Anovelantifungalhevein-typepeptide from
Triticum kiharaeseedswithaunique10-cysteine motif
Tatyana I. Odintsova
1
, Alexander A. Vassilevski
2
, Anna A. Slavokhotova
1
,
Alexander K. Musolyamov
2
, Ekaterina I. Finkina
2
, Natalia V. Khadeeva
1
, Eugene A. Rogozhin
2
,
Tatyana V. Korostyleva
1
, Vitalii A. Pukhalsky
1
, Eugene V. Grishin
2
and Tsezi A. Egorov
2
1 Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia
2 Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia
Introduction
To protect themselves against pathogens and pests,
plants have developed a variety of mechanisms, includ-
ing the creation of a physical barrier to limit pathogen
spread and the production of antimicrobial compounds
inhibiting pathogen growth and the colonization of
plant tissues. Among the defensive compounds
deployed by plants to combat infection are secondary
metabolites, phytoalexins and phytoanticipins, reactive
oxygen species and numerous proteins and peptides
that exert inhibitory activity against invaders. Some
antimicrobials are synthesized constitutively, whereas
others are induced upon challenge with pathogenic
microorganisms [1–5]. The first group contributes to
preformed (basal) resistance, whereas the second group
contributes to resistance activated in response to infec-
tion or wounding by herbivores (induced resistance).
Defense proteins produced by plants fall into two main
categories according to their size: (a) proteins of
> 100 amino acid residues and (b) smaller polypep-
tides (< 100 amino acid residues), classified as pep-
tides. Among the proteins implicated in plant defense,
the so-called pathogenesis-related proteins play a key
role [6]. They comprise a structurally and functionally
heterogeneous groups of polypeptides, among them
Keywords
antifungal peptide; chitin-binding; cysteine
motif; recombinant peptide; Triticum kiharae
Correspondence
T. Egorov, Shemyakin & Ovchinnikov
Institute of Bioorganic Chemistry, Russian
Academy of Sciences, ul. Miklukho-Maklaya
16 ⁄ 10, 117997 Moscow, Russia
Fax: +7 495 330 7301
Tel: +7 495 3364022
E-mail: ego@mx.ibch.ru
Database
The protein sequences reported in this
paper have been submitted to the
UniProtKB database under the accession
number P85966
(Received 7 April 2009, revised 1 June
2009, accepted 5 June 2009)
doi:10.1111/j.1742-4658.2009.07135.x
Two forms of anovel antimicrobial peptide (AMP), named WAMP-1a and
WAMP-1b, that differ by a single C-terminal amino acid residue and
belong to a new structural type of plant AMP were purified fromseeds of
Triticum kiharae Dorof. et Migusch. Although WAMP-1a and WAMP-1b
share similarity withhevein-type peptides, they possess 10 cysteine residues
arranged in aunique cysteine motif which is distinct from those described
previously for plant AMPs, but is characteristic of the chitin-binding
domains of cereal class I chitinases. An unusual substitution of a serine for
a glycine residue in the chitin-binding domain was detected for the first
time in hevein-like polypeptides. Recombinant WAMP-1a was successfully
produced in Escherichia coli. This is the first case of high-yield production
of a cysteine-rich plant AMP froma synthetic gene. Assays of recombinant
WAMP-1a activity showed that the peptide possessed high broad-spectrum
inhibitory activity against diverse chitin-containing and chitin-free patho-
gens, with IC
50
values in the micromolar range. The discovery of a new
type of AMP active against structurally dissimilar microorganisms implies
divergent modes of action and discloses the complexity of plant–microbe
interactions.
Abbreviations
AMP, antimicrobial peptide; CNBr, cyanogen bromide; Trx, thioredoxin.
4266 FEBS Journal 276 (2009) 4266–4275 ª 2009 The Authors Journal compilation ª 2009 FEBS
chitinases and 1,3-b-glucanases, proteinases and some
other enzymes, proteinase and a-amylase inhibitors,
thaumatin-like proteins and antimicrobial peptides
(AMPs). AMPs form a highly evolved defense arsenal
against pathogens that act in concert and form a
ubiquitous tool of the plant innate immune system
[5]. Most plant AMPs are cysteine-rich peptides con-
taining an even number of cysteine residues, all of
which are involved in the formation of intrachain
disulfide bridges, providing their molecules with high
structural stability [1,2,4]. Based on cysteine spacing
motifs and 3D structures, several AMP families have
been discriminated in plants, for example, thionins,
defensins, hevein- and knottin-like peptides, and non-
specific lipid-transfer proteins. Their mode of action
includes disruption of pathogen membranes via spe-
cific or nonspecific interactions with cell-surface
groups [7,8]. Despite a conserved scaffold of mole-
cules within the members of each family, there is
considerable variation in amino acid sequences. There
also exists considerable diversity in the processing
and subcellular targeting of different AMP types [9].
New bioinformatics-based approaches have revealed
an astonishing abundance of AMP-encoding genes in
plant genomes, with hundreds of different genes
being identified in the completely sequenced genomes
of Arabidopsis and rice [10,11]. Such biodiversity
ensures efficient defense against numerous invading
and constantly evolving microorganisms. However, it
remains unclear whether all genes identified in the
genome are expressed and functional. In addition to
being of fundamental significance in AMP research,
they attract considerable attention as candidates for
the genetic transformation of crops and as novel
therapeutics.
