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Differences in midgut serine proteinases from larvae of the bruchid beetles Callosobruchus maculatus and Zabrotes subfasciatus.

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18
Silva et al.
Archives of Insect Biochemistry and Physiology 47:18–28 (2001)
Differences in Midgut Serine Proteinases From Larvae of
the Bruchid Beetles Callosobruchus maculatus and
Zabrotes subfasciatus
Carlos P. Silva,1* Walter R. Terra,2 and Rodrigo M. Lima1
1
Laboratório de Química e Função de Proteínas e Peptídeos, Centro de Biociências e Biotecnologia,
Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, Brasil
2
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo,
Caixa Postal 26077, CEP 05599-970, São Paulo, Brasil
Proteinase activities in the larval midguts of the bruchids
Callosobruchus maculatus and Zabrotes subfasciatus were investigated. Both midgut homogenates showed a slightly acidic
to neutral pH optima for the hydrolysis of fluorogenic substrates. Proteolysis of e-aminocaproil-Leu-Cys(SBzl)-MCA was
totally inhibited by the cysteine proteinase inhibitors E-64 and
leupeptin, and was activated by 1.5 mM DTT in both insects,
while hydrolysis of the substrate Z-ArgArg-MCA was inhibited
by aprotinin and E-64, which suggests that it is being hydrolysed by serine and cysteine proteinases. Gel assays showed that
the proteolytic activity in larval midgut of C. maculatus was
due to five major cysteine proteinases. However, based on the
pattern of E-64 and aprotinin inhibition, proteolytic activity
in larval midgut of Z. subfasciatus was not due only to cysteine proteinases. Fractionation of the larval midgut homogenates of both bruchids through ion-exchange chromatography
(DEAE-Sepharose) revealed two peaks of activity against ZArgArg-MCA for both bruchid species. The fractions from C.
maculatus have characteristics of cysteine proteinases, while
Z. subfasciatus has one non-retained peak of activity containing cysteine proteinases and another eluted in a gradient of
250–350 mM NaCl. The proteolytic activity of the retained peak
is higher at pH 8.8 than at pH 6.0 and corresponds with a single
peak that is active against N-p-tosyl-GlyGlyArg-MCA, and sensitive to 250 mM aprotinin (90% inhibition).The peak contains a
serine proteinase which hydrolyzes a-amylase inhibitor 1 from
the common bean (Phaseolus vulgaris). Arch. Insect Biochem.
Physiol. 47:18–28, 2001. © 2001 Wiley-Liss, Inc.
Key words: Bruchidae; proteinases; insect digestion; seed weevil
Contract grant sponsor: CNPq; Contract grant sponsor:
FAPERJ; Contract grant sponsor: PRONEX; Contract grant
sponsor: FENORTE; Contract grant sponsor: FINEP; Contract grant sponsor: FAPESP; Contract grant sponsor: International Foundation for Science (IFS).
*Correspondence to: Carlos P. Silva, Laboratório de Química
e Função de Proteínas e Peptídeos, Centro de Biociências e
Biotecnologia, Universidade Estadual do Norte Fluminense,
CEP 28015-620 Campos dos Goytacazes, Brasil. E-mail:
capsilva@cbb.uenf.br
Received 4 October 2000; Accepted 21 January 2001
© 2001 Wiley-Liss, Inc.
Bruchid Midgut Serine Proteinases
INTRODUCTION
The digestive proteinases of larval bruchid
beetles belong to a group of putative extra cellular cathepsins with digestive function, known to
occur only in certain groups of insects, such as
coleopterans of the series Cucujiformia, members
of Heteroptera in the order Hemiptera and cyclorrhaphous Diptera (Terra and Ferreira, 1994).
