(white-tailed-jararaca) snake venom

Biochimie 89 (2007) 319e328 www.elsevier.com/locate/biochi

Purification and partial characterization of two phospholipases A2 from Bothrops leucurus (white-tailed-jararaca) snake venom D.A. Higuchi a, C.M.V. Barbosa a, C. Bincoletto a, J.R. Chagas a, A. Magalhaes c, M. Richardson c, E.F. Sanchez c, J.B. Pesquero b, R.C. Araujo a, J.L. Pesquero a,d,* a

University of Mogi das Cruzes, Av Dr Candido Xavier de Almeida Souza 200, Centro Cı´vico, CEP 08780-911 e Mogi das Cruzes, S~ao Paulo, Brazil b Biophysics Department, UNIFESP, SP, Brazil c Research and Development Center, Ezequiel Dias Foundation, MG, Brazil d Biophysics Department, UFMG, MG, Brazil Received 25 April 2006; accepted 13 October 2006 Available online 10 November 2006

Abstract Two proteins with phospholipase A2 (PLA2) activity were purified to homogeneity from Bothrops leucurus (white-tailed-jararaca) snake venom through three chromatographic steps: Conventional gel filtration on Sephacryl S-200, ion-exchange on Q-Sepharose and reverse phase on Vydac C4 HPLC column. The molecular mass for both enzymes was estimated to be approximately 14 kDa by SDS-PAGE. The N-terminal sequences (48 residues) show that one enzyme presents lysine at position 48 and the other an aspartic acid in this position, and therefore they were designated blK-PLA2 and blD-PLA2 respectively. blK-PLA2 presented negligible levels of PLA2 activity as compared to that of blD-PLA2. The PLA2 activity of both enzymes is Ca2þ-dependent. blD-PLA2 did not have any effect upon platelet aggregation induced by arachidonic acid, ADP or collagen, but strongly inhibits coagulation and is able to stimulate Ehrlich tumor growth but not angiogenesis. Ó 2006 Elsevier Masson SAS. All rights reserved. Keywords: Bothrops leucurus; PLA2; Platelet aggregation; Blood coagulation; Angiogenesis; Snake venom

1. Introduction Bothrops snakes are the commonest cause of envenomation in Latin America countries, being responsible for approximately 90% of the reported cases, followed by Crotalus and Lachesis. Snake venoms are composed by a complex mixture of active substances, mainly peptides and proteins, able to interfere with the course of several biological processes

Abbreviations: AA, arachidonic acid; ADP, adenosine 50 -diphosphate; ELISA, enzyme-linked immunosorbent assay; FPLC, fast protein liquid chromatography; FUNED, Fundacao Ezequiel Dias; Leuc-a, leucurolysin-a; PLA2, phospholipase A2; blK-PLA2 and blD-PLA2, phospholipases A2 K48 and D48 from B. leucurus venom; PRP, platelet rich plasma; PPP, platelet poor plasma; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; TLE-Bl, thrombin-like enzyme from B. leucurus. * Corresponding author. Tel./fax: þ55 11 4798 7087. E-mail address: [email protected] (J.L. Pesquero). 0300-9084/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2006.10.010

including thrombosis by affecting platelet aggregation and blood coagulation [1,2]. The main pathological effects of Bothrops venoms are characterized by hemorrhage and bleeding disorders that can be the result of clotting factors and platelet consumption [3e5]. From the medical point of view B. leucurus (white-tailed-jararaca) constitutes an important poisonous snake that inhabits the north-eastern Brazil, where it is responsible for essentially all of the envenomations reported [6]. Due to the action of various enzymes, especially phospholipases A2 (PLsA2) and metalloproteinases, the Bothrops venom produces extensive tissue damage as well as activating or inactivating effects on nearly all the phases of human hemostasis. PLsA2 catalyze the hydrolysis of the sn-2 ester bond of 1,2diacyl-3-phosphoglycerides to produce lysophosphatidylcholine and free fatty acids, including arachidonic acid [7,8]. PLsA2 are a large family of enzymes and are found in diverse mammal tissues and also in the venoms of scorpions, bees and snakes [9]. In the mammalian cells some phospholipases are

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D.A. Higuchi et al. / Biochimie 89 (2007) 319e328

