Triapsin, an unusual activatable serine protease from the saliva of the hematophagous vector of Chagas' disease Triatoma infestans (Hemiptera: Reduviidae)

Insect Biochemistry and Molecular Biology 31 (2001) 465–472 www.elsevier.com/locate/ibmb

Triapsin, an unusual activatable serine protease from the saliva of the hematophagous vector of Chagas’ disease Triatoma infestans (Hemiptera: Reduviidae) Rogerio Amino a, Aparecida Sadae Tanaka b, Sergio Schenkman a

a,*

Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Sa˜o Paulo, R. Botucatu 862 8A, Sa˜o Paulo, S.P., 04023-062, Brazil b Departamento de Bioquı´mica Escola Paulista de Medicina, Universidade Federal de Sa˜o Paulo, R. Botucatu 862 8A, Sa˜o Paulo, S.P., 04023-062, Brazil Received 25 April 2000; received in revised form 7 August 2000; accepted 28 August 2000

Abstract Salivary anticoagulant activities are widely distributed among hematophagous arthropods. Most of them are inhibitors of the serine proteases of the coagulation cascade. Here we show that the saliva of the exclusively hematophagous insect Triatoma infestans, an important vector in the transmission of Chagas’ disease, contains an uncommon trypsin-like activity, triapsin. This novel enzyme was purified and characterized. It is a serine protease that is stored as a zymogen in the luminal content of the salivary glands D2. Triapsin is activated by trypsin treatment, or when the saliva is ejected during the insect bite. The enzyme was purified 300-fold from the released saliva by anion exchange chromatography in a HiTrap Q column, followed by chromatography in Phenyl-Superose, and Superdex HR75. The purified triapsin shows an apparent molecular mass of around 40 kDa in non-reduced SDS gels and in sieving chromatography, and 33 kDa in reduced SDS-gels. Its activity is lost after incubation with dithiothreitol indicating that cysteine bridges are essential for activity. Triapsin cleaves gelatin and synthetic substrates showing preference for arginine at P1 residues. The best p-nitroanilide substrate is isoleucyl-prolyl-arginine. It does not cleave bradykinin, angiotensin and other lysine containing substrates. The triapsin amidolytic activity against chromogenic substrates is similar to plasminogen activators, such as urokinase and tissue plasminogen activator. However, it does not activate plasminogen. The fact that triapsin is released at the bite in its active form suggests that it has a role in blood feeding.  2001 Elsevier Science Ltd. All rights reserved. Keywords: Protease; Salivary; Triatoma infestans; Limited proteolysis

1. Introduction Inflammatory and hemostatic responses of vertebrates represent a key obstacle for acquisition of the blood meal by hematophagous arthropods. To obtain blood successfully, the salivary glands of bloodsucking arthropods produce a large variety of molecules capable of antagonizing the main effectors of host responses (Ribeiro, 1995). Protease inhibitors directed mainly to thrombin and factor Xa, which are central enzymes of the common pathway of coagulation cascade are widespread in the

* Corresponding author. Tel.: +55-11-5751996; fax: +55-115715877. E-mail address: [email protected] (S. Schenkman).

saliva of bloodsucking arthropods (Stark and James, 1996). These inhibitors prevent the activity of highly specific serine proteases, responsible for the fine regulation and tuning of the several activities involved in hemostasis and inflammation (Ware and Coller, 1995; Jesty and Nemerson, 1995; Cirino et al., 2000). On the other hand, insects, as most vertebrates and invertebrates, display proteolytic activities involved in the protein catabolism and digestive processes. In insects, these enzymes are localized mainly in the digestive tract, and are usually trypsin-like enzymes, which are serine proteases that cleave polypeptide chains on the carboxyl side of basic amino acids (arginine and lysine), have optimal activities at pH 8–9, and a molecular mass of around 20–35 kDa. Exceptions are some Coleoptera and Hemiptera species, in which the proteo-

0965-1748/01/$ - see front matter  2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 0 0 ) 0 0 1 5 1 - X