Non-serine digestive proteinases resembling cathepsins were at first considered to be restricted to
the hemipterans (Gooding, 1969; Houseman and
Downe, 1982, 1983), but they have now been described in other insects, mainly in phytophagous
insects of the order Coleoptera (Terra and Ferreira, 1994). Digestive cysteine proteinases resembling cathepsins were first identified outside the
hemipterans in bruchids (Xavier-Filho and Coelho, 1980; Gatehouse et al., 1985; Kitch and
Murdock, 1986; Wieman and Nielsen, 1988; Campos et al., 1989), as well as aspartic proteinases
(Lemos et al., 1990; Silva and Xavier-Filho, 1991).
Recently, Ishimoto and Chrispeels (1996)
suggested the existence of a minor serine proteinase in larval midgut of Zabrotes subfasciatus,
which may be involved in the hydrolysis of the
Phaseolus vulgaris α-amylase inhibitor 1 (α-AI1)
to detoxify the protein. The detection of this proteinase was based mainly on the degradation of
the α-amylase inhibitor by a crude midgut homogenate followed by electrophoresis, and by the effect of serine proteinase inhibitors. These authors
did not characterize the proteinase with other proteinaceous or synthetic substrates and did not describe the distribution of this proteinase in the
midgut. Thus, the detoxification role of this proteinase is not clear.
There are no detailed data on the specificity
of bruchid digestive proteinases. Curiously, only proteinaceous substrates were used in the most detailed studies referring to digestive proteinases in
bruchid beetles. This prompted us to reinvestigate
the specificity of intestinal proteinases from larvae
of two important bruchid pests using synthetic substrates that allow comparisons with other insects.
MATERIALS AND METHODS
Materials
The following substrates were used: carbobenzoxy-L-arginine-arginine-p-nitroanilide (Z-
19
ArgArg-pNA), carbobenzoxy-L-glycine-glycinearginine-4-methylcoumarin (Z-GlyGlyArg-MCA),
carbobenzoxy-L-phenylalanine-L-arginine-4methylcoumarin (Z-PheArg-MCA), ε-aminocaproil-LeuCys-(SBzl)-MCA, N-p-tosyl-GlyProArg-MCA, N-succinyl-AlaAlaProPhe-p-nitroanilide
(SAAPF-pNA), N-succinyl-AlaAlaProLeu-p-nitroanilide (SAAPL-pNA), Nα-Benzoyl-L-arginine-pnitroanilide (BApNA). The following inhibitors
were used: aprotinin, leupeptin, trans-epoxy succinyl-L-leucylamido-(4-guanidino)-butane (E-64)
and pepstatin. Dithiothreitol (DTT), sodium
dodecyl sulphate (SDS), and trichloroacetic acid
(TCA) were all purchased from Sigma-Aldrich. αAmylase inhibitor 1 (αAI-1) from the seeds of
Phaseolus vulgaris was a gift from Dr. Maria F.
Grossi de Sá (Cenargen-EMBRAPA- Brasília).
Insects
The colony of C. maculatus used in this work
was established using animals that were supplied
originally by Dr. J.H.R. Santos, Centro de Ciências Agrárias, Universidade Federal do Ceará,
Fortaleza, Brazil. A Z. subfasciatus colony was
established from insects supplied originally by
Prof. F.M. Wiendl of the Centro de Energia
Nuclear na Agricultura, Piracicaba, Brazil. Stock
cultures of both species are grown in Campos
since 1994. The insects are reared on Vigna
unguiculata seeds (cultivar Epace 10) in the dark
inside an incubator chamber maintained at 29 ±
1°C and relative humidity of 65 ± 5%. Seeds are
frozen in order to prevent infestation from the
field.
Preparation of Samples From Insects
Final instar larvae were cold immobilized,
dissected, and the whole midgut was removed in
cold 250 mM NaCl. Actively feeding larvae with
food-filled gut tract were selected for the experiments. The whole gut was then cleaned from adhering unwanted tissues, midgut walls were
pulled apart, and luminal contents were collected
into a known volume of distilled water. Pooled
midgut tissues, after being cleaned of luminal contents by rinsing in 250 mM NaCl, were homogenized in cold distilled water in a hand-held
Potter-Elvehjem homogenizer on ice. Midgut tissue homogenates were centrifuged at 20,000g for
30 min at 4°C and the supernatant (soluble frac-
20
Silva et al.
tion) collected and the sediment (membrane fraction) was dispersed in distilled water using a Potter-Elvehjem homogenizer.