Hevein-type AMPs show structural similarity to he-
vein, the 43-amino acid chitin-binding peptide isolated
from the rubber tree Hevea brasiliensis [12], and com-
prise the single hevein domain subfamily in a large
group of chitin-binding proteins [13–16] that share a
common property, the ability to bind chitin, a beta-
1,4-linked polymer of N-acetylglucosamine and related
polysaccharides containing N-acetylglucosamine or
N-acetylneuraminic acid. Because chitin does not occur
in higher plants, but is a component of fungal cell
walls and the exoskeleton of invertebrates, such as
insects and nematodes, it has been hypothesized that
chitin-binding proteins are involved in plant defense
against microorganisms and pests. All known chitin-
binding proteins contain a common structural motif of
30-45 amino acids with several cysteine and glycine
residues at conserved positions named the chitin-
binding domain, which is responsible for binding the
carbohydrate. In addition to hevein, the members of
this family are lectins, chitinases, some wound-induced
proteins and a number of AMPs, later classified as he-
vein-type. Although hevein-type AMPs share certain
sequence homology, they differ in the number of disul-
fide bonds. Most possess eight cysteine residues form-
ing four disulfide bonds [13,14] and in this respect are
close to the chitin-binding domains of class I ⁄ IV
chitinases [15,16]. ‘Truncated’ variants with only six
cysteine residues also occur [17–20]. Only a few ten-
cysteine hevein-type AMPs have been described to date
[21,22]. The functional significance of the additional
cysteine residues of hevein-type AMPs remains to be
elucidated.
In a previous study, we used N-terminal sequences
to identify several dozen novel AMPs fromseeds of
the wheat Triticumkiharae Dorof. et Migusch. [23]. In
this study, we completely sequenced one of those novel
peptides Tk-AMP-H1, in which the number of cysteine
residues was corrected and which was renamed
WAMP-1a. We show that it belongs to a new unique
structural type of hevein-like AMP. We also report
here high-yield heterologous production of WAMP-1a
in Escherichia coli and assays of recombinant WAMP-
1a activity against diverse plant pathogens, such as
chitin-containing and chitin-free fungi, and Gram-
positive and Gram-negative bacteria. The amino acid
sequence of a second peptide WAMP-1b, which differs
from WAMP-1a by an additional C-terminal arginine
residue, was also determined.
Results
Isolation and sequencing of WAMPs
To purify WAMPs from T. kiharae seeds, the acidic
extract of seeds was subjected to three-step chromato-
graphic separation. Affinity chromatography on hepa-
rin–Sepharose was the first step. The fraction eluted at
100 mm NaCl was further separated by size-exclusion
chromatography and the peptide-containing fraction
was then subjected to RP-HPLC (Fig. 1). The major
peak during RP-HPLC (Fig. 1C) contained two pep-
tides with measured monoisotopic molecular masses
(by MALDI-TOF MS) of 4431.9 and 4588.1 Da. The
peptides named WAMP-1a and WAMP-1b were puri-
fied by rechromatography on the same column with a
shallower acetonitrile gradient (10–40% B in 90 min,
B: 80% acetonitrile containing 0.1% trifluoroacetic
acid) (not shown). Both peptides were reduced and
alkylated and their molecular masses became 5482.0
and 5638.5 Da for WAMP-1a and WAMP-1b, respec-
tively, indicating the presence of 10 cysteine residues in
T. I. Odintsova et al. Unique10-cysteineantifungalpeptidefrom wheat
FEBS Journal 276 (2009) 4266–4275 ª 2009 The Authors Journal compilation ª 2009 FEBS 4267
both peptides. Alkylation of unreduced native WAMP
peptides did not result in molecular mass changes,
pointing to the involvement of all 10 thiol groups in
the formation of five disulfide bridges. The complete
amino acid sequences of reduced and alkylated
peptides were determined by automated Edman degra-
dation: WAMP-1a: AQRCGDQARGAKCPNCLCCG
KYGFCGSGDAYCGAGSCQSQCRGC; WAMP-1b:
AQRCGDQARGAKCPNCLCCGKYGFCGSGDAY
CGAGSCQSQCRGCR.
The peptides WAMP-1a and WAMP-1b consist of
44 and 45 residues, respectively, and are virtually iden-
tical; WAMP-1b differs from WAMP-1a by an addi-
tional C-terminal arginine residue. The measured
molecular masses of the peptides are in good agree-
ment with calculated values (4431.7 and 4587.8 Da for
WAMP-1a and WAMP-1b, respectively), indicating
the absence of modifications except disulfide bonds. By
contrast, the other two known 10-Cys hevein-like pep-
tides have a pyroglutamic acid at their N-termini
[21,22].
WAMPs belong to anovel type of AMP. Despite
sequence similarity with hevein and homologous pep-
tides, they possess 10 cysteine residues and thus may
be classified as 10-Cys hevein-like peptides with a
unique previously unknown cysteine motif (Fig. 2).
Only a few 10-Cys hevein-like peptides have been
described to date, isolated from the bark of the trees
Eucommia ulmoides Oliv. [21] and Euonymus ;europa-
eus L. [22]. Amino acid sequence identity between
WAMP-1a and Ee-CBP from E. europaeus, EAFP1
from Eu. ulmoides and hevein amounts to 50–56%.
Although WAMPs, Ee-CBP and EAFP1 possess 10
cysteine residues, the cysteine motif in WAMPs differs
remarkably from that of their 10-Cys homologues,
indicating that WAMPs belong to a new structural
type of hevein-like peptide (Fig. 2B,C). The uneven
distribution of basic amino acids in WAMPs should be
noted; except for a lysine residue at position 21, other
basic residues are located in the N- and C-terminal
regions of the molecules. Striking similarity with
hevein-type domains of cereal class I chitinases, both
in terms of amino acid sequences and cysteine patterns
was noticed (Fig. 2A). High homology between a
hevein-type AMP and the chitin-binding domain of the
chitinase from the same source that act synergistically
was also shown by Van den Bergh et al. [24].
The cysteine connectivities in WAMPs were deduced
from sequence alignment with hevein. Of the 10 cyste-
ines in WAMPs, eight (C1, C2, C4, C5, C6, C7, C8
and C9) are in an identical position in hevein, and
consequently by homology form the same disulfide
bridges (Fig 2B,C). Because WAMPs do not possess
free thiol groups, two additional cysteines (C3 and
C10) are concluded to be connected to each other.
