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Insect chymotrypsinschloromethyl ketone inactivation and substrate specificity relative to possible coevolutional adaptation of insects and plants.

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A r t i c l e
INSECT CHYMOTRYPSINS:
CHLOROMETHYL KETONE
INACTIVATION AND SUBSTRATE
SPECIFICITY RELATIVE TO POSSIBLE
COEVOLUTIONAL ADAPTATION
OF INSECTS AND PLANTS
Adriana R. Lopes, Paloma M. Sato, and Walter R. Terra
Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de
São Paulo, São Paulo, Brazil
Insect digestive chymotrypsins are present in a large variety of insect
orders but their substrate specificity still remains unclear. Four insect
chymotrypsins from 3 different insect orders (Dictyoptera, Coleoptera,
and two Lepidoptera) were isolated using affinity chromatography.
Enzymes presented molecular masses in the range of 20 to 31 kDa and
pH optima in the range of 7.5 to 10.0. Kinetic characterization using
different colorimetric and fluorescent substrates indicated that insect
chymotrypsins differ from bovine chymotrypsin in their primary specificity
toward small substrates (like N-benzoyl-L-Tyr p-nitroanilide) rather than
on their preference for large substrates (exemplified by Succynil-Ala-AlaPro-Phe p-nitroanilide). Chloromethyl ketones (TPCK, N- a-tosyl-L-Phe
chloromethyl ketone and Z-GGF-CK, N- carbobenzoxy-Gly-Gly-Phe-CK)
inactivated all chymotrypsins tested. Inactivation rates follow apparent
first-order kinetics with variable second order rates (TPCK, 42 to
130 M 1 s 1; Z-GGF-CK, 150 to 450 M 1 s 1) that may be remarkably
low for S. frugiperda chymotrypsin (TPCK, 6 M 1 s 1; Z-GGF-CK,
Grant sponsor: FAPESP; Grant sponsor: CNPq (PRONEX program).
Abbreviations used: B-T-pNa, N-benzoyl-L-Tyr-p-nitroanilide; B-T-ee, benzoyl-Tyr-ethyl ester; DEPC, diethyl
pyrocarbonate; DMSO, dimethyl sulfoxide; PAGE, polyacrylamide gel electrophoresis; PBA, phenylbutyl amine;
SBTI, soybean trypsin inhibitor; Suc-AAF-MCA, succynil-Ala-Ala-Phe-7-amido-4-methyl coumarin; Suc-AAPFMCA, succynil-Ala-Ala-Pro-Phe-7-amido-4-methyl coumarin; Suc-LY-MCA, succynil-Leu-Tyr-7-amido-4-methyl
coumarin; TLCK, N-a-tosyl-L-lys-chloromethyl ketone; TPCK, N-a-tosyl-L-Phe- chloromethyl ketone; Z-GGFCK, N-carbobenzoxy-Gly-Gly-Phe-chloromethyl ketone.
Adriana R. Lopes’ present address is Instituto Butantan, Laboratory of Biochemistry and Biophysics, Av.
Vital Brasil, 1500, 05503-900, São Paulo, Brazil
Correspondence to: Walter R. Terra, Departamento de Bioquı́mica, Instituto de Quı́mica, Universidade de
São Paulo, C.P. 26077, 05513-970 São Paulo, Brazil. Fax: 155-11-3091-2186. E-mail: warterra@iq.usp.br
ARCHIVES OF INSECT BIOCHEMSTRY AND PHYSIOLOGY, Vol. 70, No. 3, 188–203 (2009)
Published online in Wiley InterScience (www.interscience.wiley.com).
& 2009 Wiley Periodicals, Inc. DOI: 10.1002/arch.20289
Insect Chymotrypsins
189
6.1 M 1 s 1). Homology modelling and sequence alignment showed that
in lepidopteran chymotrypsins, differences in the amino acid residues in
the neighborhood of the catalytic His 57 may affect its pKa value. This is
proposed as the cause of the decrease in His 57 reactivity toward
chloromethyl ketones. Such amino acid replacement in the active site is
C 2009
proposed to be an adaptation to the presence of dietary ketones. Wiley Periodicals, Inc.
Keywords: chymotrypsin; substrate specificity; protein digestion; chloromethyl ketones; coevolution
INTRODUCTION
Chymotrypsin (E.C.3.4.21.1) preferentially cleaves protein chains at the carboxyl side
of aromatic amino acids. Insect chymotrypsins usually have molecular masses of
20–30 kDa and pH optima of 8–11, and they differ from their vertebrate counterparts
in their instability at acidic pH, inhibition pattern with SBTI (Terra and Ferreira,
1994), and, finally, in reacting with TPCK (see below).
Chymotrypsins, like trypsins, are widespread among insects. They have been
described in Diptera (Shukle et al., 1985; Vizioli et al., 2001; Ramalho-Ortigão et al.,
2003), Hymenoptera (Jany et al., 1983; Whitworth et al., 1998), Orthoptera (Sakal et al.,
1988; Lam et al., 1999), Dictyoptera (Baumann, 1990), Coleoptera (Girard and Jouanin,
1999; Oppert et al., 2002; Alarcón et al., 2002; Hernández et al., 2003; Oliveira Netto
et al., 2004; Elpidina et al., 2005), and Lepidoptera (Pritchett et al., 1981; Houseman and
Philogène, 1989; Houseman and Chin, 1995; Hegedus et al., 2003; Volpicella et al.,
2006). Chymotrypsin activity and genes were also identified in Hemiptera salivary glands
(Colebatch et al., 2001).The earlier failure to detect chymotrypsin activity in insect
midguts (especially among lepidopterans) was a consequence of using mammalian
chymotrypsin substrates, like B-T-pNA or B-T-ee, in the assays. Lee and Anstee (1995)
proposed that insect chymotrypsins have an extended active site justifying the need of
larger substrates, like Suc-AAPF-pNA, for their detection.
