Myotoxic phospholipases A2 isolated from Bothrops brazili

snake venom and synthetic peptides derived from their

C-terminal region: Cytotoxic effect on microorganism

and tumor cells

Tassia R. Costa a, Danilo L. Menaldo a, Clayton Z. Oliveira a, Norival A. Santos-Filho a,Sabrina S. Teixeira a, Auro Nomizo a, Andre L. Fuly b, Marta C. Monteiro c,Bibiana M. de Souza d, Mario S. Palma d, Rodrigo G. Stabeli e, Suely V. Sampaio a,Andreimar M. Soares a,*aDepartamento de Analises Clınicas, Toxicologicas e Bromatologicas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de

Sao Paulo, FCFRP-USP, Ribeirao Preto, SP, BrazilbDepartamento de Biologia Celular e Molecular (GCM), Instituto de Biologia, Universidade Federal Fluminense, Niteroi, RJ, BrazilcUniversidade Estadual do Centro-Oeste, UNICENTRO, Guarapuava, PR, BrazildDepartamento de Biologia, Instituto de Biociencias de Rio Claro, Universidade do Estado de Sao Paulo, UNESP, Rio Claro, SP, Brazile Instituto de Pesquisas em Patologias Tropicais, IPEPATRO, Universidade Federal de Rondonia, UNIR, RO, Brazil

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 6 4 5 – 1 6 5 6

a r t i c l e i n f o

Article history:

Received 7 April 2008

Received in revised form

16 May 2008

Accepted 19 May 2008

Published on line 5 June 2008

Keywords:

Cytotoxicity

Microbicide

Bothrops brazili

Synthetic peptides

Phospholipases A2

Myotoxins

Snake venom

a b s t r a c t

This paper reports the purification and biochemical/pharmacological characterization of

two myotoxic phospholipases A2 (PLA2s) from Bothrops brazili venom, a native snake from

Brazil. Both myotoxins (MTX-I and II) were purified by a single chromatographic step on a

CM-Sepharose ion-exchange column up to a high purity level, showing Mr � 14,000 for the

monomer and 28,000 Da for the dimer. The N-terminal and internal peptide amino acid

sequences showed similarity with othermyotoxic PLA2s from snake venoms, MTX-I belong-

ing to Asp49 PLA2 class, enzymatically active, and MTX-II to Lys49 PLA2s, catalytically

inactive. Treatment of MTX-I with BPB and EDTA reduced drastically its PLA2 and antic-

oagulant activities, corroborating the importance of residue His48 and Ca2+ ions for the

enzymatic catalysis. Both PLA2s inducedmyotoxic activity and dose–time dependent edema

similar to other isolated snake venom toxins from Bothrops and Crotalus genus. The results

also demonstrated that MTXs and cationic synthetic peptides derived from their 115–129 C-

terminal region displayed cytotoxic activity on human T-cell leukemia (JURKAT) lines and

microbicidal effects against Escherichia coli, Candida albicans and Leishmania sp. Thus, these

PLA2 proteins and C-terminal synthetic peptides present multifunctional properties that

might be of interest in the development of therapeutic strategies against parasites, bacteria

and cancer.

# 2008 Elsevier Inc. All rights reserved.

* Corresponding author. Tel.: +55 16 3602 4714; fax: +55 16 3602 4725.E-mail address: andreims@fcfrp.usp.br (A.M. Soares).

