Purification and characterization of the biological effects of
phospholipase A
2
from sea anemone
Bunodosoma caissarum
Rene´ D. Martins
a, Renata S. Alves
a, Alice M.C. Martins
b, Paulo Sergio F. Barbosa
a, Janaina
S.A.M. Evangelista
c, Joa˜o Jose´ F. Evangelista
a, Rafael M. Ximenes
a, Marcos H. Toyama
d,
Daniela O. Toyama
e, Alex Jardelino F. Souza
f, Diego J.B. Orts
d, Se´rgio Marangoni
f,
Dalgimar B. de Menezes
g, Manasse´s C. Fonteles
e, Helena S.A. Monteiro
a,*aDepartment of Physiology and Pharmacology – Institute of Biomedicine and Clinical Research Unit – Federal University of Ceara´, Fortaleza, Ceara´, Brazil bDepartment of Clinical and Toxicological Analysis, Federal University of Ceara´, Fortaleza, Ceara´, Brazil
cVeterinary Faculty, State University of Ceara´, Fortaleza, Ceara´, Brazil
dSa˜o Vicente Unit, Campus of Litoral Paulista, Paulista State University (UNESP), Sa˜o Paulo, Brazil eMackenzie Presbyterian University, Sa˜o Paulo, Brazil
fUNICAMP, IB, Biochemistry Department, Campinas, Sa˜o Paulo, Brazil gDepartment of Pathology, Federal University of Ceara´, Fortaleza, Ceara´, Brazil
a r t i c l e
i n f o
Article history:
Received 23 January 2009
Received in revised form 25 April 2009 Accepted 11 May 2009
Available online 20 May 2009
Keywords:
Bunodosoma caissarum
Phospholipase A2 Biological effects
a b s t r a c t
Sea anemones contain a variety of biologically active substances.Bunodosoma caissarumis a sea anemone from theCnidaria phylum, found only in Brazilian coastal waters. The aim of the present work was to study the biological effects of PLA2isolated from the sea anemone
B. caissarumon the isolated perfused kidney, the arteriolar mesenteric bed and on insulin secretion. Specimens ofB. caissarumwere collected from the Sa˜o Vicente Channel on the southern coast of the State of Sa˜o Paulo, Brazil. Reverse phase HPLC analysis of the crude extract of B. caissarum detected three PLA2 proteins (named BcPLA21, BcPLA22
and BcPLA23) found to be active inB. caissarumextracts. MALDI-TOF mass spectrometry of
BcPLA21 showed one main peak at 14.7 kDa. The N-terminal amino acid sequence of
BcPLA21 showed high amino acid sequence identity with PLA2group III protein isolated
from the Mexican lizard (PA23 HELSU, HELSU, PA22 HELSU) and with the honey bee Apis mellifera (PLA2and 1POC_A). In addition, BcPLA21 also showed significant overall
homology to bee PLA2. The enzymatic activity induced by native BcPLA21 (20mg/well) was
reduced by chemical treatment withp-bromophenacyl bromide (p-BPB) and with morin. BcPLA21 strongly induced insulin secretion in presence of high glucose concentration.
In isolated kidney, the PLA2fromB. caissarumincreased the perfusion pressure, renal
vascular resistance, urinary flow, glomerular filtration rate, and sodium, potassium and chloride levels of excretion. BcPLA21, however, did not increase the perfusion pressure on
the mesenteric vascular bed. In conclusion, PLA2, a group III phospholipase isolated from
the sea anemone B. caissarum, exerted effects on renal function and induced insulin secretion in conditions of high glucose concentration.
Ó2009 Elsevier Ltd. All rights reserved.
1. Introduction
Sea anemones contain a variety of biologically active substances, including some potent toxins. The composition of cnidarian venoms is not known in detail, but they appear *Corresponding author. Departamento de Fisiologia e Farmacologia,
Faculdade de Medicina, Universidade Federal do Ceara´, CEP-60.420-970, PO Box 3229 – Fortaleza, Ceara´, Brasil. Tel.: þ55 (085) 33668248; fax:þ55 (085) 32815212.
E-mail addresses:martinsalice@gmail.com(A.M.C. Martins),serrazul@ baydenet.com.br(H.S.A. Monteiro).
Contents lists available atScienceDirect
Toxicon
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / t o x i c o n
to contain a variety of proteinaceous (peptides, proteins, enzymes and proteinase inhibitors) and non-proteinaceous substances (purines, quaternary ammonium compounds, biogenic amines and betaines) (Malpezzi et al., 1993; Grotendorst and Hessinger, 2000; Anderluh and Macek, 2002). Nematocysts possess a high concentration of polypeptides and proteins that act as neurotoxins, hemo-lysins and enzymes, which are responsible for a variety of harmful effects (cardiotoxicity, dermatitis, local itching, swelling, erythema, paralysis, pain and necrosis) (Oliveira et al., 2006). Some of the substances isolated of the Buno-dosoma cangicum anemone include polypeptides that interact with voltage-sensitive sodium channels (Cunha et al., 2005), potassium channels (including the hERG channel), acid-sensing ion channels, as well as poly-peptides that function as pore-forming toxins (actino-porins) and protease inhibitors (Diochot et al., 1998, 2003, 2004; Bosmans and Tytgat, 2007).
