Lysophosphatidylcholine produced by the phospholipase A2 isolated from Lachesis muta snake venom modulates natural killer activity as a protein kinase C effector.

Abstract

We have showed that a phospholipase A(2) isolated from Lachesis muta snake venom, denoted LM-PLA(2)-I, had some biological effects. Here, we examined its effects on lymphocytes. Pre-incubation of human peripheral blood lymphocytes with LM-PLA(2)-I plus phosphatidylcholine (PC) stimulated the natural killer (NK) activity. This was accompanied by DNA binding of nuclear transcription factor kappaB and the increase in PKC activity with translocation of the enzyme from the cytoplasma into the plasma membrane. These effects were reproduced when lymphocytes were pre-incubated with commercial lysophosphatidylcholine (LPC) and abolished by stausrosporin or p-bromophenacyl bromide. Evaluation of phosphorylated PKC isoforms showed that pre-incubation with LPC activated the autophosphorylation of the PKCzeta isoform. Taken together, these results confirm that the enzymatic activity of the phospholipase A(2) present in L. muta venom is for the biological activity of the snake venom, and strongly suggest that the LPC produced may be acting as a modulator of PKC isoforms.

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Toxicon 50 (2007) 400–410
Lysophosphatidylcholine produced by the phospholipase A2
isolated from Lachesis muta snake venom modulates natural
killer activity as a protein kinase C effector
Andre ´ L. Fulya,?, Alexandre L. Machadob, Paulo Castrob, Agessandro Abraha ˜ ob,
Paulo Rednerc, Ulisses G. Lopesc, Jorge A. Guimara ˜ esd, Vera Lucia G. Koatzb
aDepartamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense, Campus do Valonguinho s/n,
Nitero ´i, Rio de Janeiro 24210-150, Brazil
bInstituto de Bioquı ´mica Me ´dica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
cInstituto de Biofı ´sica, Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
dCentro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
Received 16 April 2007; accepted 18 April 2007
Available online 25 April 2007
Abstract
We have showed that a phospholipase A2isolated from Lachesis muta snake venom, denoted LM-PLA2-I, had some
biological effects. Here, we examined its effects on lymphocytes. Pre-incubation of human peripheral blood lymphocytes
with LM-PLA2-I plus phosphatidylcholine (PC) stimulated the natural killer (NK) activity. This was accompanied by
DNA binding of nuclear transcription factor kB and the increase in PKC activity with translocation of the enzyme from
the cytoplasma into the plasma membrane. These effects were reproduced when lymphocytes were pre-incubated with
commercial lysophosphatidylcholine (LPC) and abolished by stausrosporin or p-bromophenacyl bromide. Evaluation of
phosphorylated PKC isoforms showed that pre-incubation with LPC activated the autophosphorylation of the PKCz
isoform. Taken together, these results confirm that the enzymatic activity of the phospholipase A2present in L. muta
venom is for the biological activity of the snake venom, and strongly suggest that the LPC produced may be acting as a
modulator of PKC isoforms.
r 2007 Elsevier Ltd. All rights reserved.
Keywords: Lysophosphatidylcholine; Phospholipase A2; Lachesis muta; Snake venom; Lymphocytes; Protein kinase C
1. Introduction
Lachesis muta (Bushmaster) is a snake widely
distributed in South America, commonly found in
sugarcane plantations, which bite can be fatal. Its
venom, like others, is formed by a complex mixture of
active substances, mainly proteins, often responsible
for the observed pharmacological and toxicological
symptoms that follow the envenomation by a snake
bite. Among these proteins, enzymes with phospho-
lipase A2 (PLA2) activity have been purified and
characterized (Fuly et al., 1997, 2003; Six and Dennis,
2000; Kini, 2003). PLA2is a family of enzymes widely
distributed in living organisms that catalyzes the
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?Corresponding author. Tel.: +552126292294;
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E-mail address: andfuly@vm.uff.br (A.L. Fuly).

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hydrolysis of phospholipids, producing free fatty
acids and lysophospholipids. They exist in secreted
and intracellular forms, of which the former has
received the most attention (Dennis et al., 1991;
Glaser et al., 1993). Many biological and toxic effects
involve these enzymes, but it is unclear whether all of
the biological properties observed are due to the
enzymatic production of lysophosphatidylcholine
(LPC) and arachidonic acid. Once released, arachi-
donic acid can be metabolized by cyclooxygenases
and lipooxygenases, forming various eicosanoids that
have their own biological effects (Balsinde et al., 2002;
Murakami et al., 1999). The other product, LPC,
stimulates the transcription of adhesion molecules in
endothelial cells, induces chemotaxis in T-lympho-
cytes and monocytes, enhances diacylglycerol-depen-
dent activation of lymphocytes, induces apoptosis
and modulates the activity of transcription factors
(for a review, see Prokazova et al., 1998).