Abbreviations: BPB, 4-bromophenacyl bromide; BthTXs, B. jararacussu bothropstoxins; CB, crotoxin B from Crotalus durissus terrificus;MTX-I, B. brazili myotoxic Asp49 PLA2; MTX-II, B. brazili myotoxic Lys49 PLA2, PLA2s, phospholipases A2.

avai lab le at www.sc iencedi rec t .com

journal homepage: www.elsev ier .com/ locate /pept ides

0196-9781/$ – see front matter # 2008 Elsevier Inc. All rights reserved.doi:10.1016/j.peptides.2008.05.021

1. Introduction

Phospholipases A2 (PLA2s, E.C. 3.1.1.4) are enzymes of high

medical-scientific interest due to their involvement in a large

number of human inflammatory diseases as well as in

envenomations by snake and bee venoms. They play

important roles in dietary lipid metabolism and in the general

metabolism of structural lipids of cell membranes. Hydrolysis

of cell membrane lipids promotes loss of their structure, thus

impairing their selective permeability. Hydrolysis occurs

specifically in the 2-acyl ester linkage of 3-sn-phospholipids,

releasing free fatty acids and lysophosphatids [2,23].

Although snake venoms contain a number of bioactive

proteins, PLA2 isoforms constitute major toxic components. It

is well known that snake venom PLA2s exhibit a variety of

physiological activities in addition to intrinsic lipolytic action.

Free fatty acids and lysophospholipids, both products of

catalytic action, represent precursors for signaling molecules

that can exert a multitude of biological functions, such as

sleep regulation, immune responses, inflammation, pain

perception and cell proliferation, survival and migration. So

far, several hundred snakes venom PLA2 enzymes have been

purified and characterized. Many of them are toxic and induce

a wide spectrum of pharmacological effects, such as neuro-

toxic, cardiotoxic, myotoxic, hemolytic, convulsive, antic-

oagulant, antiplatelet, edema inducing and tissue damage

effects, cytotoxic properties [6,24,45].

Snake venom PLA2s (svPLA2s) are secreted enzymes

belonging to groups I and II [47]. svPLA2s from the Viperidae

family are placed in class II and are subdivided into two main

groups: (i) Asp49 PLA2s, which display an Asp residue at

position 49, with relatively high catalytic activity upon

artificial substrates; (ii) Lys49 PLA2s, showing a Lys residue

at position 49, with low or no catalytic activity [2,35,47]. These

enzymes show significant similarities in their tridimensional

structures, although exhibiting different pharmacological

properties, what makes them interesting targets for many

researches [2,14,24,45,51,52].

Some works in Latin America were developed using the

venom from the Brazilian snake Bothrops brazili [34,43],

including somewith isolated components, such asmyotoxins,

a hemorrhagicmetalloprotease, an L-amino acid oxidase and a

proteolytic enzyme [3,7,18,37,38,53]. This article describes, the

isolation and biochemical/pharmacological characterization

of twomyotoxic PLA2s from B. brazili venom, an Asp49 (MTX-I)

and a Lys49 (MTX-II) PLA2, and the synthesis of 13-mer

peptides derived from their C-terminal region, also showing

their cytotoxic and antimicrobial properties.

2. Material and methods

2.1. Material

The venom from B. brazili snake (state of Paraıba, Brazil) was

acquired from Serpentario Proteınas Bioativas, Batatais, SP. Male

albine Swiss mice, weighing 18–22 g, were provided by Bioterio

Central, Universidade de Sao Paulo (USP), Ribeirao Preto, SP,

Brazil. Type I Collagen from bovine tendon was purchased

from Chrono Log Corporation. The reagents 4-bromophenacyl

bromide (BPB), ethylenediaminetetraacetic acid (EDTA), mole-

cular weight protein standards and acrylamide were obtained

from Sigma Chemical Co.

2.2. Isolation of the phospholipases A2

Lyophilized venom from B. brazili (300 mg) was fractionated on

a CM-Sepharose column (2 cm � 20 cm),whichwas previously

equilibrated with 0.05 M ammonium bicarbonate (Ambic)

buffer, pH 8.0 [31,48]. Elution was carried out with a

continuous gradient up to a concentration of 0.5 M of Ambic

at a flow rate of 0.8 ml/min. Absorbance of the effluent

solution was recorded at a wavelength of 280 nm.

