Báo cáo khoa học: Molecular characterization of a blood-induced serine carboxypeptidase from the ixodid tick Haemaphysalis longicornis docx

Molecular characterization of a blood-induced serine
carboxypeptidase from the ixodid tick Haemaphysalis
longicornis
Maki Motobu
1
, Naotoshi Tsuji
1
, Takeharu Miyoshi
1
, Xiaohong Huang
1
, M. K. Islam
1
,
M. A. Alim
1
and Kozo Fujisaki
2,3
1 Laboratory of Parasitic Diseases, National Institute of Animal Health, Ibaraki, Japan
2 National Research Centre for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Japan
3 Laboratory of Emerging Infectious Diseases, Kagoshima University, Japan
Proteases are known to play essential roles in a wide
range of biological processes, including the degrada-
tion of regulatory proteins, precursor processing, apop-
tosis and digestive processes. For both endo- and
ectoparasites, proteases are involved in parasite inva-
sion and survival [1–3]. It has been postulated that
exopeptidases may also take part in the proteolytic
cascades for hemoglobin (Hb) degradation [4].
Serine carboxypeptidases (SCPs) belong to the
a ⁄ b-hydrolase-fold enzyme superfamily and contain

a conserved amino acid triad, Ser-Asp-His, which
catalyzes hydrolysis of C-terminal residues in peptides
and proteins at acidic pH [5]. SCPs are widely distri-
buted among fungi, plants and animals. Among SCPs,
yeast serine carboxypeptidase Y (CPY) has been well
studied, and has been shown to participate in the
processing of precursors to form secreted mature
proteins [6,7]. In plants, SCPs have been shown to be
involved in growth, apoptosis, brassinosteroid signa-
ling and seed development [8–12]. In arthropods,
however, there is little information available on SCPs.
Recently, a SCP has been identified in the orange
wheat blossom midge, Sitodiplosis mosellana, and has
been shown to have dual functions as a digestive
Keywords
blood digestion; haemoglobin; hydrolysis
activity; serine carboxypeptidase; tick
Correspondence
N. Tsuji, Laboratory of Parasitic Diseases,
National Institute of Animal Health, National
Agriculture and Food Research Organization,
3-1-5 Kannonndai, Tsukuba, Ibaraki
305-0856, Japan
Fax: +81 29 838 7780
Tel: +81 29 838 7749
E-mail:
Database
The nucleotide sequence data has been
deposited in the GenBank database under
the accession number AB287330

(Received 7 February 2007, revised 16 April
2007, accepted 1 May 2007)
doi:10.1111/j.1742-4658.2007.05852.x
Ticks feed exclusively on blood to obtain their nutrients, but the gene
products that mediate digestion processes in ticks remain unknown. We
report the molecular characterization and possible function of a serine
carboxypeptidase (HlSCP1) identified in the midgut of the hard tick Haem-
aphysalis longicornis. HlSCP1 consists of 473 amino acids with a peptidase
S10 family domain and shows structural similarity with serine carboxypep-
tidases reported from other arthropods, yeasts, plants and mammals.
Endogenous HlSCP1 is strongly expressed in the midgut and is supposed
to localize at lysosomal vacuoles and on the surface of epithelial cells.
Endogenous HlSCP1, identified as a 53 kDa protein with pI value of 7.5,
was detected in the membrane ⁄ organelle fraction isolated from the midgut,
and its expression was upregulated during the course of blood-feeding. En-
zymatic functional assays revealed that a recombinant HlSCP1 (rHlSCP1)
expressed in yeast efficiently hydrolyzed the synthetic substrates specific for
cathepsin A and thiol protease over a broad range of pH and temperature
values. Furthermore, rHlSCP1 was shown to cleave hemoglobin, a major
component of the blood-meal. Our results suggest that HlSCP1 may play a
vital role in the digestion of the host’s blood-meal.
Abbreviations
CPY, yeast serine carboxypeptidase Y; E64, trans-epoxysuccinyl-
L-leucylamido-(4-guanidino) butane; Hb, hemoglobin; HlSCP, Haemaphysalis
longicornis serine carboxypeptidase; Pyr,
L-pyroglutamyl; SCP, serine carboxypeptidase; Suc, succinyl; Z, benzyloxycarbonyl.
FEBS Journal 274 (2007) 3299–3312 ª 2007 The Authors Journal compilation ª 2007 FEBS 3299
enzyme and an exopeptidase involved in degrading
vitellogenin [13].
The host’s blood-meal is the only source of energy in

ticks. Unlike blood-sucking insects, ticks make a blood
pool by rupturing blood vessels under the host’s skin
and feed on this blood for a relatively long period,
varying from several days to weeks, depending on the
life stage, the host type and the species of tick involved
[14]. Only one blood-meal is taken during each life
stage, and after completion of feeding, ticks can survive
for several months without a further blood-meal [15].
Because ticks have such unique feeding behavior, it is
speculated that they are equipped with an efficient
blood-digestion and nutrient-utilization system for sur-
vival. In addition, ticks act as vectors of disease-causing
agents in humans and animals by injecting their saliva,
which contains anticoagulants and other bioactive com-
ponents as well as pathogens, into the blood pool dur-
ing feeding [16]. Suppression of tick vector populations
is thus crucial for controlling diseases transmitted by
ticks. However, chemical acaricides, which are cur-
rently used for tick control, have the disadvantages of
causing acaricidal resistance problems [17], leading to
food animals containing chemical residues, which are a
threat to human health. Consequently, novel approa-
ches are sought to control tick populations based on
tick-specific potential biochemical pathways.
We describe here the cloning and partial characteri-
zation of a cDNA encoding a SCP from the ixoidid
tick Haemaphysalis longicornis, which has a wide geo-
graphical distribution in Russia, eastern Asia, Austra-
lia, and New Zealand, and has the potential to
transmit pathogens including viruses, rickettsia and