Consequently, it is reasonable to predict the arrange-
ment of disulfide bonds in WAMPs as follows:
C4–C19, C13–C25, C18–C32, C37–C41. An additional
fifth disulfide bond is likely to be formed between C16
Fig. 1. Isolation of WAMP peptides. (A) Affinity chromatography of
the acid-soluble extract from 5 g of T. kiharaeseeds on a 5-mL
HiTrap Heparin HP column. WAMP-containing fraction eluted at
100 m
M NaCl is indicated by a gray box. (B) Size-exclusion chroma-
tography on a Superdex Peptide HR 10 ⁄ 30 column of one half of
the 100 m
M NaCl fraction (see A). The WAMP-containing fraction is
boxed. (C) RP-HPLC of the pooled WAMP-containing fraction (see
B) on a Vydac C
18
column witha 60-min linear acetonitrile gradient
(10–50% B, B: 80% acetonitrile in 0.1% trifluoroacetic acid). The
fraction containing WAMPs is shown by an arrow.
Unique 10-cysteineantifungalpeptidefrom wheat T. I. Odintsova et al.
4268 FEBS Journal 276 (2009) 4266–4275 ª 2009 The Authors Journal compilation ª 2009 FEBS
and C44. This is supported by X-ray analysis of a
class I chitinase from rice (Y. Kezuka, Y. Nishizawa,
T. Watanabe & T. Nonaka, Nagaoka University of
Technology, Japan; PDB accession number 2DKV).
Binding of WAMPs to chitin
The amino acids forming the chitin-binding site
involved in carbohydrate recognition by hevein-type
molecules deserve special attention (Fig. 2). In this site,
several strictly conserved residues are present: serine
19, two glycines at positions 22 and 25 and three aro-
matic amino acids at positions 21, 23 and 30, respec-
tively (the numbering is according to hevein). In
WAMPs from T. kiharae seeds, however, the con-
served serine is replaced by a glycine. The chitin-bind-
ing properties of both WAMP peptides were assayed
in vitro. Purified peptides were applied to a chitin
column and the bound fraction was eluted with 0.1%
trifluoroacetic acid. RP-HPLC and mass measurements
of unbound and bound fractions showed that both
peptides eluted only in the bound fraction. Therefore,
despite the serine to glycine substitution in the carbo-
hydrate-binding site, WAMPs bind to chitin, although
the affinity of this interaction and the precise impact
of the Gly ⁄ Ser substitution are still to be investigated.
Recombinant peptide production
To provide sufficient material for functional investiga-
tions, recombinant WAMP-1a was produced in a pro-
karyotic expression system. Thioredoxin (Trx) was
chosen as the fusion partner for expression, because it is
known to ensure high yields of cystine-containing poly-
peptides with native conformation and mask the
unwanted toxic activity of the produced peptides [9,25].
A synthetic gene coding for WAMP-1a was prepared
from oligonucleotides (Table 1) and cloned into pET-32b
expression vector, and the resulting plasmid (pET-32b-
M–WAMP-1a) was used to transform E. coli BL21
(DE3) cells. Trx–WAMP-1a fusion protein production
and purification was followed by SDS ⁄ PAGE (Fig. 3).
The chimeric protein was treated with cyanogen bromide
(CNBr) and the recombinant WAMP-1a was purified by
RP-HPLC (Fig. 3). The recombinant peptide had the
same retention time and co-eluted with the native
WAMP-1a when analyzed by analytical RP-HPLC, it
also had the expected N-terminal amino acid sequence as
determined by direct Edman sequencing. The molecular
mass of the recombinant product obtained by MALDI
MS was equal to the mass measured for the native
WAMP-1a. The final yield of purified recombinant
WAMP-1a was 8mgÆL
)1
of bacterial culture.
Antimicrobial activity of the recombinant
WAMP-1a
Testing of the biological activity of the recombinant
peptide WAMP-1a against several fungi including
deiteromycetes and ascomycetes showed marked
inhibition of spore germination at micromolar concen-
trations with IC
50
ranging from 5 to 30 lm depending
on the fungus (Table 2). The highest inhibitory activity
was achieved against Fusarium solani, Fusarium
oxysporum and Bipolaris sorokiniana,IC
50
for these
fungi was 5 lm, as low as reported for Ac-AMP2, one
of the most potent antifungal peptides [17]. The
Fig. 2. Sequence alignment of WAMP-1b with selected hevein-
type antimicrobial peptides and chitin-binding domains of class I
chitinases. Conserved cysteine residues are shaded in black. The
conserved residues of the carbohydrate-binding site are marked
with asterisks; the glycine residue in WAMP-1b that substitutes
serine is boxed. (A) Manual alignment of WAMP-1b with chitin-
binding domains of class I chitinases from cereals. GenBank acces-
sion numbers are shown in parentheses for: wheat (Triticum aes-
tivum, AAR11388), rye (Secale cereale, Q9FRV1), barley
(Hordeum vulgare, P11955) and rice (Oryza sativa, 2DKV). Identical
and similar residues are shaded in gray. (B) Hevein-type peptides:
WAMP-1b from T. kiharae (P85966); Ee-CBP from the bark of
E. europaeus (AAP35269); EAFP1 from the bark of Eu. ulmoides
(P83596); hevein from H. brasiliensis (P02877); Ac-AMP2 from
Amaranthus caudatus (P27275). (C) Disulfide bond arrangement in
hevein-type AMPs. Cysteine residues found in all known hevein-like
peptides are presented. Disulfide bonds in hevein are shown by
solid line, an additional fifth disulfide in 10-Cys hevein-like peptides
is shown by dashed line in WAMPs, short-dashed line in Ee-CBP
and dashed-dotted line in EAFP1.
T. I. Odintsova et al. Unique10-cysteineantifungalpeptidefrom wheat
FEBS Journal 276 (2009) 4266–4275 ª 2009 The Authors Journal compilation ª 2009 FEBS 4269
morphological changes in two fungi, F. oxysporum and
B. sorokiniana, in the presence of WAMP-1a were also
examined. In F. oxysporum, inhibition of hyphal elon-
gation and browning of hyphae were observed. In
B. sorokiniana, the most pronounced changes occurred
in spores; destruction and discoloration of spores were
noted. The effect of the peptide on disease develop-
ment caused by the oomycete Phytophthora infestans
using potato tuber discs was also studied. Two differ-
ent strains, the highly aggressive OSV 12 strain and
the Pril 2 strain with low pathogenicity were tested.