Bunodosoma caissarum is a sea anemone from the
Cnidariaphylum, which is found only in Brazilian coastal waters, and comprises of both benthic and pelagic aquatic animals, including the members of the classes Anthozoa (hard corals, soft corals, sea pens, sea anemones), Hydrozoa (hydroids, fire corals), Scyphozoa (jellyfish) and Cubozoa (box jellyfish) (Nevalainen et al., 2004b). Sea anemones possess tentacles that are used for capturing prey and act as protection against predators (Cunha et al., 2005). These structures contain cnidocytes, which are composed of organelles known as nematocysts. These nematocysts possess harpoon-like microscopic structures (cnida) that, when fired, penetrate the surface layer of the victim to deliver a mixture of highly toxic substances (Cunha et al., 2005; Nevalainen et al., 2004b).
The purine caissarone was the first adenosine receptor antagonist described from a marine organism that elicited anomalies in sea urchin eggs and increased the intestinal motility in mammals (Freitas and Sawaya, 1986, 1990; Cooper et al., 1995). It was also reported that alcohol extracts from the whole body of B. caissarum had an antimitotic effect on sea urchin eggs (Malpezzi and Freitas, 1990). The venom extracted from the nematocysts of this anemone possesses high hemolytic activity towards erythrocytes from various verterbrate species (fish, toad, snake, mouse and rat) (Malpezzi and Freitas, 1991); this effect has been attributed to a 20 kDa protein with phos-pholipase A2 activity called caissarolysin I (Oliveira et al., 2006). This same protein also induced glutamate release from rat synaptosomes and stimulated exocytosis in bovine chromaffin cells (Migues et al., 1999; Ale´s et al., 2000).
PLA2s have been identified in marine invertebrates (McIntosh et al., 1995; MacPherson and Jacobs, 2000; Kishimura et al., 2000; Talvinen and Nevalainen, 2002), in hard corals, fire coral, crown-of-thorns starfish, sea cucumber, marine sponges and in both the acontia and tentacles of Cnidaria (Nevalainen et al., 2004a, b).
Envenomation by the sea anemonePhyllodiscus semoni
causes fulminant dermatitis and acute renal failure in humans (Mizuno et al., 2007). In some instances these effects are associated with complement (C)-activating components in the venoms, components that indirectly contribute to tissue damage (Yamamoto et al., 2002;
Bertazzi, 2003; Rodrigues et al., 2004; Tambourgi et al., 2004). While no direct association between sea anemone venoms and C activation has been reported, the sea anemone-derived toxin AvTX-60A has recently been reported to exhibit structural similarities to terminal pathway C proteins (Oshiro et al., 2004).
The aim of the present work was to study the biological effects of PLA2isolated from the sea anemoneB. caissarum on the isolated perfused kidney, the arteriolar mesenteric bed and on insulin secretion.
2. Material and methods
2.1. Crude protein extracts preparation
Specimens (60) ofB. caissarum(weighing approximately 13 grams each) were collected during periods of low tide by free diving at different rocky shores of the Sa˜o Vicente Channel on the southern coast of Sao Paulo, Brazil. The animals were transported alive and starved in an aquarium for 72 h to eliminate gastrovascular cavity contents. The tentacles of animals were removed from the body using forceps and immediately immersed in an ice-cold 0.1% trifluoroacetic acid (TFA). Tentacles were then subjected to three freeze–thaw cycles. After the last freeze–thaw cycle, the solution was centrifuged at 20,000g for 60 min at 4C. The supernatant was recovered and filtered through a 0.45
m
m filter, followed by a second ultrafiltration through a 0.22m
m filter. The protein was precipitated from the crude extract using 10% TFA at 4C and centrifuged at 4500g for 10 min at 4C. The protein pellet was then dissolved in water and subsequently lyophilized.2.2. Purification and isolation of PLA2from theB. caissarum
The lyophilized crude extract was dissolved in 200
m
L of TFA (0.1% triflouroacetic acid; buffer A) then clarified by a high-speed centrifugation step (4500gfor 3 min). The supernatant was then fractionated on a Bondapack C18 reverse phase HPLC column (0.7830 cm). Protein elution was performed using a non-linear gradient of buffer B (66.6% of acetonitrile in 0.1% TFA) at a constant flow rate of 2.0 mL/min. Chromatography was monitored at A214 nm and the desired fraction was then lyophilized. The purity of the HPLC-purified PLA2 was analyzed as described by Hernandez-Oliveira et al. (2005)using a Tricine SDS-PAGE gel and MALDI-TOFF mass spectrometry.a PICO-TAG amino acid analyzer system. The analysis of PTH-amino acid was conducted using a PICO-TAG amino acid analyzer (Waters).
