Cysteine and serine proteolytic activities in larval midgut of yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae)


Thie, NMR.; Houseman, JG.

Insect Biochemistry 20(7): 741-744

1990


The midgut of the yellow mealworm, Tenebrio molitor, contains both serine and cysteine proteolytic activities, identified using synthetic substrates, pH optima characteristic for each enzyme, diagnostic activators and inhibitors. The cysteine proteinase, detected by optimal hydrolysis of N-benzoyl-dl-arginine-ß-naphthylamide at pH 5.0, was activated by thiol compounds, inhibited by the cysteine specific proteinase inhibitors iodoacetamide (IAA) and trans-epoxysuccinyl-l-leucyl-amido(4guanidino)-butane (E-64). The serine proteinase detected by maximal hydrolysis of benzoyl-dl-arginine-p-nitroanilide at pH 8.0, was inhibited by the serine specific proteinase inhibitors aprotinin, soybean trypsin inhibitor, PMSF and activity was not affected by either IAA or E-64. Both proteinase activities were higher in gut contents compared to wall and higher cysteine proteolytic activity occurred in the anterior, compared to posterior, portion of the midgut. Serine proteolytic activity predominated in the posterior region of the midgut.

Insect
Biochem.
Vol.
20,
No.
7,
pp.
741-744,
1990
0020-1790/90
$3.00
+
0.00
Printed
in
Great
Britain.
All
rights
reserved
Copyright
©
1990
Pergamon
Press
plc
CYSTEINE
AND
SERINE
PROTEOLYTIC
ACTIVITIES
IN
LARVAL
MIDGUT
OF
YELLOW
MEALWORM,
TENEBRIO
MOLITOR
L.
(COLEOPTERA:
TENEBRIONIDAE)
NORMAN
M.
R.
TI-HE
and
JON
G.
HOUSEMAN*
Ottawa-Carleton
Institute
of
Biology,
Biology
Department,
University
of
Ottawa,
Ottawa,
Ontario,
Canada
KIN
6N5
(Received
9
January
1990;
revised
and
accepted
10
July
1990)
Abstract—The
midgut
of
the
yellow
mealworm,
Tenebrio
molitor,
contains
both
serine
and
cysteine
proteolytic
activities,
identified
using
synthetic
substrates,
pH
optima
characteristic
for
each
enzyme,
diagnostic
activators
and
inhibitors.
The
cysteine
proteinase,
detected
by
optimal
hydrolysis
of
N-benzoyl-
DL-arginine-Pnaphthylamide
at
pH
5.0,
was
activated
by
thiol
compounds,
inhibited
by
the
cysteine
specific
proteinase
inhibitors
iodoacetamide
(IAA)
and
trans-epoxysuccinyl-L-leucyl-amido(4guanidino)-
butane
(E-64).
The
serine
proteinase
detected
by
maximal
hydrolysis
of
benzoyl-DL-arginine-p-nitroanilide
at
pH
8.0,
was
inhibited
by
the
serine
specific
proteinase
inhibitors
aprotinin,
soybean
trypsin
inhibitor,
PMSF
and
activity
was
not
affected
by
either
IAA
or
E-64.
Both
proteinase
activities
were
higher
in
gut
contents
compared
to
wall
and
higher
cysteine
proteolytic
activity
occurred
in
the
anterior,
compared
to
posterior,
portion
of
the
midgut.
Serine
proteolytic
activity
predominated
in
the
posterior
region
of
the
midgut.
Key
Word
Index:
cysteine
proteinase,
serine
proteinase,
digestion,
Tenebrio
molitor,
Tenebrionidae
INTRODUCTION
The
major
protease
classes
are
serine,
cysteine,
aspar-
tic
and
metalloproteinases
and
their
identification
is
based
on
reaction
mechanism
employed
in
the
active
site
of
the
enzyme
(Barrett
and
McDonald,
1980;
McDonald
and
Barrett,
1980;
Storey
and
Wagner,
1986).
Serine
proteases
such
as
trypsin
were
at
one
time
considered
to
be
the
principle
digestive
enzymes
in
insects,
(see
reviews
by:
House,
1974;
Law
et
al.,
1977;
McFarlane,
1985).
Other
enzyme
classes
are,
however,
known
to
be
involved
in
hydrolysis
of
ingested
protein
(Applebaum,
1985).
