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Specificity of an extracellular proteinase from Conidiobolus coronatus and its inhibition by an inhibitor from insect hemolymph.

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186
Bania et al.
Archives of Insect Biochemistry and Physiology 62:186�6 (2006)
Specificity of an Extracellular Proteinase From
Conidiobolus coronatus and Its Inhibition by an
Inhibitor From Insect Hemolymph
1
2
Jacek Bania, * Jaroslaw Samborski,
2
Mieczyslawa Bogus,
3
and Antoni Polanowski
The relatively little-investigated entomopathogen Conidiobolus coronatus secretes several proteinases into culture broth. Using
a combination of ion-exchange and size-exclusion chromatography, we purified to homogeneity a serine proteinase of Mr
30,000�,000, as ascertained by SDS-PAGE. The purified enzyme showed subtilisin-like activity. It very effectively hydrolyzed N-Suc-Ala2-Pro-Phe-pNa with a Km-1.36
10
�
�
M and Kcat-11 s
17,030s
�
M
�
�
10
�
M and Kcat-24 s
�
, and N-Suc-Ala2-Pro-Leu-pNa with Km-6.65
�
. The specificity index kcat/Km for the tested substrates was calculated to be 176,340s
M
�
�
and
, respectively. Using oxidized insulin B chain as a substrate, the purified proteinase exhibited specificity to
aromatic and hydrophobic amino-acid residues, such as Phe, Leu, and Gly at the P1 position, splitting primarily the peptide
1
2
bonds: Phe -Val , Leu
15
16
-Tyr
, and Gly
23
-Phe
24
. The proteinase appeared to be sensitive to the specific synthetic inhibitors of
the serine proteinases DFP (diisopropyl flourophosphate) and PMSF (phenyl-methylsulfonyl fluoride) as well as to some naturally occurring protein inhibitors of chymotrypsin. It is worth noting that the enzyme exhibited the highest sensitivity to inhibition by
AMCI-1 (with an association constant of 3
�
10
10
�
M
), an inhibitor of cathepsin G/chymotrypsin from the larval hemolymph of
Apis mellifera, reinforcing the possibility of involvement of inhibitors from hemolymph in insect innate immunity. The substrate
specificity and proteinase inhibitor effects indicate that the purified proteinase from the fermentation broth of Conidiobolus coronatus
is a subtilisin-like serine proteinase. Arch. Insect Biochem. Physiol. 62:186�6, 2006.
� 2006 Wiley-Liss, Inc.
KEYWORDS : Conidiobolus coronatus; proteinase; substrate specificity; proteinase inhibitor; insect hemolymph
INTRODUCTION
gal virulence (Bidochka and Khachatourians, 1994;
Cole et al., 1993; Gupta et al., 1992; Samuels and
Unlike other microbial pathogens, e.g., bacte-
Paterson, 1995; St Leger et al., 1987, 1988).
ria, rickettsiae, protozoa, and viruses, that invade
On the other hand, it has been known for a num-
insects after being ingested, fungi can infect insects
ber of years that insect hemolymph contains sev-
by direct penetration of the host integument.
eral different types of polypeptides that are able to
Entomopathogenic fungi produce an array of ex-
quench the catalytic function of proteolytic enzymes
tracellular digestive enzymes, such as proteinases,
(Eguchi, 1993; Polanowski et al., 1992; Polanowski
lipases, and chitinases, which are involved in de-
and Wilusz, 1996). Without a clear elucidation of
grading cuticular compounds. Among the enzymes
the specificity and mode of action of both fungal
taking part in breaching the cuticle, the protein-
extracellular proteinases and insect host proteinase
ases are believed to play a major role, and have,
inhibitors, we can never hope to have a complete
therefore, been consider a significant factor of fun-
understanding of the pathogenic processes.
1
2
3
Department of Food Hygiene and Consumer Protection, Faculty of Veterinary Medicine, Agricultural University of Wroclaw, Wroclaw, Poland
W. Stefanski Institute of Parasitology, Polish Academy of Sciences, Warsaw, Poland
Institute of Biochemistry and Molecular Biology, University of Wroclaw, Wroclaw, Poland
*Correspondence to: Jacek Bania, Department of Food Hygiene and Consumer Protection, Faculty of Veterinary Medicine, Agricultural University of Wroclaw,
Norwida 31, 50-375 Wroclaw, Poland. E-mail: bania@ozi.ar.wroc.pl
Received 1 October 2005; Accepted 6 February 2006
� 2006 Wiley-Liss, Inc.