2.2.2. N-terminal amino acid sequencing of PLA2from the B. caissarum
Protein sequencing was performed as previously described by de Oliveira et al. (2003). Briefly, two milli-grams of the purified protein were dissolved in 200
m
L of a 6 mol/L guanidine chloride solution (Merck, Darmstadt, Germany) containing 0.4 mol/L of Tris–HCl and 2 mmol/L EDTA (pH 8.15). Nitrogen was blown over the top of the protein solution for 15 min, after which the protein solu-tion was reduced with DTT (6 M, 200m
L) and subjected to a second incubation under a nitrogen atmosphere for 90 min. After this incubation time, 80m
L of iodoacetic acid were added to the solution (50 mM of cold iodoacetic and carboxymethylated14C-iodoacetic acid), followed by a third incubation under a nitrogen atmosphere after which the reaction tube was sealed. To remove excess reagent and to purify the PLA2protein, we used a preparative C5 reverse phase column; peptides were separated by a linear gradient of acetonitrile (66% in 0.1% of TFA) at a constant flow rate of 2.5 mL/min for 90 min. Buffer A was used in the first 15 min of the HPLC run to remove the salts and reagents. The progress of the chromatographic elution was monitored at 214 nm, and the PLA2fraction active peak was lyophilized. The amino acid sequences of PLA2from theB. caissarum (BcPLA2) were determined using an Applied Biosystems model Procise f gas-liquid protein sequencer. The phenyl-thiohydantoin (PTH) derivates of the amino acids were identified with an Applied Biosystems model 450 micro-gradient PTH-analyzer.2.3. Phospholipase A2activity assay
PLA2 activity assays were conducted following the protocol described byWen-Hwa et al. (1999)and adapted byToyama et al. (2003)for a 96-well plate. The standard assay mixture contained 200
m
L of buffer (10 mM Tris–HCl, 10 mM CaCl2, 100 mM and NaCl, pH 7.8), 20m
L of substrate (4-nitro-3-octanoyloxy-benzoic acid (4N3OBA), manufac-tured by BIOMOL, USA), 20m
L of water and 20m
L of PLA2to give a final volume of 260m
L. After the addition of native PLA2(20m
g), the mixture was incubated for up to 40 min at 37C with absorbance readings performed at 10 min intervals. Enzyme activity, expressed as the initial velocity of the reaction (Vo), was calculated based on the increase in absorbance after 20 min. All assays were done in triplicate and the absorbance at 425 nm was measured by a Spec-traMax 340 multiwell plate reader (Molecular Devices, Sunnyvale, CA). The chemical treatment of PLA2from theB.caissarum (BcPLA2) with p-Bromophenacyl bromide (p-BPB) was done according to the protocol described by Landucci et al. (2000)andIglesias et al. (2005).
2.4. Insulin secretion
Insulin secretion was determined as described by Toyama et al. (2000). Briefly, rat islets were isolated by collagenase digestion of the pancreas. For static secretion,
groups of five islets were first incubated for 45 min at 37C in Krebs-bicarbonate buffer (in mmol/L: 115 NaCl, 5 KCl, 2.56 CaCl2, 1 MgCl2, 10 NaHCO3, 15 HEPES, and 5.6 glucose) supplemented with 3 g/L of bovine serum albumin and equilibrated with a mixture of 95% O2–5% CO2, pH 7.4. The medium was then replaced with fresh Krebs-bicarbonate buffer and the islets were incubated for an additional 1 h in medium containing different concentrations of glucose, in the presence or absence of native BcPLA2, morin-modified BcPLA2 (BcPLA2: morin), or p-BPB-modified BcPLA2 (BcPLA2:p-BPB). The insulin content of the medium at the end of the incubation period was measured by radioim-munoassay analysis (Nogueira et al., 2005).
2.5. Perfused kidney assay
Adult male Wistar rats (260–320 g) were fasted for 24 h with unrestricted access to water. The rats were anes-thetized with sodium pentobarbitone (50 mg/kg, i.p). After careful dissection of the right kidney, the right renal artery was cannulated via the mesenteric artery without inter-rupting blood flow as described by Bowman (1970)and modified byFonteles et al. (1983). The perfusate consisted of a modified Krebs–Henseleit solution (MKHS) of the following composition (in mmol/L): 118.0 NaCl, 1.2 KCl, 1.18 KH2PO4, 1.18 MgSO4.7H2O, 2.50 CaCl2 and 25.0 NaHCO3. Bovine serum albumin (6 g, BSA) were added to 100 mL of MKHS, and dialyzed for 48 h at 4C against 10 volumes of MKHS. Immediately before the beginning of each perfusion protocol, 100 mg of urea, 50 mg of inulin and 50 mg of glucose were added to every 100 mL of perfusate and the pH was adjusted to 7.4. In each experiment, 100 mL of MKHS were recirculated for 120 min. The perfusion pres-sure (PP) was meapres-sured at the tip of the stainless steel cannula in the renal artery. Samples of urine and perfusate were collected at 10 min intervals for analysis of sodium and potassium level by flame photometry; inulin was measured as described byWalser et al. (1955)and modified byFonteles et al. (1983); osmolality was measured using a vapor pressure osmometer (Wescor 5100C, USA). The chloride analysis was carried out using a LabTest kit. BcPLA2 was added to the system 30 min after the beginning of each perfusion. The renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR). The excretion levels of sodium (ENaþ), potassium (EKþ) and chloride (ECl) were also determined (Martinez-Maldonato and Opava-Stitzer, 1978).
2.6. Isolated perfused arteriolar mesenteric bed
114.0 mM of NaCl; 4.96 mM of KCl; 1.24 mM of KH2PO4; 0.5 mM of MgSO4$7H2O; 24.99 mM of NaHCO3; 2.10 mM of CaCl2$2H2O; and 3.60 mM of glucose. The perfusion solution was kept warmed at 37C and the mesenteric bed was perfused by a constant flow (4 mL/min) and the variable perfusion pressure was measured for 80 min after the equilibration period. In this experiment, the direct vascular effects of BcPLA2(3
m
g/mL/min.;n¼6), infused at a constant rate (0.1 mL/min), were examined and compared to the effects of infusion of the vehicle alone at the same rate.2.7. Statistical analysis
Results are shown as meanS.E.M of six experiments for each group. Differences between groups were compared by using Student’st-test or analysis of variance (ANOVA) with significance set at 5%.
