Isolation and biochemical, functional and structural characterization of a novel l -amino acid oxidase from Lachesis muta snake venom

Toxicon 60 (2012) 1263–1276

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Isolation and biochemical, functional and structural characterization of a novel L-amino acid oxidase from Lachesis muta snake venom Cristiane Bregge-Silva a, Maria Cristina Nonato a, Sérgio de Albuquerque b, Paulo Lee Ho c, Inácio L.M. Junqueira de Azevedo c, Marcelo Ribeiro Vasconcelos Diniz d, Bruno Lomonte e, Alexandra Rucavado e, Cecilia Díaz e, f, José María Gutiérrez e, Eliane Candiani Arantes a, * a

Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Departamento de Física e Química, Universidade de São Paulo, Av. do Café s/n, 14040-903 Ribeirão Preto-SP, Brazil Departamento de Análises Clínicas Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Brazil c Centro de Biotecnologia, Instituto Butantan, São Paulo, Brazil d Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Belo Horizonte, Brazil e Instituto Clodomiro Picado, Facultad de Microbiologia, Universidad de Costa Rica, Costa Rica f Departamento de Bioquímica, Escuela de Medicina, Universidad de Costa Rica, Costa Rica b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 May 2012 Received in revised form 6 August 2012 Accepted 9 August 2012 Available online 31 August 2012

The aim of this study was the isolation of the LAAO from Lachesis muta venom (LmLAAO) and its biochemical, functional and structural characterization. Two different purification protocols were developed and both provided highly homogeneous and active LmLAAO. It is a homodimeric enzyme with molar mass around 120 kDa under non-reducing conditions, 60 kDa under reducing conditions in SDS-PAGE and 60852 Da by mass spectrometry. Forty amino acid residues were directly sequenced from LmLAAO and its complete cDNA was identified and characterized from an Expressed Sequence Tags data bank obtained from a venom gland. A model based on sequence homology was manually built in order to predict its three-dimensional structure. LmLAAO showed a catalytic preference for hydrophobic amino acids (Km of 0.97 mmol/L with Leu). A mild myonecrosis was observed histologically in mice after injection of 100 mg of LmLAAO and confirmed by a 15-fold increase in CK activity. LmLAAO induced cytotoxicity on AGS cell line (gastric adenocarcinoma, IC50: 22.7 mg/mL) and on MCF-7 cell line (breast adenocarcinoma, IC50:1.41 mg/mL). It presents antiparasitic activity on Leishmania brasiliensis (IC50: 2.22 mg/mL), but Trypanosoma cruzi was resistant to LmLAAO. In conclusion, LmLAAO showed low systemic toxicity but important in vitro pharmacological actions. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: L-amino acid oxidase Lachesis muta Cytotoxic activity Enzyme structure Myotoxicity

1. Introduction Snake venoms of the genus Lachesis comprise a complex mixture of pharmacologically active substances, such as

Abbreviations: LmLAAO, L-amino acid oxidase from Lachesis muta venom; svLAAO, snake venom LAAO. * Corresponding author. Tel.: þ55 16 3602 4275; fax: þ55 16 3602 4880. E-mail address: [email protected] (E.C. Arantes). 0041-0101/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxicon.2012.08.008

metalloproteases (Rucavado et al., 1999), phospholipases A2 (Ferreira et al., 2009), serine proteases (Magalhães et al., 1997) and other important enzymes. The venom of Lachesis muta, from Brazil (Campbell & Lamar, 1989), contains Lamino acid oxidase (LAAO; EC 1.4.3.2), but its functional and structural characterization has not been performed (Sanchez and Magalhães, 1991). This venom induces tissue damage, nausea, vomiting, sweating, bradycardia, hypotension, shock, and, in severe cases, death due to neurotoxic, hemorrhagic and coagulant activities of this complex

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mixture of pharmacologically active substances (Jorge et al., 1997). LAAOs are homodimeric flavoenzymes that catalyze the stereospecific oxidative deamination of L-amino acids by reduction of cofactor FAD. This reaction generates an intermediate imino acid which produces ammonia and the corresponding a-keto acid. In a parallel reaction, the reoxidation of cofactor FAD by molecular oxygen generates hydrogen peroxide (Massey and Curti, 1967; Curti et al., 1992; Sun et al., 2010). According to Du and Clemetson (2002), snake venom LAAOs (svLAAO) have 110–150 kDa when determined by gel filtration, or 50–70 kDa as judged by electrophoresis on polyacrylamide gel with sodium dodecyl sulfate (SDS-PAGE). To exert their activity, LAAOs may be organized as dimers, therefore with molar mass between 110 and 150 kDa. Pawelek et al. (2000) showed that Calloselasma rhodostoma LAAO is a homodimer of 55 kDa monomers. Furthermore, svLAAOs may be acidic or basic proteins, showing isoelectric points ranging from 4.4 to 8.5 (Ahn et al., 1997; Curti et al., 1992; Du and Clemetson, 2002). Some svLAAO crystal structures have been determined (Moustafa et al., 2006; Zhang et al., 2004) revealing a functional dimer in which each monomer consists of a FAD-binding domain, a substrate-binding domain and a helical domain that is involved in protein dimerization. Concerning enzymatic properties, different svLAAOs have shown a preference for hydrophobic L-amino acids. This catalytic profile has been observed with LAAOs from Naja naja oxiana (Samel et al., 2008), Bothrops pirajai (Izidoro et al., 2006) and C. rhodostoma (Ande et al., 2008). Several studies were performed with svLAAO in order to determine its activities in vivo. Wei et al. (2009) showed the induction of paw edema in mice after injection of 5 mg of Bungarus fasciatus LAAO. Besides edema, they have been shown to induce hemorrhage (Zhong et al., 2009) and systemic effects such as renal toxicity (Boer-Lima et al., 1999). Unexpectedly, despite its toxicity in vivo, LAAO does not cause lethality after injection of 120 mg/30 g in Swiss-Wistar mice (Ali et al., 2000). In vitro studies with svLAAOs have shown antibacterial (Sun et al., 2010; Ciscotto et al., 2009), leishmanicidal (Rodrigues et al., 2009) and trypanocidal activities (Franca et al., 2007), toxicity upon cancer cell lines (Alves et al., 2008) and both induction and/or inhibition of platelet aggregation (Alves et al., 2008; Li et al., 1994; Sakurai et al., 2001; Sun et al., 2010; Zhong et al., 2009). It has been shown that these effects are correlated with the production of H2O2. Currently, many compounds from snake venoms have been the basis for therapeutic agents (Barros et al., 2009; Lewis and Garcia, 2003) and svLAAOs emerge as an important tool for possible pharmacological applications. Although many svLAAOs have been isolated and studied, this is the first report on the LAAO from L. muta venom. The aim of this work was to isolate this enzyme and perform its biochemical, structural and functional characterization. Two different purification protocols were developed and allowed the isolation of pure and active enzyme. Its primary structure was obtained by cloning and sequencing of its cDNA, and a model based on sequence homology was manually built in order to predict its three-dimensional structure. Additionally, LmLAAO has been kinetically