DOI: 10.1002/arch.20134
Published online in Wiley InterScience (www.interscience.wiley.com)
Archives of Insect Biochemistry and Physiology
August 2006
doi: 10.1002/arch.
Specificity of an Extracellular Proteinase From C. coronatus
The relatively little-investigated entomopatho-
creas
was
purified
in
our
laboratory.
187
Purified
gen Conidiobolus coronatus secretes a range of pro-
Beauveria bassiana proteinase was kindly provided
teinases to culture broth (Phadatare et al., 1992;
by Dr. Maria Kolaczkowska. All other reagents used
Sutar et al., 1991; Tanksale et al., 2000). In our
were of analytical grade. Conidiobolus coronatus, iso-
studies, using a combination of ion-exchange and
late number 3491, originally isolated from Dendro-
size-exclusion chromatography, we have purified
laelaps spp., was obtained from the collection of
into homogeneity a serine proteinase of Mr 30,000�
Prof. Balazy (Polish Academy of Sciences, Research
32,000, as assessed by SDS-PAGE. The purified en-
Center for Agricultural and Forest Environment,
zyme exhibits subtilisin-like activity. We show that
Poznan), was routinely maintained in 90-mm Petri
two serine proteinases derived from the entomo-
dishes at 20 C with cyclic changes of light (LD12:12)
pathogens Conidiobolus
�
coronatus and Beauveria
on Sabouraud agar medium with the addition of
bassiana are strongly inhibited by an inhibitor from
homogenized G. mellonella larvae to a final con-
Apis mellifera hemolymph. This finding supports
centration of 10% wet weight.
an as yet little documented hypothesis about the
involvement of proteinase inhibitors from hemo-
Growth Conditions
lymph in insect innate immunity.
Conidiobolus coronatus was grown on liquid medium containing 0.1% (NH4)2SO4, 0.45% KH2PO4,
MATERIALS AND METHODS
1.05% K2HPO4, 0.05% sodium citrate dihydrate,
Chemicals and Entomopathogen Strains
0.2% glucose, and 0.025% MgSO4. The medium
was sterilized by autoclaving and its pH adjusted
Sephadex
G-75
was
from
Pharmacia
Chemicals, Uppsala, Sweden. Bovine
chymotrypsin,
subtilisin
Fine
b-trypsin, a-
Carlsberg,
leupeptin,
chymostatin, soybean trypsin inhibitor (STI), Bowman-Birk trypsin inhibitor (BBI), the p-nitroanilide
(-pNa) substrates a N-Suc-Ala-Ala-Pro-Phe-pNa, NSuc-Ala-Ala-Pro-Leu-pNa, benzoyl-Arg-pNa (BApNa),
�
to 7.0. Cultivation was performed at 26 C for 10
days in 750-ml Erlenmeyer flasks containing 250
ml of medium, with mechanical shaking at 150
rpm (rotary shaker). The mycelia were removed
by filtration through Whatman no. 1 filter paper
and the cell-free filtrate served for proteinase
preparation.
N-Suc-Ala-Ala-Pro-Val-pNa, N-Suc-Ala-Ala-Ala-pNa,
and N
a-Tosyl-L-lysine
chloromethyl ketone hy-
Protein Assay
drochloride (TLCK), N-p-Tosyl-L-phenylalanine
chloromethyl ketone (TPCK), diisopropyl flouro-
Protein content was determined either with
phosphate (DFP), trinitrobenzenesulfonic acid
bicinchoninic acid (Smith et al., 1985) or by the
(TNBS), and phenyl-methylsulfonyl fluoride (PMSF)
biuret micro-method (Goa, 1953) using bovine se-
were obtained from the Sigma Chemical Company
rum albumin as a standard.
(St. Louis, MO). N,N,N�,N�-tetramethylethylenediamine (TEMED), N,N�-metylenebisacrylamide, 4-
Proteinase Activity
nitrophenyl 4-guanidinobenzoate hydrochloride
(NPGB) and HPLC grade reagents were from Fluka
AG, Buchs, Switzerland. Basic pancreatic trypsin
inhibitor (BPTI) Trascolan�, was purchased from
Proteolytic activity was measured using the
chromogenic substrate: N-suc-Ala-Ala-Pro-Phe-p-Na
in 100 mM Tris-HCl buffer, pH 8.3, containing 20
Pharmaceutical Co. Jelfa (Jelenia Gora, Poland).
mM CaCl2 and 0.005% Triton X-100. The reactions
Turkey ovomucoid (OMTKY) and a chymotrypsin/
were performed in polystyrene cuvettes containing
cathepsin G inhibitor from the hemolymph of Apis
the appropriate amount of proteinase, 50
mellifera (AMCI-1) were purified according to
strate concentration, and reaction buffer to a final
mM sub-
Bogard et al. (1980) and Bania et al. (1999), re-
volume of 2 ml. Progress curves at A412, after start-
spectively. Kazal-type inhibitor from bovine pan-
ing a reaction by adding substrate, were followed
Archives of Insect Biochemistry and Physiology
August 2006
doi: 10.1002/arch.