3. Results
3.1. Purification and isolation of PLA2from theB. caissarum
Reverse phase HPLC chromatography of crude extract revealed the presence of three active PLA2proteins named
BcPLA21, BcPLA22 and BcPLA23 (Fig. 1a). BcPLA21 was the main fraction and appeared as a 14 kDa protein after the treatment of the protein with DTT (1 M). In the absence of DTT treatment, BcPLA21 appeared as one protein of
approximately 30 kDa (Fig. 1b). The MALDI-TOF mass spectrometry revealed BcPLA21 as one main peak at 14,706 Da (Fig. 1c).
3.2. Amino acid compositon and sequencing of PLA2from B. caissarum
The N-terminal amino acid sequence of BcPLA21 showed high amino acid identity with other members of the group III PLA2 family isolated from the Mexican lizard such as PA23 HELSU, HELSU, and PA22 HELSU and with the honey bee Apis PLA2 and 1POC_A. Analysis of the N-terminal showed that BcPLA21 has both a conserved calcium binding region and a conserved catalytic site (Fig. 2a).Fig. 2b shows the amino acid composition results of BcPLA21.
3.3. Enzymatic action and insulin secretion
BcPLA21 showed a moderate enzymatic activity using 4-nitro-3-octanoyloxy-benzoic acid (4N3OBA) as a substrate. Aliquots of BcPLA2were incubated withp-BPB (3.5
m
M) or morin (3.5m
M) for 30 min followed by removal of excess reagent by reverse phase HPLC; the chemically treated PLA2 was used for experiments that involved enzymatic activity and insulin secretion. Chemical treatment of BcPLA21 (20m
g/well;n¼20) by p-BPB (PLA21:p-BPB) or by morin (PLA21: Morin) reduced the enzymatic activity of BcPLA21 (p<0.05,n¼20) (Fig. 3a). BcPLA21 strongly induced insulin secretion, which was increased in conditions of high glucose concentrations (p<0.05; n¼12; Fig. 3b). By contrast, BcPLA21 had no significant effect under conditions of lower glucose concentration (Glucose 2.8 mM). The chemical treatment of BcPLA21 with morin abolished the effect induced by native BcPLA21 (p<0.05;n¼12) whereasp-BPB reduced only the insulinotropic effect of BcPLA21 (p<0.05;n¼12) (Fig. 3b).
3.4. Renal effects of PLA2fromB. caissarum
B. caissarumPLA2altered the renal parameters studied. Treatment with 1
m
g/mL BcPLA21 increased the perfusion pressure (Fig. 4a), the renal vascular resistance (Fig. 4b), the urinary flow (Fig. 5a), and the glomerular filtration rate (Fig. 5b) at 60 min during the perfusion. The sodium (Fig. 6a), potassium (Fig. 6b), and chloride (Fig. 6c) excre-tion levels also increased significantly when compared to the control group.Treatment with 0.3
m
g/mL BcPLA21 increased the perfusion pressure (Fig. 4a) and the renal vascular resis-tance (Fig. 4b) at 60, 90 and 120 min. However, the urinary flow (Fig. 5a) and glomerular filtration rate (Fig. 5b) increased only at 90 and 120 min during the perfusion when compared of the control group. The sodium (Fig. 6a), potassium (Fig. 6b), and chloride (Fig. 6c) excretion levels increased significantly after infusion of the venom.Kidneys perfused with a lower concentration of BcPLA21 (0.1
m
g/mL) showed a similar response as the group treated with BcPLA21 at 0.3m
g/mL. The main difference was that potassium excretion levels increased only at 90 and 120 min of perfusion.3.5. Effects of PLA2fromB. caissarumon mesenteric
blood vessels
BcPLA21 (3
m
g/mL/min) did not significantly increase the perfusion pressure on the mesenteric vascular bed. The average perfusion pressure (PP) in the control group was 38.930.97 mmHg while the average perfusion pressure in the BcPLA21 infused group was 41.601.08 mmHg.As a positive control, mesenteric blood vessels were induced to contract by the administration of phenylephrine (controlPP¼38.930.97 mmHg and PhePP¼127.60 6.80 mmHg).
4. Discussion
Numerous sPLA2 have been described in venom from
both vertebrate and invertebrate animals (Davidson and Dennis, 1990; Valentin and Lambeau, 2000b). In the present study, the B. caissarum extract was shown to consist of three main PLA2isoforms. The enzymatic activity
confirmed the presence of this enzyme and identified
a group of similar proteins. Eletrophoretic analysis of BcPLA21 showed that dimerization of the BcPLA21
mono-mer increases the enzymatic activity of PLA2(Toyama et al.,
2005).
Amino acid residues at positions 6–14 (in particular W10, G12, G14) are proposed to form the Ca2þ binding segment, similar to that found in other group III secretory PLA2s from the lizards (Sosa et al., 1986), bee venoms
(Nakashima et al., 2004) and humans (Valentin et al., 2000). The amino acid residues corresponding to positions 29–40 correspond to the catalytic site, represented by H(36) and D(37) in BcPLA21 and corresponding to H(48) and D(49) in the enzymatically active pancreatic PLA2.