Homogeneity was demonstrated by reverse phase HPLC

and mass spectrometry. Evidence of high purity of the

isolated PLA2s was obtained by reverse phase HPLC using a

C18 column of 4.6 mm � 100 mm (Shimadzu) equilibrated

with solvent A (5% acetonitrile, 0.1% trifluoroacetic acid) and

eluted with a concentration gradient of solvent B (60%

acetonitrile, 0.1% trifluoroacetic acid) from 0 to 100%, at a

flow rate of 1.0 ml/min during 110 min. The peaks were

monitored through the Abs 280 nm and registered by Dataq

software (Dataq, Inc.).

2.3. Biochemical characterization

Polyacrylamide gel electrophoresis (12%, w/v) in the pre-

sence of sodium dodecyl sulfate (SDS-PAGE) followed a

previously described method [25]. Isoelectric focusing was

run according to Vesterberg [56]. Buffalyte, pH range 3.0–9.0

(Pierce IL), was used to generate the pH gradient. The

microbiuret method of Itzhaki and Gill [19] was used for

protein determination.

All mass spectrometric analysis were performed in a

triple quadrupole mass spectrometer (MICROMASS, mod.

Quattro II). The mass spectrometer was outfitted with a

standard probe electrospray (ESI - Micromass, UK,

Altrinchan). The samples were injected into electrospray

transport solvent by using a microsyringe (250 ml) coupled to

a micro-infusion pump (KD Scientific) at a flow rate of 4 ml/

min. The mass spectrometer was calibrated with intact

horse heart myoglobin and its typical cone-voltage induced

fragments to operate at resolution 4000. The samples were

dissolved in water [containing 0.1% (v/v) formic acid] to be

analyzed by positive electrospray ionization (ESI+) using

typical conditions: a capillary voltage of 3.5 kV, a cone

voltage of 30 V, a dessolvation gas temperature of 80 8C and

flow of nebulizer gas (nitrogen) of 20 l/h and drying gas

(nitrogen) 200 l/h. The spectra were obtained in the

continuous acquisition mode, scanning from m/z 100 to

4000 at a scan time of 5 s. The acquisition of raw data was

performed with MassLynx software and the data treatment

for the deconvolution of raw spectrum was performed by

using the Transform software (Micromass, UK). Gas-phase

sequencer PPSQ-21A (Shimadzu) based on automated

Edman degradation chemistry was used to perform the

amino acid sequence. The internal peptide amino acid

sequence was obtained from PLA2s previously digested

with trypsin and the tryptic peptides were analyzed by

ESI-CID-MS/MS.

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 6 4 5 – 1 6 5 61646

2.4. Peptide synthesis

Peptides (10 mg) were synthesized by Fmoc chemistry with

native endings by a commercial provider (Chiron Mimotopes,

Victoria, Australia or PepMetric Technologies Inc., Vancouver,

Canada). Their estimatedmolecularmasseswere in agreement

with corresponding calculated values,with final purity levels of

at least 95% by RP-HPLC analysis. Peptides were kept dry at

�20 8C and dissolved in 0.12 M NaCl, 40 mM phosphate-

buffered saline (PBS), pH 7.2, immediately before being tested

for their activities. Peptides were derived from the C-terminal

region 115–129 of myotoxic PLA2s, MTX-I (pepMTX-I =115RKYMAYLRVLCKK129) and MTX-II (pepMTX-I =115KKYRYHLKPLCKK129), isolated from the venom of B. brazili.

2.5. Phospholipase and anticoagulant activities

PLA2 activity of MTX-I and II (previously incubated or not with

EDTAorBPB)was evaluated invitro by indirect erythrocyte lysis

in agar containing human erythrocytes and egg yolk, as

previously described [13]. Asp49 PLA2s (BthTX-II and CB) and

PBS were used as positive and negative controls, respectively.

Stability of both PLA2s was assayed at different pH values (2.5–

10) and temperatures (4–100 8C). Anticoagulant effect was

evaluated in platelet-poor plasma (PPP) prepared by centrifuga-

tion of titrated blood twice at 1000 � g. PPP (250 ml) was

incubated with 0.5; 1; 5 and 10 mg/ml of BthTX-II, CB, MTX-I

and MTX-I + BPB (5.0 mM) for 10 min at 37 8C, 50 ml of 0.25 M

CaCl2 were added and the clotting times were recorded.

