Tài liệu Báo cáo khoa học: A zymogen form of masquerade-like serine proteinase homologue is cleaved during pro-phenoloxidase activation by Ca2+ in coleopteran and Tenebrio molitor larvae docx

A zymogen form of masquerade-like serine proteinase homologue
is cleaved during pro-phenoloxidase activation by Ca
2+
in coleopteran
and
Tenebrio molitor
larvae
Kum Young Lee
1
, Rong Zhang
1
, Moon Suk Kim
1
, Ji Won Park
1
, Ho Young Park
2
, Shun-ichiro Kawabata
3
and Bok Luel Lee
1
1
College of Pharmacy, Pusan National University, Jangjeon Dong, Korea;
2
Insect Resources Laboratory, Korea Research Institute
of Bioscience and Biotechnology, Taejeon, Korea;
3
Department of Biology, Kyushu University, Fukuoka, Japan
To elucidate the biochemical activation mechanism of the
insect pro-phenoloxidase (pro-PO) system, we purified a
45-kDa protein to homogeneity from the hemolymph of

Tenebrio molitor (mealworm) larvae, and cloned its cDNA.
The overall structure of the 45-kDa protein is similar to
Drosophila masquerade serine proteinase homologue, which
is an essential component in Drosophila muscle development.
This Tenebrio masquerade-like serine proteinase homologue
(Tm-mas) contains a trypsin-like serine proteinase domain in
the C-terminal region, exceptfor thesubstitution of Ser to Gly
at the active site triad, and a disulfide-knotted domain at the
amino-terminal region. When the purified 45-kDa Tm-mas
was incubated with CM-Toyopearl eluate solution contain-
ing pro-PO and other pro-PO activating factors, the resulting
phenoloxidase (PO) activity was shown to be independent of
Ca
2+
. This suggests that the purified 45-kDa Tm-mas is an
activated form of pro-PO activating factor. The55-kDa
zymogen form of Tm-mas was detected in the hemolymph
when PO activity was not evident. However, when Tenebrio
hemolymph was incubated with Ca
2+
, a 79-kDa Tenebrio
pro-PO and the 55-kDa zymogen Tm-mas converted to
76-kDa PO and 45-kDa Tm-mas, respectively, with detect-
able PO activity. Furthermore, when Tenebrio hemolymph
was incubated with Ca
2+
and b-1,3-glucan, the conversion of
pro-PO to PO and the 55-kDa zymogen Tm-mas to the
45-kDa protein, was faster than in the presence of Ca
2+

only.
These results suggestthat the cleavage ofthe55-kDa zymogen
of Tm-mas by a limited proteolysis is necessary for PO
activity, and the Tm-mas is a pro-PO activating cofactor.
Keywords: innate immunity; insect; Masquerade; pro-phe-
noloxidase; serine proteinase homologue.
The pro-phenoloxidase (pro-PO) activation system in
arthropods is an important part of the host immune
defence, where it functions to detect and kill invading
pathogens. It is also a good model system to elucidate the
pattern recognition mechanism of nonself pattern recogni-
tion proteins, such as peptidoglycan recognition protein or
b-1,3-glucan binding protein, which are part of the innate
immune reaction [1,2]. However, the molecular mechanism
of pro-PO activation remains poorly understood. Previously,
we reported the structures and functions of two pro-PO
activating factors (PPAF-I and PPAF-II) from the coleopt-
eran insect, Holotrichia diomphalia larvae [3–5]. PPAF-I is
an easter-type serine proteinase and PPAF-II is a masqu-
erade-like serine proteinase homologue. We have demon-
strated that they are necessary for activating the
phenoloxidase (PO) cascade, by in vitro reconstitution
experiments. However, we did not determine the biological
functions of PPAF-II during Holotrichia pro-PO activation.
Two questions remain to be answered: why is a masquer-
ade-like serine proteinase homologue a requirement for PO
activity, and how and why is there cross-talk between a
masquerade-like serine proteinase homologue and an eas-
ter-type serine proteinase during the pro-PO activation
reaction? Another key question that remains is how these

