Download Isolation and Amino Acid Sequence of Two New PR

Journal of Protein Chemistry, Vol. 20, No. 4, May 2001 (© 2001)
Isolation and Amino Acid Sequence of Two New PR-4
Proteins from Wheat
Carla Caruso,1,2 Monica Nobile,1 Luca Leonardi,1 Laura Bertini,1 Vincenzo Buonocore,1
and Carlo Caporale1
Received April 5, 2001
We have purified and characterized two new pathogenesis-related (PR) proteins from wheat belonging to the PR-4 family. We named the proteins wheatwin3 and wheatwin4 in analogy with the
previously characterized wheatwin1 and wheatwin2. Their isoelectric points were 7.1 and 8.4, respectively. We determined the complete amino acid sequence of both proteins by a rapid approach
based on the knowledge of the primary structures of the homologous wheatwin1 and wheatwin2.
Wheatwin3 differs from wheatwin1 in one substitution at position 88, while wheatwin4 differs from
wheatwin2 in one substitution at position 78. The secondary structure and solvent accessibility of
these residues were determined on the three-dimensional model of wheatwin1. Residue 88 was very
accessible and was located in a flexible region. Preliminary results indicate that, like wheatwin1 and
wheatwin2, wheatwin3 and wheatwin4 have antifungal activity.
KEY WORDS: Amino acid sequence; molecular modeling; pathogenesis-related proteins; wheat kernel.
1. INTRODUCTION
Several PR proteins show both acidic and basic isoforms generally located in the apoplast and in the vacuole, respectively. It has been well established that only
intracellular (vacuolar) PR proteins show antifungal activity (Woloshuk et al., 1991), whereas extracellular
(apoplastic) isoforms might be involved in recognition
processes releasing defense-activating signal molecules
from the wall of invading pathogens (Mauch and Staehelin, 1989). In many cases, isoforms also differ in tissuespecific expression during development and induction
pattern by endogenous and exogenous signaling compounds. These different behaviors are related to the existence of multigene families coding homologous proteins,
whose expression suggests that PR proteins might be involved in some processes extending beyond a role in
adaptation to biotic stress. A better understanding of the
Among the biochemical events following plant–pathogen
interaction is the production of a heterogeneous group of
pathogenesis-related (PR)3 proteins showing a strong antifungal activity in vitro against pathogenic fungi. These
host-coded proteins were first described in tobacco plants
infected with tobacco mosaic virus (Gianinazzi et al.,
1970; Van Loon and Van Kammen, 1970); a large number of members of this protein group has since been detected in other plant species and classified in 14 families
(Van Loon and Van Strien, 1999). Members of some
classes have an enzymatic or inhibiting activity (e.g.,
those of the PR-2 family are ␤-glucanases; PR-3, chitinases; PR-6, proteinase inhibitors; PR-9, peroxidases;
PR-10, ribonuclease-like), whereas for others the mode
of action is unknown (Van Loon and Van Strien, 1999).
3
1
Dipartimento di Agrobiologia ed Agrochimica, Università della Tuscia, 01100 Viterbo, Italy.
2
To whom correspondence should be addressed; e-mail: caruso@
unitus.it
Abbreviations: ES-MS, electrospray mass spectrometry; FPLC, fast
protein liquid chromatography; IEF, isoelectric focusing; PR, pathogenesis related; PTH, phenylthiohydantoin; PyEt, pyridylethylated;
RP-HPLC, reversed-phase high-performance liquid chromatography;
SDS–PAGE, polyacrylamide gel electrophoresis in the presence of
sodium dodecyl sulfate.
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0277-8033/01/0500-0327$19.50/0 © 2001 Plenum Publishing Corporation
328
physiological roles of PR proteins can be obtained
through the isolation and structural–functional characterization of new isoforms and the identification of their
targets and mode of action.
