A short proregion of trialysin, a pore-forming protein of Triatoma infestans salivary glands, controls activity by folding the N-terminal lytic motif Rafael M. Martins 1 , Rogerio Amino 2 , Katia R. Daghastanli 3 , Iolanda M. Cuccovia 3 , Maria A. Juliano 4 and Sergio Schenkman 1 1 Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Sa˜o Paulo, Brazil 2 Departamento de Bioquı ´ mica, Universidade Federal de Sa˜o Paulo, Brazil 3 Departamento de Bioquı ´ mica, Instituto de Quı ´ mica, Universidade de Sa˜o Paulo, Brazil 4 Departamento de Biofı ´ sica, Universidade Federal de Sa˜o Paulo, Brazil Hematophagous animals counteract physical and molecular barriers such as the epidermis and the inflammatory, hemostatic and immune systems of the hosts to fulfill their nutritional needs [1]. Therefore, their saliva has evolved for the specific task of circum- venting several biochemical cascades to facilitate blood acquisition. Triatomine insects are exclusive blood- feeders that transmit Chagas’ disease, acquiring Try- panosoma cruzi from the blood of infected mammalian hosts, and transmitting this parasite through their feces, instead of injecting protozoa during the bite, like anophelines, sand-flies, or tsetse flies [2]. In the salivary secretion of Triatoma infestans are found three differ- ent anticoagulant activities [3]: proteases [4], a sialidase [5], apyrases [6], an inhibitor of platelet aggregation [7], a Na + channel blocker [8], and a cytolytic protein named trialysin [9]. Trialysin is a pore-forming protein that permeabilizes several cell types, from bacteria to mammalian cells. Synthetic peptides based on the mature amphipathic N-terminus of trialysin (first 37 amino acids) induce bacterial, protozoal and mammalian cell membrane permeabilization, and their solution structures show characteristics of cationic amphipathic antimicrobial peptides [10]. The lytic activities presented by trialysin, as well as by other pore-forming proteins and peptides, must be well controlled in order to avoid destroying the membrane compartments during their synthesis and secretion. The cecropin and melittin antimicrobial peptides are synthesized as larger polypeptides that are Keywords membrane lysis; salivary gland; trialysin; Triatoma infestans; Trypanosoma cruzi Correspondence S. Schenkman, Departamento de Microbiologia, Imunologia e Parasitologia, Rua Botucatu 862 8 o andar 04023-062 Sa˜o Paulo, SP, Brazil Fax: +55 11 5571 58 77 Tel: +55 11 5575 19 96 E-mail: sschenkman@unifesp.br (Received 30 August 2007, revised 3 December 2007, accepted 2 January 2008) doi:10.1111/j.1742-4658.2008.06260.x Triatoma infestans (Hemiptera: Reduviidae) is a hematophagous insect that transmits the protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas’ disease. Its saliva contains trialysin, a protein that forms pores in membranes. Peptides based on the N-terminus of trialysin lyse cells and fold into a-helical amphipathic segments resembling antimicrobial peptides. Using a specific antiserum against trialysin, we show here that trialysin is synthesized as a precursor that is less active than the protein released after saliva secretion. A synthetic peptide flanked by a fluorophore and a quencher including the acidic proregion and the lytic N-terminus of the protein is also less active against cells and liposomes, increasing activity upon proteolysis. Activation changes the peptide conformation as observed by fluorescence increase and CD spectroscopy. This mechanism of activa- tion could provide a way to impair the toxic effects of trialysin inside the salivary glands, thus restricting damaging lytic activity to the bite site. Abbreviations Abz, o-aminobenzoic acid; APMSF, (4-amidinophenyl)methanesulfonyl fluoride; GST, glutathione S-transferase; LUV, large unilamellar vesicle; NL, nonlytic; PGS, peptide between glycine and serine; Q-EDDnp, Gln-n-(2,4-dinitrophenyl)-ethylenediamine. 