Hydrolase Assays and Protein Determination
Assays for the hydrolysis of methylcoumarin
derivatives were carried out using a Hitachi
F4500 fluorometer to monitor the release of free
methylcoumarin (excitation wavelength 380 nm
and emission wavelength 440 nm) in a 2-ml
thermostated cuvette (1-cm path length). Enzyme
samples (80 µl) were mixed with warmed 1.9 ml
of 100 mM citrate/phosphate buffer containing 1.5
mM DTT, 3 mM EDTA, pH 6.0. Reactions were
initiated by adding 20 µl of 1 mM methylcoumarin
substrate.
Total proteinase activity of midgut homogenates was determined using the protein substrate azoalbumin. The azoalbumin assays were
adapted from a previously described method
(Lemos et al., 1990). Extracts (100 µl per assay)
were incubated with 100 µl of a mixture of 1%
substrate (w/v) in 100 mM citrate/phosphate, pH
6.0. Proteolysis was stopped by adding 100 µl of
25% (w/v) TCA, and the reaction was incubated
for 30 min on ice. Precipitated substrate was removed by centrifugation for 5 min at 7,000g at
room temperature. Aliquots of 300 µl of the supernatant were added to 300 µl of 2N NaOH solution and absorbance was read at 440 nm in a
Spercord spectrophotometer (Zeiss).
Hydrolysis of p-nitroanilide derivatives were
assayed by the release of p-nitroaniline, which
absorbs maximally at 410 nm (Erlanger et al.,
1961). The assays used 50 µl of 4 mM substrates,
50 µl of enzyme sources, and 100 µl of 100 mM
citrate/phosphate buffer, pH 6.0, with or without
inhibitors or activator. The reaction was stopped
by adding 100 µl of 30% (v/v) acetic acid, and the
absorbance was read at 410 nm in a Spercord
spectrophotometer (Zeiss).
All assays were performed at 30°C. Buffers
(100 mM) that were used to determine pH optima were: sodium acetate, citrate-sodium phosphate, Tris-HCl ranging from 3–9, with intervals
of 0.2 pH units. Incubations were carried out for
at least four different periods of time, unless otherwise stated, and initial rates of hydrolysis were
calculated. All assays were performed under conditions in which enzyme activity was proportional
to protein concentration and to the time of incubation. One enzyme unit is defined, except for proteinaceous substrates, as the amount that
catalyzes the cleavage of 1 µmol of substrate/min.
One proteinase unit, with azoalbumin as substrate, was the amount that caused a change in
absorbance of 0.01 U/min.
Protein contents of samples were determined
by the method of Bradford (1976) using ovalbumin as a protein standard.
In Gel Assays
Proteinases from bruchid midgut preparations were detected and partially characterized
by 10 or 12% SDS-polyacrylamide gel electrophoresis containing 0.1% (w/v) gelatin (Heussen
and Dowdle, 1980). Samples were diluted twofold
in electrophoresis sample buffer (2.1 ml distilled
water, 0.5 ml 0.5 M Tris-HCl, pH 6.8, 0.4 ml glycerol, 0.8 ml 10% (w/v) SDS, 0.2 ml 1% (w/v) bromophenol blue) without 2-mercaptoethanol and
subjected to electrophoresis (Laemmli, 1970) at
150 V and 4°C without heating the samples. Following electrophoresis, the gels were transferred
to a 2.5 % (w/v) aqueous solution of Triton X-100
for 20 min at room temperature in order to allow
renaturation of the enzymes. The gels were then
incubated with a proteolysis buffer (100 mM citrate/phosphate 1.5 mM DTT, pH 6.0) for different periods of time. Proteolysis was stopped by
transferring gels to a staining solution (0.1% w/v
Coomassie Brilliant Blue in 40% v/v methanol/
10% v/v acetic acid). After a brief decoloration in
40% v/v methanol/10% v/v acetic acid, clear bands
on a blue background identified the location of
the active proteinases on the gel.