protozoan parasites that cause important human and
animal diseases [18–20]. The deduced precursor protein
contains amino acids conserved among peptidase S10
family members and SCPs. Endogenous H. longicornis
serine carboxypeptidase (HlSCP1) was strongly
expressed in the vacuoles of midgut epithelial cells,
and its expression was found to be upregulated by
the blood-digestion process. A recombinant HlSCP1
(rHlSCP1) expressed in Phicia pastoris hydrolyzed not
only synthetic peptide substrates for SCP and thiol
proteases, but also bovine Hb. These findings suggest
that HlSCP1 may be involved in digestion of the host’s
blood-meal.
Results
HlSCP1 cDNA encodes a SCP homolog
Sequence analysis revealed that HlSCP1 cDNA is
1688 bp long. The start codon is predicted at nucleo-
tides 146–148 and there is a stop codon at nucleotides
1565–1567. HlSCP1 cDNA has an ORF extending
from position 146 to position 1567, coding for 473
amino acids with a predicted molecular mass of
53 294 Da (Fig. 1). This deduced protein has a poss-
ible cleavable signal peptide of 27 amino acid residues
and the preprotein has a predicted molecular mass of
50 365 Da. The HlSCP1 sequence possesses a single
peptidase S10 family domain consisting of the evolu-
tionarily conserved regions and three catalytic residues
[21]. These serine, histidine, and aspartic acid residues
that are known to form the catalytic triad of SCPs
are found in HlSCP1 at positions 178, 450 and 397,

respectively. There are three potential sites (residues
357, 416 and 439) for N-glycosylation in the putative
polypeptide encoded by HlSCP1. A search of the
protein database using the National Center for Bio-
technology Information revealed that HlSCP1 has
sequence similarity with SCPs from Sitodiplosis mosell-
ana (GenBank accession no. AAY27740, 27%
identity), Arabidopsis thaliana (NP_194790, 32%),
Saccharomyces cerevisiae CPY (CAA56806, 28%),
chicken cathepsin A (NP_001026662, 39%), mouse
cathepsin A (AAA39982, 40%) and human cathep-
sin A (NP_000299, 39%).
Endogenous HlSCP1 is localized in midgut
epithelial cells
Two-dimensional immunoblotting was performed to
identify endogenous HlSCP1 in ticks. Mouse anti-
rHlSCP1 serum reacted with a protein having a
molecular mass of 53 kDa and a pI of 7.5 (Fig. 2A),
corresponding to the predicted size of the putative pro-
tein calculated from the HlSCP1 amino acid sequence.
To detect the localization of endogenous HlSCP1,
immunohistochemistry was performed in adult female
ticks, blood-fed for a total period of 72 h, using mouse
anti-rHlSCP1 serum. It was found that endogenous
HlSCP1 was mainly expressed in the midgut of the
female ticks (Fig. 2B). Serum from mice prior to
immunization, however, did not show any reactivity.
Subcellular localization of HlSCP1
To clarify the subcellular localization of HlSCP1 in the
midgut, immunoblot analysis of the subcellular frac-

tions of midgut tissues obtained from 72-h-fed adult
female H. longicornis was performed using mouse anti-
HlSCP1 serum. An immunoreactive band was detected
in the fraction containing membranes and membrane
organelles (Fig. 3A). To determine the endogenous
form of HlSCP1 in the midgut epithelial cells,
Tick serine carboxypeptidase M. Motobu et al.
3300 FEBS Journal 274 (2007) 3299–3312 ª 2007 The Authors Journal compilation ª 2007 FEBS
Fig. 1. Comparison of the deduced HlSCP1 amino acid sequence with several known SCPs. S.m., Sitodiplosis mosellana (GenBank acces-
sion no. AAY27740); A.t., Arabidopsis thaliana (NP_194790); Ye, Saccharomyces cerevisiae carboxypeptidase Y (CAA56806); Mo, mouse
cathepsin A (AAA39982); Hu, human cathepsin A (NP_000299); Ch, chicken (NP_001026662). Asterisks, identical or conserved residues;
colons, conservative substitutions; periods, semiconservative substitutions. Three conserved regions, characteristic of SCPs, are underlined.
Conserved catalytic triad residues are marked with rhombuses. The vertical arrow shows the signal peptide cleavage site. Numbers on the
right refer to the amino acids within the sequences.
M. Motobu et al. Tick serine carboxypeptidase
FEBS Journal 274 (2007) 3299–3312 ª 2007 The Authors Journal compilation ª 2007 FEBS 3301
immunofluorescent staining of flat sections of 72-h-fed
adult H. longicornis was performed. Examination of the
stained sections revealed that HlSCP1 is localized in the
vacuoles of midgut endothelial cells, where the ingested
host’s blood-meal is thought to be degraded by proteas-
es (Fig. 3B). These results suggest that that HlSCP1 is
localized in lysosomal vacuoles and on the cell surface.
Expression of endogenous HlSCP1 is induced
by the blood-feeding process
Expression levels of endogenous HlSCP1 were also
examined in unfed and partially fed adult ticks. Immu-
noblot analysis showed that HlSCP1 was expressed
weakly at 24 h and its expression was significantly
increased at 72 and 96 h of blood-feeding (Fig. 4A). In

accordance with the results of immunoblot analysis,
the fluorescence intensity of HlSCP1 in the midgut
epithelial cells also gradually increased as blood-
feeding progressed, reaching a maximum at 72 h of
blood-feeding (Fig. 4B). These results suggest that
endogenous HlSCP1 expression is induced by the
blood-feeding process.
Purity of the recombinant HlSCP1
The ORF of HlSCP1, except for the signal sequence,
was subcloned into the pPICZB vector (Invitrogen,
Carlsbad, CA). Recombinant HlSCP1 was expressed in
P. pastoris and found to migrate as a 54 kDa fusion
protein with a hexahistidine tag of 3 kDa by
SDS ⁄ PAGE (Fig. 5, lane 3). The molecular mass of
rHlSCP1 protein is  51 kDa, similar to the mass pre-
dicted from the amino acid sequence of HlSCP1 exclu-
ding the signal sequence. rHlSCP1 was purified by
metal chelation chromatography under native condi-
tions. The purified rHlSCP1 was used for enzyme
activity and Hb hydrolysis assays.
Recombinant HlSCP1 is an active SCP
A series of synthetic substrates for SCPs was used in
kinetic analyses to determine the values of K
m
, k
cat
and k
cat
⁄ K
m

