Practical and theoretical characterization of Inga laurina Kunitz

Comparative Biochemistry and Physiology, Part B 158 (2011) 164–172
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Comparative Biochemistry and Physiology, Part B
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p b
Practical and theoretical characterization of Inga laurina Kunitz inhibitor on the
control of Homalinotus coriaceus
Maria Lígia Rodrigues Macedo a,⁎, Maria das Graças Machado Freire b, Octávio Luiz Franco c,
Ludovico Migliolo c, Caio Fernando Ramalho de Oliveira a
a
b
c
Departamento de Tecnologia de Alimentos, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, Brazil
Laboratório de Química e Biomoléculas, Centro de Pesquisa, Institutos Superiores do CENSA, RJ, Brazil
Centro de Análises Proteômicas e Bioquímicas, Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Brasília, Brazil
a r t i c l e
i n f o
Article history:
Received 4 September 2010
Received in revised form 9 November 2010
Accepted 15 November 2010
Available online 19 November 2010
Keywords:
Coleoptera
Midgut proteases
Proteinase inhibitors
Homology modeling
Three-dimensional structure predictions
a b s t r a c t
Digestive endoprotease activities of the coconut palm weevil, Homalinotus coriaceus (Coleoptera:
Curculionidae), were characterized based on the ability of gut extracts to hydrolyze specific synthetic
substrates, optimal pH, and hydrolysis sensitivity to protease inhibitors. Trypsin-like proteinases were
major enzymes for H. coriaceus, with minor activity by chymotrypsin proteinases. More importantly, gut
proteinases of H. coriaceus were inhibited by trypsin inhibitor from Inga laurina seeds. In addition, a serine
proteinase inhibitor from I. laurina seeds demonstrated significant reduction of growth of H. coriaceus
larvae after feeding on inhibitor incorporated artificial diets. Dietary utilization experiments show that
0.05% I. laurina trypsin inhibitor, incorporated into an artificial diet, decreases the consumption rate and
fecal production of H. coriaceus larvae. Dietary utilization experiments show that 0.05% I. laurina trypsin
inhibitor, incorporated into an artificial diet, decreases the consumption rate and fecal production of
H. coriaceus larvae. We have constructed a three-dimensional model of the trypsin inhibitor complexed
with trypsin. The model was built based on its comparative homology with soybean trypsin inhibitor.
Trypsin inhibitor of I. laurina shows structural features characteristic of the Kunitz type trypsin inhibitor. In
summary, these findings contribute to the development of biotechnological tools such as transgenic plants
with enhanced resistance to insect pests.
© 2010 Elsevier Inc. All rights reserved.
1. Introduction
A majority of the areas inhabited by the coconut palm-trees (Cocos
nucifera L.), spread across 86 countries, are located in the tropical
zone, between the parallels of latitude 20° (Cuenca, 1997). For some
Asians countries, coconut palms are an important source of capital,
calories for some of the Asian nations (Sarro et al., 2005). The most
important coconut producers worldwide are the Philippines,
Indonesia and India; where Brazil is listed as ninth in this rank of
producers.
The coconut fruit is subject to attack by a number of phytophagous
insects. According to Fonseca (1962), the coconut palm-trees grown in
littoral regions or in some inland areas at altitudes of below 800 m are
susceptible to the attack of the black coconut bunch weevil, Homalinotus
coriaceus (Gyllenhal), one of the most typical pests of South America,
particularly in Brazil and Argentina. Among the different products used
⁎ Corresponding author. Departamento de Tecnologia de Alimentos e Saúde Pública,
Centro de Ciências Biológicas e da Saúde, Universidade Federal do Mato Grosso do Sul
(UFMS), Cidade Universitária S/N-Caixa Postal 549, CEP 79070-900-Campo Grande-MS,
Brazil. Tel.: +55 6733457612; fax: +55 6733457400.
E-mail address: [email protected] (M.L.R. Macedo).
1096-4959/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpb.2010.11.005
for the pest control, the insecticide, carbosulfan, belonging to the
chemical class of the N-methylcarbamates, is commonly employed in
coconut culture. However, carbosulfan is speedily metabolized in plants,
via the carbofuran conversion to 3-hydroxycarbofuran (2), which can
translocate it into the coconut fruit and contaminate the coconut water.
This occurrence is a serious problem, since coconut water is often
consumed, in natura, as a refreshing drink by the local population
(Ogawa et al., 2006).
New effective protection methods that use transgenic plants
expressing proteinase inhibitors (PIs) are based on a thorough
understanding of the organization of the midgut proteolytic complex
of target insects (Gatehouse, 2002). If protein digestion in an insect is
spatially arranged, the knowledge of physico-chemical conditions
(pH) and peptidase spectrum consequences of protein degradation
in different midgut compartments is important, with regard to the
processing of PIs in the gut (Vinokurov et al., 2009). Inhibition of
proteolysis in herbivores is used as part of the defensive system of
host plants. Research on host plant resistance to herbivores has
established that plants have evolved serine protease inhibitors as one
of the natural defensive strategies against various insect pests and
pathogens (Carlini and Grossi-de-Sá, 2002; Macedo et al., 2003;
Ramos et al., 2009; Oliva and Sampaio, 2009; Oliva et al., 2010).
M.L.R. Macedo et al. / Comparative Biochemistry and Physiology, Part B 158 (2011) 164–172
The PIs inhibit insect gut proteinases by binding tightly to the
active site, making complex formation essentially irreversible. The
inability to utilize ingested protein and to recycle digestive enzymes
results in a critical amino acid deficiency, which affects the growth,
development and survival of the herbivore (Nanasahe et al., 2008).