pro-PO activating factors can be activated in response to
microbial infection. One hypothesis is that pattern recogni-
tion proteins make a complex with pro-PO activating
enzyme(s) and microbial cell wall components, and then
activation of pro-PO activating enzyme(s) zymogen con-
verts pro-PO to active PO by limited proteolysis [6–8].
Recently we reported that in larvae of the coleopteran
insect, Tenebrio molitor, pro-PO was activated by b-1,3-
glucan and Ca
2+
, and the activated PO was involved in the
cell/clump/cell adhesion reaction as well as in the synthesis
of melanin [9]. This insect has one kind of pro-PO; however,
two kinds of pro-PO were found in H. diomphalia larvae
[9,10]. If it is possible to purify pro-PO activating factors
from T. molitor larvae, we can explain the difference
between pro-PO activation reactions in the one-pro-PO
and two-pro-PO systems in T. molitor and H. diomphalia
larvae, respectively. Also, we have reported the presence of
early staged encapsulation-relating proteins in T. molitor
larvae [11,12]. Given the crucial nature of the melanotic
Correspondence to B. L. Lee, College of Pharmacy, Pusan National
University, Jangjeon Dong, Kumjeong Ku, Busan, 609-735, Korea.
Fax: +82 51 581 1508, E-mail:
Abbreviations: PO, phenoloxidase; pro-PO, pro-phenoloxidase;
Tm-mas, Tenebrio masquerade-like serine proteinase homologue;
TCA, trichloroacetic acid; PVDF, polyvinylidene difluoride; PPAF,
pro-phenoloxidase activating factors; P-NPGB, p-nitrophenyl-
p¢-guanidinobenzoate; p-APMSF, p-amidinophenyl-methanesulfonyl
fluoride.

Note: The nucleotide sequence data reported in this paper will appear
in the DDBJ, EMBL and GenBank Nucleotide Sequence Database
with the accession number AB084067.
(Received 29 April 2002, revised 9 July 2002, accepted 29 July 2002)
Eur. J. Biochem. 269, 4375–4383 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03155.x
encapsulation response in insect immunity, it is of great
importance to purify pro-PO activating factors and to
determine the Tenebrio pro-PO activation mechanism at the
molecular level.
This paper describes the purification and cDNA cloning
of a 45-kDa protein that acts as a pro-PO activating
cofactor in T. molitor larvae. The deduced amino acid
sequence of the 45-kDa protein derived from the cDNA
revealed that this protein exhibits high sequence similarity
with Drosophila masquerade serine proteinase homologue
[13]. The biological function of the purified 45-kDa protein
was examined during pro-PO activation reaction.
MATERIALS AND METHODS
Animals, collection of hemolymph and hemocytes
T. molitor larvae (mealworm) were maintained on a labo-
ratory bench in terraria containing wheat bran. Vegetables
were placed on top of the bran to provide water. Hemo-
lymph and hemocytes were collected as described previously
[9]. Briefly, to harvest the hemolymph, larvae were injected
with 50 lL of modified anticoagulation buffer (30 m
M
trisodium citrate, 26 m
M
citric acid, 20 m
M

EDTA,
15 m
M
NaCl, pH 5.5) using a 25-G needle. The tail of each
larva was cut off using fine scissors and the extruding
hemolymph was placed in a test tube on ice. The collected
crude hemolymph was centrifuged at 203 000 g for 4 h at
4 °C. The supernatant then stored at )80 °C until use.
Assay of PO activity
PO activity was determined according to our previously
published method [3]. Briefly, 30 lL crude hemolymph
(50 lg protein) or fractionated solution from column chro-
matography were preincubated in 85 lL20m
M
Tris/HCl
pH 8.0 containing 1 lg b-1,3-glucan for 10 min at 30 °C,
andthen400 lL substrate solution (1 m
M
4-methylcatechol,
2m
M
4-hydroxyproline ethylester in 20 m
M
Tris/HCl buffer
pH 8.0, containing 5 m
M
CaCl
2
) was added to the reaction
mixture. After incubation at 30 °C for 10 min, the increase

in absorbance at 520 nm was measured using a Shimadzu
spectrophotometer. PO activity was expressed as the change
in absorbance at 520 nm per 10 min incubation (A
520
per
10 min per 30 lL). To examine the effects of the 45-kDa
protein on PO activity, a mixture of CM-Toyopearl eluate
solution (15 lg) and the purified 45-kDa protein (1 lg) were
added to 400 lL substrate solution in the presence or
absence of 5 m
M
CaCl
2
. After incubation at 30 °Cfor
10 min, the increase in absorbance at 520 nm was measured
as described above.
Purification of 45-kDa
Tenebrio
masquerade protein
(Tm-mas)
To purify 45-kDa Tm-mas from Tenebrio larvae, 50 mL
hemolymph supernatant solution were collected following
ultracentrifugation, and then concentrated by ultrafiltration
through a membrane filter (Amicon, YM-10). About 3 mL
of the concentrated solution was applied to a Toyopearl
HW-55S size exclusion column (1.5 · 50 cm) equilibrated
with buffer A (50 m
M
Tris/HCl pH 6.5, containing 5 m
M