characterized and both in vivo and in vitro assays were used to determine its pharmacological properties in different biological systems. 2. Material and methods 2.1. Venom L. muta venom was obtained from the Serpentarium Bosque da Saúde, Americana city, state of São Paulo, Brasil (IBAMA Register: 647.998). All chemicals used were of analytical grade. 2.2. Purification protocol 1 2.2.1. Gel filtration on Sephacryl S-100Ò Crude venom from L. muta (20 mg) was dissolved in 500 mL of 20 mM Tris–HCl buffer plus NaCl 0.15 M (pH 7.0) and centrifuged at 3000g for 10 min to remove insoluble material. The supernatant was applied to a Sephacryl S-100Ò (Hiprep 16/60, GE Healthcare) column preequilibrated with 20 mM Tris–HCl plus 0.15 M NaCl buffer, pH 7.0 and eluted at a flow rate of 0.5 mL/min. The fractions were monitored at 280 nm and tested for LAAO activity. 2.2.2. Ion exchange in Mono QÒ Fractions with LAAO activity were collected and immediately applied on a Mono QÒ 5/50GE Healthcare column pre-equilibrated with 20 mM Tris–HCl buffer, pH 7.0 and eluted with a stepwise gradient of 20 mM Tris–HCl plus NaCl 1 M buffer, pH 7.0, at a flow rate of 1 mL/min. The fractions were also monitored at 280 nm and tested for LAAO activity. 2.3. Purification protocol 2 2.3.1. Gel filtration on Sephacryl S-200Ò Crude venom from L. muta (200 mg) was dissolved in 3 mL of 20 mM Tris–HCl buffer plus 0.15 M NaCl, pH 7.0, and centrifuged at 3000g for 10 min to remove insoluble material. The supernatant was applied to a Sephacryl S-200Ò (GE Healthcare) column pre-equilibrated with 20 mM Tris–HCl plus 0.15 M NaCl buffer, pH 7.0, and eluted at a flow rate of 0.5 mL/min. The fractions were monitored at 280 nm and tested for LAAO activity. 2.3.2. Hydrophobic interaction on Phenyl-SepharoseÒ The fraction with LAAO activity collected from Sephacryl S-200Ò was submitted to hydrophobic interaction chromatography on Phenyl-SepharoseÒ resin equilibrated with 20 mM Tris–HCl, 1.5 M NaCl. The chromatography was performed on gradient steps with 20 mM Tris–HCl, pH 8.0, and decreasing concentrations of NaCl, ranging from 1.5 to 0 M, and finished with deionized water. The flow rate was maintained at 1 mL/min. The fractions were monitored at 280 nm and tested for LAAO activity. 2.3.3. Low pressure liquid chromatography on Affi-Gel BlueÒ The fraction with LAAO activity eluted from the hydrophobic interaction chromatography on Phenyl-SepharoseÒ

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was submitted to a new chromatographic step on Affi-Gel BlueÒ (Bio Rad). The elution buffer was 20 mM Tris–HCl, pH 8.0 (buffer A) and 1.5 M NaCl in 20 mM Tris–HCl, pH 8.0 (buffer B). The chromatography was performed using a basic segmented gradient with buffer B (0–100%) and flow rate maintained at 0.5 mL/min. The absorbance was automatically monitored at 280 nm and all fractions were tested for LAAO activity. 2.4. Chromatography of AfLm1 on C-18 reverse-phase HPLC The purified LmLAAO was submitted to a RP-HPLC chromatography on an analytical C-4 column (150  4.6 mm) in order to check its homogeneity and to remove traces of salt from the sample, which is critical for the next steps of structural and functional characterization. The protein was eluted with an acetonitrile gradient (0– 70%) containing 0.1% trifluoroacetic acid, at a flow rate of 1 mL/min.