for rHlSCP1. As shown in Table 1,
rHlSCP1 had a substrate preference for benzyloxy-
carbonyl (Z)-Phe-Leu and Z-Phe-Ala over Z-Glu-Tyr.
The k
cat
values were comparable among the substrates,
but the K
m
value for Z-Glu-Tyr was 10 times higher
than that of the other substrates. SCPs, classified as
55
36
66
A
B
31
3.5 07.0
pI
Fig. 2. Endogenous HlSCP1 in adult female H. longicornis. (A) Iden-
tification of HlSCP1 by 2D-PAGE. Fifty micrograms of tick extract
were separated using 2D pH gradient gel electrophoresis, and the
proteins were transferred to a nitrocellulose membrane. The arrow
shows endogenous HlSCP1. (B) Immunohistochemical localization
of HlSCP1. Ticks after 72 h of blood-feeding were fixed in parafor-
maldehyde and embedded in paraffin. Flat sections of a whole adult
tick were exposed to mouse anti-HlSCP1 serum (upper; scale bar,
1 mm; lower left; scale bar, 100 lm) or preimmune mouse sera
(lower right; scale bar, 100 lm). cu, cuticle; mu, muscle; mg, mid-
gut; sg, salivary gland. The area marked by a square is shown at
higher magnification.

B
Cy Me Nu Cs
A
Fig. 3. Intracellular localization of endogenous HlSCP1 in midgut epi-
therial cells. (A) Identification of HlSCP1 by immunoblotting. Fraction-
ated proteins of midgut epithelial cells were separated by
SDS ⁄ PAGE, and the proteins were then transferred to a nitro-
cellulose membrane. The membrane was reacted with mouse
anti-HlSCP1 serum diluted 1 : 150. Cy, cytosolic fraction; Me, mem-
branes and organelles; Nu, nuclear fraction; Cs, cytoskeleton. The
arrow shows endogenous HlSCP1. (B) Immunofluorescence staining
of HlSCP1. Midgut epithelial cells were collected from midguts of
72 h blood-feeding ticks. The cells were fixed in paraformaldehyde,
permeabilized with Triton X-100, and incubated with mouse anti-
HlSCP1 serum followed by visualization with Alexa FluorÒ 488
(green). Nuclei were stained with DAPI (blue) (scale bar, 50 lm).
Tick serine carboxypeptidase M. Motobu et al.
3302 FEBS Journal 274 (2007) 3299–3312 ª 2007 The Authors Journal compilation ª 2007 FEBS
carboxypeptidase Cs, have high affinity for hydropho-
bic amino acids at the P1¢ position [5]. The affinity of
members of the carboxypeptidase C family, such as
CPY and cathepsin A, for hydrophobic amino acids is
similar to that of rHlSCP1 [22,23]. In addition to SCP
activity, it was found that rHlSCP1 hydrolyzed l-pyro-
glutamyl (Pyr)-Phe-Leu-pNA, a substrate for thiol
proteases (K
m
¼ 1.46 · 10
)4
m

)1
, k
cat
¼ 20.1 s
)1
) and
showed much lower enzyme activities toward Z-Ala-
Ala-Leu-pNA, succinyl (Suc)-Ala-Ala-pNA and Bz-
(dl)-Arg-pNA, substrates for subtilisin A, elastase and
trypsin, respectively (data not shown). To determine
the optimum conditions for rHlSCP1 activity toward
the substrates Z-Phe-Leu and Pyr-Phe-Leu-pNA, the
enzyme activities were assayed at different pH values
and temperatures. The hydrolysis of Z-Phe-Leu by
rHlSCP1 was optimal at pH 6 but significantly
decreased at pH values > 7 (Fig. 6A). By contrast,
enzyme activity toward Pyr-Phe-Leu-pNA had a pH
optimum of 7 and weak activity was still seen at pH 9.
With increasing temperature, the activity was
enhanced, reaching a maximum at 45 °C for both sub-
strates (Fig. 6B). Relatively higher enzyme activities
toward Z-Phe-Leu were observed over a broad tem-
perature range (37–50 °C). The same activities were
also seen when rHlSCP1 was preincubated at 37–50 °C
kDa
A
B
Fed (h)
Unfed 7224 96
31

55
36
66
21
Fig. 4. Induction of endogenous HlSCP1
expression by the blood-feeding process. (A)
Expression pattern of endogenous HlSCP1
during the blood-feeding process. Soluble
antigens from unfed and partially fed
(24–96 h blood-feeding) adult ticks were
separated by SDS ⁄ PAGE, and the proteins
were transferred to a nitrocellulose
membrane. The membrane was reacted
with mouse anti-HlSCP1 serum diluted
1 : 150. The arrow shows endogenous
HlSCP1. (B) Immunofluorescence staining of
HlSCP1 in midgut epithelial cells. Ticks were
fixed in paraformaldehyde and embedded in
paraffin. Flat sections of a whole adult tick
were exposed to mouse anti-HlSCP1 serum
followed by visualization with Alexa FluorÒ
488 (green). Nuclei were stained with DAPI
(blue) (scale bar, 25 lm).
M. Motobu et al. Tick serine carboxypeptidase
FEBS Journal 274 (2007) 3299–3312 ª 2007 The Authors Journal compilation ª 2007 FEBS 3303
for 3 h. The effects of protease inhibitors on rHlSCP1
hydrolysis activities were examined (Table 2), and the
inhibition pattern indicated that the recombinant
enzyme belongs to the serine protease family, because
phenylmethylsulfonyl fluoride, a serine protease inhib-