994 FEBS Journal 275 (2008) 994–1002 ª 2008 The Authors Journal compilation ª 2008 FEBS processed by proteolytic cleavage, resulting in one active mature molecule [11,12]. On the other hand, one prepromagainin polypeptide generates one magainin I peptide between glycine and serine (PGS) and five mag- ainin II (PGS-Gly10-Lys22) molecules [13]. The pro- regions of magainin and melittin are composed of negatively charged residues that may inhibit their action [12,13]. Aureins [14], a-defensins (cryptidins) [15], derm- aseptins [16] and latarcins are also examples of peptides containing acidic proregions [17]. In contrast, apolar residues constitute the proregion of cecropin [18]. A large proregion of approximately 100 residues is present in cathelicidins, which are mammalian proteins with multiple functions, including antimicrobial activity [19]. There is structural evidence that the processing of cath- elicidins is performed in dimeric, domain-swapped structures that expose the cleavage site and, at the same time, control their activities, impairing the antimicrobial activity of the C-terminus [20]. Trialysin cDNA predicts a secretion signal followed by an acidic domain composed of 33 amino acids, which is not found in the mature protein. The activa- tion of protrialysin into mature trialysin was suggested to occur by limited proteolysis, as it is prevented by (4-amidinophenyl)methanesulfonyl fluoride (APMSF), which inhibits the major serine protease of T. infestans saliva [4]. However, the precursor form was not previ- ously identified, and it is unknown when it is activated and whether it prevents lysis induced by trialysin released with the saliva. To identify the precursor and further understand how a potent lytic molecule is syn- thesized and controlled, we raised antibodies that react with trialysin [205 amino acids long, the nonlytic (NL) fragment] and tested for the presence of different forms of the protein and their activity in T. infestans. We found that a precursor is stored in the salivary glands and processed only after saliva is released. A peptide containing the precursor region and the lytic N-terminus of trialysin was synthesized, containing a fluorophore and a quencher at the N-terminus and C-terminus respectively. With use of this peptide, evidence was obtained showing that the activation mechanism of trialysin involves conformational changes in this segment of the protein. Results Generation of specific anti-trialysin rabbit serum Trialysin cDNA predicts a signal sequence followed by an acidic domain, shown in bold and italicized letters in Fig. 1A, upstream of the N-terminus sequence detected in the protein isolated from T. infestans saliva [9]. In order to detect precursor forms and under- stand the mechanism of activation of T. infestans protrialysin, we raised specific antibodies capable of recognizing salivary trialysin. Attempts to obtain the full-length precursor and mature forms of the recombi- nant protein in heterologous systems to immunize animals were unsuccessful. Therefore, a C-terminal NL fragment of trialysin corresponding to the underlined letters in Fig. 1A was subcloned into the pET-14b plasmid and expressed in BL21 (DE3) pLysE Escheri- chia coli cells. The recombinant protein was only soluble in urea. It was purified by Ni-affinity and gel exclusion chromatography, generating a single band of 21 kDa, as seen by Coomassie Blue staining after SDS ⁄ PAGE (Fig. 1B, lane C). It was used to immunize rabbits, and the resulting immune serum specifically recognized the recombinant protein in immunoblots after SDS ⁄ PAGE and a 24 kDa band in the T. infe- stans saliva (Fig. 1B, lane IB). No signal was obtained with preimmune or unrelated sera (data not shown). A B Fig. 1. Sequence, expression and purification of the NL fragment of trialysin. (A) The translated cDNA predicted for preprotrialysin. The leader peptide sequence is shown in bold letters, the proregion is shown in italics, and the NL fragment is underlined. The arrow indicates the N-terminus of the proP7 peptide. Numbers show the positions of amino acids according to the N-terminus of mature tri- alysin (+1), the predicted signal sequence ()55, from M1 to A22), the acidic proregion ()33, from A23 to R55), and the cleavage site ()1, R55). (B) SDS ⁄ PAGE of the NL sequence (NL2) of trialysin expressed in E. coli BL21 (DE3) pLysE; purified by Ni-affinity chro- matography after staining with Coomassie Blue (C) and reaction with antibodies to NL2 trialysin (IB). On the right is shown the T. infestans saliva stained with antibody to NL2. On the left are shown the positions of 20 kDa and 25 kDa molecular mass mark- ers (Benchmark, Invitrogen). R. M. Martins et