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2.6.2. Molar mass The homogeneity of fractions from each chromatographic step, as well as of purified LAAO, was assessed by SDS-PAGE on 10% polyacrylamide gel as described by Laemmli (1970). Molar mass standards (PAGE RulerÔ Fermentas or GE cod. 17-0615-01) were run to allow molar mass determination. The gels were stained with Coomassie Brilliant Blue G-250. The molecular mass of LmLAAO was also determined by MALDI-TOF mass spectrometry. For protein mass determination, mixtures of 0.5 mL sinapinic acid and 0.5 mL of sample were spotted onto a MALDI target, dried, and analyzed in positive linear mode on the AB 4800-Plus instrument (ABSciex). Spectra were acquired in the m/z range of 20,000–150,000, with focus at 70,000 and at a laser intensity of 4200 and 500 shots per spectrum. Bovine serum albumin (ABSciex) was used for external calibration in linear mode. The isotope-averaged molecular mass value was obtained by centroid function of the major monocharged species.

2.5. L-amino acid oxidase activity The microplate assay for LAAO activity was conducted as described by Kishimoto and Takahashi (2001) with slight modifications. The reaction mixture contained 50 mM of Tris–HCl, pH 8.0, 5 mM, L-leucine as substrate, horseradish peroxidase (5 IU/mL) and 2 mM of orthophenylenediamine (as substrate for peroxidase). Samples were incubated for 1 h at 37  C and the reaction was stopped by adding 50 mL of 2 M H2SO4. The absorbance was determined at 492 nm by a TecanÒ Sunrise microplate reader. Hydrogen peroxide standards were used and the linear regression data calculated with the GraphPad Prism 5 Software. One unit of LAAO activity was the amount of enzyme which produces 1 mmol of H2O2 and LAAO activity was expressed as nmoles of H2O2 produced per minute. 2.6. Biochemical, functional and structural characterization of LmLAAO 2.6.1. Determination of Km and Vmax of LmLAAO Before determining the kinetics parameters (Km and Vmax) it was necessary to know the best conditions for LmLAAO activity. Thus, using the method of Kishimoto and Takahashi (2001), LmLAAO was incubated with 5 mmol/L of different substrates (L-leucine, L-isoleucine, L-methionine, L-cysteine, L-valine, L-tyrosine, L-tryptophan L-glutamine, Lthreonine, L-serine, L-lysine, L-arginine, L-phenylalanine), with different concentrations of LmLAAO, different buffers pH values and different temperatures. The determination of kinetic parameters of LmLAAO was performed using the method described by Kishimoto and Takahashi (2001), with modifications. After the standardization of the best conditions for LmLAAO (described above), the assay was performed using Tris–HCl 50 mmol/L at pH 8.0 and different concentrations of L-Leucine (0.3–2.3 mmol/L). The LmLAAO concentration remained constant at 4.4 nmol/L. The reaction was maintained at 37  C, and after 1 h, was interrupted by the addition of 50 mL of H2SO4 (2 mol/L). The absorbance was monitored at 492 nm using 630 nm as reference.

2.6.3. Isoelectric point The isoelectric point of LmLAAO was determined using the method described by Arantes et al. (1994). 2.6.4. Amino-terminal sequencing The sequence determination of the first forty residues from the N-terminus of LmLAAO was performed on Shimadzu protein sequencer Automatic System (PPSQ-33A). The sequence was obtained by the method of Edman degradation (Edman and Begg, 1967). 2.6.5. cDNA cloning and sequencing The pair of venom glands was obtained immediately after the natural death of the L. muta snake, which was kept in the Ezequiel Dias Foundation (Belo Horizonte, Brazil). Total RNA was isolated following the procedure described by Chirgwin et al. (1979). The purification of RNA was made in a column of oligo-dT cellulose (Amersham Biosciences) and its integrity was evaluated in vitro using rabbit reticulocyte lysate (Pelham and Jackson, 1976). The cDNA was synthesized from 5 mg mRNA using System for cDNA Synthesis and Cloning (Invitrogen), directionally cloned in plasmid pGEM11Zf þ (Promega) and transformed into E. coli DH5a, as described in Junqueira-de-Azevedo and Ho (2002). For DNA sequencing on a large scale (generating ESTs – Expressed Sequence Tags), random clones were cultured for 22 h in medium containing antibiotic and plasmid DNA was isolated using alkaline lysis as described by Junqueira-de-Azevedo et al. (2006). Then, the DNA was sequenced in ABI 3100 sequencer using BigDye2 kit (Applied Biosystems) primer standard M13. The ESTs generated were compared with databases such as GenBank via Blast tool (http://blast.ncbi. nlm.nih.gov), leading to the identification of these transcripts. The theoretical physical–chemical parameters were predicted and analyzed by ProtParam (www.expasy. org). The comparison of the amino acid sequence with other LmLAAO was performed using FASTA (http://www. ebi.ac.uk/Tools/fasta/index.html) and BLAST (http://blast. ncbi.nlm.nih.gov/Blast.cgi).

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2.6.6. Homology modeling In order to predict the three-dimensional structure of LmLAAO, a model based on sequence homology was manually built. Search for protein homologues was initially performed by using the BLAST search algorithm (http:// www.ncbi.nlm.nih.gov) against the protein data bank (www.rcsb.org). The crystallographic structure of L-amino acid oxidase from Agkistrodon halys pallas (PDB:1REO) (Zhang et al., 2004), which shares 90% of sequence identity with LmLAAO, has been identified and used as a template for molecular modeling. Based on the sequence alignment performed by Multalin (Corpet, 1988), amino acid substitutions were manually included in the model by using COOT (Emsley and Cowtan, 2004). Structure refinement was performed using molecular dynamics with simulated annealing in CNS (Brunger et al., 1998). Stereochemistry of the predicted model was checked by PROCHECK (Laskowski et al., 1993).

containing 100 mg of LmLAAO dissolved in PBS. Another group of 5 mice (CD-1, 18–22 g) was also injected into the right quadriceps with PBS, and used as controls. After 3 h the blood was collected from the tail of the animals and the activity of creatine kinase (CK) in plasma was determined using a commercial kit (Sigma) protocol. The activity was expressed as U/L at 30  C, considering 1 Unit as 1 mmol of NADH/min. To confirm the LmLAAO myotoxic effect, this assay was done with samples from two different samples of LmLAAO (Test 1 and Test 2), obtained by purification protocol 2. 2.7.5. Histological assays After collection of blood from the tail of CD-1 mice injected in the femoral quadriceps to determine the myotoxic activity, animals were sacrificed. The muscles injected with either PBS or LmLAAO were dissected and samples were routinely processed for histological observation, as described above.

