Although WAMP-1a and WAMP-1b share similarity with hevein-type peptides, they possess 10 cysteine residues arranged in a unique cysteine motif which is distinct from those described prev
Trang 1Triticum kiharae seeds with a unique 10-cysteine motif Tatyana I Odintsova1, Alexander A Vassilevski2, Anna A Slavokhotova1,
Alexander K Musolyamov2, Ekaterina I Finkina2, Natalia V Khadeeva1, Eugene A Rogozhin2, Tatyana V Korostyleva1, Vitalii A Pukhalsky1, Eugene V Grishin2 and Tsezi A Egorov2
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 polytides (< 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 a novel 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 from seeds of Triticum kiharae Dorof et Migusch Although WAMP-1a and WAMP-1b share similarity with hevein-type peptides, they possess 10 cysteine residues arranged in a unique 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 from a 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 IC50 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.
Trang 2chitinases 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 chitin-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 from seeds of the wheat Triticum kiharae 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
Trang 3both 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 a novel 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 kiharae seeds 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 C18column with a 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.
Trang 4and 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 8 mgÆL)1of 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 IC50ranging 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, IC50 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.
Trang 5morphological 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 concentrainhibi-tions 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 C8column 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 ACTGGGTACCATGGCTCAGCGTTGCGGTGAC
Reverse GCTAGGATCCCTAGCAACCACGGCAC
Table 2 Antifungal activity of WAMP-1a IC 50 is the concentration necessary for 50% growth inhibition.
Trang 6additional 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-consumavoid-ing gene clonavoid-ing 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 novel hevein-type chitin-binding AMPs, WAMP-1a and WAMP-1b, were purified from
T kiharae seeds 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
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.
Trang 7WAMP-1a expressed from a 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 syringaeVKM 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 kiharae seeds 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% CH3CH 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 C18 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
1 mLÆmin)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 40C 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 Ulspec-traflex
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)
Trang 8Absorption spectra were recorded on a Hitachi U-3210
spectrophotometer (Hitachi, Tokyo, Japan) Polypeptide
concentrations were determined using molar extinction
coefficients at 280 nm (e280) calculated using the gpmaw
program (Lighthouse Data, Odense, Denmark; http://www
gpmaw.com/): 3160 m)1Æcm)1 for WAMP-1a, 17 220
m)1Æcm)1for the fusion protein Trx–WAMP-1a
Chitin-binding assay
The peptide (2 nmol) was dissolved in 50 mm NH4HCO3,
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
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 37C in
Luria–Bertani medium containing 100 lgÆmL)1 ampicillin
to a culture density of D600= 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 (24C) 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 C5
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 (24C) in the dark, with a 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 C8 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 C18column 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 lL of twofold serial dilutions of the peptide and mixed with
90 lL half-strength potato–glucose broth containing
104
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· 104zoosporangiaÆmL)1) to a final peptide concentration of 1.25–20 lm and incubated at 20C 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
20C 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,
Trang 9+ 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 seeds with 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 peptide from 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
Trang 1021 Huang RH, Xiang Y, Liu XZ, Zhang Y, Hu Z &
Wang DC (2002) Two novel antifungal peptides distinct
with a five-disulfide motif from the bark of
Eucommia ulmoidesOliv 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 peptide from 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 europaeusL.) 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