al. Trialysin precursor activation FEBS Journal 275 (2008) 994–1002 ª 2008 The Authors Journal compilation ª 2008 FEBS 995 Protrialysin is processed after saliva is secreted We first investigated whether mature trialysin was already processed when saliva was ejected. Small amounts of saliva were collected in ice and analyzed by immunoblot. As shown in Fig. 2A, a protein larger than 24 kDa (the size of the purified trialysin) was detected. After incubation at room temperature for 10 min, the 26 kDa protein was converted to the size of mature trialysin that was further processed at room temperature, as judged by the lower molecular mass bands that appeared at 45 and 60 min. This result indi- cates that trialysin is processed after saliva ejection. The precursor form released in saliva is found in the salivary glands To detect protrialysin stored in the salivary glands, and determine whether it would retain lytic activity, glands were extracted in the presence, or absence, of APMSF, previously shown to inhibit triapsin activity, a protease found in saliva that is proposed to be responsible for trialysin activation [9]. The 26 kDa precursor was mainly detected when the glands were prepared in the presence of APMSF (Fig. 2B, lane APMSF+), or when the glands were directly boiled in SDS ⁄ PAGE loading buffer, whereas the 24 kDa form was observed in the absence of the inhibitor (Fig. 2B, lane APMSF)). Concomitantly, the lytic activity against trypanosomes and erythrocytes was consider- ably inhibited in the glands homogenized with APMSF (Fig. 3). Inhibition of activity was not caused by the APMSF itself, as glands previously prepared in the absence of APMSF had the same lytic activity if the inhibitor was added later. A BC Fig. 2. SDS ⁄ PAGE and immunoblot of samples in different condi- tions using anti-NL2 trialysin serum. (A) Aliquots from saliva incu- bated at room temperature were taken at different time points (0, 5, 10, 20, 30, 45 and 60 min). The arrow indicates protrialysin and the arrowhead indicates mature trialysin. (B) Salivary glands of T. infestans dissected and solubilized in the absence ()) or pres- ence (+) of APMSF, or directly boiled in SDS ⁄ PAGE sample buffer (boil). The lane (rec-tria) contained a GST–protrialysin recombinant pretreated with thrombin. (C) An immunoblot with the recombinant GST–protrialysin untreated ()) and treated (+) with purified triapsin. The open arrow indicates the preprotrialysin size after removal of the leader sequence; the black arrow indicates the protrialysin found in the salivary glands, and the arrowhead shows the size of mature trialysin detected in the saliva. Molecular masses are indi- cated on the left. A B Fig. 3. Lytic activities of T. infestans salivary gland homogenates depend on proteolytic activation. Trypomastigote forms of Try. cruzi (3 · 10 6 ÆmL )1 ) (A) or human erythrocytes (3 · 10 7 ÆmL )1 ) (B) were incubated at 37 °C for 1 h and 2.5 h respectively with different amounts of salivary gland homogenates prepared in the presence (d) or absence (s) of APMSF. In (A) the number of surviving cells was determined in a hemocytometer, and the percentage of lysis was calculated relative to the control. In (B), permeabilization per- centage was obtained relative to hemoglobin release of control by treating the cells with 0.2% Triton X-100. Trialysin precursor activation R. M. Martins et al. 996 FEBS Journal 275 (2008) 994–1002 ª 2008 The Authors Journal compilation ª 2008 FEBS To identify which form of the protein would represent the active trialysin, a glutathione S-transferase (GST)– protrialysin fusion protein was expressed in E. coli. This protein contained GST, a thrombin cleavage site, and the protrialysin from amino acid ) 33 to the C-terminus (see Fig. 1A). It was obtained from soluble E. coli extracts and purified by chromatography in a glutathi- one–Sepharose column. The fusion protein was largely unstable, and it did not show lytic activity, precipitating as the protein concentration increased in solution. It could be processed by thrombin, generating a 30 kDa protein band in SDS ⁄ PAGE (Fig. 2C, lane triapsin)). The processed recombinant protein was also unable to promote lysis. When partially purified triapsin was added to the thrombin-cleaved 30 kDa protein, it was processed to a 24 kDa band (Fig. 