Toxicon 53 (2009) 330–341
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Antigenic, microbicidal and antiparasitic properties of an L-amino acid oxidase isolated from Bothrops jararaca snake venom P. Ciscotto a, R.A. Machado de Avila a, E.A.F. Coelho a, J. Oliveira a, C.G. Diniz b, L.M. Farı´as b, M.A.R. de Carvalho b, W.S. Maria c, E.F. Sanchez c, A. Borges d, C. Cha´vez-Olo´rtegui a, * a
´gicas, Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627, Departamento de Bioquı´mica e Imunologia, Instituto de Cieˆncias Biolo 30161-970 Belo Horizonte, Minas Gerais, Brazil b ´gicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil Departamento de Microbiologia, Instituto de Cieˆncias Biolo c ˜o Ezequiel Dias, Rua Conde Pereira Carneiro 80, 30550-010 Belo Horizonte, MG, Brazil Fundaça d ´rio de Toxinas Animales, Centro de Biociencias y Medicina Molecular, Instituto de Estudios Avanzados, Apartado 17606, Caracas 1015-A, Venezuela Laborato
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 September 2008 Received in revised form 3 December 2008 Accepted 4 December 2008 Available online 11 December 2008
Venoms from the bee Apis mellifera, the caterpillar Lonomia achelous, the spiders Lycosa sp. and Phoneutria nigriventer, the scorpions Tityus bahiensis and Tityus serrulatus, and the snakes Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi, Crotalus durissus terrificus, and Lachesis muta were assayed (800 mg/mL) for activity against Staphylococcus aureus. Venoms from B. jararaca and B. jararacussu showed the highest S. aureus growth inhibition and also against other Gram-positive and Gram-negative bacteria. To characterize the microbicidal component(s) produced by B. jararaca, venom was fractionated through gel exclusion chromatography. The high molecular weight, anti-S. aureus P1 fraction was further resolved by anion exchange chromatography through Mono Q columns using a 0–0.5 M NaCl gradient. Bactericidal Mono Q fractions P5 and P6 showed significant LAAO activity using L-leucine as substrate. These fractions were pooled and subjected to Heparin affinity chromatography, which rendered a single LAAO activity peak. The anti-S. aureus activity was abolished by catalase, suggesting that the effect is dependent on H2O2 production. SDS-PAGE of isolated LAAO indicated the presence of three isoforms since deglycosylation with a recombinant N-glycanase rendered a single 38.2 kDa component. B. jararaca LAAO specific activity was 142.7 U/mg, based on the oxidation of L-leucine. The correlation between in vivo neutralization of lethal toxicity (ED50) and levels of horse therapeutic antibodies anti-LAAO measured by ELISA was investigated to predict the potency of Brazilian antibothropic antivenoms. Six horses were hyperimmunized with Bothrops venoms (50% from B. jararaca and 12.5% each from B. alternatus, B. jararacussu, B. neuwiedii and B. moojeni). To set up an indirect ELISA, B. jararaca LAAO and crude venom were used as antigens. Correlation coefficients (r) between ED50 and ELISA antibody titers against B. jararaca venom and LAAO were 0.846 (p < 0.001) and 0.747 (p < 0.001), respectively. The hemolytic and leishmanicidal (anti-Leishmania amazonensis) activity of LAAO was also determined. Ó 2008 Published by Elsevier Ltd.
Keywords: Bothrops jararaca L-Amino acid oxidase Microbicidal Leishmanicidal Neutralizing potency
1. Introduction
* Corresponding author. Tel.: þ55 3134992645; fax: þ55 3134992614. E-mail address:
[email protected] (C. Cha´vez-Olo´rtegui). 0041-0101/$ – see front matter Ó 2008 Published by Elsevier Ltd. doi:10.1016/j.toxicon.2008.12.004
L-Amino acid oxidases (LAAOs, EC 1.4.3.2) are flavoenzymes catalyzing the stereo-specifically oxidative deamination of a wide range of L-amino acids, which generate the corresponding a-keto acids, H2O2 and
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ammonia. LAAOs are amongst the most abundant proteins in ophidian, particularly hemorrhagic venoms (Du and Clemetson, 2002; Gue´rcio et al., 2006) and are capable of inducing apoptosis of various cell types, including vascular endothelial cells (VEC). Although the apoptosis mechanism is not yet clear, it involves the production of H2O2 which is achieved by oxidation of some VEC plasma membrane proteins (Suhr and Kim, 1996; Torii et al., 1997, 2000). Other activities from LAAOs include induction or inhibition of platelet aggregation (Li et al., 1994; Sakurai et al., 2001; Lu et al., 2002), anticoagulant activity (Sakurai et al., 2003), stimulation of edema formation (Wei et al., 2002; Sta´beli et al., 2004; Izidoro et al., 2006), hemorrhage (Sta´beli et al., 2004) and antibacterial, antiviral, and leishmanicidal functions (Lu et al., 2002; Wei et al., 2002; Zhang et al., 2003; Izidoro et al., 2006). Venoms from viperid snakes belonging to the Neotropical genus Bothrops are specially enriched in LAAOs (Pessatti et al., 1995; Tan and Ponndurai, 1991). Significant antitumoral (da Silva et al., 2002a,b), anti-Leishmania major, and anti- Trypanosoma cruzi (Gonçalves et al., 2002) activities have been found in venom from the Brazilian Bothrops jararaca, which could be related to its LAAO-mediated production of H2O2, as has been shown for Bothrops moojeni (Tempone et al., 2001). LAAOs from Bothrops alternatus (Sta´beli et al., 2004), B. moojeni (Sta´beli et al., 2007), and Bothrops pirajai (Izidoro et al., 2006) have been characterized biochemically and functionally. Given the diversity of LAAOs in terms of molecular mass, substrate specificity, interaction with platelets, induction of hemorrhage and apoptosis, and antibacterial and antiparasitic activities (Du and Clemetson, 2002), we undertook the characterization of the enzyme from B. jararaca not only for a better understanding of its role in the envenoming mechanism, but also its biotechnological potential as immunochemical reagent to develop an in vitro technique for estimating the neutralizing potency of horse Brazilian antibothropic antivenoms. Antivenoms are considered to be the only specific treatment for envenoming by snakes. These therapeutic antivenoms are traditionally prepared from hyperimmunized horse plasmas. The neutralization ability of snake antivenoms is still assessed by the traditional in vivo lethality assay (minimum effective dose: ED50), performed in mice (World Health Organization (WHO), 1981). Besides its inherent aggressiveness to the animals, this procedure is expensive, cumbersome, and time consuming. Reproducibility is quite difficult to achieve, and it is strongly dependent on qualified and trained personnel. In vitro alternative assays that may replace, at least in part, the use of animals are urgently sought and is the main subject of this paper. Enzyme-linked immunosorbent assays (ELISAs) have been used in studies to assess antivenom potency against snakes (Theakston and Reid, 1979; Rungsiwongse and Ratanabanangkoon, 1991; Barbosa et al., 1995; Heneine et al., 1998; Rial et al., 2006) and scorpion venoms (Maria et al., 2005). In this work, a screening of the microbicidal potency of thirteen insect, arachnid and ophidian venoms rendered B. jararaca as the most active. Such potency was related to its LAAO activity, which was isolated and further characterized biochemically. B. jararaca LAAO is a potent