In order to establish the effect of pH on the
proteolytic activities in the gel assays, strips of
the gels, after the renaturation procedure, were
incubated in different buffers containing 1.5 mM
DTT (and 100 mM of sodium acetate, citrate-sodium phosphate, Tris-HCl, and pHs ranging from
3– 9, and intervals of 0.5 pH units).
Ion-Exchange Chromatography
Samples containing 250 midguts were homogenized in 2 ml of a solution containing 10 µM
E-64 and 5 µg/ml pepstatin by using a PotterElvehjem homogenizer. The homogenate was centrifuged at 20,000g for 30 min at 4°C. The
Bruchid Midgut Serine Proteinases
supernatant was applied to a DEAE-Sepharose
column (10 × 0.5 cm i.d.) equilibrated with 10 mM
imidazole buffer, pH 6.0, containing 10 µM E-64
and 5 µg/ml pepstatin, using an Econo System
(BioRad, Richmond, CA) apparatus.
The column was washed with 25 ml of the
same buffer and then eluted with 50 ml of a linear gradient (0-1M NaCl) in imidazole buffer, followed by 10 ml of isocratic elution in this NaCl
containing buffer. The flow rate was 1.0 ml/min
and fractions of 1.0 ml were collected. Recoveries
of the activities from the column were 70–80%.
In Vitro Proteolysis of the a-Amylase Inhibitor
Proteolysis of the αAI-1 by the isolated midgut serine proteinase from Z. subfasciatus was
determined by incubating 15 mg of the purified
α-amylase inhibitor with the proteinase fraction
in the presence of 10 µM E-64 and 5 µg/ml pepstatin at 30°C for 16 h. The reaction was stopped
by addition of sample buffer and boiling the mixture for 5 min before being electrophoresed on
SDS-PAGE (15% acrylamide) according to Laemmli (1970). Incubating αAI-1 with water in place
of the proteinase fraction was used as a control.
RESULTS
Specificities of the Digestive Proteinases From
Larvae of C. maculatus and Z. subfasciatus
The pH optima for the hydrolysis of fluorogenic substrates by crude midgut homogenates
from both bruchid species were found for all
fluorogenic substrates and enzyme sources between 6–7 (Figs.1 and 2). Similar profiles were
also observed for the chromogenic substrates
(data not shown).
Crude larval midgut homogenates of C. maculatus contain 5 times more activity than homogenates from larvae of Z. subfasciatus against
Z-PheArg-MCA, while larvae of Z. subfasciatus contained 4 times more protease activity against ZArgArg-MCA than larvae of C. maculatus (Table
1). Figures for the hydrolysis of Z-Arg-MCA do not
present differences in terms of absolute activity, but
in terms of specific activity, C. maculatus presents
the double of the activity observed for Z. subfasciatus (Table 1). Another important difference also
shown in Table 1 is the hydrolysis of the substrate
N-p-tosyl-GlyProArg-MCA, where larvae of Z.
21
subfasciatus contain larger absolute and specific activities (8 to 5 times, respectively) than larvae of C.
maculatus.
Enzymatic activities were the highest against
ε-aminocaproil-LeuCys-(SBzl)-MCA, for both C.
maculatus and Z. subfasciatus. The activity of C.
maculatus protease was higher than Z. subfasciatus
(Table 1). The activity against this substrate was
not inhibited by aprotinin, whereas E-64, antipain,
and leupeptin completely inhibited this activity
(Tables 2 and 3). The activity against ε-aminocaproil-LeuCys-(SBzl)-MCA was enhanced by 1.5
mM DTT (Tables 2 and 3). Protease activities from
the two bruchid species toward the other fluorogenic
substrates, as well as the activities against azoalbumin, were highly inhibited by E-64, antipain, and
leupeptin (Tables 2 and 3).
