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Purification and properties of trypsin-like enzyme from the midgut of Morimus funereus (coleoptera cerambycidae) Larvae.

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A r t i c l e
PURIFICATION AND PROPERTIES
OF TRYPSIN-LIKE ENZYME FROM
THE MIDGUT OF Morimus
funereus (COLEOPTERA,
CERAMBYCIDAE) LARVAE
Nikola Lončar and Zoran Vujčić
Department of Biochemistry, Faculty of Chemistry, University
of Belgrade, Belgrade, Serbia
Nataša Božić
Centre of Chemistry, Institute of Chemistry, Technology and Metallurgy,
University of Belgrade, Belgrade, Serbia
Jelisaveta Ivanović and Vera Nenadović
Department of Insect Physiology and Biochemistry, Institute of Biological
Research ‘‘Sinisˇa Stankovic´,’’ Belgrade, Serbia
Trypsin-like enzyme (TLE) from the anterior midgut of Morimus
funereus larvae was purified by anion exchange chromatography and
gel filtration chromatography and characterized. Specific TLE activity
was increased 322-fold by purification of the crude midgut extract.
The purified enzyme had a pH optimum of 9.0 (optimum pH range
8.5–9.5) and temperature optimum of 451C with the KM ratio
of 0.065 mM for benzoyl-arginine-p-nitroanilide (BApNA). Among
a number of inhibitors tested, the most efficient was benzamidine
(KI value of 0.012 mM, Ic50 value of 0.204 mM) while inhibition
of TLE activity by SBTI, TLCK, and PMSF was partial. Almost
all divalent cations tested enhanced the enzyme activity, amongst
them Co21 and Mn21 stimulated TLE activity for 2.5 times. The
purified TLE (after gel-filtration on Superose 12 column) had
a molecular mass of 37.5 kDa with an isoelectric point over
9.3. Sodium dodecylsulphate-polyacrylamide gel electrophoresis
Grant sponsor: Serbian Ministry of Science and Technological Development; Grant number: 142026B.
Correspondence to: Zoran Vujčić, Department of Biochemistry, Faculty of Chemistry, University of
Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia. E-mail: zvujcic@chem.bg.ac.rs
ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 74, No. 4, 232–246 (2010)
Published online in Wiley InterScience (www.interscience.wiley.com).
& 2010 Wiley Periodicals, Inc. DOI: 10.1002/arch.20371
Trypsin-Like Enzyme from Morimus funereus Larvae
233
(SDS-PAGE) revealed one band of 38 kDa, suggesting that the
C 2010 Wiley Periodicals, Inc.
enzyme is a monomer. Keywords: trypsin; midgut; inhibitor; Cerambycid beetle; Morimus
funereus; xylophagous larvae
INTRODUCTION
Serine-type endopeptidases are the primary digestive peptidases in most insect groups
(Terra et al., 1996), with the exception of some hemipteran (Houseman and Downe,
1983; Houseman et al., 1985) and coleopteran species (Murdock et al., 1987; Božić
et al., 2003) in which cysteine, aspartic, and aminopeptidases are predominant.
Trypsins (EC 3.4.21.4), one of the major insect serine endopeptidases, are involved in
the initial phases of protein digestion, preferentially cleaving internal bonds of
polypeptide chains on the carboxyl side of the basic L-amino acids, arginine or lysine.
Beside that, they take part in a number of physiological processes such as coagulation,
immunity, fibrinolysis, and embryonic development (Liu et al., 2009).
Trypsins have been isolated from midguts of various insect species and typically
exhibit molecular masses (MM) from 20 to 35 kDa and alkaline pH optima (Terra and
Ferreira, 1994; Muhlia-Almazán et al., 2008). Insect digestive trypsins show similarities
to vertebrate trypsins with respect to cleavage site specificity (Miller et al., 1974; Milne
and Kaplan, 1993), and sequence homology in the regions of catalytic site (Muller
et al., 1993). However, they differ in some other properties; for example, insect
trypsins are unaffected by calcium ions and are often unstable in acidic pH (Lam et al.,
2000). More recent data, obtained with semi-purified trypsin preparations acting on a
limited range of synthetic substrates, suggest that trypsin may differ among insect
groups as well (Lopes et al., 2004).
The purification and characterization of proteolytic enzymes from insects can play
an important role in building insect-resistant plants through transgenesis (Gatehouse
et al., 1994). The purified enzymes can be used as ligands in affinity chromatography
systems for the isolation of more effective inhibitors from plant tissues (Marchetti et al.,
1998). For this purpose, peptidases with some novel properties could be of special
interest. Despite the significant literature on trypsin sequences from other insect
groups, coleopteran serine-peptidases have not been thoroughly characterized and
rarely purified (Magalhães et al., 2007).
Cerambycid beetle, M. Funereus, inhabits an environment rich in deciduous and
coniferous trees and has a long life span with development over a 3–4-year period.
Tree mortality is normally not associated with long-horned beetle infestation
although damage to oak lumber may be economically important throughout its
range.
It is known from our previous work on the Morimus funereus that trypsin-like
activity is present in the midgut of larvae in two isoforms (ÐurXević et al., 1997) and
distribution of trypsin-like peptidases along the midgut showed that most of the
enzyme is present in the anterior part of the midgut (Lončar et al., 2009). In the
present study, we describe the purification of the more abundant trypsin-like enzyme
(TLE) isoform from the anterior midgut and its molecular and enzymological
characterization in order to better understand the biochemical organization of the
digestive process in M. funereus larvae.