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microbicidal (anti-Staphylococcus aureus) and leishmanicidal (anti-Leishmania amazonensis) component with glycosylated isoforms which differ from the snake venom LAAOs studied thus far. We have shown also, that LAAO can be used as antigen coated to ELISA plates to develop an in vitro technique for estimating the potency of horse Brazilian antibothropic antivenoms. 2. Materials and methods 2.1. Venoms and antivenoms Crude venoms from the bee Apis mellifera, the caterpillar Lonomia achelous, the spiders Lycosa sp. and Phoneutria nigriventer, the scorpions Tityus bahiensis and Tityus serrulatus, the snakes B. alternatus, B. jararaca, Bothrops jararacussu, B. moojeni, Bothrops neuwiedi, Crotalus durissus terrificus and Lachesis muta muta were provided by the ‘‘Seça˜o de Animais Peçonhentos’’ of Fundaça˜o Ezequiel Dias (FUNED), Belo Horizonte, Minas Gerais, Brazil and kept lyophilized at 20 C, at the Laboratory of Toxin Immunochemistry, Instituto de Ciencias Biolo´gicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil. Hyperimmune horse antibothropic plasmas (n ¼ 6) were obtained following the standard immunization schedules of the Immunobiological Production Unit at FUNED. The antigen for producing the antibothropic plasmas consisted of 50% of B. jararaca venom and 12.5% each of B. alternatus, B. jararacussu, B. neuwiedii and B. moojeni venoms. One week after the last injection of antigen, all horses were bled by venipuncture. Blood samples were kept in test tubes and, after clotting, the serum was separated and stored at 20 C. 2.2. Protein determination Protein content in crude venoms and isolated fractions were determined according to the method of Bradford (1976) utilizing bovine serum albumin (Sigma Chemicals) as a standard. 2.3. Antibacterial activity Agar diffusion assays for bactericidal activity were carried out at the Laboratory of Oral Microbiology, Department of Microbiology, Universidade Federal de Minas Gerais, according to the agar diffusion method described by National Committee for Clinical Laboratory Standards (2001). Briefly, bacteria were suspended in saline (0.85% w/ v) and homogenously inoculated on Petri dishes containing Brain-Heart Infusion media (5.2% w/v) and yeast extract (0.5% w/v). Depending on the nutritional requirements of the tested microorganism, the growth medium was supplemented with 5 mg/mL of menadione and hemine. Protein samples (40 mg in 50 mL of saline) were applied onto wells molded on the agar. Fifty mL of potassium metabisulphite (50 mg/mL) and NaCl 0.87% w/v were used as positive and negative controls, respectively. Plates were incubated at 37 C until bacterial growth was detected. Bactericidal activity was assessed according to the inhibition halo formed around the well. All experiments were performed in duplicates using separate plates. Various Gram-positive and
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Gram-negative bacteria were tested for their growth sensitivity upon incubation with various venoms. Aerobic bacteria were Escherichia coli (ATCC 25922), Listeria monocytogenes (ATCC 15313), Pseudomonas aeruginosa (ATCC 10145), Salmonella typhimurium (ATCC 14028), S. aureus (ATCC 33591), Staphylococcus epidermidis (ATCC 12228), which were incubated at 37 C, whereas anaerobic or facultative bacteria (Actinobacillus actinomycetencomitans ´ides fragilis (ATCC 25285), Eikenella (ATCC 29523) Bactero corrodes, Enterococcus fecalis (ATCC 19433), Eubacterium lentum, Peptostreptococcus anaerobius (ATCC 27337), Porphyromonas gingivalis (ATCC 33277), Prevotella interme´dia (ATCC 25611), Propionibacterium acnes (ATCC 6919) and Streptococcus mutans (ATCC 25175)), were incubated in anaerobic chambers at 37 C. 2.4. Polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE) Protein samples were subjected to SDS-PAGE according to the method of Laemmli (1970) in the absence or presence (reducing conditions) of b-mercaptoethanol. 2.5. L-Amino acid oxidase (LAAO) activity LAAO activity was assessed in B. jararaca chromatographic fractions or crude venom according to the method of Sakurai et al. (2001) with modifications. Briefly, a reaction mixture containing horseradish peroxidase (50 mg/ mL), 100 mM L-leucine, 10 mM o-dianisidine in 100 mM Tris– HCl (pH 7.8), and the experimental sample (2 mg) in a final volume of 1 mL was incubated at 25 C for 30 min. The increase in absorbance at 436 nm was recorded in a Shimadzu – UV 160A spectrophotometer. One unit of enzymatic activity is defined as the oxidation of 1 mM L-leucine per min. Assays were performed in duplicates. The following substrates were used to determine the substrate specificity of the isolated B. jararaca LAAO: L-alanine, L-arginine, L-asparagine, L-cysteine, L-phenylalanine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-proline, L-threonine, L-tryptophan, L-serine, and L-valine. The reaction mixture was set as described above, at an amino acid concentration of 100 mg/mL and 2.0 mg/mL of B. jararaca LAAO. 2.6. Chromatographic separation of B. jararaca crude venom 2.6.1. Gel filtration chromatography Crude B. jararaca venom was initially resolved through a gel filtration semipreparative column (SuperdexÔ 75 HR10/30 Pharmacia, 10 mm diameter) operated by a Fast Performance Liquid Chromatography (FPLC, Pharmacia) system. Venom (500 mg protein) was solubilized in 0.5 mL of elution buffer (0.15 M ammonium formiate, pH 6.0) and applied to the equilibrated column (10 mg per chromatographic run), which was subsequently eluted at 0.5 mL/ min, and the eluate collected in 1-mL fractions. Absorbance was recorded at 280 nm. Fractions were pooled according to the chromatographic profile, lyophilized and stored at 80 C until performance of the bactericidal assays.
