 4126.66) (Fig. 4). This measured mass low is compared with the calculated mass of 4129.72 and is commensurate with the peptide having two disulfide bonds. This is supported by the fact that no free S H groups were detected with DTNB. However, PDMS analysis also indicated the presence of another species (10-20%) with a n average M , of 4073.47 (Fig. 4; see "Discussion"). An identicalresult was obtainedwithsyntheticMBP-1(not shown). The CD spectrum of MBP-1 (Fig. 5a) showed minima at approximately 210 and 220 nm, which is fairly typical of ahelical proteins (Greenfield and Fassman, 1969). Calculation of the secondary structure content from the least squares fit to the experimental spectrum indicated the following: a-helix

Antimicrobial from Peptide

18818

Zea mays Kernels

playing antimicrobial properties in uitro. In mature seed the protein is found predominantly in the embryo portion of the kernel. The molecular mass of MBP-1 estimated by SDS-PAGE under reducing conditions (3740 Da) agrees closely with the molecular weight determined by PDMS ( M r= 4127). The fact that synthetic MBP-1 showed biological and chemical properties that were identical to the naturally occurring peptide provides a final verification that the deduced sequence for MBP-1 is correct. Some questions remain, however, about other species in the naturally occurring MBP-1 sample. The presence of the M, 4073.47 species, corresponding to a difference of 53.61 atomic mass units (amu)from MBP-1, suggests the deletion of the C-terminal glycine residue, perhaps through a carboxypeptidase-like activity. However, removing the glycine ought to reduce the mass by 57.12 amu, the residue weight of glycine. The observed difference, 53.61, is therefore off by 3.51 amu and suggests that the des-glycine species is actually larger than expected by about 4 amu,possibly due to W a d e w t h (nm) reduction of the disulfides. The 4073.47-amu species could FIG.5. a, far-UV CD spectrum of MBP-1 in PBS (solid line). also have arisen as a fragment ion during PDMS analysis Protein concentration was 0.137 mg/ml. The computed CD spectrum (Marshak and Binns, 1990). Alternatively, the des-glycine of MBP-1 using the Jasco secondary structureestimation software is species could also be amidatedthrough an exopeptidasealso shown (dashedline). b, fluorescence spectrum ofMPB-1. linked amidation reaction. Precedents for this kind of reaction exist for certain peptides in brain tissue (Loh et al.,1985; = 35.1%; @-sheet = 20.5%; @-turn= 17.6%; random coil = Mains et al., 1984). 26.8% (total = 100). Interestingly, MBP-1 is similar to the thionins from other The fluorescence spectrum of MBP-1 (Fig. 5b) showed a monocot seeds in its antimicrobial activity, small size, and Amax of emission at 350 nm when excited at 280 nm or 295 basic charge; however, the maize peptide differs from thionins nm. This spectrum is similar to that of free tryptophan in both inprimary sequence, distribution of cysteines, and overwater and suggests that the single tryptophan in MBP-1 is all amino acid content. One of several partially characterized exposed to a polar environment, althoughthis effect could be maize seed proteins previously reported by Jones andCooper attributedtootherinteractionsas well (Kronmanand (1980) has asimilarcation exchange elution profile and Holmes, 1971). overall amino acid composition to that of MBP-1. Jones and MBP-1 showed activity as an inhibitor of both bacterial Cooper (1980) also concluded that these maize proteins were and fungal growth in uitro (Figs. 6 and 7; Tables I1 and 111). not thionins, based on their distinct amino acid composition In bactericidal assays, more than 99.9% of E. coli cells were and their lack of toxicity to tobacco hornworm (Manduca killed by 3 pglmlpeptide (Table 11).By contrast, only 81%of sextu). C. michiganense ssp. nebraskense cells were killed by 30 pg/ A searchof the EMBL andSwiss protein databases yielded ml peptide (Table 111). Bacteriostasis was observed at this no sequences with significant homology to MBP-1. In size concentration, however (Table 111). When tested against a and amino acid composition, MBP-1 resembles the argininerange of fungi representing both pathogens and nonpathogens and cysteine-rich mammaliandefensins (Lehrer et al., 1990b) of maize, MBP-1 varied widely in its ability to prevent coni- more closely than the thionins. Its secondary structure, howdial germination/growth of hyphae. Whereas growth of F. ever, appears to be quite different than that of the defensins, graminearum was almost completely inhibited by 60 pg/ml which have almost no a-helical content (Bach et al., 1987; (score = 3), growth of A. fluvus was not significantly inhibited Pardi et al.,1988), whereas MBP-1 hasa high a-helical index. at this concentration (score = 0) (Fig. 7). In this respect MBP-1 is similar to thioninswhich also have The synthetic form of MBP-1 was equally as active as the approximately 40% a-helical content (Nimmo et al., 1974). native peptide against F. graminearum (not shown). Since many monocots, including sorghum, contain proteins The tissue localization of MBP-1 was investigated by ex- related to thionins, it may be asked whether peptides other tracting embryo and endosperm (plus seed coat) tissues sep- than MBP-1found in acid extracts of maize seed are thioninarately, as described under “Experimental Procedures.’’ The like. Based on data from Jones and Cooper (1980) plus our SDS-PAGE profiles of whole kernel, embryo, and endosperm preliminary amino acid analysis of other low M , proteins extracts from comparable numbers of seeds (Fig. 2, lanes C- extracted underacidic conditions,’ it appears likely that these E, respectively) indicate that most of the -4000-dalton Coo- other basic proteins are not thionins but rather a family of massie-staining materialis in the embryo fraction, as opposed arginine-rich peptides, which share with thionins a high PI t o endosperm seed coat. On reverse phase HPLC, an Azm and antimicrobial properties. peak corresponding to MBP-1 could be detected in all three The function of MBP-1 in the seed is unknown. Based on extracts but was at least &fold greater in peak area in the its relatively low prevalence in the seed (0.014% of total embryo and total kernel fractions (not shown). protein), it is unlikely to serve as a major source of rapidly mobilized nitrogen during germination, as was suggested for DISCUSSION a small linear, arginine-richpeptide from pumpkin seed (CuMaize kernels were shown to contain several low molecular curbita maxima) (Naisbittet al., 1988). The antimicrobial weight, basic polypeptides, which could be extracted a t acidic activity of MBP-1 in uitro includes both Gram-negative and pH and resolved by cation exchange chromatography. We Gram-positive bacteria and several filamentous fungi. This have determined oneof these, MBP-1, to be an arginine- and raises the possibility thatthe peptide (along with other, glutamate-rich peptide containing 4 cysteines/molecule, with J. P. Duvick and A. G. Rao, unpublished data. a significant a-helical content in aqueous solution, and dis-