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MATERIALS AND METHODS
Reagents
All reagents and solvents used were of the highest available purity and at least
analytical grade. They were purchased unless otherwise stated, from Merck
(Darmstadt, Germany) and Sigma-Aldrich (St. Louis, MO).
Insects
Actively feeding sixth instar M. funereus larvae were used in the experiments. Larvae
were continuously reared at 231C and at approximately 50% humidity using a
modified artificial diet for Drosophila sp. (Roberts, 1986).
Preparation of Crude Midgut Extracts
After decapitation, the midguts were isolated on ice, divided into three equal parts
(anterior, middle, and posterior), weighed and homogenized using a pre-chilled
mortar and pestle in 2 volumes (g/mL) of ice-cold 0.9% NaCl, 20 mM acetate buffer,
pH 6.0, with the addition of quartz powder. The homogenate was centrifuged for
5 min at 5000 g at 41C. Lipids were removed by combining the resulting supernatant
with an equal volume of carbon tetrachloride followed by centrifugation for 2 min at
5000 g at 41C. This procedure was repeated three times before the final supernatant
was desalted on Sephadex G-25 column. The concentration of proteins was
determined by the Bradford assay with bovine albumin as a standard (Bradford,
1976) before dividing the desalted supernatant into smaller aliquots for storage
at 201C. The concentration of proteins was determined by the Bradford assay at all
purification stages as well.
Trypsin-Like Activity Assays
Enzyme activity toward BApNA was determined spectrophotometrically by measuring
the absorbance at 410 nm (Erlanger et al., 1961). Reaction mixture consisted of 5 mL of
crude midgut extract or purified enzyme in 0.5 mL of 50 mM ethanolamine buffer pH
9.0 and 1.0 mM BApNA in 2% N,N-Dimethyl-formamide (DMF). Incubations (at 301C)
lasted 10 and 20 min for crude midgut extract and purified enzyme, respectively. All
reactions were terminated by adding 0.1 mL 30% acetic acid. Enzyme activity was
expressed in U, which was defined as the amount of enzyme hydrolyzing 1 mmol of
BApNA per min at 301C.
Under the same experimental conditions with prolonged incubation times, TLE
activity was tested toward specific substrates for chymotripsyn-like and elastase-like
enzymes. For this purpose, N-succinyl-L-phenylalanine-p-nitroanilide (SFpNA);
N-glutaryl-L-phenylalanine-p-nitroanilide (GFpNA); N-benzoyl-L-tyrosine-p-nitroanilide (BTpNA); N-succinyl-L-alanyl-L-alanyl-L-alanine-p-nitroanilide (SA3pNA); and
N-succinyl-L-alanyl-L-alanyl-L-proline-p-nitroanilide (SA2PpNA) were used.
Trypsin-Like Purification
To separate TLE activities from the crude enzyme extract, the anterior section of the
midgut was used since it was previously shown that more than 90% of TLE activity was
found in this part of the midgut (Lončar et al., 2009). A total amount of 4 mL (9.51 mg
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235
protein/mL, 4.96 U TLE activity units) of crude extract of anterior section of midgut,
corresponding to ten anterior midguts, was loaded onto Q-Sepharose FF column
(Pharmacia XK 26/20, Uppsala, Sweden). The column was equilibrated with 20 mM
Tris HCl buffer solution, pH 9.0, at a flow rate of 1.1 mL/min. Two milliliters of flowthrough fractions were collected and assayed for activity.
Fractions containing TLE activities were pooled and concentrated by lyophilization. Two milliliters of obtained TLE (0.17 mg/mL, 3.48 U) was subjected to gel
filtration on Superose 12 column (16 500 mm) on a fast protein liquid chromatography (FPLC) system (Pharmacia). The column was previously equilibrated with 0.9%
NaCl, 20 mM acetate buffer, pH 6.0, at a flow rate of 0.5 mL/min. Fractions (2 mL)
were collected and assayed for TLE activity. Purified TLE was stored at 201C in the
acetate buffer, pH 6.0. Freezing and thawing with a subsequent electrophoresis pattern
of purified TLE confirmed the lack of TLE autodegradability.
Isoelectric Point and Molecular Mass
Isoelectric focusing was performed using a Multiphor II electrophoresis system
(Pharmacia-LKB Biotechnology) according to the manufacturer’s instruction.
Focusing was carried out on a 7.5% actylamide gel with ampholytes in a pH range
3.0–10.0, at 7W constant powers for 1.5 h at 101C. Broad pI kit (GE Healthcare,
Uppsala, Sweden) was used as isoelectric point (pI) markers. After the run, the part of
the gel with purified enzyme was divided into three parts. The first part of the gel was
silver stained. The second part of the gel was washed twice (5 min each) with distilled
water and then equilibrated with 50 mM Tris-HCl buffer, pH 8.0, twice (each 5 min
duration). Thereafter, the gel was overlaid with NC membrane and left under pressure
for 30 min. After capillary transfer of proteins, NC membrane was dipped into 2 mM
BApNA solution in 50 mM Tris HCl buffer, pH 8.0, and incubated at 301C for 10 min.
Zymogram detection of TLE was further performed using diazotization of
p-nitroaniline and derivatization with 1-naphtylamine as previously published for
leucyl aminopeptidase (Božić and Vujčić, 2005).