EDTA), and eluted with buffer A at a flow rate of
12 mLÆh
)1
. Fractions specifically showing PO activity in the
presence of b-1,3-glucan and Ca
2+
were pooled. The pooled
solution was named HW-55S solution and was used to
purify 45-kDa protein. Fifty mL HW-55S solution (300 mg)
were loaded onto a Toyopearl CM-650M cation exchange
column (1 · 11 cm) equilibrated with buffer A, and washed
with buffer A until no absorbance could be detected at
280 nm. The flow-through fraction and washing solution
were combined, and concentrated by ultrafiltration. The
resulting ultrafiltered concentrated solution (3 mL) was
diluted 10 times with buffer A and loaded onto a Blue-
Sepharose (1.0 · 5.2 cm) hydrophobic column equilibrated
with buffer A. The column was washed with the same buffer
at a flow rate of 0.2 mLÆmin
)1
, until there was no
absorbance at 280 nm, and then it was eluted with buffer
A containing 0.2
M
NaCl. The eluted solution was concen-
trated by repeated ultrafiltration to exclude NaCl. To
confirm the presence of the 45-kDa protein, PO activity was
examined by incubating together the concentrated 0.2
M
NaCl elution solutions from the Blue-Sepharose and

Toyopearl CM-650M columns. For further purification of
the 45-kDa protein, the concentrated elution solution
(7 mg) from Blue-Sepharose was loaded onto a Butyl-
Toyopearl FPLC column equilibrated with buffer B [50 m
M
phosphate pH 7.0, containing 1.7
M
(NH
4
)
2
SO
4
]. The
column was eluted with a linear gradient of ammonium
sulfate (from 1.7 to 0
M
) at a flow rate of 0.3 mLÆmin
)1
.The
fractions containing an approximately 45-kDa protein as
determined by SDS/PAGE (reducing conditions) were
pooled and concentrated by ultrafiltration kit (YM-10
membrane). Fractions containing 45-kDa protein from the
Butyl-Toyopearl FPLC column were pooled and applied to
a Mono-Q FPLC anion exchange column equilibrated with
buffer C (20 m
M
Tris/HCl pH 7.4). The absorbed proteins
were eluted with a linear gradient of 0–1

M
NaCl containing
buffer C. Fractions exhibiting PO activity when incubated
with 0.2
M
NaCl eluate from the Toyopearl CM-650M
column, were pooled and concentrated by ultrafiltration.
The purified Tm-mas was reduced and S-pyridylated
according to a previously published method [14]. The
S-pyridylated 45-kDa Tm-mas was digested with trypsin,
and the resulting peptides were separated by HPLC on a C
18
reverse-phase column (Gilson). To determine the N-termi-
nal sequence of the Tm-mas, it was subjected to SDS/PAGE
under reducing conditions. The band of Tm-mas was
blotted onto a poly(vinylidene difluoride) (PVDF) mem-
brane (Millipore), cut out, and subjected to automated
amino acid sequence analysis [15]. The protein concentra-
tions were determined by the method of Lowry et al.[16]
using BSA as a standard.
Molecular cloning of 45-kDa Tm-mas
A cDNA library from T. molitor larvae was constructed by
using a ZAP-cDNA synthesis kit (Stratagene). To screen the
Tm-mas clones, we performed immunoscreening by using
the affinity-purified 45-kDa Tm-mas antibody. Following
isopropyl-b-
D
-thiogalactoside induction, 5 · 10
4
plaques

were screened with an affinity-purified antibody raised
against the purified 45-kDa Tm-mas. A secondary antibody
(alkaline phosphatase-conjugated anti-rabbit IgG, Bio-Rad)
was used at a dilution of 1 : 1000. Phage DNA was isolated
from phage lysates by using a lambda DNA preparation kit
4376 K. Y. Lee et al. (Eur. J. Biochem. 269) Ó FEBS 2002
(Biometra) according to the manufacturer’s instructions.
We analysed all of the plaques showing positive signals on
immunoscreening. We sequenced the clones according to
the dideoxy chain-termination method of Sanger et al. [17].
The amino acid sequence of the 45-kDa Tm-mas was
compared with the protein sequence database of the
National Center for Biotechnology Information using
GENETYX
(Software Development Co., Ltd, Tokyo).
Antibody and immunoblotting
Antibody against the Tm-mas was raised by injecting 10 lg
of the purified protein into a male albino rabbit with
complete Freund’s adjuvant and giving a booster injection
of the same amount of protein 14 days later [18]. The
resulting antibody was affinity-purified as described previ-
ously [11]. For immunoblotting, the proteins separated by
SDS/PAGE were transferred electrophoretically to a PVDF
membrane which was then blocked by immersion in 5%
skimmed milk solution containing 1% horse serum for 12 h.
The membrane was then transferred to rinse solution I
(20 m
M
Tris/HCl pH 7.5, containing 150 m
M