The peptide induced stable inhibition of disease devel-
opment over 120 h of observation, followed by a slight
decrease by 144 h in both instances. Complete inhibi-
tion was not achieved at the concentrations tested.
However, the degree of inhibition was higher with the
less aggressive strain (Table 3). The peptide was also
tested for inhibition of bacterial growth against
both Gram-positive (Clavibacter michiganense ) and
Gram-negative (Pseudomonas syringae and Erwinia
carotovora) bacteria; the effect was most pronounced
for the Gram-positive bacterium C. michiganense
(Table 4). The antifungal activity of WAMP-1a is
likely to be associated with its chitin-binding activity,
whereas the inhibitory effect on the oomycete P. infe-
stans and bacteria, which are devoid of chitin, implies
the existence of some other mechanism.
Discussion
In this study, we purified and completely sequenced
two novel highly homologous antimicrobial peptides,
WAMP-1a and WAMP-1b, from T. kiharae seeds,
which we previously discovered in this wheat species
[23], and showed that they belong to a new structural
type of plant AMP. Both peptides are almost identical,
the WAMP-1b peptide possesses an additional arginine
at the C-terminus. The WAMP peptides from T. kiha-
rae seeds obviously belong to the hevein-type AMPs,
as judged by sequence homology with hevein and
homologous peptides and conserved location of eight
cysteine residues and several other amino acids of the
chitin-binding site (Fig. 2). The striking similarity
between WAMPs and the chitin-binding domains of
class I chitinases deserves special attention. Despite
similarity with hevein and related proteins, WAMPs
represent a new structural type of plant AMP with a
specific 10-Cys motif. The characteristic feature of
their molecular scaffold is the presence of an
Fig. 3. Expression and purification of Trx–WAMP-1a fusion protein.
RP-HPLC of the fusion protein Trx–WAMP-1a ( 0.5 mg) cleaved
with CNBr on a Luna C
8
column. Fraction corresponding to WAMP-
1a is indicated with an arrow. The inset shows expression and puri-
fication of Trx–WAMP-1a fusion protein as followed by SDS ⁄ PAGE
(10%). Lane 1, molecular mass markers (LMW-SDS Marker Kit
from GE Healthcare), the corresponding M
r
values are labeled in
kDa; lane 2, whole-cell lysate of E. coli BL21(DE3) cells carrying the
plasmid pET-32b-M–WAMP-1a before isopropyl b-
D-thiogalactoside
treatment; lane 3, induced with 0.2 m
M isopropyl b-D-thiogalacto-
side; lane 4, soluble protein fraction; lane 5, fusion protein purified
by metal-affinity chromatography on TALON Superflow resin.
Table 1. Synthetic oligonucleotides used for WAMP-1a gene con-
struction. KpnI and BamHI restriction sites are underlined, the
methionine codon is shown in bold, the stop codon is in bold and
italics.
Primer
name
Sequence
Forward ACTG
GGTACCATGGCTCAGCGTTGCGGTGAC
2f CAGGCTCGTGGTGCTAAATGCCCGAACTGCCTGTGCTGTG
3f GTAAGTACGGCTTCTGCGGTTCTGGTGACGCTTACTGTGG
4f CGCTGGTTCTTGCCAGTCTCAGTGCCGTGGTTGCTAG
GGAT
1r TTTAGCACCACGAGCCTGGTCACCGCAACGCTGAGC
2r CGCAGAAGCCGTACTTACCACAGCACAGGCAGTTCG
3r GACTGGCAAGAACCAGCGCCACAGTAAGCGTCACCA
Reverse GCTA
GGATCCCTAGCAACCACGGCAC
Table 2. Antifungal activity of WAMP-1a. IC
50
is the concentration
necessary for 50% growth inhibition.
Fungi IC
50
(lgÆmL
)1
)
Bipolaris sorokiniana 5
Botrytis cinerea 20
Fusarium oxysporum 5
Fusarium solani 5
Fusarium verticillioides 30
Neurospora crassa 10
Unique 10-cysteineantifungalpeptidefrom wheat T. I. Odintsova et al.
4270 FEBS Journal 276 (2009) 4266–4275 ª 2009 The Authors Journal compilation ª 2009 FEBS
additional fifth disulfide bond between C3 (Cys16) and
C10 (Cys44), which is located differently from other
known 10-Cys hevein-like peptides (Fig. 2B,C). This
fifth disulfide bond brings together the N- and C-ter-
minal regions of the polypeptide chain, enriched in
basic amino acids (Arg3, Arg9, Lys12, Arg42 and
Arg45 in WAMP-1b). We therefore suggest the exis-
tence of a cluster of basic amino acid residues in
WAMPs formed by the above-mentioned basic resi-
dues of the N- and C-termini. However, elucidation of
the 3D structure of WAMPs is necessary to confirm
this hypothesis. The second structural peculiarity of
WAMPs that discriminates these peptides from all
known chitin-binding polypeptides is the unique struc-
ture of the chitin-binding site, in which a conserved
serine residue at position 20 is substituted for glycine,
although three aromatic residues (Tyr22, Phe24 and
Tyr31) are well conserved. To the best of our
knowledge, this is the first communication on such a
replacement in the chitin-binding site. Analysis of chi-
tin-binding properties of WAMPs by in vitro assays
showed that both peptides bind chitin, demonstrating
that the serine ⁄ glycine substitution is not crucial for
binding, although its precise role in the efficiency of
binding remains to be explored.