2.7. In vivo assays of LmLAAO Animal care was in accordance with ethical recommendations of the International Guiding Principles for Biomedical Research Involving Animals of the Council of International Organizations of Medical Sciences (CIOMS) and was approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUA) of the University of Costa Rica (no CICUA-012-08). 2.7.1. Hemorrhagic activity The hemorrhagic activity was determined by intradermal injection of 50 mg of LmLAAO dissolved in 50 mL of PBS solution in the abdominal region of three mice (strain CD-1, 18–22 g). After 3 h, mice were sacrificed, and the skin was observed for hemorrhagic halo formation (Gutiérrez et al., 1985). 2.7.2. Edema-inducing activity A group of 6 mice (CD-1, 18–22 g) were injected with 10 mg LmLAAO subcutaneously in the subplantar region of the right footpad. The left footpad was used as control and injected with PBS. At different time intervals (15 min, 1, 3 and 24 h), the thickness of the mice paws was measured with a low-pressure spring caliper (Lomonte et al., 1993). The formation of edema was expressed as a percentage of increment in footpad thickness. 2.7.3. Systemic toxicity The systemic toxicity was evaluated in a group of 6 mice (CD-1, 18–22 g) by intravenous injection of 100 mg of LmLAAO. A group of 6 mice (CD-1, 18–22 g) injected with PBS was used as control. Animal behavior was monitored during the first 3 h after injection. After 24 h of injection, animals were sacrificed and the tissues of heart, lung and kidney were removed, dissected, and samples were processed for histological observation, embedded in paraffin, cut to 4 mm thick and stained with hematoxylin and eosin. 2.7.4. Myotoxic activity A group of 5 mice (CD-1, 18–22 g) was injected intramuscularly in the right quadriceps with a solution

2.8. In vitro assays of LmLAAO 2.8.1. Cytotoxic activity The cytotoxic activity of LmLAAO was tested on the breast adenocarcinoma line (MCF-7), and on a gastric adenocarcinoma line (AGS). The cells were maintained in Dulbecco’s essential medium supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 100 IU/mL penicillin and amphotericin B at 37  C in a humidified atmosphere containing 7% CO2. For the experiments, cells were cultured in 96-well plate (15,000 cells/well) and left overnight. LmLAAO at different concentrations (75, 37.5, 18.8, 9.4, 4.7, 2.3 and 1.2 mg/mL) was added to adhered cells and incubated for 24 h. Assays were performed in the presence of catalase (0.1 mg/mL). The assessment of LmLAAO for toxic activity on the cells was performed using the technique of MTT colorimetric reduction (Mosmann, 1983). The tests were performed in triplicate and expressed as percentage of cell lysis. The half inhibitory concentration (IC50), mean and standard deviation were calculated using the software Graphpad Prism 5.0. 2.8.2. Antiparasitic activity 2.8.2.1. Leishmania braziliensis. Promastigote forms (L. braziliensis) were cultivated in medium 199 supplemented with 10% fetal bovine serum, penicillin and streptomycin and maintained at 22  C. Parasites in the stationary phase were deposited in 96-well microplates at a concentration of 1  106 parasites/mL and incubated with different concentrations of LmLAAO (0.5, 2, 8 and 32 mg/mL). Controls were performed with water, medium 199, catalase (0.1 mg/mL) and the parasites strain. After 24 h, the parasite viability was determined using the technique of MTT colorimetric oxidation as described by Muelas-Serrano et al. (2000). The tests were performed in triplicate for each concentration of LmLAAO and controls. Results were expressed as percentage of cell lysis (%CL) and the IC50, mean and standard deviation were calculated by Graphpad Prism 5.0 software.

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2.8.2.2. Trypanosoma cruzi. Trypomastigotes forms of B5 clone of CL Brener strain were grown in culture of LLCMK2 cells and the assays were performed according to the methodology described by Vega et al. (2005), in which the parasites were incubated in 96-well microplate in the presence of different concentrations of LmLAAO (0.5, 2, 8, 32 mg/mL), at 4  C, for 24 h. After this period, 50 mL of the solution of CPRG (chlorophenol red b-D-galactopyranoside; 400 mM in 0.3% Triton X-100, pH 7.4) were added to the wells and the plate incubated at 37  C for 6 h. Controls were performed with water, in the presence of RPMI 1640, catalase (0.1 mg/mL) and the parasites strain. The assays were performed in triplicate for each concentration of LmLAAO and controls. Results were expressed as percentage of cell lysis (%CL) and the mean and standard deviation were calculated by Graphpad Prism 5.0 software. 2.9. Statistical analysis All statistical analyses were performed using the software SPPS 17.0 for Windows or GraphPad Prism 5.0. p < 0.05 values were considered statistically significant. 3. Results and discussion 3.1. LmLAAO purification Both purification protocols resulted in highly pure and active LmLAAO (Fig. 1 and Fig. 2). The homogeneity of LmLAAO after purification by protocols 1 and 2 was confirmed by the presence of a single band in SDS-PAGE upon reducing conditions (Fig. 1B insert and Fig. 2C insert), by a single peak in RP-HPLC (Fig. 3A) and by mass spectrometry analysis (Fig. 3B). In protocol 1, the purification of LmLAAO was successfully carried out by two chromatographic steps, whereas the second protocol required three chromatographic steps. The initial amount of venom used in the first protocol was only 20 mg, while for the second protocol 200 mg was used. LmLAAO activity recovery after both purification procedures (Table 1) was shown to be very similar (41.4% and 39.9%). However the yield of protein obtained by protocol 1 (4.35%) was found to be half the value obtained by protocol 2 (8.57%). This result can be explained in terms of the total amount of soluble protein used as the starting material. At higher concentrations, as used in protocol 2 (200 mg/3 mL), the insoluble fraction is expected to be higher when compared to protocol 1 (19.3 mg/0.5 mL). As a consequence, considering that LmLAAO displays higher solubility than other venom components, we consider that the initial ratio of LAAO compared to the total amount of solubilized proteins is expected to be higher in protocol 2. This hypothesis also explains the lower specific activity of venom solution used in the protocol 1 (111 U/mg) compared to protocol 2 (364 U/mg). Finally, the specific activity of LmLAAO obtained by protocol 1 (1160 U/mg) was slightly lower than the obtained by protocol 2 (1692 U/mg), suggesting that the latter procedure, despite involving three chromatographic steps, was effective in isolating highly active enzyme. The development of two different purification protocols for this enzyme offers greater versatility to