Intestinal homogenates from larvae of Z.
subfasciatus were two times more active toward
p-nitroanilide derivatives, which are substrates
for serine proteinases (N-SAAPL-pNA and NSAAPF-pNA), than C. maculatus homogenates.
Conversely, higher activity against BApNA was
observed in larvae of C. maculatus (Table 1).
The spatial distribution of activities against
the synthetic substrates was determined by assaying activities in the luminal contents and in
the soluble and particulate fractions of the midgut epithelium. The results showed that 80 to 90%
of the proteolytic enzymes that were tested on
all the substrates were found in the luminal contents in the two bruchid species.
In Gel Activities of Larval Midgut Homogenates
From C. maculatus and Z. subfasciatus
Profiles of gelatinolytic activity bands of intestinal homogenates from larvae of C. maculatus
and Z. subfasciatus are different. It was possible
to distinguish at least 5 bands from samples of
C. maculatus, with RF between 0.20 and 0.60,
whereas in Z. subfasciatus one major band corresponding to a RF 0.15–0.30 and several less distinct bands with RF between 0.35 and 0.45 were
found (Fig. 3). The major bands in C. maculatus
had a higher electrophoretic mobility than the
major band of Z. subfasciatus (Fig. 3).
Midgut homogenates from both bruchid species showed pH optima in the range 6–7, as assayed by mildly-denaturing electrophoresis,
similarly to the determinations using synthetic
22
Silva et al.
Fig. 1. Effect of pH on the hydrolysis of fluorogenic substrates by larval midgut homogenates of Callosobruchus
maculatus. The used buffers were: citrate/phosphate (circles),
phosphate (squares), and Tris/HCl (triangles). The results
are representative of three determinations.
substrates. C. maculatus exhibited very low
acitivity below pH 5, while Z. subfasciatus had a
diffused band that peaked at pH 5.0. The major
bands of proteolytic activity from the two bruchids
could be observed up to pH 9.0 (Fig. 3).
Most of the proteinases of larval midgut
homogenates from C. maculatus appears to be
cysteine proteases, because incubating the gels
inhibitor E-64 abolished the gelatin hydrolysis
(Fig. 4) and incubating the gels with 1.5 mM DTT
caused gelatinase activation (Fig. 5). These results are in agreement with the results obtained
by incubating the enzymes with inhibitors and
DTT and assaying with synthetic substrates
Bruchid Midgut Serine Proteinases
Fig. 2. Effect of pH on the hydrolysis of fluorogenic substrates by larval midgut homogenates of Zabrotes subfasciatus. The used buffers were: citrate/phosphate (circles),
23
phosphate (squares), and Tris/HCl (triangles). The results
are representative of three determinations.
TABLE 1. Digestive Proteinase Activities Present in Midgut of Larval Callosobruchus maculatus and
Zabrotes subfasciatus Reared on Vigna unguiculata Seeds*
Callosobruchus maculatus
Substrates
Z-Arg-MCA
Z-ArgArg-MCA
Z-PheArg-MCA
Z-GlyGlyArg-MCA
N-p-GlyProArg-MCA
ε-LeuCys-(SBzl)-MCA
SAAPL-pNA
SAAPF-pNA
BApNa
nmol/min/gut
400
500
2,500
77
43
4,650
65
33
133
nmol/min/mg P
6,250
7,700
39,000
1,165
670
72,600
795
394
1,628
Zabrotes subfasciatus
nmol/min/gut
420
2,000
570
130
500
2,500
145
75
100
nmol/min/mg P
3,200
14,600
4,400
2,027
3,800
19,200
1,771
910
1,010
*Figures are the means calculated from four assays performed in each of five different preparations obtained from 20
animals. Specific activities are expressed in nmol/min per mg of gut protein. SEM were found to be 5–10% of the means.
24
et al.