NaCl, 0.1%
Tween-20, 2.5% skimmed milk) containing the affinity-
purified antibody against the 45-kDa protein (50 ngÆmL
)1
)
and incubated at 4 °C for 2 h. The bound antibody was
identified using the Western Blot Chemiluminescence
Reagent kit (NEN
TM
Life Sciences).
The gel mobility changes of 45-kDa Tm-mas
and pro-PO during pro-PO activation
To examine the biological functions of the 45-kDa Tm-mas
during pro-PO activation, the PO activity was measured by
using HW-55S solution in the presence of b-1,3-glucan and
Ca
2+
. To examine the cleavage of 55-kDa Tm-mas at
different times, reaction mixture showing PO activity was
precipitated by trichloroacetic acid (TCA) and 45-kDa
Tm-mas was identified by Western blotting. To examine the
effects of serine proteinase inhibitors on the proteolysis of
55-kDa Tm-mas and PO activity, HW-55S solution was
preincubated for 30 min with 200 l
M
p-nitrophenyl-p¢-
guanidinobenzoate (p-NPGB) and 200 l
M
p-amidino-
phenyl-methanesulfonyl fluoride (p-APMSF), and then

reaction mixture was further incubated in the presence of
b-1,3-glucan and Ca
2+
. As a control, HW-55S solution was
preincubated without inhibitors. After 60 min incubation,
PO activity was measured and the cleaved 45-kDa Tm-mas
was identified by Western blotting.
RESULTS
Purification of 45-kDa Tm-mas
The purification procedures of Tm-mas are shown in
Fig. 1A. To isolate the pro-PO activating factors (PPAFs)
from Tenebrio larval hemolymph, we first prepared HW-
55S solution showing PO activity in the presence of b-1,3-
glucan and Ca
2+
, by using a Toyopearl HW-55S column.
As shown in Fig. 1B, HW-55S solution exhibited the most
rapid increase in PO activity in the presence of b-1,3-glucan
and Ca
2+
. Under the same conditions, PO activity was not
observed with b-1,3-glucan only. However, Ca
2+
in the
absence of b-1,3-glucan activated pro-PO, but more slowly
than a combination of b-1,3-glucan and Ca
2+
. This result
suggests that HW-55S solution contained all the necessary
PPAFs, pro-PO and b-1,3-glucan recognition protein(s). To

purify PPAF, HW-55S solution was first subjected to a
Toyopearl CM-650 column chromatography and the
flow-through fraction was purified by hydrophobic chro-
matography on Blue-Sepharose CL-6B followed on Butyl-
Sepharose. The active fractions showing PO activity were
further purified by Mono-Q FPLC column (indicated as a
bar in Fig. 2A). The purified protein migrated as a single
band of  45-kDa on SDS/PAGE (12% acrylamide) under
reducing conditions (Fig. 2B). Two-hundred mL hemo-
lymph from 4000 larvae with a protein content of 2000 mg
yielded 30 lg of the purified 45-kDa Tm-mas. The purified
Tm-mas was used for raising polyclonal antibody. The
Tenebrio 79-kDa pro-PO was purified according to our
previously published methods [9].
Fig. 1.
11
(A) Procedures used to purify 45-kDa Tm-mas from the
hemolymph of T. molitor larvae and (B) the PO activity of HW-55S
solution. POactivitywasmeasuredbyincubationwith30lLHW-55S
solution with Ca
2+
and b-1,3-glucan (d), Ca
2+
only (m), b-1,3-glucan
only (j) and with neither Ca
2+
nor b-1,3-glucan (s).
Ó FEBS 2002 Masquerade-like serine proteinase homologue (Eur. J. Biochem. 269) 4377
In vitro
reconstitution experiments

To confirm the possibility of the purified 45-kDa Tm-mas as
Tenebrio PPAF, we performed in vitro reconstitution
experiments by using collected fractions or the purified
proteins from column chromatography. As shown in
Fig. 3A, when Toyopearl CM-flow-through and Toyopearl
CM-eluate were incubated in the presence of b-1,3-glucan
and Ca
2+
, PO activity was clearly shown to be dependent
on b-1,3-glucan and Ca
2+
(column 5). Tenebrio pro-PO was
localized in Toyopearl CM-eluate solution by Western
blotting analysis (data not shown). Toyopearl CM-flow-
through solution was further purified by Blue-Sepahrose
and Mono-Q FPLC column. When the purified Tenebrio
pro-PO from Toyopearl CM-eluate and the purified
Tm-mas from Toyopearl CM-flow-through were incubated,
PO activity was not observed (Fig. 3B, column 4). However,
when Toyopearl CM-eluate and the purified Tm-mas were
incubated, PO activity was shown to be independent of
Ca
2+
(column 5 and 6). These results suggested two things:
first, that another protein(s) in Toyopearl CM-eluate might
be necessary for PO activity; second, that the 45-kDa
Tm-mas already seemed to be activated from its zymogen
form, during the process of column chromatography. We
examined this latter possibility by Western blotting analysis.
As shown in Fig. 3C, the affinity-purified antibody raised