Fig. 1. Purification protocol 1. (A) Chromatogram of L. muta venom on Sephacryl S-100Ò. The venom (20 mg) was dispersed in elution buffer, applied on the column equilibrated with 20 mM Tris–HCl, pH 7.2, at 25  C, and eluted at the flow rate of 0.5 mL/min. Insert: SDS-PAGE under both reducing (R) and non-reducing conditions (NR) of the fraction with LAAO activity, named SLm1. (B) Chromatogram of SLm1 on MonoQÒ column. The sample was eluted with a stepwise gradient of 1 M NaCl (0–100%) in 0.02 M Tris–HCl, pH 7.2 (Buffer B), at 25  C, at the flow rate of 1 mL/min. Insert: SDS-PAGE of SLm1 under reducing conditions (R).

researchers who need to isolate the enzyme in future works. 3.2. Physicochemical properties of LmLAAO LmLAAO appeared as a single band in SDS-PAGE under reducing conditions (Fig. 1B insert and Fig. 2C insert), showing an estimated molar mass of 60 kDa. The molar mass of LmLAAO determined by MALDI-TOF (60.852 Da) was different from calculated mass predicted by the software Protparam, based on the protein sequence deduced from the cDNA sequence (56.538 Da) (Fig. S1 and S2). This difference might be explained by the presence of posttranslational modification, such as N-glycosylation, as observed for the LAAO from C. rhodostoma, which is responsible for a mass increment of 3.7 kDa (Geyer et al., 2001), and the presence of cofactor FAD (0.785 kDa). LmLAAO has a molar mass (60.8 kDa) which is very similar to the values determined for the LAAO from Agkistrodon halys pallas (60.7 kDa) (Zhang et al., 2004) and LAAO from Vipera libetina (60.9 kDa) (Tonismagi et al., 2006). The isoeletric point of LmLAAO (pI 6.28) predicted by the software Protparam also showed a difference when compared with the experimentally determined pI (5.1) by

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et al., 2006) and LAAO from Bothrops atrox (pI 4.4) (Alves et al., 2008). 3.3. Enzymatic properties of LmLAAO

Fig. 2. Purification protocol 2. (A) Chromatogram of L. muta venom on Sephacryl S-200Ò. The venom (200 mg) was dissolved in the elution buffer, applied on the column equilibrated with 20 mM Tris–HCl, pH 7.2, at 25  C, and eluted at a flow rate of 0.5 mL/min. Insert: SDS-PAGE under both reducing (R) and non-reducing conditions (NR) of the fraction with LAAO activity, named SpLm1. (B) Chromatogram of SpLm1 on Phenyl SepharoseÒ. The elution was performed with 1.5 M NaCl in 20 mM Tris–HCl, pH 8.0, and decreasing concentrations of NaCl, ranging from 1.5 M to 0 (deionized water), at a flow rate of 1 mL/min. Insert: SDS-PAGE of FSLm1, the fraction with LAAO activity, under reducing (R) and non-reducing conditions (NR). (C) Affinity chromatography of FSLm1 on Affi-gelÒ blue gel. The fraction FSLm1 was dialyzed, applied to the column previously equilibrated with 20 mM Tris–HCl, pH 8.0 (buffer A) and eluted with a gradient (0–100%) of 1.5 M NaCl in 20 mM Tris–HCl (buffer B), at 25  C, at a flow rate of 0.5 mL/ min. Insert: SDS-PAGE of AfLm1, the isolated LAAO, under reducing (R) and non-reducing conditions (NR). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

isoelectric focusing (results not shown). This discrepancy can be explained by the outward orientation of charged amino acids in its three-dimensional structure or possibly by charges introduced by glycosylations. The acidic characteristic of LmLAAO is also observed in LAAOs from other snake venoms such as LAAO from B. pirajai (pI 4.9) (Izidoro