The third part of the gel was subjected to zymogram detection in situ. The gel was
washed twice (5 min each) with distilled water and then equilibrated with 50 mM
Tris-HCl buffer, pH 8.0, twice (each 5 min duration). Thereafter, the gel was dipped
into 2 mM BApNA solution in 50 mM Tris HCl buffer, pH 8.0, and incubated at 301C
for 10 min. The yellow colored part of the gel, obtained as a result of enzymatically
released p-nitroaniline, was excided and used as a sample for 2D SDS PAGE.
The apparent molecular mass of TLE subunits was determined by 2D
electrophoresis, i.e., after SDS PAGE (second dimension) by comparison with
standards. A sample slice of IEF gel was soaked in reducing sample buffer (0.0625
M Tris, pH 6.8, 2% SDS, 5% beta-mercapthoethanol, 10% glycerol, and 0.002%
bromophenol blue) and heated for 3 min in a boiling water bath. Electrophoresis was
carried out according to Laemmli (1970) using 14% acrylamide. LMW-SDS marker kit
(GE Healthcare) was used as molecular mass standard. After the run, the gel was silver
stained.
The molecular mass of native TLE was determined by gel filtration on an FPLC
Superose 12 column (10 300 mm). The column was previously equilibrated with
0.9% NaCl, 20 mM acetate buffer, pH 6.0, at a flow rate of 36 mL/h. Fractions (150 mL)
were collected and assayed for TLE activity. The column was calibrated using bovine
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serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and
cytochrome C (13 kDa) (GE Healthcare).
pH Optimum Studies
To determine the pH optimum of TLE activity toward BApNA, 2 mL of the purified
enzyme and a series of 50-mM buffers in the pH range from 4.0 to 11.0 were used
[acetate, pH 4.0–6.5; phosphate I, pH 6.5–8.5; ethanolamine, pH 8.5–10.0; Tris-HCl,
pH 7.0–9.0; phosphate II, pH 10.0–11.0). Controls containing only substrate in
appropriate buffer were used to subtract the absorbance that was a result of the
substrate hydrolysis. This was the case with the highly alkaline buffers (pH410).
Temperature Optimum Studies
To determine the optimum temperature of TLE activity against BApNA, 2 ml of the
purified enzyme was assayed in 50 mM ethanolamine buffer, pH 9.0, in the
temperature range of 21 to 701C.
K M and V max Values
Initial reaction rates were determined using BApNA substrate in the concentration
range from 0.02 to 1.00 mM. Incubation mixtures contained 2 mL of purified enzyme
in 0.2 mL of 50 mM ethanolamine buffer, pH 9.0, and appropriate BApNA
concentration. Substrate hydrolysis was monitored at 410 nm at 30-sec intervals
during 20 min at 301C. The data were processed using non-linear regression analysis
by the GraphPad Prism 5.0 program (Božić et al., 2003).
Inhibitor Studies
The effects of benzamidine, Na-Tosyl-lysil-chloromethyl ketone (TLCK), Tosylphenylalanyl-chloromethyl ketone (TPCK), phenylmethylsulphonyl fluoride (PMSF),
Soybean trypsin inhibitor (SBTI), aprotinin, (2S,3S)-3-(N-(S)-1-[N-(4-guanidinobutyl)carbamoyl]3-methylbutylcarbamoyl)oxirane-2-carboxylic acid (E-64), and ethylenediaminetetraacetic acid (EDTA) on TLE activity were studied. The purified TLE was
preincubated with a different concentration of inhibitors for 20 min at 41C prior to
addition of BApNA (final substrate concentration was 1 mM in 50 mM ethanolamine
buffer, pH 9.0) or gelatin (final substrate concentration was 0.5% in 50 mM carbonatebicarbonate buffer, pH 9.0). The hydrolysis of BApNA was monitored at 410 nm at
30-sec intervals during 20 min at 301C. Gelatin hydrolysis was performed by
incubation with TLE for 2 h at 371C. After that, the reaction was terminated by
adding 0.5 ml of 15% trichloroacetic acid (TCA). The supernatants were used to
determine the content of free amino groups, using the trinitrobenzenesulfonic acid
(TNBS) method (Kwan et al., 1983). Briefly, 0.2 ml of the sample were mixed with
0.9 ml of 0.2 M borate buffer, pH 9.2, and 0.5 ml of 4 mM TNBS. The reaction was
stopped after 30 min by adding 0.5 ml of 2 M NaH2PO4 and 18 mM Na2SO3, and A420
was measured. KI and Ic50 values were calculated using the GraphPad Prism
5.0 program.
Effect of Divalent Metal Cations
CaCl2, CuCl2, ZnSO4, CdCl2, MgSO4, MnCl2, NiSO4, HgCl2, and CoCl2 were used as
the sources of divalent metal cations. Each cation (final concentration were 0.2 and
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Trypsin-Like Enzyme from Morimus funereus Larvae
237
2.5 mM) was first incubated with TLE at 301C for 10 min before BApNA was added to
the reaction mixture. The residual TLE activity was monitored at 410 nm at 30-sec
intervals during 20 min at 301C.
Protein Substrate Hydrolysis
The ability of TLE to hydrolyze protein substrates was tested using gelatin, casein,
bovine serum albumin (BSA), and hemoglobin. Each substrate (final substrate
concentration was 1% in 50 mM carbonate-bicarbonate buffer, pH 10.0) was incubated
with 10 mL of TLE for 2 h at 371C. After that, the reaction was terminated by adding
0.5 ml of 15% TCA. The supernatants were used to determine the content of free
amino groups, using the TNBS method as described above.