2.6.2. Anion exchange chromatography The gel filtration chromatography fraction exhibiting bactericidal activity was solubilized in elution buffer (20 mM HEPES, pH 8.0), and applied onto an anion exchange column (Mono QR HR 5/5, Pharmacia), operated by an FPLC (Pharmacia) system (5 mg protein per run). A total of 147 mg of protein was fractionated. The Mono Q column was equilibrated with elution buffer and eluted at 1 mL/min using a linear 0–0.5 M NaCl gradient. Absorbance of the eluate (collected in 1 mL fractions) was recorded at 280 nm. Fractions were pooled according to the chromatographic profile, lyophilized and stored at 80 C until performance of the bactericidal assays. 2.6.3. Affinity chromatography through HiTrap Heparin columns Anion exchange chromatography fractions exhibiting bactericidal activity were pooled and dialyzed against 5 mM Mes, 5 mM Tris, 1 mM Benzamidine, pH 6.0 (solution A), at 4 C for 18 h. Subsequently, the protein sample (11 mg) was applied onto a HiTrap Heparin column (0.7 2.5 cm) previously equilibrated with solution A. The column was then developed with a NaCl linear (0–0.2 M) gradient. Fractions (1 mL) were collected at a flow rate of 0.5 mL/min at room temperature. Absorbance was recorded at 280 nm. All fractions were assayed for LAAO activity according to the procedure outlined in Section 2.5. The active fractions were pooled, lyophilized and stored at 80 C until performance of bactericidal activity assays. 2.7. Deglycosylation of B. jararaca LAAO LAAO obtained from affinity chromatography columns as described in Section 2.6 was subjected to deglycosylation by treating the enzyme with recombinant Glycosidase F from Flavobacterium meningosepticum (PNGase F) as previously described by Magalhaes et al. (2007). Briefly, 200 mg of purified protein were dissolved in 90 mL of denaturing buffer (0.5% SDS, 1% m-mercaptoethanol) and denatured by boiling for 10 min. Ten ml of reaction buffer (0.05 M phosphate, pH 7.5) was then added, together with 10 mL of 10% NP-40 and 2 ml of recombinant PNGase F. The resulting digestion was analyzed by SDS-PAGE as described in Section 2.4. 2.8. Leishmanicidal activity Strain IFLA/BR/1967/pH-8 of Leishmania (Leishmania) amazonensis was used. The assay for antiparasitic activity was carried out according to the procedure of Tempone et al. (2001) with major modifications. Briefly, promastigotes were cultured in Schneider’s complete medium (Sigma Chemicals), supplemented with 20% inactivated fetal bovine serum (Sigma), 20 mM L-glutamine, 50 mg/mL gentamicin, 200 U/mL penicillin and 100 mg/mL streptomycin, at pH 7.4. Parasites were cultured at 23 C for 5 days. Viability was assessed by quantification in a Neubauer chamber. L. (L.) amazonensis promastigotes were incubated in 96-well CostarÒ microtitration plates at 4 105 cells/well, together with the experimental sample at 0.8 mg/mL, in the absence or presence of catalase (0.3 mg/mL). Incubation was performed in Dulbecco’s Modified Eagle’s Medium (DMEM,
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Sigma) containing 20% inactivated fetal bovine serum, 4.5 g/ L glucose, 20 mg/mL gentamicin sulphate, 100 U/mL penicillin and 50 mg/mL streptomycin at pH 7.4, in a final volume of 150 mL/well for 18 h at 23 C. Negative (without sample) and positive (with H2O2) controls were also included. Promastigotes were also incubated in complete Hanks Balanced Salt Solution (HBSS), characterized by its poor amino acid content, under the same conditions. Cell viability was assessed by the oxidation of MTT (3-[4,5-dimethylthiazol-21]-2,5diphenyl-tetrazolium bromide) according to Machado et al. (2007). Results are the mean of two independent experiments performed in triplicates. 2.9. Hemolytic activity Hemolytic activity was determined in TSA solid culture medium (1.5% bacto tryptone, 0.5% bacto soytone, 0.5% NaCl, 1.5% bacto agar) containing 5% horse blood. Samples (in 50 mL) were applied onto wells made on the solidified medium and the plates were incubated at 37 C for 6 h, when the erythrocyte lysis could be detected by the formation of a halo. Hemolysis areas were characterized as b-hemolysis, partial hemolysis, or b-hemolysis zones. Hemolytic activity assays were performed in duplicates. 2.10. Lethality of B. jararaca venom and in vivo neutralization assays Lethality of B. jararaca crude venom was determined by i.p. injection into Swiss mice. The LD50 of B. jararaca crude venom, used throughout this study was 42 mg per 20 g mouse weight. Deaths were recorded up to 48 h. A fixed amount (5 LD50) of Bothrops venom was incubated with varying amounts of the respective antivenom for 30 min at 37 C. Each mixture (0.5 mL) was injected i.p. into groups of eight Swiss mice (18 22 g) and deaths were recorded up to 48 h. Results were analyzed using the Probit test and neutralization was expressed as effective dose 50% (ED50), defined as the amount (mg) of venom neutralized by 1.0 mL of antivenom (volume needed to prevent death in 50% of the injected mice).
