Toxicity to cotton boll weevil Anthonomus grandis of a trypsin inhibitor from chickpea seeds.

Abstract

Cotton (Gossypium hirsutum L.) is an important agricultural commodity, which is attacked by several pests such as the cotton boll weevil Anthonomus grandis. Adult A. grandis feed on fruits and leaf petioles, reducing drastically the crop production. The predominance of boll weevil digestive serine proteinases has motivated inhibitor screenings in order to discover new ones with the capability to reduce the digestion process. The present study describes a novel proteinase inhibitor from chickpea seeds (Cicer arietinum L.) and its effects against A. grandis. This inhibitor, named CaTI, was purified by using affinity Red-Sepharose Cl-6B chromatography, followed by reversed-phase HPLC (Vydac C18-TP). SDS-PAGE and MALDI-TOF analyses, showed a unique monomeric protein with a mass of 12,877 Da. Purified CaTI showed significant inhibitory activity against larval cotton boll weevil serine proteinases (78%) and against bovine pancreatic trypsin (73%), when analyzed by fluorimetric assays. Although the molecular mass of CaTI corresponded to alpha-amylase/trypsin bifunctional inhibitors masses, no inhibitory activity against insect and mammalian alpha-amylases was observed. In order to observe CaTI in vivo effects, an inhibitor rich fraction was added to an artificial diet at different concentrations. At 1.5% (w/w), CaTI caused severe development delay, several deformities and a mortality rate of approximately 45%. These results suggested that CaTI could be useful in the production of transgenic cotton plants with enhanced resistance toward cotton boll weevil.

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Toxicity to cotton boll weevil Anthonomus grandis
of a trypsin inhibitor from chickpea seeds
Ange ´lica de P.G. Gomesa,b, Simoni C. Diasb,c, Carlos Bloch Jr.b, Francislete R. Melob,d,
Jose ´ R. Furtado Jr.a, Rose G. Monnerata, Maria F. Grossi-de-Sa ´b, Octa ´vio L. Francoa,*
aUniversidade Cato ´lica de Brası ´lia, Po ´s-Graduac ¸a ˜o em Cie ˆncias Geno ˆmicas e Biotecnologia, SGAN Quadra 916, Mo ´dulo B,
Av. W5 Norte 70.790-160-Asa Norte Brası ´lia/DF, Brazil
bCentro Nacional de Recursos Gene ´ticos e Biotecnologia-Cenargen/Embrapa, Brası ´lia-DF, Brazil
cDepartamento de Biologia Celular, Universidade de Brası ´lia, Brası ´lia-DF, Brazil
dUnia ˜o Pioneira de Integrac ¸a ˜o Social, UPIS, Brası ´lia-DF, Brazil
Received 25 March 2004; received in revised form 21 October 2004; accepted 27 October 2004
Abstract
Cotton (Gossypium hirsutum L.) is an important agricultural commodity, which is attacked by several pests such as the cotton boll weevil
Anthonomus grandis. Adult A. grandis feed on fruits and leaf petioles, reducing drastically the crop production. The predominance of boll
weevil digestive serine proteinases has motivated inhibitor screenings in order to discover new ones with the capability to reduce the
digestion process. The present study describes a novel proteinase inhibitor from chickpea seeds (Cicer arietinum L.) and its effects against A.