itor, significantly inhibited the hydrolytic activity of
rHlSCP1 toward both substrates (Table 2). It is nota-
ble that pepstatin A, an aspartic protease inhibitor,
showed different inhibitory effects between the sub-
strates Pyr-Phe-Leu-pNA and Z-Phe-Leu: it inhibited
the hydrolysis of Pyr-Phe-Leu-pNA, a thiol substrate,
more potently than cysteine protease inhibitors such
as trans-epoxysuccinyl-l-leucylamido-(4-guanidino)
butane (E64) and leupeptin, whereas only a slight
inhibitory effect of pepstatin A on the hydrolysis of
kDa
12
66
55
36
31
3
21
14
Fig. 5. SDS ⁄ PAGE analysis of rHlSCP1 expressed in P. pastoris.
The proteins expressed by pPICZ B ⁄ HlSCP1 were detected by sil-
ver staining. Lane 1, P. pastoris lysates before induction; lane 2,
P. pastoris lysates 7 h after induction with methanol; lane 3,
rHlSCP1 after purification with a Ni
+
-chelating column under native
conditions. The arrow shows rHlSCP1.
Table 1. Kinetic constants for peptide substrate hydrolysis by
rHlSCP1. HlSCP1 (0.1 mg) was incubated in 25 m
M citrate ⁄ phos-

phate buffer (pH 6) with 0.15–3 m
M of the substrates at 45 °C.
Results shown are the means from duplicate experiments.
Substrate
K
m
(M)
k
cat
(s
)1
)
k
cat
⁄ K
m
(M
)1
Æs
)1
)
Z-Phe-Leu 1.3 · 10
)4
5.7 · 10
)3
43.8
Z-Phe-Ala 2.0 · 10
)4
3.3 · 10
)3

16.5
Z-Glu-Tyr 1.6 · 10
)3
5.4 · 10
)3
3.4
Relative activity (%)
Temperature (ºC)
30 35 40 45 50 55 60
0
25
50
75
100
Pyr-Phe-Leu-pNA
Z-Phe-Leu
Pyr-Phe-Leu-pNA
Z-Phe-Leu
4 5 6 7 8 9
0
25
50
75
100
A
B
pH
Relative activity (%)
Fig. 6. Effect of pH (A) and temperature (B) on rHlSCP1 activity.
Enzyme activity was assayed using Z-Phe-Leu or Pyr-Phe-Leu-pNA

as a substrate in 25 m
M citrate ⁄ 50 mM phosphate buffer (pH 4–7)
or 50 m
M sodium phosphate buffer (pH 8–9). Data are expressed
as the mean percent enzyme activity relative to the maximum
activity ± SD (n ¼ 3).
Table 2. Inhibition of rHlSCP1 enzyme activity toward Z-Phe-Leu
and Pyr-Phe-Leu-pNA by proteinase inhibitors. rHlSCP1 (0.1 mg)
was incubated in 25 m
M citrate ⁄ 50 mM phosphate buffer (pH 6 for
Z-Phe-Leu or pH 7 for Pyr-Phe-Leu-pNA) at 45 °C with the protease
inhibitors. Inhibitory effect is indicated as percentage relative to the
maximum hydrolytic activity of rHlSCP1.
Inhbitor
Conc.
(m
M)
%inhibition
relative to
control
Z-Phe-Leu Pyr-Phe-Leu-pNA
E64 0.1 5 12
Leupeptin 0.1 14 17
Antipain 0.1 19 15
Pepstatin A 0.1 20 50
Phenylmethylsulfonyl
fluoride
186 90
Tick serine carboxypeptidase M. Motobu et al.
3304 FEBS Journal 274 (2007) 3299–3312 ª 2007 The Authors Journal compilation ª 2007 FEBS

Z-Phe-Leu was observed. Antipain, a trypsin-like ser-
ine protease inhibitor, slightly inhibited the hydrolysis
of both synthetic peptides.
Recombinant HlSCP1 hydrolyzes Hb
To assess the hydrolyzing efficiency of HlSCP1, bovine
Hb was incubated with rHlSCP1 for 9 h and was sub-
jected to SDS ⁄ PAGE analysis. As shown in Fig. 7A,
rHlSCP1 hydrolyzed Hb at 45 °C in a dose-dependent
manner. A major portion of Hb was degraded after
6 h of incubation at this temperature (Fig. 7B).
rHlSCP1 was shown to hydrolyze Hb efficiently in the
pH range 5–6 (Fig. 7C,D). In inhibition experiments,
pepstatin A was shown to inhibit Hb hydrolysis by
rHlSCP1 significantly more potently than phenyl-
methylsulfonyl fluoride, whereas other protease inhibi-
tors showed only weak inhibitory effects (Fig. 7E).
Discussion
SCPs are known to play important roles in precursor
processing, growth and apoptosis in bacteria and
plants. However, very little is known about SCPs in
arthropods, including hematophagous ticks. We have
isolated a full-length cDNA encoding an SCP from the
hard tick H. longicornis, whose amino acid sequence
shows similarity with mammalian cathepsin A
sequences. HlSCP1 possesses the catalytic triad and
conserved consensus sequence motifs of the SCPs;
however, substitution of an alanine for a cysteine was
observed in a conserved region (81WLNGGPG-
ASS90). In humans, a mutant cathepsin A, in which
cysteine was replaced by threonine in the

WLNGGPGCSS region, was enzymatically inactive,
and accumulated in the rough endoplasmic reticulum,
suggesting that the cysteine residue in the conserved
region has an important role in creating a proper con-
formation for the interaction with substrates and intra-
cellular transport [24]. However, the same substitution
is observed in SCP of S. mosellana and vitellogenic
carboxypeptidase of Aedes aegypti, which has similar-
ity with SCP [25]. These findings indicate that the alan-
ine residue in the WLNGGPGASS region is conserved
evolutionarily in arthropods and may not be involved
in the enzymatic activity. In the case of cathepsin A, a
0 0.1 0.2 0.5 1
rHlSCP1 (µg/reaction)
A
0369
Incubation time (h)
B
0.0
0.5
1.0
Ratio
Co 25 37 45 55
Temperature (°C)
C
45678Co
pH
D
Ratio
Ratio