For this reason, the incorporation of genes encoding proteinase
inhibitors into transgenic grain has been proposed as a method for
preventing seed damage by insect pests, while presenting few or no
side effects in vertebrates (Ferry et al., 2005). Insects may overcome
the effect of PIs by proteolytically inactivating these inhibitors or by
expressing inhibitor-insensitive proteases (Chi et al., 2009). The
ability of PIs to interfere with the growth and development of insects
has been attributed to their capacity to bind to and inhibit the action
of insect digestive proteinases (Volpicella et al., 2003). There are also a
number of examples of protease inhibitors expressed in transgenic
plants, which confer partial resistance to insect pests.
Multiple molecular forms of proteinase inhibitors have been
characterized from microorganisms, plants, and animals and variations in both sequence and structure have been found in such
proteins. Thus, the necessity to correlate structural and functional
aspects has prompted the study of structural characteristics by
modeling (Sattar et al., 2004). Previous workers (Garcia et al.,
2004; Macedo et al., 2007) reported that seeds of I. laurina contain an
inhibitor of trypsin (ILTI), a protein with a single polypeptide chain
containing 180 amino acids, the sequence of which was clearly
homologous to the Kunitz family of serine protease plant protein
inhibitors, and also showed significant similarity to the seed storage
proteins, sporamin and miraculin. However, ILTI displayed major
differences to most other Kunitz inhibitors in that it contained only
one disulfide bridge, and did not have two polypeptide chains, as
observed for the majority of other Kunitz inhibitors purified from
Mimosoideae species (do Socorro et al., 2002).
Characterization of digestive proteolysis in this insect would enable
a more rational choice of PIs for use in plant protection strategies. No
information is currently available on the digestive proteases present in
H. coriaceus. The aim of this study is to characterize gut protease
activities in H. coriaceus larvae. In addition, we examined the effect of
the inhibitor from I. laurina seeds on the growth and development of
H. coriaceus, and evaluated the 3D structure of ILTI.
165
2.3. Isolation of I. laurina trypsin inhibitor (ILTI)
ILTI was prepared, according to Macedo et al. (2007) with some
modifications. I. laurina seeds, free of integument and defatted with
hexane, were ground in a coffee mill. A crude extract (CE)
preparation was obtained by extraction of this meal with 0.1 M
phosphate buffer, pH 7.6 (1:10, w/v) at 4 °C overnight, with
subsequent centrifugation at 7500 g for 30 min at 8 °C. The
supernatant fraction was lyophilized against distilled water for
24 h at 4 °C and lyophilized. The lyophilized fraction (50 mg) was
dissolved in 50 mM Tris–HCl buffer, pH 8.0, and applied to a DEAESepharose column (2 × 20 cm), equilibrated in the same buffer, and
eluted with a linear gradient of NaCl (0–1 M) in the same buffer, at a
flow rate of 30 mL/h. The peak (5 mg) containing inhibitory activity
was subjected to ion-exchange rechromatography on a HiTrap Q
Sepharose column (5 mL), equilibrated with 20 mM of Tris–HCl, pH
8.0 and eluted with the same buffer containing NaCl (0–1 M), at a
flow rate of 30 mL/h. Proteins were detected by monitoring the
absorbance at 280 nm.
2.4. Midgut preparation
Fourth instar larvae were cold-immobilized and the midgut, along
with its contents, was removed in cold 150 mM NaCl and stored
frozen (− 20 °C). Guts from H. coriaceus larvae were subsequently
homogenized in 150 mM NaCl, centrifuged at 10 000 g for 10 min at
4 °C, and the supernatants were collected and used as a source of
enzymes for enzymatic assays. The samples were stored at − 20 °C, to
prevent alteration of proteolytic activity.
2.5. Fecal pellet preparations
Proteinases were obtained from the midguts of fourth-instar
larvae, according to Macedo et al. (2010). Feces of the caterpillars
were collected during the experiment and frozen (−20 °C). When
necessary, they were macerated, homogenized in 200 mM Tris–HCl
buffer, pH 8.5, centrifuged at 20 000 g for 30 min at 4 °C and
supernatants were used for in vitro enzymatic assays.
2.6. pH estimation of the gut
2. Materials and methods
2.1. Materials
I. laurina (Willd.) (Leguminosae) seeds were collected in the city
of Campo Grande, in the State of Mato Grosso do Sul (Brazil). SDSPAGE molecular weight markers, acrylamide, bis-acrylamide and
other electrophoresis reagents were purchased from Sigma-Aldrich
(St. Louis, MO, USA). Chromatography supports were from Pharmacia
(Uppsala, Sweden), all chemicals and reagents used were of
analytical grade.
2.2. Insects
The colony of Homalinotus coriaceus was supplied by Dr. M.G.M.
Freire (Laboratório de Química e Biomoléculas, ISECENSA, Campos dos
Goytacazes, RJ, Brazil) and was maintained in our laboratory,
according to Freire et al. (2008). The insects were housed at 25 ±
2 °C, at a relative humidity of 60–70% and 12 h photophase and
maintained on a substrate of cane sugar. After seven days of
oviposition, the sugar cane was opened manually to obtain eggs and
placed in humidified Petri dishes, until hatching. The neonate larvae
were placed individually in 10 mL of the artificial diet developed by
Machado and Berti Filho (1999).