grandis. This inhibitor, named CaTI, was purified by using affinity Red-Sepharose Cl-6B chromatography, followed by reversed-phase
HPLC (Vydac C18-TP). SDS-PAGE and MALDI-TOF analyses, showed a unique monomeric protein with a mass of 12,877 Da. Purified
CaTI showed significant inhibitory activity against larval cotton boll weevil serine proteinases (78%) and against bovine pancreatic trypsin
(73%), when analyzed by fluorimetric assays. Although the molecular mass of CaTI corresponded to a-amylase/trypsin bifunctional
inhibitors masses, no inhibitory activity against insect and mammalian a-amylases was observed. In order to observe CaTI in vivo effects, an
inhibitor rich fraction was added to an artificial diet at different concentrations. At 1.5% (w/w), CaTI caused severe development delay,
several deformities and a mortality rate of approximately 45%. These results suggested that CaTI could be useful in the production of
transgenic cotton plants with enhanced resistance toward cotton boll weevil.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Serine proteinase inhibitor; Cicer arietinum; Anthonomus grandis; Plant defense; Cotton; Mass spectrometry; Trypsin; Digestive enzymes
1. Introduction
Cotton crops (Gossypium hirsutum L.) are extremely
important in providing income to millions of farmers in
tropical and subtropical areas of the New World. Cotton
serves as an engine of economic growth and thus contributes
to food security worldwide. Cotton boll weevil Anthonomus
grandis is an important insect pest responsible for severe
cotton crop damage in several countries of America. Adults
feed on fruit and leaf petioles, reducing crop production and
damaging cotton fibers (Haynes and Smith, 1992; Alves et
al., 1993). The larval phase is endophytic and is not
efficiently controlled by pesticides and adult insects are
1096-4959/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpc.2004.10.013
Abbreviations: AgPL, Anthonomus grandis proteinase larvae; AoPL,
Acanthoscelides obtectus proteinase larvae; BBI, Bowman-Birk inhibitor;
BPC, Bovine pancreatic chymotrypsin; BPT, Bovine pancreatic trypsin;
BTCI, Black-eyed pea trypsin chymotrypsin inhibitor; CaRP, Cicer
arietinum Red-sepharose retained peak; CaTI, Cicer arietinum trypsin
inhibitor; CmPL, Callosobruchus maculatus proteinase larvae; CpTI,
Cowpea trypsin inhibitor; HPLC, High performance liquid chromatog-
raphy; HPT, Human pancreatic trypsin; MALDI-TOF, Matrix assisted laser
desorption ionizated-time of flight; OCI, Oryzacystatin inhibitor; PPA,
porcine pancreatic a-amylase; RASI, Rice a-amylase/subtilisin inhibitor;
SDS-PAGE, Sodium dodecyl sulphate polyacrylamide gel electrophoresis;
SKTI, soybean Kunitz trypsin inhibitor; SfPL, Spodoptera frugiperda
proteinase larvae.
* Corresponding author. Tel.: +55 61 448 7220; fax: +55 61 347 4797.
E-mail address: ocfranco@pos.ucb.br (O.L. Franco).
Comparative Biochemistry and Physiology, Part B 140 (2005) 313–319
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able to move to safe areas, escaping from toxic chemicals.
Under favorable conditions, insects move back to the crop
area to complete their life cycle (Franc ¸a, 1993).
Insect digestive proteinases are promising targets in the
control of several insect pests since insects obtain essential
amino acids by using extra-cellular proteinases secreted into
the midgut lumen, digesting soluble and structural proteins.
Despite the predominance of digestive cysteine proteinase-
like activity in some coleopterans as the bean bruchids A.
obtectus, Z. subfasciatus and C. maculatus (Wielman and
Nielsen, 1988; Zhu-Salzman et al., 2003; Melo et al., 2003),
the Colorado potato beetle Leptinotarsa decemlineata, the
black vine weevil Otiorynchus sulcatus (Michaud et al.,
1996) and phytophagous beetle, Phaedon cochleariae
(Girard and Jouanin, 1999); the cotton boll weevil A.
grandis and others pests including the coleopteran Tenebrio
molitor (Lee et al., 2002), Holotrichia diomphalia (Kwon et
al., 2000), the lepidopterans Manduca sexta (Johnson et al.,
1989), Heliothis zea (Broadway and Duffey, 1986),
Spodoptera litura (Yeh et al., 1997) and the dipteran Lucilia
cuprina (Reed et al., 1999) have large quantities of digestive
serine proteinases. For this reason several plant serine
proteinase inhibitor screenings have been undertaken in
order to identify inhibitors highly effective against digestive
enzymes (Leple ´ et al., 1995; Gatehouse and Gatehouse,
1998; Bode and Huber, 2000; Franco et al., 2004).