0.0
E
0.5
1.0
Co
rHlSCP1
+Pepstatin A
+PMSF
+E64
+Leupeptin
+Antipain
Ratio
0.0
0.5
1.0
Ratio
0.0
0.5
1.0
0.0
0.5
1.0
Fig. 7. Effect of rHlSCP1 concentration (A),
incubation time (B), temperature (C), pH (D)
and inhibitor (E) on hydrolysis of bovine Hb.
Bovine Hb (1 lg) was incubated with
rHlSCP1 in 25 m
M citrate ⁄ 50 mM phosphate
buffer (pH 4–7) or 50 m
M phosphate buffer

(pH 8). Co, reaction buffer containing bovine
Hb without addition of rHlSCP1 and inhibi-
tors.
M. Motobu et al. Tick serine carboxypeptidase
FEBS Journal 274 (2007) 3299–3312 ª 2007 The Authors Journal compilation ª 2007 FEBS 3305
54 kDa precursor is cleaved into a mature heterodimer
of 32 and 20 kDa subunits, which are linked by disul-
fide bonds [26–28], and a 34 and 20 kDa form also
exists as a transient processing intermediate [29]. No
proteolytic cleavage site except for the signal peptide
site was detected at the amino acid sequence level in
HlSCP1 using a cleavage site prediction server (http://
bp.nuap.nagoya-u.ac.jp/sosui). In addition, the het-
erodimer form was not observed when immunoblot
analysis was performed under reducing or nonreducing
conditions (data not shown), indicating that HlSCP1 is
a single-chain enzyme, like CPY.
In hematophagous arthropods, it has been suggested
that proteases induced by blood-feeding in the midgut
may play a crucial role in blood digestion [30–32]. In
ticks, digestion occurs intracellularly in the midgut epi-
thelium and is accomplished by lysosomal hydrolytic
enzymes in vacuoles [33–35]. Various kinds of protease
activities, including exopeptidase, have been described
in the midgut of the cattle tick Boophilus microplus
[36]. In this study, we have shown that endogenous
HlSCP1 was strongly expressed in vacuoles of midgut
epithelial cells. Furthermore, its expression was upreg-
ulated after blood-feeding. These results imply that
HlSCP1 may function as a lysosomal protease in the

process of blood digestion. Based on the results of a
search using sosui, a structure prediction program for
membrane proteins [37] ( />sosui/), HlSCP1 is speculated to be a membrane pro-
tein. Although endogenous HlSCP1 was detected in
the membrane ⁄ organelle fraction extracted from mid-
guts after 72 h of blood-feeding, it has not been con-
firmed whether HlSCP1 exists as a membrane protein
in the vacuoles of midgut epithelial cells. Detailed
studies of HlSCP1 localization will be performed in
the future.
rHlSCP1 showed optimum enzyme activity at acidic
pH and had a substrate preference for Z-Phe-Leu,
properties which are consistent with those of cathep-
sin A [28,38]. In addition to SCP activity, it has been
reported that cathepsin A has deamidase ⁄ esterase activ-
ity, which catalyzes the hydrolysis of bioactive peptide
hormones which contain hydrophobic amino acids at
the C-terminus at neutral pH [39,40]. It has also been
demonstrated that cathepsin A hydrolyzes Suc-Leu-
Leu-Val-Tyr-AMC [41] and Suc-Phe-Leu-Phe-thio-
benzyl ester [42], suggesting that cathepsin A attacks
N-blocked substrates having an aromatic amino acid at
their C-terminus. Therefore, it is thought that deami-
dase ⁄ esterase preferentially cleaves peptides where the
P1¢ and⁄ or P1 position is a hydrophobic amino acid
[39–41]. Similar results were observed with rHlSCP1,
which hydrolyzed a thiol substrate, Pyr-Phe-Leu-pNA,
efficiently at pH 7, and this activity was shown to be
inhibited by phenylmethylsulfonyl fluoride. In addition,
rHlSCP1 partially hydrolyzed Z-Ala-Ala-Leu-pNA and

Suc-Ala-Ala-pNA, but not Bz-(dl)-Arg-pNA (data not
shown), indicating that rHlSCP1 also has a preference
for hydrophobic amino acids in the P1 position. These
results suggest that HlSCP1 has enzyme activity
towards various substrates.
It is noteworthy that rHlSCP1 has enzymatic activ-
ity over a wide range of temperatures (37–50 °C), and
this feature has not been reported for other SCPs
except plant SCP having enzymatic activity at 37–
55 °C [12]. This enzymatic property may be related to
the feeding season of H. longicornis and body tempera-
ture of its host. In general, the feeding activity of ticks
occurs from spring through summer, and the body
temperature of hosts such as cattle, dogs and poultry
is higher than 37 °C. To digest blood-meals efficiently
under different environmental conditions, a broad tem-
perature dependency of the enzyme activity would be
required.
It was observed that HlSCP1 has potent ability to
hydrolyze bovine Hb under the same reaction condi-
tions as used for the hydrolysis of synthetic peptides.
However, it is unclear why the Hb hydrolysis was signi-
ficantly inhibited by pepstatin A, an aspartic protease
inhibitor. Because rHlSCP1 was extracted from P. pas-
toris producing rHlSCP1 and purified using metal
chelation chromatography, the possibility of contamin-
ation by aspartic proteases derived from P. pastoris is
ruled out. It has been reported that CPY and human
cathepsin A have different preferences for SCP inhibi-
tors, implying that nonconserved amino acid residues in

the active sites may contribute to the preference of
inhibitors [43]. Although HlSCP1 possesses the con-
served regions and catalytic triad of the SCPs, its over-
all sequence similarity with other SCPs is < 50%.
Therefore, nonconserved regions may be important
factors determining the preference of inhibitors.
Previous studies have shown that an aspartic prote-
ase and serine protease derived from H. longicornis
hydrolyzed rabbit Hb or BSA, suggesting that those
proteases are involved in blood digestion in ticks
[44,45]. Because it is postulated that host Hb is degra-
ded by multiple proteases, including aspartic, cysteine
and metalloproteases, in blood-feeding parasites [4],
the hard tick H. longicornis also might have such a
Hb-degradation cascade involving multiple proteases.
In endoparasites, aspartic, cysteine and metalloproteas-
es are found in the intestine, and the analysis of Hb
proteolysis by those recombinant proteases indicates
that Hb would be initially degraded by aspartic and
cysteine proteases, followed by metalloproteases [46].
Tick serine carboxypeptidase M. Motobu et al.
3306 FEBS Journal 274 (2007) 3299–3312 ª 2007 The Authors Journal compilation ª 2007 FEBS
More detailed studies of the Hb digestion cascade have
been performed in the malaria parasite Plasmodium
falciparum [47,48]. Those studies showed that Hb deg-
radation occurs in the food vacuole, where plasmepsin,
a member of the aspartic protease family, initially
cleaves in the conserved hinge region of the Hb alpha
chains [49,50]. Falcipain, a member of the cysteine
protease family, degrades the denatured globin [50,51],