It has been described that LAAOs substrate binding sites comprise three hydrophobic subsites, presenting one or two methyl/methylene carbons, and an amino binding subsite (Tan, 1998; Zhong et al., 2009). This explains the catalytic preference of LmLAAO by hydrophobic amino acids (L-Met, L-Leu, L-Phe, L-Trp, L-Tyr and L-Ile). For other amino acids, the catalytic activity was very low or even absent (Fig. 4A). These results are in accordance with other previously characterized svLAAOs (Alves et al., 2008; Ciscotto et al., 2009; Izidoro et al., 2006; Rodrigues et al., 2009; Samel et al., 2008; Tonismagi et al., 2006; Zhong et al., 2009), showing that the catalytic site has a conserved structure among snake species. When the relative enzymatic activity of LmLAAO was measured between pH 7.0 and 9.0, it exhibited optimal hydrolysis of L-Leu at pH 8.0 (Fig. 4B), which is similar to results found for other svLAAOs (Dineshkumar and Muthuvelan, 2011; Zhong et al., 2009). Buffers with pH values above 9.0 and below 7.0 can cause structural changes in both LmLAAO and horseradish peroxidase, interfering with the assay. The catalytic activity of LmLAAO was also evaluated at different temperatures. LmLAAO showed only 5.9% of its activity after freezing-thawing at 70  C for 1 h. After 30 min at 100  C, the enzyme had lost 100% of its enzymatic activity. The best temperature for storing LmLAAO was 4  C (Fig. 4C). The determination of Km and Vmax for the substrate L-leucine was performed by derivation of the Michaelis– Menten elliptic curve by GraphPad Prism 5.0 program (Fig. 4D). This software was used for setting the reliability of data in nonlinear regression. LmLAAO presented a Km value of 0.97  0.07 mmol/L and Vmax de 0.063  0.002 mmol min1 for L-Leucine. The 95% confidence interval was 0.81–1.14 for Km and from 0.059 to 0.068 for Vmax (8 degrees of freedom). Concerning the kinetics parameters, the low value of Michaelis–Menten constant for the amino acid L-Leucine confirms the catalytic preference of the LmLAAO for hydrophobic amino acids. This catalytic preference might be explained by the presence of amino acids that promote a non-polar environment in the catalytic site. 3.4. N-terminal sequencing, cDNA cloning and sequencing and molecular modeling The sequence of the first forty residues from the Nterminal of LmLAAO determined by Edman degradation was ADDRNPLGECFRETDYEEFLEIAKNGLRATSNPKHVVIGA, showing that it is a new enzyme from L. muta venom. The complete amino acid sequence of LmLAAO (Figs. S1 and S2) was deduced by Expressed Sequence Tags (ESTs) sequencing (Fig. S1). The obtained ESTs were subsequently aligned with the LAAOs from other snakes, leading to the identification of these transcripts. Among the identified transcripts, twenty ESTs showed high similarity with other

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Fig. 3. RP-HPLC and mass spectrometry of LmLAAO. (A) Chromatogram of AfLm1 on C-4 Reverse-Phase HPLC. Adsorbed protein was eluted with an acetonitrile gradient (0–70%) at a flow rate of 1 mL/min. Insert: SDS-PAGE of LmLAAO under reducing conditions (R). (B) Molar mass of LmLAAO determined by MALDI–TOF mass spectrometry.

snake LAAOs. The complete sequence of the cDNA of L. muta LAAO was resolved by the superposition of these twenty ESTs and confirmed manually. The complete deduced cDNA was named LMUT0069C. The overall proteomic profile of L. muta venom reported by Sanz et al. (2008) showed that L. muta venom contains a single LAAO molecule. This information, along with the N-terminal (ADDRNPLGECFRETDYEEFL) and internal sequences reported by them (SAGQLYEESLGK and KFWEDDGIR, corresponding to LmLAAO amino acid residues 152–163 and 334–342, respectively), are also evidences that the cDNA-deduced protein sequence

reported now may actually correspond to the venom expressed protein. LmLAAO showed high sequence identity with LAAOs from other snake venoms, such as Sistrurus catenatus edwardsii (91%), Crotalus atrox (91%), A. halys pallas (90%), Crotalus adamanteus (90.6%), Trimeresurus stejnegeri (89%) and Calloselasma rhodostoma (88%) (Fig. S2). In fact, the high sequence identity shared by L. muta and A. halys pallas LAAOs (Fig. S2) allowed us to predict the tertiary structure of the monomeric form of LmLAAO (Fig. 5). The final model consists of a 486 amino acid polypeptide chain and one FAD molecule. The fourteen last

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Table 1 Protein and LAAO activity recovery of each active fraction obtained by purification protocols 1 and 2. Sample Protocol 1

Protocol 2

c

LmSV SLm1 Sephacryl S-100Ò MQ2 (LmLAAO) MonoQÒ LmSVc SpLm1 Sephacryl S-200Ò FSLm1 Phenyl SepharoseÒ AfLm1 (LmLAAO) Affi-gelÒ

Protein (mg)

Protein (%)

LAAO Activity (U)a

LAAO Activity (%)

Specific Activity (U/mg)b

19.3 1.41

100 7.25

2150 1045

100 48.6

111 741

0.84

4.35

890

41.4

1060

200 78.50

100 31.71

72,750 67,159

100 92.3

364 856

40.56

20.28

41,966

57.7

1035

17.14

8.57

29,005

39.9

1692

The figures in boldface compare the found yield of two purification protocols. a LAAO activity unit (U): amount of protein able to release 1.0 mmol of H2O2 per minute. b Specific activity: amount of H2O2 (mmol) released per minute per mg of protein. c LmSV: L. muta venom.