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lytic digestion seems to rely on the action of cysteine and aspartic proteases (Terra et al., 1996). Triatoma infestans (Hemiptera: Reduviidae) a vector of Chagas’ disease, which is caused by the protozoan Trypanosoma cruzi, is an anthropophilic and exclusively hematophagous insect. It obtains the blood meal by injecting its maxilla in vertebrate’s skin searching for a vessel (Lavoipierre, 1965). We have recently identified and characterized a sialidase in the salivary glands of the T. infestans (Amino et al., 1998). By investigating the function of the sialidase and the presence of anticoagulant factors in the saliva of the T. infestans, we noticed a strong amidolytic activity against isoleucylglutamyl-glycyl-arginyl substrate, used in the in vitro identification of factor Xa activity. We identified this unexpected activity as a specific trypsin-like enzyme. As endoproteolytic activity has not been found in the released saliva of hematophagous insects, we describe the purification and the biochemical characterization of this novel trypsin-like activity.

2. Materials and methods 2.1. Saliva and salivary glands T. infestans were reared at the Laborato´rio de Xenodiagno´stico, Instituto Dante Pazzanese de Cardiologia, Sa˜o Paulo, Brazil, in glass cylinders at 28°C. The insects were fed on ducks between each moult. Male adults were used in this study. One week after feeding, the insects were taken ventrally to the collector who gently blew air at their rostrum to liberate the maxilla with a drop of saliva. This small drop was collected immediately by using a glass capillary. The luminal contents of D1, D2 and D3 salivary glands (Amino et al., 1998), were collected individually by dissection under a stereo microscope. The luminal content of each gland was removed by syringe puncture and the soluble material obtained after centrifugation was used in the assays. 2.2. Activity measurements Enzymatic activity was determined by the release of p-nitroanilide (pNA) from the synthetic substrate Benzoyl-Ile-Glu-Gly-Arg-pNA (S-2222), which is a chromogenic substrate for factor Xa (Chromogenix). Activity was measured at 405 nm by using an ELISA plate reader (Labsystem Multiskan, MS). The assays were performed in 96-well tissue culture plates in 50 µl of 100 mM Tris–HCl, pH 8.0, and 0.4 mM of S-2222. The samples were incubated at 37°C and the reaction was stopped by the addition of 25 µl of 30% acetic acid. One enzymatic unit was defined by the release of 1 µmol of pNA per min at 37°C at pH 8.0. Other pNA substrates (Chromogenix) were used as indicated. Proteolytic

activity was visualized by using agarose plates (1% agarose, 100 mM Tris–HCl pH 8.0) containing 74 µM of the fluorogenic substrate N-t-Boc-Ile-Glu-Gly-Arg-7Amido-4-methylcoumarin (Sigma), observed under UV trans-illumination. The activity was also detected in SDS-PAGE under non-reducing conditions. After electrophoresis, the gel was washed twice with distilled water and twice with 100 mM Tris–HCl pH 8.0, 2% Triton X100 to remove SDS. The gel was cut in 3 mm slices with a surgery razor blade, the slices were homogenized in the presence of 50 µl of 0.4 mM of S-2222 in 100 mM of Tris–HCl, pH 8.0 and incubated at 37°C. The reaction was stopped with acetic acid and the released pNA was measured at 405 nm as above. The molecular mass standards used in the gel were albumin (66 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa) and β-lactoglobulin (17.8 kDa). Alternatively, a 12.5% SDS-PAGE was prepared containing 3.0 mg/ml of gelatin (DIFCO) to detect the proteolytic activity in the gel (Hanspal et al., 1983). After electrophoresis, the gels were washed twice (30 s) with 2% Triton X100 to remove SDS, and incubated at 37°C in 100 mM Tris– HCl, pH 8.0. After incubation, the gel was stained with Coomassie Blue R250 in ethanol:water:acetic acid (4:4:1), and destained with a solution of etanol:water:acetic acid (4:4:1). 2.3. Triapsin purification Two hundred microlitres of saliva were diluted with 400 µl of Milli Q water and centrifuged for 10 min at 14,000 g. The supernatant was chromatographed in a HiTrap Q column (Pharmacia) equilibrated in 20 mM Tris–HCl, pH 8.0. The activity was eluted with a gradient up to 1.0 M NaCl in the equilibration buffer. Two amidolytic peaks were obtained (see Section 3). The second amidolytic activity peak was pooled, mixed with the same volume of 3.4 M (NH4)2SO4, and applied into a Phenyl-Superose column (Pharmacia) equilibrated with 20 mM Tris–HCl, pH 8.0, 1.7 M (NH4)2SO4. The column was eluted with a gradient of up to 20 mM of Tris– HCl, pH 8.0. The fractions containing activity were pooled, concentrated using a Centricon 10 (Amicon) filter to 250 µl, and applied into a Superdex HR75 column (Pharmacia) equilibrated in 20 mM of Tris–HCl, pH 8.0, 150 mM NaCl. 2.4. Plasminogen activation Plasminogen (Sigma) was pre-incubated with either saliva or tissue plasminogen activator (t-PA, Boehringer) for 5 min at 37°C. After this pre-incubation, the plasmin substrate S-2251 H-D-Val-Leu-Lys-pNA (Chromogenix) was added to 0.8 mM and the mixture incubated for 10 min at 37°C. The amounts of saliva and t-PA used in