and the resultant globin peptides are target molecules
for the zinc metalloprotease falcilysin [52]. Further-
more, P. falciparum aminopeptidases possess enzymatic
activity to hydrolyze peptides derived from the endo-
proteolytic digestion of hemogloblin to amino acids
[53], supporting the idea that aminopeptidases take
part in the terminal stages of hemoglobin degradation.
HlSCP1, having the SCP activity to release amino
acids sequentially from the C-terminus of peptide
chains, may function at the terminal stage of blood
digestion following aspartic and serine proteases.
In this study, our results suggested that SCP identi-
fied in the hard tick H. longicornis takes part in the
host’s blood-digestion process. The protease cascade
for Hb digestion would be crucial for the survival of
ticks, for which the blood-meal from the host is the
only source of energy. Elucidation of the function of
HlSCP1 in the hard tick may contribute a better
understanding of the physiology of blood digestion
and development, and thus improved tick control.
Experimental procedures
Ticks
Adults of H. longicornis obtained from the parthenogenetic
Okayama strain maintained at the Laboratory of Parasitic
Diseases, National Institute of Animal Health (Tsukuba,
Ibaraki, Japan), were bred by feeding on rabbits as
described previously [18].
Animal ethics
All animals used in this study were acclimatized to the
experimental conditions for 2 weeks prior to the experi-

ment. Animal experiments were conducted in accordance
with the protocols approved by the Animal Care and Use
Committee, National Institute of Animal Health (Approval
nos. 441, 508, and 578).
Cloning and sequencing of HlSCP1
HlSCP1 was identified from expressed sequence tags con-
structed from the midgut cDNA libraries of H. longicornis
as described previously [45]. Briefly, the plasmids containing
HlSCP1 gene-encoding inserts were extracted using the
Qiagen DNA Purification kit (Qiagen, Valencia, CA). The
nucleotide sequences of the cDNAs were determined by
the big-dye terminator method on an ABI PRISM 3100
automated sequencer (Applied Biosystems, Foster City, CA).
BioEdit sequence alignment editor (Isis Pharmaceuticals,
Inc, Carlsbad, CA) and the BLAST network server of the
National Center for Biotechnology Information (National
Institutes of Health, Bethesda, MD) were used to analyze
the nucleotide sequence and deduce the amino acid
sequences for determining similarities with previously repor-
ted sequences in GenBank. A primary sequence motif was
identified using the PROSITE network server at EMBL.
Analysis of the signal sequence was performed using signal
ip 3.0 at the Center for Biological Sequence Analysis
( />Generation of anti-HlSCP1 serum
Anti-HlSCP1 serum was obtained from a mouse immun-
ized with Escherichia coli-expressed recombinant HlSCP1
(rHlSCP1). One set of oligonucleotide primers derived from
an ORF of the HlSCP1 gene was used: a sense primer
(5¢-GGGGTACCCCAATGTATACGGTAACCATG-3¢),
corresponding to nucleotides 146–163 of the HlSCP1

nucleotide sequence and an antisense primer (5¢-CGGA
ATTCCGACTAAAGTGGCTTATTGGC-3¢), correspond-
ing to nucleotides 1551–1567 of the HlSCP1 nucleotide
sequence. The nucleotide sequence of these primers
contained KpnI and EcoRI restriction sites, respectively.
Amplified product was inserted into the pTrcHis B plasmid
(Invitrogen, Carlsbad, CA) E. coli expression vector after
digestion with KpnI and EcoRI. The resulting plasmid
(pTrcHis B ⁄ HlSCP1) was transformed into E. coli
(Top10F¢, Invitrogen) using standard techniques. Expres-
sion of the HlSCP1 cDNA in E. coli was performed essen-
tially as described [54]. rHlSCP1 was purified using metal
chelation chromatography (Invitrogen) under denaturing
conditions as described in the manufacturer’s protocol.
Proteins eluted with imidazole were concentrated using
Centrisart (molecular mass cut-off, 10 000 Da; Sartorius,
Goettingen, Germany) and then dialyzed against NaCl ⁄ P
i
using a Slide-A-Lyzer Dialysis Cassette (Pierce Biotechno-
logy, Rockford, IL). Protein concentration was determined
using micro-BCA reagent (Pierce). Antisera against
rHlSCP1 were generated in BALB ⁄ c mice (Japan SLC,
Inc., Hamamatsu, Japan) by subcutaneous injection with
100 lg of rHlSCP1 emulsified with complete (first injec-
tion) or incomplete (second and later injections) Freund’s
adjuvant at 2-week intervals. Mice were bled 2 weeks after
the fourth injection. Antisera from the mice were stored at
)20 °C until used. Animal studies were approved by the
Animal Care and Use Committee, National Institute of
Animal Health (Approval no. 569).