The pH of the gut content was estimated using narrow range pH
indicator paper (Merck, Darmstadt, Germany). Immediately after
dissection, midguts were split lengthwise and the papers introduced
in the middle region. Measurements were obtained from four
midguts, and each determination was carried out in triplicate. The
pH paper was compared to the values obtained using standard
solutions of known pH (Walker et al., 1998).
2.7. Enzyme assays
Total proteolytic activity was determined by the method of
Marchetti et al. (1998), with some modifications. Typically, the
sample (20 μL containing 10 μg protein) and 0.1 M Tris buffer, pH 8.0
(360 μL), were pre-incubated for 5 min at 37 °C before the addition of
20 μL 2% azocasein (w/v, in glass-distilled water). After 20 min of
incubation, the reaction was stopped using 400 μL 10% trichloroacetic
acid (w/v). Tubes were kept on ice for 10 min and then centrifuged at
5 000 g for 5 min; 500-μL aliquots of the supernatant were withdrawn
and mixed in a cuvette with 500 μL 1 M NaOH and absorbance at
420 nm was determined. Blanks (test tubes without samples) were
run in all cases. Protein contents were determined by Coomassie blue
staining (dye-binding method) (Bradford, 1976). BSA (1 mg/mL) was
used as standard.
Trypsin-like activity was assayed using the chromogenic substrate,
BApNA (N-benzoyl-L-arginine-p-nitroanilide). Briefly, 0.1 M Tris
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buffer, pH 8.0 (1.35 mL), and the sample (10 μL containing 10 μg
protein) were pre-incubated for 5 min at 37 °C before the addition of
0.2 mL 7.8 mM BApNA (in 13% dimethyl sulfoxide; 1 mM final
concentration) to start the reaction. After 20 min of incubation, the
reaction was stopped with 0.75 mL 30% acetic acid and absorbance
was measured at 410 nm. Assays were carried out in triplicate and
appropriate blanks were run in all cases.
Chymotrypsin-like activity was assayed using the chromogenic
substrate SAAPFpNA (N-succinyl-ala-ala-pro-phe p-nitroanilide),
specific for chymotrypsin-like proteinases (Sigma). Stock substrates
of SAAPFpNA (100 mg/mL in dimethyl sulphoxide) were diluted
25 μl mL− 1 in 0.1 M Tris buffer, pH 8.0, and assays were carried out as
previously described for trypsin-like activity.
The effect of ILTI on the development of H. coriaceus larvae was
determined by the method of Macedo et al. (2010), with some
modifications. To evaluate the bioinsecticidal effects of ILTI on
H. coriaceus development, neonate larvae were placed individually
in glass pipes (8.5 × 2.5 cm) covered with cotton using three
concentrations of ILTI (0.00 to 0.1% w/w) mixed with the artificial
diet during its preparation. Each treatment presented fifteen
replicates with three larvae (n = 45). At the end of the fourthinstar, the weight and number of larvae, larval consumption and
fecal output were analyzed for the elaboration of nutritional
parameters.
2.8. Optimal pH
2.12. Nutritional parameters
All assays were carried out in triplicate and blanks were used to
account for spontaneous breakdown of substrates. Reaction buffers
were: 0.1 M citric acid-NaOH (pH 2.0–3.0), 0.1 M citrate (pH 3.0–
6.0); 0.1 M phosphate (pH 6.0–7.0); 0.1 M Tris–HCl (pH 7.0–9.0);
0.1 M glycine-NaOH (pH 9.0–11.0); and 0.05 M Na2HPO4–NaOH
(pH 11.0–12.0). All buffers contained 0.15 M NaCl and 5 mM MgCl2,
except Na2HPO4–NaOH, which only contained 0.15 M NaCl, since
MgCl2 is not maintained in highly alkaline solutions (Hernández
et al., 2003).
Unless otherwise stated, all protease activities were measured for
30 min at their optimum pH in a 1 mL reaction mixture that contained
10 μg of gut extract. Non-specific protease activity was assayed with
0.1% sulfanylamide-azocasein; trypsin-like activity with 1 mM
BApNa; and chymotrypsin-activity with 1 mM SAAPFpNA. Activities
were assayed as previously described. Specific activity was defined as
mmoles nitroaniline released per min per gut equivalent (extinction
coefficient of 8800 M− 1 cm− 1).
A number of nutritional parameters were compared among
fourth-instar larvae exposed to either ILTI-treated or a control diet.
The larvae, feces, and remaining uneaten food were separated using a
microscope, dried and weighed. Nutritional indices of consumption,
digestion and utilization of food were calculated, as described by
Farrar et al. (1989). The nutritional indices, namely efficiency of
conversion of ingested food (ECI), efficiency of conversion of digested
food (ECD) and approximate digestibility (AD) were calculated as
follows: ECI (ΔB/I) × 100; ECD [ΔB/(I − F)] × 100; and AD [(I − F)/
I] × 100, where I = weight of food consumed, ΔB is change in body
weight, and F = weight of feces produced during the feeding period.
Metabolic cost (CM) was calculated as: 100ECD.
2.9. Effect of temperature
The effect of temperature was determined by the method of
Pereira et al. (2005), with some modifications. Briefly, 0.1 M Tris, pH
8.0 buffer and samples were pre-incubated for 5 min at given
temperatures before the addition of the substrate to start the reaction.
Assays were carried out, as previously described. To assess the
stability of the enzyme, the buffer and the sample were pre-incubated
at given temperatures for 5 or 30 min. Test tubes were then
transferred to a water bath at 30 °C for 2 min. Following thermal
equilibration, the activity was assayed as previously described.