residues are missing in the protein model due to the lack of information on template structure. Analysis of Ramachandran plot revealed that 95.9% residues are in most favored, 3.1% in additionally allowed, and 1.0% in disallowed regions. The overall fold of snake venom LAAOs consists of three domains: a FAD-binding domain, the substrate binding domain and the a-helical domain (Fig. 5). The FAD cofactor is found inside a cavity formed between cofactor binding and the substrate binding domains. In terms of overall structure, no major structural differences have been found when comparing the

simulated LmLAAO structure with the template model (PDB entry: 1REO). In fact, structural comparison of all LAAO crystal structures available at the protein data bank (PDB entries: 1REO, 3KVE, 2IID, 1TDN) suggests a high degree of sequence identity and structural similarity amongst snake venoms LAAOs (Fig. S2). They share over 80% of sequence identity and the root mean square deviation (RMSD) of aligned Ca atoms is found to be in average 0.6  A. Out of a total of 49 substitutions, only 6 positions were found to be occupied by uncommon residues when

Fig. 4. LmLAAO enzymatic properties. (A) Substrate specificity of LmLAAO (B) Dependence of pH for LmLAAO activity determined by colorimetric assay. Results are expressed as mean  SD (n ¼ 3). (C) Dependence of temperature for L-amino oxidase activity. Results are expressed as mean  SD (n ¼ 3). (D) Kinetic parameters of LmLAAO for L-Leu as substrate: Km ¼ 0.9737 mmol/L  0.0736 mmol/L and Vmax ¼ 0.06345 mmol min1  0.00209 mmol min1.

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Fig. 5. Three dimensional structure model for LmLAAO. FAD-binding domain (pink), the substrate binding domain (blue) and the a-helical domain (wheat). FAD molecule is represented in yellow and the glycosilation site (Asn172) is found in red. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

considering snake venoms LAAOs: Glu54, His113, Glu135, Gln141, Pro284 and Thr303 (Fig. 6). Nevertheless, those random punctual amino acid substitutions do not introduce any significant changes in charge or volume distribution over the protein structure. The majority of amino acid substitutions are solvent exposed and spread out over the protein surface (Fig. 6). They are found to be either invariant or in agreement with the sequence variation observed amongst snake venoms LAAOs, which shows that specific positions can be occupied by amino acids of different chemical properties and shape (Fig. S2). When comparing the predicted model for LmLAAO to the template structure, A. halys pallas LAAO, we found that FAD cofactor binding and the substrate binding domains as

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well as the glycosylation sites are kept intact. These results showed the conservation of regions important for enzyme function. For example, the presence of His223 and Arg322 residues that are found conserved in LAAOs sequences is important for the catalytic reaction mediated by LAAO. The side chain of His223 undergoes a conformational change that allows the transfer of the pair of nitrogen electrons from the imidazole ring to the nitrogen of a carbon present in the L-amino acid substrate. This is followed by concomitant loss of hydride and for the formation of imino acids (Moustafa et al., 2006). The conserved Lys326 (Pawelek et al., 2000), makes a hydrogen bond with a water molecule that interacts with the N5 from aloxazine ring in the FAD cofactor. The presence of Lys326 would help the attack of water on the intermediate imino acid, transforming it in the corresponding a-keto acid by non-enzymatic cleavage. Moreover, the residues Phe328, Ile370, Tyr372, Tyr356, Met89 and Leu86 are also conserved in svLAAOs and form a hydrophobic environment around the Lys326 (Pawelek et al., 2000). Finally both the Asn172 and Asn361 residues are maintained, and these positions are usually found glycosylated (Georgieva et al., 2011; Pawelek et al., 2000). These two glycosylation sites, found solvent exposed (Fig. 5) are described to be important for interaction between LAAOs and cell surface, increasing the concentration of hydrogen peroxide at the region of interaction, leading to cellular toxicity (Geyer et al., 2001).

3.5. In vivo effects of LmLAAO Intradermal injection of 50 mg of LmLAAO in the abdominal region of mice did not cause hemorrhage. Likewise, no paw edema was induced in mice injected with 10 mg of enzyme, when compared to PBS (P ¼ 0.518), used as negative control (results not shown). Furthermore, no morphological changes were observed in mice heart, lung and kidney tissues after 24 h of intravenous injection of

Fig. 6. LmLAAO surface indicating the conserved (dark pink) and not conserved (blue) substitutions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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100 mg of LmLAAO, compared to tissues from mice injected with PBS (Fig. 7). Conversely, two different samples of LmLAAO (Test 1 and Test 2), obtained by purification protocol 2, were injected in quadriceps muscle of two groups of 5 mice and a significant increase in serum creatine kinase activity was observed, when compared with the control group injected with PBS (U ¼ 2.745, p ¼ 0.006 in Test 1, and U ¼ -2.739, p ¼ 0.006 in Test 2). There were no significant differences between the results of Test 1 and Test 2 (U ¼ 12, p ¼ 0.917), indicating that the purification protocol 2 shows good reproducibility and reliability, since the two different samples showed similar activities (Fig. 8A). The mild myonecrosis induced by LmLAAO was confirmed by histological alterations observed in muscle (Fig. 8C), when compared with the control (Fig. 8B). Thus, it was

demonstrated that LmLAAO is an enzyme able to induce low toxicity in vivo. 3.6. In vitro toxicity 3.6.1. Cytotoxic activity LAAOs from snake venoms are described as enzymes with antitumor and apoptotic effects in various types of cells (Alves et al., 2008; Rodrigues et al., 2009). The LmLAAO median cytotoxic concentration (IC50) for AGS cell line (Fig. 9A) was 22.7 mg/mL (95% confidence interval: 11.6 mg/mL to 44.5 mg/mL). Likewise, LmLAAO induced dose-dependent cytotoxicity in MCF-7 cell line (Fig. 9B), with an IC50 of 1.4 mg/mL (95% confidence interval: 1.2 mg/mL to 1.7 mg/mL). The cytotoxic effect of LmLAAO was mainly attributed to the release of hydrogen

Fig. 7. Light micrographs of the heart muscle, lung and kidney after intravenous injection of LmLAAO. Tissues were collected 24 h after i.v. injection of 100 mL of PBS (controls) or 100 mg of LmLAAO dissolved in 100 mL of PBS. After routine processing and paraffin embedding, sections were prepared and stained with hematoxylin-eosin. 400. (I A) Cardiac muscle from mice injected with PBS and (I B) Cardiac muscle from mice injected with LmLAAO. (II A) Lung tissue from mice injected with PBS and (II B) lung tissue from mice injected with LmLAAO. (III A) Kidney tissue from mice injected with PBS and (III B) Kidney tissue from mice injected with LmLAAO. III B shows a detail of the renal capsule. Notice the absence of pathological alterations in tissues injected with LmLAAO.