The serine proteinase inhibitor family is composed of
abundant, small and stable molecules found in plant storage
tissues such as seeds, tubers, leaves and fruits (Richardson,
1977, 1991; Haq and Khan, 2003). Various functions have
been suggested for these plant proteinase inhibitors, includ-
ing their action as storage proteins (Xavier-Filho, 1992),
regulators of endogenous proteolytic activity (Ryan, 1991)
and components of programmed plant cell death mecha-
nisms (Solomon et al., 1999). Mostly serine proteinase
inhibitors are known for their function in response to abiotic
stresses (Franco and Melo, 2000) and in plant defense
processes against pests and pathogens (Gatehouse and
Gatehouse, 1998) reacting with cognate enzymes, binding
to catalytic sites in a canonical fashion (Laskowski and
Kato, 1980; Gru ¨tter et al., 1990). All canonical inhibitors
possess an exposed loop that simulates the substrate, with a
rapidbindingandaslowdissociationmechanism(Laskowski
and Kato, 1980; Gru ¨tter et al., 1990; Richardson, 1992).
Despite strong efforts dedicated to understanding the
structure and specificity of canonical serine proteinase
inhibitors, there are still unanswered questions awaiting
detailed investigation concerning mechanistic aspects of
inhibitors in plant defense. Due to the complexity of
enzyme/inhibitor interactions, the choice of an efficient
inhibitor will determine the success of pest a control strategy.
Our main goals in this study were the purification and
characterization of a novel serine-proteinase inhibitor (CaTI)
from chickpea seeds (C. arietinum), and evaluation of the in
vitro and in vivo activity of this inhibitor against the cotton
boll weevil A. grandis.
2. Materials and methods
2.1. Purification of C. arietinum trypsin inhibitor (CaTI)
Chickpea seeds (C. arietinum) were triturated into a fine
textured flour. The flour was defatted with acetone 50% and
was extracted with a solution of 0.5 M Tris–HCl pH 7.0
containing 100 mM NaCl in the proportion of 1:3 (w/v) for
4 h under agitation at 4 8C. This extract was centrifuged at
5000?g for 40 min at 4 8C. Precipitate was discarded and
the supernatant was dialyzed against distilled water. Crude
extract was precipitated with ammonium sulphate (100%),
following further centrifugation. Precipitate was dissolved
and dialyzed against distilled water. This fraction was
applied onto a Red-Sepharose Cl-6B affinity column
equilibrated to 0.1 M Tris–HCl buffer, pH 7.0 containing
5.0 mM CaCl2. The retained peak (CaRP) was eluted using
a single step of 0.1 M Tris–HCl buffer, pH 7.0 containing
3.0 M NaCl. Fractions of 3.0 mL were collected and the
optical density was measured at wavelength of 280 nm.
After dialysis and lyophilization, 4.0 mg of CaRP fraction
was dissolved in 250 AL of 0.1% trifluoroacetic acid and
applied onto a semi-preparative reversed phase HPLC
column (Vydac C18-TP) and eluted with a liner acetonitrile
gradient (0–100%) generating several peaks. The purified
Cicer arietnum trypsin inhibitor, which was named CaTI,
composed one of them.
2.2. Isolation of fluid from larval pest midgut
A. grandis and Spodoptera frugiperda larvae were
obtained from the Biological Control Department of
EMBRAPA/Cenargen (Brası ´lia-DF, Brasil). Larvae were
reared on an artificial diet (Monnerat et al., 1999) at 25 8C
and 55% relative humidity. Furthermore, Callosobruchus
maculatus and Acanthoscelides obtectus were reared in
cowpea and common bean seeds, respectively. The guts
were dissected from larvae and placed into an iso-osmotic
saline (0.15 M NaCl). Midgut tissues were homogenized
and centrifuged for 10 min at 16,000?g at 4 8C. The
supernatant was removed and used for enzymatic assays.
2.3. Proteinase inhibitory assays
Bovine and human pancreatic trypsin (BPT and HPT), as
well bovine pancreatic chymotrypsin were purchased from
Sigma, St. Louis, MO, USA, and proteinase extracted from
larvae of A. grandis (AgPL), S. frugiperda (SfPL), A.
obtectus (AoPL) and C. maculatus (CmPL) were used for
enzymatic assays. Proteolytic inhibitory activities were
tested against AgPL, SfPL, CmPL, AoPL and BPT using
10 mM of fluorogenic peptide Z-CBZ-Phe-Arg-7-MCA
(Sigma) and against bovine pancreatic chymotrypsin (BPC)
using 10 mM of fluorogenic peptide Ala-Ala-Pro-Phe-
MCA. Assays were performed in 25 mM Tris–HCl, pH
6.5 and 20 mM DMSO according to Solomon et al. (1999).