Depending on the stimulus, lymphocytes can
proliferate or exert a spontaneous natural killer
(NK) activity. NK cells are large-granule lympho-
cytes, providing natural resistance to microbial infec-
tions, and to tumor growth and metastases (Cifone
et al., 1993; Whalen et al., 1999; Fabbri et al., 2003).
In addition to their cytotoxic role, NK cells produce
cytokines involved in signaling of hematopoiesis and
in the response of the adaptive immune system to
different agents (Fabbri et al., 2003). Interaction of
NK cells with sensitive tumor target cells can initiate a
sequence of biochemical events involving the genera-
tion of second messengers such as diacylglycerol and
inositol triphosphate, leading to activation of protein
kinase C (PKC) (Chow et al., 1988; Steele and
Brahmi, 1988; Fabbri et al., 2003). NK activation
may also require the release of arachidonic acid from
membrane phospholipids, mainly phosphatidylcho-
line, by the activation of PLA2(Hoffman et al., 1981;
Deem et al., 1987; Whalen et al., 1999). In this paper,
we show that the LPC generated by the enzymatic
activity of a PLA2isolated from the snake L. muta
venom can modulate both lymphocyte spontaneous
NK and PKC activities. We also show that these
effects were blocked by staurosporine, suggesting a
direct effect of LPC on PKC pathway.
2. Materials and methods
2.1. Materials
The antibodies anti-PKC aPKCz and anti-
cPKCabg were purchased from Santa Cruz Biothec-
nology, Santa Cruz, CA, the oligonucleotides
and RNAse were from DNAgency (USA), T4
polynucleotide kinase kit from Biolabs (UK),
[g-32P] ATP (45000Ci/mmol, Amersham Interna-
tional Biosciences, UK) and the PKC assay kit
and fetal bovine serum (FBS) were from Gibco
BRL (Life Technologies). All other reagents used
were purchased from Sigma Chemical Co. (St.
Louis, MO, USA) and L. muta snake venom,
as well. Phosphatidylcholine (PC), phosphatidylgly-
cerol (PG),phosphatidylethanolamine
phosphatidic acid (PA) and LPC were sonicated
at 41C for 10min prior to add to the assay
medium.
(PE),
2.2. Cells
Peripheral blood mononuclear cells (PBMC)
from healthy volunteers or from the local blood
bank (Hospital Universita ´ rio Clementino Fraga
Filho, Universidade Federal do Rio de Janeiro)
were obtained by Ficoll–Hypaque density gradient
centrifugation, as described elsewhere (de Moraes
et al., 1989). The peripheral blood mononuclear
cells obtained were resuspended in RPMI medium
and depleted of phagocyte cells by adherence onto
plastic flasks. After 1h at 371C and 5% CO2, the
non-adherent cells, considered as peripheral blood
lymphocytes (PBL), were adjusted to a concentra-
tion of 5?106cells/mL and used as effector cells.
The K562 cell line derived from human erythroleu-
kemia was used as target cells.
2.3. NK activity assay
Before the assay, target cells K562 cells (106cells/
mL) were labeled with 150mCi Na2
90min at 371C and 5% CO2. After two washes
with RPMI containing 5% FBS, the cells were
resuspended at a concentration of 5?104cells/mL
in the same culture medium. Cytotoxicity was
determined by measuring
effector: target ratios of 100:1 (5?106cells/mL:
5?104cells/mL), 50:1 and 25:1, as described by de
Moraes et al. (1989). After a 4h-incubation period
at 371C and 5% CO2, 100mL of supernatant were
collected and radioactivity assessed in a gamma
counter. The effector:target ratio of 100:1 was
chosen to be used throughout this study. The
percentage of specific lysis was calculated from the
following formula:
51CrO4 for
51Cr release by using
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Specific lysis ¼Experimental51Cr release ? spontaneous51Cr release
Total51Cr release ? spontaneous51Cr release
? 100.
2.4. Purification of phospholipase A2(PLA2) and
protein assay
A purified PLA2, denoted by LM-PLA2-I was
isolated from L. muta snake venom by gel-filtering
crude venom on Sephacryl S-200HR followed by
reverse-phase chromatography on an FPLC as
described elsewhere (Fuly et al., 1997). Protein
determination was carried out by the procedure of
Lowry et al. (1951).