Observationswere carried out for amaximumperiod of 45 min.

2.6. Myotoxic activity

Groups of five male Swiss mice (18–22 g) were injected in the

right gastrocnemius muscle with MTX-I and II (6.25; 12.5; 25

and 50 mg in 50 ml of PBS), BthTX-I, BthTX-II and CB (50 mg in

50 ml of PBS) or PBS alone (50 ml). After 3 h, blood was collected

from the tail in heparinized capillary tubes and centrifuged for

plasma separation. The activity of creatine kinase (CK) was

then determined using 4 ml of plasma, which was incubated

for 3 min at 37 8C with 1.0 ml of the reagent according to the

kinetic CK-UV protocol from Bioclin, Brazil. The activity was

expressed in U/l, one unit corresponding to the production of

1 mmol of NADH per minute [54].

2.7. Edema-inducing activity

Groups of five male Swiss mice (18–22 g) were injected in the

subplantar region with MTX-I and II, BthTX-I, BthTX-II and CB

(25 mg in 50 ml of PBS) or PBS alone (50 ml). After 0.5, 1.0 and

1.5 h, the paw edema was measured with the aid of a low-

pressure spring caliper (Mitutoyo, Japan). The zero-time

values were then subtracted and the differences expressed

in mean (%) � S.D. [54].

2.8. Cytotoxic activity

2.8.1. Upon tumor cells

Tumor cytotoxic activity of PLA2s on human T-cell leukemia

(JURKAT) lines was assayed by 3-(4,5-dimethylthiazol-2-yl)-

2,5-diphenyltetrazolium bromide (MTT) staining as described

by Mosmann [32]. Cells were disposed in 96-well plates at a

density of 1 � 105 cells per well. After 24 h of culture, the

medium was removed and fresh medium, with or without

different concentrations of indicated compounds (MTX-I,

MTX-II, pepMTX-I, pepMTX-II or methotrexate), was added

to the wells and incubated for 24 h [54].

2.8.2. Upon microbial cells

Escherichia coli (ATCC 29648) and Candida albicans (ATCC 24433)

were dispersed in 0.01 M sodium phosphate pH 7.4 buffer

containing 1%peptone. Thesemicroorganisms, harvested from

fresh agar plates and adjusted to 4 � 105 CFU/ml, were utilized

as targets for determining microbicidal activity. For that,

4 � 105 cells were incubated with different concentrations of

MTX-I, MTX-II, pepMTX-I and pepMTX-II for 30 min at 37 8C, in

PBS plus 1% peptone. Surviving microorganisms were counted

by plate dilution technique as previously described [54].

2.8.3. Upon Leishmania spp.

The direct cytotoxic effect of the myotoxins and synthetic

peptides against Leishmania species (L. amazonensis and L.

braziliensis) was measured. Parasites (3 � 106/well) were

incubated in M199 medium supplemented with 10% heat-

inactivated fetal calf serum (FCS) in the presence of different

concentrations of MTX-I, MTX-II, pepMTX-I and pepMTX-II for

4 h, then pulsed with 0.5 mCi/well [3H] thymidine. The

incorporation of radioactivity by viable parasites was deter-

mined after 16 h using a b-counter [54].

2.9. Statistical analysis

Results are presented as the mean � S.D. obtained with the

indicated number of tested animals. The statistical signifi-

cance of differences between groups was evaluated using

ANOVA test. A p value <0.05 was considered to indicate

significance.