2.10. Effects of protease inhibitors and activators, in vitro
The proteolytic activities of gut extracts were assayed in the
presence of the following protease inhibitors; the serine protease
inhibitors, ILTI, SKTI (Soybean Kunitz trypsin inhibitor), SBBI
(Soybean Bowman-Birk inhibitor), BZD (Benzamidine) and PMSF
(phenylmethylsulfonyl fluoride); the trypsin inhibitors, TLCK
(Na-p-tosyl-L-lysine chloromethyl ketone); the chymotrypsin inhibitor,
TPCK (N-tosyl-L-phenylalanine chloromethyl ketone); the cysteine protease inhibitors, E-64 (L-trans-epoxysuccinylleucylamido-(4-guanidino)butane) and IAA (iodoacetamide); the metalloproteinase, EDTA
(ethylenediaminetetraacetic acid disodium salt) and the aspartic protease inhibitor, pepstatin-A. The cysteine protease activators, L-cysteine
and DTT (dithiothreitol), were also investigated.
Gut extracts were pre-incubated with the protease inhibitors and
activators for 15 min at 30 °C, prior to addition of substrate. All
compounds were added in 100 μL 0.15 M NaCl, except TPCK and
pepstatin-A, which were added in 20 μL DMSO. Doses according to
the effective concentrations were recommended by Beynon and
Salvesen (1989).
2.11. Effects of ILTI on the development of H. coriaceus larvae
2.13. Molecular modeling
Primary sequences of ILTI were obtained by Macedo et al. (2007).
PSI-BLAST was used for templates data mining (Farrar et al., 1989).
Soybean Kunitz inhibitor (PDB: 1avw), which shows 30% identity, was
chosen as a template. The ILTI three-dimensional model was
constructed by using crystal atomic coordinates of free SKTI (Macedo
et al., 2007) at a resolution of 1.9 Å. Thirty models were constructed
using Modeller v9.6 (Altschul et al., 1990; Arnold et al., 2006), which
models protein tertiary structures by satisfaction of spatial restraints,
considering energy minimization, which was conducted by default
parameters (Eswar et al., 2007). Predicted ILTI model evaluation, i.e.,
geometry, stereochemistry, and energy distributions in the models,
was performed using PROSA II to analyze packing and solvent
exposure characteristics and PROCHECK for additional analysis of
stereochemical quality (Laskowski et al., 1993). In addition, RMSD
was calculated by superposition of Cα traces and backbones onto the
template crystal structure through the program 3DSS (Sumathi et al.,
2006). The protein structures were visualized and analyzed on a SPDB
viewer v.3.7 (Guex and Peitsch, 1997) and Delano Scientific's PYMOL
(http://pymol.sourceforge.net/).
3. Results
To determine the conditions in the gut that affect digestive
proteolysis, larvae of H. coriaceus were dissected and midguts
homogenized and analyzed. The pH of the midgut was mildly
basic-neutral (7.1 ± 0.2) and the pH dependence of the proteolytic
activities of the gut extracts from H. coriaceus larvae are presented in
Figs. 1 and 2. The general proteinaceous substrate azocasein (Fig. 1)
was hydrolyzed over a broad range of pH, while total proteolytic
activity of midgut extracts for azocasein hydrolysis increased when
pH was increased from 5.0 to 10.0. Two major groups of activity were
observed in this pH range, with a low activity at pH 5 to 6.5, and a
high activity at pH 8.0 to 11.0, with optimum activity at pH 10.0,
respectively (Fig. 1).
M.L.R. Macedo et al. / Comparative Biochemistry and Physiology, Part B 158 (2011) 164–172
Table 1
Effect of protease inhibitors and activators on the hydrolysis of protein and synthetic
substrates by gut extracts from H. coriaceus larvae.
100
Activity, %
80
Residual activity (%)
60
40
20
0
2
4
6
8
10
12
pH
Fig. 1. Effects of pH on the activity of extracts from H. coriaceus larval midgut, assayed
with azocasein substrate.
To further characterize the digestive proteases present in H.
coriaceus, inhibition studies were carried out using a series of classspecific chemical inhibitors, activators and protein protease inhibitors. Using synthetic specific substrates and chemical inhibitors,
different types of proteases were identified in gut homogenates from
H. coriaceus (Table 1). The trypsin substrate, BApNA, was hydrolysed
to a greater degree than the chymotrypsin substrate, SA2PFpNA
(Fig. 2). The pH for maximal hydrolysis of BApNA was 10.0, equivalent
to the alkaline pH for maximal hydrolysis of azocasein. SA2PFpNA was
hydrolysed at low levels in all buffers. The pH for maximal hydrolysis
of SA2PFpNA was 9.0.
Some inhibitors tested were also effective against the general
protein substrate, azocasein. Serine inhibited the hydrolysis of
azocasein to some extent (Table 1). The general inhibitor of cysteine
protease activity, E64, the IAA and TPCK, essentially failed to inhibit.
EDTA, a metalloproteinase inhibitor, also failed to inhibited hydrolysis
of this substrate. The serine, cysteine, metalloproteinase and aspartic
protease inhibitors resulted in minimal inhibition of this substrate. It
is known that, outside of their effective concentration range, such
inhibitors may become non-specific, although results suggest a
complex protease profile made up of multiple activities. More specific
inhibition studies were therefore undertaken with the synthetic
substrates, SA2PFpNA and BApNA (Table 1).