involved in the processes of remodelling of membranes, digestion, cellular signalling and blood coagulation [10,11]. PLsA2 from snake venoms can be neurotoxic [12], myotoxic [13], cardiotoxic [14] and can inhibit platelet aggregation and blood coagulation [15]. They are classified in cytosolic (cPLsA2) or secretory (sPLsA2) based on their cellular localisation. These forms also can be subdivided into dependent or independent of calcium. Several authors verified that cPLsA2 found in the cytosol of mammal cells need sub-micromolar concentrations of Ca2þ to be catalytically active, while others have indicated that an increase in intracellular calcium availability is a dispensable signal for cPLA2 activation [16,17]. Four cPLsA2 were described in mammals namely cPLsA2a, cPLsA2b, cPLsA2g and cPLsA2d with Mrs in the range 61e110 kDa [18e21]. cPLsA2a the most well characterized preferentially hydrolyzes phospholipids containing arachidonic acid [22] and has been implicated in many physiological and pathological processes [10]. The Mrs of sPLsA2 are in the range of 14e19 kDa [11]. They are highly expressed in mammals and venoms and require concentrations of Ca2þ in the order of micro- to millimolar for their catalytic activity [10,11]. The sequence DXCCXXHD involving the His48-Asp49 catalytic dyad and the Ca2þ-binding loop (XCGXGG) between residues 27e32 (numbering system from ref. [23]) are highly conserved in sPLsA2. Ten catalytically active sPLsA2 (IB, IIA, IIC, IID, IIE, IIF, III, V, X, and XIIA) have been identified in mammals. Calcium-independent phospholipases (iPLsA2), are also high Mr proteins (w85 kDa) and are found in the cytosol. The hydrolysis caused by iPLsA2 generally results in the release of lysophospholipids [24]. The hydrolysis of the glycerophospholipids by PLsA2 has great importance in the release of lipid mediators [22,25], however, high phospholipase activity can lead to pathological implications. More recently the phospholipase activity has been associated with angiogenesis, apoptosis and cancer [26e30]. Arachidonate is metabolized into prostaglandins and to leukotrienes, potent mediators of immune suppression, cellular proliferation, tumor motility as well as invasion and angiogenesis [26,31e33] regulating tumor vascularization and metastasis in animal models. In this communication we report the isolation and partial characterisation of two enzymes with PLA2 activity from the venom of B. leucurus (blPLA2). Based on their N-terminal sequences, the enzymes were termed blD-PLA2 and blK-PLA2. Results regarding the effect of blD-PLA2 upon platelet aggregation, blood coagulation and cytotoxicity are also presented. Since there is growing evidence for the participation of PLsA2 in the development of tumors we also determined the effect of the blD-PLA2 upon growth of Ehrlich ascites tumor implanted in mice. The effect of the enzyme upon angiogenesis was also investigated by using the sponge implant model.

from Grace Vydac (Columbia, MD, USA). 1-O-hexadecanoyl-2-O-(10-(1-pyrenyl)decanoyl)-sn-glyceryl-3-phosphorylcholine from Cayman Chemical (Michigan, USA). All other chemicals were of analytical grade. 2.2. Venom and antibodies Commercial horse bothropic antivenom was obtained from the Immunological Production Unit of Ezequiel Dias Foundation (FUNED, MG, Brazil). The antigenic pool is composed of venoms of B. jararaca (50%), B. alternatus (12.5%), B. jararacussu (12.5%), B. neuwiedi (12.5%) and B. moojeni (12.5%). Anti-leuc-a and anti-thrombin-like enzyme (TLE-Bl) immunoglobulins were raised by immunization of two rabbits (one for each enzyme) with the purified enzymes leucurolysin-a or TLE-Bl from B. leucurus respectively, as described [34]. Venom from the snake B. leucurus was collected by manual milking of adult B. leucurus of both sexes, lyophilized and stored at 4e8 C until used. 2.3. Animals Swiss male mice, 25e30 g body weight were used for biological assay of blD-PLA2. The experiments reported here were done within the guidelines established by the Brazilian College for Animal Experimentation (COBEA) and overseen by local animal Ethics Committee. 2.4. Purification of PLsA2 Crude venom (1.9 g) was dissolved in 15 mL of 50 mM ammonium acetate buffer pH 7.3 containing 0.3 M NaCl and centrifuged at 6000 g to remove insoluble material. The solution was applied to two columns in series (2.5 100 cm, each) packed with Sephacryl S-200, equilibrated and eluted with the same buffer at a flow rate of 7 mL/h and 7 mL fractions were collected. The fractions showing PLA2 activity were pooled, dialyzed against distilled water, lyophilized and further resuspended in 20 mM TriseHCl buffer pH 8.4 and loaded on a column of Q-Sepharose (0.5 2 cm) on a FPLC system (GE Healthcare, Uppsala, Sweden). The column was equilibrated and eluted with 20 mM TriseHCl buffer pH 8.4 at a flow rate of 1 mL/min. Adsorbed proteins were eluted with a stepwise gradient of NaCl concentration (200, 300, 500 and 1000 mM in 50 mL each step) in the 20 mM TriseHCl buffer pH 8.4. The active fractions were pooled and 1 mg of protein was loaded on a Vydac C4 column (250 4.6 mm). The column was eluted with solvents A (0.1% trifluoroacetic acid in water) and B (acetonitrile containing 0.1% trifluoroacetic acid) at a flow rate of 1 mL/min as follows: 5 mL solvent A and then a gradient from 0 to 100% B in 60 mL.