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the assay were normalized by the activity toward 0.2 mM H-D-Ile-Pro-Arg-pNA (S-2288, Chromogenix).

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the insertion of the insect maxilla (Fig. 2). No fluorescence was seen in uncoated plates. This result shows that a protease is released with ejected saliva in active form.

3. Results 3.1. Identification of a proteolytic activity in T. infestans saliva A sample of saliva released by T. infestans was fractionated by SDS-PAGE containing gelatin, and the presence of proteolytic activity was determined after the removal of SDS using Triton-X100, incubation at 37°C, and staining with Coomassie Blue. As shown in Fig. 1A, a strong proteolytic activity was found in a band migrating with a molecular mass of around 40 kDa. In a sample run in a parallel gel, activity was detected in a similar position by using the S-2222, a factor Xa substrate (Fig. 1B). This salivary proteolytic activity was denominated triapsin. Similar results were obtained in gelatin gels by using saliva of males, females, or fourth and fifth stage nymphs. 3.2. Triapsin protease is released at moment of the bite To verify whether the protease was released with saliva during the insect bite, we took advantage of the fact that when starved, T. infestans bites any warm surface releasing saliva. Thus, when a warmed agarose plate, bitten by three insects, was coated with the fluorogenic substrate, N-t-Boc-Ile-Glu-Gly-Arg-7-amido-4-methylcoumarin, fluorescent spots were detected at the place of

Fig. 1. Detection of proteolytic and amidolytic activity from T. infestans saliva separated by SDS-PAGE. (A) Saliva (5 µl) was loaded in a SDS-PAGE (12.5% polyacrylamide) containing 3.0 mg/ml of gelatin. After electrophoresis, the gel was washed, incubated at 37°C for 30 min and stained with Coomassie Blue R250. (B) Saliva (2.5 µl) was run in a SDS-PAGE without gelatin and the gel cut into 3 mm slices. The pieces were tested for amidolytic activity using H-D-IleGlu-Gly-Arg-pNA. The standards (쐌) are described in Section 2.

3.3. Triapsin is stored in D2 salivary gland as zymogen Very low amidolytic (Fig. 3A) and proteolytic (Fig. 3B) activities were detected in the luminal contents of D1, D2 and D3 salivary glands in comparison with the activity found in the released saliva, suggesting saliva release activates the protease. When immobilized trypsin was added to the luminal contents of each gland, a large increase in the activity was found in the D2 gland by using either the amidolytic or the gelatin gel assay. These results suggest that inactive triapsin is stored in the lumen of the D2 gland, and an activation mechanism, based on limited proteolysis, occurs during saliva release. Increase in activity was not due to contamination with trypsin from beads, as the proteolytic activity detected in the zymogram of Fig. 3B migrated differently from trypsin (Fig. 3D). Fig. 3B also shows that the luminal contents of the D2 salivary gland processed after trypsin-Sepharose treatment was similar to the protein pattern found in the ejected saliva (Fig. 3C). 3.4. Triapsin purification To further characterize triapsin, saliva was diluted with water and chromatographed in a strong anion exchanger HiTrap Q column at pH 8.0. Two peaks containing amidolytic activity eluted with ionic strength of 200 and 400 mM of NaCl (Fig. 4A). The largest activity peak eluted at 400 mM of NaCl and corresponded to about 64% of the total activity. As the activity in the second peak was 215-fold enriched compared to the original saliva (Table 1), in which most of proteins with

Fig. 2. Amidolytic activity is released at the insect bite site. An agarose plate was bitten by starved T. infestans and immediately photographed under UV exposition after the addition of the fluorogenic substrate Boc-Ile-Glu-Gly-Arg-AMC (74 µM). The black arrows show some of the bright dots, which correspond to hydrolysis of the substrate at the place of the maxilla insertion.