2.5. Chemical modification
Modification of the LM-PLA2-I enzyme was
performed by incubating 50–80mg/mL protein in
20mM Tris–HCl, pH 7.5 with 1mM p-bromophe-
nacyl bromide (p-BPB) dissolved in 0.5% dimethyl-
sulfoxide (final concentration). Incubation was
carried out overnight at 41C and then the excess
of the reagent was removed by dialysis against
Tris–buffer, pH 7.5. Immediately after that proce-
dure, modified or unmodified LM-PLA2-I was used
on experimental analysis. The unmodified LM-
PLA2-I was achieved by mixing native LM-PLA2-I
with dimethylsulfoxide or Tris–buffer (Fuly et al.,
2003).
2.6. Stimulation of NK activity
PBL (5?106cells/mL) were pre-incubated in the
presence of 10 or 20nM TPA, 45mg/mL LM-PLA2-I
alone or plus 60mg/mL PC, or different concentra-
tions of commercial LPC. After 1h at 371C and 5%
CO2, cells were washed three times with RPMI
containing 5% of FBS, resuspended to the initial
concentration and used as effector cells in the
citotoxic assay, as described in Section 2.3. The final
concentrations of LM-PLA2-I and PC used in this
study were determined in a previous set of experi-
ments. When indicated, 2mM staurosporine or 1mM
p-BPB (final concentrations) was also added to the
culture medium.
2.7. PKC assay
The PKC assay was based on phosphorylation of a
synthetic peptide from myelin basic protein (MBP),
which acts as a specific substrate for PKC (Yasuda
et al., 1990). PBL (5?106cells/mL) were incubated for
1h at 371C and 5% CO2with 20nM TPA, 60mg/mL
LPC, 45mg/mL LM-PLA2-I alone or plus 60mg/mL
PC. After this period, cells were washed three times
with saline, resuspended in a lysis buffer and PKC
activity was determined in 1–2mgprotein/mL, accord-
ing to the kit. The reaction media contained 20mM
Tris, pH 7.5; 20mM MgCl2; 1mM CaCl2; 20mM
ATP; 50mM MPB with or without phosphatidylserine
(12.5ng/mL). The [g-32P] ATP (20mCi/mL) was added
to the tubes and after 10min at 301C, an aliquot
(25mL) was removed and applied onto phosphocellu-
lose paper. The paper disc was immersed in 1% (v/v)
phosphoric acid, followed by three washes with water
to remove unreacted ATP.32P incorporation into the
substrate MPB was determined in a beta counter.
Non-specific PKC radioactivity was evaluated in the
absence of protein.
2.8. Subcellular fractionation
Soluble and particulate cellular fractions were
prepared as described by Keenan et al. (1997), with
minor alterations. Briefly, PBL (5?108cells) pre-
incubated as described in Section 2.6 were harvested
and washed in ice-cold 0.15M NaCl, then lysed in a
buffer containing 20mM Tris–HCl, 2mM EDTA,
5mM EGTA, 50mg/mL leupeptin, 2mM PMSF,
10mM benzamidine, pH 7.5. The cell suspension was
sonicated for 2min in an ice bath and the lysate
centrifuged at 100,000?g for 30min at 41C. The
resulting supernatant was used as the cytosolic
fraction, and the pellet resuspended in 0.1% (v/v)
Triton X-100 in the Tris–buffer described above.
After a 20-min incubation at 41C, the samples
were centrifuged at 12,000?g for 20min at 41C,
yielding the solubilized particulate fraction in the
supernatant. Protein determination was carried out
by the procedure of Lowry et al. (1951). Identification
of cytosolic and membrane fractions was done
by measuring lactate dehydrogenase (LDH) and
Na+K+-ATPase activities, respectively, in the iso-
lated fractions.
2.9. LDH activity
The reduction of pyruvate by NADH catalyzed
by LDH was followed at 340nm on Ultrospec 3000
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(Pharmacia-LKB) spectrophotometer, at room tem-
perature. The reaction was triggered by adding the
reaction mixture consisted of 1mM sodium pyr-
uvate, 0.3mM NADH, 0.1% (v/v) Triton X-100
and 86mM phosphate buffer saline at pH 7.5 (final
concentrations) into the medium containing PBL
(5?106cells/mL). As a control, cells were sonicated
for 5min and their total LDH content evaluated.
2.10. Na+K+-ATPase activity
The ATPase activity was determined by measur-
ing the hydrolysis of [g-32P]ATP. The nucleotide
was trapped on charcoal suspended in 0.1M HCl,
and the
natant after centrifugation. The Na+K+-ATPase
activityofmembrane
(200mgprotein/mL) was assayed at 371C in a
medium containing 20mM BTP-HCl, 20mM KCl,
5mM MgCl2, 120mM NaCl, 2mM EGTA, 10mM
NaN3, 2mM Na+-ATP, pH 7.0. The Na+K+-
ATPase activity was calculated by subtracting the
activity obtained in the presence of 10mM ouabain
from the total ATPase activity.