ILTI, SKTI, TLCK, E-64, IAA, pepstatin-A, and EDTA had a relatively
low effect on the hydrolysis of the synthetic substrates, SA2PFpNA (0–
15%) (Table 1), suggesting that previous inhibition against the general
100
80
Activity, %
167
60
40
20
0
2
4
6
8
10
12
pH
Fig. 2. Effects of pH on the activities of extracts from larval midgut, assayed with serine
peptidase substrates. (■) BAPNA; (●) SA2PFpNA.
Azocasein
BAPNA
SA2PPpNA
pH 5.0
pH 10.0
pH 8.0
Inhibitor
ILTI (1.0 μM)
SKTI (1.3 μM)
SBBI (4.0 μM)
Benzamidine (0.5 mM)
TLCK (1.0 mM)
TPCK (1.0 mM)
E64 (10 μM)
IAA (1 mM)
Pepstatin-A (10 μg)
Phenantroline (1 mM)
PMSF (1 mM)
EDTA (1 mM)
EGTA (1 mM)
81.2
79.0
84.2
72.0
80.0
ne
ne
92.0
ne
92.0
98.0
ne
99.3
47.0
54.1
82.1
62.1
67.2
85.1
99.2
102.2
ne
105.0
98.7
ne
98.9
88.0
86.2
69.3
56.0
97.1
74.7
101.8
102.6
ne
100.5
99.8
ne
100.9
Activator
DTT (1 mM)
L-cysteine (1 mM)
Mg+
Ca+
100.6
101.3
102.4
98.7
98.5
100.5
98.9
97.7
95.5
99.8
105.1
102.5
Values are mean ± SD of triplicate measurements from a unique pool of gut extracts
treated with an inhibitor or activator vs. their corresponding controls without them. No
effect (ne) was considered for activities between 95 and 110%.
substrate was the result of loss of selectivity, owing to the high
concentrations used and not to the presence of different mechanistic
classes of protease. SBTI, Benzamidine and TPCK had a significant
inhibitory effects on SA2PFpNA (31%, 44% and 26%, respectively),
providing strong evidence for the presence of chymotrypsin (Fig. 2;
Table 1). BApNA (a trypsin and cathepsin substrate) was also
susceptible to inhibition by ILTI (47%), SKTI (54%), Benzamidine
(62%) and TLCK (67) (Table 1), suggesting a trypsin activity as the
main among all the enzymes. However, both substrates were also
significantly inhibited by SKTI, SBBI and Benzamidine. In agreement
with this, pepstatin-A and EDTA failed to cause significant levels of
inhibition of any of the substrates tested (Table 1).
The in vitro analysis of gut proteinases and synthetic substrates
indicated that H. coriaceus larvae contain digestive proteinases that
are active in acidic (weakly) and alkaline (strongly) pH buffers. Based
on the hydrolysis of a trypsin substrate by crude gut extracts, trypsinlike proteinases are predicted to be important enzymes for H.
coriaceus food digestion, with minor activity by chymotrypsin
proteinases.
Proteolytic activity, as determined by BApNA hydrolysis, was
temperature dependent and maximum activity was obtained at 40 °C
(Fig. 3). The thermal stability of the trypsin-like activity in midgut
samples that were pre-incubated for 5 min remained unchanged at
temperatures of up to 40 °C. However, preincubation for 30 min at
temperatures of 40 °C, or more, sharply reduced the proteolytic
activity, which was almost abolished at 60 °C (Fig. 4).
The antimetabolic effects of a trypsin inhibitor from I. laurino seeds
(ILTI) were tested against H. coriaceus larvae by incorporating ILTI in
an artificial diet at a level of 0–0.25% of total protein. The effect of ILTI
on the development of H. coriaceus was assessed by determining the
number and weight of surviving fourth-instar larvae fed on a diet
containing increasing amounts of ILTI. The dose–response effect of ILTI
on the growth and mortality of the insect larvae is shown in Fig. 5. The
survival (Fig. 5A) and weight (Fig. 5B) of larvae fed on control seeds
(represented by the y-intercept value) were approximately 100% and
275.4 mg, respectively, whereas diets containing 0.25%, 0.1% and
0.05% of ILTI caused 100%, 60% and 10% mortality, respectively and a
60% and 96% decrease in weight at 0.05% and 0.1% of ILTI, respectively.
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100
Activity, %
80
60
40
20
0
10
20
30
40
Temperature, °C
50
60
Fig. 3. Effect of temperature on trypsin-like activity from the larval midgut of H. coriaceus.
BAPNA (1 mM) hydrolysis was measured at given temperatures in 0.1 M Tris, pH 10.0, for
15 min. Data are reported as means± SEM of three independent determinations.
Regression analysis showed that for each 0.01% increase in the ILTI
dose, there was a 2.62 mg decrease in weight (R2 = 0.98).
Nutritional analyses revealed that ILTI presented a toxic effect
when ingested by larvae. ILTI, when incorporated in an artificial diet at
0.05%, reduced ECI and ECD and increased AD and metabolic cost
(CM) for H. coriaceus larvae, when compared with the control
(Table 2). Both ECI and ECD showed significant decreases of 30.7%
and 35.2%, respectively, and AD and CM were increased by 20.0% and
10.0%, respectively, when compared with larvae of H. coriaceus that
were reared on control diets. As shown in Fig. 6, the consumption of
the artificial diet with the incorporation of 0.05% of ILTI by H. coriaceus
larvae demonstrated a decrease of 52% when compared to the
consumption of the control diet by H. coriaceus. Fecal production of H.
coriaceus larvae, reared on the artificial diet containing 0.05% ILTI, was
approximately 80% lower than that of the control group, when
compared with larvae of the control group (Fig. 6). These results
suggest that ILTI acts on the insect's intestinal tract or interferes with
digestion.