Chloromethyl ketones like TPCK alkylate His 57 (bovine chymotrypsin numbering), which is part of the catalytic triad of serine proteinases (Barrett et al., 2004). In
accordance with this, TPCK inactivation (in conditions that are effective for bovine
chymotrypsin) have been described for chymotrypsins from insects belonging to
different orders such as: Orthoptera (Locusta migratoria, Sakal et al., 1988), Coleoptera
(Lissorhopterus brevirostris, Hernández et al., 2003), and Hymenoptera (Solenopsis invicta,
Whitworth et al., 1998). Data for lepidopteran chymotrypsins were more controversial.
Whereas TPCK inactivation was reported for Ostrinia nubilalis (Houseman and
Philogène, 1989) and Galleria mellonella (Hamed and Attias, 1987), no effect was
observed in the case of Heliothis virescens (Johnston et al., 1995), Spodoptera littoralis (Lee
and Anstee, 1995), Heliothis zea (Mazumdar-Leighton and Broadway, 2001),
Choristoneura fumiferana (Valaitis et al., 1999), Manduca sexta (Peterson et al., 1995),
and Bombyx mori (Kotani et al., 1999).
This report describes the purification of chymotrypsins from 4 insects pertaining
to 3 different orders and shows that insect chymotrypsins differ from mammalian ones
in their primary specificity toward small substrates rather than in their preference for
large substrates. Furthermore, the differential reactivity toward TPCK and DEPC
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Archives of Insect Biochemstry and Physiology, March 2009
observed among lepidopteran chymotrypsins was attributed to amino acid replacements in the neighbourhood of His 57 affecting its pKa.
MATERIALS AND METHODS
Animals
Periplaneta americana (Dictyoptera) were laboratory-reared, feeding on chayote (Sechium
sp.) and oats (Avena sativa) and maintained under a natural photoregime at room
temperature. Adult males were used in the experiments. Males were used because they
fed routinely, did not lay eggs (that may affect results), and have similar sizes.
Stock cultures of the yellow mealworm, Tenebrio molitor L. (Coleoptera), were
cultured under natural photoregime conditions on wheat bran at 24–261C and a
relative humidity of 70–75%. Fully grown larvae (each weighing about 0.12 g), having
midguts full of food, of both sexes were used.
Diatraea saccharalis were laboratory reared according to Parra and Mishfeldt
(1992). The larvae were contained in glass vials with the diet and maintained at a
temperature of 251C and a photoperiod of 14:10 h (photophase:scotophase). The diet
was composed of soybean meal (Glycine max), sucrose, wheat germ, a vitamin complex,
ascorbic acid, Wesson salts, choline chloride, agar, and microbial inhibitors. Adults
were fed a 10% honey solution. Fifth (last) instar larvae of both sexes were used in the
experiments.
Spodoptera frugiperda (Lepidoptera: Noctuidae) were laboratory reared according
to Parra (1986). The larvae were individually contained in glass vials with a diet based
on kidney bean (Phaseolus vulgaris), wheat germ, yeast, and agar and were maintained
under a natural photoregime at 251C. Fifth (last) instar larvae of both sexes were
used in the experiments.
Chemicals
Inhibitors, substrates, DMSO (see Abbreviations footnote), and bovine chymotrypsin
were purchased from Sigma-Aldrich; buffer salts and detergents were obtained from
Calbiochem-Merck.
Enzyme Samples
P. americana adults were immobilized in a carbon dioxide chamber for 10 min. The
antennae, legs, and wings were removed, the insects placed on ice and then dissected
in cold 220 mM NaCl. Crop and midgut with their contents were isolated and
homogenized in double distilled water with the aid of a Potter-Elvehjem homogenizer
and centrifuged at 20,000 g for 30 min at 41C. The resulting supernatant was filtered in
glass wool and stored at -201C until use. Activities remain unchanged in this condition
for at least 6 months.
T. molitor larvae were immobilized on ice, after which they were dissected in cold
342 mM NaCl. After removal, the midgut was divided into three sections (anterior,
middle, and posterior) of identical length. The posterior section was homogenized and
processed like P. americana samples.
D. saccharalis and S. frugiperda larvae were immobilized by placing them on ice,
after which they were rinsed in water and blotted with filter paper. Their guts were
dissected in cold 125 mM NaCl, and the peritrophic membrane with contents was
Archives of Insect Biochemstry and Physiology
Insect Chymotrypsins
191
pulled apart. Midgut contents were homogenized and processed like P. americana
samples, in double distilled water with the aid of a Potter-Elvehjem homogenizer, and
centrifuged at 25,000 g for 30 min at 41C. The resulting supernatant was filtered in
glass wool and stored at 201C until use. Activities remain unchanged in this condition
for at least 6 months.
Hydrolase Assays Protein Determination
Routine assays of chymotrypsin were accomplished at 301C using as substrate 10 mM
Suc-AAF-MCA (initially solubilized in 1% DMSO) in 0.1 M Tris-HCl buffer, pH 8.5, or
0.83 mM Suc-AAPF-pNa in 1% DMSO and 0.1 M Tris-HCl buffer, pH 8.5. Methyl
coumarin and p-nitroaniline released were measured by the method of Alves et al.
(1996) and Erlanger et al. (1961), respectively. For this, fluorimetric assays used 10 mL
of enzyme samples in 1.0 mL of 10 mM of substrate in 0.1 M Tris-HCl, pH 8.5. Assays
were terminated with 200 mL of 30% acetic acid and fluorescence readings were
accomplished in a F2000 Hitachi Spectrofluorimeter (excitation 320 nm, emission
420). Colorimetric assays used 50 mL of enzyme sample added to 250 mL of the same
buffer as before. Assays were terminated with 50 mL of 30% acetic acid and absorbance
readings were carried out in an Autofill Pharmacia Spectrophotometer at 410 nm.