32P released was measured in the super-
or cytosolicfractions
2.11. DNA fragmentation assay
Analysis of DNA fragmentation was performed
as previously described by Compton et al. (1990),
with modifications. Briefly, the genomic DNA was
isolated from PBL (2?107cells) pre-incubated as
described in Section 2.6, after treatment with a
buffer solution containing 5mM Tris–HCl, 5mM
EDTA, 0.5% Triton X-100, pH 7.4, following by
treatment with 0.15M NaCl, and an extraction with
phenol/chloroform. The DNA was precipitated by
the addition of isopropanol and 0.15M NaCl, into
the medium and incubated for 16h at ?201C. After
this period, 10mg of RNAse was added into the
medium and the mixture incubated at room
temperature for 1h. Samples were taken, applied
to a 1.8% agarose gel and electrophoresis per-
formed. DNA was visualized under ultraviolet light
after staining with ethidium bromide.
2.12. Electrophoretic mobility shift assay (EMSA)
Nuclear
(2?107cells) pre-incubated as described in Section
2.6, and EMSA was performed as described by
Castro et al. (2004). Nuclear extracts from culture
cells 70Z3 stimulated with 15mg/mL lipopolysac-
extracts preparedfromPBL
charide (E. coli serotype 055:B5, Sigma Chem. Co,
USA) for 1h were used as positive control. The
binding reaction between the nuclear factor NF-kB
consensus sequence 50-AGT TTG ATG AGT CAG
CCG-30and 30-CGG CTG ACT CAT CAA ACT-50
with nuclear protein (10mg) was performed in a final
volume of 30mL in 8mM HEPES, 10% glycerol
(v/v), 20mM KCl, 4mM MgCl2, 1.0mg polydl-dC
(pH 7.0). The oligonucleotides were 50-end labeled
with T4 polynucleotide kinase kit, and [g-32P]
ATP. relevant double-stranded oligonucleotides
50,000cpm were used per reaction. Binding was
allowed to proceed for 30min at room temperature.
Samples were applied into a 6% polyacrylamide
non-denaturating gel with 0.5?TBE, and electro-
phoresis performed. Specificity was determined by
addition of 50-fold excess unlabeled oligonucleotide
(COLD). The gels were dried and radioactivity was
quantifiedinPhosphorimager
namics, Ltd., USA), and expressed as relative
optical density arbitrary units.
(Molecular Dy-
2.13. Immunoprecipitation of PKC isoforms and
phosphorylation
Immunoprecipitation was performed based on
Even-Faitelson and Ravid (2006). Briefly, PBL
(5?107cells) pre-incubated as described in Section
2.6 were centrifuged and incubated with
(100mCi) for 4h at 371C and 5% CO2. After this
period, cells were washed in culture medium and
incubated with 10nM TPA or 60mM LPC for 1h.
Then, cells were washed and resuspended in the lysis
buffer consisting of 50mM Tris–HCl, pH 7.5, 1%
NP-40, 0.5% deoxycholic acid, 50mM sodium
pyrophosphate, 100mM NaF and a mixture of
proteases inhibitors, and incubated overnight with
anti-PKC antibodies described in Section 2.1. After
this period, the samples were separated in SDS-
PAGE. Western blots were visualized using ECL
system. Bands were analyzed using ImageQuant
software (Molecular Dynamics Ltd., UK) and
expressed as relative optical density arbitrary units.
32P
3. Results
3.1. Stimulation of NK activity by LM-PLA2-I plus
PC, LPC or TPA
To investigate an effect of the PLA2activity on
the cytoxicity exerted by NK cells, 45mg/mL of the
isolated LM-PLA2-I plus 1.3mM PC were added to
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the medium containing effector and target cells
K562 for the effector:target ratio of 100:1 at
the beginning of the cell culture period (Section
2.3). After a 4h-incubation, the presence of
the enzyme plus its substrate did not alter the
spontaneousNKactivity
However, when effector cells were pre-incubated
for 1h with LM-PLA2-I plus PC at 371C before
being mixed with target cells, there was an
increase in the NK activity similar to what was
found when cells were pre-incubated with 10nM
TPA, the selective activator of PKC (Fig. 1A). This
enhancement on NK activity was not observed
when PC was replaced by PA, PE or PG (data not
shown).