against the 45-kDa Tm-mas recognized a 45-kDa single
band in the Mono-Q fraction (lane 4). However, a 55-kDa
protein was recognized in crude hemolymph (lane 1) and
Toyopearl CM-flow-through (lane 2). Both the 55-kDa and
45-kDa proteins were recognized in the Blue-Sepharose
fraction (lane 3). This result suggests that the purified
45-kDa protein is activated from a precursor 55-kDa
zymogen form by limited proteolysis during Blue-Sepharose
column and Mono-Q FPLC column chromatography.
Isolation of cDNA clone for Tm-mas
To determine the whole amino acid sequence of the purified
Tm-mas, we first determined three partial amino acid
sequences by trypsin digestion as follows: NSQGIDFNLI,
GNLYNDIALL and NPNRYLQVGIVA. However, an
N-terminal sequence could not be obtained due to blockage.
To isolate a cDNA clone for the 45-kDa Tm-mas, we
immunoscreened the cDNA library of T. molitor larvae
Fig. 2. (A) Elution pattern of Mono-Q FPLC column and (B) SDS/
PAGE pattern of each column step during 45-kDa Tm-mas purification.
(A) The Mono-Q column fractions indicated by bars were collected
and used for subsequent experiments (see Materials and methods). (B)
Lane 1, HW-55S solution; lane 2, the eluate solution of Toyopearl
CM-650; lane 3, flow-through fractions of Toyopearl CM-650; lane 4,
eluate solution of Blue-Sepharose CL-6B column, lane 5, active frac-
tion of Mono-Q FPLC column. The proteins were analysed by SDS/
PAGE (10% acrylamide) under reducing conditions.
Fig. 3. (A) PO activities of HW-55S and fractions of Toyopearl
CM-650 column, (B) PO activities between the purified pro-PO and the
eluate solution of Toyopearl CM-650, and (C) Western blotting of
products during 45-kDa Tm-mas purification. (C) Proteins were pre-

cipitatedwithTCAandsubjectedtoSDS/PAGEandthenimmu-
noblotting with affinity-purified antibodies raised against 45-kDa
Tm-mas. Lane 1, 10 lg hemolymph protein; lane 2, 10 lgflow-
through solution of Toyopearl CM-650 column; lane 3, 5 lgeluate
solution of Blue-Sepharose column; lane 4, 1 lg of the active fractions
of Mono-Q FPLC column. The molecular size markers are indicated
to the right in kDa.
4378 K. Y. Lee et al. (Eur. J. Biochem. 269) Ó FEBS 2002
with an affinity-purified antibody against the 45-kDa Tm-
mas, and obtained 10 positive clones. The nucleotide
sequence and the deduced amino acid sequence of one of
these clones, named Tm-mas, are shown in Fig. 4. This
cDNA contained an open reading frame of 1332 nucleotides
corresponding to 444 amino acid residues with a precise
mass of 48 814 Da. The apparent mass of 55-kDa for Tm-
mas on SDS/PAGE is slightly larger than that of the
deduced sequence. This mobility pattern is similar to several
other masquerade proteinase homlogue’s [5,19] where the
mass of the purified masquerade-like proteins is higher
under nonreducing than reducing conditions suggesting that
disulfide bonding is responsible for the higher mass. The
three chemically determined partial amino acid sequences of
the 45-kDa Tm-mas coincided with the deduced amino acid
sequences in this open reading frame (dotted lines). There-
fore, we concluded that this is a cDNA for the Tm-mas. The
Tm-mas has two domains, an N-terminal domain and a
serine proteinase-like domain. The hydrophobic first 22
amino acids of the N-terminal end of the protein probably
constitute a signal peptide sequence with a putative signal
peptidase cleavage site between Ala22 and Ile23 (arrow-