The same conditions, unless otherwise specified, were used for the other substrates.
In each determination, incubations were continued for at least four different periods of
time and the initial rates were calculated. All assays were performed so that the
measured activity was proportional to protein and to incubation time, as established in
control experiments. Controls without enzyme or without substrate were included. One
unit of enzyme (U) is defined as the amount that hydrolyses 1 mmol of substrate/min.
Protein was determined according to Bradford (1976), using ovalbumin as a
standard.
Chymotrypsin Purification
Enzyme samples from P. americana, T. molitor, D. saccharalis, and S. frugiperda were
applied onto an affinity (phenyl-butylamine-agarose, Pharmacia Biotech) column
(1.0 4.0 cm) equilibrated with 20 mM Tris-HCl buffer, pH 8.0, containing 0.2 M NaCl
and run in a FPLC System (Pharmacia Biotech). After application, samples were left
for 15 min at room temperature in order to increase enzyme-inhibitor interactions (see
details in Barrett et al., 2004). Afterwards, the column was washed with 20 mL of
20 mM Tris-HCl buffer, pH 8.0, containing 0.2 M NaCl, to remove nonspecifically
bound protein, followed by elution with 25 mL of equilibrating buffer with 0.1 M
phenylbutylamine at a flow rate of 1.0 mL/min. Fractions of 1.0 mL were collected and
those containing chymotrypsin activity (see Hydrolase Assays Protein Determination
section) were pooled. Soon before kinetic determinations, this pool was applied onto a
Hitrap desalting column (GE Healthcare) to remove the inhibitor. This procedure is
necessary to prevent autolysis of the purified protein.
Sodium Dodecyl Sulfate (SDS)-Polyacrylamide Gel Electrophoresis (PAGE)
Samples with approximately 3 mg of protein were combined with sample buffer
containing 60 mM Tris-HCl buffer, pH 6.8, 2.5% SDS, 0.36 mM b-mercaptoethanol,
10% (v/v) glycerol, and 0.005% (w/v) bromophenol blue. The samples were heated for
5 min at 95oC in a water bath, before being loaded onto a 12% (w/v) polyacrylamide gel
slab containing 0.1% SDS (Laemmli, 1970). The gels were run at a constant voltage of
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200 V at room temperature and silver-stained for proteins (Blum et al., 1987).
Molecular masses were calculated using the following standards: lysozyme (14.4 kDa),
trypsin inhibitor (21.5 kDa), carbonic anydrase (31.0 kDa), ovoalbumin (45.0 kDa),
serum albumin (66.2 kDa), phosphorylase b (97.4 kDa).
CHEMICAL MODIFICATION STUDIES
Purified chymotrypsins from P. americana, T. molitor, D. saccharalis, and S. frugiperda and
the soluble portion of different insect midgut homogenates were incubated at 301C with
at least three different concentrations of TPCK or Z-GGP-CK (initially solubilized in
DMSO). Samples were collected at different periods of time (total of 30 or 330 min,
depending on the chymotrypsin studied) and the reaction was stopped by a hundredfold
dilution with 0.1 M Tris-HCl buffer, pH 8.5. The remaining activity was measured with
Suc-AAPF-MCA as substrate. The soluble portion of the midgut luminal contents from
D. saccharalis and S. frugiperda was also incubated at 301C with 1.0 mM DEPC (initially
solubilized in ethanol) in 0.1 M phosphate buffer, pH 6.4, in the presence or absence of
Suc-AAPF-pNa. Samples were collected at different periods of time (total of 60 min) and
the reaction was stopped by a 20-fold dilution with 100 mM imidazole buffer, pH 6.5.
This mixture was then diluted 5-fold in 0.1 M phosphate buffer, pH 8.0. The remaining
activity was measured with Suc-AAF-MCA as substrate. Calculations were performed
(means and SEM) with the help of the software Enzfitter (Elsevier, Biosoft).
Kinetic Studies
The effect of substrate concentration on chymotrypsin activity from different insects
was determined using at least 10 different substrate concentrations. Km and Vm
values (mean and SEM) were determined by linear regression using the software
Enzfitter (Elsevier, Biosoft).
The effect of pH on chymotrypsin activity was studied with different buffers
(100 mM) containing 0.2 M NaCl: Tris-HCl (pH 7–9), Gly-NaOH (pH 9–10.5), and
phosphate-NaOH (10.5–12).
Multiple Sequence Analysis and Homolog Modelling
All insect chymotrypsin sequences deposited at Genbank (www.ncbi.nlm.nih.gov) (up
until February 2008) were submitted to multiple sequence alignment performed with
ClustalX version 1.81 PPC (Thompson et al., 1999).
The 3-D structure of the chymotrypsin from Spodoptera frugiperda (GenBank access
number AY251276) was modeled using the Swiss-Model (Guex and Peitsch, 1997;
Schwede et al., 2003) and taking bovine chymotrypsinogen A (PDB number: 2CGA) as
a template. The structure was visualized with Deep View (Swiss Pdb. Viewer).
RESULTS
Chymotrypsin Purification and Properties
Insect chymotrypsins undergo strong autolysis. Because of that, the partial purification
was carried out in a single chromatographic step using an affinity chromatographic
PBA-Sepharose column (Fig. 1). The active eluted fractions (substrate Suc-AAPFArchives of Insect Biochemstry and Physiology
Insect Chymotrypsins
193
MCA) were pooled and used in all subsequent experiments after passing through a
Hitrap desalting column to remove PBA and change buffer.
SDS-PAGE of P. americana partial purified chymotrypsin results in the resolution of
bands of 31 and 29 kDa, in addition to others with masses lower than 14 kDa (Fig. 2A).
The same result was obtained with T. molitor samples (Fig. 2B). In the case of
D. saccharalis, a single band of 27 kDa is observed (Fig. 2C), whereas for S. frugiperda
several bands are resolved in addition to those of masses around 27 kDa (Fig. 2D). The
bands of about 14 kDa become more visible if the samples were previously stored,
indicating that they result from autolysis. The other bands may correspond to multiple
chymotrypsins or to inactive proteins.