Statistical Analysis
Each data point represents the mean of three independent assays. The data (see
Figs. 3–5 and Tables 2 and 3) are presented as the mean7standard error of the mean
(SEM). The data (see Tables 2 and 3) are presented as percentages, taking the control
value as 100%.
RESULTS
Purification of TLE
The purification of TLE from the anterior section of the midgut of M. funereus larvae
was monitored by the ability of TLE to hydrolyze the substrate BApNA. The
purification procedure consisted of two steps: Ion-exchange chromatography and
subsequent gel filtration on Superose 12 column. The results of the purification are
summarized in Table 1 and Figure 1. Trypsin-like enzyme was purified 322-fold with a
yield of 42% and was homogenous according to IEF (Fig. 1, Lane 3) and 2D
electrophoresis (Fig. 2, Lane 1). The enzymatic purity was confirmed by in-gel activity
assay after IEF with a subsequent detection of enzymatic activity band (Fig. 1, Lane 4).
Characteristics of the TLE
Activity of TLE was detected after isoelectric focusing and in-gel activity staining in the
alkaline region (Fig. 1). After comparing with standard protein pI values, the pI for
TLE was over 9.3.
The apparent MM of the enzyme was estimated to be 38 kDa according to 2D SDSPAGE (Fig. 2). The molecular mass of purified TLE rechromatographed on the FPLC
Superose 12 was calculated from the plot of log MM versus Kav using standard
Table 1. Purification of Major TLE from the Anterior Section of Midgut of M. funereus Larvae
Purification stage
Crude midgut extract
Q-Sepharose FF
FPLC Superose 12
Total protein
(mg)
Total activity
(U)
Specific activity
(U/mg)
Purification
(-fold)
Yield
(%)
38.07
0.33
0.05
4.96
3.48
2.09
0.13
10.55
41.80
1
81
322
100
70
42
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Figure 1. Purification of M. funereus TLE (IEF profiles). Lane 1: Crude extract of anterior section of
midgut. Lane 2: Proteins after ion-exchange chromatography on Q-Sepharose FF. Lane 3: Purified TLE
obtained after FPLC Superose 12 gel filtration. Lane 4: In-gel activity staining. BApNA was used as a
substrate for in gel activity staining. Lane pI: Positions of standard proteins pI values. Arrow indicates
positions of the band referred to TLE.
Figure 2. 2D electrophoresis of purified M. funereus midgut TLE. Lane 1: Purified TLE. Lane 2: Standard
proteins (LMW-SDS marker kit). Lane kDa: Molecular masses of standard proteins.
proteins as markers after elution from a Superose 12 FPLC column (results not
shown). The molecular mass of TLE was estimated to be 37.5 kDa. These data suggest
that the major M. funereus TLE is a monomer.
Amongst the number of p-nitroanilide derivatives tested, activity of TLE
was detected only toward specific substrate for trypsin-like enzyme, BApNA, while
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239
against specific substrates for chymotrypsin-like and elastase-like enzymes, SFpNA,
GFpNA, BTpNA, SA3pNA, and SA2PpNA, no activity was observed (results not
shown).
Optimum TLE activity using BApNA as a substrate was observed between pH
8.5 and 9.5. Maximum TLE activity at pH 9.0 was observed in ethanolamine buffer
(Fig. 3A). No enzymatic activity was detected in buffers with a pH below pH 4.0 or
above 11.0.
Temperature-dependant TLE activity was determined within a broad range (21 to
701C). TLE showed maximal activity at 451C and was completely denatured at 701C
(Fig. 3B).
Reaction kinetics of TLE using BApNA as a substrate was according to MichaelisMenten principles. The calculated KM value was 0.065 mM and the calculated Vmax
value was 0.261 mmol min 1 mL 1. The KM/Vmax ratio was 4.02.
We found that the most efficient inhibitor of TLE activity was benzamidine
(KI value of 0.012 mM, Ic50 value of 0.204 mM) (Fig. 4), which, in the concentration of
5 mM, inhibited around 90% of TLE activity against BApNA as a substrate (Table 2).
SBTI was also a reasonable inhibitor decreasing the TLE activity by 60% with gelatin as
a substrate. TLCK and PMSF inhibited 25 and 30% of TLE activity, respectively, while
TPCK reduced 12% of TLE activity when gelatin was used as a substrate. On the other
hand, aprotinin, as well as EDTA and E-64, had no effect on TLE activity. However,
some of the inhibitors (SBTI, TPCK, and aprotinin) increased the activity of TLE
when BApNA was used as substrate.
Amongst the divalent cation tested, 2.5 mM Hg21 completely inhibited TLE
activity (Fig. 5) while Cu21 was also a reasonable inhibitor. Higher concentrations of
other cations, especially Mn21 and Co21, were activators of TLE activity. Co21 and
Mn21 (2.5 mM) enhanced the TLE activity for about 2.5 times.
The best protein substrate for TLE amongst those tested was gelatin (Table 3). The
ability of TLE to hydrolyze different proteins was tested at pH values from 7.0 to 10.0.
Maximum activity against all protein substrates used was at pH 10.0 (results not
shown). Compared to gelatin, hydrolysis of casein at the same reaction conditions was
45% while the hydrolysis of BSA and hemoglobin was 10 to 15 times slower.
Figure 3. A: Effect of pH on M. funereus TLE activity. Buffers used: & , acetate; ., phosphate I; ,
ethanolamine; m, Tris HCl; b, phosphate II. B: Effect of temperature on M. funereus TLE activity. Each
data point represents the mean of three independent assays (the standard errors were less than 5% of
the means).