head) [20]. One putative disulfide-knotted motif is present in
the N-terminal domain of the protein. The putative catalytic
domain, from Asn176 (arrow) to Glu444, is characteristic of
that found in serine proteinase homologues, as is the
presence of a Gly residue instead of a Ser residue in the
catalytic site. The residues of a serine proteinase substrate
binding pocket (open diamond in Fig. 5), which determine
the substrate specificity of active serine proteinases, were
present in the 45-kDa Tm-mas. The six cysteine residues
(closed circles in Fig. 5), which form three disulfide bridges
in most serine proteinases, were conserved in this 45-kDa
Tm-mas. There were two potential N-glycosylation sites
(Asn-Xaa-Set/Thr, indicated by closed-diamonds in Fig. 4).
A homology between the Tm-mas and those in the NCBI
database showed that the deduced amino acid sequence of
the Tm-mas was similar to Holotrichia 45-kDa protein (Hd-
PPAF-II, 59.1%) [5], Tachyplus factor D (Td-D, 38.9%)
[21], masquerade of Drosophila melanogaster (Dm-mas,
35.2%) [13], masquerade-like protein of crayfish Pacifasta-
cus leniusculus (Pl-mas, 41.2%) [22], mosquito infection-
response serine proteinase-like protein (Ag-ispl5, 34.1%)
[23], Tenebrio 45-kDa (Tm-45, 46.6%) [5], as well as
Holotrichia PPAF-I (Hd-PPAF-I, 33.3%) [4] and Tachyplus
proclotting enzyme (Td-PCE, 31.8%) [24] as shown in
Fig. 5. The proteins Ag-ispl5, Td-D, Pl-mas, and Dm-mas
showed amino acid substitutions of Ser with Gly (or Ala) in
the active site triad (arrow). In conclusion, a schematic
comparison of the main structural features of Tm-mas with
Hd-PPAF-II, AP-ispl5, Tm-45, Td-D and Dm-mas showed
a modified serine proteinase domain at the C terminus, and

a disulfide-knotted motif present in the N terminus.
Previously we found that a disulfide-knotted motif of
Hd-PPAF-I was present in big defensin [25], an antimicro-
bial peptide purified from Tachyleus hemocytes. Six cysteine
residues of the disulfide-knotted motif of Tm-PPAF-I are
also conserved with those of other disulfide-knotted motifs.
Fig. 4. Nucleotide and deduced amino acid
sequences of cloned cDNA encoding the 45-kDa
Tm-mas. Numbers of nucleotides starting
from the first base at the 5¢ end are shown on
the left of each line; the deduced amino acid
sequence is numbered from the initiating Met
residue on the right of each line. The chemi-
cally determined three partial amino acid
sequences of the 45-kDa Tm-mas are under-
lined by dots. The potential attachment sites
for N-linked carbohydrate chains are indicat-
ed by r sites of the catalytic triad of serine
proteinases are shown by s; the arrowhead
indicates the putative cleavage site for the
signal peptide; the arrow indicates the putative
start residue of catalytic domain.
Ó FEBS 2002 Masquerade-like serine proteinase homologue (Eur. J. Biochem. 269) 4379
However, the biological function of this motif has not yet
been determined. Finally, the purified Tm-mas exhibited no
amidase activity against a variety of commercially available
peptidyl-NH-Mec substrates tested (data not shown).
Determination of Tm-mas localization
To examine the localization of Tm-mas, we firstly prepared
fat body, plasma, hemocyte lysate and hemolymph from

T. molitor larvae.AsshowninFig.6,noappreciable
amount of 55-kDa zymogen Tm-mas was detected in the
hemocyte lysate or fat body (lanes 2 and 3). A significant
amount of protein, however, was detected in the plasma and
hemolymph (lanes 4 and 5), indicating that Tm-mas was
localized in the plasma.
Biochemical characteristics of Tm-mas during pro-PO
activation
To determine the biological function of the purified 45-kDa
Tm-mas in the Tenebrio pro-PO system, we used SDS/
PAGE and Western blotting to assess whether changes in
PO activity were reflected in changes in the gel mobility of
Tenebrio pro-PO and the 55-kDa zymogen Tm-mas. As
showninFig.7A, 25% of Tenebrio pro-PO from HW-
55S was activated to PO in the presence of Ca
2+
after
45 min incubation (lane 6); however, when HW-55S
solution was incubated with b-1,3-glucan and Ca
2+
,
 50% of pro-PO was activated after 45 min (lane 11).
Under the same conditions, all of the 55-kDa zymogen form
of Tm-mas had converted to 45-kDa Tm-mas by 45 min, in
the presence of b-1,3-glucan and Ca
2+
(lane 11 in Fig. 7B).
The activation ratio of the 55-kDa zymogen form in the
presence of b-1,3-glucan and Ca
2+