Unusual structural characteristics of WAMPs sug-
gested unique biological properties, however, limited
amounts of the peptides recovered from T. kiharae
seeds were insufficient for large-scale assays of their
biological activity. To produce the target peptide in a
correctly folded soluble form and eliminate possible
toxic effects on the host cells, thioredoxin, a natural
soluble component of E. coli cells, was chosen as a
fusion partner. Preliminarily, the synthetic gene
encoding WAMP-1a was constructed. As a result, the
biologically active 10-Cys recombinant peptide
WAMP-1a was successfully produced in E. coli with
high yields (8 mgÆL
)1
of culture). To the best of our
knowledge, for the first time, a synthetic gene con-
struction was used to generate a 10-Cys AMP in
E. coli. This approach is indispensable for the rapid
production of AMPs with unknown functions avoid-
ing time-consuming gene cloning. Moreover, it effec-
tively allows introducing codons optimized for
E. coli. The biological activity of the recombinant
WAMP-1a was assayed against fungi, oomycetes and
bacteria. The results showed that the peptide has
broad inhibitory activity both against chitin-contain-
ing and chitin-free pathogens. The peptide not only
inhibited spore germination of the deiteromycetes and
ascomycetes tested, but also caused morphological
changes in the fungi. The activity of the peptide both
against chitin-containing and chitin-free pathogens
may result from the unique structural features of
WAMPs detailed above. We speculate that the inhibi-
tion of morphogenesis in chitin-containing fungi is
associated with its chitin-binding activity, whereas the
effect on bacteria may result from interactions
between the cluster of basic amino acid residues in
WAMP molecules and negatively charged phospholip-
ids of the bacterial membranes, as postulated for
membrane-active amphiphilic AMPs. The positive
charge ensures accumulation at polyanionic microbial
cell surfaces that contain acidic polymers, such as
lipopolysaccharide and cell-wall-associated teichoic
acids in Gram-negative and Gram-positive bacteria,
respectively. By insertion into the membrane, amphi-
philic AMPs disrupt the integrity of the bilayer
through membrane thinning, transient poration
and ⁄ or disruption of the barrier function, or translo-
cate across the membrane and act on internal targets
[7,26].
In summary, two novelhevein-type chitin-binding
AMPs, WAMP-1a and WAMP-1b, were purified from
T. kiharaeseeds and sequenced. The peptides consist
of 44 and 45 amino acids, respectively, and differ by a
single amino acid residue at the C-terminus. The
WAMP peptides belong to a new structural type of
hevein-like AMP with sequence similarity to chitin-
binding domains of cereal class I chitinases. Testing of
the biological activities of the recombinant peptide
Table 4. Antibacterial activity of WAMP-1a.
Peptide
concentration
(lgÆ50 lL
)1
)
a
Inhibition zone in cm including the size of the
peptide application zone
b
Clavibacter
michiganense
Erwinia
carotovora
Pseudomonas
syringae
10 1.7 (3.6) 1.5 (3.4) 1.3 (1.4)
5 1.5 (3.2) 1.3 (2.7) 1.2 (1.2)
2.5 1.3 (3.0) 1.1 (1.1) 0.9 (1.0)
a
Sample volume 50 lL.
b
Size of the peptide application zone
0.5 cm. The size of the inhibition zone caused by claforan is shown
in parentheses.
Table 3. The effect of WAMP-1a on Phytophthora infestans dis-
ease development. Peptide concentrations in micromoles are
shown in brackets.
Strain 96 h 120 h 144 h
PRIL 2 +++ (5.0) +++ (5.0) ++ (5.0)
OSV 12 ++ (5.0) + (5.0) no effect
OSV 12 ++ (10) ++ (10.0) + (10.0)
T. I. Odintsova et al. Unique10-cysteineantifungalpeptidefrom wheat
FEBS Journal 276 (2009) 4266–4275 ª 2009 The Authors Journal compilation ª 2009 FEBS 4271
WAMP-1a expressed froma synthetic gene in E. coli
showed potent antifungal and antibacterial effects at
micromolar concentrations. The discovery of WAMPs
expands our knowledge on the molecular diversity of
AMPs produced by plants to combat pathogenic
microorganisms.
Experimental procedures
Biological material and chemicals
Seeds of T. kiharae Dorof. et Migush. (Poaceae, Magno-
liophyta) were obtained from the collection of the Institute
of General Genetics of the Russian Academy of Sciences
(Moscow, Russia). Fungi and bacteria, F. solani VKM F-
142, F. verticillioides VKM F-670, F. oxysporum TSA-4,
B. cinerea VKM F-85, Neurospora crassa VKM F-184,
Ps. syringae VKM B-1546, C. michiganense subsp. michigan-
ense VKM Ac-1144 and Er. carotovora subsp. carotovora
VKM B-1247 were obtained from the All-Russian Collec-
tion of Microorganisms (Pushchino, Russia). The fungus
B. sorokiniana, strain 6 ⁄ 10 was from the Timiryazev Agricul-
tural Academy (Moscow, Russia). The oomycete P. infestans
strains Pril 2 and OSV 12 were from the Institute of Plant
Protection (Priluki, Minsk District, Bellorussia). E. coli
BL21(DE3) strain and the expression vector pET-32b (Nov-
agen, Madison, WI, USA) were purchased from Rusbiolink
(Moscow, Russia). Restriction enzymes, T4 DNA ligase,
PCR reagents, PCR clean-up system and DNA purification
system were from Promega (Madison, WI, USA). TALON
Superflow Metal Affinity Resin (Clontech, Mountain View,
CA, USA) was used for affinity chromatography. Bacterial
cultures were grown using the safety recommendations from
All-Russian Collection of Microorganisms. Chemicals were
from Sigma-Aldrich (St. Louis, MO, USA), Merck (Darm-
stadt, Germany) and UV-grade acetonitrile was from
Cryochrom (St Petersburg, Russia). 4-Vinylpyridine (Sigma-
Aldrich) was vacuum distilled under argon.