2C, lane triapsin+). In some experiments, lytic activity was detected, although the resulting protein precipitated and became inactive, as observed with the trialysin purified from the saliva [9]. The same processed bands were obtained when the GST fusion protein was directly incubated with triapsin, confirming that the 24 kDa molecule found in saliva is the active form of trialysin, and sug- gesting that the presence of 10–15 amino acids of the proregion of protrialysin found in the glands (instead of the predicted 33-mer acidic propiece) is sufficient to inhi- bit lytic activity. A synthetic peptide containing the proregion and the N-terminus of trialysin is structured The amino acid sequence of the N-terminus of the tri- alysin precursor has not been determined so far either by Edman degradation or by MS. To investigate how a small additional negative prosequence could decrease the lytic activity, a peptide containing 12 residues from the proregion and 27 residues downstream of the N-terminus of mature trialysin was synthesized. This 12 residue segment roughly corresponds to the differ- ence observed between the mature and precursor forms. The 27-mer segment was previously character- ized as the lytic peptide P7 [10]. The new peptide (proP7) is represented in Fig. 4A, and was synthesized containing at the N-terminus the fluorescent probe o-aminobenzoic acid (Abz), and at the C-terminus a fluorescence quencher group Gln-N-(2,4-dinitrophe- nyl)ethylenediamine (Q-EDDnp). When proP7 was treated with endoproteinase Arg-C, which cleaves at the unique Arg of proP7, it released the negative prosequence from the P7 portion, and this was accom- panied by an increase in fluorescence over time (Fig. 4B). A similar increase in fluorescence was observed when triapsin or saliva was added (not shown). As quenching of fluorogenic peptides longer than 40 amino acids is minimal, unless they are folded [21], this increase in fluorescence suggests that proP7 is folded before being processed. The fact that proP7 fluorescence in 6 m guanidine hydrochloride was higher than the fluorescence in nondenaturing conditions con- firms this hypothesis, although part of the increase in fluorescence may be due to the peptide hydrolysis, which abolishes intramolecular quenching. Evidence that proP7 is structured and that it unfolds after Arg-C treatment was also obtained by CD spectros- copy (Fig. 4C). ProP7 contains 36% a-helix, decreas- ing to 7% after Arg-C treatment. Lysis increases after cleavage of proP7 Next, the lytic activities of proP7 and Arg-C-processed peptide were compared by using artificial liposome membranes (20 : 80 cardiolipin ⁄ phosphatidylcholine) containing 6-carboxyfluorescein, which is a fluorophore that autosuppresses its fluorescence at higher concen- trations. Upon permeabilization, these liposomes release the entrapped quenched 6-carboxyfluorescein, diluting the fluorophore in the sample, and fluores- cence increases. As expected, the Arg-C-processed peptide was more effective at promoting liposome permeabilization than proP7 (Fig. 4D,E). Similar results were obtained when the lysis of trypanosomes was assayed (Fig. 4F). These results indicate that a small acidic portion of the proregion decreases the lysis efficiency of trialysin in the model using a lytic synthetic peptide based on the trialysin N-terminus. Discussion Here we describe trialysin processing from its accumu- lation in the salivary glands of T. infestans until its release during saliva ejection, when the full lytic capac- ity of the protein is achieved. Judging by the cDNA sequence, protrialysin should contain a 22 amino acid prosequence migrating in SDS ⁄ PAGE as a 32 kDa protein. However, we only detected a shorter protein (26 kDa) that accumulates in the glands and is released during salivation. This 26 kDa precursor is less active than mature trialysin, due to no more than 15 amino acid residues in the proregion. This finding was con- firmed using an engineered synthetic peptide contain- ing 12 amino acids from the proregion followed by the trialysin N-terminus. Upon proteolytic processing, the precursor-like synthetic peptide unfolds, as observed in the CD spectra, and lytic activity increases. We pro- pose that the presence of a short acidic sequence affects the trialysin