Among the less extensively studied PR proteins are
those belonging to the PR-4 family. Their action mechanism and potential for enhancing plant resistance to pathogenic fungi and amplifying the antifungal effects of
other well-described PR proteins (osmotin, chitinase,
glucanase) have not been explored. Genes encoding PR-4
proteins were studied in potato, tomato, tobacco, Arabidopsis, and rubber tree (Broekaert et al., 1990;
Friedrich et al., 1991; Linthorst et al., 1991; Potter et al.,
1993; Stanford et al., 1989), while mature proteins have
been characterized only from tomato (Joosten et al.,
1990), tobacco (Ponstein et al., 1994), and the monocot
barley (Hejgaard et al., 1992; Svensson et al., 1992).
A further classification for this protein family has been
proposed based on the presence (class I) or absence
(class II) of a cysteine-rich domain (hevein-like) corresponding to mature hevein, a small antifungal protein
isolated from the rubber tree (Hevea brasiliensis) latex
(Broekaert et al., 1990). The hevein-like domain, also referred as chitin-binding domain, is also present in genes
coding lectins (Raikhel et al., 1993; Van Damme et al.,
1998) as well as basic chitinases of class I (PR-3)
(Collinge et al., 1993); in these PR-3 proteins the chitinbinding domain is fused to a chitinolytic C-terminal portion unrelated to the domain coding class II PR-4
proteins (Beintema, 1994).
We previously isolated and sequenced two PR-4
proteins from wheat kernels, named wheatwin1 and
wheatwin2, that inhibit phytopathogenic fungi with wide
host range (e.g., Botrytis cinerea) and host-specific
pathogens (e.g., Fusarium culmorum, F. graminearum)
(Caruso et al., 1993, 1996) and demonstrated that these
proteins are specifically induced in wheat seedlings infected with Fusarium culmorum (Caruso et al., 1999a).
Recently, we have isolated two cDNA clones coding
wheatwin1 and wheatwin2 by PCR amplification of
cDNA obtained by a reverse transcriptase reaction using
primers specifically designed on the known sequence of
wheatwin1 (Caruso et al., 1999b). Furthermore, on the
basis of the knowledge of the tertiary structure in solution of barwin (Ludvigsen and Poulsen, 1992a, b), a
highly homologous protein from barley seeds (Svensson
et al., 1992) showing a six-stranded, double-psi beta barrel (Castillo et al., 1999), the three-dimensional model of
wheatwin1 has been designed and experimentally validated (Caporale et al., 1999).
In this paper we report the isolation and structural
characterization of two new members of the wheatwin
family that we named wheatwin3 and wheatwin4, in anal-
Caruso et al.
ogy with wheatwin1 and wheatwin2. The existence of
further isoforms of wheat PR-4 proteins might represent
a selective advantage toward a wide range of potential
pathogens. Their characterization should contribute to a
better understanding of the structure–function relationship of this protein group.
2. MATERIALS AND METHODS
2.1. Materials
Triticum aestivum, variety San Pastore, was kindly
supplied from the Istituto Nazionale per la Cerealicoltura (S. Angelo Lodigiani, Italy). Fast protein liquid
chromatography (FPLC) procedures were carried out on
a Pharmacia apparatus. Q-Sepharose was from Pharmacia. Reverse-phase HPLC (RP-HPLC) procedures were
carried out on a Beckman GOLD apparatus equipped
with a variable-wavelength monitor Model 166. The
␮-Bondapak C18 column (0.39 ⫻ 30 cm) was from WatersMillipore (Milford, MA). Pulsed liquid-phase automatic
sequencer (Model 477A) equipped on-line with phenylthiohydantoin (PTH) amino acid analyzer (Model 120A)
and relative reagents were from Applied Biosystems.
The mass spectrometer (model API-100) equipped with
an ionic electrospray source (ES-MS) was from Perkin
Elmer (Norwalk, CT). Bovine serum albumin, trifluoroacetic acid, dithiothreitol, and 4-vinylpyridine were from
Sigma Chemical Co. (St. Louis, MO). Sequencing-grade
trypsin (EC 3.4.21.4) was from Boeringher Mannheim
(Italia SpA). Reagents for SDS–PAGE, including lowmolecular weight markers, were from Bio-Rad Italy);
reagents for isoelectric focusing were from Pharmacia
(Uppsala, Sveden). All other reagents were of the highest
purity commercially available.