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The reaction was stopped with 1.9 mL of 0.2 M Na2CO3.
The endpoint reaction was measured after 30 min in a
DyNA Quant 500 fluorescence reader (Amersham Pharma-
cia), with excitation at 365 nm and emission at 460 nm.
Crude chickpea extract, CaRP and CaTI were tested at a
standard concentration of 75 Ag mL?1. Inhibitory activities
were calculated using the fluorescence reduction. The
inhibitory activities were direct compared to free MCA
produced by trypsin-like enzymes. One relative unit
corresponds to 0.5 mM of free MCA produced for 30 Ag
mL?1of proteinases utilized, after 30 min of reaction. The
blank fluorescence readings (minus substrate) were sub-
tracted. Assays were carried out in triplicate, with variability
in endpoint fluorescence values not exceeding 10%.
2.4. Molecular mass analysis
Peaks obtained in HPLC were analyzed by 15% SDS-
PAGE (Laemmli, 1970) and by mass spectrometry using the
method of Franco et al. (2000). Freeze-dried samples of the
peaks from HPLC were prepared for Matrix-Assisted Laser
Desorption Time of Flight Analysis (MALDI-TOF) on a
Voyager-DE STR Bioworkstation (PerSeptive Biosystems,
Framingham, MA, USA). Samples were dissolved in
trifluoracetic acid (TFA) 1.0% and a-cyan (a saturated
solution dissolved in acetonitrile/0.1% TFA 1:1, v/v) from
Sigma. The solution was then vortex mixed and 1 mL
aliquots were applied onto the Voyager Bioworkstation
sample plate. Samples were air-dried at room temperature.
The spectrometer, equipped with a delayed-extraction
system, was operated in linear mode. Sample ions were
evaporated by irradiation with a N2laser at a wavelength of
337 nm, and accelerated at 23 kV potential in the ion source
with a delay of 150 ns. Samples were ionized with 100–200
shots of a 3 ns pulse width laser light. The signal was
digitalized at a rate of 500 MHz and averaged data was
presented to a standard Voyager data system for manipu-
lation. MALDI-ToF was calibrated using a Saquazyme
calibration mixture (Applied Biosystems) consisting of
bovine insulin (5734 Da), E. coli thioredoxin (11,674 Da)
and horse apomyoglobin (16,952 Da).
2.5. Chickpea rich fraction feeding tests on cotton boll
weevil
Bioassays were performed in 40 ml sterilized artificial
diet (Monnerat et al., 1999). CaRP was incorporated in the
artificial diet at standard concentrations of 0.5%, 1.0% and
1.5% (w/w). Purified CaTI was also used for feeding tests,
being incorporated in the artificial diet at the concentration
of 0.5%. Protein concentration was calculated according to
Bradford (1976). The artificial diet was added to Petri dishes
and larvae 48 h post-hatch were placed in pits created in
solid diet. After 7 days, the dead larvae were counted and
the size of surviving larvae was measured. In negative
control treatment, bovine serum albumin (BSA) was added
to the artificial diet at the same concentration of inhibitor.
Each treatment was repeated three times and each replicate
used 15 larvae. The bioassay was maintained under
controlled conditions at 28 8C and 55% stable humidity.
3. Results and discussion
3.1. Cicer arietinum trypsin inhibitor (CaTI) purification
Lyophilized chickpea crude extract was precipitated with
ammonium sulphate 100%, dialyzed and subjected to a Red-
Sepharose CL-6B affinity column, originating a single
retained peak (CaRP) (Fig. 1A). Proteins with positive
charge surface, generally generated by residues of lysine
and/or arginine, bind to Red-Sepharose resin (Melo et al.,
2002). This resin, when associated to HPLC reversed phase
columns, has been shown to be an efficient means of
purification of plant defense proteins, such as thionins
(Bloch and Richardson, 1991), a-amylase inhibitors (Melo
et al., 1999) and proteinase inhibitors (Melo et al., 2002).
CaRP was applied onto an HPLC column, generating
several peaks, in which only three of them demonstrated
proteolytic inhibitory activity (1, 2 and 3) (Fig. 1B).
Fraction 3 was selected due to its higher activity against
our main target AgPL.