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2.11. ELISA ELISA was performed by coating the plates (Nunc/ Denmark) overnight at 5 C with 100 ng/well of crude B. jararaca venom. After blocking and washing, the horse antibothropic sera corresponding to a final dilution of 1:40,000 were added and incubated for 1 h at 37 C. ELISA was performed by the method of Maria et al. (1998). The absorbance values were determined at 492 nm with a Titertek Multiscan spectrophotometer. All measurements were made in duplicate and the results were expressed as the median of two assays. Correlation and regression analysis were performed by the least-squares means method using standard software (Excel and Instat). 3. Results 3.1. Screening of insect, arachnid, and ophidian venoms for bactericidal activity We assayed for antibacterial activity of thirteen venoms from various insect, arachnid and ophidian sources utilizing S. aureus as target bacterium. Fig. 1 shows representative plates with inhibition haloes indicating that snake venoms from the genus Bothrops are the most potent anti-S. aureus bactericidal agents at the concentration tested. Noticeably, venoms from B. jararacussu and B. jararaca contained the highest activity.
3.2. Gram-positive and Gram-negative bacteria sensitivity to B. jararacussu and B. jararaca venoms Given that the anti-S. aureus tests rendered B. jararacussu and B. jararaca as the bothropic venoms exhibiting the highest bactericidal activity, both venoms were assayed against an ample spectrum of representative Gram-positive and Gram-negative bacterial strains. Table 1 shows the results of inhibition tests performed on seven Grampositive and seven Gram-negative bacteria responsible for various human pathologies. The venoms were capable of
Fig. 1. Growth inhibition of S. aureus by insect, arachnid, and snake venoms (0.8 mg/mL). 1. Apis mellifera, 2. Lonomia achelous, 3. Lycosa sp., 4. Phoneutria nigriventer, 5. Tityus bahiensis, 6. T. serrulatus, 7. Lachesis muta muta, 8. Bothrops jararacussu, 9. B. alternatus, 10. B. moojeni, 11. B. neuwiedii, 12. Crotalus durrisus terrificus, 13. B. jararaca. (Cþ) Positive control, potassium metabisulphite (50 mg/mL). (C) Negative control, NaCl 0.87% w/v. Plates were incubated for approx. 7 h at 37 C.
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Table 1 Sensitivity of Gram-positive and Gram-negative bacteria to B. jararaca and B. jararacussu venoms; (þ) bacterial inhibition; () without bacterial inhibition. Pathology
Bacterial strain
Inhibition of bacterial growth by Bothrops venomsa B. jararaca
B. jararacussu
Gram-positive
Eubacterium lentum Peptostreptococcus anaerobius (ATCC 27337) Propionibacterium acnes (ATCC 6919) Staphylococcus aureus (ATCC 33591) Staphylococcus epidermidis (ATCC 12228) Enterococcus fecalis (ATCC 19433) Streptococcus mutans (ATCC 25175)
þ þ þ þ þ
þ þ þ þ þ
Urine infection Respiratory tract infection Skin infection Hospitalary infection Skin infection Hospitalary infection Periodontal disease
Gram-negative
Porphyromonas gingivalis (ATCC 33277) Prevotella interme´dia (ATCC 25611) Pseudomonas aeruginosa (ATCC 10145) Salmonella typhimurium (ATCC 14028) Bacteroides fragilis (ATCC 25285) Eikenella corrodes Escherichia coli (ATCC 25922)
þ þ þ þ
þ þ þ þ
Periodontal disease Periodontal disease Hospitalary infection Food poisoning Skin infection Respiratory tract infection Food poisoning
a
Assays were performed at 0.8 mg/mL venom concentration.
equally inhibiting the growth of five Gram-positive and four Gram-negative species. Noticeably, E. faecalis and E. coli were resistant to both venoms at the dose tested. 3.3. Gel filtration chromatography of B. jararaca venom Based on the anti-S. aureus activity of B. jararaca venom (Fig. 1), we undertook its fractionation by gel filtration chromatography to initiate isolation and characterization of the bactericidal component(s). Fig. 2A shows the elution profile through Superdex columns (equilibrated in 0.15 M ammonium formiate, pH 6.0) of B. jararaca soluble venom (10 mg). A total of 500 mg of protein were fractionated using this protocol. The high molecular weight fraction P1 contained approx. 30% of B. jararaca whole venom bactericidal (anti-S. aureus) activity (Fig. 2B). P1 exhibited LAAO activity (approx. 80% of the whole venom activity), based on the assay for oxidation of L-leucine (Fig. 2C). 3.4. Anion exchange chromatography To further purify the bactericidal components with LAAO activity, gel filtration fraction P1 (5 mg) was suspended in 20 mM HEPES buffer (pH 8.0) and applied onto a Mono Q HR 5/5 column previously equilibrated with the same solution. A total of 140 mg of fraction P1 were fractionated using such protocol. Fig. 3A shows the chromatographic profile of P1 proteins eluted using a linear 0–0.5 M NaCl gradient. LAAO activity was contained in fractions P5 and P6 which together correspond to 8% of the protein content of gel filtration fraction P1. P5 and P6 were pooled into fraction TAP5/6 for further fractionation procedures. TAP5/6 retained most of the bactericidal activity of fraction P1 (Fig. 3B). 3.5. Affinity chromatography Active anion exchange fraction TAP5/6 (11 mg) was dialyzed against 10 mM Mes, 10 mM Tris, 2 mM Benzamidine
(pH 6.0) and applied onto Heparin columns for affinity chromatography on HiTrap Heparin columns, equilibrated with the same solution and eluted with a linear 0–0.4 M NaCl gradient. This approach has been used previously for isolation of LAAOs from other snake venoms. Fig. 4A shows the elution profile, indicating that LAAO activity eluted in the fraction 1 from the column. This fraction was named HTP1. After dialysis against 0.05 M Tris–HCl (pH 7.8), it was confirmed that HTP1 was active against S. aureus (Fig. 4B). With the goal of determining whether HTP1 bactericidal activity was related to the hydrogen peroxide produced via LAAO, anti-S. aureus activity was determined after incubation of the fraction with catalase (0.3 mg/mL). Catalase (EC 1.11.1.6) catalyzes the conversion of H2O2 to water and O2. It was found that the HTP1 bactericidal activity was abolished after such incubation with catalase (Fig. 4B, well 3).