Considering the limited sequence identity between ILTI and the
templates used for its modeling, objective validation provides
suggestive results of reliable models. The ILTI model construction
started with PSI-BLAST analysis, which was used to select the best
template. The ILTI sequence was directly compared to amino acid
residue sequences that possess structures that have been experimentally resolved and deposited in the Protein Data Bank (PDB) (Berman
et al., 2000). The Kunitz Inhibitor from Glycine max presented 30%
identity with ILTI, and this was chosen as the template.
Alignments among sequences and structure were carried out with
the ClustalW program (Thompson et al., 1994) to analyze the profile
of the primary sequence inhibitor (Fig. 7). The inhibitor presented 65%
of similarity to the primary sequence of the template soybean
inhibitor. After alignment analysis, atomic coordinates were transferred to the ILTI primary structure. The construct model displayed
110
100
90
80
Activity, %
Fig. 5. Effect of dietary ILTI on H. coriaceus when administered in an artificial diet.
(A) Survival and (B) weight, using an artificial diet bioassay. Insert: (1) larva fed on the
control diet; (2) larvae fed on 0.05% ILTI. Each point has an n = 75. Error bars indicate
standard error of the mean. The same letters indicate that there were no significant
statistical differences (p b 0.05; Student's t-test).
70
60
50
40
30
20
Table 2
Nutritional indices of H. coriaceus fourth-instar larvae on 0.05% ILTI-treated and control
diets.
10
0
10
20
30
40
Temperature, °C
50
60
Fig. 4. Thermal stability of trypsin-like enzymes from the larval midgut of H. coriaceus.
Samples were pre-incubated at given temperatures in 0.1 M Tris, pH 10.0, for 5 min
(filled circles) and 30 min (open circles). After 2 min of thermoequilibration at 30 °C,
BAPNA (1 mM) was added to start the reaction (15 min). Data are reported as means ±
SEM of three independent determinations.
Nutritional indices (mean ± SE)
Treatment (%)
ECI (%)
ECD (%)
AD (%)
CM (%)
Control
0.05
12.31 ± 1.1a
8.52 ± 1.22b
15.01 ± 0.94a
9.73 ± 0.7b
74.92 ± 1.38a
89.56 ± 0.26b
84.99 ± 0.89a
92.70 ± 0.75b
Means within a column followed by the same letter are not significantly different,
p b 0.05; based on Tukey's test.
M.L.R. Macedo et al. / Comparative Biochemistry and Physiology, Part B 158 (2011) 164–172
Dry weight (mg)
(A)
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Diet
0
0.05
ILTI dose (%, w/w)
(B)
1.2
1.1
Feces
Dry weight (mg)
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
169
96.2% of amino acid residues were in the most favorable region and
only four residues (Ile30, Ser122, Ser124 and Ala170) were in the
disallowed regions. These residues were presented within loops and,
as such, were not expected to affect the predicted ILTI structure.
Structural differences between the crystal structure of SKTI and
predicted three-dimensional structure of the ILTI model were
calculated by superimposing both structures. The RMSD values
between the crystal structure of SKTI and homology model of ILTI,
calculated for Cα traces, and the main chain atom were 0.49 . The
RMSD values and low variability among the experimental structure
templates and the structure modeled reflect the presence of strong
restraints in most regions and emphasize a similar folding pattern
among these inhibitors. Furthermore, the lower score acquired for
PROSA II in the case of the ILTI was of −4.85, indicating the quality of
model. This result indicated that the constructed ILTI model presented
its amino acid residues of the average of the observed parameters. On
the other hand, the structure of the lateral chains was considered to be
well located, when compared with the experimental structures with
the same resolution.
The complex between ILTI and the trypsin structure (PDB: 1fn6)
(Deepthi et al., 2001) was used for the study of the enzyme–inhibitor
interaction (Fig. 9). The mode of interaction showed an identical in
vitro mechanism of inhibition of the competitive type as those
observed for other Kunitz inhibitors. The reactive site presented
blockade access, as shown by the interaction of the inhibitor in the
catalytic site. The inhibitor presented an interface surface area of
708 Å2; this fact hinders substrate access to the enzyme. The O− atom
of the backbone in the Lys64 residue interacts with the OH− atoms of
Ser195, forming hydrogen bound with 2.92 Å. This interaction is
favored because of the attraction that the His57 and Asp102 produce
around Ser195.
0.2
4. Discussion
0.1
0.0
0
0.05
Dry weight (mg)
Fig. 6. Physiological parameters measured for H. coriaceus larvae. Larvae were fed on
control diets, or diets containing 0.05% ILTI. (A) Diet consumption by larvae (mg; dry
weight basis). (B) Fecal production by larvae (mg; dry weight basis). Different letters
denote a significant difference between the treatments (ANOVA, p b 0.05).
internal three-fold symmetry, as seen in SKTI, and one polypeptide
chain, as encountered in the majority of Kunitz inhibitors of the
Fabaceae family (Fig. 8).
The amino acids were structurally organized in loops connecting
consecutive β-strand patterns, as observed in Kunitz type inhibitors.
