Cattle tick Boophilus microplus salivary gland contains a thiol-activated metalloendopeptidase displaying kininase activity

Insect Biochemistry and Molecular Biology 32 (2002) 1439–1446 www.elsevier.com/locate/ibmb

Cattle tick Boophilus microplus salivary gland contains a thiolactivated metalloendopeptidase displaying kininase activity Michele Bastiani ab, Sandro Hillebrand b, Fabiana Horn ab, Tarso Benigno Ledur Kist ab, Jorge Almeida Guimara˜es ac, Carlos Termignoni ad,∗ a

Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul. Caixa Postal 15005, 91501-970, Porto Alegre, Brazil Departamento de Biofı´sica, Universidade Federal do Rio Grande do Sul. Caixa Postal 15005, 91501-970, Porto Alegre, Brazil c Departamento de Biotecnologia, Universidade Federal do Rio Grande do Sul. Caixa Postal 15005, 91501-970, Porto Alegre, Brazil d Departamento de Bioquı´mica, Universidade Federal do Rio Grande do Sul. Caixa Postal 15005, 91501-970, Porto Alegre, Brazil b

Received 2 November 2001; received in revised form 9 April 2002; accepted 12 April 2002

Abstract This work reports on the characterization of a metalloendopeptidase kininase present in Boophilus microplus salivary glands. Using the guinea pig ileum assay, salivary gland whole extracts (SGE) were found to have a potent kininase activity. Ion-exchange chromatography separated two kininase activities from SGE. The major enzymatic component, eluted at lower ionic strength, was named BooKase (Boophilus Kininase). Analysis of the hydrolysis products by capillary electrophoresis identified Phe5-Ser6 as the only hydrolyzable peptide bond in bradykinin after BooKase treatment. This is the same specificity as the mammalian thimet oligoendopeptidase (EC 3.4.24.15). Like this enzyme, BooKase is also a metallo-peptidase (requires Mn2+) and is activated by SH protecting reagents. In addition, BooKase was partially inhibited by cFP-AAF-pAB, a specific inhibitor of thimet oligopeptidase. Contrary to other kininases, BooKase had no actitivy upon angiontensin I. Our results show that BooKase behaves as a typical peptidase with kinase activity.  2002 Elsevier Science Ltd. All rights reserved. Keywords: Kininase; Boophilus; Tick salivary glands; Thimet oligoendopeptidase

1. Introduction The tick Boophilus microplus is the major bovine ectoparasite in Australia and Latin America. It causes great economical losses to cattle breeding, directly due to bovine weight loss and indirectly due to transmission of babesiosis and anaplasmosis. Unlike other ticks, B. microplus remains fixed on the host during all parasitic stages, around 21 days. Salivary glands and salivary gland secretions play a vital role in

∗

Corresponding author. Abbreviations: Bk, Bradykinin; AbzBkGlnEDDnp, orto-aminobenzoyl-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Gln-(2,4,dinitrophenyl) ethylenediamine; cFP-AAF-pAB, (N-[1(RS)-carboxy-3-phenyl-propyl]-Ala-Ala-Phe-p-aminobenzoato; BooKase, Boophilus microplus kininase; E-64, (2S,3S)-trans-epoxysuccinil-L-Leucilamido-3-ethylbutane; PMSF, phenylmethylsulfonyl-fluoride; EDTA, ethylenediaminetetraacetic acid E-mail address: [email protected] (C. Termignoni).

this long-term association, since they are the organs responsible for the tick fluid homeostasis, and for the secretion of an array of molecules with pharmacological properties that evade the host defense mechanisms against tick attachment (Ribeiro, 1995). A better comprehension of B. microplus salivary glands is likely to throw light into the mechanisms of B. microplus successful parasitism. We have already reported on a salivary anticoagulant from B. microplus (Horn et al., 2000), but much of what is postulated for B. microplus salivary glands have been inferred from what is known for other ixodid ticks (for a review see Sauer et al. (2000)). Bradykinin (Bk) is a biologically active nonapeptide (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) discovered by Rocha e Silva and co-workers (Rocha e Silva et al. (1949), with a key role in blood pressure control (Bhoola et al., 1992) and in inflammatory response (Calixto et al., 2000). These actions are mediated by B1 and B2 receptors displaying typical properties (Regoli and Barabe, 1980). The physiological actions of Bk mediated by