wasfasterthaninthe
presence of Ca
2+
only (compare lane 6 with lane 11 in
Fig. 7B). This result suggests that the activation of the
55-kDa zymogen form to the 45-kDa Tm-mas is a
requirement for PO activity in the Tenebrio Pro-PO
activation system. Furthermore, we examined the effects
of serine proteinase inhibitors against proteolysis of the
55-kDa Tm-mas and PO activity by using two kinds of
serine proteinase inhibitors, such as p-NPGB and
p-APMSF. As shown in Fig. 7C and D, when HW-55S
Fig. 6. Localization of the 55-kDa Tm-mas determined by immuno-
blotting. Hemolymph was obtained from 150 larvae and centrifuged at
700 g
2
at 4 °C for 10 min. Precipitated hemocytes were washed with
500 lL anticoagulation buffer (pH 5.5) and suspended again with
500 lL anticoagulation buffer. One hundred lL of this suspension
provided the total haemocyte component. The remaining solution
(400 lL) was sonicated for 15 s at 4 °Candcentrifugedat
15 000 r.p.m. (22 000 g)at4°C for 10 min The supernatant was used
as haemocyte lysate. The soluble proteins were precipitated with TCA
and subjected to SDS/PAGE and then immunoblotting with affinity-
purified antibody raised against the 45-kDa Tm-mas. Lane 1, 10 lg
protein of flow-through solution from Toyopearl CM-650 column;
lane 2, 10 lg of soluble fat body protein; lane 3, 10 lg of soluble
haemocyte lysate protein; lane 4, 10 lgofplasmaprotein;lane5,10 lg
of haemolymph protein.
Fig. 5. Alignment of the catalytic domains of Tm-mas and Holotrichia 45-kDa protein (Hd-PPAF-II) with catalytic domains of known serine

proteinase homologues. Tachyplus factor-D (Tt-D), Drosophila masquerade (Dm-mas), crayfish masquerade (Pl-mas), Anopheles immune response
serine proteinase-like protein (Ap-ispl5) and known serine proteinase, such as Holotrichia PPAF-I (Hd-PPAF-I) and Tachyplus proclotting enzyme
(Tt-PCE). Numbers refer to the predicted protein sequence. Stars indicate the residues of the catalytic triad of serine proteinase. The conserved
cysteine residues are indicated by d; residues conserved in all sequences are shown within boxes; e indicate the positions of residues known to
occupy the substrate binding pockets of trypsin; the arrow indicates the substitution residue of the catalytic triad of serine proteinase. Gaps were
introduced to obtain maximal sequence similarity.
4380 K. Y. Lee et al. (Eur. J. Biochem. 269) Ó FEBS 2002
solution was preincubated with p-NPGB and p-APMSF,
andthencalciumandb-1,3-glucanwereaddedtoHW-55S
solution, PO activity and proteolysis of the 55-kDa Tm-mas
were not observed (Fig. 7C, column 3 and Fig. 7D, lane 3).
However, PO activity and the cleavage of the 55-kDa
Tm-mas were clearly shown when calcium and b-1,3-glucan
were added to HW-55S solution in the absence of inhibitors
(Fig. 7C, column 2 and Fig. 7D, lane 2). These results
suggest that the cleavage of the 55-kDa Tm-mas might be
induced by unidentified serine proteinase. Also, it was
confirmed that PO activity is shown when the 55-kDa
Tm-mas is cleaved to the 45 kDa Tm-mas.
DISCUSSION
In this study, we isolated a novel 45-kDa protein from the
hemolymph of T. molitor larvae, which showed high
homology (35% sequence identity) with Drosophila mas-
querade serine proteinase homologue. We have called this
protein Tenebrio masquerade-like proteinase homologue
(Tm-mas). This is also the first report to demonstrate that a
novel masquerade-like serine proteinase homologue zymo-
gen (55-kDa Tm-mas) is cleaved to 45-kDa Tm-mas as a
prerequisite for PO activity. Several kinds of serine
proteinase homologue have been purified already from

vertebrates and invertebrates, and they have been suggested
to perform different biological functions, including antim-
icrobial activities (e.g. horseshoecrab factor D [21] and
human azurocidin [26]), or by acting as adhesion molecules
(e.g. Drosophila masquerade [13], Pacifastacus masquerade-
like protein [22], glutacin [27] and neurotactin [28]), immune
molecules (e.g. mosquito ispl5 [23], Holotrichia PPAF-II
[5]), growth factors (e.g. human hepatocyte growth factor
[29]) or as pattern recognition proteins in crayfish [19]. As
their name indicates, all known serine proteinase homo-
logues are very similar to serine proteinases, differing only in
the substitution of their catalytic residues.
It is suggested that the pro-PO activating system, which
functions in nonself recognition and defence responses in
invertebrates, is composed of an enzyme cascade consisting
of pattern recognition proteins, several serine proteinases
and pro-PO [1,2]. Recently our group and three other
groups have reported that Drosophila easter-type serine
proteinases with disulfide-knotted domain(s) are involved in
the pro-PO activation system [4,30–32]. Recently we have
reported that Holotrichia masquerade-like PPAF-II (Hd-
PPAF-II) is also engaged in Holotrichia pro-PO activation
and is cleaved at Arg99–Glu100 by an easter-type serine
Fig. 8. Amino acid sequence comparison of the cleavage sites for
Tm-mas and Hd-PPAF-II. The arrow indicates the cleavage site of
Hd-PPAF-II by Hd-PPAF-I.
Fig. 7. Western blotting of (A) Tenebrio 79-kDa pro-PO, and (B)
55-kDa Tm-mas in HW-55S solution by incubation with Ca
2+
and