In order to analyze the purification degree and molecular
masses, SDS-PAGE of crude extract, CaRP fraction and
HPLC fractions 1 and 2 were undertaken (Fig. 1C). CaRP
analyses indicated the presence of a strong band of
molecular mass of approximately 13 kDa and some minor
contaminants. When this fraction was applied onto HPLC,
fractions 1 and 2 showed several bands with different
molecular masses (Fig. 1C). Although both fractions are
able to inhibit AgPL, the presence of different contaminants
and their lower inhibitory activity (Fig. 1B) led to the choice
of fraction 3. Other inhibitor fractions from chickpea seeds
warrant future study. In fraction 3, contaminants were
eliminated using reversed-phase HPLC and a single band of
approximately 12 kDa was observed by SDS-PAGE (Fig.
2B). The protein purified in peak 3 was named C. arietinum
trypsin inhibitor (CaTI) and its molecular mass was
calculated using mass spectrometry (MALDI-TOF) (Fig.
2A). This method showed a unique protein of 12,877.44 Da
observed in monomeric form. A lower presence of dimeric
form was also detected (Fig. 2A). Wheat a-amylase
inhibitors (Feng et al., 1996; Franco et al., 2000) and rye
bifunctional (a-amylase/proteinase) inhibitors (Iulek et al.,
2000) are known to be multimeric hydrolytic enzyme
inhibitors. Proteinase inhibitor from the Kunitz family with
different molecular masses from CaTI occurs in soybean
(19.0 kDa) and mustard seeds (20.0 kDa) (Mandal et al.,
2002) when compared to CaTI (Fig. 2). Only Cajanus cajan
trypsin inhibitor from the Kunitz family (Haq and Khan,
2003), with approximately 14 kDa, showed a similar
molecular mass to CaTI. Furthermore, bifunctional inhib-
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itors that act against a-amylases and trypsin (Xavier-Filho,
1992; Iulek et al., 2000; Franco et al., 2002) also have
molecular masses near 13 kDa. Additionally, a peptide mass
fingerprint (data not shown) indicated, by two peptides
produced by trypsin cleavage that matched to rice amylase/
subtilisin inhibitor (RASI), that this inhibitor probably
belongs to a-amylase/trypsin bifunctional class. No peptide
produced by trypsin cleavage matched to Kunitz inhibitors.
The similarities between CaTI and bifunctional inhibitors
prompted us to test CaTI against a-amylases. We first
looked for inhibitory activity against porcine pancreatic a-
amylase (PPA) and found that CaTI did not inhibit PPA
(data not shown). CaTI was also assayed against insect a-
amylases from A. grandis and bean bruchids and no
inhibitory activity was observed (data not shown). While
it is possible that CaTI inhibits other a-amylases, our results
suggested that this inhibitor is only capable to inhibit
proteinases.
3.2. Enzymatic assays of CaTI against digestive proteinases
from A. grandis larvae
Two trypsin-like serine proteinases (AgPL and BPT)
were used to monitor the chickpea inhibitory activity (Fig.
1B, data not shown). Purified CaTI inhibited approximately
73% of AgPL and 51% of SpPL, strongly suggesting that
this inhibitor is a trypsin-like serine proteinase inhibitor
since both pests synthesize enhanced quantities of digestive
trypsin-like enzymes (Franco et al., 2004). This result was
confirmed by inhibition of BPT (78%) and HPT (82%) (Fig.
3). CaTI also did not show any inhibitory activity against
AoPL and CmPL (Fig. 3). This may be due a low level of
trypsin-like serine proteinase and a higher rate of cysteine-
like proteinases in bruchids midguts (Melo et al., 2003)
suggesting that CaTI was unable to inhibit this proteolytic
enzyme class. Furthermore, CaTI was incapable of inhibit-
ing BPC, confirming that this inhibitor does not pertain to
Bowman-Birk family. Although cotton boll weevil is
capable of synthesizing a wide range of digestive protei-





Fig. 1. (A) Elution profile on Red-Sepharose CL-6B of trypsin-like serine
proteinase inhibitor from C. arietinum seeds. Approximately 25 mg of
protein was applied onto the column equilibrated with 0.1 M Tris–HCl
buffer pH 7.0 containing 5.0 mM CaCl2. Fractions were eluted with a single
step of 3.0 M NaCl (black arrow) and they were monitored at 280 nm. (B)
Analytical reverse-phase HPLC of the retained peak. The separation was
carried out on a Vydac C18-TP analytical column using a flow rate of 1 ml
min?1. TFA (0.1%) was used as an ion-pairing agent and the dashed line
indicates the acetonitrile gradient. The sample applied contained 1.0 mg of
CaRP. Fractions I, II, III, IV and V were tested against AgPL. (C) SDS-
PAGE analysis of crude extract (CE), C. arietinum Red-Sepharose
Retained-peak (CaRP) and fractions 1 (F1), 2 (F2), 3 (F3) and 4 (F4).