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B2 receptors are strictly dependent on the molecule’s full integrity. Thus, enzymes that hydrolyze any peptide bond in Bk abolish its activity and can play a regulatory role in Bk responses through B2 receptors (Regoli and Barabe, 1980). These enzymes, named kininases, together with the enzymes (kallikreins) that release Bk from kininogens, its circulating precursor, have been extensively studied because of their medical importance (Bhoola et al., 1992). Here we demonstrate the presence of a kininase in B. microplus salivary gland and characterize its activity.

Guinea pigs weighed 180–200 g. A 2 cm terminal ileum segment was prepared isotonically with a tension of 0.9 g. The preparation was allowed to rest for 60 min and was then regularly stimulated with Bk or with each sample at 3-min-intervals. Resulting contractions were recorded through a transducer (Transducer Amplifier, Harvard) and registered using a one-channel recorder (LKB, Sweden). For assaying kininase activity, 500 or 1000 ng of Bk were incubated with the SGE, column fractions or the kininase final preparation in 20 mM TrisHCl pH 7.5 at 37 °C and Bk hydrolysis was monitored using the ileum bioassay.

2. Materials and methods

2.5. Kininase assay

2.1. Materials

To analyze the effect of divalent metals on kininase activity, 3 µg of the partially purified enzyme were dialyzed against 20 mM EDTA, 10 mM sodium phosphate pH 7.5, and then against 10 mM sodium phosphate pH 7.5. This sample was preincubated with 2.5 mM of the indicated cations in 10 mM phosphate for 60 min at room temperature. Two micrograms of Bk were added to the preparations and after 45 min at 37 °C remaining Bk was analyzed by the ileum bioassay. Assays to verify thiol activation were also done using the guinea pig ileum assay. Kininase final preparation (2.15 µg protein) was incubated with 500 ng of Bk in the presence of dithiothreitol at several concentrations (0.025, 0.05, 0.1, 0.3, 0.4, 0.5, 1.0, 2.5, 5.0 or 10.0 mM) in 100 µl of 20 mM Tris-HCl, pH 7.5. After 30 min at 37 °C, an aliquot was taken and the remaining Bk was measured by the ileum bioassay. Activity in the absence of dithiothreitol was considered as 100%. For testing inhibition by the Orlowsky inhibitor (cFPAAF-pAB), 4.2 µg of the enzyme final preparation were incubated with 4.5, 45 or 90 µM of cFP-AAF-pAB in 95 µl of 20 mM Tris-HCl, pH 7.5 for 30 min at 37 °C. Bk (500 ng in 5 µl) was then added and the samples were incubated at 37 °C. After 45 min, remaining Bk was measured by the guinea pig ileum bioassay.

Bradykinin was purchased from Sigma-Aldrich; fragments of bradykinin (Bk 1-7, Bk 1-5) were purchased from Bachem (USA). Quenched fluorescence bradykinin analog, orto-aminobenzoyl-Arg-Pro-Pro-Gly-Phe-SerPro-Phe-Arg-Gln-(2,4-dinitrophenyl)-ethylenediamine, (AbzBkGluEDDnp), and Orlowsky inhibitor ((N[1(RS)-carboxy-3-phenyl-propyl]-Ala-Ala-Phe-paminobenzoato) were a kind gift from Dr. Maria A. Juliano and Dr. Durval Rosa Borges (EPM, Universidade Federal de Sa˜ o Paulo, Brasil), respectively. 2.2. Preparation of tick salivary gland extract Salivary gland extract (SGE) was obtained by dissecting salivary glands from partially engorged female ticks. Salivary glands were collected, rinsed in cold PBS and sonicated at 500 W 5×4 s in Tris-HCl 20 mM, pH 7.5. Cell debris was removed by centrifugation at 12,000 g for 10 min and the supernatant fraction was stored at ⫺20 °C until use. Protein determination of SGE was done using the BCA method (Pierce), according to the manufacturer’s instructions. 2.3. Kininase partial purification