In contrast to, when LM-PLA2-I was mixed with
human plasma or hen’s egg yolk emulsion, the
stimulatory effect was recovered (data not shown)
as well as observed when PC was used as substrate
(Fig. 1A).
Similar results were obtained in the presence of
crescent concentrations of commercial LPC, show-
ing stimulation of NK activity in a dose-dependent
manner (Fig. 1B). Pre-incubation with LM-PLA2-I
or PC alone did not affect NK activity (data not
shown).
(data notshown).
3.2. Inhibition of stimulated NK activity by LM-
PLA2-I plus PC, LPC or TPA by staurosporine and
p-bromophenacyl bromide
To investigate whether activation of NK cells by
LM-PLA2-I was associated to PKC activity, cells
were pre-incubated for 1h at 371C with 45mg/mL
LM-PLA2-I plus 1.3mM PC, 20nM TPA or 45mM
LPC, in the absence or in the presence of 2mM
staurosporine, a PKC inhibitor. After this period,
cells were washed three times in culture medium,
adjusted to a final concentration of 5?106cells/mL
and used as effector cells in the NK assay (Section
2.3). As shown in Fig. 2A, pre-incubation with
staurosporine alone did not inhibit spontaneous
NK activity (column 2). However, it suppressed the
enhancement of NK activity elicited by LPC
(column 4) or TPA (column 6), similarly to what
was found with LM-PLA2-I plus PC (data not
shown). To confirm the role of the enzymatic
activity of LM-PLA2-I in such effect, the enzyme
was chemically modified with the specific PLA2
inhibitor, (p-BPB). The modified and unmodified
(native) enzyme was mixed with PC for 1h at 371C,
and then assayed for cytotoxicity, as described in
Fig. 1. Fig. 2B shows that pre-incubation with
unmodified LM-PLA2-I plus PC stimulated NK
activity, but the modified enzyme did not. These
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A.L. Fuly et al. / Toxicon 50 (2007) 400–410
Fig. 1. Activation of natural killer (NK) activity by LM-PLA2-I,
LPC and TPA. (A) PBL (5?106cells/mL) were pre-incubated for
1h at 371C and 5% CO2either in RPMI (1), 10nM TPA (2),
45mg/mL LM-PLA2-I plus 1.3mM PC (3), LM-PLA2-I alone (4)
or PC alone (5). (B) Lymphocytes were pre-incubated as above,
with RPMI (1) or with 25mM (2), 45mM (3) or 90mM (4) of
commercial LPC. After this period, cells were washed in RPMI,
adjusted to 5?106cells/mL and incubated with K562 cells
(5?104cells/mL) for 4h at 371C and 5% CO2. NK activity
was determined by measuring51Cr liberation from K562 lysis, as
described in Section 2.3. Values are the means7S.E.M. of four
experiments.
Fig. 2. Inhibition of activation of NK activity by staurosporine
and p-BPB. PBL (5?106cells/mL) were pre-incubated for 1h at
371C and 5% CO2in different conditions. (A) RPMI (1), 2mM
staurosporine (2), 45mM LPC (3), LPC plus staurosporine (4),
20nM TPA (5) and TPA plus staurosporine (6). (B) RPMI (1),
native LM-PLA2-I (2) or modified LM-PLA2-I (3). After this
period, cells were washed and adjusted to be used in the NK assay
as described in Fig. 1. Values are the means7S.E.M. of three
individual experiments.
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experiments indicated that the modulation of NK
activity was entirely dependent on the enzymatic
activity of LM-PLA2-I, and directly associated with
the formation of LPC. Native or modified LM-
PLA2-I alone did not affect NK activity (data not
shown). It is known that for some enzymes of this
class, the biological activity depends on the presence
of a source of phospholipids in the medium as they
do not hydrolyze phospholipids present in cell
membranes (Diaz et al., 2001).
3.3. Activation of PKC activity by LM-PLA2-I plus
PC, LPC or TPA
In the next step, we investigated the direct effect
of LPC on PKC activity after pre-incubating PBL
for 1h with the LM-PLA2-I plus PC, LPC or TPA
as described in Fig. 2. After this period, cells were
washed three times and used to assay PKC activity
(Section 2.7). As shown in Fig. 3, pre-treatment of
lymphocytes with LM-PLA2-I plus PC led to
stimulation of PKC activity as TPA did. This effect
was also observed in the presence of LPC, but not
when LM-PLA2-I was mixed with p-BPB (data not
shown).