M. Motobu et al. Tick serine carboxypeptidase
FEBS Journal 274 (2007) 3299–3312 ª 2007 The Authors Journal compilation ª 2007 FEBS 3307
2D electrophoresis
Tick extracts from unfed and partially fed adults were pre-
pared as previously described [55]. Tick extracts were treated
with an equal volume of urea mixture composed of 9 m urea,
4% Nonidet P-40, 0.8% ampholine (pH 3.5–10; GE Health-
care, Piscataway, NJ) and 2% 2-mercaptoethanol, and then
subjected to 2D PAGE. Non-equilibrium pH gradient elec-
trophoresis was performed in the first dimension using a rect-
angular gel electrophoresis apparatus (AE-6050A; ATTO,
Tokyo, Japan). After electrophoresis at 400 V for 2 h, gels
were incubated in equilibration buffer for 10 min on a sha-
ker. Electrophoresis in the second dimension was performed
on 12.5% SDS ⁄ PAGE gels under reducing conditions. The
proteins were transferred to nitrocellulose membranes.
Immunoblot analysis
Immunoblot analysis was performed as previously des-
cribed [44]. Tick extracts or rHlSCP1 separated by 1D or
2D electrophoresis were transferred onto nitrocellulose
membranes, and the membranes were incubated for 30 min
with 5% skim milk. For the detection of endogenous
HlSCP1 or rHlSCP1, antiserum against rHlSCP1 diluted
1 : 150 was used. After the membranes were washed with
Tris-buffered saline containing 0.1% Tween-20 (NaCl ⁄ Tris-
T), they were incubated with alkaline phosphatase-conju-
gated goat anti-(mouse IgG) (Invitrogen) as a secondary
antibody. After the membranes were washed, the proteins
bound to the secondary antibody were visualized with
Nitro Blue Terazolium ⁄ 5-bromo-4-chloro-3-indolyl phos-

phate (Invitrogen).
Immunohistochemistry
Immunohistochemistry was performed with peroxidase-
labeled goat anti-(mouse IgG) secondary antibody as des-
cribed previously [44]. Flat sections of whole partially fed
adults were exposed to mouse antirHlSCP1 serum diluted
1 : 150 overnight at 4 °C. After washing with NaCl ⁄ P
i
, the
sections were reacted with peroxidase-labeled mouse IgG
secondary antibody and the substrate 3¢,3¢-diaminobenzi-
dine tetrahydrochloride (Sigma FastÒ DAB set; Sigma
Aldrich, St Louis, MO).
Preparation of protein fractions
Midguts collected from adult ticks after 72 h of blood-
feeding were subjected to stepwise extraction of cytosolic,
membrane, nuclear and cytoskelton proteins using a Pro-
teoExtract subcellular proteome extraction kit and follow-
ing the manufacturer’s instruction (S-PEK; Calbiochem,
San Diego, CA). Expression of endogenous HlSCP in each
fraction was analyzed by immunoblot analysis as described
above.
Immunofluorescence staining
For intracellular localization of HlSCP1, midguts were
collected from adult ticks after 72 h of blood-feeding, and
midgut cells were prepared by teasing midguts through a
stainless steel mesh. To remove cell debris and host’s eryth-
rocytes, the cells were fractionated by centrifugation on a
Percoll density gradient (GE Healthcare). Isotonic Percoll
solution was made with 10 · NaCl ⁄ P

i
(pH 7.4) and Percoll
(1 : 9 v ⁄ v), and diluted in NaCl ⁄ P
i
containing 1% fetal
bovine serum. A Percoll gradient was made by placing 50
and 80% isotonic Percoll in the centrifuge tube, and then
the midgut cell suspension was slowly placed on top of the
gradient and centrifuged for 20 min at 1700 g at room tem-
perature. The 50% Percoll fraction was collected and
washed with NaCl ⁄ P
i
, and the cells were attached to a glass
slide using Shandon cytospinÒ (Thermo Electron, Walt-
ham, MA). After fixation with 4% paraformaldehyde in
NaCl ⁄ P
i
for 20 min at room temperature, the cells were
permeabilized in NaCl ⁄ P
i
containing 0.1% Triton X-100
for 20 min at room temperature. After washing with
NaCl ⁄ P
i
, cells were blocked with 10% goat serum (MP Bio-
medicals, Irvine, CA) for 30 min at room temperature, and
then incubated with mouse anti-rHlSCP1 serum diluted
1 : 150 for 1 h at room temperature. The cells were washed
three times with NaCl ⁄ P
i

, then incubated with green fluor-
escence-labeled mouse IgG secondary antibody [Alexa
FluorÒ 488 goat anti-(mouse IgG) (H + L); Invitrogen]
for 1 h at room temperature. Immunofluorescence staining
of flat sections was performed as described previously [56].
Flat sections of whole unfed or partially fed adult ticks
were exposed to mouse anti-rHlSCP1 serum diluted 1 : 150
overnight at 4 °C. Slides were rinsed thoroughly with
NaCl ⁄ P
i
and incubated with Alexa FluorÒ 488 (Invitrogen)
for 1 h at room temperature. After washing with NaCl ⁄ P
i
,
slides were mounted with VECTASHIELDÒ mounting
medium with DAPI (Vector Laboratories, Burlingame,
CA), covered with glass cover slips, and then observed
under a fluorescence microscope (Leica, Wetzlar, Ger-
many).
Functional expression of rHlSCP1
in Pichia pastoris
Expression of rHlSCP1 in Pichia pastoris was conducted
using an EasySelectÒ Pichia expression kit (Invitrogen).
Primers used to generate rHlSCP1 in P. pastoris were: sense
primer (5¢-CGGAATTCCGAATAATGTCTCAGGGACC
TGCTGAGGAC-3¢), corresponding to nucleotides 227–244
of the HlSCP1 nucleotide sequence, and antisense primer
(5¢-GGGGTACCCCAAGTGGCTTATTGGC-3¢), corres-
ponding to nucleotides 1551–1567 of the HlSCP1 nucleotide
sequence. The nucleotide sequence of these primers con-