188
Bania et al.
using an HP8452 diode-array spectrophotometer.
gel and 4% stacking gel with 0.1% sodium dodecyl
A unit of activity was defined as the amount of
sulfate (SDS), pH 8.9. Coomassie brilliant blue
enzyme that caused an increase in the absorbance
staining was used to visualize protein bands. Mo-
by 0.1 per min.
lecular weight standards bovine serum albumin (66
kDa), egg ovoalbumin (43 kDa), soybean trypsin
inhibitor (21 kDa), and basic pancreatic trypsin in-
Proteinase Purification
hibitor (6.5 kDa) served as reference proteins. The
The cell-free filtrate was concentrated about 30-
apparent Mr value of purified protein was calcu-
fold with an Amicon hollow fiber cartridge concen-
lated from semi-log plots of standard protein Mr
trator (model H1P10). The retentate was applied
vs. migration distances.
to a Sephadex G-75 column (45
� 600 mm) equili-
brated with 50 mM sodium acetate buffer, pH 5.0.
Proteinase pH Stability
The column was eluted at of 8 ml/h. Fractions (3
ml) were collected and assayed for protein at 280
nm and for enzyme activity. Fractions exhibiting
proteinase activities were pooled, concentrated, and
then desalted using an Amicon YM10 membrane.
This preparation of active proteins was subjected
to HPLC using a Waters SP 5PW 8
� 75 mm ion-
exchange column, equilibrated with 50 mM Tris-
The enzyme (1
� 10
�
M) was incubated for 30
min in 100 mM buffers of pH 2� (citrate-phosphate buffer for pH抯 2� Tris-HCl buffer for pH抯
7� carbonate-bicarbonate buffer for pH抯 9�).
After incubation, the remaining activity of the proteinase was measured by the method described
above.
HCl buffer, pH 8.0. Proteins were eluted with a
linear gradient of 0�M NaCl in the equilibrating
buffer for 50 min at 0.7 ml/min. Absorbance was
monitored at 280 nm.
pH Optimum
The effect of pH on the proteinase activity toward N-suc-Ala-Ala-Pro-Phe-p-Na was assayed with
100 mM buffers of pH 7�. The reaction was car-
Determination of Proteinase Concentration
ried out in polystyrene cuvettes in 2 ml of the appropriate buffer, containing proteinase and substrate
The C. coronatus proteinase concentration was
determined by spectrophotometric titration with
the inhibitor AMCI-1. The following procedure was
used to estimate the concentration of AMCI-1: the
at concentrations of 1
� 10
�
M and 50
mM, respec-
tively. Activity was determined as the release of pnitroaniline, and was continuously monitored at
412 nm.
molar concentration of a stock solution of bovine
b-trypsin (dissolved in 1 mM HCl, 20 mM CaCl ),
2
Effects of Inhibitors
was determined by titration with p-nitrophenyl pguanidinobenzoate
HCl
(NPBG)
according
to
The enzyme (1
� 10
�
M) was allowed to com-
Chase and Shaw (1969). This standardized trypsin
plex with an inhibitor (10 mM) in a final volume
solution was used to titrate BPTI, which in turn
of 2 ml in 100 mM Tris-HCl buffer, pH 8.3, con-
served as a secondary standard for determining the
taining 20 mM CaCl2 and 0.005% Triton X-100.
activity of bovine
After preincubation for 30 min, the residual pro-
a-chymotrypsin
that was used
for determining AMCI-1 concentration.
teinase activity was measured toward 50
mM N-suc-
Ala-Ala-Pro-Phe-p-Na as described above.
SDS-Polyacrylamide Gel Electrophoresis and
Molecular Mass Determination
Gel
electrophoresis
was
Kinetic Constants kcat and Km
the
Kinetic constants were determined for the hy-
method of Laemmli (1970) using 15% separating
carried
out
by
drolysis of N-suc-Ala-Ala-Pro-Phe-p-Na and N-suc-
Archives of Insect Biochemistry and Physiology
August 2006
doi: 10.1002/arch.