b-1,3-glucan and the effects of serine proteinase inhibitors on (C) PO
activity and (D) proteolysis of 55-kDa Tm-mas. PO activity of HW-55S
solution was measured as described in Fig. 1. The reaction mixtures at
different times were precipitated with TCA and subjected to SDS/
PAGE and then immunoblotting with the affinity-purified antibody
raised against the Tenebrio 79-kDa pro-PO and 45-kDa Tm-mas. (A)
The 79-kDa and 76-kDa bars indicate Tenebrio pro-PO and PO,
respectively. (B) The 55-kDa and 45-kDa bars indicate the zymogen
form and cleaved protein of 45-kDa Tm-mas, respectively. Lane 1,
30 lL HW-55S solution was incubated with neither Ca
2+
nor b-1,3-
glucan for 60 min; lane 2, incubation with b-1,3-glucan only for
60 min; lane 3, 4, 5, 6 and 7, incubation with Ca
2+
only for 10, 20, 30,
45, 60 min, respectively; lane 8, 9, 10, 11 and 12, incubation with Ca
2+
and b-1,3-glucan for 10, 20, 30, 45, 60 min, respectively. (C) Incuba-
tion conditions with serine proteinase inhibitors were described in
Materials and methods. PO activity of HW-55S solution with or
without inhibitors was measured as described in Fig. 1. Column 1,
after 0 min incubation without inhibitors; column 2, after 60 min
incubation without inhibitors; column 3, after 60 min incubation with
inhibitors. D, The reaction mixtures of Fig. 7C at different times were
precipitated with TCA and subjected to SDS/PAGE and then
immunoblotting with the affinity-purified antibody raised against the
45-kDa Tm-mas.
Ó FEBS 2002 Masquerade-like serine proteinase homologue (Eur. J. Biochem. 269) 4381
proteinase [5]. To address the possibility that Tm-mas also

has a similar cleavage site to Holotrichia masquerade-like
PPAF-II, we have compared the Tm-mas amino acid
sequence with the sequence in the vicinity of the Hd-PPAF-
II cleavage site. As shown in Fig. 8, the tentative cleavage
site of Lys98-Glu99 in Tm-mas is perfectly conserved in
Holotrichia masquerade-like PPAF-II, suggesting that a
serine proteinase cleaves the Lys98–Glu99 site of Tm-mas.
However, in this study, the identity of the serine proteinase
that cleaves the 55-kDa zymogen form, has not been
elucidated. Further studies focusing on this unidentified
serine proteinase will provide clues to understanding the
biological function of serine proteinase homologues in the
pro-PO activation system.
Previously, we reported the cDNA sequence of another
masquerade-like serine proteinase homologue (Tm-45
protein), obtained from a Tenebrio cDNA library that
had been cloned following screening with Holotrichia
PPAF-II antibody [5]. The deduced amino acid sequence
of Tm-45 protein has 46.6% similarity with the purified
45-kDa Tm-mas described in the present report (data not
shown). Although the Tm-45 protein has not yet been
purified, any differences we may be able to detect between
the biological functions of Tm-45 and Tm-mas will be
important for understanding the details of the Tenebrio
pro-PO system.
One interesting point regarding masquerade-like serine
proteinase homologues, is that horseshoecrab factor D
copurified with a serpin during purification procedures [21],
which suggests that masquerade-like serine proteinases
might make a complex with serpin. During insect pro-PO

activation in response to invasion by a microbial pathogen,
there is a possibility that zymogen Tm-mas could be released
from a serpin, and that the released zymogen form of
Tm-mas could be cleaved at the Arg/Lys–Glu site by an
easter-type serine proteinase. The cleaved Tm-mas would
then be able to cause pro-PO activation by acting as a
cofactor. Further studies will be required to test this
hypothesis.
ACKNOWLEDGEMENTS
This work was supported mainly by research grants No. R01-1999-
00118 from the KOSEF to B. L. Lee. This work was also supported by
KOSEF grant (No. 20005-209-02-2) to B. L. Lee.
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