tained an EcoRI and a KpnI restriction site, respect-
ively. The amplified product was inserted into pPICZB
Tick serine carboxypeptidase M. Motobu et al.
3308 FEBS Journal 274 (2007) 3299–3312 ª 2007 The Authors Journal compilation ª 2007 FEBS
P. pastoris expression vector (Invitrogen) after digestion
with EcoRI and KpnI. The resulting plasmid
(pPICZB ⁄ HlSCP1) was transformed into E. coli (Top10F¢,
Invitrogen). The pPICZB ⁄ HlSCP1 was linearized using
PmeI and transformed into P. pastoris strain GS115 by
electroporation (Bio-Rad GenePulser; Bio-Rad Laborator-
ies, Hercules, CA). The transformed cells were spread onto
YPDS plates (1% yeast extract, 2% peptone, 2% dextrose,
and 1 m sorbitol) containing 100 lgÆmL
)1
zeocin and incu-
bated for 3 days at 30 °C. To examine whether the trans-
formants were methanol utilization phenotypes (Mu
+
), a
number of colonies were picked up randomly from the
YPDS plates and plated on both an MMH plate [1.34%
yeast nitrogen base (YNB), 4 · 10
)5
% biotin, 0.5% meth-
anol, 0.004% histidine] and an MDH plate (1.34% YNB,
4 · 10
)5
% biotin, 2% dextrose, 0.004% histidine), and
incubated for 3 days at 30 °C. Selected Mu
+

transformant
cells were grown to D
600
¼ 2.0 at 30 °C in MGYH (1.34%
YNB, 1% glycerol, 4 · 10
)5
% biotin, 0.004% histidine).
The cells were centrifuged at 3000 g for 5 min at room tem-
perature and resuspended to D
600
¼ 1.0 in MMH. The cul-
ture was grown for 7 h at 30 °C. P. pastoris cells were
pelleted and resuspended in 50 mm sodium phosphate buf-
fer (pH 7.4) containing 1 mm EDTA and 5% glycerol. The
cell suspension was disrupted by vortexing with an equal
volume of acid-washed glass beads. The lysate was centri-
fuged at 15 000 g for 30 min at 4 °C, and the rHlSCP1 pro-
tein in the supernatant was purified using metal chelation
chromatography (Invitrogen) under nondenaturing condi-
tions as described in the manufacturer’s protocol. Proteins
eluted with imidazole were concentrated and dialyzed as
described for rHlSCP1 expressed in E. coli.
Enzyme assay
SCP activity was measured using a modification of the
method of Galjart et al. [27]. Briefly, rHlSCP1 (0.1 lg) was
incubated in 100 lLof25mm citrate ⁄ 50 mm phosphate
buffer (pH 6) at 45 °C with different concentrations (0.15–
3mm) of N-blocked dipeptides Z-Phe-Leu (Sigma-Aldrich),
Z-Phe-Ala (Fluka; Sigma-Aldrich) or Z-Glu-Tyr (Peptide
Institute, Osaka, Japan). To analyze pH stability, rHlSCP1

(0.1 lg) was incubated with 1.5 mm Z-Phe-Leu in 50 lLof
buffer (25 mm citrate ⁄ 50 mm phosphate buffer, pH 4–7 or
50 mm sodium phosphate buffer, pH 8–9) at 45 °C. The
temperature dependency of the enzyme was determined by
incubating the recombinant protein with Z-Phe-Leu in
25 mm citrate ⁄ 50 mm phosphate buffer (pH 6) at various
temperatures (30–60 °C). Reactions were stopped by addi-
tion of an equal volume of 10% (v ⁄ v) trichloroacetic acid.
Precipitates were removed by centrifugation at 15 000 g for
10 min at room temperature, and the supernatants were col-
lected to measure the concentration of amino acids released
using the fluorimetric method [26,57]. For inhibition studies,
the recombinant protein was preincubated in 25 mm
citrate ⁄ 50 mm phosphate buffer (pH 6) with E64, antipain,
leupeptin, pepstatin A (100 lm; Peptide Institute) or phenyl-
methylsulfonyl fluoride (1 mm; Nacalai, Kyoto, Japan) at
45 °C for 15 min. Enzyme activity was monitored using a
Sectrafluor (TECAN, Ma
¨
nnedorf, Switzerland) with wave-
length pairs of 360 and 465 nm for emission and excitation,
respectively. Enzyme activity was defined as lmol of leucine,
alanine or tyrosine released per min per mg of protein. The
kinetic parameters k
cat
and K
m
were obtained from Hanes–
Woolf plots. To examine whether rHlSCP1 has the ability to
hydrolyze specific substrates for other proteases, rHlSCP1

was incubated with 200 lm Pyr-Phe-Leu-pNA, Z-Ala-Ala-
Leu-pNA, Suc-Ala-Ala-Ala-pNA or Bz-(dl)-Arg-pNA (Pep-
tide Institute) as described previously [56].
Hemoglobin hydrolysis assay
To examine whether rHlSCP1 has the ability to hydrolyze
Hb, a Hb hydrolysis assay was conducted. Unless otherwise
indicated, 1 lg of bovine Hb (Sigma) was incubated with
rHlSCP1 (0.1 lg) in 25 mm citrate ⁄ 50 mm phosphate buffer
(pH 6) at 45 °C for 9 h. For the analysis of pH depend-
ency, the assay was performed in different buffers (25 mm
citrate ⁄ 50 mm phosphate buffer, pH 4–7 or 50 mm sodium
phosphate buffer, pH 8). For inhibitor studies, E64, anti-
pain, leupeptin, pepstatin A or phenylmethylsulfonyl fluor-
ide was added to the rHlSCP1 mixture at the same
concentration as described for the enzyme assay. The reac-
tion was stopped by the addition of reducing SDS ⁄ PAGE
sample buffer, and samples were stored at )20 °C until
electrophoresis on 15% polyacrylamide gels. After electro-
phoresis, gels were stained using a silver staining kit (Dai-
ichi-Pure Chemical, Tokyo, Japan). The densities of stained
bands were measured using image j 1.36b (National Insti-
tutes of Health) and the ratio of the density divided by that
of the control was determined.
Acknowledgements
The authors thank H. Shimada and M. Kobayashi for
help in preparing tick paraffin sections. This study was
supported by the 21st Century COE Program (A-1, to
KF) and Grant-in-Aids (to NT and KF) from the
Ministry of Education, Culture, Sports, Science, and
Technology of Japan. This study was also supported

by a grant (to NT and KF) for Promotion of Basic
Research Activities for Innovative Biosciences from
the Bio-oriented Technology Research Advancement
Institution.
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