According to our amino acid alignment results, both these segments are highly conserved, sharing an amino acid sequence identity of approximately 90%. The presence of 10 half cystein, as determined by amino acid analysis, suggests that BcPLA21 forms 5 disulfide bridges. These results
suggest that BcPLA21 is a novel PLA2of the same family as
the PLA2from the bees (A. mellifera) and lizards (Heloderma suspectum). The primary structure of group III PLA2s is
distinct from that of group I and II PLA2s, having only the
Ca2þ-loop and the active site conserved between group III PLA2members. The overall three dimensional structure of
bee venom group III PLA2reveals the striking features of
the PLA2 fold, which includes a well-defined Ca2þ-loop,
three large
a
-helices and ab
-wing-like structure. Thus, BcPLA2may have the same molecular properties as those ofbee (Rizzo et al., 2000; Nakashima et al., 2003, 2004) and lizard PLA2(Sosa et al., 1986).
In the present study, we found that BcPLA21 stimulated
insulin secretion only in conditions of high glucose concentration. Nogueira et al., 2005 demonstrated the insulinotropic effect induced byCrotalus durissus terrificus
venom. The secretion of insulin by glucose induction depends on the metabolism of this sugar to increase the ATP/ADP ratio, which blocks KþATP channels and causes B-cell membrane depolarization. The resulting massive influx of Ca2þ, which occurs primarily through the Ca2þ voltage-dependent L-type channels, increases cytosolic Ca2þ concentration and stimulates insulin secretion (Nogueira et al., 2005).
Despite numerous studies characterizing PLA2s, the
molecular basis for the selective and specific pharmaco-logical action of PLA2s in theB. caissarumvenom is still
unclear. One would predict that the primary targets for PLA2s would be located outside the cell. Therefore,
poten-tial mechanisms of action could involve: 1) the intrinsic catalytic activity of venom PLA2, i.e., its ability to release
potent biologically active fatty acids and lysophospholipids from membrane lipids; 2) the interfacial binding of PLA2to
the membrane lipid bilayer, which may affect cellular function by perturbing the cellular membrane independent of phospholipid hydrolysis; and 3) the binding of venom PLA2 to specific proteins located at the cell surface
Fig. 3.Enzymatic action of PLA2fromBunodosoma caissarumand insulin secretion. (a) Enzymatic activity of BcPLA21 and that of BcPLA21 chemically treated with morin andp-BPB. (b) insulinotropic effect of BcPLA21 and that of chemically treated BcPLA21. BcPLA21¼Bunodosoma caissarumPLA21.
30 60 90 120 0
100 200
Control
BcPLA21 0.1µg/mL
BcPLA21 0.3µg/mL
BcPLA21 1µg/mL
*
*
* *
*
*
*
Time (min) Pe rfu s io n p re s s u re (m m H g )30 60 90 120 0.0 2.5 5.0 7.5 10.0 Control
BcPLA21 0.1µg/mL
BcPLA21 0.3µg/mL
BcPLA21 1µg/mL
*
*
*
*
*
Time (min) Va s c u la r re n a l re s is ta n c e (m m H g /m L /g /m in )*
*
b
a
Fig. 4.Effects of PLA2fromB. caissarum(BcPLA21) on perfusion pressure (a) and renal vascular resistance (b) Data are expressed as meanSEM from six different animals. *p<0.05 compared to the corresponding control group for each interval. BcPLA21¼Bunodosoma caissarumPhosfolipase A21.
30 60 90 120 0.0 0.1 0.2 0.3 0.4 Control
BcPLA21 0.1 µg/mL
BcPLA21 0.3 µg/mL
BcPLA21 1 µg/mL
*
*
*
*
*
*
Time (min)a
b
U ri n a ry F lo w (m L /g /m in )*
30 60 90 120 0
1 2
Control
BcPLA21 0.1 µg/mL
BcPLA21 0.3 µg/mL
BcPLA21 1 µg/mL
*
*
*
*
*
*
Time (min) G lo m e ru la r F il tr a ti o n ra te (m L /g /m in )(Lambeau and Lazdunski, 1999; Valentin and Lambeau, 2000a). Our results using morin treated BcPLA2 suggest
that the enzymatic activity of BcPLA21 is not required for its
pharmacological activity. Both morin andp-BPB modifica-tions strongly decreased the enzymatic activity of PLA2; p-BPB specifically binds to histidine residues located in the active site of PLA2(Landucci et al., 2000); whereas morin
strongly inhibits the enzymatic activity of PLA2by altering the three dimensional structure of the PLA2as shown by
Iglesias et al. (2005).
Several venoms and toxins from snakes, spiders, and other venomous animals have been described that target the kidney (Tu, 1987; Burdmann et al., 1996; Luciano et al., 2004). The kidney is often exposed to higher levels of toxic substances than most organs (Prozialeck and Edwards, 2007). Mizuno et al. (2007) showed that the kidney is a target of the protein toxin from the sea anemone
P. semoni. BcPLA21 produced renal vascular and glomerular
alterations such as increase in perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate and electrolytes transport.
Results from the mesenteric blood vessel model showed that BcPLA21 did not alter basal perfusion pressure in the vascular bed. This result suggests that the increase in renal perfusion pressure and vascular resistance that was previ-ously observed in isolated perfused rat kidney did not occur by the direct action of BcPLA21 on renal vasculature.
In conclusion, PLA2, a group III phospholipase isolated from the sea anemoneB. caissarum,might promote indirect renal effects, and under conditions of high glucose content, may induce insulin secretion.