2. Materials and methods 2.5. N-terminal sequence determination 2.1. Materials Q-Sepharose and Sephacryl S-200 were purchased from Amersham-GE Healthcare (Uppsala, Sweden). Vydac C4

The N-terminal amino acid sequences of the native proteins blK-PLA2 and blD-PLA2 (10 nmol each) were determined by automated Edman degradation using a Shimadzu PPSQ-21A


(Tokyo, Japan) protein sequencer according to the manufacturer’s instructions. 2.6. PLA2 activity assay PLA2 activity was determined fluorometrically using as substrate phospholipids labeled at the sn-2-acyl position with 10-pyrenyldecanoic acid [35]. Fluorescence intensity was monitored in a thermostatic Hitachi F-2000 spectrofluorometer (Midland, ON, Canada). Wavelengths were set at 345 nm for excitation and 398 nm for emission. The reaction was carried out in 50 mM TriseHCl pH 7.5 buffer containing 100 mM NaCl, 0.1% fatty acid-free bovine serum albumin, 6 mM CaCl2 and 60 nM 1-O-hexadecanoyl-2-O-(10-(1-pyrenyl)decanoyl)-sn-glyceryl-3-phosphorylcholine as substrate. The fluorescence of the reaction medium was recorded during 5 min and then the reaction was initiated by addition of the enzyme. The kinetic parameters were determined by measuring the initial rate of hydrolysis. The fluorescence signal of the product 10-pyrenyldecanoic acid (46,830 fluorescence units/nmol) was obtained by determination of the maximal fluorescence at the end of reaction. Specific activity was expressed as activity units (AU)/mg of protein. One AU was defined as 1 nmol/min of liberated fluorescent product. Protein concentration of samples was estimated by the Coomassie Blue method [36]. 2.7. Cytotoxity Cellular viability was determined by an adaptation of the method described by Boschert et al. [37]. Human leukaemia cells (K-562) obtained from the Rio de Janeiro Cell Bank/ RJ, Brazil, were cultivated in RPMI 1640 medium (Sigma Chemical Co, MO, USA) supplemented with 10% fetal calf serum containing 100 IU/mL penicillin (Sigma Chemical Co, MO, USA) and 100 mg/mL streptomycin (Sigma Chemical Co, MO, USA) in a humidified atmosphere at 37 C in 5% CO2. After resuspension, 3 104 cells per well (Corning, Acton, MA, USA) were incubated for 24 h at 37 C and 5% CO2 atmosphere with blD-PLA2 at concentrations of 2, 10, 25 and 100 mg/mL. After 24 h, K562 cells were stained with 0.4% Trypan Blue (Gibco, CA, USA) vital stain that allows evaluation of the structural integrity of the cellular membrane. The number of viable cells was counted with a hemocytometer under 400 magnification. 2.8. Platelet aggregation The blD-PLA2 was tested for inhibitory effect on platelet aggregation using fresh human platelet rich plasma (PRP) as described by Sanchez et al. [38]. A PACKS-4 platelet aggregation chromogenic kinetic system (Helena Laboratories, Beautmont, TX, USA) was used to monitor platelet aggregation with 0.5 mL PRP. Inhibition of adenosine 50 -diphosphate (ADP), arachidonic acid (AA) and collagen-induced platelet aggregation was conducted at 37 C by adding blD-PLA2 (100e400 nM, final concentration) 3 min before the addition of the agonist (final concentrations: ADP, 10 mM; AA, 30 mg/mL; and collagen, 5 mg/mL).

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2.9. Enzyme-linked immunosorbent assay (ELISA) ELISA assay was used with the purpose of evaluating the antigenic cross-reactivity of blD-PLA2 with horse bothropic antivenom, rabbit anti-leuc-a and anti-TLE-Bl immunoglobulins, which were prepared from rabbit plasma after immunization with the purified enzymes leuc-a and TLE-Bl according to ref. [34]. Microtitration plates (Micro Test IIITm flexible assay plate, Becton and Dickinson and Co, Oxnard, USA) were coated with 0.5 mg/well of B. leucurus crude venom or purified blD-PLA2 in 50 mM sodium bicarbonate pH 9.6 (100 mL, standard volume) and kept at 4 C overnight. After washing with 0.05% Tween/saline, the wells were blocked with 2% casein in PBS (phosphate buffered saline, pH 7.4) for 1 h at room temperature. Sera from pre-immune and immune (horse and rabbits) were added and incubated for 1 h at 37 C at 1:200e1:6400 dilutions. The plates were then washed and incubated with anti-horse and anti-rabbit IgGs conjugated with alkaline phosphatase (Sigma-Aldrich, St Louis, MO, USA), diluted 1:4000 for 1 h at 37 C. The wells were washed with 0.05% Tween/saline and 100 mL of o-phenylenediamine (0.33 mg/mL in citrate buffer, pH 5.2 in the presence of 0.04% hydrogen peroxide) was added and the color reaction developed for 1 h at 37 C. Other details were described by Camey et al. [39]. All ELISA tests were performed in triplicate. 2.10. Clotting activity The blood clotting activity of samples was analyzed by the recalcification time of the citrated human platelet poor plasma induced by the sample as described by Valenzuela et al. [40]. In 1.5 mL tubes were added 50 mL of platelet-poor human citrated plasma and 50 mL of 10 mM Hepes buffer pH 7.4 containing 150 mM NaCl. After 2 min incubation at 37 C, 50 mL of 25 mM CaCl2 were added followed by 50, 100 or 500 ng of blD-PLA2. The absorbance at 650 nm was determined at 30 s intervals. An increase of 0.03 in the absorbance was indicative of the beginning of coagulation. 2.11. Angiogenesis Angiogenesis was determined indirectly by the sponge implant model [41]. The hemoglobin levels in a sponge were used as a measure of the vascularization level. Pieces of polyurethane sponge (Vitafoam Ltd, Lancashire, UK) of 8 mm diameter and 5 mm thickness were used as the matrix for fibrovascular tissue growth. The sponges had been kept in 70% ethanol for 24 h before implantation. Only sponge pieces that had weights around 20 mg (20 1.0) were used. At the moment of the implantation pieces were washed in distilled water and boiled for 15 min. The animals (25e30 g mice) were anesthetized with 2,2,2-tribromoethanol (Sigma-Aldrich, St Louis, MO, USA) 1 mg kg1; i.p. After surgery, the animals were kept under artificial heating until complete reestablishment of their vital functions. The animals with implant were randomly divided in four groups (n ¼ 6). Treatment was initiated 24 h after the implantation with daily injections of 100 mL