N-terminus, preventing its positive R. M. Martins et al. Trialysin precursor activation FEBS Journal 275 (2008) 994–1002 ª 2008 The Authors Journal compilation ª 2008 FEBS 997 charges from interacting with the phospholipid head groups on target membranes that are required to pro- mote lysis. One possible reason for this inhibition is that this acidic region interacts with the basic residues in the amphipathic N-terminus, preventing its interaction with phospholipid head groups on the target mem- brane. The observed residual lytic activity for proP7, and protrialysin, might occur because the acidic seg- ment could not block all available cationic surfaces. Alternatively, lysis could be inhibited by the formation of dimers, in which the proregion of one molecule would interact with the cationic surface of another molecule. It has been shown in the case of cathelicidins (stored in neutrophil granules) that impairment of activity can be accomplished by domain-swapping, in which two molecules fold themselves together as a dimer [20]. In the case of trialysin, we have no evidence for dimerization, as the precursor molecule isolated from the gland in the presence of APMSF behaves as a monomer in gel exclusion chromato- graphy (not shown). We cannot exclude the possibility, however, that when stored at high concentration in the salivary glands, protrialysin dimerizes. Structural data on the N-terminus of trialysin pep- tides show that the very first amino acids are folded in a nonrigid structure that could be part of the bending region of the hairpin in the proposed model [10]. This flexibility could allow the acidic domain to interact with positive charges in the context of the protein, but much less in the case of a short synthetic peptide, as secondary interactions with the protein C-terminal domain are absent, suggesting that the protein struc- ture might also have additional roles in the inactiva- tion of protrialysin. In fact, the increase in fluorescence of proP7 after specific proteolysis by A B D C F E Fig. 4. ProP7 design, activation and lytic activity. (A) Schematic representation of the proP7 peptide with the fluorescence donor Abz at the N-terminus and the quencher Q-EDDnp at the C-terminus. (B) Time course of fluorescence increase of proP7 (3.6 l M) after incubation without (s) or with (d)5lgÆmL )1 endoproteinase Arg-C at 37 °C. Fluorescence readings from endopro- teinase Arg-C-treated ( ) and untreated (D) proP7 in 6 M guanidine hydrochloride are shown. (C) CD (50 l M) spectra of proP7 treated (dashed line) or not treated (solid line) with Arg-C. (D, E) The leakage of carboxyfluorescein from liposomes (phosphatidylcholine ⁄ cardiolipin, 80 : 20 w : w) incubated with the indicated concentrations of proP7 or Arg-C-treated proP7 respectively. (F) Lysis of Try. cruzi trypomastigotes after 30 min of incubation with the indicated concentrations of proP7 pretreated (d) or not preteated (s ) with Arg-C. Trialysin precursor activation R. M. Martins et al. 998 FEBS Journal 275 (2008) 994–1002 ª 2008 The Authors Journal compilation ª 2008 FEBS Arg-C indicates that it is folded, as fluorescence quenching is not possible for a long, unfolded peptide [21]. This is confirmed by fluorescence readings obtained from both peptides in 6 m guanidine hydro- chloride showing that intramolecular quenching is very low when proP7 is unstructured. The CD data support the notion that the proregion stabilizes a folded struc- ture at the N-terminus of protrialysin, as Arg-C-trea- ted proP7 and the N-terminus-spanning peptides are poorly structured in water solution. The results obtained using phospholipid liposomes also indicate that unfolding and activation are directly correlated with an increase in lysis, and that no other molecules of the parasite are necessary for its activation. We have previously observed that small variations in the sequence of the trialysin N-terminus peptides can modify its specificity for target cells [10]. The mobility of the N-terminus seems to prevent lysis of erythrocytes, as substitution of Gly and Pro residues in this peptide end increases activity for these cells, but not for trypanosomes. This could explain why the acidic portion of trialysin, interacting with the basic amino acids in the amphipathic helix, is less effective in inhibiting lysis of