+


18819

0 = No inhibition

...

1. Slight inhibition

2 = Moderate inhibition

3 = Strong inhibition

4 = Complete inhibition

FIG. 6. Photomicrographs of F. graminearum conidia after 24 h incubation with increasing concentrations of MBP-1. Panels indicate typical levels of growth inhibition that correspond to scores 0-4 (0 = no observable inhibition relative to water control; 1 = slight inhibition; 2 = substantial inhibition; 3 = almost complete inhibition; 4 = complete inhibition).

I

I

, ~

I related peptides not completely characterized but also pos- (Neucere and Godshall, 1991; Roberts et al., 1988; Vigers et sessing antimicrobial activity) may contribute to the resist- al., 1991). At present it is difficult to assess therole of any of ance of the kernel toinfection by plant pathogenic fungi and these compounds in limiting pathogen spread in the maize bacteria. Three of the fungi tested in the present study (F. ear. Genetic transformation of maize may eventually allow graminearum, F. moniliforme, and A. flavus) are major causal the function of putative antimicrobial factors to be assessed agents of ear rots in field-grown maize as well as maize seed by overexpressing them or inhibiting their expression. It is in storage. The bacterium C. michiganense ssp. nebraskense likely that a combination of physical,developmental, and causes a severe wilt diseaseon susceptiblemaize varieties and biochemical factors areresponsible for resistance to ear pathis thought to be seed-borne (Shurtleff, 1980). The precise ogens. localization of MBP-1 and other small basic proteins within Apart from its function in maize, the MBP-1 peptide is of the seed, as well as during thecourse of seed development, is interest for its antimicrobial properties invitro. This activity unknown. The association of the maize basic peptides with is in a range (5-30 pg/ml) similar to that of several other the embryo of the seed distinguishes them from the monocot small, basic antimicrobial peptides from both plant (Bohlthionins, which reside largely in the endospermof the respec- mann andApel, 1991; Cammue etal., 1992; Garcia-Olmeda et tive species (Carbonero et al., 1980). Althoughhistological al., 1989) and non-plant (Lehrer et al., 1990a) sources. The studies of maize ear rots have been made (Smart et al., 1990), general mechanism for activityof these small, basic peptides verylittleis known about how the seed and surrounding is thought to involve interaction with the plasma membrane tissues respond to and are protected againstmicrobial path- (Christensen et al., 1988). The diversity of structure of pepogens during kernel development. Other proteins having an- tides with similar activities suggests that a specific receptor tifungal properties in vitro have been isolatedfrom maize seed is not involved, although interaction with specific membrane

18820


4

I

Acknowledgments-We thank Drs. Ben Bowen, Kurt Timmerman, and Nancy Tomes for critical reading of the manuscript, Dr. Berne Jones (United States Department of Agriculture Cereal Crops Research Unit, Madison, WI) for helpful discussions, Xia-Ying Zhou and Shirley Elliott (Iowa State University) for protein sequencing and amino acid analysis, and Prof. Ken Neet (UHS/Chicago Medical School) and Dave Timm (Case Western Reserve University, Cleveland) for the circular dichroism measurements.