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Figure 4. Dose-dependent inhibition of M. funereus TLE activity by benzamidine. BApNA was used a
substrate. KI and Ic50 values were calculated using the GraphPad Prism 5.0 program. Each data point
represents the mean of three independent assays (the standard errors were less than 5% of the means).
Table 2. The Effect of Inhibitors on M. funereus Midgut TLE Activity
Inhibitor
Benzamidine
SBTI
PMSF
TLCK
TPCK
Aprotinin
E-64
EDTA
Final
concentration
Residual TLE activity
against BApNA (%)
Residual TLE activity
against gelatin (%)
5 mM
5 mM
10 mM
0.1 mM
0.1 mM
5 mM
10 mM
5 mM
8.370.1
239.873.1
74.771.5
81.670.9
156.672.3
270.373.9
96.571.2
93.871.2
15.770.8
39.972.2
70.770.7
75.370.4
87.970.1
93.172.8
94.571.7
97.871.3
BApNA and gelatin were used as substrates. Each value represents the mean of three independent assays
7standard error value (the standard errors were less than 5% of the means).
DISCUSSION
Peptidases are the major digestive enzymes in the insect gut. They are responsible for a
continuous supply of amino acids and energy from the food source for development.
Although serine peptidases of the elastase type are the major digestive endopeptidase
in the midgut of M. funereus larvae (Božić et al., 2003), trypsin-like enzymes are the
major digestive endopeptidases in the anterior midgut of M. funereus larva. Its
dominant status in the anterior section of midgut is assumed from its high level of
activity detected relative to other endopeptidase activities (Lončar et al., 2009). In
Coleoptera species, unlike in some other insect orders, distribution of TLE through
the midgut varies among the species. In Rhyzopertha dominica, more BApNAase activity
was also located in the anterior region compared to that in the posterior region of
the midgut (Zhu and Baker, 1999); in Tenebrio molitor larvae, TLE was found in
the posterior midgut (Nation, 2002) while in rice water weevil, TLE was evenly
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241
Figure 5. The effect of metal divalent cations on M. funereus midgut TLE. BApNA was used a substrate.
Each bar represents the mean of three independent assays (the standard errors were less than 5% of the
means for lower and less than 10% of the means for higher cation concentrations).
Table 3. Hydrolysis of Different Protein Substrates by M. funereus TLE
Substrate
Gelatin
Casein
BSA
Hemoglobin
Hydrolysis rate (%)
100.071.5
45.371.2
11.170.8
6.070.6
Hydrolysis of proteins was presented as a percentage in relation to gelatin hydrolysis, which was taken as 100%
after treatment with TLE for 2 h at 371C. Each value represents the mean of three independent assays7standard
error value (the standard errors were less than 5% of the means).
distributed among the anterior, middle, and posterior portions of the gut (Hernández
et al., 2003).
We have purified to homogeneity and characterized a novel cationic form of
trypsin-like activity from the anterior section of midgut of M. funereus larvae. Activity of
the purified enzyme was monitored in vitro using a chromogenic, synthetic substrate
L-BApNA. Separation of M. funereus TLE was achieved after the crude enzyme extract
was subjected to Q Sepharose ion exchange chromatography. Thereafter, a major
isoform was purified by gel filtration on a Superose 12 column. Using this procedure,
the TLE was homogenous according to 2D-PAGE, IEF, and in-gel activity determination.
Most insect midgut trypsin-like MM values are in the range 20 to 35 kDa (Terra
and Ferreira, 1994). In terms of molecular masses M. funereus TLE bears resemblance
to other insect midgut trypsin-like enzymes and compared with other coleopteran
species it is most similar to Pyrearinus termitilluminans TLE with a MM of 39 kDa
(Colepicolo-Neto et al., 1986). Since the apparent MM of M. funereus TLE was 38 kDa
and of enzyme native form was 37.5 kDa, it is concluded that the M. funereus TLE is
a monomer.
Although pI values for most of the insect midgut trypsin-like enzymes are in the
range 4–5 (Terra and Ferreira, 1994), several TLEs displaying pI values higher than 8,
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Archives of Insect Biochemistry and Physiology, August 2010
like TLE from M. funereus larvae, have also been reported (Levinsky et al., 1977; Milne
and Kaplan, 1993).
The purified TLE exhibited optimal activity around pH 9.0, as is typical for insect
digestive serine peptidases (Christeller et al., 1989; Terra and Ferreira, 1994; Lam
et al., 2000). However, in our previous work we showed that the pH in the lumen of
the anterior midgut of M. funereus larvae was 5.5–6.0 (Lončar et al., 2009). The
physiological meaning of this is yet to be determined. It is possible that TLE may be
synthesized in the anterior midgut but is activated only when it is translocated with the
food bolus to the alkaline pH of the middle or posterior midgut, similar to T. molitor
larvae (Vinokurov et al., 2006). On the other hand, the existence of anterior midgut
compartments, with the local pH values more alkaline than the average pH, cannot be
excluded.
The optimal temperature for TLE activity was 451C. Similar temperature optima,
from 401 to 451C, have been reported for other M. funereus midgut enzymes purified
and characterized so far, leucyl aminopeptidase (Božić et al., 2003) and a-amylase
(Dojnov et al., 2008).