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Fig. 3. Triapsin, localized in the D2 salivary gland, is activated upon saliva released, or by trypsin treatment. D1, D2 and D3 salivary glands were removed from 10 insects and their contents were collected separately in 60 µl of phosphate buffered saline. (A) One half of the collected material was incubated with 4 µl of trypsin-Sepharose beads for 5 min at room temperature. The beads were removed by centrifugation and the supernatant was used on the assays. The trypsin-Sepharose treated (hatched bars) or untreated (open bars) samples were incubated with H-D-Ile-Glu-Gly-Arg-pNA. (B) The other half of the sample was loaded into a SDS-PAGE 12.5% containing gelatin. (C) A sample with 1.5 µl of ejected saliva. (D) Soluble trypsin sample (10 ng).

size below 30 kDa were removed (compare lanes a and b, Fig. 5), we continued the purification with this fraction. The purification was followed with an hydrophobic column of Phenyl-Superose (Fig. 4B). This column also resolves the amidolytic activity in two peaks. These peaks might correspond to the same protease processed at different sites, as they produce in gelatin gels, bands of similar size (not shown). To further characterize the protease and to obtain a highly purified preparation to be used in functional assays, we decided to use the fraction with the highest activity eluted from the PhenylSuperose column. In this fraction, although the specific activity decreased (Table 1), most of the contaminants with size similar to the triapsin were removed (Fig. 5, lane c). The use of a large pool resulted in a worse preparation. The single fraction was concentrated by ultrafiltration and fractionated by using a molecular sieving column (Fig. 4C). Triapsin eluted with a size corresponding to 40 kDa, yielding a 300-fold final purification, with 7% of the initial activity, and specific activity of 12 units/mg (Table 1). A single band was

Fig. 4. Triapsin purification. Two hundred microlitres of saliva was diluted in water and the insoluble material was removed by centrifugation. (A) The supernatant was chromatographed in an anion exchanger HiTrapQ column and eluted with 20 ml of a linear gradient to NaCl 1.0 M. The second peak of amidolytic activity (pool A) was collected and applied in a Phenyl Superose column (B). The bound material was eluted with a decreasing linear gradient of (NH4)2 SO4 1.7 M. The peak of amidolytic activity was concentrated by centrifugation in a Centricom 10 and chromatographed in a Superdex HR75 column (C). The dotted lines represent the absorbance at 280 nm, the open circles represent the amidolytic activity using H-D-Ile-Glu-Gly-Arg-pNA, and the traced lines are the salt gradients.

obtained in a silver stained gel (Fig. 5A, lane d), which corresponded to the protein size detected in the zymogram (Fig. 5B). Triapsin was further chromatographed in an HPLC column (C8) in a 0.1% TFA–acetonitrile system, and a single peak was obtained. 3.5. Effect of protease inhibitors As shown in Table 2, triapsin was inhibited by aminophenyl-methyl-sulfonyl fluoride (APMSF), soybean

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469

Table 1 Triapsin purification steps Step

Saliva HiTrapQ Phenyl-Superose Superdex a

Volume

Protein

Activity

(ml)

(µg)

(%)

(unitsa)

0.6 4.0 0.5 1.0

10,500 28 3.75 2.0

100 0.27 0.036 0.019

0.379 0.241 0.029 0.025

Purification fold (%)

(units/mg)

100 63.6 7.7 6.6

0.04 8.60 7.70 12.33

1.0 215 193 308

1 unit=1 µmol of pNA released per min at 37°C.