2.6. Fluorimetric assays The SGE was applied onto a Mono Q HR 5/5 column (Pharmacia) equilibrated with 20 mM Tris-HCl pH 7.5. Samples were eluted with a gradient of 0–0.4 M NaCl (from 0 to 25 ml) followed by a gradient of 0.4–1 M NaCl (25–45 ml) at 0.5 ml/min and monitored by absorbance at 280 nm. Column fractions (1 ml) were tested for kininase activity in the guinea pig ileum bioassay. 2.4. Smooth muscle guinea pig ileum bioassays The smooth muscle guinea pig ileum bioassays were done in a bath using Tyrode’s solution at 37 °C and infused with CO2 and O2 (Webster and Prado, 1970).

Inhibition assays were done using a quenched fluorescent substrate (Oliveira et al., 1992). Kininase preparation (0.7 µg protein) was preincubated with 1 mM PMSF, 10 µM E-64, 5 mM EDTA or 1 µM captopril in 50 mM Hepes pH 7.0 for 30 min at 37 °C, in a final volume of 90 µl. Ten µl of AbzBkGlnEDDnp (50 µM, final concentration) were then added. The reaction was monitored (λex=320 nm/λem=430 nm) for 240 min at intervals of 75 s using a fluorometer microplate reader (Fmax, Molecular Devices). For optimum pH determination, 0.7 µg of kininase preparation were preincubated in (1) 50 mM sodium phosphate pH 4.0, (2) 50 mM sodium phosphate pH 5.0,

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(3) 50 mM sodium phosphate pH 6.0, (4) 50 mM sodium phosphate pH 7.0, (5) 20 mM Tris-HCl pH range 7.5 to 10.0, all in a final volume of 90 µl. After 10 min at 37 °C, 10 µM (10 µl) AbzBkGluEDDnp were added and the reaction was monitored at 430/320 nm for 180 min at 60 s-intervals in a fluorometer microplate reader (Fmax, Molecular Devices). 2.7. Hydrolysis of Bk In order to determine the bonds of Bk that are hydrolyzed by SGE or the kininase preparation, 5 µg of SGE or 2 µg of kininase preparation were incubated with 105 µg of Bk in 10 mM sodium phosphate pH 7.5 at 37 °C (final volume of 100 µl). At different intervals, aliquots of 10 µl were taken and boiled for 5 min, to stop the reaction. The aliquots were kept at ⫺20 °C until analysis by capillary electrophoresis. Experiments using the bradykinin fragments Bk 1-7 and Bk 1-5 as substrates were performed in the same way. To test for hydrolysis of angiotensin I, 1.2 µg of the kininase preparation (in 20 µl of 20 mM Tris-HCl pH 7.5 150 mM NaCl) were incubated with 26 µg of angiotensin I (in 30 µl of PBS pH 7.4) at 37 °C. After 10 h of incubation, the reaction was boiled for 5 min and kept at ⫺20 °C until analysis by capillary electrophoresis.

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using a level difference of 12 cm between reservoirs) and then subjected to electrophoresis by applying 10,000 Volts (243.3 Volts/cm). The hydrolysis products were identified by comparing them to standard peptides. Bk itself was considered an internal standard to correct electroosmotic flow fluctuations. When Bk was not used as substrate, it was added as an internal standard after the enzymatic reaction and before fluorescamine labeling.

3. Results 3.1. Demonstration of kininase activity in B. microplus salivary gland To test for the presence of kininase activity in B. microplus salivary gland, different concentrations of salivary gland extract (SGE) were incubated with Bk, and the remaining bradykinin was tested in the guinea pig ileum assay. As shown in Fig. 1, Bk was inactivated by B. microplus SGE in a time dependent-manner. Bk degradation was also monitored by capillary electrophoresis. Analysis of the hydrolysis products showed that Bk inactivation is due to its fragmentation in at least five peptides (Fig. 2). Such a number of hydrolysis pro-

