A short proregion of trialysin, a pore-forming protein of Triatoma infestans salivary glands, controls activity by folding the N-terminal lytic motif

A short proregion of trialysin, a pore-forming proteinof Triatoma infestans salivary glands, controls activityby folding the N-terminal lytic motifRafael M. Martins1, Rogerio Amino2, Katia R. Daghastanli3, Iolanda M. Cuccovia3, Maria A. Juliano4

and Sergio Schenkman1

1 Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Sao Paulo, Brazil

2 Departamento de Bioquımica, Universidade Federal de Sao Paulo, Brazil

3 Departamento de Bioquımica, Instituto de Quımica, Universidade de Sao Paulo, Brazil

4 Departamento de Biofısica, Universidade Federal de Sao 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 8o andar 04023-062 Sao

Paulo, SP, Brazil

Fax: +55 11 5571 58 77

Tel: +55 11 5575 19 96

E-mail: [email protected]

(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

B C

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 · 106ÆmL)1) (A) or human erythrocytes (3 · 107ÆmL)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 lM) after incubation without (s) or with

(d) 5 lgÆmL)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 lM) 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 ⁄Pi. 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 ⁄Pi. 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 ⁄Pi 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 A600 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 MgCl2, 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, Sao

Paulo, Brazil) equilibrated with 20 mm Tris ⁄HCl (pH 8.0),

300 mm NaCl and 8 m urea in an AktaPurifier system

(GE). The purified protein was visualized by SDS ⁄PAGE,

and selected samples were dialyzed twice against 1 L of

NaCl ⁄Pi at 4 �C to remove urea.

Protrialysin fused with GST was obtained from DH5acells 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¢-CGGATCCGCTGAATATGAACTTG-3¢) and ECOR-

13LYS (5¢-CGAATTCTTAATCAATTTCAACTTC-3¢).Cells were grown at 37 �C in LB medium. At D600 nm = 1.5,

the culture was induced with 0.1 mm isopropyl b-d thioglu-

copyranoside, with subsequent growth overnight at 30 �C at

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 lL of

NaCl ⁄Pi 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 ⁄Pi 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 ⁄Pi, 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 ⁄Pi 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 Akta 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)1Æcm)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 kem = 420 nm after excitation at

kexc = 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

5 mm 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 cm2Ædmol)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 N2 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 (kex = 490 nm

and kem = 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 Rogerio de

Oliveira for assistance with cell cultures, Dr Izaura

Ioshico Hirata for performing amino acid analysis, and

Dr Luis Juliano Neto for helpful suggestions. This

work was supported by grants from Fundacao de

Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)

and Conselho Nacional de Desenvolvimento Cientıfico

e Tecnologico (CNPq) from Brazil.

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