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of sterile saline containing 1, 5 or 10 mg of purified blD-PLA2 per animal per day. The treatment lasted nine days with subcutaneous injections. The control group received daily injections of 100 mL saline (0.9% NaCl). At 10 days treatment, the implant bearing mice were anesthetized by ether inhalation and killed by cervical dislocation; the sponge was removed, dissected free from adherent tissue, weighed and homogenized for hemoglobin quantitation. Alternatively the sponges were analyzed for histological assessment. Central slices from the sponge were cut perpendicular to the long axis and fixed in 10% neutral buffered formalin. After routine embedding in paraffin, 5-mm thick sections were selected and stained with hematoxylin-eosin and periodic acid Schiff stains. All the procedure was repeated three times. 2.12. Ehrlich tumor The Ehrlich ascitic tumor, derived from a spontaneous murine mammary adenocarcinoma, was maintained in the ascitic form by passages in syngeneic BALB/c mice by transplantation of 5 105 tumor cells [42]. Ten days after intraperitoneal inoculation of cells, the ascitic tumor was removed and centrifuged for 3 min at 3000 g. After washing the cells with saline, the cellular viability was determined using Trypan Blue (Gibco, CA, USA). Samples that presented cellular viability lower than 90% were discarded. Cells (2.5 106) were inoculated subcutaneously in mice to generate solid tumor. The animals with tumor were randomly divided in four groups (n ¼ 6). Treatment was initiated in the eighth day after inoculation with daily injections of 100 mL of sterile saline containing 1, 5 or 10 mg of purified blD-PLA2 per animal per day. The treatment lasted nine days with subcutaneous injections. On the tenth day after the beginning of the treatment, the animals were sacrificed; the tumor was removed and weighed. The control group received daily injections of 100 mL saline. All the procedure was repeated three times. 2.13. Hemoglobin levels The method used was based on the oxidation of iron by potassium ferricyanide under weakly alkaline conditions, to form metahemoglobin, which is converted to cyanmetahemoglobin [43]. The sponge was homogenized in 5 mL of Drabkin reagent (Labtest, SP, Brazil). After homogenization, the material was centrifuged at 10 000 g for 30 min, the supernatant was filtered through a 0.22 mm filter (Millipore, Billerica, USA) and the levels of the cyanmetahemoglobin in the supernatant determined spectrophotometrically at 540 nm. The amount of hemoglobin was determined by comparison with a standard curve assayed in parallel and the results expressed as mg Hb mg1 of wet tissue. 2.14. Statistics A two-tailed, unpaired Student’s t-test was performed to determine the statistical significance by the probability of difference between the means. P < 0.05 was considered statistically significant.