TABLE Silva
2. Effect
of Inhibitiors and One Activator on the Hydrolysis of Synthetic Substrates and on One
Proteinaceous Substrate by Larval Midgut Homogenate From Callosobruchus maculatus*
Substrates
Z-ArgArg-AMC
Z-PheArg-AMC
ε-LeuCys-(SBzl)-MCA
Azoalbumin
DTT
100
776
1,084
695
E-64
0
0
0
5
Effectors (% relative activity)
Aprotinin
Leupeptin
50
46
100
60
0
0
0
0
Antipain
0
0
0
5
*The figures are percentages in relation to the measured activity in the controls for each substrate. In the case of DTT, the
activity in the control was measured using an assay medium without the activatior, whereas for the inhibitors, the assays
were made in the presence of 1.5 mM DTT. Triplicate measurements showed a variation of 2–5% for all cases.
TABLE 3. Effect of Potential Inhibitors and One Activator on the Hydrolysis of the Synthetic Substrates and
on One Proteinaceous Substrate by Larval Midgut Homogenate From Zabrotes subfasciatus*
Substrates
Z-ArgArg-AMC
Z-PheArg-AMC
ε-LeuCys-(SBzl)-MCA
Azoalbumin
DTT
100
423
2,000
142
E-64
0
0
0
23
Effectors (% relative activity)
Aprotinin
Leupeptin
30
45
100
78
0
0
0
24
Antipain
0
0
0
nd
*The figures are percentages in relation to the measured activity in the contorls for each substrate. In the case of DTT, the
activity in the control was measured using an assay medium without the activator, whereas for the inhibitor, the assays were
made in the presence of 1.5 mM DTT. Triplicate measurements showed a variation of 3–5% for all cases. nd, not determined.
(Table 2). Similarly, the activity observed in gel
assays for larvae of Z. subfasciatus was also completely abolished by E-64 and there was a significant activation of the major band by 1.5 mM
DTT (Figs. 4 and 5).
Isolation of a Serine Proteolytic Enzyme From
Larvae of Z. subfasciatus
Fig. 3. Effect of pH on the hydrolysis of gelatin by larval digestive proteinases from Callosobruchus maculatus (A) and
Zabrotes subfasciatus (B). Samples containing 0.4 gut equivalents were resolved on gelatin-containing polyacrylamide gels
and allowed to hydrolyse gelatine at 30°C for 4 h (C. maculatus)
and 15 h (Z. subfasciatus) at different pH values. Buffers used
in the experiment were citrate/phosphate (3.0–7.0); phosphate
(6.0–8.0), and Tris-HCl (8.0–9.0). The pH values from left to
right were: 3.0, 4.0, 5.0, 6.0, 7.0, 7.5, 8.0, 8.5, and 9.0.
A single peak of activity against the proteinase substrates Z-ArgArg-MCA or Z-GlyGlyArg-MCA
was obtained by DEAE-Sepharose chromatography
of intestinal homogenates of Z. subfasciatus in the
chromatography performed in the presence of 20
mM E-64 and 5 µg/ml pepstatin (Fig. 6). The activity in the retained peak is higher at pH 8.8, which
favors the action of serine proteinases, than at pH
6.0, which favors the action of cysteine proteinases
(data not shown) and was inhibited by 250 mM
aprotinin (Fig. 6), suggesting that it is a serine proteinase.
Incubation of a purified α-amylase inhibitor
1 from seeds of P. vulgaris with the isolated serine
proteinase fraction resulted in the cleavage of the
inhibitor, as demonstrated by SDS-PAGE (Fig. 7).