3.6. Electrophoretic composition of purified fractions and effect of deglycosylation To assess the purity and subunit composition of the bactericidal, LAAO-active HTP1 fraction obtained by affinity chromatography, SDS-PAGE gels were performed. Fig. 5 shows the presence of three distinct bands with molecular masses 80, 60.8 and 48.1 kDa (Fig. 5A). To determine whether the above components could be glycosylated by variants of a unique B. jararaca LAAO enzyme, we subjected fraction HTP1 to digestion with Glycosidase (PNGase) F (see Section 2.7) and electrophoresed by SDS-PAGE. Such procedure has been used previously to study the glycan moieties of LAAO from Calloselasma rhodostoma (Geyer et al., 2001). PNGase F is an amidase that cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins (Maley et al., 1989). After digestion of HTP1, a single electrophoretic component of 38.2 kDa was obtained (Fig. 5B), attesting to the purity of the fraction.
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A
A
2
P1
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0.5
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P4 P3 P5
0.5
P6 0
5
10
15
Abs 280 nm (
1
P6
0.3
P8 0.2 P5
20 P4
Volume (mL)
P1
P2
P3 P2 0
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P4
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P3
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0.04
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Abs 280 nm
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1
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P1
P7 20
35
0
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P6
B C+ C+
C
P2
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P6
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P4
0.06 0.05
Abs 436 nm
P1
P7
P8
0.04 0.03 0.02 0.01 0.00 Crude venom
P1
P2
P3
P4
P5
P6
Fig. 2. Gel exclusion chromatography of B. jararaca venom and LAAO and bactericidal activity of pooled fractions. (A) Representative elution profile of B. jararaca crude venom (10 mg protein) chromatography in a SuperdexÔ 75 HR10/30 column, using an FPLC system. Elution buffer was 0.15 M ammonium formiate, pH 6.0; flow rate 0.5 mL/min at room temperature. (B) Bactericidal activity of gel filtration chromatographic fractions. Each fraction (P1–P6) was assayed at 0.8 mg/mL on wells made on BHI plates inoculated with S. aureus. B. jararaca venom was used as positive control (Cþ). Plates were incubated for approx. 7 h at 37 C. (C) LAAO activity of chromatographic fractions performed as outlined in materials and methods (Section 2.5).
3.7. Leishmanicidal and hemolytic activity of B. jararaca crude venom and LAAO-active fractions Taking into account the observed bactericidal activity of fraction HTP1 (also identified as an LAAO-active fraction), we wished to determine its antiparasitic (leishmanicidal) and hemolytic potency. It is known that other ophidian
Fig. 3. Anion exchange chromatography of B. jararaca bactericidal fractions obtained by gel filtration. (A) Elution profile of fraction P1 subjected to anion exchange chromatography. Gel filtration fraction P1 (see Fig. 2A) was suspended in 20 mM HEPES, pH 8.0, applied to a Mono Q anion exchange column and eluted with a linear 0–0.5 M NaCl gradient (in cinder). Flow rate was 1 mL/min; 1 mL fractions. Elution was monitored at 280 nm (d). Each fraction was assayed for LAAO activity using L-leucine as substrate; activity was recorded at 436 nm (- - - - - -). Asterisks (*) indicate active fractions P5 and P6. (B) Bactericidal activity of Mono Q fractions. Each fraction (P1–P8) was assayed at 0.8 mg/mL on wells made on BHI plates inoculated with S. aureus. B. jararaca venom was used as positive control (Cþ). Plates were incubated for approx. 7 h at 37 C.
LAAOs exhibit such activities as a result of enzymecatalyzed H2O2 production (Du and Clemetson, 2002). Fig. 6 shows the results of assays carried out to study the effect of HTP1 on the viability of L. (L.) amazonensis promastigotes. Upon incubation with B. jararaca whole venom, 69% viability was obtained, whereas 47.5% viability was observed after incubation with HTP1. Inclusion of catalase abolished the leishmanicidal activity of both crude venom and HTP1. After incubation with H2O2 (as a positive control) 71% of promastigotes remained viable. Incubation of cells with complete HBSS medium produced 75% viability
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A
0.6
by L-arginine (82.2 U/mg), L-tryptophan (76.2 U/mg), and L-phenylalanine (58.2 U/mg). Oxidation of L-asparagine and L-serine by HTP1 was poor and L-proline was not oxidized at all.
0.1
HTP1
0.5 0.08
Abs 280 nm ( )
0.06 0.3 0.04 0.2
Abs 436 nm (- - - -)
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0 2
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16
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3.9. Indirect ELISA for potency estimation of antibothropic antivenom The potency of five Bothrops antivenoms samples and one pre-immune serum horse as negative control in protecting against lethality in mice and the ELISA antibody titers against crude venom and LAAO antigens is shown in Table 2 and Fig. 8. Antivenoms protected against lethality of B. jararaca with ED50 ranging from 1 to 9 mg/mL. A best correlation was found between ELISA titers and neutralizing of lethal activity (ED50) when using LAAO to coat the plates. Correlation coefficients (r) between ED50 and ELISA antibody titers against B. jararaca crude venom and LAAO were 0.747 (p < 0.001) and 0.846 (p < 0.001), respectively (Fig. 6A and B). 4. Discussion
B 1
3
2
4
Fig. 4. Affinity chromatography of active fractions obtained from Mono Q anion exchange columns. (A) Elution profile of pooled fractions P5 and P6 on HiTrap Heparin columns using an FPLC system. Sample (11 mg) was applied onto a column equilibrated with 10 mM Mes, 10 mM Tris, 2 mM Benzamidine, pH 6.0, and eluted using a linear 0–0.4 M NaCl gradient (in cinder). Flow rate was 0.5 mL/min; 1 mL fractions. Elution was monitored at 280 nm (d). Each fraction was assayed for LAAO activity using L-leucine as substrate; activity was recorded at 436 nm (- - -). (B) Bactericidal activity of fraction HTP1 obtained by affinity chromatography. Samples (0.8 mg/mL) were assayed for activity against S. aureus. 1. B. jararaca crude venom, 2. HTP1 fraction, 3. HTP1 fraction including catalase (0.3 mg/mL), 4. Catalase (0.3 mg/mL).