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REFERENCES Anderson, B. L., Berry, R. W., and Telser, A. (1983) Anal. Biochem. 132,365375 Apel, K., Bohlmann, H., and Reimann-Philipp, U. (1990) Physiol. Plant. 80, 315-321 0 Bach, A. C., Selsted, M. E., and Pardi, A. (1987) Biochemistry 26,4389-4397 Berkowitz, B. A., Bevins, C. L., and Zasloff, M. A. (1990) Biochem. Phurmacol. 39,25-629 Bloch, C. J., and Richardson, M. (1991) FEBS Lett. 2 7 9 , 101-104 Bohlmann, H.,and Apel, K. (1991) Annu. Reu. Plant Physiol. Mol. Biol. 4 2 , FIG. 7. Inhibition of six phytopathogenic fungi by MBP-1. 227-240 Conidia or hyphal pieces were incubated with various concentrations Cammue, B. P. A., De Bolle, M. F. C., Terras, F. R. G., Proost, P., Van Damme, of the peptide in dilute growth medium, and growth was evaluated J.. Rees. S. B.. Vanderlevden. " , J... and Broekaert. W. F. (1992) . , J. Biol. Chem. 267,222a2233 visually using an inverted microscope, using a scale of 0-4 to rate the Carbonero, P., Garcia-Olmedo, F., and Hernandez-Lucas, C. (1980) J. Agric. effect of added peptide (see Fig.6). Fungi tested were F. graminearurn Food Chem. 28,399-402 (H),F. moniliforme (O), A . flauus (O), A . longipes (O),S. sclerotiorurn Christensen, B., Fink, J., Merrifield, R. B., and Mauzerall, D. (1988) Proc. Natl. (V),and S. trifoliorurn (A).Each data point is the average of two Acud. Sci. U. S. A. 86,5072-5076 Diamond, G., Zasloff, M., Eck, H., Brasseur, M., Maloy, W., and Bevins, C. independent experiments with two replications per experiment. (1991) Proc. Natl. Acud. Sci. U. S. A. 88,3962-3956 Fields, C., Lloyd, D., Macdonald, K., Otteson, K., and Noble, R. (1991) Peptide Res. 4,95-101 TABLE I1 Garcia-Olmedo, F., Salcedo, G., Sanchez-Moinge, R., Gomez, L., Royo, J., and Bactericidal effect of MBP-1 on E. coli cells Carbonero, P. (1987) Oxford Suru. Plant Mol. Cell Biol. 4,275-334 Garcia-Olmeda, H.,Rodriguez-Palenzuela, P., Hernandez-Lucas, C., Ponz, C., Data represent average of 2-4 indeDendent experiments. Marana, C., Carmona, M. J., Lopez-Fando, J., Fernandez, J. A., and CarboMBP-1 Colonv formine units nero, P. (1989) Oxford Suru. Plant Mol. Cell Biol. 6, 31-60 Greenfield, N., and Fasman, G. (1969) Biochemistry 8,4108-4116 % control MIml Hernandez-Lucas, C., Caleya, R. F. d.,and Carbonero, P. (1974)Appl. Microbiol. 28,165-158 1 10.8 Hill, C., Yee, J., Selsted, M. E., and Eisenherg, D. (1991) Science 2 6 1 , 14813 1.2 1485 10 0.1 Huynh, Q. K., Hironaka, C. M., Levine, E. B., Smith, C. E., Borgmeyer, J. R., and Shah, D. M. (1992) J.Biol. Chem. 267,6635-6640 30 c0.002 Jones, B. L., and Cooper, D. B. (1980) J.Agric. Food Chem. 28,904-908 Kini, R. M., and Evans, H. J. (1989) Znt. J. Peptide Protein Res. 3 4 , 277-286 Kohayashi, Y., Sato, A,, Takashima, H., Tamaoki, H., Nishimura, S., Kyogoku, TABLE 111 Y., Ikenaka, K., Kondo, T., Mikoshiba, K., Hojo, H., Aimoto, S., and Moroder, Effect of MBP-1 on C. nebraskense ssp. nebraskense cells L. (1991) Neuroehem. Int. 16,525-534 Kronman, M., and Holmes, L. (1971) Photochem. Photobiol. 