3.4. Effect of LM-PLA2-I plus PC, LPC or TPA on
cellular distribution of PKC
It has been shown that activation of PKC by TPA
produces a redistribution of PKC activity from
cytosolic to membrane fractions (Keenan et al.,
1997). As shown in Fig. 4A, pre-incubation of PBL
with LM-PLA2-I plus PC or LPC in the same
conditions used in Fig. 2 led to a redistribution of
PKC activity as TPA did. The decrease of cytosolic
PKC activity was concomitant with an increase in
the PKC activity of membrane fractions. Fig. 4B
shows that in all cases, the translocation of PKC
was inhibited by staurosporine. When LM-PLA2-I
was treated with p-BPB, PKC redistribution was not
observed (data not shown).
3.5. Activation of NF-kB by LM-PLA2-I plus PC,
LPC or TPA
It is well known from the literature that activation
of lymphocytes involves the PKC pathway leading
to phosphorylation of kinases, such as those bound
to the nuclear transcription factor kB (NF-kB).
After phosphorylation, the factor is released from
its inhibitor, leaving the cytoplasm into the nucleus,
where it will bind to others proteins in order to
orchestrate gene transcription. To investigate for an
effect on this step, nuclear extracts were prepared
from PBL pre-incubated with LM-PLA2-I plus PC,
LPC or TPA as described in Fig. 2, and transloca-
tion measured by the mobility shift assay (Section
2.12). As seen in Fig. 5, stimulation by LPC or TPA
led to the DNA-binding NF-kB, similar to what was
observed in the presence of LM-PLA2-I plus PC
(data not shown). The figure shows one representa-
tive of two independent experiments.
3.6. Effect of LM-PLA2-I plus P, LPC or TPA in
the phosphorylation of PKC isoforms
PKC enzymes represent a large family of proteins
which contain autophosphorylation sites (Nishizu-
ka, 2003). Depending on the stimulus used, different
isoforms can be autophosphorylated, which may be
a means of regulating the function of PKC family
members. To investigate this possibility on lympho-
cyte activation, cells were pre-incubated with LPC
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A.L. Fuly et al. / Toxicon 50 (2007) 400–410
Fig. 3. Stimulation of PKC activity by LM-PLA2-I and LPC.
PBL (5?106cells/mL) were pre-incubated for 1h at 371C and
5% CO2with RPMI (1) 45mg/mL LM-PLA2-I plus 1.3mM PC
(2) or with 40mM LPC (3). After this period, cells were washed
three times with saline, resuspended in a lysis buffer and PKC
activity was determined according to Section 2.7. Values are the
means7S.E.M. of three individual experiments.
405

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or TPA in the same conditions as described in Fig. 2
and autophosphophorylation of classical isoforms
of PKCabg or the atypical PKCz isoform was
detected by specific antibodies in a Western blotting
assay (Section 2.13). As shown in Fig. 6, activation
of lymphocytes by LPC led to the autophosphor-
ylation of the aPKCz, differently from TPA,
whereas no difference was found for cPKCabg.
The data show one representative of two indepen-
dent experiments. To further investigate the differ-
ence between LPC and TPA effect, PBL were
cultivated for 4, 24, 48 and 96h with RPMI alone
or in the presence of LPC, TPA or LPC plus TPA,
as described in Fig. 2. The results showed that in
culture medium alone or containing LPC, lympho-
cytes progressively entered in apoptosis, in a time
dependence manner, whereas in the presence of
TPA alone or plus LPC this effect was not observed
up to 96h incubation (data not shown).
4. Discussion
In this report, we show that the enzymatic activity
of the PLA2purified from the L. muta snake venom
stimulated NK activity of lymphocytes, during a
step preceding the killer action, when PKC activity
seems to be essential. These data were confirmed
when commercial LPC was used.
It has been described that in victims of enveno-
mation by snakes, myotoxins with PLA2molecular
structure are responsible for the muscle necrosis
detected (Kini, 1997). Recently, we showed that an
acidic PLA2 isolated from L. muta snake venom
(LM-PLA2-I) displayed hemolytic, inhibition on
platelet aggregation and myotoxic activities (Fuly et
al., 2000). And, all of these biological effects were
dependent on enzymatic activity of LM-PLA2-I,
thus suggesting a participation of LPC formed by
this enzyme upon a substrate. And, here, when
commercial PC was mixed with the PLA2, a
stimulatory effect on lymphocyte’s NK activity
was observed. This, in turn corroborates the data
which formation of LPC could be important for the
studied biological actions. When lymphocytes were
exposed with commercial LPC in concentrations of
50–80mM, enhancement on NK activity was also
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A.L. Fuly et al. / Toxicon 50 (2007) 400–410
Fig. 5. Activation of NF-kB from peripheral blood lymphocytes
by LM-PLA2-I, LPC and TPA. On the left side, the figure shows
the eletrophoretic mobility shift assay (Section 2.12) of the
positive control (+) obtained from nuclear kB DNA-binding
from 70Z3 cells after incubation with 15mg/mL lipopolysacchar-
ide for 1h, and negative control obtained after competition with
cold ATP (COLD). On the right side, figure shows the nuclear kB
DNA-binding from nuclear
(2?107cells) pre-incubated for 1h at 371C and 5% CO2with
RPMI (CTR) 10nM TPA or 45mM LPC. The results are
representative of two independent experiments.