whereas incubation with 1 M Tris–HCl did not influence cell viability (Fig. 6). Regarding hemolytic activity, all bactericidal fractions and the crude B. jararaca venom produced significant lysis of horse blood. Fig. 7 shows formation of a greenish halo around the wells indicative of the occurrence of b-hemolysis. 3.8. B. jararaca LAAO specific activity towards amino acid substrates Table 3 presents the results of a study carried out to determine the specific activity of fraction HTP1 towards various amino acid substrates. Previously, the specific activity of HTP1 towards oxidation of L-leucine had been shown (Figs. 3 and 4). Activity was highest for L-leucine (142.5 U/mg) and L-methionine (136.5 U/mg), followed
4.1. Screening of antimicrobial activity of arthropod and ophidian venoms Our original goal was to identify insect, arachnid, or ophidian venoms that could be used as primary source of antibacterial and/or antiprotozoal components suitable for structure-based drug design. Initially, our work revealed that venoms from bees, scorpions, and spiders belonging to genera Apis, Tityus, Lycosa, and Phoneutria, respectively, are not active in inhibiting the growth of the Gram-positive bacterium S. aureus at the dose tested (0.8 mg/mL). These results agree with previous reports documenting that antimicrobial peptides derived from scorpions (Opistophtalmus carinatus and Parabuthus schlechteri) (Moerman et al., 2002) and Lycosa spiders preferentially inhibited the growth of Gram-negative bacteria (Yan and Adams, 1998). On the other hand, Moerman et al. (2002) have shown that melittin, a major peptide of A. mellifera venom, is more effective against Gram-positive bacteria (such as S. aureus) in the same concentration range. Our result indicating the bee venom lower or null microbicidal activity is probably expected since we used crude A. mellifera venom extracts. Our work demonstrated the antimicrobial potency of Neotropical ophidian venoms, particularly those from the genus Bothrops as shown before in several reports (Paramo et al., 1998; Rodrigues et al., 2004; Sta´beli et al., 2004). In general, venoms from the Elapidae and Viperidae families are the most active against bacteria (Stiles et al., 1991). Noticeably, we found that among all Bothrops venoms tested, B. jararacussu and B. jararaca exhibited the highest inhibitory activity against S. aureus. As it will be discussed later, antimicrobial activity of snake venoms has been associated with their LAAO and/or phospholipase A2 activities. Pessatti et al. (1995) reported a higher LAAO activity of B. neuwiedii venom from the Amazon region (655.97 U/mL) than those of B. jararaca and B. jararacussu from southeast Brazil (314.95 and 391.87 U/mL, respectively). Similarly, Tan and Ponndurai (1991) found a higher
P. Ciscotto et al. / Toxicon 53 (2009) 330–341
A
1
2
3
4
337
B
207.0 92 .0 -
55.0 -
35.0 -
Fig. 5. Molecular mass and isoform composition of B. jararaca LAAO. (A) SDS-PAGE of active chromatographic fractions. 1. Crude B. jararaca venom, 2. Gel filtration fraction P1 (see Fig. 2), 3. Anion exchange chromatography fractions P5 and P6 (pool) (see Fig. 3), 4. HiTrap Heparin fraction HTP1 (Fig. 4). Samples (2 mg) were subjected to electrophoresis in 12.5% gels which were subsequently silver-stained. (B) SDS-PAGE (12.5% gel) of a sample of fraction HTP1 (230 mg) subjected to deglycosylation as described in Section 2.7 of materials and methods.
LAAO activity in B. neuwiedii, and also considerable individual variation in the enzyme levels among B. jararaca specimens. It is known the considerable influence of geography and ontogeny variation in protein content and quality in snake venoms (see for example Meier, 1986). The consistent lower bactericidal potency detected by us in Bothrops venoms (including B. neuwidii) other than B. jararacussu and B. jararaca could well be related to a combination of these factors. Regardless of the basis of such differences, both these venoms were active not only on S. aureus but on other Gram-positive (Gþ) and Gram-negative (G) bacteria. For instance, S. typhimurium and P.
aeruginosa (both G) were sensitive to B. jararacussu and B. jararaca venoms, whereas E. coli (G)remained refractory to venom action, an observation consistent with other reports on the activity of viperid venoms (see Blaylock, 2000 and Stiles et al., 1991). Out of these two most active venoms, B. jararacussu has been studied intensively regarding the microbicidal activities of isolated phospholipases (Barbosa et al., 2005) and the molecular cloning of LAAO (Franca et al., 2007). We then choose B. jararaca as the model Bothrops venom for isolation of biomolecules with antimicrobial properties. 4.2. Purification of B. jararaca microbicidal components
Celular Viability ( )
100 80 60 40 20 0
A
B
C
D
E
F
G
H
I
Fig. 6. Characterization of the leishmanicidal activity of B. jararaca venom. (A) L. (L.) amazonensis promastigotes in complete DMEM; (B) promastigotes (4 105 cells/well) in DMEM þ 5 mM H2O2; (C) promastigotes in DMEM þ B. jararaca crude venom (0.8 mg/mL); (D) promastigotes in DMEM þ 0.8 mg/mL B. jararaca crude venom (0.8 mg/mL) þ catalase (0.3 mg/mL); (E) promastigotes in DMEM þ affinity chromatography fraction HTP1 (0.8 mg/mL); (F) promastigotes in DMEM þ affinity chromatography fraction HTP1 (0.8 mg/ mL) þ catalase (0.3 mg/mL); (G) promastigotas in DMEM þ Tris–HCl 1 M; (H) promatigotes in complete HBSS medium; (I) promastigotes in HBSS þ affinity chromatography fraction HTP1 (0.8 mg/mL). Viability (median of experiments performed in triplicates) is expressed as a percentage of absorbance readings at 570 nm of control promastigotes suspended in culture media.
