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: tsujin@affrc.go.jp 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] (http://bp.nuap.nagoya-u.ac.jp/ 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 (http://www.cbs.dtu.dk/services/SignalP/). 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. 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