2.2. Protein Purification
The PR-4 protein family (MW 13,000 Da) was prepared from wheat flour as previously reported (Carrano
et al., 1989) up to the gel filtration chromatography step.
The Q-Sepharose ion exchange chromatography step,
leading to the purification of wheatwin1 and wheatwin2
(Carrano et al., 1989; Caruso et al., 1996), was modified
to isolate wheatwin3 and a peak containing wheatwin2
and wheatwin4. The column (1 ⫻ 40 cm) was equilibrated with 20 mM buffer piperazine–HCl, pH 9.71, at a
flow rate of 0.75 ml/min and developed with the following elution program: washing with 75 ml of the equilibration buffer; linear gradient of 0–35 mM NaCl in
the same buffer (265 ml of each); isocratic elution with
75 ml of 35 mM NaCl in the piperazine buffer; linear
Two New PR:4 Proteins from Wheat
gradient of 35–55 mM NaCl in the piperazine buffer
(150 ml of each); and washing with 75 ml of the piperazine buffer containing 100 mM NaCl. Elution was
monitored for absorbance at 280 nm. Wheatwin1 and
wheatwin3 were eluted in different peaks. Wheatwin2
and wheatwin4 were eluted in the same peak and then
separated by RP-HPLC on a ␮-Bondapak C18 column.
Eluent A was aqueous 0.1% trifluoroacetic acid and
eluent B was 0.07% trifluoroacetic acid in acetonitrile.
The following elution program was used at a flow rate
of 1 ml/min: isocratic elution with 28% B for 5 min followed by a linear gradient from 28% to 36% B in 55 min.
Elution was monitored for absorbance at 220 nm. Peaks
were manually collected and freeze-dried.
The protein concentration was determined by the
Bradford assay (Bradford, 1976) using bovine serum
albumin as a standard.
2.3. Electrophoresis, Immunoblotting, and
Isoelectric Focusing
Polyacrylamide gel electrophoresis (PAGE) of the
native proteins was carried out in 7.5% polyacrylamide
gel at pH 8.1 using a mini-Protean apparatus (Bio-Rad).
SDS–PAGE on a 15% acrylamide gel was carried out according to Laemmli (1970). Immunoblotting using a
polyclonal antiserum raised against wheatwin1 (Caruso
et al., 1999a) was performed according to Towbin et al.,
(1979). Proteins from SDS–PAGE were electrophoretically transferred onto nitrocellulose membranes; membranes were blocked in PBS buffer (6.5 mM Na2HPO4,
1.5 mM KH2PO4, 3 mM KCl, 0.15 mM NaCl, pH 7.4)
containing 0.5% Triton X 100 and 3% BSA and incubated for 2–24 hr at room temperature with the antiserum.
Isoelectric focusing (IEF) was performed in the pH range
3.5–10 on a 0.4-mm polyacrylamide gel containing 5%
(w/v) acrylamide cross-linked with bis-acrylamide and
5% ampholyte (pH 3.5–10.0, Pharmacia), using a miniIEF apparatus from Bio-Rad according to the manufacturer’s protocol. The pI markers (Sigma), ranging from pI
3.6 to 9.3, were electrophoresed to estimate the pIs of the
isoforms. After IEF, the gels were both stained with
Coomassie blue and subjected to Western blotting.
329
The reaction was stopped by freezing and reagents in excess were removed by RP-HPLC on the ␮-Bondapak C18
column. Eluents A and B were those described in Section
2.2. The elution was carried out at a flow rate of 1 ml/min
by the following program: isocratic elution for 10 min at
15% B followed by a linear gradient from 15% to 50%
B in 40 min. The S-pyridylethylated (PyEt) proteins were
manually collected and freeze-dried.