The pH optima of the purified chymotrypsins were: P. americana, 7.5; T. molitor,
8.0; and D. saccharalis, 9.8.
Substrate Specificity of Insect Chymotrypsins
Chymotrypsin substrate specificities were studied with supernatants of insect midgut
homogenates (Table 1). Vmax/Km values are the best parameters to compare the
efficiency with which the enzymes hydrolyze different substrates (Segel, 1975). As
A
1500
6000
Emission 460 nm ( )
Emission 460nm ( )
1300
1100
900
700
500
300
5000
4000
3000
2000
1000
0
0
10
20
30
fractions
40
50
C
7000
10
20
30
fractions
10
20
40
50
D
1400
Emission 460nm ( )
5000
4000
3000
2000
1000
0
0
1600
6000
Emission 460 nm ( )
B
7000
1200
1000
800
600
400
200
0
10
20
30
40
fractions
50
0
0
30
40
50
fractions
Figure 1. Affinity chromatographic purification of P. americana (A), T. molitor (B), D. saccharalis (C), and S.
frugiperda (D) digestive chymotrypsin. Chromatographies of the soluble fraction of midgut homogenates on
PBA-Agarose equilibrated with 0.02 M Tris-HCl buffer, pH 8.0, containing 0.2 M NaCl. Elution with the
equilibration buffer containing 0.1 M PBA. The arrows indicate elution start. Substrate used: Suc-AAPF-MCA.
Archives of Insect Biochemstry and Physiology
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Archives of Insect Biochemstry and Physiology, March 2009
Figure 2. SDS-PAGE of P. americana (A1), T. molitor (B1), D. saccharalis (C1), and S. frugiperda (D1) purified
digestive chymotrypsin. The resolved bands were silver stained. Lane M, molecular weight markers. The
major bands found in the purified preparations are indicated by arrows.
mentioned above, some of the studied insects may have more than one midgut
chymotrypsin. In spite of that, Lineweaver-Burk plots of enzyme activity versus substrate
concentration were rectilinear in all cases. This favors the proposal that from the kinetic
Archives of Insect Biochemstry and Physiology
Insect Chymotrypsins
195
Table 1. Substrate Specificity of Insect and Bovine Chymotrypsins
Source
Suc-F-pNA
Km
(mM)
P. americana
571
T. molitor
2.370.2
D. saccharalis
371
S. frugiperda 2.470.4
Bovine
180720
Suc-AAPF-pNA
Relative
Vmax/Km
0.8.10
7.3.10
3.6.10
4.6.10
6.10 3
3
3
3
3
Suc-AAF-MCA
Suc-LY-MCA
Km
(mM)
Relative
Vmax/Km
Km
(mM)
Relative
Vmax/Km
Km
(mM)
Relative
Vmax/Km
1.1070.4
0.3070.02
0.5070.03
0.2070.02
0.670.1
38
18
47.3
85
87
6.070.4
1071
1372
2375
100710
100
100
100
100
100
10.070.9
3272
2272
2573
80730
0.35
19.6
2.1
4.5
0.8
Insect samples are supernatants of centrifuged midgut homogenates. Bovine chymotrypsin was supplied by
Sigma-Aldrich. B-T-pNA was assayed with all samples, but was hydrolyzed only by bovine chymotrypsin with a
relative Vmax/Km of 1.1.
point of view there is a single chymotrypsin (or very similar chymotrypsins) in all studied
samples. Furthermore, kinetic data obtained with partially purified chymotrypsins from
P. americana, T. molitor, D. saccharalis, and S. frugiperda with Suc-LY-MCA as substrate were
nearly the same as the ones displayed in Table 1 (data not shown).
In contrast with bovine chymotrypsin, insect chymotrypsins were unable to
hydrolyze B-T-pNA, but like the former chymotrypsin, prefer longer substrates,
exemplified by Suc-AAF-MCA (Table 1). However, insect chymotrypsins differ in their
specificities. Thus, T. molitor chymotrypsin hydrolyzed Suc-LY-MCA and Suc-AAPFpNa with efficiencies (relative Vmax/Km) clearly different from the other chymotrypsins. The same is true for the hydrolyzing efficiencies of the lepidopteran
chymotrypsins on Suc-AAPF-pNA and Suc-LY-MCA.
Chymotrypsin Inactivation by Chloromethyl Ketones and DEPC
T. molitor chymotrypsin was inactivated according to apparent first-order kinetics for at
least 3 half-lives by the addiction of TPCK (Fig. 3) or Z-GGF-CK (Fig. 4). These
chloromethyl ketones specifically alkylate the catalytic His residue close to the reactive
site Ser of chymotrypsins (Barrett et al., 2004).
A plot of log (kobs, s 1) of TPCK modification against log [TPCK] (Fig. 3, inset), as
well as a plot of log (kobs,s 1) of Z-GGF-CK modification against log [Z-GGP-CK] (Fig. 4,
inset), result in a straight line with a slope (reaction order) in the range of 0.9 to 1.0.
These data indicate that there is a single chymotrypsin species (or chymotrypsins with
identical active sites) in the T. molitor midgut preparation. Similar data were obtained for
the other chymotrypsins studied, revealing that the other insects studied also have a
single kinectically recognizable midgut chymotrypsin (data not shown). The secondorder rate constants of the chloromethyl ketone inactivation of the insect chymotrypsins
are presented in Table 2. The rate constants are nearly the same whatever midgut
homogenates or partially purified enzymes were used (Table 2). This reinforces the
inference that there is a single recognizable midgut chymotrypsin in the insects studied.
Table 2 also shows that chloromethyl ketones react remarkably slower with the
chymotrypsins from S. frugiperda in comparison with the other chymotrypsins.