Table 2 Effect of protease inhibitors on the triapsin activity. Partially purified triapsin (after Phenyl-Superose column) was incubated for 30 min at 37°C with 0.24 mM of H-D-Ile-Glu-Gly-Arg-pNA in the presence of the indicated concentration of protease inhibitor. The table shows the inhibition relative to the control incubated in the absence of the indicated reagents Inhibitor

Concentration (µM) Inhibition of activity (%)

APMSF SBTI 2T Antipain E-64 o-phenanthroline EDTA Pepstatin

200 1.6 0.7 30 100 10×103 10×103 145

a

80 34 72 97 n.i.a n.i. n.i. n.i.

n.i.=not inhibited.

Fig. 5. Protein and proteolytic activity profile during triapsin purification. (A) Silver stained gel (12.5% SDS-PAGE) of total saliva (lane a) and fractions containing amidolytic activity eluted from Hitrap Q (lane b), Phenyl Superose (lane c) and Superdex (lane d) columns. (B) A gel run in parallel containing gelatin, was washed and incubated at 37°C for 2 h and stained with Coomassie Blue R250 to determine proteolytic activity of the samples. The loading in both gels corresponds respectively to 0.17%, 0.38%, 4% and 4% of the total of each fraction.

trypsin inhibitor (SBTI) and antipain, showing a typical inhibition pattern of serine proteases. Triapsin was also inhibited by LDTI-2T, a thrombin inhibitor from leech selected by using a phage display library, which has an arginine residue at the P1 position (Tanaka et al., 1999). No inhibition was found for EDTA, o-phenanthroline, E64 and pepstatin. Addition of APMSF, an irreversible inhibitor of serine proteases, before SDS-PAGE was effective in inhibiting the activity of non-purified and purified triapsin (Fig. 6). In contrast antipain, which is a reversible competitive inhibitor of some serine proteases, was unable to block the activity in the zymogram (not shown). These data reinforce the hypothesis that triapsin is a serine protease.

Fig. 6. The proteolytic activity of Triapsin is inhibited by APMSF. Total saliva and purified triapsin were incubated with APMSF and loaded into a SDS-PAGE (12.5%) containing gelatin. To analyze the proteolytic activity of the samples the gel was incubated for 1 h at 37°C and stained with Coomassie Blue R250.

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Fig. 7. Effect of DTT on the activity and migration of purified triapsin. The purified triapsin was incubated in the presence and absence of 2 mM DTT. (A) The enzyme activity was measured by hydrolysis of H-D-Ile-Glu-Gly-Arg-pNA. (B) Detection of the proteolytic activity on gelatin. (C) DTT treated and untreated samples visualized after silver staining.

Triapsin activity was lost when treated with the reducing agent dithiothreitol, as measured by the amidolytic and zymogram assay (Fig. 7A,B). Activity inhibition was accompanied by change in the migration in SDSPAGE (Fig. 7C), suggesting that S–S bridges are important for the maintenance of triapsin tertiary structure. 3.6. Triapsin substrate specificity The substrate specificity of triapsin was tested against substrates used to detect serine protease activity in vertebrate blood. As shown in Table 3, the best cleaved substrate was the H-D-Ile-Pro-Arg-pNA, followed by the AC-Ile-Glu-Gly-Arg-pNA, H-D-Phe-Pip-Arg-pNA, HD-Pro-Phe-Arg-pNA, and H-D-Val-Leu-Arg-pNA. No hydrolysis was detected with H-D-Val-Leu-Lys-pNA (Table 3), bradykinin, and angiotensin (data not show). Therefore, triapsin shows high specificity for arginine containing substrates. Table 3 Triapsin activity measured with synthetic substrates. Triapsin purified up to the Phenyl-Superose column was incubated with the synthetic substrates listed below in a concentration of 0.3 mM in Tris–HCl, 100 mM pH 8.0, at 37°C. The numbers are mean ± standard deviation of triplicate measurements Substrate

Amidolytic activitya

H-D-Ile-Pro-Arg-pNA Ac-Ile-Glu-Gly-Arg-pNA H-D-Phe-Pip-Arg-pNA H-D-Pro-Phe-Arg-pNA H-D-Val-Leu-Arg-pNA H-D-Val-Leu-Lys-pNA

27.21±0.54 6.31±0.11 3.71±0.08 3.21±0.03 3.06±0.35 0

a

µmol of pNA released per min per mg of protein.