Out of the six fractions obtained by gel exclusion chromatography of B. jararaca venom, the highest molecular mass fraction P1 contained most crude venom’s microbicidal activity. Maria et al. (1998) obtained a similar elution profile for B. jararaca venom, reporting that the fraction equivalent to P1 contained components which were lethal to mice upon intraperitoneal injection. To further resolve fraction P1 microbicidal/antiprotozoal components, we used a combination of anion exchange and affinity chromatography steps, which rendered active fraction HTP1, which corresponds to approx. 1.1% of the crude venom’s protein content. Such fraction exhibited both anti-S. aureus and LAAO activity which is in agreement with previous findings on the bactericidal activity of LAAOs from the genus Bothrops (Sta´beli et al., 2004). 4.3. Characterization of fraction HTP1 containing B. jararaca LAAO After non-reducing SDS-PAGE, HTP1 was shown to contain three protein bands, which are unlikely isoforms of
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P. Ciscotto et al. / Toxicon 53 (2009) 330–341 Table 3 Specific activity of B. jararaca LAAO towards amino acid substrates. Amino acid
Specific activity (U/mg)a
L-Leucine
142.5 136.5 82.3 76.2 58.2 58.2 54.1 50.1 18.0 16.0 16.0 12.0 6.0 4.0 0.0
L-Methionine L-Arginine L-Tryptophan L-Phenylalanine L-Valine L-Histidine L-Isoleucine L-Cysteine L-Lysine L-Alanine L-Threonine L-Asparagine L-Serine L-Proline a
One unit of enzyme activity is defined as the oxidation of 1 mM for min.
L-Leucine
Fig. 7. Hemolytic activity of B. jararaca and bactericidal fractions. Samples (20 mg) were applied onto wells made of TSA agar prepared with horse blood as indicated in Section 2.8. (A) After 6 h of incubation at 37 C; (B) After 18 h of incubation at 37 C; 1. Crude venom from B. jararaca; 2. Gel filtration fraction P1; 3. Mono Q column fraction TAP5/6 (pool of P5 and P6); 4. HiTrap Heparin column fraction HTP1; 5. HTP1 fraction including catalase (0.3 mg/ mL) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
the LAAO protein moiety itself since the venom used for fractionation was a pool of several specimens. Under such conditions, we expect that individual variations should be minimal. Accordingly, a single, symmetrical peak was obtained after subjecting HTP1 to reverse phase HPLC (data Table 2 Potency of bothropic antivenoms in protecting against lethality and ELISA antibody reactivity of the Bothrops jararaca crude venom and LAAO. Antivenom Sample No
Lethality(ED50) mg/mLa
ELISA reactivity (492 nm) B. jararaca venom
LAAO
1 Control 2 3 4 5 6
0 1 3 5 7 9
0.190 0.250 0.220 0.350 0.890 0.720
0.115 0.180 0.190 0.250 0.650 0.950
a Amount of antivenom need to protect half of the mice injected with 5 LD50 of B. jararaca crude venom.
Fig. 8. Correlation between ELISA antibody level (absorbance at 492 nm) and in vivo neutralizing potency of B. jararaca antivenoms. Ninety-six-well microtiter plates were coated with crude B. jararaca venom (A) and LAAO (B) and 16 antivenoms were used at 1:40,000 dilution. Neutralizing potency was expressed as effective dose 50% (ED50), defined as the amount (mg) of venom neutralized by 1.0 mL of antivenom (volume of antivenom needed to prevent death of 50% of the injected mice). All data points are the means of two experiments.
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not shown), indicating protein homogeneity. Curti et al. (1968) working on the inactivation process of LAAO from Crotalus adamanteus also found three protein bands for the active and inactive forms of the enzyme. Hayes and Wellner (1969), also working on the enzyme from C. adamanteus, found three bands corresponding to enzymatically active LAAO variants; each of the these bands rendered 5–7 components when subjected to isoelectric focusing. Since LAAOs are FAD-dependent glycoproteins, its carbohydrate moieties could contribute to differential electrophoretic migration. Such carbohydrate moieties play a functional role in the LAAO-induced cytoxicity, as shown in the case of C. rhodostoma (Ande et al., 2006), although glycosylation has shown to be homogenous in this case (Geyer et al., 2001). To explore such possibility, we decided to subject HTP1 to digestion with Peptide:N-Glycosidase F, an amidase that cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins (Maley et al., 1989). Sugar removal of HTP1 by PNGase F resulted in the appearance of a single band (38.2 kDa) in non-reducing SDS-PAGE, suggesting that B. jararaca LAAO heterogeneity resides in the glycan moiety. We are currently investigating the nature of such moieties and its functional implications. Further proof of the identity of LAAO as the microbicidal enzyme contained in HTP1 relies on its substrate specificity. Kinetic studies indicate that B. jararaca LAAO is active against hydrophobic amino acids, such as L-Phe, L-Tyr, L-Leu, L-Ile and L-Trp. It is accordance with the substrate specificity of LAAOs from C. rhodostoma, Naja naja kaouthia (Tan and Swaminathan, 1992), and B. moojeni (Sta´beli et al., 2007) venoms. Also, these results agree with the findings of Pessatti et al. (1995) who determined that L-Met and L-Leu were the substrates oxidized at a higher rate by LAAO from Bothrops cotiara. B. jararaca LAAO isolated by us differs considerably from other characterized ophidian oxidases on the basis of its molecular mass (38.2 kDa). LAAO from Crotalus atrox possesses a mass of 60 kDa (Masuda et al., 1997), whereas Sta´beli et al. (2004) reported a molecular mass of 66 kDa for the enzyme obtained from B. alternatus. For B. moojeni, Sta´beli et al. (2007) reported a mass of 64.8 kDa. In general, ophidian LAAOs are homodimers (50– 70 kDa subunit mass) associated non-covalently (Du and Clemetson, 2002). Since a single type of venom may contain more than one LAAO (Stiles et al., 1991), further experiments are needed to determine whether the enzyme we isolated from B. jararaca venom is an atypical ophidian LAAO. In this sense, molecular cloning or N-terminal sequencing should be carried out to compare the primary structures of the B. jararaca enzyme with those isolated and sequenced from the genus Bothrops. 4.4. Hydrogen peroxide-mediated antiparasitic and hemolytic activity of B. jararaca LAAO Both B. jararaca crude venom and fraction HTP1 (characterized as containing a single 38.2 kDa component) showed activity against L. (L.) amazonensis promastigotes and promoted partial lysis of horse erythrocytes. Previous reports have indicated that B. jararaca venom inhibited growth of T. cruzi and of L. major promastigotes (Gonçalves
