peptides 29 (2008) 1645–1656
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/peptides
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, André L. Fuly b, Marta C. Monteiro c,
Bibiana M. de Souza d, Mário S. Palma d, Rodrigo G. Stábeli e, Suely V. Sampaio a,
Andreimar M. Soares a,*
a
Departamento de Análises Clı́nicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de
São Paulo, FCFRP-USP, Ribeirão Preto, SP, Brazil
b
Departamento de Biologia Celular e Molecular (GCM), Instituto de Biologia, Universidade Federal Fluminense, Niterói, RJ, Brazil
c
Universidade Estadual do Centro-Oeste, UNICENTRO, Guarapuava, PR, Brazil
d
Departamento de Biologia, Instituto de Biociências de Rio Claro, Universidade do Estado de São Paulo, UNESP, Rio Claro, SP, Brazil
e
Instituto de Pesquisas em Patologias Tropicais, IPEPATRO, Universidade Federal de Rondônia, UNIR, RO, Brazil
article info
abstract
Article history:
This paper reports the purification and biochemical/pharmacological characterization of
Received 7 April 2008
two myotoxic phospholipases A2 (PLA2s) from Bothrops brazili venom, a native snake from
Received in revised form
Brazil. Both myotoxins (MTX-I and II) were purified by a single chromatographic step on a
16 May 2008
CM-Sepharose ion-exchange column up to a high purity level, showing Mr 14,000 for the
Accepted 19 May 2008
monomer and 28,000 Da for the dimer. The N-terminal and internal peptide amino acid
Published on line 5 June 2008
sequences showed similarity with other myotoxic PLA2s from snake venoms, MTX-I belonging to Asp49 PLA2 class, enzymatically active, and MTX-II to Lys49 PLA2s, catalytically
Keywords:
inactive. Treatment of MTX-I with BPB and EDTA reduced drastically its PLA2 and antic-
Cytotoxicity
oagulant activities, corroborating the importance of residue His48 and Ca2+ ions for the
Microbicide
enzymatic catalysis. Both PLA2s induced myotoxic activity and dose–time dependent edema
Bothrops brazili
similar to other isolated snake venom toxins from Bothrops and Crotalus genus. The results
Synthetic peptides
also demonstrated that MTXs and cationic synthetic peptides derived from their 115–129 C-
Phospholipases A2
terminal region displayed cytotoxic activity on human T-cell leukemia (JURKAT) lines and
Myotoxins
microbicidal effects against Escherichia coli, Candida albicans and Leishmania sp. Thus, these
Snake venom
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: [email protected] (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.
0196-9781/$ – see front matter # 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.peptides.2008.05.021
1646
1.
peptides 29 (2008) 1645–1656
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 neurotoxic, cardiotoxic, myotoxic, hemolytic, convulsive, anticoagulant, 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 some with isolated components, such as myotoxins,
a hemorrhagic metalloprotease, 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 two myotoxic 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 Paraı́ba, Brazil) was
acquired from Serpentário Proteı́nas Bioativas, Batatais, SP. Male
albine Swiss mice, weighing 18–22 g, were provided by Biotério
Central, Universidade de São Paulo (USP), Ribeirão Preto, SP,
Brazil. Type I Collagen from bovine tendon was purchased
from Chrono Log Corporation. The reagents 4-bromophenacyl
bromide (BPB), ethylenediaminetetraacetic acid (EDTA), molecular 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), which was 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 presence 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.
peptides 29 (2008) 1645–1656
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 estimated molecular masses were 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 phosphatebuffered 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 =
1 15
RKYMAYLRVLCKK 1 29 )
and
MTX-II
(pepMTX-I =
115
KKYRYHLKPLCKK129), 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
EDTA or BPB) was evaluated in vitro 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 centrifugation 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.
Observations were carried out for a maximum period of 45 min.
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.
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% heatinactivated 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 determined after 16 h using a b-counter [54].
Statistical analysis
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.
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. These microorganisms, 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].
2.9.
2.6.
1647
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 lowpressure 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)-
Results are presented as the mean S.D. obtained with the
indicated number of tested animals. The statistical significance 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 with Mr of approximately
14 kDa for the monomer 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 spectrum (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
1648
peptides 29 (2008) 1645–1656
Fig. 1 – Purification of PLA2s from Bothrops brazili snake venom. 300 mg of the crude venom were 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—reduced MTX-I (14,000); 3—reduced MTX-II (14,000); 4—non-reduced MTXII (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.
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. 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.
1649
peptides 29 (2008) 1645–1656
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
MH+ matched
Delta Da
Start
End
Asp49 MTX-I
859.4268
960.4971
1367.7028
1556.7018
859.9196
961.1779
1368.6478
1557.8641
0.13
0.12
0.13
0.14
35
106
106
84
42
112
116
97
(R)GPKDATDR(C)
(R)KYMAYLR(V)
(R)KYMAYLRVLCK(K)a
(K)QICECDKAAAVCFR(E)
Lys49 MTX-II
861.4499
1076.5047
1380.7245
1565.7484
862.0418
1077.1888
1382.6403
1566.9126
0.12
0.13
0.13
0.14
53
64
107
84
60
71
116
97
(K)KLTGCDPK(K)a
(R)YSYSWKDK(T)
(K)KYRYHLKPLCK(K)a
(K)ELCECDKAVAICLR(E)
m/z Submitted
a
Met was modified by oxidation.
Sequence
1650
peptides 29 (2008) 1645–1656
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 dosedependent anticoagulant activity and that activity was also
inhibited by chemical modification with BPB (Fig. 7B).
MTX-I and II induced a prompt increase of plasma CK 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 activity 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
of myotoxins 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.
peptides 29 (2008) 1645–1656
1651
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 means W S.D. (n = 6).
crude venom by ion-exchange chromatography on CMSepharose gave rise to 10 fractions at 280 nm, the two last
being the basic myotoxins, named MTX-I (B-6) and MTX-II (B7). This rapid procedure showed high yield, producing 5–9 mg
of the proteins with high purity levels. SDS-PAGE showed that
both isolated PLA2s have Mr 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 by mass 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].
1652
peptides 29 (2008) 1645–1656
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 means W S.D. (n = 6).
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 by hemorrhagins 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 on membranes
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 edema 30 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. 7 – Pharmacological effects induced by isolated phospholipases A2. (A) Time-course of edema induced by B. brazili MTXI, 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 brazili MTX-I (in the presence and absence
of 5.0 mM BPB), B. jararacussu BthTX-II and C. d. terrificus CB. Results are presented as means W S.D. (n = 6).
peptides 29 (2008) 1645–1656
1653
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).
[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 preeclampsia, general anesthetic action, treatment of rheumatoid 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. 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).
1654
peptides 29 (2008) 1645–1656
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 comprising 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 organisms, 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. brazili myotoxins might be exerted in a similar way
to that proposed for other cationic peptides, by displaying
metal ions as Ca2+ and Mg2+ from negatively 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 functional characterization of these enzymes will provide better
insights of their action mechanism and make possible the use
of these molecules as therapeutic agents and drug design for
future medicines.
Acknowledgements
The authors express their gratitude to Fundação de Amparo à
Pesquisa do Estado de São Paulo (FAPESP) and Conselho
Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq)
for financial support.
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