erythrocytes as compared to try- panosomes. Perhaps the different negative charges in these membranes would compete differently for the basic domain, and the protein or peptides would end up inserting in the target with variable efficiency. Other larger forms of trialysin precursors could not be detected in the salivary glands, indicating that pro- cessing must occur rapidly after protein synthesis, and the stored precursor is nontoxic for the gland. Attempts to express either the full-length predicted protrialysin, or trialysin itself, in bacteria or in the yeast Pichia pastoris have been unsuccessful so far. Our results have indicated that both the predicted pro- region and the whole N-terminus (the first two pre- dicted amphipathic a-helices) need to be ablated or fused to a bigger protein (NusA protein or GST; histi- dine tags are insufficient) in order to be translated. On the other hand, the obtained recombinant proteins (GST–protrialysin or NL fragment) were unstable or quickly precipitated during purification. These findings suggest that protrialysin might require a proper envi- ronment to first fold into an inactive protein, to be further activated by proteolysis. The possibility cannot be excluded that another salivary molecule would pre- vent protrialysin activity, releasing trialysin after saliva dilution. The lipids present in T. infestans salivary glands could control trialysin activity. Cytochemical analyses have shown a high lipid content in the D1 sal- ivary glands [22], the gland where hemolytic activity accumulates [23]. Acidic portions are also found in other cytolytic protein toxins. For example, some pore-forming bacte- rial toxins are synthesized as precursors with acidic propieces: proaerolysin is synthesized and secreted by Aeromonas hydrophila as a dimer that binds the glycan core of glycosylphosphatidyl inositol-anchored proteins on the cell surface, and it is processed by host pro- teases, releasing a small C-terminal peptide, thus enabling the toxin to oligomerize into the heptameric channel [24]. The El Tor hemolysin of Vibrio cholerae is also processed by many host proteases in different sites at the acidic ⁄ apolar propiece [25]. In this work, we have provided evidence that the acidic portion of a pore-forming protein precursor controls the lytic activity of the mature molecule. A synthetic peptide that mimics lysis inhibition and is suitable for proteolytic activation might be useful in designing regulated antimicrobial compounds. Experimental procedures Insects and cells T. infestans (males and females) were maintained at room temperature and fed twice weekly on mice anesthetized with 0.2% (w ⁄ v) ketamine chlorhydrate and 0.12% (w ⁄ v) xyla- zine chlorhydrate in NaCl ⁄ P i . Trypomastigote forms of the Y strain of Try. cruzi human erythrocytes were obtained as previously described [9]. Saliva extraction and salivary glands extracts Saliva was collected as previously described [9] from both male and female insects 2 days after feeding. Salivary glands were obtained by dissecting the insects by pulling off the rostrum and exposing the thoracic viscera. The glands were isolated from the esophagus and ducts, and kept in ice-cold NaCl ⁄ P i . For SDS ⁄ PAGE analysis, glands were readily homogenized in SDS ⁄ PAGE loading buffer containing 1% 2-mercaptoethanol, and boiled for 5 min before electrophoresis. Otherwise, glands were mechanically disrupted in ice-cold NaCl ⁄ P i containing or not containing 200 lg of the serine protease inhibitor APMSF (Roche Diagnostics, Indianapolis, IN, USA) per mL, and centrifuged for 5 min at 14 000 g. The collected supernatants were used for activity assays and SDS ⁄ PAGE analysis. The protein concentration was determined by the Bradford technique, using BSA as standard [26]. Activity assays Lysis of trypomastigotes and permeabilization assays of erythrocytes were performed as previously described [9,10], R. M. Martins et al. Trialysin precursor activation FEBS Journal 275 (2008) 994–1002 ª 2008 The Authors Journal compilation ª 2008 FEBS 999 using twofold dilutions of salivary glands extracts, or stock solutions of the peptide proP7. Recombinant protein expression and purification An NL region of preprotrialysin (spanning the C-terminal region between amino acids Met89 and Asp260, NL2) was