2.5. Trypsin Digestion and HPLC Analyses
PyEt-wheatwin proteins (5 nmol of each) were digested with trypsin (enzyme/substrate 1:50, w/w) for 8 hr
at 37°C in 0.1 ml of 0.1% ammonium bicarbonate buffer,
pH 8.0. The digests were freeze-dried and dissolved in
0.1 ml aqueous 0.1% trifluoroacetic acid. Each fragment
mixture was resolved by RP-HPLC on a ␮-Bondapak
C18 column. Eluents A and B were those described in
Section 2.2. The elution was performed at a flow rate of
1 ml/min by using the following program: 10 min 5% B
followed by two linear gradients from 5% to 27% B in
60 min and 27% to 30% B in 30 min. Elution was monitored for absorbance at 220 nm. Peaks were manually
collected and freeze-dried.
2.6. Sequence and Mass Spectrometry Analyses
Peptide fragments (100–200 pmol of each) showing
different retention times in the HPLC chromatograms
of the four protein digests were submitted to sequence
analysis using a pulsed liquid-phase automatic sequencer.
Samples were loaded onto a trifluoroacetic acid-treated
glass-fiber filter, coated with polybrene, and washed according to the manufacturer’s instructions. Analysis data
were processed by the instrument software. The theoretical initial yield of the coupling reaction was 60–70%.
The average and combined repetitive amino acids yields
were not lower than 90%.
The native wheatwin proteins (1 mg in 0.1 ml of
water/acetonitrile 1/1 v/v, 1% acetic acid) were submitted to ES mass spectrometry analysis. Data were
processed by the software Bio-multiwaver (Perkin Elmer,
Norwalk, CT). Lysozyme was used as a molecular weight
standard.
2.4. Protein Reduction and Alkylation
2.7. Molecular Modeling
Wheatwin proteins (15 nmol) were treated with 0.75
␮mol of dithiothreitol in 0.15 ml of 0.25 M Tris–HCl
buffer, pH 8.0, 2 mM EDTA, 6 M guanidine–HCl, for
2 hr at 37°C under nitrogen atmosphere. Alkylation was
performed with 4-vinylpyridine (25 ␮mol) for 2 hr at
room temperature in the dark under nitrogen atmosphere.
The 3D model of wheatwin1 was constructed as
previously described (Caporale et al., 1999) using the
spatial coordinates of the homologous protein barwin
(Ludvigsen and Poulsen, 1992b) stored in the Protein
Data Bank (pdb code: 1bw3). Solvent accessibility of
amino acids was evaluated by the program NACCESS
330
Caruso et al.
(Hubbard et al., 1991) calculating the atomic accessible
surface defined by rolling a probe of 1.40 Å around the
van der Waals surface of the protein models. Figures
were drawn with the RasMol (Sayle and Milner-White,
1995) and Swiss PDB viewer (Guex and Peitsch, 1997)
programs.
3. RESULTS AND DISCUSSION
3.1. Protein Purification and Characterization
The modification of the ion-exchange chromatography previously utilized for the purification of
wheatwin1 (Carrano et al., 1989) allowed us to elute
five well-separated protein peaks (Fig. 1A). As assessed
by polyacrylamide gel electrophoresis, wheatwin1,
which has been already characterized (Caruso et al.,
1993, 1999b), was associated to peak ww1, while the
new proteins wheatwin3 and wheatwin4 were contained
in the peaks ww3 and ww4 ⫹ ww2, respectively. Peak
ww2 ⫹ ww4 also contained wheatwin2, whose sequence has already been determined (Caruso et al.,
1996, 1999b); the other two peaks contained ␣-amylase
inhibitors (Carrano et al., 1989). Wheatwin4 was present in minor amount and separated from wheatwin2 by
RP-HPLC (Fig. 1B). The protein level in the seeds is
about 10 ␮g/g grain for wheatwin3 and 2 ␮g/g grain for
wheatwin4. SDS–PAGE showed that all the proteins
were obtained in homogeneous form and their molecular weights were about 13,000 Da (data not shown). The
isoelectric points of wheatwin3 and wheatwin4 were
7.1 and 8.4, respectively, as estimated by isoelectric focusing. These values are very similar to those obtained
for wheatwin1 and wheatwin2 (Caruso et al., 1993,
1996), respectively, indicating that the amino acid compositions of wheatwin3 and wheatwin1, as well as those
of wheatwin4 and wheatwin2, should be closely related.