The M. funereus TLE KM value of 0.065 mM using BApNA as a substrate was more
similar to the value 0.04 mM reported for trypsin-like enzymes isolated from T. molitor
(Tsybina et al., 2005) than to that (0.37 mM) reported for Costelytra zealandica
(Christeller et al., 1989).
Many general serine-peptidase inhibitors including PMSF inhibit trypsin, but a
greater specificity for enzymes with trypsin-like activities, is shown by leupeptin,
benzamidine, and TLCK as well as with protein inhibitors such as SBTI and aprotinin
(Barrett et al., 1998). This is in agreement with M. funereus midgut TLE with the
exception of TLCK. TLE activity was more affected by benzamidine and SBTI than by
PMSF and TLCK. Examples of BApNAse activities that were also less affected by PMSF
and TLCK can be found for coleopteran species T. molitor and Rhynchophorus ferrugineus
(Thie and Houseman, 1990; Alarcón et al., 2002). After inhibition of T. molitor
BApNAse by 0.5 mM TLCK and 5 mM PMSF, residual enzyme activities were 81 and
68%, respectively, while for R. ferrugineus BApNAse 82 and 39% of activities were
retained.
The purified M. funereus TLE was most sensitive to inhibition by benzamidine.
KI obtained was the same as KI for TLE from larvae of Ostrinia nubilalis (Bernardi et al.,
1996). TLE was not completely inhibited by trypsin-specific plant inhibitor SBTI.
Although this is not very common with most of the TLE isolated from insects, a similar
occurrence of limited sensitivity to SBTI (50% of trypsin-like activity was inhibited) was
found for TLE from the midgut of Anoplophora glabripennis, also a member of the
cerambycid family (Bian et al., 1996) and for BApNAse activity in gut extract from
coleopteran larvae Lissorhoptrus brevirostris Suffrian (Hernández et al., 2003).
Despite apparent similarities among insect trypsin subsites in the active site, their
preferences between polar and hydrophobic residues are markedly different.
Comparing their hydrophobicities, it was recently shown that all subsite hydrophobicities increase from less to more evolved insects (Lopes et al., 2006). The authors
explained the evolutionary meaning of this trend as an adaptation of efficient plant
feeders to decrease the impairment caused by ingesting plant material rich in trypsin
inhibitors. The plant trypsin inhibitors have at their active sites hydrophylic sequences
that do not bind well to the hydrophobic subsites of trypsin from more evolved insects.
Regarding subsite hydrophobicity, modeling showed that there are several amino acid
replacements that would affect the nature of the interactions between insect trypsins
Archives of Insect Biochemistry and Physiology
Trypsin-Like Enzyme from Morimus funereus Larvae
243
and SBTI. This may be the case with M. funereus TLE as well. The fact that 0.5M NaCl
enhanced TLE activity against BApNA 2 times (results not shown), supports the thesis
that hydrophobic interactions play important roles in substrate hydrolysis by
M. funereus TLE.
Plant proteinase inhibitors (PIs) are common, natural plant products that can
inhibit midgut endopeptidases from herbivorous insects. Some species overcome the
effect of ingested PIs by altering the complement of trypsins in their midgut following
ingestion of PI such that the majority of trypsin activity originates from proteins that
are not susceptible to PIs (Mazumdar-Leighton and Broadway, 2001). Insensitivity to
inhibition by aprotinin and partial insensitivity to inhibition by SBTI may suggest that
M. funereus larvae poses as an inherently resistant trypsin-like enzyme to a bulky
inhibitor as a result of surface incomplementarity. This might explain why SBTI was
an inhibitor of TLE when gelatin, a large protein substrate, was used for hydrolysis,
while it was not an inhibitor when the small substrate BApNA was used. It is very
interesting to point out here that in Diptera, the arrangement of the midgut keeps
trypsin PIs ingested with food in the anterior, away from the site of protein digestion,
which occurs in the posterior midgut (Nation, 2002). Hence, the fact that M. funereus
TLE occurs in the anterior and is insensitive to some PIs might be explained as an
evolutionary selective pressure.
Trypsin-like activity has been increased with the presence of 2.5 mM Ca21, Mg21,
and Zn21 by almost 50%. Our data are in contrast to results from other insect trypsins
that were unaffected by Ca21 (Levinsky et al., 1977; Johnston et al., 1991). It is not very
common to study the effect of divalent cations on enzymes that do not belong to the class
of metallopeptidases. Nevertheless, it can be of physiological importance, keeping in
mind that the food consists of macro- and microelements beside the nutritive monomers
and polymers. Co21 (2.5 mM) activated TLE, enhancing the enzyme activity by almost
150%. In one previous study where the influence of various trace elements on tryptic
hydrolysis was examined, cobalt accelerated the enzyme’s capacity for hydrolysis at all
concentrations used (Wieninger-Rustemeyer et al., 1980). In another study Ca21, Mg21,
Mn21, Co21, and Cd21 were activators, while Hg21 and Cu21 were inhibitors of trypsin
(Green and Neurath, 1953) as was the case in this study as well.
Interesting results related to the discovery of the serine-peptidase member,
functionally different from those found in other insect species, have arisen from this
study. M. funereus TLE was partially inhibited by SBTI, even less by TLCK and PMSF,
almost completely inhibited by benzamidine, and was insensitive to aprotinin while a
broad range of divalent ions, including calcium, enhanced enzyme activity. The fact
that M. funereus TLE differs from other insect TLEs with respect to their behaviour in
the presence of inhibitors justifies the necessity to study the properties of specific insect
digestive enzymes. Future studies concerning M. funereus TLE with particular
emphasis on enzyme compartmentalisation, together with other purified enzymes
from M. funereus (Božić et al., 2008; Dojnov et al., 2008), will make our understanding
of the digestive processes within this polyphagous Cerambycid beetle possible.