3.7. Plasminogen activation Triapsin shows a substrate specificity similar to the plasminogen activators, t-PA and urokinase (Table 4). To test if triapsin could activate plasminogen, we preincubated plasminogen with saliva, or with t-PA and tested for hydrolysis of H-D-Val-Leu-Lys-pNA, which is not a substrate for triapsin, or t-PA. The same activity towards H-D-Ile-Pro-Arg-pNA was used for both activators. While t-PA activated plasminogen, no plasmin activity was obtained with total saliva. We also tested partially purified triapsin (the peak of the Phenyl-Superose column) and even after 5 h of incubation (not shown) no plasmin activity was observed.

4. Discussion We have identified a powerful trypsin-like protease that is released with the saliva of the exclusively hematophagous insect. This protease was named triapsin, and is present in the D2 salivary gland as an inactive precursor. Saliva ejection, stimulated by biting, promotes triapsin activation. This activation seems to rely on limited proteolysis, since the treatment of luminal contents of D2 salivary glands with trypsin beads generates the active form of triapsin found in ejected saliva. Active triapsin purified from the ejected saliva was unable to induce triapsin activation, or to change the protein pattern from the luminal contents of D2 salivary glands (data not shown). Therefore, a second trypsinlike activity may be responsible for the triapsin activation upon saliva release. A careful examination of the zymogram of trypsin-treated luminal contents of D2 salivary glands reveals a minor proteolytic activity in the

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471

Table 4 Comparison of hydrolysis profile of trypsin, blood and T. infestans proteasesa Substrates

Serine proteaseb TRY

Ac-Ile-Glu-GlyArg-pNA H-D-Phe-Pip-ArgpNA H-D-Ile-Pro-ArgpNA H-D-Pro-Phe-ArgpNA H-D-Val-Leu-ArgpNA H-D-Val-Leu-LyspNA

THR

FXa

KAL

PLA

aPC

URO

t-PA

Triapsin

1.00

0.01

1.00

0.03

0.14

⬍0.01

0.23

0.80

0.23

0.37

1.00

0.04

0.04

0.05

0.97

0.07

0.13

0.14

0.48

0.58

0.42

0.53

1.00

1.00

1.00

1.00

1.00

0.10

0.06

0.20

1.00

0.95

0.27

⬍0.01

0.10

0.12

0.15

0.01

⬍0.01

0.03

0.05

0.48

0.03

0.13

0.11

0.04

⬍0.01

⬍0.01

0.04

0.95

–

⬍0.01

0.10

⬍0.01

a

Data were extracted from Chromogenix commercial catalog and were normalized as 1 towards the best substrate. Trypsin (TRY), thrombin (THR), factor Xa (FXa), kallicrein (KAL), plasmin (PLA), activated protein C (aPC), urokinase (URO), tissue plasminogen activator (t-PA), T. infestans salivary protease (triapsin). b

range of 60–70 kDa. This activity could be the protease responsible for triapsin activation. Alternatively, it could be an active, but not fully processed triapsin or another zymogen, as this 60–70 kDa protease is just seen in freshly released saliva, and disappears as the saliva is stored. In addition, another protease with undetectable amidolytic activity and unable to cleave gelatin could be the activator. Interestingly, besides triapsin activation, most of the D2 salivary proteins appears to undergo proteolytical changes, as the gel pattern of saliva is only similar to the protein pattern of the D2 salivary glands after trypsin treatment. This proteolytic processing is restricted to D2 salivary glands. The protein pattern of D1 salivary glands remains unaltered when compared with the pattern of the ejected saliva. Perhaps, such a kind of processing regulates other activities, which could influence the blood acquisition mechanism. Therefore, it might be interesting to investigate how triapsin activation occurs, as well as the mechanism that led to activation upon saliva ejection. Triapsin was purified to homogeneity, as judged by silver stained SDS-PAGE, and has a molecular mass expected for trypsin-like proteases (苲40 kDa). A second isoform of triapsin could be seen in SDS-PAGE migrating with the same size, and seems to correspond to the first peak of the HiTrap column. This enzyme could be an isoenzyme, present in a sub-population of insects, it could represent another product of activation of the same pro-enzyme, or it could be another protease. The present characterization will allow us to generate enough enzyme, by using the most abundant form, in order to obtain amino acid sequence information and solve this issue. Based on the inhibition profile of the purified triapsin