Isolation of WAMPs
Isolation of WAMPs from T. kiharaeseeds mainly fol-
lowed the earlier developed procedure [23]. Briefly, flour
was extracted with 5 volumes of mixture of 1% trifluoro-
acetic acid, 1 m HCl, 5% HCOOH and 1% NaCl in the
presence of the proteinase inhibitor cocktail for plant cell
extracts (Sigma-Aldrich). Extracted proteins and peptides
were precipitated with acetone. The pellet was dissolved in
0.1% trifluoroacetic acid, desalted by RP-HPLC and dried
on a SpeedVac concentrator, whereupon it was subjected
consecutively to three types of HPLC: affinity, size-exclu-
sion and reversed-phase. First, the desalted fraction was
solubilized in 10 mm Tris ⁄ HCl, pH 7.2 (buffer A) and
subjected to affinity chromatography on a 5-mL HiTrap
Heparin HP column (GE Healthcare, Little Chalfont,
UK) equilibrated with buffer A. After elution of the unad-
sorbed fraction, proteins and peptides were eluted with a
step-wise NaCl gradient in buffer C: 50, 100 and 500 mm
NaCl at a flow rate of 1 mLÆmin
)1
. Proteins and peptides
were detected at 280 nm. The obtained fractions were
desalted and dried as described above. The fraction eluted
at 100 mm NaCl during affinity chromatography was sep-
arated by size-exclusion chromatography on a Superdex
Peptide HR 10 ⁄ 30 column (GE Healthcare). Proteins and
peptides were eluted with 5% CH
3
CH in 0.05% trifluoro-
acetic acid at a flow rate of 15 mLÆh
)1
and detected at
214 nm. The peptide fraction was further separated by
RP-HPLC on a Vydac 218TP54 C
18
column (4.6 · 250
mm; Separations Group, Hesperia, CA, USA) with a
60-min linear acetonitrile gradient (10–50% B, B: 80%
acetonitrile in 0.1% trifluoroacetic acid) at a flow rate of
1mLÆmin
)1
and detection at 214 nm.
Reduction and alkylation of peptides
Reduction with dithiothreitol and alkylation with 4-vinyl-
pyridine were accomplished essentially as earlier described
[23]. Shortly, the protein was dissolved in 20 lL of 0.5 m
Tris ⁄ HCl buffer, pH 7.6, containing 6 m guanidine hydro-
chloride and 2 mm EDTA (disodium salt). One microliter
of freshly prepared 1.4 lm aqueous dithiothreitol solution
was added to the mixture. The reaction was allowed to
proceed under nitrogen for 4 h at 40 °C. After reduction,
2 lL of 50% 4-vinylpyridine in 2-propanol were added,
mixed and allowed to react for another 20 min under
nitrogen at room temperature in the dark. After the
reaction, the mixture was diluted twofold with 0.1%
trifluoroacetic acid and separated by RP-HPLC on a
Vydac column as described above. The number of cysteine
residues was estimated from the mass difference between
the reduced and alkylated and nonalkylated polypeptide.
Analytical methods
Peptides were analyzed by MALDI-TOF MS. Mass spec-
tra were acquired on a model Ultraflex MALDI-TOF-
TOF mass spectrometer (Bruker Daltonics, Bremen,
Germany). Calibration was performed using a Proteo-
Mass peptide and protein MALDI-MS calibration kit
(mass range 700–66 000 Da; Sigma-Aldrich). Molecular
masses were determined in linear or reflector positive-ion
mode using samples prepared using the dried-droplet
method with a-cyano-4-hydroxycinnamic acid (10 mgÆmL
)1
in 50% acetonitrile with 0.1% trifluoroacetic acid) matrix
(Sigma-Aldrich).
Amino acid sequences were determined by automated
Edman degradation on a model 492 Procise sequencer
(Applied Biosystems, Foster City, CA, USA).
Unique 10-cysteineantifungalpeptidefrom wheat T. I. Odintsova et al.
4272 FEBS Journal 276 (2009) 4266–4275 ª 2009 The Authors Journal compilation ª 2009 FEBS
Absorption spectra were recorded on a Hitachi U-3210
spectrophotometer (Hitachi, Tokyo, Japan). Polypeptide
concentrations were determined using molar extinction
coefficients at 280 nm (e
280
) calculated using the gpmaw
program (Lighthouse Data, Odense, Denmark; http://www.
gpmaw.com/): 3160 m
)1
Æcm
)1
for WAMP-1a, 17 220
m
)1
Æcm
)1
for the fusion protein Trx–WAMP-1a.
Chitin-binding assay
The peptide (2 nmol) was dissolved in 50 mm NH
4
HCO
3
,
pH 7.8, and loaded onto a chitin column (0.5 mL) equili-
brated with the same buffer. Elution of the unadsorbed
fraction was performed until the absorbance of the eluate
at 280 nm reached the value of 0.01, the bound fraction
was eluted with 0.1% trifluoroacetic acid. Both fractions
were analyzed by MS and RP-HPLC.
Expression vector construction
Antimicrobial peptide expression was achieved essentially
by following a previously elaborated procedure [25]. The
DNA sequence encoding WAMP-1a was constructed from
a number of synthetic oligonucleotides (Table 1) using the
PCR technique. The target PCR fragment was amplified
using a forward primer containing a KpnI restriction site
and a Met codon for CNBr cleavage, and a reverse pri-
mer containing a BamHI restriction site and a stop
codon. The PCR fragment was gel purified, digested by
suitable restriction enzymes and cloned into the expression
vector pET-32b (Novagen) to produce pET-32b-
M–WAMP-1a. The resulting construct was checked by
sequencing.