3. Results 3.1. PLsA2 purification PLsA2 from B. leucurus venom were purified by a threestep procedure involving gel filtration on Sephacryl S-200, ion exchange chromatography on Q-Sepharose and reverse phase HPLC on Vydac C4 column (Fig. 1AeC). The fractionation of B. leucurus venom by gel filtration resulted in seven main protein peaks (P1 to P7, Fig. 1A). PLA2 activity was concentrated in peak 6. Tubes corresponding to the higher activity fractions were pooled (P6). SDS-PAGE of P6 (Fig. 1D, left) showed that one band with Mr estimated around 14 kDa was the major component, accounting for more than 80% of protein content of this fraction. The volume of P6 was dialyzed against 20 mM TriseHCl buffer pH 8.4 and loaded on a Q-Sepharose column which was eluted in a stepwise manner with different concentrations of NaCl. The profile of protein elution in this chromatography is shown in Fig. 1B. As can be seen, the majority of protein content (w75%) eluted in the first peak (P1q), which corresponded to un-adsorbed proteins. By SDS-PAGE (Fig. 1D, right), the protein eluted in the first peak was shown to be homogeneous with a Mr estimated as 14 kDa. When a sample of P1q was loaded on a Vydac C4 HPLC column, two main peaks of protein, P3 and P4, eluted at 32 and 36.5% acetonitrile respectively (Fig. 1C). Both peaks showed PLA2 activity. The specific phospholipase A2 activity was 25.0 4.7 and 503 26.7 AU/mg for P3 and P4, respectively. No PLA2 activity was observed in the absence of CaCl2 for both enzymes. Table 1 shows the parameters for all the purification steps of these enzymes. For determination of specific activity the experiments were repeated at least three times. 3.2. N-terminal sequence The first forty-eight amino acids at the N-terminal sequence of each protein, eluted at 32 and 36.5% acetonitrile on the Vydac C4 HPLC column were determined. As these proteins contained Lys (P3) and Asp (P4) at position 48 (position 49 of catalytic dyad according numbering of Renetseder et al. [23]), they were named blK-PLA2 and blD-PLA2 respectively. Alignments of the N-terminal sequences of blK-PLA2 and blDPLA2 with those of other PLsA2 from snake venoms are shown in Fig. 2. The sequence of blK-PLA2 presented 93.8% homology with myotoxin II and 89.6% homology with bothropstoxin I, two K-PLsA2 isolated from the venoms of B. moojeni [44] and B. jararacussu [45] respectively. The sequence of blD-PLA2 presented 100% homology with a putative PLA2 of B. jararacussu whose coding sequence was deduced from a cDNA library [46] and 89.6% homology with a PLA2 from the venom of B. pirajai which presents moderate phospholipase and anticoagulant activities and high myotoxic activity [47]. 3.3. Cytotoxicity The cytotoxicity analysis was carried out to verify whether blD-PLA2 is able to produce lethal effect in the human


B

6

4

Optical density 280 nm

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10 2 5

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Elution volume (mL)

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Fraction

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NaCl (M)


A

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kDa

M

M P1q

P6 kDa

50 1.0

50 30 20

30

0.5 2 1

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Time (min)

15

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15 10

Fig. 1. blPLsA2 purification steps. A, gel filtration chromatography of crude venom of B. leucurus on Sephacryl S-200 (2.6 100 cm). The horizontal line refers to the pooled active fractions of peak 6. B, ionic exchange chromatography on Q-Sepharose (0.5 2 cm). C, reverse phase HPLC on Vydac C4 (250 4.6 mm). D, polyacrylamide gel electrophoresis (12%) under reducing conditions of active samples from gel filtration (P6) and ionic exchange (P1q) chromatographies. The gel was silver stained. M, molecular mass marker (BenchMarker Ladder, Invitrogen).

leukaemia cell line K-562. Cells were submitted to blD-PLA2 at concentrations of 2, 10, 25 and 100 mg/mL. The results are shown in Fig. 3. At the concentration of 25 mg/mL, during a 24-h period, blD-PLA2 was able to reduce cellular viability by almost 50%. When incubated with blD-PLA2 at the concentration of 100 mg/mL, the cellular viability of K-562 cells was reduced to 30%. In addition, under the microscope dead cells presented morphological alterations, typical of cellular membrane integrity disruption, suggesting a necrotizing effect (data not shown). 3.4. Immunoreactivity The results presented in the Fig. 4 show that the crude venom of B. leucurus was partially recognized by anti-leucTable 1 Parameters of B. leucurus phospholipases A2 purification procedure Step

Total protein (mg)

Specific activitya

Total activityb

Recovery (%)

Crude venom Sephacryl S200 Q-Sepharose

1900 106.4 80.3

39.7 5.1 120.0 13.2 453.3 28.0

75,430 12,768 36,376

100 16.9 48.2

HPLC/C4 P3 (blK-PLA2) P4 (blD-PLA2)

42.0 34.3

25.0 4.7 503.0 26.7

1050 17,253

1.4 22.9

a and anti-TLE-B1 antibodies. However, no reactivity was observed with the polyvalent anti-bothropic antiserum. Under our experimental conditions, blD-PLA2 showed moderate cross-reaction with the anti-TLE-Bl antibody. 3.5. Platelet aggregation The platelet aggregation inhibitory activity of blD-PLA2 on human PRP was assayed with 10 mM ADP, 30 mg/mL AA or 5 mg/mL collagen as stimulators. The results presented in Fig. 5 show that blD-PLA2 under these conditions did not interfere with platelet aggregation. 3.6. Clotting activity As some PLsA2 of snake venoms exhibit anticoagulant effect, we tested whether blD-PLA2 exhibit this type of activity. Different amounts of blD-PLA2 were incubated with citrated human platelet-poor plasma in the presence of CaCl2, the aggregation inductive agent, and then the coagulation time was determined in each situation. The results are presented in the Fig. 6 and show that blD-PLA2 at a dose of 50 ng was capable of delaying the onset of coagulation, when compared with the control (9.0 0.5 vs 12.0 0.7 min), while at the dose of 100 ng blD-PLA2 completely inhibited the plasma coagulation. 3.7. Tumorigenesis

a

Expressed as AU per milligram of protein. One AU is defined as one nanomol of fluorescent product (46,830 fluorescence units) generated per minute. b Total activity ¼ specific activity x total protein. Expressed as AU.