3. Results

This work reports the isolation and characterization of

myotoxic phospholipases A2 isolated from B. brazili snake

venom. This crude venom was applied on a CM-Sepharose

ion-exchange column, previously equilibrated with 0.05 M

Ambic, pH 8.0, and then eluted with a continuous gradient up

to a concentration of 0.5 M Ambic. Fractions B-6 and B-7,

which induced myotoxicity, were named MTX-I and II,

respectively (Fig. 1A), and were obtained with high purity

levels analyzed by RP-HPLC (Fig. 1B and C), showing a single

slightly diffuse electrophoretic band withMr of approximately

14 kDa for themonomer and 28 kDa for the dimer (Fig. 1A) and

pI 8.0 and 8.2. Electrospray positive mass spectra showed a

molecular mass of 13,870.4 Da for MTX-I (Fig. 2A) and

13,965.5 Da for MTX-II (Fig. 2B). B. brazili PLA2s were digested

with trypsin and its tryptic peptides were detected and

characterized by mass spectrometry. MALDI-TOF-MS spec-

trum (Fig. 3A) demonstrated a peptide mass profiling that was

submitted to databank search using MS-FIT algorithm at

Protein Prospector web. Some of these peptides were

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 6 4 5 – 1 6 5 6 1647

submitted to tandem mass spectrometry and internal amino

acid sequence was obtained (Table 1). The amino acid

sequence was deduced from pattern of ion fragmentation b

and y ions of peptide ions by ESI-CID-MS/MS (Fig. 3B). A

comparison of the N-terminal and internal amino acid

sequence of MTX-I showed similarity with other myotoxic

Asp49-PLA2s from Bothrops genus and MTX-II have structure

similarities with Lys49-PLA2s of the same genus (Fig. 4).

Fig. 1 – Purification of PLA2s from Bothrops brazili snake venom. 300mg of the crude venomwere applied on a CM-Sepharose

ion-exchange column, previously equilibrated with 0.05 M Ambic, pH 8.0, and then eluted with a continuous gradient up to

a concentration of 0.5 M Ambic. Fractions B-6 and B-7 induced myotoxicity and were named MTX-I and II, respectively. (A)

SDS-PAGE 12%, Lanes: 1—B. brazili venom; 2—reducedMTX-I (�14,000); 3—reducedMTX-II (�14,000); 4—non-reduced MTX-

II (�28,000); 5—standard molecular weight markers (Sigma Chem. Co) were: phosphorylase b (97,000), bovine serum

albumin (66,000), albumin egg (45,000), glyceraldehyde-3-phosphate dehydrogenase (36,000), carbonic anhydrase (29,000),

soybean trypsin inhibitor (20,100), and a-lactalbumin (14,200). Evidence of high purity of the isolated PLA2s, MTX-I (B) and

MTX-II (C), were obtained by reverse phase HPLC using a C18 column.

Fig. 2 – Raw and deconvoluted electrospray positive mass spectra of the proteins eluted in peaks MTX-I (A) and MTX-II (B),

both in the native state.

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 6 4 5 – 1 6 5 61648

Fig. 3 – Peptide mass fingerprint by MALDI-TOF-MS (A) and ESI-CID-MS/MS of ion m/z 650 [M+3H+] (Mr � 1370.1) (B).

Table 1 – Peptide mass fingerprint of MTXs obtained from tryptic peptides by MALDI-TOF-MS

m/z Submitted MH+ matched Delta Da Start End Sequence

Asp49 MTX-I

859.4268 859.9196 0.13 35 42 (R)GPKDATDR(C)

960.4971 961.1779 0.12 106 112 (R)KYMAYLR(V)

1367.7028 1368.6478 0.13 106 116 (R)KYMAYLRVLCK(K)a

1556.7018 1557.8641 0.14 84 97 (K)QICECDKAAAVCFR(E)

Lys49 MTX-II

861.4499 862.0418 0.12 53 60 (K)KLTGCDPK(K)a

1076.5047 1077.1888 0.13 64 71 (R)YSYSWKDK(T)

1380.7245 1382.6403 0.13 107 116 (K)KYRYHLKPLCK(K)a

1565.7484 1566.9126 0.14 84 97 (K)ELCECDKAVAICLR(E)

a Met was modified by oxidation.