When submitted to immunoblotting, wheatwin3 and
wheatwin4 were recognized by polyclonal antibodies to
anti-wheatwin1 (Caruso et al., 1999a), thus enforcing
their classification as members of the PR-4 family (data
not shown).
3.2. Amino Acid Sequence
The knowledge of the sequences of wheatwin1 and
wheatwin2 allowed us to use a rapid approach for the
determination of the differences in the sequence of the
homologous wheatwin3 and wheatwin4. The strategy
consists in digesting the four proteins with the same hy-
Fig. 1. (A) FPLC of the 13,000-Da protein family; (B) RP-HPLC of
the peak ww2 ⫹ ww4.
drolytic agent, comparing the HPLC chromatograms of
the digestion mixtures, sequencing just the peptidic fragments showing different retention times, and, finally,
confirming the differences by mass spectrometric analysis performed on the native proteins.
All the proteins were reduced, pyridylethylated, and
digested with trypsin. The digestion mixtures were resolved by RP-HPLC and the chromatograms compared.
The slope of the elution gradient was very mild, allowing a good separation of peptides (Fig. 2 and 3). Only the
two peaks, indicated by the arrows, showed different retention times in the chromatograms of wheatwin1 and
wheatwin3 (Fig. 2), indicating that the only differences
in the sequence of the two proteins should be contained
in these peptides. In fact, the sequence analysis of the
Two New PR:4 Proteins from Wheat
331
Fig. 2. RP-HPLC of digestion mixtures of wheatwin1 and wheatwin3.
Peaks showing different retention times are indicated by the arrows.
Fig. 3. RP-HPLC of digestion mixtures of wheatwin2 and wheatwin4.
Peaks showing different retention times are indicated by the arrows.
wheatwin1 fragment identified the peptide 82–101 containing asparagine (N) at position 88, as already reported
(Caruso et al., 1993, 1999b). The wheatwin3 fragment
showed the same sequence, but aspartic acid (D) was
found at position 88. Wheatwin1 contains 8 asparagine
and 9 glutamine residues and most of them have a high
solvent accessibility (Caporale et al., 1999); although
possible, it seems unlikely that wheatwin3 is produced
by deamidation of wheatwin1 during the purification
procedure, since the reaction should occur just on asparagine 88.
Similarly, the only peaks showing different retention
times in the chromatograms of the digestion mixtures of
wheatwin2 and wheatwin4 are those indicated by the arrows in Fig. 3. Their sequence analyses identified the
peptides 69–81, but isoleucine was confirmed to be present at position 78 of wheatwin2 (Caruso et al., 1996,
1999b), while valine was found at the same position
of wheatwin4. These structural differences were further
validated by electrospray mass spectrometry analyses
performed on the native proteins (Fig. 4). This type of
mass spectrometry is able to appreciate minimal differences in molecular weights, furnishing extremely precise
mass values. Wheatwin1 showed a molecular weight of
13,642.44 Da, in agreement with the weight deduced
from the sequence including the pyroglutamate residue
(Z) at the N-terminus (Caruso et al., 1993), while
wheatwin3 showed a molecular weight of 13,643.59 Da.
The difference of 1 Da in the mass of the two proteins
confirmed that the only variance in their sequences is due
to the substitution of one asparagine residue in wheatwin1 with aspartic acid in wheatwin3. As assessed by the
sequence analysis, this substitution is at position 88.
Wheatwin2 showed a molecular weight of 13,654.25 Da,
while the molecular weight of wheatwin4 was 13,640.38
Da. Also in this case, the mass difference is in agreement with the substitution of an isoleucine residue in
wheatwin2 with a valine residue in wheatwin4 which
occurs at position 78. Figure 5 shows the complete
sequences of the wheatwin and barwin proteins. The
332
Caruso et al.
Fig. 4. ES mass spectra of native wheatwin proteins.