The effect of blD-PLA2 upon tumor growth was examined by injections of 1, 5 and 10 mg of the enzyme subcutaneously


324

A blK49-PLA2 bmK49-PLA2 bjK49-PLA2 baK49-PLA2 batK49-PLA2 caK49-PLA2 tmK49-PLA2 tpK49-PLA2

1 10 SLFELGKMIL SLFELGKMIL SLFELGKMIL SLFELGKMIL SLVELGKMIL SLVELGKMIL SLIELGKMIF SVIQLGKMIL

20 QETGKNSVKS QETGKNPAKS QETGKNPAKS QETGKNPAKS QETGKNPLTS QETGKNPITS QETGKNPVKN QETGKNPVKY

30 YGVYGCNCGV YGVYGCNCGV YGAYGCNCGV YGAYGCNCGV YGAYGCNCGV YGIYGCNCGV YGLYLCNCGV YGAYGCNCGP

40 GGRGKPKDAT GGRGKPKDAT LGRGKPKDAT LGRGKPKDAT GGRGKPKDAT GSRHKPKDGT GNRGKPVDAT LGRRKPLDAT

DKCCYVHK DRCCYVHK DRCCYVHK DRCCYVHK DRCCYVHK DRCCFVHK DRCCFVHK DRCCYMHK

ref 44 45 56 * 57 58 59

1 10 DLWQFGQMIL DLWQFGQMIL DLWQFGKMIL SLIEFAKMIL NLWQFREMIK NLFQFAKMIN SLVQFETLIM SLVQFETLIM

20 KETGKLPFPY KETGKLPFPY KETGKLPFPY EETKRLPFPY EATGKEPLTT GKLGAFSVWN KIAGRSGVWY KIAKRSGVWF

30 YTTYGCYCGW YTTYGCYCGW YVTYGCYCGV YTTYGCYCGW YLFYACYCGW YISYGCYCGW YGSYGCYCGS YGSYGCFCGS

40 GGQGQPKDAT GGQGQPKDAT GGRGGPKDAT GGQGQPKDAT GGRGEPKDAT GGQGTPKDAT GGQGRPQDAS GGQGRPQDAS

DRCCFVHD DRCCFVHD DRCCFVHD DRCCFVHD DRCCFVHD DRCCFVHD DRCCFVHD DRCCFVHD

46 47 60 * * 61 *

B blD49-PLA2 bjD49-PLA2 bpiD49-PLA2 baD49-PLA2 pmD49-PLA2 vaD49-PLA2 beD49-PLA2 bpD49-PLA2

Fig. 2. Alignments of the amino terminal sequences of blK-PLA2 (A) and blD-PLA2 (B), two subtypes of PLsA2 from B. leucurus (bl, this work) with those of other PLsA2 from snake venoms. ba, B. asper; bat, B. atrox; be, B. erythromelas; bj, B. jararacussu; bm, B. moojeni; bp, B. pictus; bpi, B. pirajai; ca, Crotalus atrox; pm, Protobothrops mucrosquamatus; tm, Trimeresurus mucrosquamatus; tp, Trimeresurus puniceus; va, Vipera aspis aspis. Amino acids as single letter codes. Different amino acids are underlined. Numbering refers to residues of B. leucurus blK-PLA2 and blD-PLA2. *Accession number in http://www.ncbi.nlm.nih.gov (batK49-PLA, AY431026.1; bpD49-PLA, AF288754.1; pmD49-PLA, X77646.2; vaD49-PLA, AJ580285).

4. Discussion In South America, Bothrops snakes have been well studied due to their predominance and medical importance in countries such as Brazil, Colombia, Equator, Venezuela, Bolivia and Peru. The venom of the Bothrops snakes is a complex

A Optical density 492 nm

into mice bearing Ehrlich tumors. The results are presented in the Fig. 7A. The tumor mass of the group that received 1 mg was not different from that of the control group (0.20 0.03 vs 0.22 0.02 g). However, in the groups injected with 5 or 10 mg the tumor mass was significantly larger, 0.35 0.04 and 0.40 0.05 g (P < 0.05), respectively. To verify the possibility of increased angiogenesis, the hemoglobin levels were determined in the homogenate of sponges after 10 days of implantation on the back of mice (Fig. 7B). The hemoglobin levels in the sponge homogenates for the treated group were not different to those from animals receiving only saline. The histological analysis showed that the vascularization level in sponge slices from control and treated groups was very low and not different between groups (data not shown).