amplified by PCR using primers NDE15P15 89 (5¢-CCATATGAAGAAAGGAGCAGC-3¢) and Bam-LYS30 reverse (5¢-CGGGATCCTTAATCAATTTCAACTTC ATC-3¢), and the protrialysin cDNA cloned in pGEM-T Easy (Promega, Madison, WI, USA) as template [9] in order to insert NdeI and BamHI restriction sites at the 5¢-terminus and 3¢-terminus. The amplified fragment was inserted in the cloning vector pCR 2.1-TOPO (Invitrogen, Carlsbad, CA, USA), and the reaction was used to trans- form chemically competent E. coli DH5a. After sequence confirmation, the obtained plasmid was digested with restriction enzymes NdeI and BamHI (Fermentas Interna- tional, Burlington, Canada), and the insert was purified from agarose gel and ligated into pET-14b (Novagen, EMD, Madison, WI, USA) previously digested with the same restriction enzymes using a Rapid DNA Ligation Kit (Promega). The ligation reaction was used to transform E. coli DH5a, and the recovered plasmid (pET14b-NL2) was used to transform BL21 (DE3) pLysE. The recombi- nant protein expression was obtained in 300 mL of LB medium cultures at 37 °C induced at A 600 nm @ 0.6 with 0.6 mm isopropyl b-d-thioglucopyranoside (Sigma Chemical Co., St Louis, MO, USA). Bacteria were collected after overnight incubation by centrifugation at 3000 g for 10 min. The bacterial cell pellet was resuspended in 20 mm Tris ⁄ HCl (pH 8.0), 6 mm MgCl 2 , and 0.1% Triton X-100, and lysis was obtained by three freeze–thawing cycles. The lysate was centrifuged (15 000 g, 15 min, 4 °C), and the insoluble pellet was extracted with 8.0 m urea. The insolu- ble material was removed by centrifugation, and urea-solubilized NL2 was purified by chromatography in Ni–nitrilotriacetic acid agarose resin (Qiagen Inc., Chats- worth, CA, USA) after elution with 100 mm sodium phosphate, 10 mm Tris ⁄ HCl, and 8 m urea (pH 4.3). NL2- containing fractions were pooled, and the recombinant protein was further purified by gel filtration in a Super- dex HR200 column (GE Health Care do Brasil LTDA, Sa ˜ o Paulo, Brazil) equilibrated with 20 mm Tris ⁄ HCl (pH 8.0), 300 mm NaCl and 8 m urea in an A ¨ ktaPurifier system (GE). The purified protein was visualized by SDS ⁄ PAGE, and selected samples were dialyzed twice against 1 L of NaCl ⁄ P i at 4 °C to remove urea. Protrialysin fused with GST was obtained from DH5a cells transformed with the vector pGEX-2T containing pro- trialysin cDNA. This construct was obtained after inserting the restriction sites at the flanking regions of protrialysin by PCR amplification. The template was the same as above, and the reaction included oligonucleotides BAMH15P15 (5¢-CGGATCCGCTGAAT ATGAACTTG-3¢) and ECOR- 13LYS (5¢-CGAATTCTTAATCAATTTCAACTTC-3¢). Cells were grown at 37 °C in LB medium. At D 600 nm = 1.5, the culture was induced with 0.1 mm isopropyl b-d thioglu- copyranoside, with subsequent growth overnight at 30 °Cat 200 r.p.m. Afterwards, the culture was centrifuged at 3000 g for 10 min, and the cell pellet was subjected to 10 pulses (20 s each, at maximum power) of sonication in a Branson Sonifier 450 (Branson Ultrasonics Corporation, Danbury, CT, USA) in 20 mm Tris ⁄ HCl (pH 8.0) and 5 mm EDTA containing 0.1% Triton X-100 (v ⁄ v). Soluble proteins were collected after centrifugation at 15 000 g for 20 min, and the resulting supernatant was incubated with 1 mL of gluta- thione–Sepharose 4B (GE) previously equilibrated in the buffer used for cell lysis. The column was washed with 50 mL of lysis buffer, and bound proteins were eluted with the same buffer containing 20 mm reduced glutathione after an overnight incubation at 4 °C. Antiserum production and immunoblotting A suspension containing 100 lg of NL2 in 300 lLof NaCl ⁄ P i was emulsified with the same volume of complete Freund’s adjuvant (Sigma) and subcutaneously injected throughout the dorsum of a female rabbit. Two consecu- tive boosts in incomplete Freund’s adjuvant (Sigma) at 3 week intervals were administered, and blood was col- lected from the ear marginal vein. For immunoblots, SDS ⁄ PAGE gels were wet-transferred to nitrocellulose membranes (Hybond C-extra; GE), and total blotted proteins were visualized by Ponceau S staining. The mem- brane was incubated for 1 h in NaCl ⁄ P i containing 5% nonfat dry milk and for 1 h with the antiserum diluted 1 : 5000 in the same solution. After three 10 min