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et al., 2002). Also, Tempone et al. (2001) have verified the leishmanicidal potency of B. moojeni venom and associated such activity to an LAAO enzyme with a molecular mass of 69 kDa (through SDS-PAGE) and 140 kDa by gel filtration chromatography. The anti-Leishmania activity of fraction HTP1 is dependent on the free amino acid content of the culture media since venom effect on promastigotes grown in complete HBSS medium (lacking amino acids) was null. Therefore, the B. jararaca venom leishmanicidal action is probably exerted through its LAAO activity given the substrate dependence of such effect. The hemolytic properties of ophidian LAAOs have been little explored. Detected hemolytic activity in the crude venom of Eristocophis macmahoni and its purified LAAO on sheep eryhrocytes. Ours is the first report of such activity associated to an LAAO isolated from the genus Bothrops and is most probably related to its H2O2 producing activity. Addition of catalase abolished the microbicidal, leishmanicidal, and hemolytic activities of fraction HTP1 suggesting that H2O2, a product of the LAAO-mediated reaction, is involved in such effects. This result is in accordance with the findings of Tempone et al. (2001) who were able to suppress the leishmanicidal activity of B. moojeni venom upon incubation with catalase. Leishmania promastigotes lack production of catalase and superoxide dismutase which renders parasites extremely sensitive to H2O2 produced by macrophages (Tempone et al., 2001). The enzymes OHAP-1 (Trimeresurus flavoviridis) and apoxin I (C. atrox) lost its apoptotic capacity upon addition of antioxidants such as catalase and reduced glutathione (Sun et al., 2003; Torii et al., 1997). Wei et al. (2002) also observed that the platelet aggregation-mediated activity of LAAO isolated from Trimeresurus mucrosquamatus was also lost upon incubation with catalase. These results provide further support for our proposal that the enzyme we have isolated from B. jararaca venom is an L-amino acid oxidase acting as an indirect microbicidal/leishmanicidal agent which promotes cell death through the oxidizing action of H2O2 on biological membranes. Minimal inhibitory concentration determinations and in vivo assays will be needed in the future to assess the possibility of utilizing B. jararaca LAAO as an antimicrobial and/or leishmanicidal reagent. According to Sorg (2004), microorganism in general are several fold more sensitive to reactive oxygen species (ROS) than human tissues, implying that there is a bactericidal window where ROS concentrations sufficient to abolish bacterial growth are harmless to the host cells. 4.5. In vitro technique for estimating the potency antibothropic antivenoms Since the LAAOs have different physiological effects (Wei et al., 2007) and a possible role in the functional toxicity of snakebites (e.g. inhibition of platelet aggregation; To˜nisma¨gi et al., 2006), we explored the possibility of developing an enzyme-linked immunosorbent assay (ELISA) to test the potency of antibothropic antivenoms based on the reactivity towards LAAO. We have previously shown that ELISA utilizing the toxic, highest molecular weight gel filtration fraction of B. jararaca venom can be used as an in vitro technique for estimating the potency of
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antibothropic antivenoms (Maria et al., 1998). In this study, purified LAAO was used to coat microtiter plates as antigen on the indirect ELISA type and a good correlation was observed in entire antibody titers and neutralization of the venom lethal activity. Our results indicate that this kind of ELISA should be adequate to follow antibody titters during immunization procedures as well as during different stages of the plasma fractionating process and the application of the in vivo assay to be restricted due that, this procedure requires the use and sacrifice of a large number of mice, unacceptable in many countries due to new and rapidly evolving legislation prohibiting the production of pain and suffering in the animal involved. As conclusion our finding showed that LAAO from the B. jararaca venom plays important pharmacological and antimicrobial properties and this protein may involve in some pharmacological effect of the whole venom and also their possible biotechnological use as molecular marker for in vitro stimation of neutralizing potency of horse antibothropic antivenoms. Acknowledgements This research was supported by the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) and Fundaça˜o de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG), Brazil. Conflict of interest The authors declare that there is no conflict of interest. References Ande, S.R., Kommoju, P.R., Draxl, S., Murkovic, M., Macheroux, P., Ghisla, S., Errando-May, E., 2006. Mechanisms of cell death induction by L-amino acid oxidase, a major component of ophidian venom. Apoptosis 11, 1439–1451. Barbosa, C.F., Rodrigues, R.J., Olortegui, C.C., Sanchez, E.F., Heneine, L.G., 1995. Determination of the neutralizing potency of horse antivenom against bothropic and crotalic venoms by indirect enzyme immunoassay. Braz. J. Med. Biol. Res. 28, 1077–1080. Barbosa, O.S., Martins, A.M., Havt, A., Toyama, D.O., Evangelista, J.S., Ferreira, D.P., Joazeiro, P.P., Beriam, L.O., Toyama, M.H., Fonteles, M.C., Monteiro, H.S., 2005. Renal and antibacterial effects induced by myotoxin I and II isolated from Bothrops jararacussu venom. Toxicon 46, 376–386. Blaylock, R.S., 2000. Antibacterial properties of KwaZulu Natal snake venoms. Toxicon 38, 1529–1534. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Curti, B., Massey, V., Zmudka, M., 1968. Inactivation of snake venom L-amino acid oxidase by freezing. J. Biol. Chem. 243, 2306–2314. Du, X.Y., Clemetson, K.J., 2002. Snake venom L-amino acid oxidases. Toxicon 40, 659–665. Franca, S.C., Kashima, S., Roberto, P.G., Marins, M., Ticli, F.K., Pereira, J.O., Astolfi-Filho, S., Stabeli, R.G., Magro, A.J., Fontes, M.R., Sampaio, S.V., Soares, A.M., 2007. Molecular approaches for structural characterization of Bothrops L-amino acid oxidases with antiprotozoal activity: cDNA cloning, comparative sequence analysis, and molecular modeling. Biochem. Biophys. Res. Commun. 355, 302–306. Geyer, A., Fitzpatrick, T., Pawelek, P.D., Kitzing, K., Vrielink, A., Ghisla, S., Macheroux, P., 2001. Structure and characterization of the glycan moiety of L-amino acid oxidase from the Malayan pit viper Calloselasma rhodostoma. Eur. J. Biochem. 268, 4044–4053. Gonçalves, A.R., Soares, M.J., de Souza, W., DaMatta, R.A., Alves, E.W., 2002. Ultrastructural alterations and growth inhibition of
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