1.6 1.2 0.8 0.4 0

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80 0.8 0.6 0.4 0.2 0 C

2

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25

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Concentration ( g/mL) Fig. 3. Cytotoxic effect of blD-PLA2 upon K-562 cells. Cytotoxity was evaluated by incubating 3 104 cells without (C) or with blD-PLA2 at concentrations of 2, 10, 25 and 100 mg/mL. Cellular viability was determined by Trypan Blue. *P < 0.05 versus control.

0.2

0.4

0.8

1.6

3.2

6.4

Antibody dilution x 10-3 Fig. 4. Reactivity of B. leucurus venom and blD-PLA2 assessed by indirect ELISA. 0.5 mg B. leucurus crude venom (A) and 0.5 mg purified blD-PLA2 (B) against antibothropic (-), anti-leuc-a (B) and anti-TLE-B1 (:) sera at different dilutions in ELISA. All data points are the means of triplicate.

Platelet aggregation ( ) Platelet aggregation ( )

*

60 40 20

* 0.30

0.15

0.00

S

1

5

10

Optical density at 540 nm

80

0.3

0.2

0.1

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S

P

Fig. 7. A, Effect of saline (S) and blD-PLA2 (1, 5 or 10 mg) on the mass of Ehrlich tumor implanted on mice. B, Hemoglobin levels on the sponge implanted in mice treated with 5 mg blD-PLA2 (P). Hemoglobin levels determined as described in material and methods. *P < 0.05 versus control (S).

100 80 60 40 20

C 100 80 60 40 20 0

100

200

300

400

500

Time (s) Fig. 5. Effect of blD-PLA2 on platelet aggregation. Platelets-rich plasma were pre-incubated at 37 C for 3 min without (-) and in the presence of blD-PLA2 at concentrations of 100 (:), 200 (>) and 400 nM () before the addition of agonists, 10 mM ADP (A), 30 mg/mL arachidonic acid (B) or 5 mg/mL collagen (C).

0.8

Optical density at 650 nm

B

0.45

100

B

0.6

0.4

0.2

0

325

A

A

120

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Platelet aggregation ( )


1

5

9

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Time (min) Fig. 6. Effect of blD-PLA2 on the recalcification time. Citrated human plateletpoor plasma was incubated with 25 mM CaCl2 in the absence (,) and presence of 50 (C), 100 (B) and 500 ng (:) of blD-PLA2.

mixture of active proteins (about 90% of the dry mass) with different toxic properties. Bothropic envenomation can clinically be classified in accordance with the presence of local or systemic manifestations. Among the systemic manifestations are coagulation disorders, hemorrhage, alteration in platelet function and depletion of fibrinogen [48]. The effects can be caused by myotoxins, disintegrins, vasoactive peptides and enzymes such as metalloproteinases, phospholipases, hyaluronidases and serineproteinases. Little is known about the venom components from B. leucurus. Recently a fibrinolytic metalloproteinase with no hemorrhagic effect and a fibrinogen clotting enzyme (thrombin-like) have been isolated from this venom and partially characterized in our laboratory [49,50]. In this work we present results that show the presence of two PLsA2 in the B. leucurus venom. These enzymes have been purified and some biological and physicochemical properties determined. PLsA2 from snake venoms exhibit different isoforms and distinct isoelectric points and therefore are classified as basic, neutral or acidic. For the isolation of B. leucurus PLsA2, a three step procedure involving gel filtration, ionic exchange and reverse phase chromatographies was used. From the 14 kDa Mr and elution from the ionic exchange column, we can conclude that the PLsA2 from B. leucurus venom are basic, since they eluted from the column with a low ionic strength buffer. Our results show that the fraction showing PLA2 activity represents more than 30% of the protein content of B. leucurus crude venom. Therefore, after the first chromatographic step, of gel filtration, the PLA2 fraction showed a relatively high degree of purity. The active fraction of this chromatography had more than 80% of the protein content as PLA2. This was confirmed during the ionic exchange chromatography, in which the main protein peak which failed to bind to the ionic exchanger contained the majority of the total phospholipase activity loaded on this column and accounted for 75% of the totality of the protein content eluted from the column. After reverse phase chromatography we verified that this fraction contained two main components, which were both PLsA2. One of these contained the aminoacid Asp at position 48 in its amino acid sequence and the other had Lys in the same