Specificity of an Extracellular Proteinase From C. coronatus
Ala-Ala-Pro-Leu-p-Na by Conidiobolus proteinase.
�
�
189
tilisin Carlsberg were added to a 5% ground cu-
M
ticle suspension in 0.1 M Tris-HCl, pH 8.3. A unit
of Conidiobolus proteinase and increasing concen-
of subtilisin Carlsberg activity was defined as the
The 2-ml reaction mixtures contained 1
�
�
10
M in 100 mM
amount of enzyme that caused 0.1 A412 per min
Tris-HCl buffer, pH 8.3, with 20 mM CaCl2 and
increase under the conditions described for deter-
0.005% Triton X-100. After starting the reaction by
mining C. coronatus proteinase activity. The mix-
adding the appropriate substrate, the release of p-
tures were incubated at 37 C for 3, 6, and 24 h,
nitroaniline was continuously monitored at A412.
then 0.5 ml of 1% SDS was added and the tubes
Absorbance rates were converted to the substrate
were heated at 75 C for 15 min. The enzyme was
concentration changes using p-nitroaniline molar
added to the negative control (5% ground cuticle
extinction coefficient of 8,800/M/cm, and a graphic
suspension in 0.1 M Tris-HCl, pH 8.3), after pre-
representation of substrate concentration vs. reac-
heating the tube to 75 C. The reaction mixtures
tion rate was prepared. The Km was determined
were centrifuged at 13,000 rpm, and supernatants
using nonlinear regression analysis of the result-
were subsequently assayed for free amino groups
ing curve fitted to the Michaelis朚enten equation
according to Adler-Nissen (1979). An appropriate
by GraFit software. kcat was calculated as a quo-
volume of the reaction mixture was completed to
tient of Vmax and the enzyme concentration (E0).
250
trations of substrate up to 2
10
�
�
�
ml
ml of 0.1 M sodium
500 ml of 0.1% trinitro-
with water, then 250
The Vmax value was calculated by analysis of the
tetraborate, pH 9.6, and
curve representing experimental points fitted to the
benzenesulfonic acid were added. The samples
Michaelis� Menten equation by GraFit software.
were incubated at 37 C for 1 h in the dark and
The E0 value was determined in an independent
the A420 was read. The absorbance of the negative
experiment as described previously.
controls was subtracted from the samples. A unit
�
of proteolytic activity was defined as the amount
Digestion of the Oxidized Insulin B-Chain
of enzyme able to release free amino groups corresponding to 1 mg of alanine.
Oxidized insulin B chain 1 mg (Sigma Chemical Company) dissolved in 0.5 ml of Tris-HCl
Association Constant of Conidiobolus coronatus
buffer, pH 8.0, and was incubated with 1 mol% of
Proteinase and Beauveria bassiana Basic Proteinase
Conidiobolus proteinase at 37 C for an appropriate
With AMCI-1
�
reaction time. At intervals of 15, 35, and 60 min,
tein was withdrawn and added to 100
mg of digested proml 10% TFA
timated using the method described by Empie and
to stop the reaction. Products of digestion were
Laskowski (1982). The reactions were conducted
an aliquot containing about 50
separated via HPLC using a Waters m-Bondpak 3.9
� 150 mm, C-18 column. Each fraction of the pep-
The equilibrium association constants were es-
in a polystyrene cuvette in a final volume of 2.0
ml of 0.1 M Tris-HCl buffer, pH 8.3, with 20 mM
tide peaks was collected and analyzed using an
CaCl2 and 0.005% Triton X-100. The enzyme con-
amino-acid sequencer (Applied Biosystems) to lo-
centrations used [E0] complied with the equation:
碖
calize cleavage sites on the oxidized insulin B-
2 < [E0]
chain.
tor [I0] ranged from 0 to 2
a
< 50 and the concentration of inhibi-
�[E ]. A constant amount
0
of an enzyme reacted with an increasing amount
�
Hydrolysis of Insect Cuticle With
of inhibitor at 22 C for a predetermined period of
C. coronatus Proteinase
time to reach equilibrium, followed by the addition of substrate, whose final concentration in the
Exuviae from Dendrolimus pini were washed with
reaction did not exceed 0.2
碖
. The hydrolysis of
m
0.1 M Tris-HCl, pH 8.3, then air dried for 24 h. C.
p-nitroanilide was monitored at 412 nm for 120
coronatus proteinase (20 U) or an equivalent of sub-
sec and the free enzyme concentration was calcu-
Archives of Insect Biochemistry and Physiology
August 2006
doi: 10.1002/arch.