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 6 4 5 – 1 6 5 6 1649

Only MTX-I displayed significant enzymatic activity in the

analyzed concentrations (1.0 and 5.0 mg), showing effects

comparable to the positive controls, BthTX-II and CB (Fig. 5A).

This myotoxin showed to be a stable enzyme at temperatures

from 25 8C to 60 8C (Fig. 5B) and pH from 2.5 to 7 (Fig. 5C). The

enzymatic activity of MTX-I was abolished by EDTA and after

treatment with BPB (Fig. 5D). MTX-I was able to induce dose-

dependent anticoagulant activity and that activity was also

inhibited by chemical modification with BPB (Fig. 7B).

MTX-I and II induced a prompt increase of plasmaCK levels

(Fig. 6) and edema in mouse paw (Fig. 7A). These myotoxins

also displayed cytotoxic activity against JURKAT cell lines

(Fig. 8A), antimicrobial activity inhibiting E. coli and C. albicans

growth (Fig. 8B) and effective parasiticidal activity against two

species of Leishmania (Fig. 8C). Both synthetic peptides

evaluated, pepMTX-I and pepMTX-II, showed cytotoxic activ-

ity against tumor, bacteria and parasite cell lines as well

(Fig. 9).

4. Discussion

Due to complications, mainly local (edema, hemorrhage and

necrosis), usually occurred following ophidian accidents with

Bothrops snakes [15,20], studies involving PLA2s became very

important, since they are the main venom components

responsible for necrosis and inflammatory response [15,35,52].

The purification procedure for basic PLA2s developed by

Soares et al. [48] showed to be also efficient for the obtainment

ofmyotoxins from B. brazili snake venom. Fractionation of this

Fig. 4 – Comparison of the N-terminal and internal amino acid sequences of the tryptic peptides of B. brazili PLA2s (MTX-I

and II) with other snake venom PLA2s. Multiple alignment of phospholipases A2 from Bothrops genus.

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 6 4 5 – 1 6 5 61650

crude venom by ion-exchange chromatography on CM-

Sepharose gave rise to 10 fractions at 280 nm, the two last

being the basic myotoxins, named MTX-I (B-6) and MTX-II (B-

7). This rapid procedure showed high yield, producing 5–9 mg

of the proteins with high purity levels. SDS-PAGE showed that

both isolated PLA2s haveMr of �14 kDa for the monomers and

�28 kDa for the dimers, similarly to basic PLA2s isolated from

other venoms [14,21,33,41,42,49,52]. The molecular masses

obtained bymass spectroscopy showed to be similar to that of

other snake venom PLA2s [14,52].

Comparison of the N-terminal sequence of MTXs showed

similarity with other myotoxic PLA2s from Bothrops genus

(Fig. 4). For MTX-II, one substitution was found in the

hydrophobic channel where Trp19, which is also reported

as part of the interfacial binding surface [46,52], is replaced by

an Ala19 residue.

Enzymatic activity showed that only MTX-I presented

significant results (Fig. 5A), agreeing with previous studies

in PLA2s, where the presence of catalytic activity was

detected only among Asp49 PLA2s [5,14,17,48,52,55,58]. The

enzymatic activity of MTX-I was abolished by EDTA, a

chelating agent of divalent ions, confirming that Ca2+ ions

are important for catalysis of these enzymes [2,55].

Similarly, MTX-I also looses its enzymatic activity after

treatment with BPB (Fig. 5D), corroborating results of other

works that showed the importance of His48 in their catalytic

activity [51]. The high stability of snake venom PLA2s,

including MTX-I (Fig. 5B and C), is probably due to the

relatively small molecular size of these proteins (�121

amino acid residues) and the presence of 7 disulfide bridges

in their structures makes them active along a large range of

temperatures and pH [52].