DEPC reacts with His residues (Miles, 1977), like chloromethyl ketones, but is a
smaller molecule. DEPC inactivates D. saccharalis chymotrypsin according to apparent
first-order kinetics and this inactivation is suppressed in the presence of excess SucArchives of Insect Biochemstry and Physiology
196
Archives of Insect Biochemstry and Physiology, March 2009
log kobs
remaining activity (%)
100
3
2
1
0
4.5
5
5.5
6
log [TPCK], M
10
0
10
20
30
Time, min
Figure 3. Inactivation of T. molitor chymotrypsin in the presence of 0 (); 2.5 mM (&); 7.5 mM (J); 10.0 mM
(~); and 15.0 mM ( & ) TPCK dissolved in DMSO and diluted to appropriate concentrations in 0.1 M TrisHCl buffer, pH 8.5. The inset shows a plot of log kobs (s 1) versus log of TPCK concentration. Suc-AAF-MCA
was used as substrate.
3
10
log kobs
remaining activity (%)
100
2
1
0
5
5.5
6
log [Z-GGF-CK], M
1
0
5
10
15
20
25
30
Time, min
Figure 4. Inactivation of T. molitor chymotrypsin in the presence of 0 (); 1.0 mM (&); 3.0 mM (J); 5.0 mM
(~) and 7.0 mM ( & ) Z-GGF-CK dissolved in DMSO and diluted to appropriate concentrations in 0.1 M TrisHCl buffer pH 8.5. The inset shows a plot of log kobs (s 1) versus log of Z-GGF-CK concentration. Suc-AAFMCA was used as substrate.
Table 2. Second Order Rate Constants (M
Z-GGF-CK
S
1
) of Chymotrypsin Inactivation by TPCK and
Midgut homogenates
Source
P. Americana
T. molitor
D. saccharalis
S. frugiperda
Bovine
1
TPCK
Z-GGF-CK
Purified
TPCK
4275
4573
130710
671
–
150710
150720
450760
6.170.6
620770a
3779
40710
8873
1071
80720
a
Purified bovine chymotrypsin.
AAPF-pNA, confirming that the modified His residue is in the active site (Fig. 5A).
Otherwise, S. frugiperda chymotrypsin is not affected by DEPC (Fig. 5B).
Homology modeling of the S. frugiperda sequence deposited in GenBank
(accession number AY251276) showed that the following residues are in the
Archives of Insect Biochemstry and Physiology
Insect Chymotrypsins
197
remaining activity (%)
A
100
10
1
0
15
30
45
60
Time, min
remaining activity (%)
B
100
10
1
0
15
30
Time, min
45
60
Figure 5. Inactivation of D. saccharalis and S. frugiperda chymotrypsin activity in the presence of DEPC. A:
Inactivation of D. saccharalis chymotrypsin activity, in 0 mM (&), 1.0 mM DEPC (), and 1.0 mM DEPC plus
0.25 mM Suc-AAPF-pNa (~). B: Inactivation of S. frugiperda chymotrypsin activity in 0 mM (&) and 1.0 mM
() DEPC. Suc-AAF-MCA was used as substrate.
neighborhood of the catalytic His 57 residue (bovine chymotrypsin numbering): 56,
58, 59, 102 (3Å for His 57); 55, 94, 195, 213, 214 (4Å from His 57); 90, 99, 103,196,
212, 215 (6Å form His 57). Residues 102 (Asp) and 195 (Ser) are catalytic residues that
with C58 and G196 are conserved in all chymotrypsins. A multiple alignment was
performed with 54 insect chymotrypsin sequences deposited in GenBank from 33
insects pertaining to 5 different orders, from which 34 representative sequences are
shown in Figure 6. Regarding bovine chymotrypsin, the enzyme from S. frugiperda
shows amino acid replacements (in bold in Fig. 6) near the catalytic His 57, as
previously described for the Solenopsis invicta enzyme (Botos et al., 2000). Some of
those replacements are characteristics of lepidopteran chymotrypsins and may be
responsible for the differential ketone reactivity of most of them. Best residue
candidates to explain low ketone reactivity of S. frugiperda chymotrypsin are: W59,
M90, V103, and T213.
DISCUSSION
Insect Chymotrypsin Substrate Specificity
The chymotrypsins from P. americana, T. molitor, D. saccharalis, and S. frugiperda were
purified in a single step by affinity chromatography to avoid extensive autolysis. The
molecular masses found are those usual for chymotrypsins (Terra and Ferreira, 1994)
Archives of Insect Biochemstry and Physiology
Figure 6. Alignment of insect and bovine chymotrypsin sequences (GenBank, 2008). D. saccharalis sequences are contigs found in a random sequencing of a cDNA
library prepared with D. saccharalis midguts (Silva et al., unpublished data). Residues are shown from 53 (bovine chymotrypsin numbering) through 216, which
include all those in the neighborhood (up to 6 Å) of the catalytic His 57. Residues in bold in the sequence of S. frugiperda are those around His 57 that may affect
chymotrypsin reactivity to methyl ketones.
198
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199
and the pH optima determined for the different chymotrypsins conform to what is
expected from the taxa they belong to (Terra and Ferreira, 1994).
The same purification procedure described above has been used in several
instances (Jany et al., 1974; Sakal et al., 1988; Peterson et al., 1995; Botos et al., 2000;
Muharsini et al., 2001), although there are reports regarding non-lepidopteran insects
in which a successful purification of chymotrypsins was attained by ion-exchange
chromatography (Terra and Ferreira, 2005). It is worth-mentioning that Elpidina et al.
(2005) purified a chymotrypsin from T. molitor midgut that differs from the one
described here in being stable during ion-exchange chromatography. This and TPCK
inactivation discussed below indicates that Elpidina et al.’s and our T. molitor
chymotrypsin are different.