190
Bania et al.
lated. The association constant Ka was calculated
by fitting the experimental data to the following
equation:
E =
1
2
�
([E0] � F[I0] � K a +
2
� ([E0] � F[I0] � K�a ) � 4[E0]F[I0])
where [E] is the residual enzyme concentration, [E0]
and [I0] are the total enzyme and inhibitor concentrations, respectively, and F is an enzyme-inhibitor equimolarity factor.
RESULTS
Purification of
Conidiobolus Proteinase
The C. coronatus grown as described above produced an extracellular proteinase that reached its
maximal activity on the 10th day of cultivation.
The level of activity did not increase beyond 10
days of cultivation. Enrichment of the culture medium with casein or yeast extract as a source of
carbon resulted in a significant decrease in proteinase concentration (data not shown).
The proteinase was purified from the culture
broth after removing the fungi by filtration through
filter paper. The filtrate volume was reduced with
an Amicon
Hollow
Fiber Cartridge H1P10-20 to
about 100 ml, then concentrated using an Amicon
YM10 membrane to a final volume of 5 ml. The
purification method is summarized in Table 1. The
concentrated cell-free medium was first applied on
a Sephadex G-75 column at pH 5.0. The concentrated medium was resolved into several peaks, of
which only one showed proteinase activity (Fig.
1). SDS-PAGE analysis of the proteolytically active
fractions showed several bands (data not shown);
Fig. 1.
A: Elution profile of C. coronatus proteins from a
Sephadex G-75 column at pH 5.0. The protein peak exhibiting proteolytic activity is marked by a horizontal bar.
B: Elution profile of proteinase collected from a Sephadex
G-75 on SP 5PW ion-exchange column at pH 8.0. Horizontal bar indicates proteinase activity.
therefore, they were pooled, concentrated using an
Amicon YM10 membrane, and applied to an HPLC
ing all the proteinase activity was eluted from the
Waters SP 5PW ion-exchange column at pH 8.0.
column after about 16 min post-injection. SDS-
As shown in Figure 1B, the protein peak contain-
PAGE analysis of this peak revealed a single band
TABLE 1.
Summary of
Conidiobolus Proteinase Purification
Protein concentration
Purification step
Culture fluid concentrate
Sephadex G-75
HPLC ion-exchange
Volume (ml)
Specific activity
Yield (%)
Purification factor
5
3.6
410
22
�
�
10
0.4
240
60
58
0.33
185
616
45
0.9
(mg/ml)
Activity (U)
(U/mg protein)
Archives of Insect Biochemistry and Physiology
2.7
28
August 2006
doi: 10.1002/arch.
Specificity of an Extracellular Proteinase From C. coronatus
191
for 30 min at pH抯 of 6 to 10 had no effect on its
activity. A significant loss of activity could be observed during incubation of proteinase at pH below 5. Proteinase incubations at pH 3 or below
irreversibly loses its activity (Fig. 3).
The pH dependence of the amidase activity for
Fig. 2.
SDS-PAGE of Con-
purified enzyme is shown in Figure 4. The opti-
idiobolus protease after ion-
mal pH抯 for the digestion of N-Suc-Ala-Ala-Pro-
exchange chromatography
Phe-pNa by
on
to be 8� At pH below 8 and above 9, its activity
an
5PW 8
HPLC
�
Waters
SP
75 mm column.
Conidiobolus
proteinase was determined
drastically decreases (Fig. 4).
The protein was resolved
in
15%
polyacrylamide
gel, and then stained with
Coomasie brilliant blue.
of a Mr of 30 kDa (Fig. 2). The enzyme was purified to homogeneity with an overall purification
factor of 28-fold and a final yield of over 45%.
Effect of Proteinase Inhibitors on
Conidiobolus
Proteinase Activity
A range of synthetic and naturally occurring inhibitors was used to determine the nature of the
active site of
Conidiobolus
proteinase. The protein-
ase was inhibited by PMSF and DFP, indicating the
Effect of pH on the Activity and Stability of the
presence of serine and histidine residues in the ac-
Conidiobolus Proteinase
tive site. As expected, specific inhibitors of other
The
Conidiobolus
classes of proteinases, i.e., aspartyl, metallo, and
proteinase was stable over a
wide range of pH values. Incubation of proteinase
cysteine proteinases, did not affect its activity. These
results confirm that
Conidiobolus
Fig. 3.
proteinase belongs
Effect of pH on the stabil-
ity of proteinase from C. coronatus.