ACKNOWLEDGMENTS
This work was supported by the Serbian Ministry of Science and Technological
Development (project grant number 142026B).
Archives of Insect Biochemistry and Physiology
244
Archives of Insect Biochemistry and Physiology, August 2010
LITERATURE CITED
Alarcón FJ, Martinez TF, Barranco P, Cabello T, Diaz M, Moyano FJ. 2002. Digestive proteases
during development of larvae of red palm weevil, Rhynchophorus ferrugineus (Olivier, 1790)
(Coleoptera: Curculionidae). Insect Biochem Mol Biol 32:265–274.
Barrett AJ, Rawlings ND, Woessner JF. 1998. Serine and threonine peptidases. In: Barrett AJ,
Rawlings ND, Woessner JF, editors. Handbook of proteolytic enzymes. New York: Academic
press. p 1–22.
Bernardi R, Tedeschi G, Ronchi S, Palmieri S. 1996. Isolation and some molecular properties of
a trypsin-like enzyme from larvae of European corn borer Ostrinia nubilalis Hübner
(Lepidoptera: Pyralidae). Insect Biochem Mol Biol 26:883–889.
Bian X, Shaw BD, Han Y, Christeller JT. 1996. Midgut proteinase activities in larvae of
Anoplophora glabripennis (Coleoptera: Cerambycidae) and their interaction with proteinase
inhibitors. Arch Insect Biochem Physiol 31:23–37.
Božić N, Vujčić Z. 2005. Detection and quantification of leucyl aminopeptidase after native
electrophoresis using leucine-p-nitroanilide. Electrophoresis 26:2476–2480.
Božić N, Vujčić Z, Nenadović V, Ivanović J. 2003. Partial purification and characterization of
midgut leucyl aminopeptidase of Morimus funereus (Coleoptera: Cerambycidae) larvae.
Comp Biochem Physiol 134B:231–241.
Božić N, Ivanović J, Nenadović V, Bergström J, Larsson T, Vujčić Z. 2008. Purification and
properties of major midgut leucyl aminopeptidase of Morimus funereus (Coleoptera,
Cerambycidae) larvae. Comp Biochem Physiol 149B:454–462.
Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:
248–254.
Christeller JT, Shaw BD, Gardiner SE, Dymock J. 1989. Partial purification and characterization
of the major protease of grass grub larvae (Costelytra zealandica, Coleoptera: Scarabaeidae).
Insect Biochem 19:221–231.
Colepicolo-Neto P, Bechara EJH, Ferreira C, Terra WR. 1986. Evolutionary consideration of the
spatial organization of digestion in the luminescent predaceous larvae of Pyrearinus
termitilluminans (Coleoptera: Elateridae). Insect Biochem 16:811–817.
Dojnov B, Božić N, Nenadović V, Ivanović J, Vujčić Z. 2008. Purification and properties of
midgut a-amylase isolated from Morimus funereus (Coleoptera: Cerambycidae) larvae. Comp
Biochem Physiol 149B:153–160.
ÐurXević A, Vujčić Z, Jankov RM, Nenadović V, Ivanović J. 1997. Trypsin-like enzymes from the
midgut of Morimus funereus larvae (Coleoptera: Cerambycidae). Arch Biol Sci Belgrade
49:19P–20P.
Erlanger BF, Kokowsky N, Cohen W. 1961. The preparation and properties of two new
chromogenic substrates of trypsin. Arch Biochem Biophys 95:271–278.
Gatehouse AMR, Hilder VA, Powell KS, Wang M, Davison GM, Gatehouse LN, Down RE,
Edmonds HS, Boulter D, Newell CA, Merryweather A, Hamilton WDO, Gatehouse JA.
1994. Insect-resistant transgenic plants: choosing the gene to do the ‘‘job.’’ Biochem Soc
Trans 22:944–949.
Green NM, Neurath H. 1953. The effects of divalent cations on trypsin. J Biol Chem
204:379–390.
Hernández CA, Pujol M, Alfonso-Rubı́ J, Armas R, Coll Y, Pérez M, González A, Ruiz M,
Castañera P, Ortego F . 2003. Proteolytic gut activities in the rice water weevil, Lissorhoptrus
brevirostris Suffrian (Coleoptera: Curculionidae). Arch Insect Biochem Physiol 53:19–29.
Houseman JG, Downe AER. 1983. Cathepsin D-like activity in the posterior midgut of
hemipteran insects. Comp Biochem Physiol 75B:509–512.
Archives of Insect Biochemistry and Physiology
Trypsin-Like Enzyme from Morimus funereus Larvae
245
Houseman JG, Morrison PE, Downe AER. 1985. Cathepsin B and aminopeptidase in the
posterior midgut of Phymata wolffii (Hemiptera: Phymatidae). Can J Zool 63:1288–1291.
Johnston KA, Lee MJ, Gatehouse JA, Anstee JH. 1991. The partial purification and
characterisation of serine protease activity in midgut of larval Helicoverpa armigera. Insect
Biochem 21:389–397.
Kwan KKH, Nakai S, Skura J. 1983. Comparison of four methods for determining protease
activity in Milk. J Food Sci 48:1418–1421.
Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 227:680–685.