These
include,
for
example,
cysteine
and
aspartic
proteases
in
Hemiptera
(Houseman
and
Downe,
1980,
1981,
1982,
1983)
and
pepsin-like
aspartate
proteinases
in
Diptera
(Greenberg
and
Paretsky,
1955;
Sinha,
1975;
Pendola
and
Greenberg,
1975).
Serine
proteinases,
with
either
trypsin
or
chymo-
trypsin-like
characteristics,
have
been
identified
in
the
following
carabid
species:
Pterostichus
melan-
arius
(Gooding
and
Huang,
1969),
Calosoma
calidum,
Carabus
taedatus
(Cheung
and
Gooding,
1970),
Carabus
auronitens,
Carabus
punctatoauratus,
Carabus
lineatus
and
Carabus
splendens
(Vaje
et
al.,
1984).
Similar
proteinases
are
found
in
the
elaterid
beetle
Pyrophorus
divergens
(Colepicolo-Neto
et
al.,
1987),
the
dermestid
beetle
Attegenus
megatoma
(Baker,
1981),
Sitophilus
weevils
S.
oryzea,
S.
zeamais
and
S.
granarius
(Baker,
1982),
the
sweet
potato
weevil,
Cyclas
formicarius
elegantus
(Baker
et
al.,
1984)
and
the
scarab
beetle
Costelytra
zealandi
(Christellar
et
al.,
1989).
*Author
to
whom
all
correspondence
should
be
addressed.
Initial
investigations
with
bruchid
beetles
Calloso-
bruchus
chinensis
and
Acanthoscelides
obtectus
re-
vealed
only
weak
proteolytic
activity
(Applebaum,
1964)
when
assayed
using
the
general
protein
sub-
strate
azocasein
at
pH
7.6.
Subsequent
investigations
identified
cysteine
proteinase
activity
in
Calloso-
bruchus
maculatus
(Campos
et
al.,
1989)
and
A.
obtectus
(Wieman
and
Nielson,
1988)
which
was
similar
to
cathepsin
B
in
C.
maculatus
(Gatehouse
et
al.,
1985;
Kitch
and
Murdock,
1986).
Cysteine
proteinases
were
detected
in
the
Colorado
potato
beetle,
Leptinotarsa
decemlineata
(Wolfson
and
Murdock,
1987)
and
activity
was
attributed
to
cathepsin
B
and
H,
as
well
as
aspartic
proteinase
cathepsin
D
(Thie
and
Houseman,
1990).
Murdock
et
al.
(1987),
based
on
a
survey
of
11
different
species,
suggest
that
cysteine
proteinases
may
be
common
in
the
Coleoptera.
In
Tenebrio
molitor
maximal
proteolysis
of
casein
occurred
at
pH
6.2-6.4
(Birk
et
al.,
1962).
Trypsin,
carboxypeptidase
and
aminotripeptidase
activity
were
identified,
at
pH
6.5,
using
a
variety
of
synthetic
substrates
and
differential
sensitivities
to
soybean
trypsin
inhibitor
(Applebaum
et
al.,
1964).
Tenebrio
molitor
proteolytic
activity
was
further
resolved
into
an
a-
and
/3-protease.
The
a-protease
differed
from
vertebrate
trypsin
and
chymotrypsin,
while
the
1
6
-
protease
shared
many
vertebrate
trypsin
character-
istics
and
only
its
cleavage
specificity
towards
insulin
and
dodeca-peptide
differed
(Zwilling,
1968;
Zwilling
et
al.,
1972).
Mealworm
trypsin
also
shared
similar
inhibitory,
molecular
weight,
substrate
specificity
and
optimal
activity
characteristics
with
bovine
trypsin,
with
differences
only
being
conformational
(Levinsky
et
al.,
1977).
The
initial
investigation
with
T.
molitor
741
25
20
15
10
5
Bz-
Arg
-
NHNe
E.
U.
/
mg
p
ro
te
in
0
742
NORMAN
M.
R.
THIE
and
JON
G.
HOUSEMAN
preparations
found
that
thiol
compounds
and
chelating
agents
activated
or
stabilized
exopeptidase
activity,
and
that
the
soybean
inhibitor
resistant
preparations
contained
an
unidentified
activity
that
could
be
either
carboxypeptidase
B
or
cathepsin
B
(Applebaum
et
al.,
1964).
The
objective
of
this
study
is
to
determine,
using
synthetic
substrates,
whether
both
cysteine
and
serine
proteinases
are
present
in
the
alimentary
tract
of
T.
molitor
by
determining
the
pH
for
maximal
substrate
hydrolysis
and
the
potential
effects
of
diagnostic
inhibitors
and
activators
on
substrate
hydrolysis.