Proteinase in a concentration of 1
� 10
�
M was incubated for 30 min
at the pH indicated. Then the remaining activity was assayed with
N-suc-Ala-Ala-Pro-Phe-p-Na in 100
mM Tris-HCl buffer, pH 8.3, containing 20 mM CaCl2 and 0.005%
Triton X-100.
Archives of Insect Biochemistry and Physiology
August 2006
doi: 10.1002/arch.
192
Bania et al.
Fig. 4.
Determination of pH opti-
mum for C.
coronatus proteinase
activity. A constant amount of proteinase was assayed toward N-SucAla-Ala-Pro-Phe-pNa in a range of
pH values (100 mM buffers containing 20 mM CaCl2 and 0.005% Triton X-100).
to the family of serine proteinases. Some protein
teract irreversibly with serine proteinases exhibit-
inhibitors of serine proteinases are useful in dem-
ing chymotrypsin-like and trypsin-like specificities,
onstrating the preferences of proteinase for the in-
respectively, were examined. In view of previous
hibitor P1 position, and also help in determining
results, the lack of inhibition by trypsin-specific
the enzyme specificity. From the range of inhibi-
TLCK was not surprising, although it was striking
tors tested, AMCI-1, OMTKY, and the microbial-
that TPCK also appeared to be ineffective.
derived inhibitors leupeptin and chymostatin all
An additional set of inhibitors known to in-
showed strong inhibition. The inhibition profile
hibit chymotrypsin-like enzymes, namely BPTI,
Conidiobolus proteinase has a subtili-
STI, and BBI at concentrations of 10 mM, inhib-
sin-like specificity. To prove this, the effect of two
ited proteinase activity by 50, 60, and 80%, re-
synthetic inhibitors, TLCK and TPCK, known to in-
spectively (Table 2).
suggests that
In contrast, the Kazal-type inhibitor from boTABLE 2.
Effect of Selected Inhibitors on the Activity of
Conidiobolus
Proteinase*
trypsin-like proteinases, had no effect on
Inhibitors
% proteinase activity
DFP
Conidio-
bolus proteinase activity.
0
PMSF
0
TPCK
100
TLCK
100
Leupeptin
0
STI
40
BBI
20
BPTI
50
OMTKY
10
AMCI-1
0
Kazal type inhibitor from bovine pancreas
*The enzyme (1
Proteinase Specificity
10
Chymostatin
�
10
�
From the range of synthetic substrates tested,
Conidiobolus proteinase hydrolyzed only chymotrypsin-specific substrates with phenylalanine and
leucine residues at the P1 position, and did not
generate observable cleavage of the substrates with
100
arginine, valine, or alanine at that position. An
M) was allowed to complex with an inhibitor (10 mM).
After preincubation for 30 min, the residual proteinase activity was measured toward
vine pancreas, known to inhibit exclusively the
N-suc-Ala-Ala-Pro-Phe-p-Na.
analysis of the enzyme kinetics showed that
Conidio-
bolus proteinase hydrolyzed N-Suc-Ala-Ala-Pro-Phe-
Archives of Insect Biochemistry and Physiology
August 2006
doi: 10.1002/arch.
Specificity of an Extracellular Proteinase From C. coronatus
pNa with Km = 1.36
s
�
�
� 10
�
M and a kcat value of 24
and N-Suc-Ala-Ala-Pro-Leu-pNa with Km = 6.65
�
10
M and a kcat value of 11 s
�
. The specificity
193
Association Constant of Conidiobolus coronatus
Proteinase and Beauveria bassiana Basic Proteinase
With AMCI-1
indices kcat/Km for the tested substrates were calculated to be 176,340 s
�
�
Inhibition studies showed (Table 2) that the
purified enzyme exhibited the highest sensitivity
proteinases, such as BApNa (trypsin), N-Suc-Ala-Ala-
to AMCI-1, a chymotrypsin/cathepsin G inhibitor
Pro-Val-pNa (human leukocyte elastase), and N-Suc-
from the larval hemolymph of the honeybee (Apis
Ala-Ala-Ala-pNa (porcine pancreatic elastase) were
mellifera) (Bania et al., 1999). Considering the
not cleaved by Conidiobolus proteinase.
physiological significance of AMCI-1 presence in
substrates
for
M
�
, re-
Specific
and 17,030 s
�
serine
spectively.