Superposition of these domains showed a high degree of similarity of
the β-strands, but not for the connecting loops presenting a classical
structure in the barrel shape. A procheck summary of ILTI showed that
The alfalfa weevil, Hypera postica (Wilhite et al., 2000), the black
vine weevil, Otiorhynchus sulcatus (Michaud et al., 2002), and the boll
weevil, Anthonomus grandis (Murdock et al., 1997), have slightly
acidic midguts and cysteine proteases provide the major midgut
endoproteolytic activity. Nevertheless, aspartic and/or serine proteases have been identified in some of these species (Purcell et al.,
1992).
We found that gut extracts of the H. coriaceus larvae have
azocaseinolytic activity within a broad range of pH values, from acid
to alkaline (Fig. 1). Specific inhibitors of serine proteases were
effective inhibitors of azocasein hydrolysis at alkaline pH, suggesting
that this species has a digestive system based on proteases of serine
mechanistic class. A wide pH range of proteolytic activity against
proteinaceous substrates has also been reported for other curculionid
species, such as the boll weevil, A. grandis (Wolfson and Murdock,
1990), the black vine weevil, O. sulcatus (Michaud et al., 2002), the
Fig. 7. Multiple sequence alignment between template (PDB: 1avw) and the Kunitz inhibitor from Inga laurina. Asterisks represent identical amino acids; one and two points, are
amino acids with weak and strong similarities, respectively.
170
M.L.R. Macedo et al. / Comparative Biochemistry and Physiology, Part B 158 (2011) 164–172
Fig. 8. Diagram of the ILTI model constructed by comparative modeling. View of the
superficial layer of ILTI. Lys64 represents the amino acid situated in the reactive loop.
rice weevil, Sitophilus oryzae and the maize weevil, Sitophilus zeamais
(Baker, 1982), the cabbage seed weevil, Ceutorhynchus assimilis
(Girard et al., 1998), a sugar beet weevil, Aubeonymus mariaefranciscae (Ortego et al., 1999), and the weevil Baris coerulescens (BonadéBottino et al., 1999), rice water weevil, Lissorhoptrus brevirostris
(Hernández et al., 2003) and banana weevil, Cosmopolites sordidus
(Montesdeoca et al., 2005).
Although the pH of H. coriaceus luminal contents was mildly basicneutral, azocasein and BAPNA hydrolysis was highest in alkaline
buffer. Differences in values of the luminal pH and optimal pH for
azocasein hydrolysis may be attributed to several factors. Gradients in
pH have been documented within the insect lumen, and proteinases
are often localized to specific regions (Berenbaum, 1980). Alternatively, the luminal extract contains a mixture of proteins, and the
properties of isolated proteinases may differ. Proteinases are active
over a range of pH values and may be more stable at pH values other
than their respective maxima (Oppertl et al., 2002).
Larvae and adults of most curculionid species examined rely on
complex proteolytic systems for protein digestion. Serine-, cysteine-,
and aspartylproteases have been identified in the guts of S. oryzae and
S. zeamais (Alfonso-Rubí et al., 2003). There are also some curculionids with serine protease activity and alkaline pH optima, such as the
red palm weevil, Rhynchophorus ferrugineus (Alarcon et al., 2002), and
the citrus weevil, Diaprepes abbreviatus (Yan et al., 1999).
The trypsin-like activity was strongly temperature dependent and
was similar to that reported for other Coleoptera larvae (Tsybina et al.,
2005). The effect of temperature on the metabolism of these insects
and, consequently, on their life cycle is well known. Leppla et al.
(1977) have reported that, at 21.1 °C, larvae require almost twice the
time to complete their development to pupae when compared to
larvae reared at 32.2 °C under laboratory conditions.
Despite extensive studies on the efficiency of PIs as a defense
against insects, has been hardly any study dealing with H. coriaceus.
Since serine proteases forms a major component of the total
digestive proteolytic machinery in both the larvae and adults of
H. coriaceus, ILTI based studies were performed to record its potency
against the insect gut proteases. When incorporated into the
artificial diet to a level of 0.05%, ILTI decreased weight gain by 60%
(Fig. 5B) and thus reduced the larval survival to 10% (Fig. 5A).
Survival larvae fed on the ILTI at 0.1% were strongly reduced (60%)
and presented a decreased weight of 96%. The concentration of ILTI
used (0.0–0.1%, w/w) corresponded to levels in legume seeds and
Fig. 9. Diagram of the ILTI model, constructed by comparative modeling. (A) View of the
superficial layer of ILTI. Lys64 represents the amino acid situated in the reactive loop.
(B) View of the interaction between ILTI in carton (black) and trypsin in ribbon (gray).
The Lys64 and catalytic triad are represented in stick.
were similar to inhibitors expressed in transgenic plants (review in
Jongsma and Bolter 1997). Several authors have demonstrated that
ingestion of protease inhibitors in natural or artificial diets increased
mortality and delayed the developmental time in larvae of H. postica
(Elden, 1995), S. oryzae (Pittendrigh et al., 1997), and A. mariaefranciscae (Ortego et al., 1999), Anagasta kuehniella (Macedo et al.,
2009; Macedo et al., 2010).
Despite a digestive proteolytic system, based on proteases of
different mechanistic classes, curculionids are among the insects that
have shown the highest susceptibility to protease inhibitors.