2.8. Capillary electrophoresis Capillary electrophoresis (CE) analyses were made using equipment developed in our laboratory, with a laser-induced fluorescence detection system (Kist et al., 1994). We used a pulsed nitrogen laser (operated at a rate of 20 pulses/s and 50 µJ energy/pulse) to induce a fluorescent emission from the analytes. This fluorescent light was collected by a microscope objective and detected by a photomultiplier tube (PMT). PMT analog output pulses were integrated by a boxcar and collected by an A/D card on a PC. The instrument was equipped with a fused silica capillary with an internal diameter of 50 µm and 41.1 cm (35.4 cm to detector) in length. The capillary was conditioned at the beginning of every day by rinsing a 1 M NaOH solution for 10 min, followed by water for 10 min, and running buffer (20 mM sodium tetraborate, pH 9.5, 15% (v/v) methanol) for another 10 min. Between runs the capillary was rinsed with the running buffer for 10 min. Peptides resulting from enzymatic hydrolysis, as well as standards, were labeled with fluorescamine. The amine-reactive reagent fluorescamine reacts with aliphatic primary amine groups, including the amine terminus of proteins and α-amino group of lysines, producing fluorescent derivatives. Typically, the reaction was made mixing 4 µl of the samples with 2 µl of running buffer and 2 µl of 25 mM fluorescamine in acetone. The samples were then hydrodynamically injected (for 20 s

Fig. 1. Bradykinin inactivation by SGE. SGE (5 µg protein) was incubated with Bk (1000 ng) in 50 mM Tris-HCl, pH 7.0 at 37 °C. At the indicated incubation times, a sample (corresponding to 50 ng of Bk) was taken from the incubation mixture, and the remaining Bk was immediately measured by the guinea pig ileum bioassay. Inset: representative tracings of contractions of guinea pig ileum during the assay described above: 1) 5 min; 2) 10 min; 3) 15 min; or 4) 30 min of incubation; 5) 50 ng of Bk.

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Fig. 3. SGE fractionation by ion-exchange chromatograpy. SGE (9.4 mg protein) were applied onto a Mono Q HR 5/5 (1.0 ml) column previously equilibrated with 20 mM Tris-HCl, pH 7.5. After washing the column with the same buffer, the sample was eluted with a linear gradient of 0–0.4 M NaCl (25 ml), followed by a second gradient of 0.4–1.0 M NaCl (20 ml). Protein was estimated by absorbance at 280 nm. Fractions of 1.0 ml were collected and tested for kininase activity by incubating a 5 µl-aliquote with Bk (1000 ng) in 95 µl 20 mM TrisHCl pH 7.5 buffer, for 30 min at 37 °C. Remaining Bk was assayed in the guinea pig ileum bioassay. Fractions indicated by the dark bar and dashed bar were able to inactivate Bk.

Fig. 2. Bradykinin hydrolysis by SGE. SGE (5 µg protein) was incubated with 1 mM Bk (105 µg) in 10 mM sodium phosphate, pH 7.5 at 37 °C. After 0 h (panel A) and 6.5 h (panel B), samples were taken and boiled for 5 min, labeled with fluorescamine and analyzed by capillary electrophoresis. Bk indicates bradykinin migration time.

ducts suggests that (1) there is one kininase that is able to degrade Bk in several fragments, and/or (2) that the salivary gland has more than one peptidase acting upon Bk. 3.2. Separation of salivary gland kininases To find if B. microplus salivary gland contains one or more kininase activities, salivary gland extract (9.4 mg protein) was subjected to ion-exchange chromatography on MonoQ column. Bk hydrolysis was determined using the guinea pig ileum bioassay. Two peaks of kininase activity were detected: the first one eluted from the column at 0.15 M NaCl, while the second eluted at 0.37 M (Fig. 3). The first peak accounts for 67% of the total salivary gland kininase activity, and the second for the remaining 33%. The B. microplus kininase eluted at lower ionic strength, hereafter referred to as BooKase, was further studied. After this single purification step,