M
other
Cleavage specificity of Conidiobolus proteinase
the hemolymph of the honeybee, we studied its
was also determined by using oxidized insulin B-
possible role in insect anti-fungal responses. Since
chain as a substrate. The hydrolyzed peptide bonds
proteinase inhibitors in insect hemolymph are be-
are indicated by arrows in Figure 5. After 15-min
lieved to protect the insect against fungal infection,
digestion, it was found that Conidiobolus protein-
we tested the susceptibility of fungal proteinases
1
,
to AMCI-1. It was shown that both Conidiobolus
. Secondary cleavage
coronatus proteinase and the basic proteinase, pu-
ase cleaved the substrate at positions Phe 朧al
15
Leu
朤yr
16
, and Gly
23
朠he
24
2
24
�
rified from another enthomopathogen Beauveria
. Incubation times up to 1 h
bassiana, were strongly inhibited by AMCI-1. The
did not change the elution profile pattern from the
association constants of AMCI-1/Conidiobolus pro-
C-18 column, seen at 35 min, indicating a lack of
teinase and AMCI-1/Beauveria basic proteinase were
additional digestion sites (Fig. 5).
determined to be 3
sites were found after 35 min at positions Phe
25
Phe
and Phe
25
朤hr
26
To prove that C. coronatus proteinase may be
� 10
10
M
�
and 4
� 10
9
M
�
, re-
spectively.
involved in the penetration of the insect body via
the cuticle, its ability to hydrolyze the insect cu-
DISCUSSION
ticle was tested. Hydrolysis was assayed by measuring the free amino groups released during the
C. coronatus secretes several proteinases into the
reaction. It was found that both C. coronatus pro-
culture medium with molecular weight ranging
teinase and subtilisin Carlsberg, used as a refer-
from 6.8 to 32 kDa (Sutar et al., 1991). The mo-
ence enzyme, effectively hydrolyzed the insect
lecular masses of the better-described proteinase I
cuticle. Within 24 h, the enzymes from C. coron-
and proteinase II were found to be 23 and 19 kDa,
atus and Carlsberg at the amount specified pro-
respectively. Their similar properties, including
duced a free amino group from 50 mg of cuticle
amino-acid composition, substrate specificity, and
corresponding to 28 and 20 mg of alanine, re-
antigenic properties suggest that both of them are
spectively.
presumably the products of the same gene, and
Fig. 5.
Cleavage pattern of oxidized insulin B-chain di-
gested by
Conidiobolus
proteinase. Digestion was performed
Archives of Insect Biochemistry and Physiology
August 2006
doi: 10.1002/arch.
in 0.1 M. Tris-HCl buffer, pH 8.0, for 30 min. Vertical arrows show cleavage sites after 15 and 35 min of digestion.
194
Bania et al.
proteinase II appears to be the result of auto-
teinases used confirms that Conidiobolus protein-
proteolysis of proteinase I (Phadatare et al., 1992).
ase represents a subtilisin-like specificity. The in-
Another subtilisin-like proteinase isolated from the
hibitors of both trypsin and chymotrypsin (BPTI,
culture broth of C. coronatus of molecular weight
STI, BBI, and OMTKY, but also AMCI-1 and chymo-
27�.5 kDa has been described by Tanksale et al.
statin, specific inhibitors of chymotrypsin-like en-
(2000).
zymes) efficiently inhibit Conidiobolus proteinase.
A novel proteinase of molecular weight 30-32
The Kazal-type inhibitor from bovine pancreas,
kDa was purified from the culture supernatant of
known to inhibit trypsin-like enzymes exclusively,
C. coronatus. The optimization of broth composi-
did not affect the activity of Conidiobolus protein-
tion towards the proteinase yield was not a major
ase.
goal of our study, but, in contradiction to other
known to inhibit primarily cysteine proteinases,
authors demonstrating that the addition of selected
has
organic compounds clearly enhances the produc-
Conidiobolus proteinase activity. Some examples of
tion of proteinases (Phadatare et al., 1993; Sutar
serine
et al., 1991), we noted a significant reduction in
known (Fujishiro et al., 1980) and it has been
proteinase concentration in the presence of casein
shown that a cysteine residue near the active site
and/or other organic sources of carbon in the cul-
histidine in some subtilisins renders these enzymes
ture medium. The proteinase reduction may be the
susceptible to cysteine protease inhibitors, such as
result of either decreased proteinase production or
leupeptin (Barrett and Rawlings, 1991).
disturbed proteinase secretion to the medium.