Lam W, Coast GM, Rayne RC. 2000. Characterisation of multiple trypsins from the midgut of
Locusta migratoria. Insect Biochem Mol Biol 30:85–94.
Levinsky H, Birk Y, Applebaum SW. 1977. Isolation and characterization of a new trypsin-like
enzyme from Tenebrio molitor L. larvae. Int J Pept Prot Res 10:252–264.
Liu Y, Sui Y-P, Wang J-X, Zhao X-F. 2009. Characterization of the trypsin-like protease
(HA-TLP2) constitutively expressed in the integument of the cotton bollworm, Helicoverpa
armigera. Arch Insect Biochem Physiol 72:74–87.
Lončar N, Božić N, Nenadović V, Ivanović J, Vujčić Z. 2009. Characterization of trypsin-like
enzymes from the midgut of Morimus funereus (Coleoptera: Cerambycidae) larvae. Arch Biol
Sci Belgrade 61:713–718.
Lopes AR, Juliano MA, Juliano L, Terra WR. 2004. Coevolution of insect trypsin and inhibitors.
Arch Insect Biochem Physiol 55:140–152.
Lopes AR, Juliano MA, Marana SR, Juliano L, Terra WR. 2006. Substrate specificity of
insect trypsins and the role of their subsites in catalysis. Insect Biochem Mol Biol 36:
130–140.
Magalhães CP, Fragoso RR, Souza DSL, Barbosa AEAD, Silva CP, Finardi-Filho F, da Silva MCM,
Rocha TL, Franco OL, Grossi-de-Sa MF. 2007. Molecular and structural characterization of
a trypsin highly expressed in larval stage of Zabrotes subfasciatus. Arch Insect Biochem
Physiol 66:169–182.
Marchetti S, Chiaba C, Chiesa F, Bandiera A, Pitotti A. 1998. Isolation and partial
characterization of two trypsins from the larval midgut of Spodoptera littoralis (Boisduval).
Insect Biochem Mol Biol 28:449–458.
Mazumdar-Leighton S, Broadway RM. 2001. Transcriptional induction of diverse midgut
trypsins in larval Agrotis ipsilon and Helicoverpa zea feeding on the soybean trypsin inhibitor.
Insect Biochem Mol Biol 31:645–657.
Miller JW, Kramer KJ, Law JH. 1974. Isolation and partial characterization of the larval midgut
trypsin from the tobacco hornworm, Manduca sexta Johannson (Lepidoptera: Sphingidae).
Comp Biochem Physiol 48B:117–129.
Milne R, Kaplan H. 1993. Purification and characterization of a trypsin-like digestive enzyme
from spruce budworm (Choristoneura fumiferana) responsible for the activation of
d-endotoxin from Bacillus thuringiensis. Insect Biochem Mol Biol 23:663–673.
Muhlia-Almazán A, Sánchez-Paz A, Garcı́a-Carreño FL. 2008. Invertebrate trypsins: a review.
J Comp Physiol 178B:655–672.
Muller H-M, Crampton JM, Della Torre A, Sinden R, Crisanti A. 1993. Members of a trypsin
gene family in Anopheles gambiae are induced in the gut by blood meal. EMBO J
12:2891–2900.
Murdock LL, Brookhart G, Dunn PE, Foard DE, Kelly S, Kitch L, Shade RE, Shukle RH,
Wolfson JL. 1987. Cysteine digestive proteinases in Coleoptera. Comp Biochem Physiol
87B:783–787.
Nation JL. 2002. Digestion. In: Nation JL, editor. Insect biochemistry and physiology. Boca
Raton, Florida: CRC Press LLC. p 27–65.
Archives of Insect Biochemistry and Physiology
246
Archives of Insect Biochemistry and Physiology, August 2010
Roberts DB. 1986. Basic Drosophila care and techniques. In: Roberts DB, editor. Drosophila: a
practical approach. Oxford: IRL Press. p 15–19.
Terra WR, Ferreira C. 1994. Insect digestive enzymes: properties, compartmentalization and
function. Comp Biochem Physiol 109B:1–62.
Terra WR, Ferreira C, Jordão BP, Dillon RJ. 1996. Digestive enzymes. In: Lehane MJ,
Billingsley PF, editors. Biology of the insect midgut. London: Chapman & Hall. p 153–194.
Thie NMR, Houseman JG. 1990. Cysteine and serine proteolytic activities in larval midgut of
yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae). Insect Biochem
20:741–744.
Tsybina TA, Dunaevsky YE, Belozersky MA, Zhuzhikov DP, Oppert B, Elpidina EN. 2005.
Digestive proteinases of yellow mealworm (Tenebrio molitor) larvae: purification and
characterization of a trypsin-like proteinase. Biochemistry (Moscow) 70:300–305.
Vinokurov KS, Elpidina EN, Oppert B, Prabhakar S, Zhuzhikov DP, Dunaevsky YE,
Belozersky MA. 2006, Diversity of digestive proteinases in Tenebrio molitor (Coleoptera:
Tenebrionidae) larvae. Comp Biochem Physiol 145B:126–137.
Wieninger-Rustemeyer R, Kirchgessner M, Steinhart H. 1980. Influence of various trace
elements on tryptic hydrolysis. Nutr Metab 24:343–351.
Zhu Y-C, Baker JE. 1999. Characterization of midgut trypsin-like enzymes and three
trypsinogen cDNAs from the lesser grain borer, Rhyzopertha dominica (Coleoptera:
Bostrichidae). Insect Biochem Mol Biol 29:1053–1063.
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