the specific activity increased 24 fold, from 83 U/mg protein in the salivary gland extract to 2000 U/mg protein. Further purification was not attempted due to limitation of starting material. 3.3. BooKase activity characterization The BooKase preparation thus obtained was able to inactivate Bk in a time-dependent manner (Fig. 4). In order to identify which bradykinin bond(s) was(were) hydrolyzed, we incubated the enzyme preparation with Bk and analyzed the fragments that were generated. Fig. 4 shows that bradykinin disappears concomitantly as only two fragments were formed, in a time-dependentmanner. Degradation of Bk monitored by capillary electrophoresis correlates precisely with the inactivation of Bk detected in the ileum assay (Fig. 4B, open squares). By capillary electrophoresis analyses, one fragment was identified as Bk 1–5, therefore the other is Bk 6–9. This result shows that the enzyme cleaves Bk at the Phe5-Ser6 peptide bond. The Bk fragment Bk 1–7 was never found, irrespective of the incubation time (Fig. 4A). Although BooKase does not generate Bk 1– 7 from Bk, the enzyme was able to use Bk 1–7 as substrate, in this case also yielding Bk 1–5 (Fig. 5A). In a separate experiment, BooKase was incubated with Bk 1– 5 and no hydrolysis was detected (Fig. 5B), confirming that Bk 1–5 is not further hydrolyzed by BooKase.

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Fig. 5. BooKase activity upon Bk 1–7 and Bk 1–5. (A) Bk 1–7 or (B) Bk 1–5 were incubated with BooKase (2 µg protein) in 10 mM sodium phosphate, pH 7.5 at 37 °C. After 5 h, an aliquot was taken, boiled and analyzed by capillary electrophoresis. Int. Std., internal standard; BK 1–7, bradykinin 1–7; BK 1–5, bradykinin 1–5.

Fig. 4. Bradykinin inactivation by BooKase. (A) Time course of bradykinin hydrolysis by BooKase. Bk (1 mM, 105 µg) was incubated with BooKase (2 µg protein) in 10 mM sodium phosphate, pH 7.5 at 37 °C. At different intervals, aliquots were taken, boiled for 5 min and analyzed by capillary electrophoresis as indicated in Material and Methods. Bk and fragment 1–5 (Bk 1–5) were identified by comparison with standard peptides. (B) Conversion of Bk into Bk 1–5 by BooKase. The concentration of Bk (closed squares) and Bk 1–5 (closed circles) were calculated from the respective peak areas obtained in the capillary electrophoresis. At the same intervals, remaining Bk was also measured by the ileum bioassay (open squares).

Since several kininases are able to hydrolyze angiotensin I, we tested whether BooKase also had this activity. Fig. 6 shows that even after a long incubation, double the time BooKase takes to completely hydrolyze the same quantity of bradykinin, no fragmentation of angiotensin I was detected. BooKase has an optimum pH around 7.0 (Fig. 7). The enzyme was partially inhibited by captopril, EDTA, PMSF and cFP-AAF-pAB, but it was not inhibited by E-64 (Table 1). Inhibition of the enzyme activity by

Fig. 6. Incubation of angiotensin I with BooKase. Angiotensin I (26 µg) was incubated with BooKase (1.2 µg protein) at 37 °C (see Materials and Methods). At 0 h and after 10 h of incubation, samples from the incubation mixture were collected, boiled and analyzed by capillary electrophoresis. AngI, angiotensin I.

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Table 2 Effect of divalent metals on BooKase activity. BooKase (3 µg protein) was dialyzed against 20 mM EDTA, 10 mM sodium phosphate pH 7.5, and then against 10 mM sodium phosphate pH 7.5. This sample was preincubated with 2.5 mM of each of the indicated cations for 60 min at room temperature. Two micrograms of Bk were added to each incubation and, after 45 min at 37 °C, remaining Bk was analyzed by the biological assay BooKase treatment

Bk Hydrolysis (%)

Non-dialyzed Dialyzed plus CoCl2 plus MgCl2 plus CuCl2 plus CaCl2 plus ZnCl2 plus MnCl2

Fig. 7. Influence of pH on BooKase activity. BooKase (0.7 µg protein) was preincubated at different pHs (see Materials and Methods) at 37 °C for 10 min, before the addition of AbzBkGlnEDDnp (10 µM, final concentration). After substrate addition, the kinetics of substrate hydrolysis was measured (320 nm ex./430 nm em.) in a fluorescence microplate reader.

dialysis against EDTA was partially recovered by Co2+, Zn2+ and totally recovered by Mn2+ (Table 2). BooKase was activated by DTT, reaching a peak of maximum activation (3-fold compared to control) around 0.5 mM. The activation rate fell off as the concentration of DTT increases, but even at 10 mM DTT BooKase activity remained higher than without DTT (Fig. 8).