DISCUSSION
Most of the proteolytic activities from crude
midgut homogenates of the two bruchid species
assayed with synthetic substrates or by using in
Bruchid Midgut Serine Proteinases
25
Fig. 4. Effect of the inhibitor E-64 on the gelatinolytic activity of larval midgut proteinases from Callosobruchus
maculatus and Zabrotes subfasciatus as revealed by in gel
assay. After migration and a renaturation step, proteinase
activities were assayed at 30°C for 4 h in 50 mM citratephosphate buffer containing 1.5 mM DTT at pH 6.0 in the
absence or in the presence of 10 µM E-64.
gel assays showed pH optima in the range 6–7
(Figs. 1, 2, and 3), which corresponds to the pHs
found in the midguts of the following bruchids C.
maculatus (Kitch and Murdock, 1986; Silva et al.,
1999), Acanthoscelides obtectus (Osuala et al.,
1994), and Bruchus pisorum (Lagadic, 1994). The
use of in gel assays to determine pH optima
showed that Z. subfasciatus possessed at least one
proteinase that is stable during the mildly-denaturing electrophoresis and has a pH optimum
lower than the pH found in the luminal contents.
On the other hands, the major proteinases from
both bruchids are maximally active at the higher
pH range up to pH 9.0 (Fig. 3). Gel assays also
revealed a greater diversity of proteinase forms
as compared with column chromatography. Incubating the gels with 5 µg/ml pepstatin did not
inhibit the gelatinase activity (data not shown).
It is probable that the activity of aspartic proteinases cannot be observed on our zymograms,
because these enzymes are inactivated at alkaline pHs (Barrett and Kirschke, 1981).
Differences in activities using N-p-tosylGlyProArg-MCA, Z-ArgArg-MCA, N-SAAPL-pNA,
Fig. 5. Effect of DTT on the gelatinolytic activities of larval midgut proteinases from Callosobruchus maculatus and
Zabrotes subfasciatus using in gel assay. After migration and
a renaturation step, proteinase activities were assayed at
30°C for 4 h in 50 mM citrate-phosphate buffer at pH 6.0 in
the absence or in the presence of 1.5 mM DTT.
and N-SAAPF-pNA and intestinal homogenates
of larvae from C. maculatus and Z. subfasciatus
suggest a larger amount of serine proteinase activities in Z. subfasciatus than in C. maculatus
(Table 1). C. maculatus larvae contain more cysteine proteinases than larvae of Z. subfasciatus,
and this was demonstrated by the hydrolysis of
the substrate ε-aminocaproyl-LeuCys-(SBzl)-MCA
(Table 1). The effects of the activator DTT and
the inhibitor E-64 strengthen this observation
(Figs. 4 and 5). The lack of activation by DTT on
the activity against the synthetic substrates ZArgArg-MCA and N-CBZ-GlyProArg-MCA, in addition to the partial inhibition of these activities
by aprotinin, suggest that these substrates are
being hydrolyzed by serine proteinases (Tables 2
and 3). The complete inhibition of the enzymatic
activities of midgut homogenates from larvae of
C. maculatus and Z. subfasciatus with E-64 sug-
26
Silva et al.
Fig. 6. Effect of the inhibitor
aprotinin (100 mM) on the proteolytic activity (assayed with ZGlyGlyArg-MCA) of fractions
obtained from the DEAE-Sepharose column. Circles, activity
against Z-GlyGlyArg-MCA at pH
8.8 without inhibitor; diamonds,
activity in the presence of 100
mM aprotinin. The column was
equilibrated with 10 mM imidazole buffer pH 6.0 containing 10
µM E-64 and 5 µg/ml pepstatin,
washed with 25 ml of the same
buffer and then was eluted with
50 ml of a linear gradient (0-1M
NaCl) in imidazole buffer, followed by 10 ml of isocratic elution in this NaCl containing
buffer. The flux was 1.0 ml/min
and fractions of 1.0 ml were collected. Recoveries of the activities applied to the column were
70–80%.
Fig. 7. In vitro hydrolysis of the α-amylase inhibitor 1 from
seeds of Phaseolus vulgaris by the intestinal serine proteinase
fraction from Zabrotes subfasciatus isolated by ion-exchange
chromatography. The amylase inhibitor was incubated with the
proteolytic fraction for 16 h. The reaction was stopped by the
addition of sample buffer, boiled, and run on SDS-PAGE. The
gel was stained with coomassie blue dye. The arrow points to
the band corresponding to nonhydrolyzed α-amylase inhibitor,
while the asterisks show the hydrolytic products. Lane 1: Control without the proteolytic fraction. Lane 2: Proteolysis in
the presence of the minor serine proteinase.
gests the existence of cysteine proteinases in these
species (Table 2, Fig. 4).