Fig. 5 – Enzymatic characterization of Asp49 PLA2s from Bothrops brazili in function of pH and temperature. (A) PLA2 activity

of MTX-I (1.0 and 5.0 mg) and MTX-II (5.0 mg) from B. brazili venom and Asp49 PLA2s from B. jararacussu (acidic BthTX-II,

5.0 mg) and Crotalus d. terrificus venom (basic CB, 5.0 mg); (B) MTX-I at different temperatures; (C) MTX-I at different pH values

(37 8C); (D) MTX-I incubated with EDTA and BPB. Controls: EDTA (30 mM), BPB (5.0 mM) and MTX-I (5.0 mg) Results are

presented as meansW S.D. (n = 6).

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 6 4 5 – 1 6 5 6 1651

Myotoxicity induced by snake venoms, including B. brazili,

may result from the direct action of myotoxins on the plasma

membranes of muscle cells, or indirectly, as consequence of

vessel degenerations and ischemia caused byhemorrhagins or

metalloproteases [14,35,52]. The myotoxic and enzymatic

activities seem to be dissociated, since MTX-I (an Asp49

PLA2) and MTX-II (a Lys49 PLA2) are myotoxic. Myotoxic PLA2s,

both Asp49 and Lys49, affect directly the plasma membrane

integrity of muscle cells, originating an influx of Ca2+ ions to

the cytosol that starts several degenerative events with

irreversible cell injures [14,35,52]. The binding sites of

myotoxins on the plasma membranes are not clearly

established, although two types have been proposed: (a)

negatively charged phospholipids [11], present onmembranes

of several cell types, explaining the high in vitro cytotoxic

action of these enzymes [29], and (b) protein receptors present

on muscle cells that make these cells more susceptible to

myotoxin action [30].

Bothrops snake venoms induce local edema in humans and

experimental animals [12]. Besides B. brazili myotoxins,

several other svPLA2s also induce edema30 min after injection

[4,22,41,50]. Studies have been directed trying to understand

the mechanisms involved in the inflammatory response

induced by myotoxic PLA2s from several snake venoms

Fig. 6 – Myotoxic activity of MTX-I (A) and MTX-II (B) of Bothrops brazili in mice. Plasma creatine kinase (CK) increase after the

intramuscular injection (in male Swiss mice, 18–22 g) of MTX-I and II (6.25; 12.5; 25 and 50 mg in 50 ml of PBS), BthTX-I,

BthTX-II and CB (50 mg in 50 ml of PBS) or PBS alone (50 ml). Results are presented as meansW S.D. (n = 6).

Fig. 7 – Pharmacological effects induced by isolated phospholipases A2. (A) Time-course of edema induced by B. brazili MTX-

I, B. jararacussu BthTX-II and Crotalus d. terrificus CB in the paw of 18–22 g male Swiss mice. PBS was included as a control.

(B) Anticoagulant activity induced by enzymatically active Asp49 PLA2s: Bothrops braziliMTX-I (in the presence and absence

of 5.0 mM BPB), B. jararacussu BthTX-II and C. d. terrificus CB. Results are presented as meansW S.D. (n = 6).

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 6 4 5 – 1 6 5 61652

[26,27,28,58]. However, the relationship between enzymatic

activity and edema is contradictory [57]. It is assumed that

myotoxic and edematogenic activities can be induced by

different structural domains in these PLA2s, or that a partial

overlapping of these domains occur [52,58].

The anticoagulant activity produced by MTX-I was fully

abolished by BPB, suggesting the importance of the catalytic

activity for this pharmacological effect. Similar results were

observed for basic Asp49 PLA2s from snake venoms, including

the association between the catalytic and anticoagulant

effects [51]. Alkylation of MTX-I His48 residue with BPB

reduced its myotoxic activity, thus suggesting a dissociation

or partial overlapping of the enzymatic and toxic effects

(results not shown). The action mechanism by which BPB

disturbs the toxic and pharmacological properties of the Lys49

PLA2 myotoxins remains to be determined [51,52].