Conflict of interest
The authors declare that there are no conflicts of interest.
References
Ale´s, E., Gabilan, N.H., Cano-Abad, M.F., Garcia, A.G., Lo´pez, M.G., 2000. The sea anemone toxin Bc2 induces continuous or transient exocy-tosis, in the presence of sustained levels of high cytosolic Caþþin chromaffin cells. J. Biol. Chem. 275 (48), 37488–37495.
Anderluh, G., Macek, P., 2002. Cytolytic peptide and protein toxins from sea anemones (Anthozoa: Actiniaria). Toxicon 40, 111–124. Bertazzi, 2003. Effect ofTityus serrulatusscorpion venom and its major
toxin, TsTX-I, on the complement systemin vivo. Toxicon 41 (4), 501–508.
Bosmans, F., Tytgat, J., 2007. Sea anemone venom as a source of insecti-cidal peptides acting on voltage-gated Naþchannels. Toxicon 49 (4), 550–560.
Bowman, R.H., 1970. Gluconeogenesis in the isolated perfused isolated rat kidney. J. Biol. Chem. 245, 1604–1612.
Burdmann, E.A., Antunes, I., Saldanha, L.B., Abdulkader, R.C., 1996. Severe acute renal failure induced by the venom ofLonomiacaterpillars. Clin. Nephrol. 46, 337–339.
Cooper, R.A., de Freitas, J.C., Porreca, F., Eisenhour, C.M., Lukas, R., Huxtable, R.J., 1995. The sea anemone purine, caissarone: adenosine receptor antagonism. Toxicon 33 (8), 1025–1031.
Cunha, R.B., Santana, A.N., Amaral, P.C., Carvalho, M.D., Carvalho, D.M., Cavalheiro, E.A., Maigret, B., Ricart, C.A., Cardi, B.A., Sousa, M.V., Carvalho, K.M., 2005. Primary structure, behavioral and electroen-cephalographic effects of an epileptogenic peptide from the sea anemoneBunodosoma cangicum. Toxicon 45 (2), 207–217. Davidson, F.F., Dennis, E.A., 1990. Evolutionary relationships and
impli-cations for the regulation of phospholipase A2from snake venom to human secreted forms. J. Mol. Evol. 31 (3), 228–238.
Diochot, S., Schweitz, H., Be´ress, L., Lazdunski, M., 1998. Sea anemone peptides with a specific blocking activity against the fast inactivating potassium channel Kv3.4. J. Biol. Chem. 273 (12).
Diochot, S., Loret, E., Bruhn, T., Be´ress, L., Lazdunski, M., 2003. APETx1, a new toxin from the sea anemone Anthopleura elegantissima, blocks voltage-gated human ether-a-go-go-related gene potassium chan-nels. Mol. Pharmacol. 64 (1), 59–69.
Diochot, S., Baron, A., Rash, L.D., Deval, E., Escoubas, P., Scarzello, S., Salinas, M., Lazdunski, M., 2004. A new sea anemone peptide, APETx2, inhibits ASIC3, a major acid-sensitive channel in sensory neurons. EMBO J. 23 (7), 1516–1525.
de Oliveira, D.G., Toyama, M.H., Martins, A.M.C., Havt, A., Nobre, A.C.L., Marangoni, S., Camara, P.R., Antunes, E., de Nucci, G., Beliam, L.O.S., Fonteles, M.C., Monteiro, H.S.A., 2003. Structural and biological characterization of a crotapotin isoform isolated from Crotalus durissus cascavellavenom. Toxicon 42 (1), 53–62.
Freitas, J.C., Sawaya, M.I., 1986. Anomalies in sea-urchin egg development induced by a novel purine isolated from the sea-anemone Bunodo-soma caissarum. Toxicon 24 (8), 751–755.
Freitas, J.C., Sawaya, M.I., 1990. Increase of mammalian intestinal motility by the iminopurine caissarone isolated from the sea anemone
Bunodosoma caissarum. Toxicon 28 (9), 1029–1037.
Fonseca, F.V., Antunes, E., Morganti, R.P., Monteiro, H.S.A., Martins, A.M.C., Toyama, D.O., Marangoni, S., Toyama, M.H., 2006. Characterization of a new platelet aggregating factor from crotoxinCrotalus durissus cascavellavenom. Protein J. 25 (3), 183–192.
Fonteles, M.C., Cohen, J.J., Black, A.J., Wertheim, S.J., 1983. Support of kidney function by long-fatty acids derived from renal tissue. Am. J. Physiol. 244, 235–246.
30 60 90 120 0 10 20 30 40 Control
BcPLA21 0.1 µg/mL
BcPLA21 0.3 µg/mL BcPLA21 1 µg/mL
* *
*
*
*
*
*
*
*
*
Time (min) So d iu m Ex c re ti o n (µ Eq ./ g /m in )*
0.0 0.5 1.0 1.5 ControlBcPLA21 0.1 µg/mL
BcPLA21 0.3 µg/mL
BcPLA21 1 µg/mL
*
*
*
*
Time (min) Po ta s s iu m Ex c re ti o n (µ Eq ./ g /m in )*
*
30 60 90 120
30 60 90 120 0 10 20 30 40 Control
BcPLA21 0.1 µg/mL
BcPLA21 0.3 µg/mL
BcPLA21 1 µg/mL
*
*
*
*
*
*
Time (min) C h lo ri d e Ex c re ti o n (µ Eq ./ g /m in )b
c
a
Grotendorst, G.R., Hessinger, D.A., 2000. Enzymatic characterization of the major phospholipase A2 component of sea anemone (Aiptasia
pallida) nematocyst venom. Toxicon 38, 931–943.