washes in NaCl ⁄ P i , bound antibodies were detected after 1 h of incubation with peroxidase-conjugated goat anti-(rabbit IgG) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and three 10 min NaCl ⁄ P i washes, and detected by enhanced chemoluminescence (Pierce, Rockford, IL, USA), using Hyperfilm-ECL (GE) for detection. Peptide synthesis and purification The fluorescence resonance energy transfer peptide based on the presumptive N-terminus of protrialysin found in the salivary glands including the P7 region of mature trialysin [10] was produced by solid-phase synthesis [27]. An auto- mated benchtop simultaneous multiple solid-phase peptide synthesizer (PSSM 8 system; Shimadzu, Japan) was used to synthesize peptides, using the Fmoc procedure. All peptides obtained were purified by semipreparative HPLC on an Ecosil C-18 column using an Econosil C18 column (10 lm; 22.5 · 250 mm) and the following two-solvent system: sol- vent A, 0.1% trifluoroacetic acid in water; and solvent B, Trialysin precursor activation R. M. Martins et al. 1000 FEBS Journal 275 (2008) 994–1002 ª 2008 The Authors Journal compilation ª 2008 FEBS 0.1% trifluoroacetic acid in 90% acetonitrile and 10% water. The molecular mass and purity of synthesized peptides were checked by amino acid analysis and MALDI- TOF MS, using a Tof-Spec-E from Micromass, Manches- ter, UK. Further purification was performed using a lRPC C2 ⁄ C18 reverse-phase column in the A ¨ kta Purifier system with 0.1% trifluoroacetic acid and a linear gradient to 100% acetonitrile. Stock solutions of peptides were pre- pared in dimethylsulfoxide ⁄ water (20 : 80), and the peptide concentrations were determined spectrophotometrically using a molar extinction coefficient of 17.300 m )1 Æcm )1 at 365 nm. Fluorimetric measurements Stock solutions of the peptide were diluted in the indicated buffer solutions at 37 °C incubated with partially purified triapsin (step 2 of [4]), or with 1 mgÆmL )1 trypsin (type VI, bovine; Sigma) or 5 lgÆmL )1 Arg-C endoproteinase (Calbiochem, EMD, San Diego, CA, USA). The proteo- lytic cleavage of proP7 peptide was monitored by measuring the fluorescence at k em = 420 nm after excitation at k exc = 320 nm in a Synergy HT plate-reader spectrofluo- rimeter (BioTek Instruments, Winooski, VT, USA). CD CD experiments were performed using a Jasco J-810 spec- tropolarimeter (Jasco International Co. Ltd, Tokyo, Japan), coupled to a peltier Jasco PFD-425S system for tempera- ture control. ProP7 (50 lm) was digested with Arg-C in 5mm Tris ⁄ HCl (pH 7.4) at 37 °C for 16 h. After treatment, NaCl was added to 10 mm and CD measurements were carried out using a 0.1 mm cell in the spectral range 190– 260 nm, at 37 °C. Each spectrum is the average of four consecutive scans. After baseline correction, the observed ellipticity, h (mdeg) was converted to mean residue molar ellipticity (h) (deg cm 2 Ædmol )1 ). The a-helix content was calculated as previously described [28]. Liposome preparation and carboxyfluorescein leakage assay Large unilamellar vesicles (LUVs) were prepared from egg phosphatidylcholine and bovine heart cardiolipin (80 : 20, weight) dissolved in methanol and dried under an N 2 flow. The lipid film on the tube was hydrated in 10 mm Tris ⁄ HCl and 50 mm carboxyfluorescein, previously purified [29], and adjusted to pH 8.0. This suspension was extruded through 11 rounds in a LiposoFast (Avestin Inc., Ottawa, Canada) system containing two polycarbonate membranes (100 nm) and applied to a Sephadex G-25 medium column equili- brated in 10 mm Tris ⁄ HCl (pH 8.0) and 0.3 m NaCl to remove free carboxyfluorescein from LUVs. The phospho- lipid content was determined according to Rouser [30]. LUVs were diluted in 1 mL of 10 mm Tris ⁄ HCl (pH 8.0) and 0.3 m NaCl, and fluorescence was measured in an Hitachi F-2000 (Japan) spectrofluorimeter (k ex = 490 nm and k em = 512 nm) after addition of peptide solutions. At the end of each experiment, total carboxyfluorescein fluores- cence was recorded by the addition of 10% Triton X-100. Acknowledgements The authors would like to thank Claudio Roge ´ rio de Oliveira for assistance with cell cultures, Dr Izaura Ioshico Hirata for performing amino acid analysis, and Dr Luis Juliano Neto for helpful suggestions. 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