The data presented here confirm that insect chymotrypsins prefer longer than
shorter substrates but the same is also true for bovine chymotrypsin. Perhaps the
proposal that chymotrypsins (insect but also mammalian ones) have an extended active
site (Lee and Anstee, 1995) is only part of a more general rule according to which
hydrolases acting on polymers and oligomers prefer longer rather than shorter
substrates. Examples are the enzymes hydrolyzing polymers such as amylases (Robyt
and French, 1967; Hiromi et al., 1973), b-1,3-glucanases (Genta et al., 2007), chitinases
(Aronson et al., 2003), and trypsin (Lopes and Terra, 2003) and the enzymes active on
oligomers like aminopeptidases (Ferreira and Terra, 1986) and b-glycosidases
(Ferreira et al., 2001). At least regarding small substrates, data in this report showed
that insect chymotrypsins prefer Phe at subsite P1 (nomenclature of Schechter and
Berger, 1967) and are almost inactive if Tyr is at that position. The reverse is true for
mammalian chymotrypsin. Thus, the past failure of chymotrypsin detection in insect
midguts apparently relies more on the differences of substrate specificities between
insect and mammalian chymotrypsins than on the size of the substrates employed.
Chymotrypsin Ketone Inactivation and Dietary Adaptation
Quantitative data on chloromethyl ketone inactivation reported here confirmed that
chymotrypsins from insects pertaining to orders other than Lepidoptera are readily
inactivated, whereas those from Lepidoptera vary.
Why does His 57 reactivity vary among lepidopteran chymotrypsins? The most
obvious answer is to attribute the cause to differences in the neighbourhood of His 57.
Actually, Peterson et al. (1995), referring to M. sexta chymotrypsin, suggested that its
His 57 would be in an environment different from that of bovine chymotrypsin, thus
explaining TPCK inactivation only at pH 9.75, instead of at the standard pH 7.5–8.5.
Homology modeling and multiple alignment of insect midgut chymotrypsin sequences
(Fig. 6) show that, regarding bovine chymotrypsin, those enzymes may have amino
acid replacements near the catalytic His 57. Most lepidopteran chymotrypsins
characteristically have amino acid replacements at the positions 59, 90, 103, and
213. Notice that 59 and 90 are the only residues around His 57 that differ between
D. saccharalis 1 and 2. One of those sequences may correspond to the TPCK-sensitive
D. saccharalis chymotrypsin found in midguts. D. saccharalis 1 should correspond to the
sensitive one because it lacks W59, which is typical of lepidopteran chymotrypsins,
including S. frugiperda.If that is true, residue 59 is the most important in affecting His
57 reactivity towards TPCK, at least among lepidopteran chymotrypsins. Thus, this
replacement may cause the differences found between D. saccharalis and S. frugiperda
chymotrypsins, regarding the reactivity with TPCK, Z-GGF-CK, and also DEPC.
Archives of Insect Biochemstry and Physiology
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Archives of Insect Biochemstry and Physiology, March 2009
These differences would result in the reduction of the access of substrates and
inhibitors to the active site due to steric hindrance. Another possibility is that changes
in the neighbourhood of His 57 affect its pKa value, thus decreasing His reactivity with
ketones and other reactants, as suggested by Peterson et al. (1995).
The putative His 57-pKa change may be an adaptation to the high pH found in
lepidopteran midgut contents or a protection against plant ketones the effects of which
may be mimicked by TPCK in vitro. For example, plant 2-cyclopentenones, which are
very prone to nucleophilic addition reactions (Spencer, 1988), may result from the
enzymatic hydrolysis of cyclopentenoid b-cyanogenic glucosides. Cyanogenic bglucosides are produced by more than 2,500 plant species (Zagrobelny et al., 2004)
and together with other toxic b-glucosides are present in concentrations as high as 0.5
to 1% of the organ weight (Spencer, 1988).
The presence of chymotrypsins with a different ketone sensibility might be the
result of selective pressures due to differences in food habit. There are Lepidoptera
species that feed on at least 10 different families of plants while others use a single
plant family as host (Robinson et al., 2001, 2002). In accordance with this point of view,
the chymotrypsins of S. frugiperda (a polyphagous species) and that of D. sacharalis (a
monophagous species) react slowly or rapidly with TPCK, respectively.
The proposed adaptation is a new example of the interplay between insects and
plants during their evolutionary arms race and deserves more attention through sitedirected mutagenesis of recombinant chymotrypsins, starting with the candidate
residues mentioned above. Besides that, a systematic study of subsite specificities in
insect chymotrypsin, as already done with trypsin (Lopes et al., 2006), showed
differences among insect species (Sato et al., 2008) and may help in evaluating the
chymotrypsin role in plant inhibitor resistance and in understanding the evolution of
insect chymotrypsins.
ACKNOWLEDGMENTS
We are indebted to Dr. C. Ferreira for helpful discussion. A.R. Lopes and P.M. Sato
were graduate fellows of FAPESP and W.R. Terra is a staff member of his department
and a research fellow of CNPq.
LITERATURE CITED
Alarcón FJ, Martı́nez TF, Barranco P., Cabello T, Dı́az M, Moyano F. 2002. Digestive proteases
during development of larvae of red palm weevil, Rhynchophorus ferrugineus (Olivier, 1790)
(Coleoptera: Curculionidae). Insect Biochem Mol Biol 32:265–274.
Alves LC, Almeida PC, Franzoni L, Juliano L, Juliano MA. 1996. Synthesis of Na-protected
aminoacyl 7-amino-4-methyl-coumarin amide by phosphorous oxychloride and preparation of specific fluorogenic substrates for papain. Peptide Res 9:92–96.
Aronson NN, Halloran BA, Alexyev MF, Amable L, Madura JD, Pasupulati L, Worth C, Van
Roey P. Family 18 chitinase-oligosaccharide of Serratia marcerans chitinase A. Biochem J
376:87–95.