4. Discussion Using the guinea pig ileum bioassay, a very sensitive biological method, we showed that Bk was inactivated by B. microplus salivary gland extract (Fig. 1). Bradykinin fragmentation in several peptides (Fig. 2) suggests

100 40 80 50 50 50 80 100

(i) the presence of a kininase able to hydrolyze several bradykinin peptide bonds; (ii) the presence of more than one peptidase acting upon Bk; or (iii) that once the first bond is hydrolyzed, one or more of the newly formed peptides are subsequently hydrolyzed by other peptidases. To test these hypotheses, SGE was fractionated by ion-exchange chromatography. SGE kininase activity was separated in two fractions, suggesting the presence of two kininases. The major enzyme peak, named BooKase, was further characterized. Comparing Bk hydrolysis products generated by BooKase action with standard Bk fragments, we concluded that the Bk bond hydrolyzed by BooKase is Phe5-Ser6 (Fig. 4A). BooKase activity appears to be specific for that peptide bond, since disappearance of Bk occurred simultaneously to the formation of BK1–5, in a 1:1 ratio (Fig. 4B). Moreover, formation of Bk 1–7 was never observed. However, Bk 1–7 was recognized as substrate and the enzyme converts it into Bk 1–5 (Fig. 5A). Thus, Bk1–5 is the end product of the BooKase activity upon Bk (Fig. 5B). BooKase specificity for the Phe5-Ser6 bond is the same as the thiol activated metallo-oligopeptidase (thimet oligoendopeptidase, EC 3.4.24.15) (Camargo et

Table 1 Inhibition of BooKase activity. BooKase (0.7 µg) was preincubated with protease inhibitors for 30 min at 37 °C in 50 mM Hepes pH 7.0. Fifty µM of AbzBkGluEDDnp were added and the reaction was monitored for 240 min Inhibitor Captopril EDTA E64 PMSF cFP-AAF-pABa cFP-AAF-pABa cFP-AAF-pABa a

Concentration (mM) 0.001 5 0.01 1 0.0045 0.045 0.090

Inhibition (%) 70 50 0 50 0 30 45

Inhibition by cFP-AAF-pAB was tested in the biological assay as described in Materials and Methods.

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Fig. 8. BooKase activation by DTT. BooKase (2.15 µg protein) was incubated with DTT in 20 mM Tris-HCl, pH 7.5 at 37 °C. After 10 min, Bk (500 ng) was added and the mixture was further incubated for 30 min at 37 °C. The remaining Bk was measured by the guinea pig ileum bioassay. Activity in the absence of dithiothreitol was considered 100%.

al., 1983a); (Dando et al., 1993). Other similarities with this enzyme include the fact that BooKase is a metalloenzyme (Table 2) and is activated by thiol (Fig. 8), although the metal ion activator and sensitivity to DTT are not the same. In contrast to thimet oligopeptidase, in which the strongest activator is Zn2+ (Barrett and Brown, 1990), BooKase main activator is Mn2+. Both enzymes are activated by DTT, but while thimet is maximally activated at 0.1 mM (Barrett and Brown, 1990), BooKase is maximally activated at 0.5 mM DTT (Fig. 8). After this maximal activation, similarly as thimet oligopeptidase (Barrett and Brown, 1990), BooKase activation rate decreases with higher DTT concentrations. BooKase was also partially inhibited by the well known thimet oligoendopeptidase inhibitor cFP-AAF-pAB (Table 1) (Orlowski et al., 1988), although with a lower sensitivity. Similarly to the rat liver thimet oligoendopeptidase, BooKase was inhibited by captopril (Molina et al., 2000). Whether BooKase is the B. microplus counterpart of the mammalian thimet oligopeptidase remains to be determined, but the fact that BooKase is a thiol-activated, metalloendopeptidase allow us to consider them similar enzymes. The specificity of BooKase for the Phe5-Ser6 bond shows that BooKase is distinct from other well known bradykinin degrading enzymes, such as kininase II (the same as angiotensin converting enzyme, ACE, EC 3.4.21.1), neutral endopeptidase 24.11 (Erdo¨ s, 1990) and prolyl endopeptidase (Quinto et al., 2000). All of these