14, 113-134 Bactericidal assay LeGendre, N., and Matsudaira, P. (1988) BioTechniques 6, 154-159 MBP-1 Bacteriostatic assay Lehrer, R. I., Gam, T., and Selsted, M. E. (1990a) Cell 64,229-230 Expt. 1 Expt. 2 Lehrer, R. I., Ganz, T., and Selsted, M. E. (1990b) ASM News 66,315-318 Loh, Y., Parish, D., and "deja, R. (1985) J. Biol. Chem. 260,7194-7205 MIml % control Mains, R., Glembotski, C., and Eipper, B. (1984) Endocrinology 1 1 4 , 1522, 1 89 Marshak, D., and Binns, G. (1990) in Current Research cn Protetn Chemrstry (Villafrance, J., ed) pp. 127-138, Academic Press, New York 3 47 98 Naisbitt, G. H., Lu, M.-R., Gray, W. R., and Vernon, L. P. (1988) Plant Physiol. 10 49 33 57 88, 770-773 30 19 1 Neucere, J. N., and Godshall, M. A. (1991) Mycopathobgin 113,103-108 100 12 Nimmo, C., Kasarda, D., and Lew, A. J.-L. (1974) J. Sci. Food Agric. 26,607617 Osoreo e Castro, V., and Vernon, L. P. (1989) Toxicon 27,511-517 A., Hare, D. R., Selsted, M. E., Morrison, R. D., Bassolino, D. A., and proteins is not precluded (Osoreo e Castroand Vernon, 1989). Pardi, Bach, A. C. (1988) J. Mol. Biol. 201,625-636 Some structural motifs that may contribute to theproperties Pearson, W., and Lipman, D. J. (1988) Proc. Natl. Acud. Sci. U. S. A. 86,24442448 of these peptides have been identified (Kini andEvans, 1989; Raikhel, N.V., and Wilkins, T. A. (1987) Proc. Natl. Acud. Sci. U. S. A. 8 4 , Kobayashi et al., 1991). Interestingly, MBP-1 contains two 6745-6749 Riddles, P., R., and Lerner, B. (1983) Methods Enzymol. 91.49-60 paired cysteine motifs (C-X-X-X-X-C and C-X-X-C) similar Roberts. W.Blakely, K.. Laue. B. E.. and Selitrennikoff. C. P. (1988) Ann. N . Y.Acud. to those identified by Kobayashi et al. (1991) for several Sei. 6 4 4 , 141-151 ' ' H., and von Jagow, G. (1987) Anal. Biochem. 166,366-379 membrane-active neurotoxins with a-helical content. Distinct Schiigger, Selsted, M. E., Szklarek, D., and Lehrer, R. I. (1984) Infect. Zmmun. 4 6 , 150but highly conserved cysteine residue motifs exist for both 154 defensin and thionin protein families (Bohlmann and Apel, Shurtleff, M. (1980) Compendium of Corn Diseases, The Disease Compendia Series, 2nd Ed., American Phytopathological Society, St. Paul, MN 1991; Lehrer et al.,1990a). In a recent report (Hillet al.,1991) Smart, M. G., Wicklow, D. T., and Caldwell, R. W. (1990) Phytopathology 80, 1287-1294 defensin HNP-3 was shown by x-ray crystallography to exist T., and Witkop, B. (1967) Methods Enzynol. 11,498-506 as a dimer in aqueous solution, leading the authors to propose Spande, Steiner, H., Andreu, D., and Merrifield, R. B. (1988) Biochim. Biophys. Acta 939,260-266 that ion-mediated dimer formation may be required for inserM. M., Mazer, J. A., and L'Italien, J. J. (1981) Biochemistry 20,5437tion of HNP-3 into the membrane. This property may also Teeter, 5443 explain the salt sensitivity of activity of this protein (Hill et Vigers, A. J., Roberts, W.K., and Selitrennikoff, C. P. (1991) Mol. PlantMicrobe Interact. 4,315-323 al.,1991). It is possible that MBP-1also forms a multimerin Wall, J., and Paulis, J. (1987) in Aduances in Cereal Science and Technology solution, since its activity also is sensitive to salt concentra(Pomeranz, Y., ed) Vol. 2, pp. 135-219, American Association of Cereal Chemists, St. Paul, MN ti~n.~ Wilson, C. (1987) in Corn: Chemistry and Technology (Watson, A., and Raustad, P., eds) pp. 273-310, American Association of Cereal Chemistry, St. Paul, MN J. P. Duvick and T. Rood, unpublished data.