Barrett AJ, Rawlings ND, Wosner J.F. 2004. Handbook of proteolytic enzymes. 2nd ed. London:
Elsevier Academic Press.
Baumann E. 1990. Isolation and partial characterization of a chymotrypsin-like endoprotease
from cockroach intestinal system. Insect Biochem 20:761–768.
Archives of Insect Biochemstry and Physiology
Insect Chymotrypsins
201
Blum H, Beier H, Gross HJ. 1987. Improved silver staining of plant proteins, RNA, and DNA in
polyacrlamide gels. Electrophoresis 8:93–99.
Botos I, Meyer E, Nguyen M, Swanson SM, Koomen JM, Russell DH, Meyer EF. 2000. The
structure of an insect chymotrypsin. J Mol Biol 298:895–901.
Bradford mM. 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.
Colebatch G, East P, Cooper P. 2001. Preliminary characterisation of digestive proteases of the
green mirid, Creontiades dilutus (Hemiptera: Miridae). Insect Biochem Mol Biol 31:415–423.
Elpidina EN, Tsybina TA, Dunaevsky YE, Belozersky M, Zhuzhikov DP, Oppert B. 2005. A
chymotrypsin-like proteinase from the midgut of Tenebrio molitor larvae. Biochimie 87:771–779.
Erlanger BF, Kokowsky N, Cohen W. 1961. The preparation and properties of two new
chromogenic substrates of trypsin. Arch Biochem Biophys 95:271–278.
Ferreira AHP, Marana SR, Terra WR, Ferreira C. 2001. Purification, molecular cloning, and
properties of a beta-glycosidase isolated from midgut lumen of Tenebrio molitor (Coleoptera)
larvae. Insect Biochem Mol Biol 31:1065–1076.
Ferreira C, Terra WR. 1986. Substrate specificity and binding loci for inhibitors in an
aminopeptidase purified from the plasma membrane of an insect (Rhynchosciara americana )
larva. Arch Biochem Biophys 244:478–485.
Genta FA, Dumont AF, Marana SR, Terra WR, Ferreira C. 2007. The interplay of processivity,
substrate inhibition and a secondary substrate binding site of na insect exo-b-1,3-glucanase.
Biochim Biophys Acta 1774:1079–1091.
Girard C, Jouanin L. 1999. Molecular cloning of cDNAs encoding a range of digestive enzymes
from a phytophagous beetle Phaedon cochleariae. Insect Biochem Mol Biol 29:1129–1142.
Guex N, Peitsch MC. 1997. Swiss-Model and the Swiss-Pdb Viewer: an environment for
comparative modeling. Electrophoresis 18:2714–2723.
Hamed MBB, Attias J. 1987. Isolation and characterization of two alkaline proteases of the
greater wax moth Galleria mellonella (L.). Insect Biochem 5:653–658.
Hegedus D, Baldwin D, O’Grady M, Braun L, Gleddie S, Sharpe A, Lydiate D, Erlandson M.
2003. Midgut proteases from Mamestra configurata (Lepidoptera: Noctuidae) larvae:
characterization, cDNA cloning, and expressed sequence tag analysis. Arch Insect Biochem
Physiol 53:30–47.
Hernández CA, Pujol M, Alfonso-Rubi J, Armas R, Coll Y, Perez 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.
Hiromi K, Nittta Y, Numata C, Ono S. 1973. Subsite affinities of glucoamylase: examination of
the validity of the subsite theory. Biochim Biophys Acta 302:362–375.
Houseman JG, Chin PS. 1995. Distribution of digestive proteinases in the alimentary tract of the
European corn borer Ostrinia nubilalis (Lepidoptera: Pyralidae). Arch Insect Biochem
Physiol 28:103–111.
Houseman JG, Philogène BJR. 1989. Partial characterization of proteinase activity in the larval
midgut of the European corn borer, Ostrinia nubilalis Hübner (Lepidoptera: Pyralidae). Can
J Zool 67:864–868.
Jany KD, Pfleider G, Molitori HP. 1974. Purification and some physical properties of a
chymotrypsin-like protease of larva of hornet, Vespa orientalis. Eur J Biochem 42:419–428.
Jany KD, Bekelar K, Pfleiderer G, Ishay J. 1983. Amino acid sequence of an insect chymotrypsin
from the larvae of the hornet, Vespa orientalis. Biochem Biophys Res Commun 110:1–7.
Johnston KA, Lee MJ, Brough C, Hilder V.A., Gatehouse AMR, Gethouse J A. 1995. Protease
activity in the larval midgut of Heliothis virescens: Evidence for trypsin and chymotrypsin-like
enzymes. Insect Biochem Mol Biol 25:375–383.
Archives of Insect Biochemstry and Physiology
202
Archives of Insect Biochemstry and Physiology, March 2009
Kotani E, Niwa T, Suga K, Sugimura Y, Oda K, Mori H, Furusawa T. 1999. Cloning and
sequencing of a cDNA for a highly basic protease from the digestive juice of the silkyworm,
Bombyx mori. Insect Mol Biol 8:299–304.
Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 227:680–685.
Lee MJ, Anstee JH. 1995. Endoproteases from the midgut of larval Spodoptera littoralis include a
chymotrypsin-like enzyme with an extended binding site. Insect Biochem Mol Biol
25:49–61.
Lopes AR, Terra WR. 2003. Purification, properties and substrate specificity of a digestive
trypsin from Periplaneta americana (Dictyoptera) adults. Insect Biochem Mol Biol
33:407–415.
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
Mazumdar-Leighton S, Broadway RM. 2001. Identification of six chymotrypsin cDNAs from
larval midguts of Helicoperva zea and Agrotis ipsilon feeding on the soybean (Kunitz) trypsin
inhibitor. Insect Biochem Mol Biol 31:633–644.
Miles EW. 1977. Modification of histidyl residues in proteins by diethylpyrocarbonate. Methods
Enzymol 47:431–442.