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position. For this reason they were named as blD-PLA2 and blK-PLA2 respectively. Considering the 48 sequenced residues (this work), blKPLA2 showed high level homology (75.0e93.8%) with many Lys-PLsA2, from different snakes species. blD-PLA2 exhibited 100% homology with a PLA2 from B. jararacussu venom whose sequence was deduced from a cDNA library [46] and 89.6% homology with a PLA2 from B. pirajai venom that shows moderate phospholipase and anticoagulant activities and high myotoxic activity [47]. The highly conserved sequences XCGXGG and DXCCXXHD responsible for the Ca2þ-binding loop and the catalytic site of Asp49 PLsA2 (numbering system from ref. [23]) respectively are present in the sequence of both enzymes with the respective substitution (D48K) in the blK-PLA2. Although the data in the literature suggest the necessity of an Asp residue at position 49 for catalytic activity, blK-PLA2 showed negligible calcium-dependent phospholipase activity, which was approximately 20times lower than that presented by blD-PLA2. The enzymes seemed to be completely separated by reverse phase chromatography, and when the K-form of the protein was sequenced the yields of the amino acid derivatives obtained (>3e5 nmol per cycle, during the first ten cycles) were of sufficient strength to have enabled the detection of a 50-fold lower concentration of any contaminating D-form. In this study some experiments were carried out with the objective to better characterizing the properties of blDPLA2. The indirect ELISA assays showed moderate cross-reaction between the purified blD-PLA2 and antibodies raised against the TLE-Bl, suggesting that both blD-PLA2 and the TLE-Bl isolated from the same B. leucurus venom may share similar epitopes. It is however, interesting to consider that the reactivity seen with the anti-TLE-Bl antibodies may be due to the presence of low levels of PLsA2 in the antigen sample used to prepare the antisera as well as the presence of high affinity epitopes on PLsA2 molecules. By contrast, no cross-reaction was observed between blD-PLA2 and bothropic antivenom and anti-leuc-a antibodies. It is known that bothropic antivenom gives low titers against B. leucurus venom, and the results obtained here are consistent with the earlier study [39]. On the other hand, leuc-a is a 23 kDa metalloproteinase present in the venom of B. leucurus and is structurally different from PLsA2 [49]. These data and that of Camey and collaborators [39] indicate the small similarity in the immunogenic properties of the B. leucurus venom components, when compared with other Bothrops venoms species such as B. jararaca, B. moojeni, B. jararacussu, B. alternatus and B. neuwiedi. Very clearly, there is an urgent need to develop B. leucurus specific antivenom which might prove much more efficient than the polyvalent bothropic antivenom. A number of PLsA2 of snake venoms exhibit anticoagulant effects, being capable to inhibiting the activity of the prothrombin complex and or factor X [51]. At a concentration of 33 ng/mL, blD-PLA2 was able to delay the plasma coagulation time from 9 to 12 min when compared with the control, and at 66 ng/mL coagulation was not observed. We can compare this anticoagulant activity with that presented by Angulo

et al. [52], which had observed that 50 mg or even larger amounts of myotoxin II of B. nummifer were unable to inhibit the coagulation of the platelet poor plasma. Diaz et al. [53] isolated a basic PLA2 from B. asper venom that presented anticoagulant activity only at concentration of 40 mg/mL or higher. Therefore, blD-PLA2 was shown to be a powerful coagulation inhibitor. Other PLsA2 isolated from snake venoms are capable of inhibiting platelet aggregation. This effect is probably more due to multiple signal transduction pathways than the release of ADP from platelets or by a thrombin-like proaggregating effect [1]. blD-PLA2 at concentrations of 100, 200 and 400 nM, under induction with ADP, collagen or arachidonic acid did not interfere with platelet aggregation. However blD-PLA2 exhibited cytotoxic activity, verified by an in vitro assay. blD-PLA2, in addition to presenting anticoagulant activity can also be classified as cytotoxic, since it was able to diminish the K-562 cellular viability, in a dose-dependent manner, causing disruption of cellular membrane integrity when analyzed after staining with Trypan Blue and viewed under the microscope. Although these results suggest that dead cells were necrotizing, further studies are necessary to clarify the mechanisms involved in the cytotoxicity induced by blDPLA2. More recently some authors have shown that PLsA2, via arachidonic acid, exhibit angiogenic activity with consequent participation in the tumor growth. The oncogenic action of secretory PLsA2 have been reported [26,27]. In addition, the use of phospholipases inhibitors as drugs for anticancer therapy has been suggested [30,31,54]. However, there are some controversies in this respect, since Dong et al. [55] observed an important participation of a cPLA2 in the TNF-induced apoptosis in colon malignant cells. These authors observed that the suppression of arachidonic acid production by the inhibition of PLA2 favors the growth of colon cancer. Furthermore, in an elegant paper, Ilsley et al. [30] have shown that the deletion of the gene of a cPLA2 from mouse genome resulted in a transgenic model that had enhanced tumor sensitivity. These authors suggest that this altered sensitivity is due to a lower frequency of apoptotic cells caused by reduced ceramide levels, as a result of an alternative pathway involving the processing of arachidonic acid. In the model used by us, blD-PLA2 was able to induce tumor growth. We used the Ehrlich solid tumor implanted in the back of mice as a model. The treatment of mice with daily injection of 5-mg blD-PLA2 per animal was enough to increase by more than 50% the tumor mass after 10 days of treatment, as compared to the control. As the tumoral growth may be related to an angiogenic process, we also measured the levels of hemoglobin and the vascularization levels induced by blD-PLA2 in a sponge implanted in experimental animals. After treatment, the vascularization levels, as confirmed by histology and the hemoglobin levels in the sponges were not different from those of the control group. By this model, our data do not support the potential role for PLA2 in stimulating angiogenesis. From these results we may draw two conclusions: a) PLA2 is angiogenic in a tumor model but not for the sponge one. It could


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