Fusion protein production and purification
E. coli BL21(DE3) cells transformed with the expression
vector pET-32b-M–WAMP-1a were cultured at 37 °Cin
Luria–Bertani medium containing 100 lgÆmL
)1
ampicillin
to a culture density of D
600
= 0.4–0.8. Expression was
induced by adding isopropyl b-d-thiogalactoside to a con-
centration of 0.2 mm. Cells were cultured at room tempera-
ture (24 °C) overnight (16 h) and harvested (centrifuged for
20 min at 5000 g). The cell pellet was resuspended in the
start buffer for affinity chromatography (1 g of wet weight
cells in 10 mL of 300 mm NaCl, 20 mm Tris ⁄ HCl buffer,
pH 7.5) and ultrasonicated. Lysed cells were centrifuged for
15 min at 20 000 g to remove all insoluble particles. The
supernatant was applied to a preliminarily equilibrated
TALON Superflow resin (volume of 3 mL; Clontech) and
the fusion protein (Trx–WAMP-1a) was purified according
to the protocol supplied by the manufacturer (washed with
the buffer containing 5 mm imidazole, 500 mm NaCl, 5%
glycerol, 0.1% Triton X-100, 20 mm Tris ⁄ HCl, pH 7.5, and
eluted with the final elution buffer containing 150 mm imid-
azole, 300 mm NaCl, 20 mm Tris ⁄ HCl, pH 7.5).
Fusion protein cleavage and target peptide
isolation
The hybrid protein was quickly desalted on a Jupiter C
5
column (4.6 · 150 mm; Phenomenex, Torrance, CA,
USA), using a step of acetonitrile concentration (0–70%)
in 0.1% trifluoroacetic acid. The collected fusion protein
was dried on a vacuum concentrator at room temperature
and dissolved in 0.1 m HCl. Protein cleavage with CNBr
was performed overnight (16 h) at room temperature
(24 °C) in the dark, witha protein-to-CNBr molar ratio
of 1 : 1000. The solvent and excess CNBr were removed
on a SpeedVac concentrator. Recombinant WAMP-1a was
purified by RP-HPLC on a Luna C
8
column (4.6 · 150
mm; Phenomenex) in a linear gradient of acetonitrile (5–
25% in 30 min, 25–60% in 10 min) in 0.1% trifluoroacetic
acid; detection was performed by measuring effluent absor-
bance at 280 nm. The purity of the target peptide was
checked by MS, as well as by N-terminal sequencing, and
the concentration was measured by optical absorption at
280 nm. Chromatographic retention times of recombinant
and natural WAMP-1a were compared by co-injecting
samples onto a Vydac C
18
column and running a shallow
acetonitrile gradient.
Antifungal assays
The antifungal activity of the peptides was tested against
several fungi using microtiter-plate assays essentially as
described previously [27]. Wells were filled with 10 lLof
twofold serial dilutions of the peptide and mixed with
90 lL half-strength potato–glucose broth containing
10
4
sporesÆmL
)1
. The inhibition of spore germination
was evaluated by measuring the absorbance at 620 nm.
Morphological changes were recorded using a light micro-
scope.
The biological activity of peptides was also assayed by
estimating the degree of inhibition of the oomycete P. infe-
stans development on potato tuber discs. Two potato tuber
discs of similar size were placed in each Petri dish. Peptide
samples were mixed with 50 lL of zoosporangium suspen-
sion in distilled water (2 · 10
4
zoosporangiaÆmL
)1
) to a final
peptide concentration of 1.25–20 lm and incubated at 20 °C
for 2 h. The peptide sample was applied to the center of
each potato tuber disc. Potato discs infected with zoospo-
rangium suspension without peptide served as controls. Petri
dishes with infected potato tuber discs were incubated at
20 °C for 120 h. The severity of the disease was assayed 96,
120 and 144 h after inoculation by measuring the infected
area of each disc and scored from 0 to ++++, with 0
denoting the absence of inhibition compared with controls,
T. I. Odintsova et al. Unique10-cysteineantifungalpeptidefrom wheat
FEBS Journal 276 (2009) 4266–4275 ª 2009 The Authors Journal compilation ª 2009 FEBS 4273
+ denoting low inhibitory activity (disease development 20–
40%), ++ designating moderate inhibitory activity (disease
development 10–20%,), +++ denoting strong inhibition
(disease development below 10%), and ++++ represent-
ing complete inhibition (no disease symptoms are observed).
Ten discs were analyzed in each of three independent experi-
ments. The morphological changes in the fungi were also
recorded using a light microscope.
Antibacterial assays
The antibacterial activity of peptides was assayed against
several Gram-positive and Gram-negative bacteria using
radial diffusion assay. Petri dishes with Luria–Bertani agar
were seeded with test bacteria. The peptide solutions
(50 lL) were applied to the wells (5 mm in diameter)
punched into the agar, and the Petri dishes were incubated
at room temperature for 24–48 h. The antibacterial activity
was evaluated by the size of the inhibition zone formed
around the wells with the peptide solution. The antibiotic
claforan and sterile water were used as controls.
Acknowledgements
This work was supported in part by the Biodiversity
Program of the Russian Academy of Sciences and
grants from the Russian Foundation for Basic
Research (no. 08-04-00783 and no. 09-04-00250). We
also thank Bert Billen (KU Leuven, Belgium) for help
with manuscript preparation.
References
1 Broekaert WF, Cammue BPA, De Bolle MFC, Thevissen
K, De Samblanx GW & Osborn RW (1997) Antimicro-
bial peptides from plants. Crit Rev Plant Sci 16, 297–
323.
2 Garcia-Olmedo F, Molina A, Alamillo JM & Rodri-
guez-Palenzuela P (1998) Plant defense peptides. Bio-
polymers 47, 479–491.
3 Selitrennikoff CP (2001) Antifungal proteins. Appl Envi-
ron Microbiol 67, 2883–2894.
4 Garcia-Olmedo F, Rodriguez-Palenzuela P, Molina A,
Alamillo JM, Lopez-Solanilla E, Berrocal-Lobo M &
Poza-Carrion C (2001) Antibiotic activities of peptides,
hydrogen peroxide and peroxynitrite in plant defence.
FEBS Lett 498, 219–222.
5 Manners JM (2007) Hidden weapons of
microbial destruction in plant genomes. Genome Biol 8,
225, doi:10.1186/gb-2007-8-9-225.
6 Sels J, Mathys J, De Coninck BM, Cammue BP &
De Bolle MF (2008) Plant pathogenesis-related (PR)
proteins: a focus on PR peptides. Plant Physiol Biochem
46, 941–950.