Likewise, larvae of the weevil A. mariaefranciscae fed on diets
containing the serine protease inhibitors SBBI, soybean Kunitz
trypsin inhibitor (SKTI), turkey egg white trypsin inhibitor, or lima
bean trypsin inhibitor present lower survival rates and display
significant delays in the developmental time to pupation and to adult
emergence. Interestingly, the most significant levels of mortality
(about 90%) occurred with larvae fed on diets containing a
combination of two or three inhibitors, suggesting a synergistic
toxicity (Ortego et al., 1999).
Dietary utilization experiments show that 0.05% ILTI, incorporated
into an artificial diet, decreases the consumption rate and fecal
production of H. coriaceus larvae (Fig. 6A and B). An index of dietary
utilization showed that ECI and ECD decreased when a 0.05% ILTI diet
was employed. In the present study, the AD value for larvae of H.
coriaceus was increased throughout the feeding period of the
experiment.
M.L.R. Macedo et al. / Comparative Biochemistry and Physiology, Part B 158 (2011) 164–172
A greater AD would help to meet the increased demand for
nutrients (Nathan et al., 2005) and compensate for the deficiency in
foodstuff conversion (reduction in ECI and ECD), perhaps by diverting
energy from biomass production into detoxification. This behavior has
also been observed by others (Macedo et al., 2010). A drop in ECI
indicates that more food is being metabolized for energy and less is
being converted to body mass, i.e., growth of insect (Koul et al., 2003).
ECD also decreases as the proportion of digested food metabolized for
energy increases (Coelho et al., 2007). Confirming these results,
Table 2 demonstrates an increase of CM in H. coriaceus larvae. We
suggest that the reduction in ECD is likely to result from a reduction in
the efficiency to convert foodstuffs into growth, perhaps by a
diversion of energy from production of biomass into detoxification
of ILTI, i.e. an increase in costs.
The use of protease inhibitors to protect plant against insect pests
is complicated by the ability of insects to circumvent plant defenses.
Available biochemical and molecular evidence indicates that some
insects adapt to the presence of protease inhibitors by overproducing
existing digestive proteases (Lopes et al., 2004), whereas others adapt
to soybean proteinase inhibitors by changing the expression of trypsin
and chymotrypsin activities (Paulillo et al., 2000; Brito et al., 2001)
others suggest the synthesis of a new iso/enzyme in response to
protease inhibitor (Macedo et al., 2010) or the selective induction of
inhibitor-insensitive proteases (Cloutier et al., 2000). Digestive
proteolysis, mediated by direct proteolytic fragmentation, is another
strategy used by insects to minimize the adverse effects of plant
defense proteins on food digestion and nutrient uptake (Zhu-Salzman
et al., 2003).
Transgenic plants have already proven useful against several
curculionid pests. For example, transgenic expression of the trypsin
inhibitor, BTI-CMe, from barley in rice seeds confers resistance to S.
oryzae (Alfonso-Rubí et al., 2003). Likewise, Irie et al., (1996),
reported that a corn cystatin encoding gene expressed in rice seeds
was able to inhibit S. zeamais gut proteases. In addition, Atkinson et al.
(2004) published the first report of transgenic resistance against a
major pest or disease of banana by a rice cystatin. This technology
offers the potential for developing transgenic banana and plantain
plants with enhanced resistance to C. sordidus as a result of the
expression of a suite of protease inhibitors that are matched to the
digestive enzymes of this pest (Montesdeoca et al., 2005).
Protein–protein interface server analysis (PPIS) of the most
favored ILTI-trypsin and template reveals several properties involving
the relation of the interface and surface area. The SKTI-trypsin
complex presented 803 Å2, compared to 708 Å2, in the ILTI-trypsin.
Other PPIS parameters are nearly identical, with a single exception.
Having established the benchmark for acceptable quality of the ILTI
model, the best PROSA II scores were used in docking experiments
with the HEX v5.1 suite of programs (Gabb et al., 1997; Moont et al.,
1999). The top 50 docking solutions of 500 interaction results were
screened for the model with the PPIS. These data compare well with
values for all known enzyme–inhibitor complexes of 785 Å2 ± 75
(mean ± SD) with surprising complementation (Jones and Thornton,
1996). The 3D interaction models of ILTI-trypsin showed a competitive type of inhibitory mechanism, in agreement with data presented
in vitro.
In summary, the inhibitor from I. laurina seeds presented a reliable
3D structure, after several validations. In silico studies have shown
that ILTI interacts with trypsin trough the classic model, in which the
64th residue from the inhibitor interacts with the serine (195) present
on trypsin. The interaction sites of ILTI form a binary complex
observed by in silico methods. This inhibitor possesses a substitution
in the residue 64 of the reactive site (Arg→Lys). This similar
substitution promotes the trypsin inhibition in accordance with the
Kunitz type inhibitor family, thus, these findings contribute to the
development of biotechnological tools such as transgenic plants with
enhanced resistance to insect pests.
171
5. Conclusions
In conclusion, the main digestive enzyme of the midgut digestive
protease system of H. coriaceus is trypsin. ILTI inhibitors strongly
inhibited the gut proteinases and growth of H. coriaceus. The
inhibitory effects of ILTI on the activity of proteinases and larval
growth, and its 3D structure make these proteins and their genes
promising candidates for the development of transgenic plants to
enhance plant defenses against insects.
Acknowledgements
This work was supported by CNPq (Conselho Nacional de
Desenvolvimento Científico e Tecnológico), FUNDECT (Fundacão de
Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado
do Mato Grosso do Sul), and PROPP/UFMS (Pró-Reitoria de Pesquisa e
Pós-graduação da Universidade Federal de Mato Grosso do Sul).
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