Hernandez-Oliveira, S., Toyama, M.H., Toyama, D.O., Marangoni, S., Hyslop, S., Rodrigues-Simioni, L., 2005. Biochemical, pharmacological and structural characterization of a new PLA(2) fromCrotalus durissus terrificus (South American rattlesnake) venom. Protein J. 24 (4), 233–242.
Iglesias, C.V., Aparı´cio, R., Rodrigures-Simioni, L., Camargo, E.A., Antunes, E., Marangoni, S., Toyama, D.D., Beriam, L.O.S., Monteiro, H.S. A., Toyama, M.H., 2005. Effects of morin on snake venom phospho-lipase A(2) (PLA(2)). Toxicon 46 (7), 751–758.
Kishimura, H., Ojima, T., Hayashi, K., Nishita, K., 2000. cDNA cloning and sequencing of phospholipase A2 from the pyloric ceca of the starfish
Asterina pectinifera. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 126 (4), 579–586.
Landucci, E.C.T., Castro, R.C., Toyama, M.H., Giglio, J.R., Marangoni, S., de Nucci, G., Antunes, E., 2000. Inflammatory oedema induced by the Lys-49 phospholipase A2homologue piratoxin-I in the rat and rabbit: effect of polyanions and p-bromophenacyl bromide. Biochem. Pharmacol. 59, 1289–1294.
Lambeau, G., Lazdunski, M., 1999. Receptors for a growing family of secreted phospholipases A2. Trends Pharmacol. Sci. 20, 162–170. Luciano, M.N., da Silva, P.H., Chaim, O.M., dos Santos, V.L., Franco, C.R.,
Soares, M.F., Zanata, S.M., Mangili, O.C., Gremski, W., Veiga, S.S., 2004. Experimental evidence for a direct cytotoxicity ofLoxosceles inter-media(brown spider) venom in renal tissue. J. Histochem. Cytochem. 52, 455–467.
McGregor, D.D., 1965. The effect of sympathetic nerve stimulation on vasoconstrictor responses in perfused mesenteric blood vessels of the rat. J. Physiol. 177, 21–30.
Macpherson, J.C., Jacobs, R.S., 2000. An 18.5 kDa protein from the amebocyte of Limulus pholyphermus, homologous to the previously described amebocyte aggregating factor, expresses alternative phospholipases A2activity. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 127 (1), 31–44.
Malpezzi, E.L., Freitas, J.C., 1990. Antimitotic effect of an extract of the sea anemoneBunodosoma caissarumon sea urchin egg development. Braz. J. Med. Biol. Res. 23 (9), 811–814.
Malpezzi, E.L., Freitas, J.C., 1991. Hemolytic activity of the nematocyst venom from the sea anemoneBunodosoma caissarum. Braz. J. Med. Biol. Res. 24 (12), 1245–1249.
Malpezzi, E.L., Matsui, D.H., Groote, S.C., Freitas, G.M., Santelli, G.M., Fernandes, J.B., 1993. Antitumoral activity in an organic extract of the sea anemoneBunodosoma caissarum. Toxicon 33, 291.
Martinez-Maldonato, M., Opava-Stitzer, S., 1978. Free water clearance curves during saline, mannitol, glucose and urea. J. Physiol. 280, 487–497. Mcintosh, J.M., Ghomashchi, F., Gelb, M.H., Dooley, D.J., Stoehr, S.J.,
Giordani, A.B., Naisbitt, S.R., Olivera, B.M., 1995. Conodipine-M, a novel phospholipase A2isolated from the venom of the marine snail
Conus magus. J. Biol. Chem. 270 (8), 3518–3526.
Migues, P.V., Leal, R.B., Mantovani, M., Nicolau, M., Gabilan, N.H., 1999. Synaptosomal glutamate release induced by the fraction Bc2 from the venom of the sea anemoneBunodosoma caissarum. NeuroReport 10, 67–70.
Mizuno, M., Nozaki, M., Morine, N., Suzuki, N., Nishikawa, K., Morgan, B.P., Matsuo, S., 2007. A protein toxin from the sea anemonePhyllodiscus semoni
targets the kidney and causes a severe renal injury with predominant glomerular endothelial damage. Am. J. Pathol. 1 (2), 402–414.
Nakashima, S., Ikeno, Y., Yokohama, T., Kuwana, M., Bolchi, A., Ottonello, S., Kitamoto, K., Arioka, M.T., 2003. Secretory phospholipases A2induce neurite outgrowth in PC12 cells. Biochem. J. 376, 655–666. Nakashima, S., Kitamoto, K., Arioka, M., 2004. The catalytic activity, but
not receptor binding, of sPLA(2)s plays a critical role for neurite outgrowth induction in PC12 cells. Brain Res. 1015, 207–211. Nevalainen, T.J., Quinn, R.J., Hooper, J.N., 2004a. Phospholipase A2in
Por-ifera. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 137 (3), 413–420. Nevalainen, T.J., Peuravuori, H.J., Quinn, R.J., Llewellyn, L.E., Benzie, J.A., Fenner, P.J., Winkel, K.D., 2004b. Phospholipase A2in cnidaria. Comp. Biochem. Physiol. B Biochem Mol. Biol. 139 (4), 731–735.