PLA2s are multifunctional proteins able to participate as

mediators in several inflammatory diseases and can be used in

some applied areas of medicine, as detection of severe pre-

eclampsia, general anesthetic action, treatment of rheuma-

toid arthritis, as bactericidal agents in lacrimal glands and

other tissues, as a new class of HIV inhibitors by blocking the

host cell invasion and as potential antimalarial agents

[40,52,54]. MTX-I and II also displayed cytotoxic activity

against JURKAT lines, antimicrobial activity against E. coli

and C. albicans, and effective parasiticidal activity against

Fig. 8 – Antitumoral and microbicidal effects induced by B. brazili MTXs in vitro. (A) Antitumoral activity of MTXs on human

acute T-cell leukemia (JURKAT) lines. Different concentrations of myotoxins were incubated with JURKAT lines.

Methotrexate (Metx, 100 mg) was used as positive control. (B) Microbicidal activity induced by different concentrations of

MTXs on E. coli and C. albicans. (C) Cellular viability of Leishmania species after treatment with MTXs. Cytotoxicity was

expressed as percentage (%). Results are presented as means W S.D. (n = 6).

Fig. 9 – Antitumoral and microbicidal effects of synthetic peptides in vitro. (A) Antitumoral activity of synthetic peptides on

human acute T-cell leukemia (JURKAT) lines. Different concentrations of peptides were incubated with JURKAT lines.

Methotrexate (Metx, 120 mg) was used as positive control. (B) Microbicidal activity induced by different concentrations of

synthetic peptides on E. coli (&) and C. albicans (*). (C) Cellular viability of Leishmania amazonensis and L. braziliensis, after

treatment with synthetic peptides. Cytotoxicity was expressed as percentage (%). Results are presented as means W S.D.

(n = 3).

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 6 4 5 – 1 6 5 6 1653

Leishmania sp. Those activities of B. brazili myotoxins were

independent of their catalytic activity, since MTX-II is a Lys49

myotoxin and hence catalytically inactive. Some authors

propose that cytotoxic activity on tumor cell lines is associated

with apoptosis induction, considering the fact that PLA2

enzymes have been proposed to play a role in mediating

apoptosis in various models, including cell lines [8]. The PLA2

activity is proposed to accelerate turnover of phospholipids,

which may influence membrane changes that occur during

apoptosis [36].

In this work, we also report that 13-mer synthetic peptides

derived from the cationic/hydrophobic C-terminal region of

MTX-I and II display cytotoxic properties against tumor cell

lines and antimicrobial properties as well. Some studies

involving a Lys49-PLA2 isolated from Bothrops asper snake

venom have demonstrated that the C-terminal region com-

prising amino acids from 115 to 129 is related to the cytotoxic

and bactericidal effects of this protein [39].

Cationic peptides are widely distributed in living organ-

isms, playing a variety of functions. They are often referred to

as antimicrobial peptides, due to their well-characterized role

in innate immunity against infectious agents [10,16,39,44]. The

possible bactericidal mechanism of the synthesized peptides

based on B. brazilimyotoxinsmight be exerted in a similar way

to that proposed for other cationic peptides, by displaying

metal ions as Ca2+ andMg2+ fromnegatively charged groups of

the cell surface, promoting membrane destabilization and

allowing insertion of the hydrophobic domain of the peptides

into the bilayer, followed by membrane permeabilization and

cell death [39]. A number of studies have reported antitumoral

effects of individual proteins isolated from snake venoms

[8,9,54], but there is few information on the use of short

segments derived from a snake toxin, in the form of synthetic

peptides [1].

Snake venoms PLA2s are multifunctional proteins with

promising pharmacological applications. Isolation and func-

tional characterization of these enzymes will provide better

insights of their action mechanism andmake possible the use

of these molecules as therapeutic agents and drug design for

future medicines.

Acknowledgements

The authors express their gratitude to Fundacao de Amparo a

Pesquisa do Estado de Sao Paulo (FAPESP) and Conselho

Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq)

for financial support.

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