MATERIALS
AND
METHODS
Insects
Adult
Yellow
mealworms,
Tenebrio
molitor,
were
reared
on
wheat
bran
at
25°C,
50%
r.h.
with
a
16:8
(light:dark)
photoperiod.
Midguts
from
third
instar
larvae
were
col-
lected,
homogenized
in
0.15
M
NaCl
and
supernatants,
after
centrifugation
at
13,500
g
for
10
min
at
4°C,
were
used
for
protein
and
proteinase
activity
determinations.
Anterior
and
posterior
gut
samples
were
collected
by
dividing
the
alimen-
tary
tract
into
two
equal
portions
by
cutting
midway
between
the
most
anterior
region
and
the
insertion
of
the
Malpighian
tubules.
Midgut
contents
were
separated
from
the
gut
wall
by
placing
an
intact
gut
in
0.15
M
NaCl
and
splitting
the
wall
longitudinally,
using
fine
forceps,
to
free
the
intact
peritrophic
membrane
and
its
contents.
Enzyme
assays
All
substrates,
potential
activators
and
inhibitors
were
purchased
from
Sigma
Chemical
Co.
Assays
were
carried
out
in
duplicate
at
30°C
using
a
thermoregulated
Beckman
DU
65
spectrophotometer.
Buffers,
0.1
M
in
the
final
reaction
mixture
for
optimal
pH
determination,
were
succinate,
acetate,
bis-Tris[bis(2-hydroxyethypimino-
Tris(hydroxymethypmethanel,
bis-Tris
propane
(1,3bis-
[Tris-(hydroxymethyl)methyl-amino]propane)
and
Tris
[Tris-(hydroxymethyl)aminomethanel,
all
containing
1
mM
DTT
(dithiothreitol),
and
either
1.5
mM
or
3
mM
EDTA
(ethylenediamine
tetraacetic
acid)
for
Bz-Arg-NHNa
(N-benzoyl-DL-arginine-fl-naphthylamide)
and
Bz-Arg-Np
(benzoyl
DL-arginine-p-nitroanilide)
hydrolysis,
respect-
ively.
The
synthetic
substrates,
Bz-Arg-NHNa
and
Bz-Arg-Np,
were
used
for
detection
of
cysteine
and
serine
proteolytic
activities
respectively
using
the
methods
of
Thie
and
Houseman
(1990).
The
molar
extinction
coefficients
of
27,000
for
Bz-Arg-NHNa
hydrolysis
(Barrett,
1977),
and
8800
for
Bz-Arg-Np
hydrolysis
(Erlanger
et
al.,
1961)
were
used
to
determine
nanomoles
of
substrate
hydrolyzed
and
one
enzyme
unit
was
defined
as
hydrolysis
of
1
nmol
of
substrate
per
min
per
mg
protein.
Proteolytic
activities
were
further
identified
using
poten-
tial
activators
incubated
at
30°C
for
5
min
with
midgut
homogenate
before
addition
of
substrate.
Buffers were
0.1
M
succinate,
pH
5.0,
for
Bz-Arg-NHNa
and
0.1
M
Tris,
pH
8.0,
at
30°C,
for
Bz-Arg-Np.
Thiol
compounds,
DTT,
cysteine,
glutathione
and
mercaptoethanol,
chelating
agents
EDTA
(ethylene
diamine
tetraacetic
acid),
EGTA
[ethylene
glycol
bis
(fl-aminoethyl
ether)N,N,N,N-tetraacetic
acid],
1,10-phenanthroline
and
divalent
cations,
CaCl
2
and
MgCl
2
were
tested
using
the
procedures
described
by
Thie
and
Houseman
(1990).
To
further
identify
the proteinases
re-
sponsible
for
hydrolysis
of
the
two
substrates,
inhibitors
that
are
potentially
diagnostic
for
the
different
protease
classes
were
tested.
For
the
serine
proteases,
PMSF
(phenyl-
methyl
sulfonyl
floride),
added
in
50
pll-propanol
for
a
final
concentration
of
5
mM,
200
µg
aprotinin
(from
bovine
lung)
and
10
pg
STI
(soybean
trypsin
inhibitor)
were
included
4
5
6
7
5
9
10
PH
Fig.
1.
The
effect
of
pH
on
hydrolysis
of
Bz-Arg-NHNa
(open
symbols)
and
Bz-Arg-Np
(solid
symbols)
by
larval
midgut
homogenates
from
T.
molitor.