Muharsini S, Dalrymple B, Vuocolo T, Hamilton S, Willadsen P, Wijffels G. 2001.Biochemical
and molecular characterization of serine proteases from larvae of Chrysomya bezziana, the Old
World Screw worm fly. Insect Biochem Mol Biol 31:1029–1040.
Oliveira-Neto O B, Batista JAN, Rigden DJ, Fragoso RR, Silva RO, Gomes EA, Franco OL,
Dias SC, Cordeiro CMT, Monnerat RG, Grossi-de-Sá MF. 2004. A diverse family
of serine proteinase genes expressed in cotton boll weevil (Anthonomus grandis): implications
for the design of pest-resistant transgenic cotton plants. Insect Biochem Mol Biol 34:
903–918.
Oppert B, Hartzer K, Zuercher M. 2002. Digestive proteinases in Lasioderma serricorne
(Coleoptera: Anobiidae). Bull Entomol Res 92:331–336.
Parra JRP. 1986. Raising insects for pathogen studies. In: Alves SB, editor. Microbiological
Control of Insects. São Paulo: Editora Manole. p 348–373.
Parra JRP, Mishfeldt LJ. 1992. Comparison of artificial diets for rearing the sugarcane borer. In:
Anderson TE, Leppla NC, editors. Advances in insect rearing for research and pest
management. Boulder, CO: Westview Press.
Peterson AM, Fernando GJP, Wells MA. 1995. Purification, Characterization and cDNA
Sequence of an Alkaline Chymotrypsin from the Midgut of Manduca sexta. Insect Biochem
Mol Biol 25:765–774.
Pritchett DW, Young SY, Green CR. 1981. Proteolytic activity in the digestive fluid of larvae of
Trichplusia ni. Insect Biochem 11:523–526.
Ramalho-Ortigão JM, Kamhawi S, Rowton ED, Ribeiro JMC, Valenzuela JG. 2003. Cloning and
characterization of trypsin-and chymotrypsin-like proteases from the midgut of the sand fly
vector Phlebotomus papatasi. Insect Biochem Mol Biol 33:163–171.
Robinson GS, Ackery PR, Kitching IJ, Beccaloni GW, Hernandez LM. 2001. Hostplants of the
moth and butterfly caterpillars of the Oriental Region. Kuala Lumpur: Southdene Sdn Bhd.
744p.
Robinson GS, Ackery PR, Kitching IJ, Beccaloni GW, Hernandez LM. 2002. Hostplants of the
moth and butterfly caterpillars of America north of Mexico. Gainesville, FL: Memoirs of the
American Entomological Institute 69:1–824.
Robyt JF, French D. 1967. Multiple attack hypothesis of alpha-amylase action - action of porcine
pancreatic, human salivary, and Aspergillus oryzae alpha-amylases. Arch Biochem Biophys
122:8–16.
Archives of Insect Biochemstry and Physiology
Insect Chymotrypsins
203
Sakal E, Applebaum SW, Birk Y. 1988. Purification and characterization of Locusta migratoria
chymotrypsin. Int J Peptide Res 32:590–598.
Sato PM, Lopes AR, Juliano L, Juliano MA, Terra WR. 2008. Subsite substrate specificity of
midgut insect chymotrypsins. Insect Biochem Mol Biol 38:628–633.
Schechter I, Berger A. 1967. On the size of active site in proteases. Biochem Biophys Res
Commum 27:157–162.
Schwede T, Kopp J, Guex N, Peitsch MC. 2003. SWISS-MODEL: an automated protein
homology-modeling server. Nucl Acids Res 31:3381–3385.
Segel IH. 1975. Enzyme kinetics. New York: Wiley.
Shukle RH, Murdock LL, Gallun RL. 1985. Identification and partial characterization of a major
gut proteinase from larvae of the Hessian fly, Mayetiola destructor (Say) (Diptera:
Cecidomyidae). Insect Biochem 15:93–101.
Spencer KC. 1988. Chemical mediation of coevolution in the Passiflora-Heliconius interaction.
Chemical mediation of coevolution. New York: Academic Press, Inc. 609p.
Terra WR, Ferreira C. 1994. Insect digestive enzymes: properties, compartmentalization and
function. Comp Biochem Physiol 109B:1–62.
Terra WR, Ferreira C. 2005. Biochemistry of digestion. In: Gilbert LI, Iatrov K, Gill S, editors.
Comprehensive molecular insect science. vol. 4. New York: Oxford. p 171–224.
Thompson JD, Plewniak F, Poch O. 1999. A comprehensive comparison of multiple sequence
alignment programs. Nucleic Acids Res 27:2682–2690.
Valaitis A, Augustin S, Clancy K. 1999. Purification and characterization of the western spruce
budworm larval midgut proteinases and comparison of laboratory-reared and fieldcollected insects. Insect Biochem Mol Biol 29:405–415.
Vizioli J, Catteruccia F, della Torre A, Reckmann I, Muller HM. 2001. Blood digestion in the
malaria mosquito Anopheles gambiae: molecular cloning and biochemical characterization of
two inducible chymotrypsins. Eur J Biochem 268:4027–4035.
Volpicella M, Cordewener J, Jongsma MA, Gallerani R, Ceci lR, Beekwilder J. 2006.
Identification and characterization of digestive serine proteases from inhibitor-resistant
Helicoverpa zea larval midgut. J Chromatogr B 833:26–32.
Whitworth ST, Blum MS, James T. 1998. Proteolytic enzymes from larvae of the fire ant,
Solenopsis invicta. Isolation and characterization of four serine endopeptidases. J Biol Chem
273:14430–14434.
Wu YR, Llewellyn D, Mathews A, Dennis ES. 1997. Adaptation of Helicoverpa armigera
(Lepidoptera: Noctuidae) to a proteinase inhibitor expressed in transgenic tobacco. Mol
Breed 3:371–380.
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