7 Shai Y (2002) Mode of action of membrane active anti-
microbial peptides. Biopolymers 66, 236–248.
8 Thevissen K, Ferket KK, Francois IE & Cammue BP
(2003) Interactions of antifungal plant defensins
with fungal membrane components. Peptides 24,
1705–1712.
9 Vassilevski AA, Kozlov SA & Grishin EV (2008) Anti-
microbial peptide precursor structures suggest effective
production strategies. Recent Pat Inflamm Allergy Drug
Discov 2, 58–63.
10 Silverstein KA, Graham MA, Paape TD &
VandenBosch KA (2005) Genome organization of more
than 300 defensin-like genes in Arabidopsis. Plant
Physiol 138, 600–610.
11 Silverstein KA, Moskal WA Jr, Wu HC, Underwood
BA, Graham MA, Town CD & VandenBosch KA
(2007) Small cysteine-rich peptides resembling antimi-
crobial peptides have been under-predicted in plants.
Plant J 51, 262–280.
12 Van Parijs J, Broekaert WF, Goldstein IJ & Peumans
WJ (1991) Hevein: an antifungal protein from
rubber-tree (Hevea brasiliensis) latex. Planta 183,
258–264.
13 Koo JC, Lee SY, Chun HJ, Cheong YH, Choi JS,
Kawabata S, Miyagi M, Tsunasawa S, Ha KS, Bae
DW et al. (1998) Two hevein homologs isolated from
the seed of Pharbitis nil L. exhibit potent antifungal
activity. Biochim Biophys Acta 1382, 80–90.
14 Li SS & Claeson P (2003) Cys ⁄ Gly-rich proteins
with a putative single chitin-binding domain from oat
(Avena sativa) seeds. Phytochemistry 63, 249–255.
15 Raikhel NV, Lee HI & Broekaert WF (1993) Structure
and function of chitin-binding proteins. Annu Rev Plant
Physiol Plant Mol Biol 44, 591–615.
16 Beintema JJ (1994) Structural features of plant chitinas-
es and chitin-binding proteins. FEBS Lett
350, 159–163.
17 Broekaert WF, Marien W, Terras FR, De Bolle MF,
Proost P, Van Damme J, Dillen L, Claeys M, Rees SB,
Vanderleyden J et al. (1992) Antimicrobial peptides
from Amaranthus caudatus seedswith sequence
homology to the cysteine ⁄ glycine-rich domain of
chitin-binding proteins. Biochemistry 31, 4308–4314.
18 Huang X, Xie W & Gong Z (2000) Characteristics and
antifungal activity of a chitin binding protein from
Ginkgo biloba. FEBS Lett 478, 123–126.
19 Nielsen KK, Nielsen JE, Madrid SM & Mikkelsen JD
(1997) Characterization of a new antifungal chitin-bind-
ing peptidefrom sugar beet leaves. Plant Physiol 113,
83–91.
20 Lipkin A, Anisimova V, Nikonorova A, Babakov A,
Krause E, Bienert M, Grishin E & Egorov T (2005) An
antimicrobial peptide Ar-AMP from amaranth
(Amaranthus retroflexus L.) seeds. Phytochemistry 66,
2426–2431.
Unique 10-cysteineantifungalpeptidefrom wheat T. I. Odintsova et al.
4274 FEBS Journal 276 (2009) 4266–4275 ª 2009 The Authors Journal compilation ª 2009 FEBS
21 Huang RH, Xiang Y, Liu XZ, Zhang Y, Hu Z &
Wang DC (2002) Two novelantifungal peptides distinct
with a five-disulfide motiffrom the bark of
Eucommia ulmoides Oliv. FEBS Lett 521, 87–90.
22 Van den Bergh KP, Proost P, Van Damme J,
Coosemans J, Van Damme EJ & Peumans WJ (2002)
Five disulfide bridges stabilize a hevein-type
antimicrobial peptidefrom the bark of spindle tree
(Euonymus europaeus L.). FEBS Lett 530, 181–185.
23 Egorov TA, Odintsova TI, Pukhalsky VA & Grishin
EV (2005) Diversity of wheat anti-microbial peptides.
Peptides 26, 2064–2073.
24 Van den Bergh KP, Rouge P, Proost P, Coosemans J,
Krouglova T, Engelborghs Y, Peumans WJ & Van
Damme EJ (2004) Synergistic antifungal activity of two
chitin-binding proteins from spindle tree (Euonymus
europaeus L.). Planta 219, 221–232.
25 Shlyapnikov YM, Andreev YA, Kozlov SA,
Vassilevski AA & Grishin EV (2008) Bacterial pro-
duction of latarcin 2a, a potent antimicrobial peptide
from spider venom. Protein Expr Purif 60, 89–95.
26 Hancock RE & Sahl HG (2006) Antimicrobial and
host-defense peptides as new anti-infective therapeutic
strategies. Nat Biotechnol 24, 1551–1557.
27 Broekaert WF, Terras FRG, Cammue BPA &
Vanderleyden J (1990) An automated quantitative assay
for fungal growth inhibition. FEMS Microbiol Lett 69,
55–59.
T. I. Odintsova et al. Unique10-cysteineantifungalpeptidefrom wheat
FEBS Journal 276 (2009) 4266–4275 ª 2009 The Authors Journal compilation ª 2009 FEBS 4275
. CGCTGGTTCTTGCCAGTCTCAGTGCCGTGGTTGCTAG
GGAT
1r TTTAGCACCACGAGCCTGGTCACCGCAACGCTGAGC
2r CGCAGAAGCCGTACTTACCACAGCACAGGCAGTTCG
3r GACTGGCAAGAACCAGCGCCACAGTAAGCGTCACCA
Reverse GCTA
GGATCCCTAGCAACCACGGCAC
Table. A novel antifungal hevein-type peptide from
Triticum kiharae seeds with a unique 10-cysteine motif
Tatyana I. Odintsova
1
, Alexander A. Vassilevski
2
,