Leupeptin,
also
been
a
microbial-derived
shown
proteinases
to
exert
inhibited
some
by
inhibitor,
effect
leupeptin
on
are
The substrate preferences of Conidiobolus pro-
The proteinase inhibition effects, as well as the
teinase were determined by its cleavage sites on
substrate specificity studies, showed that the new
oxidized insulin B-chain. The proteinase affinity
Conidiobolus proteinase may be classified as a sub-
seems to be restricted exclusively to the aromatic
tilisin-like serine proteinase. The proteinase cleaves
Phe and aliphatic Leu and Gly residues. Interest-
substrates with phenylalanine (N-Suc-Ala-Ala-Pro-
ingly, the proteinase cleaved at all the phenylala-
Phe-pNa) and leucine (N-Suc-Ala-Ala-Pro-Leu-
nine residues present in the peptide, while only
pNa) residues at the P1 positions with kcat and Km
one of the four leucines present in the insulin B-
values similar to known chymotrypsins and sub-
chain were cleaved. This may indicate that the se-
tilisins (Nakajima et al., 1979; Yamagata et al.,
quence of the residues neighboring the P1 site of
1995). The Conidiobolus proteinase was unable to
leucine may strongly influence the proteinase speci-
hydrolyze the amide substrates containing Arg, Val,
ficity. In turn, the cleavage at the Gly residue is a
or Ala at P1 position. Other known proteinases
characteristic trait of pancreatic elastase (Merops
from C. coronatus were shown to be able to cleave
database http://merops.sanger.ac.uk). One unusual
the ester substrates with both Tyr and Arg at P1,
cleavage site after the first Phe residue was found.
but failed to hydrolyze its amide counterparts
The proteinases from pathogenic fungi are poorly
(Phadatare et al., 1992). The authors of the study
characterized in their ability to cleave such well-
conclude that the investigated proteinases simply
defined peptide substrates as insulin B-chain, and
lack the amidase activity toward the tested sub-
consequently the presence of such an activity in
strates. We have shown that the amide substrates
the class of serine proteinases cannot be excluded.
with Phe and Leu at P1 may be successfully cleaved
The enzymes currently known to be able to simul-
by the novel Conidiobolus proteinase, confirming
taneously act as endo- and exopeptidases and to
its amidase activity. This may indicate that the ori-
cleave the Phe/Val bond in insulin B-chain belong
gin of the observed differences may result from dif-
to the class of aspartic or metallo proteinases
ferent specificities of proteinases and not from the
(Merops database http://merops.sanger.ac.uk).
lack of its activity towards the amide substrates.
The range of natural inhibitors of serine pro-
Entomopathogenic fungi secrete a range of hydrolases that facilitate the penetration of the fun-
Archives of Insect Biochemistry and Physiology
August 2006
doi: 10.1002/arch.
Specificity of an Extracellular Proteinase From C. coronatus
195
gus into the insect body. The insect cuticle is com-
these proteinases for AMCI-1 are relatively high.
posed of chitin fibers buried in protein matrix. As
To our knowledge, the only well-characterized in-
was confirmed by a number of studies, the fungal
teraction between a fungal proteinase and an in-
serine proteinases are able to degrade the insect
sect proteinase inhibitor is that of the proteinase
cuticle and are thus especially important in fungal
from Aspergillus melleus and the FPI-F inhibitor
pathogenesis (Bidochka and Khachatourians, 1994;
from Bombyx mori. The association constant of this
Cole et al., 1993; Gupta et al., 1992; Samuels and
interaction was determined to be 1.1
Paterson, 1995; St Leger et al., 1987, 1988). Sub-
(Eguchi et al., 1993). AMCI-1 is able to bind pro-
strate specificity and inhibition studies show that
teinases from Conidiobolus and Beauveria two or-
C. coronatus proteinase displays a broad specificity
ders of magnitude more tightly than FPI-F, with
characteristic of the fungal proteinases involved in
Ka抯 of 3
the degradation of insect cuticle (Samuels and
This reinforces the hypothesis, that inhibitors from
Patterson, 1995). The demonstration of its ability
insect hemolymph may act as protective agents
to degrade insect cuticle suggests that the protein-
against fungal invasions.
�
10
10
M
�
and 4
�
9
10
M
�
�
10
7
M
�
, respectively.
ase may be a virulence factor of C. coronatus.
In contrast, the hemolymph of insects contains
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