Buffers
were
succinate,
pH
4.0-6.0
(circles),
sodium
acetate,
pH
4.0-6.0
(inverted
triangles),
bis-Tris,
pH
5.5-7.0
(squares),
bis-Tris
propane,
pH
6.5-9.0
(triangles),
and
Tris,
pH
7.0-9.0
(diamonds).
in
the
assays.
Cysteine
protease
inhibitors
iodoacetamide,
0.5
mM,
E-64
[trans
-epoxysuccinyl-L-leucyl-amido(4guani-
dino)butane],
0.5
p
g
and
pepstatin,
an
aspartate
protease
inhibitor,
10µg
in
2S
µl
methanol,
were
added
to
the
reaction
mixture.
The
effects
of
the
chloromethyl
ketone
TLCK
(tosyl-L-lysine
chloromethyl
ketone),
0.5
mM
and
0.75
pg
leupeptin,
both
of
which
are
inhibitors
of
cysteine
and
serine
proteases,
were
also
tested.
Protein
determination
Protein
was
determined
using
the
method
of
Bramhall
et
al.
(1969)
with
bovine
serum
albumin
(Fraction
V,
Sigma
Chemical
Co.)
as
a
standard.
RESULTS
Maximum
hydrolysis
of
Bz-Arg-NHNa
in
the
pres-
ence
of
1
mM
DTI'
and
1.5
mM
EDTA
occurred
at
pH
5.0
with
succinate
and
acetate
as
buffers
(Fig.
1).
In
the
presence
of
1.0
mM
DTT
and
3
mM
EDTA,
optimal
Bz-Arg-Np
hydrolysis
occurred
at
pH
8.0
and
8.5
with
Tris
and
bis-Tris
propane,
respectively,
as
buffers
(Fig.
1).
Chelating
agents
EGTA,
EDTA
and
1,10-phenan-
throline
and
the
divalent
cations
CaCl
2
and
MgCl
2
had
no
effects
on
the
hydrolysis
of
either
substrate
(data
not
shown).
Effects
of
other
potential
activators
and
inhibitors
are
shown
in
Table
1.
All
four
thiol
compounds
tested
activated
Bz-Arg-NHNa
hydroly-
sis
with
DTT
and
cysteine
being
better
activators
than
glutathione
or
mercaptoethanol.
Bz-Arg-Np
hydrolysis
increased
in
the
presence
of
DTT
but
substrate
hydrolysis
was
not
affected
by
the
other
thiols
tested.
Pepstatin
had
no
effect
and
leupeptin
inhibited
hydroylsis
of
both
substrates.
Aprotinin
and
PMSF
inhibited
midgut
hydrolysis
of
the
Bz-
Arg-Np
substrate
and
both
iodoacetamide
and
E-64
inhibited
only
Bz-Arg-NHNa
hydrolysis.
STI
was
an
inhibitor
of
Bz-Arg-Np
hydrolysis,
however, its
effects
on
Bz-Arg-NHNa
could
not
be
determined
due
to
formation
of
a
precipitate
in
the
reaction
mixture
when
the
color
reagent
was
added.
Bz-Arg-
Np
hydrolysis
was
inhibited
by
TLCK,
however,
its
effects
on
Bz-Arg-NHNa
hydrolysis
could
not
be
observed
because
it
chemically
interferes
with
assay
conditions
(Barrett,
1972).
150
125
100
M
75
50
25
0
3
~\
~
Mealworm
proteinases
743
Table
1.
The
effect
of
potential
activators
and
inhibitors
on
substrate
hydrolysis
by
larval
midgut
homogenate
from
the
yellow
mealworm,
Tenebrio
molitor.
Buffers,
unless
stated
otherwise
were:
0.1
M
succi-
nate,
pH
5.0,
for
Bz-Arg-NHNa,
0.1
M
Tris,
pH
8.0,
for
Bz-Arg-Np,
both
at
30°C
containing
1
mM
DTT
and
3.0
mM
EDTA
%
Relative
activity
Bz-Arg-NHNa
Bz-Arg-Np
Buffer
only
100 100
2.5
mM
DTT•
689'
171
d
5.0
mM
cysteine*
610°
112
d
5.0
mM
glutathione*
347°
110
d
5.0
mM
mercaptoethanol•
294°
109d
0.5
mM
TLCK
81'
0.5
mM
IAA
5 "
95'
10
pg
pepstatin
103"
99<
200
pg
aprotinin
95
b
13'
0.75
pg
leupeptin
23"
46
<
0.5
pg
E-64
6"
100'
5.0
mM
PMSF
108°
68
f
10
pg
STI
14'
The
following
values
(mean
±
range/2
in
E.U.
per
mg
protein)
represent
100%
activity.