extractsprepared fromPBL
Fig. 4. Redistribution of protein kinase C activity in lymphocyte subcellular fractions after pre-incubation with LM-PLA2-I, LPC and
TPA. PBL (5?106cells/mL) were pre-incubated for 1h at 371C and 5% CO2in different conditions. (A) RPMI (1), 20ng/mL TPA (2),
45mg/mL LM-PLA2-I plus 1.3mM PC (3) or 40mM LPC (4) and (B) in the presence of 2mM staurosporine. After this period, cells were
washed three times with saline, resuspended in a lysis buffer and PKC activity was determined according to Section 2.7. Values are the
means7S.E.M. of three individual experiments.
406

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noticed. The same effect on lymphocytes were also
observed when PC was replaced by different natural
sources of phospholipids, such as hen’s egg yolk or
human plasma, suggesting that the concentration of
LPC enzymatically formed was enough to produce
such effect on those cells. The LPC formed as a
result of hydrolysis of PC present in membranes by
PLA2 and that formed during oxidation of low-
density lipoproteins (LDL), may regulate a broad
range of cellular activity (Kabarowski et al., 2002).
LPC concentrations in body fluids are high,
probably up to 100mM, and therefore it exists
mainly as inactive form, such as in micelles, bound
to hydrophobic serum proteins, incorporated into
plasma membranes or in lipoprotein complexes
(Croset et al., 2000; Xu, 2002). During pathological
processes, its concentration may reach 200mM or
more (Schilling et al., 2004) Some authors stated
that the real content of LPC in cells or in tissues,
such as in plasma is very hard to determine. Early
studies demonstrated that high concentrations of
LPC (above 100mM) could disrupt plasma mem-
brane integrity through micelle formation, suggest-
ing that its biological activity was exclusively
cytolytic and structural, as a detergent-like sub-
stance (Pestronk et al., 1982). On the other hand, a
recent work demonstrated that different lysopho-
spholipids, as LPC could induce a reversible skeletal
paralysis at a 150mM-LPC concentration (Caccin et
al., 2006). And also in this work, LPC esterified with
fatty acids of different lengths and saturation
produced this paralysis with different potencies. In
our work, to eliminate the possibility of a lytic
effect, activity of cytosolic LDH from lymphocytes
or K562 cells was evaluated in LPC-treated cells.
There was no LDH activity in the supernatants of
cultivated cells up to 96h of the culture (data not
shown). More recently, specific effects of LPC in
low concentrations have been reported. Some
studies showed that the physiological and patholo-
gical processes elicited by LPC are mediated by the
G-protein-coupled receptor (Zhu et al., 2001). Two
specific LPC receptors have been identified in
hematopoietic cells such as T-lymphocytes, and
one of them shows an important immunoregulatory
function and exhibits higher affinity binding to the
ARTICLE IN PRESS
A.L. Fuly et al. / Toxicon 50 (2007) 400–410
Fig. 6. Autophosphorylation of aPKCz induced by LM-PLA2-I, LPC and TPA. PBL were pre-incubated for 1h at 371C and 5% CO2
with RPMI (CTR) 10nM TPA or 45mM LPC as described in Section 2.13. The upper part of the figure shows a representative western blot
obtained after immunoprecipitation of cell lysates with anti-PKC antibodies. In the lower part, bands obtained were analyzed by using the
ImageQuant software and expressed as relative optical density arbitrary units.
407

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lysophospholipids (Kabarowski et al., 2001, 2002;
Zhu et al., 2001). The binding of LPC to their
receptors may regulate signaling events leading to
lymphocyte proliferation or inactivation, cellular
migration and cytotoxicity, as we observed for NK
activity towards the lysis of tumor cells, after
treatment with active enzyme PLA2or LPC. This
agrees with the data obtained before with commer-
cial LPC (Whalen et al., 1999; Rigoni et al., 2005).