Recently, it was reported that E-64 is also
capable of inhibiting activities of some trypsinlike enzymes in addition to cysteine proteinases
(Sreedharan et al., 1996; Novillo et al., 1997). The
inhibition pattern of the cathepsins of the bruchids C. maculatus and Z. subfasciatus by E-64
is more in line with the presence of cysteine proteinases, because incubating the enzymes with
the inhibitor led to a total loss of the enzymatic
activity, while the minor serine proteinase detected in this paper and by Ishimoto and Chrispeels (1996) was not inhibited by E-64. The data
reported in this paper using in gel assays and
synthetic substrates are in agreement with our
previous data showing that C. maculatus has
more cysteine proteinases than Z. subfasciatus
(Silva and Xavier-Filho, 1991).
The presence of a serine proteinase in intestinal homogenates from Z. subfasciatus was confirmed with synthetic substrates and by column
chromatography isolation of a fraction that is insensitive to E-64, inhibited by aprotinin, and
shows a higher activity against Z-ArgArg-MCA
and Z-GlyGlyArg-MCA at pH 8.8 than at pH 6.0.
The isolated proteinase is a serine proteinase capable of degrading αAI-1 (Fig. 7) as was
shown by Ishimoto and Chrispeels (1996). These
Bruchid Midgut Serine Proteinases
authors reported that intestinal homogenates
from larvae of Z. subfasciatus contain a serine
proteinase, which is capable of detoxifying the αamylase inhibitor. We determined the spatial distribution of the digestive proteinases in both
bruchid species. Assays performed in samples
from luminal contents and epithelium revealed
that most of the digestive proteinases of both weevils are found in the luminal contents, and therefore they may have digestive or detoxifying roles
of potentially toxic proteins.
In summary, our results suggest that the larvae of C. maculatus rely on a larger variety of
cysteine proteinases, while the larvae of Z.
subfasciatus use, in addition to the aspartic and
cysteine proteinases, serine proteinases. Larvae
of Z. subfasciatus seem to have a greater variety
in classes of intestinal proteinases and, possibly
due to this fact, they are able to use many more
hosts than C. maculatus.
We speculated that the specialization to
grow inside seeds led the bruchid beetles to lose
the digestive serine endopeptidases of the chymotrypsin family (C1, as defined by Rawlings
and Barrett, 1993), and that these insects rely
only on the cysteine or aspartic cathepsin-like
proteinases (Campos et al., 1989; Terra and
Ferreira, 1994). These enzymes are appropriate
to digest the seed storage proteins and are not
affected by the major serine proteinase inhibitors of the seeds. The description of a minor
serine proteinase in larvae of Z. subfasciatus
seems to challenge this hypothesis, unless the
observed serine proteinase is a cathepsin G-like
proteinase that also belongs to the family of the
chymotrypsins (Rawlings and Barrett, 1993).
The enzyme that we isolated, which should correspond to the enzyme described by Ishimoto and
Chrispeels (1996), is also capable of acting on
the extended binding substrates used to assay
chymotrypsin and elastase. Because these substrates are also susceptible to cathepsin G, we
speculate that this minor enzyme could be a
serine proteinase with a lysosomal origin, similar to the mammalian cathepsin G (Barrett,
1981). Unfortunately, the low activity level and
its instability have been hindering the preparation of this enzyme in a homogeneous state and
in a large enough amount to further characterization and to test this hypothesis.
27
ACKNOWLEDGMENTS
We thank Dr. Richard I. Samuels for his comments on the manuscript and for the English revision. R. M. Lima is an undergraduate fellow
from CNPq. C.P. Silva and W.R. Terra are staff
members of their respective departments and are
also research fellows of CNPq.
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