Two New PR:4 Proteins from Wheat
333
Fig. 5. Homology alignment of the amino acid sequences of barwin and wheatwin proteins. Positions showing
different residues are labeled by a dash and are numbered.
structural differences among all the wheatwins are localized at positions 62, 68, 78, and 88. Barwin is different
in four additional residues located at positions 4, 5, 57,
and 58. It is identical in 118, 119, 120, and 121 of 125
residues to wheatwin3, wheatwin1, wheatwin4, and
wheatwin2, respectively.
3.3. Homology Modeling
The 3D model of wheatwin1 based on the knowledge of the three-dimensional structure of barwin (Loudvigsen and Poulsen, 1992b) has been already constructed
and validated. Wheatwin1 structure consists of a main
beta-sheet of four antiparallel strands, two short parallel
beta strands constituting a small independent beta-sheet,
and few short helices (Caporale et al., 1999). Figure 6
shows the surface position and solvent accessibility of
the residues located at positions 62, 68, 78, and 88. Ser
62 is hidden (5% accessibility), Asn 88 is the most accessible (89.1%) and is located at the opposite side of
Gln 68 and Ile 78, which both show a medium accessibility (27% and 22%, respectively). Furthermore, Asn 88
and Ser 62 should have a high flexibility since they are
located in random coil loop regions, while Gln 68 and Ile
78 should be less flexible since they are located in ␤-sheet
regions (Hubbard et al., 1991, 1994) (Fig. 7). On the
basis of these findings, the substitution of Asn 88, located in a very accessible and flexible region, could be
most important in differentiating the biological activity
of wheatwin proteins. We are planning to construct and
compare 3D models of all wheatwin proteins to further
validate this hypothesis.
4. CONCLUSION
An accurate study on the antifungal activity of
wheatwin3 and wheatwin4 using specific and nonspecific
wheat pathogens is currently in progress. Such study will
be directed to determine differences as well as synergistic
334
Caruso et al.
Fig. 7. Ribbon view of wheatwin1 model. The spatial positions of the
residues 62, 68, 78, and 88 are labeled; secondary structures are marked
by different colors.
Fig. 6. Space-filling frontal (A) and back (B) views of the wheatwin1
model. The surface positions of residues 62, 68, 78, and 88 are shown;
the solvent accessibility is also indicated.
effects in the antifungal activity of all wheatwin proteins. Preliminary results indicate that, as expected,
wheatwin3 and wheatwin4 have antifungal activity. In
the present paper we have reported the isolation and
structural characterization of two new PR-4 proteins
from wheat. The approach utilized to determine the
amino acid sequence is not expensive or time-consuming
and can be considered of general utility when the primary structure of protein isoforms is to be assessed. In
our case, just four sequence and mass analyses were
necessary to determine the complete sequence of both
wheatwin3 and wheatwin4. Although other rapid methods
using sequence and mass data of digestion mixtures have
been suggested for determining the amino acid sequence
of isoproteins (Caporale et al., 1991, 1996, 1998; Caporale, 1998), the present approach should represent a
good alternative when the studied molecules have the
N-terminus blocked, as in wheatwins. In fact, no sequence data of the N-terminal peptide are available in
this case, while methods based on the analysis of fragment mixtures need full information on each peptide to
reconstruct the complete sequence.
The knowledge of the sequence of further wheatwin proteins is fundamental to determine their secondary
and tertiary structures. Micro-differences in the threedimensional folding which could be determined by comparative homology modeling should furnish information
on particular domains implicated in the action mechanism since wheatwin proteins have different antifungal
activities (Caruso et al., 1996). The understanding of
how PR-4 proteins inhibit fungal growth and spore germination is still incomplete, though preliminary results
suggest that it is more likely that these molecules interact with cell membranes than with cell walls. The determination of the primary structure of each individual
isoform is the first step to comprehending the mechanism
of antifungal action of PR-4 proteins so that their full
potential as defense molecules can be realized.
Two New PR:4 Proteins from Wheat
ACKNOWLEDGMENTS
This research was supported by a grant from the
Ministero per le Politiche Agricole (MIPA, former
MIRAAF), Piano Nazionale Biotecnologie Vegetali,
Progr. N. 152.
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