Gel was stained with silver.
Fig. 2. (A) Mass spectrum (MALDI-TOF) and (B) SDS-PAGE analyses of
fraction 3 (CaTI inhibitor). Electrophoresis was stained with silver.
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316

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nases, most of them belong to the trypsin-like serine
proteinases class (Purcell et al., 1992). Inhibition caused
by CaTI from chickpea seeds (Fig. 3) to midgut extracts of
A. grandis was similar to results observed with SKTI from
soybean seeds (soybean Kunitz trypsin inhibitor) (Purcell et
al., 1992; Franco et al., 2004) and BTCI from cowpea seeds
(black eyed-pea trypsin/chymotrypsin inhibitor) (Franco et
al., 2003). Other proteinases inhibitors shown to have
inhibitory activity against pest enzymes have been consid-
ered to be natural agents of plant defense (Broadway and
Duffey, 1986; Hilder et al., 1987). Among them, rice
oryzacystatin (OCI) and soybean Bowman-Birk inhibitors
(BBI) showed inhibition of Phaedon cochleariae digestive
enzymes (Girard et al., 1998). Nevertheless, is important to
remember that a substantial in vitro inhibition of digestive
proteinases is not enough to determine an efficient
resistance factor against an insect-pest. Pests are able to
adapt to the presence of inhibitors, modifying the compo-
sition of their digestive proteinases, altering their concen-
trations or inducing the expression of novel proteinases
(Jongsma et al., 1995). In order to measure the CaTI
inhibitory efficiency, feeding tests were also carried out.
3.3. Feeding tests
Due to the in vitro activity of CaTI against trypsin-like
enzymes from cotton boll weevil midgut (Fig. 3), bioassays
were conducted in order to demonstrate the potential of
chickpea serine proteinase inhibitors as a protective factor.
Bioassays revealed an enhanced mortality rate and delayed
insect development when A. grandis larvae where fed on
diet with three different CaRP concentrations (Fig. 4A and
B). Using a CaRP concentration of 1.5% we observed a
mortality rate of 43.33% (Fig. 4A) and 40% of surviving
larvae had retarded development (Fig. 4B). Feeding test
using purified CaTI was also carried out, using a single
concentration of 0.5%, causing a mortality rate of 48% (data
not shown). Increased mortality at 0.5% protein concen-
tration could be caused by the high purity of CaTI used.
Furthermore, several larvae that consumed CaRP and CaTI
showed severe deformities. The concentration used, 0.5–
1.5% (w/w), corresponds to the expressed levels in legume
seeds (Ryan, 1991) and was very similar to that used by
other researchers (Gatehouse et al., 1999; Franco et al.,
2003; Oppert et al., 2003). Pests fed artificial diet containing
proteinase inhibitors with specificity to their main class of
digestive proteinases at similar concentrations suffered a
significant delay in their development and an increase in
mortality (McManus and Burgess, 1995). The potential of
serine proteinase inhibitors against larvae and adult insects
of the cotton boll weevil has been demonstrated previously.
Two inhibitors (SKTI and BTCI) showed deleterious effects
Fig. 4. Effects of CaRP on development (A) and on mortality (B) of cotton
boll weevil A. grandis. Each measurement was done in triplicate and each
replicate does not differ by more than 10%.
Fig. 3. Inhibitory activities of CaTI toward A. grandis and S. frugiperda
proteinase larvae (AgPL and SfPL), bovine and human pancreatic trypsin
(BPT and HPT), bovine pabcreatic chymotrypsin (BPC) and proteinases
from A. obtectus and C. maculatus larvae (AoPL and CmPL). Each
measurement was done in triplicate and each replicate does not differ by
more than 10%.
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