we conclude that it is a serine protease, with high specificity towards arginine at the P1 site. Triapsin could be involved in the painless response when the Triatome sp inject and keep for a few minutes its maxilla in the vertebrate skin. However, triapsin is unable to cleave angiotensin, lysine containing substrates, and bradykinin, which could mediate pain sensibility. By comparing the substrate preference of the triapsin with blood serine proteases involved in homeostasis, we found that triapsin is similar to the plasminogen activators, t-PA and urokinase, suggesting, on the other hand, that it could act as a plasminogen activator (Table 4). These enzymes have preferences for Ile-Pro-Arg-pNA, followed by Ile-GluGly-Arg, and very weak activity against the other arginine containing substrates. Our data, however, show that triapsin does not activate plasminogen, and may therefore be directed to other substrates. As plasminogen activators have been found in the saliva of an entomophagic insect of the Reduviidae family (Ben Hamouda and Ammar, 1984; Ammar and Ben Hamouda, 1985) it is possible that these proteases had evolved from a common ancestry. This could explain the resemblance of triapsin with t-PA and urokinase. The lack of plasminogen activation may reflect a functional divergence between this salivary enzymes driven by their feeding habits. Proteases have also been described in the saliva of Platymeris rhadamnthus (Hemiptera, Reduviidae), another insect predator (Edwards, 1961). Molecular cloning is required to further establish any similarity between these enzymes. Triapsin does not inhibit the recalcification time of platelet-poor plasma and does not have fibrinolytic activity in fibrin-agar plates (data not shown). However, there are several other possible targets for triapsin required for appropriate blood acquisition. For example,

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triapsin could hydrolyse members of the superfamily of Proteinase Activated G protein-coupled Receptors (PAR). The PARs regulate growth, development, inflammation, and responses to injury, and they are activated by a single cleavage in the N-terminal site (LDPR↓SF —PAR1, SKGR↓SL — PAR 2, LPIK↓TF — PAR 3, PAPR↓GY — PAR 4) by proteases such as thrombin, trypsin and tryptase (Bohm et al., 1996). The fact that triapsin is inhibited much better by the 2T, which contains PRVLWA, than by the 5T (PRVLSL), or 10T (PRLLDI) (Tanaka et al., 1999) (data not shown), suggests that a highly specific substrate may be the target for triapsin. A digestive role for triapsin is unlikely because the proteolytic digestion of Hemiptera seems to depend on the action of cysteine and aspartic proteases, which possess a slight acidic optimum pH, compatible with the pH of the digestive tube of T. infestans (Terra et al., 1996). Triapsin has a more alkaline optimal pH. Importantly, endoproteolytic activity has not been described in the saliva of hematophagous insects. Moreover, these organisms have been considered to lack proteases in the saliva and have several protease inhibitors (Jacobs et al., 1990; Perez et al., 1998; Cappello et al., 1998; Stark and James, 1998; Waidhet-Kouadio et al., 1998; Valenzuela et al., 1999). Therefore, acquisition of a powerful protease with a fine specificity by T. infestans, may represent an adaptive advantage related to its blood feeding abilities. In summary, we have demonstrated the presence of an activatable serine protease that is injected in an active form in vertebrate skin. It does not have an apparent digestive role and its function remains obscure. We have also shown that a part of the repertoire of molecules of salivary glands is processed upon saliva release by limited proteolysis. This processing could be more disseminated in salivary glands, which would have implications for disease transmission by hematophagous vectors. Acknowledgements We thank Vera Lucia Pereira-Chioccola and Margarida Bezerra, from the Laborato´rio de Xenodiagno´stico do Instituto Dante Pazzanese, Sa˜o Paulo, for providing the T. infestans, and Dr Claudio A.M. Sampaio (UNIFESP) for reading the manuscript. This work was supported by grants from the Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo (FAPESP), and Conselho Nacional de Desenvolvimento Cientı´fcio e Tecnolo´gico (CNPq), Brazil. References Amino, R., Porto, R.M., Chammas, R., Egami, M.I., Schenkman, S., 1998. Identification and characterization of a sialidase released by

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