'5.0
±
0.3;
b
12.7
±
0.1;
'1.1
±
0.1;
°
21.5
±
0.2;
'48.7
±
2.3;
'39,1
±
1.3.
*Tested
in
the
absence
of
EDTA.
The
anterior
gut
portion
contained
71.3%
of
cys-
teine
proteinase
activity
but
only
33.1%
of
the
serine
proteolytic
activity
which
was
predominant
in
the
posterior
gut
portion.
Both
proteinases
were
found
at
higher
levels,
93.6
and
90.2
for
cysteine
and
serine
proteinases
respectively,
in
the
gut
contents
com-
pared
to
the
gut
wall
(Table
2).
DISCUSSION
The
midgut
of
the
yellow
mealworm,
Tenebrio
molitor,
contains
both
serine
and
cysteine-like
pro-
teinases.
The
serine
proteinase
hydrolyzes
Bz-Arg-Np
optimally
under
alkaline
conditions
and
is
inhibited
by
PMSF,
aprotinin,
leupeptin,
TLCK
and
STI.
These
properties,
characteristic
of
tryptic
activity
(Barrett
and
McDonald,
1980),
are
consistent
with
previous
identifications
of
mealworm
trypsin
(Levinsky
et
al.,
1977;
Zwilling,
1968;
Zwilling
et
al.,
1972).
The
hydrolysis
of
Bz-Arg-NHNa,
thiol
acti-
vation,
optimal
activity
at
pH
5.0,
and
inhibition
by
cysteine
proteinase
inhibitors
IAA,
E-64
and
leu-
peptin
(which
does
not
inhibit
cathepsin
H),
are
all
consistent
with
the
identification
of
cathepsin
B
(Barrett
and
McDonald,
1980)
in
the
alimentary
tract
of
T.
molitor.
Cysteine
proteolytic
activities
are
present
as
digestive
enzymes
within
T.
molitor
and
this
confirms
the
identity
of
the
unknown
protease
detected
in
soybean
inhibitor
resistant
fraction
reported
by
Applebaum
et
al.
(1964).
Cysteine
and
serine
class
proteases
require
different
pH
conditions
and
activation
requirements
for
maximal
activity.
Thiol
compounds,
or
a
reducing
environment,
required
for
optimal
activity
of
the
cysteine
proteases,
are
capable
of
inhibiting
trypsin
but
in
the
mealworm
tryptic
activity
is
either
acti-
vated
or
not
affected
by
thiol
compounds.
These
catalytic
differences
may
be
accommodated
by
the
presence
of
the
cysteine
proteinase
in
the
anterior
midgut
while
the
serine
proteinases
are
restricted
to
the
posterior
portion.
Both
proteinase
activities
are
higher
in
midgut
contents
and
this
is
consistent
with
the
extracellular
digestion
in
insects.
IB
20/7-F
Table
2.
Distribution
of
Bz-Arg-NHNA
and
Bz-Arg-Np
hydrolytic
activities
in
the
larval
midgut
of
the
yellow
mealworm,
Tenebrio
molitor
Bz-Arg-NHNa
(cysteine
proteinase)
Bz-Arg-Np
(serine
proteinase)
E.U./gut
portion
Anterior
portion
14.4±
1.3
14.5±
1.3
Posterior
portion
5.8
+
0.6*
29.3
+
3.6*
Gut
wall
1.1
+
0.1
1.3
+
0.3
Gut
contents
16.0
+
0.7*
12.0+
1.9*
*Significantly
different,
P
0.01,
from
corresponding
gut
portion.
This
is
the
first
case
where
both
serine
and
cysteine
digestive
proteinases
are
found
together
in
an
insect
midgut.
These
results
suggest
that
various
Coleopteran
species
can
be
divided
into
at
least
three
different
groups
based
on
digestive
proteolysis
by
either
serine
or
cysteine
digestive
proteinases
or
a
combination
of
both.
Further
investigations
will
be
required
in
order
to
determine
whether
or
not
the
presence
of
both
is
a
unique
occurrence
within
Coleopteran
insects.
Acknowledgement-This
work
was
supported
by
an
operating
grant
from
the
Natural
Sciences
and
Engineering
Research
Council
of
Canada
(A3618)
awarded
to
J.
G.
Houseman.
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