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latter enzymes are dipeptidylcarboxypeptidases and cleave Bk at the Pro7-Phe8 bond. Most important, BooKase is distinct from Bm91, a B. microplus protein able to confer some immunological protection, because Bm91 is a protein with high similarities to ACE and capable of hydrolyzing angiotensin I (Jarmey et al., 1995). In agreement with this conclusion, BooKase was unable to hydrolyze angiotensin I, even after a long incubation time (Fig. 6). Although Bm91 activity upon Bk has not been reported, it is possible that the second peak of kininase activity from B. microplus salivary glands (Fig. 3) contains Bm91, since this protein was found in salivary glands (Riding et al., 1994) and Bk is also hydrolyzed by ACE (Ferreira, 1985). At the present, we do not know the physiological role of BooKase. Bk was not inactivated when saliva was used instead of SGE (results not shown), suggesting that, in B. microplus, any kininase present in salivary glands is absent from saliva. Thus, BooKase may not be playing a role in the parasite-host interaction, as in the case of the well-characterized salivary kininase in Ixodes scapularis (Ribeiro and Mather, 1998). However, since we tested saliva from adult semi-engorged females we cannot discard the possibility that BooKase were secreted into the saliva of some early tick stage, such as recently fixed larvae. Despite the fact that Bk is an excellent substrate for BooKase, we cannot exclude the possibility that BooKase has another unknown, physiological substrate, which would be hydrolyzed with the same specificity. Saccharomyces cerevisiae, for example, also has a metalloendopeptidase that hydrolyzes Bk at the Phe5-Ser6 bond, yet this free-living organism usually has no access to Bk and therefore Bk cannot be a natural substrate (Hrycyna and Clarke, 1993). Likewise, the physiological function for the mammalian thimet oligoendopeptidase, even after several investigation efforts, is not completely elucidated. Based on its activity upon some peptides and proteins, several roles have been proposed for this enzyme. Early results have shown that circulating Bk is inactivated in the liver (Borges et al., 1979), and there is now evidence that this clearance activity is mainly from thimet oligoendopeptidase action (Molina et al., 2000). Other proposed functions for thimet oligoendopeptidase include (1) a role in some neuropeptide regulation (Camargo et al., 1983b); (2) chemotatic peptide degradation (Lesser et al., 1996); (3) Alzheimer’s amyloid-β peptide degradation (Yamin et al., 1999); and (4) degradation of proteosome peptide products in order to leave only stable MHC class I epitopes (Portaro et al., 1999). All these proposed activities wouldn’t involve Bk, and Bk hydrolysis would just be a consequence of the enzyme specificity to substrates not related to Bk. BooKase could also have a role in tick peptide processing. This hypothesis is supported by observations that an angiotensin-converting enzyme from Musca domestica

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is able to cleave, in addition to the mammalian peptides Bk and angiotensin I, several insect neuropeptides (Lamango et al., 1997). Besides, as already mentioned, BooKase might well be secreted into the saliva of an early tick stage, thus modulating tick-host interaction. The elucidation of BooKase physiological function will certainly add to a better understanding of B. microplus salivary gland, therefore widening the range of potential targets for tick control. In this work, we have demonstrated that B. microplus salivary gland contains an endopeptidase able to hydrolyze Bk at the Phe5-Ser6 bond. This is the same specificity of the mammalian thimet oligoendopeptidase, an enzyme with which BooKase shares several similarities.

Acknowledgements We are thankful to Prof. Maria Aparecida Juliano (Universidade Federal de Sa˜ o Paulo) for the kind supply of quenched fluorescence bradykinin analogue and to Prof. Durval Rosa Borges (Universidade Federal de Sa˜ o Paulo) for the supply of cFP-AAF-pAB. We acknowledge the financial support of CNPq, PRONEX/FINEP and FAPERGS.

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