Curiously, stimulation of NK activity occurred
during a step before NK cells being in contact with
target cells, similarly to TPA, the selective activator
of PKC. The activation of PKC has been the
primary mechanism implicated in several biological
effects of LPC; however, the molecular basis for
that activation, especially the upstream signal for
the phosphoinositide turnover and the production
of diacylglycerol that is required for the activation
of PKC, is poorly understood. The human PKC
enzymes represent a large family of proteins with
differentstructuresand
grouped as classical (a, bI, bII and g), novel (d, e,
Z and y) and atypical (z and i/l), usually existing as
inactive cytosolic forms, which may be differently
activated depending on the lipid mediator present in
the medium (Keenan et al., 1997; Nishizuka, 2003;
Littler et al., 2006; Even-Faitelson and Ravid,
2006). In our conditions, both LPC and TPA
stimulated PKC activity of lymphocytes, leading
to a translocation of the activated enzyme to
membranes and the transcription factor NF-kB
into the nucleus. A biphasic regulation of NF-kB in
human endothelial cells by LPC through the action
of PKC has been observed before, differently from
other PKC activators, such as diacylglycerol and
fatty acids, which stimulate PKC over a wide range
of concentration (Sugiyama et al., 1998; Kazuhiko
et al., 1998; Masamune et al., 2001). Activation of
lymphocytes by LPC or by the mixture LM-PLA2-I
and PC were as effective as TPA; and, in all cases,
staurosporine, the selective inhibitor of PKC
(Fabbri et al., 2003), inhibited the PKC activation,
suggesting a direct effect of LPC on native PKC.
Furthermore, LM-PLA2-I treated with p-BPB was
unable to stimulate NK activity, PKC activity and
translocation of PKC into lymphocyte membranes.
Autophosphorylation of residues may be a
common feedback mechanism in various PKC
isoforms and may function as a control of the
enzyme activity (Even-Faitelson and Ravid, 2006;
Littler et al., 2006). The classical PKC isoforms a,
bI, bII and g require phosphatidylserine (PS),
differentactivators,
calcium
whereas the atypical isoforms (z and i/l) requires
only PS. Our data show that stimulation of NK cells
by LPC led to autophosphorylation of aPKCz
isoform, stimulating NK cells, which resulted in
the increment of the lymphocyte spontaneous killer
activity. Among playing a role in many cell-
signalingpathways,PKC
important regulator in cytoskeletal function. The
cross-talk between the signaling triggered when
NK cells binds to target cells and cytoskeletal
systems may be an important step to the killing
process, such as the release of cytotoxic granules
from NK cells (Graves et al., 1986). PKC activity
seems to be an important step to modulate
cytoskeletal function (Keenan and Kelleher, 1998),
and it has been demonstrated that PKCz regulates
myosin phosphorylation and filament assembly,
constituting an important link between signaling
systems(Even-Faitelson
This may explain the effect of LPC produced by
LM-PLA2-I in activating NK cells to the killer
action, after binding to its receptors in lymphocyte
membrane, although the similarity found between
the effect of LPC and TPA to stimulate NK activity
may indicate that other PKC isoforms may be
involved inthe whole
whereas LPC action was achieved after binding to
its receptors, TPA by passes the need for receptor-
mediated signaling to stimulate NK cytotoxicity
(Zhu et al., 2001), leading to different responses of
PKC isoforms as observed before (Keenan et al.,
1997). We also observed (data not shown) that
differently form LPC, TPA triggered lymphocyte
proliferation and also protected cells to enter in
apoptosis, suggesting different activation mechan-
isms at PKC isoform level that remain to be
elucidated.
Taken together, our data show that a purified
PLA2from L. muta snake venom produces LPC,
which may modulate NK activity. This effect is
entirely dependent on the integrity of LM-PLA2-I’s
active site, with significant participation of a PKC-
dependent pathway. These results provide a new
approach to physiological effects of the PLA2
family and may contribute toward understanding
the mechanism of PLA2and LPC biological effects.
And, the local production of physiologically rele-
vant LPC concentrations and the substrate specifi-
city of the enzyme in realizing LPC with a given
type of fatty acid moiety are, in our opinion,
strong arguments favoring the hypothesis of the
ions and diacilglycerolas activators,
appearstobe an
and Ravid,2006).
process.Furthermore,
ARTICLE IN PRESS
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PLA2 being unequivocally responsible for the
biological effects we described.
Acknowledgments
This work was supported by the following
Brazilian agencies: UFF/PROPP, FAPERJ, FUJB,
FAPERGS and CNPq. We thank Dr. Martha
Sorenson for careful revision of the manuscript
and Dr. Carmen Nogueira (UFRJ) for providing
the blood bags used throughout this study.
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