Nogueira, T.C.A., Ferreira, F., Toyama, M.H., Spoppiglia, L.F., Maragoni, S., Boschero, A.C., Carneiro, E.M., 2005. Characterization of the
insulinotropic action of a phospholipase A(2) isolated fromCrotalus durissus collilineatus rattlesnake venom on rat pancreatic islets. Toxicon 45 (2), 243–248.
Oliveira, J.S., Zaharenko, A.J., de Freitas, J.C., Konno, K., de Andrade, S.A., Portaro, F.C., Richardson, M., Santanna, O.A., Tambourgi, D.V., 2006. Caissarolysin I (Bcs I), a new hemolytic toxin from the Brazilian sea anemoneBunodosoma caissarum: purification and biological charac-terization. Biochim. Biophys. Acta. 1760 (3), 453–461.
Oshiro, N., Kobayashi, C., Iwanaga, S., Nozaki, M., Namikoshi, M., Spring, J., Nagai, H., 2004. A new membrane-attack complex/perforin (MACPF) domain lethal toxin from the nematocyst venom of the Okinawan sea anemoneActineria villosa. Toxicon 43 (2), 225–228.
Prozialeck, W.C., Edwards, J.R., 2007. Cell adhesion molecules in chemi-cally-induced renal injury. Pharmacol. Ther. 114 (1), 74–93. Rizzo, M.T., Nguyen, E., Aldo-Benson, M., Lambeau, G., 2000. Secreted
phospholipase A(2) induces vascular endothelial cell migration. Blood 96, 3809–3815.
Rodrigues, F.G., PetretskiI, J.H., Kanashiro, M.M., Lemos, L., da Silva, W.D., Kipnis, T.L., 2004. The complement system is involved in acute inflammation but not in the hemorrhage produced by aBothrops atroxsnake venom low molecular mass proteinase. Mol. Immunol. 40 (16), 1149–1156.
Sosa, B.P., Alago´n, A.C., Martin, B.M., Possani, L.D., 1986. Biochemical characterization of the phospholipase A2purified from the venom of the Mexican beaded lizard (Heloderma horridum horridum Wieg-mann). Biochemistry 25 (10), 2927–2933.
Talvinen, K.A., Nevalainen, T.J., 2002. Cloning of a novel phospholipase A2 from the cnidarianAdamsia carciniopados. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 132 (3), 571–578.
Tambourgi, D.V., de Fernandes Pedrosa, M., Van Den Berg, C.W., Gonçalves de Andrade, R.M., Ferracini, M., Paixa˜o Cavalcante, D., Morgan, B.P., Rushimere, N.K., 2004. Molecular cloning, expression, function and immunoreactivities of members of a gene family of sphingomyelinases fromLoxoscelesvenom glands. Mol. Immunol. 41 (8), 831–840.
Toyama, M.H., Carneiro, E.M., Marangoni, S., Barbosa, R.L., Corso, G., Boschero, A.C., 2000. Biochemical characterization of two crotamine isoforms isolated by a single step RP-HPLC fromCrotalus durissus terrificus(South American rattlesnake) venom and their action on insulin secretion by pancreatic islets. Biochim. Biophys. Acta 1474, 56–60.
Toyama, M.H., de Oliveira, D.G., Beriam, L.O., Novello, J.C., Rodrigues-Simioni, L., Marangoni, S., 2003. Structural, enzymatic and biological properties of new PLA2 isoform from Crotalus durissus terrificus venom. Toxicon 41 (8), 1033–1038.
Toyama, M.H., Toyama, D.O., Joazeiro, P.P., Carneiro, E.M., Beriam, L.O.S., Marangoni, L.S., Boschero, A.C., 2005. Biological and structural char-acterization of a new PLA(2) from theCrotalus durissus collilineatus
venom. Protein J. 24 (2), 103–112.
Tu, A.T., 1987. Biotoxicology of sea snake venoms. Ann. Emerg. Med. 16, 1023–1028.
Valentin, E., Lambeau, G., 2000a. Increasing molecular diversity of secreted phospholipases A2and their receptors and binding proteins. Biochim. Biophys. Acta 1488, 59–70.
Valentin, E., Lambeau, G., 2000b. What can venom phospholipases A(2) tell us about the functional diversity of mammalian secreted phos-pholipases A(2). Biochimie 82 (9), 815–831.
Valentin, E., Ghomashchi, F., Gelb, M.H., Lazdunski, M., Lambeau, G., 2000. Novel human secreted phospholipase A(2) with homology to the group III bee venom enzyme. J. Biol. Chem. 17, 275 (11), 7492–7496.
Wen-Hwa, L., Toyama, M.H., Soares, A.M., Giglio, J.R., Marangoni, S., Polikarpov, I., 1999. Crystallization and preliminary X-ray diffraction studies of piratoxin III, a D-49 phospholipase A(2) from the venom of
Bothrops pirajai. Acta Crystallogr. 55, 1229–1230.
Walser, M., Davidson, D.G., Orloff, J., 1955. The renal clearance of alkali-stable inulin. J. Clin. Invest. 34, 1520–1523.
Yamamoto, C., Tsuru, D., Oda Ueda, N., Ohno, M., Hattori, S., Kim, S.T., 2002. Flavoxobin, a serine protease from Trimeresurus flavoviridis