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Fig. 8. Myotoxic activity of LmLAAO. (A) Creatine kinase activity in the plasma of mice injected in the quadriceps muscle with two different samples of LmLAAO (100 mg) purified by protocol 2 (Test 1 and Test 2). Results are expressed as mean  SD (n ¼ 5). *P < 0.05. (B) Light micrograph of a section of quadriceps muscle injected with 100 mL of PBS (control). (C) Light micrograph of a section of quadriceps muscle injected with 100 mL of LmLAAO (1 mg/mL). Muscle samples were collected 3 h after injection and processed routinely for paraffin embedding, sectioning and staining with hematoxylin-eosin. Notice a mild myotoxic effect in muscle injected with LmLAAO, evidenced by the presence of some necrotic cells (arrow). 400.

peroxide to the medium since the presence of catalase at concentration of 0.1 mg/mL completely abrogated the toxic action of LmLAAO in both cell lines. In the presence of catalase, which destroys hydrogen peroxide released, this effect is significantly reduced or abolished (Torii et al., 1997). The inhibitory effect of LAAO on tumor growth has been demonstrated on different cell lines, such as human promyelocytic leukemia HL-60, HeLa, glioma, human ovary carcinoma A2780, endothelial cells from the human umbilical cord, mouse NR-3 endothelial cells, murine EL-4 lymphoma cells, SKBR-3 cells, Jukart cells and Eat cells (Ciscotto et al., 2009; Kanzawa et al., 2004; Iijima et al., 2003; Souza et al., 1999; Sun et al., 2003; Torii et al., 1997). Moreover, this is the first study showing the cytotoxic effects of LAAO on AGS and MCF-7 cell lines. 3.7. Antiparasitic activity Fig. 9C shows the dose-dependent inhibitory activity (IC50: 2.2 mg/mL; 95% confidence interval: 1.9–2.6 mg/mL) of LmLAAO on the promastigote form of L. braziliensis. The addition of catalase completely inhibited LAAO activity. Leishmanicidal studies have demonstrated that LmLAAO is not as toxic as LAAO from Bothrops moojeni. Tempone et al. (2001) used 1.44 mg/mL of B. moojeni LAAO to reach the IC50 for L. braziliensis whereas 2.2 mg/mL of LmLAAO is required to obtain the same result.

It was not possible to determine the median inhibitory concentration of LmLAAO on the T. cruzi Brener strain (Fig. 9D), since maximum concentration of LmLAAO used (32 mg/mL) was not able to induce death of 50% of the parasites. These results indicate that this strain of Trypanosoma cruzi is somewhat resistant to the cytotoxic action of LmLAAO. Parasite resistance was also observed in assays performed with LAAO from B. atrox, since a dose of 32 mmol/L was necessary to kill 41.7  2.4% of trypomastigotes (Alves Paiva et al., 2011). In conclusion, LmLAAO shows a low toxicity in vivo when compared with other enzymes or toxins from snake venoms, but it might be used as cytotoxic tool toward pathogens or cancer cells, as verified by in vitro toxicity experiments. Additionally this study showed, for the first time, the cytotoxic effects of LAAO on AGS cell line (gastric adenocarcinoma) and MCF-7 cell line (breast adenocarcinoma). Furthermore, our analyses show evolutionary sequence and structural conservation of LAAOs across snake species, suggesting the existence of selective pressures in the evolution of this enzyme. Therefore, the biochemical, structural and functional characterizations of LmLAAO, demonstrates that it is a novel LAAO molecule with several important biological functions. Funding source The work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil,

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Fig. 9. In vitro cytotoxic and antiparasitic activity of LmLAAO. (A) Cytotoxic activity of LmLAAO upon AGS gastric carcinoma line. Cells were incubated for 24 h in the presence of different concentrations of LmLAAO (75, 37.5, 18.75, 9.37, 4.68, 2.34 and 1.17 mg/mL). Controls were performed in the presence of catalase (0.1 mg/ mL). Results are expressed in percentage of cell lysis (mean  SD; n ¼ 3). (B) Cytotoxic activity mediated by LmLAAO on MCF-7 breast carcinoma line. LmLAAO at different concentrations of (75, 37.5, 18.75, 9.37, 4.68, 2.34 and 1.17 mg/mL) was added to adhered cells in microplate, and incubated for 24 h Controls were performed in the presence of catalase (0.1 mg/mL). Results are expressed in percentage of cell lysis (mean  SD; n ¼ 3). (C) Antiparasitic activity of LmLAAO upon Leishmania braziliensis line. Parasites in the stationary phase were incubated for 24 h in the presence of LmLAAO at different concentrations (0.5, 2, 8 and 32 mg/mL). Controls were performed in the presence of catalase (0.1 mg/mL). Results are expressed in percentage of cell lysis (mean  SD; n ¼ 3). (D) Antiparasitic activity of LmLAAO upon Trypanosoma cruzi line. Parasites were incubated for 24 h in the presence of LmLAAO at different concentrations (0.5, 2, 8 and 32 mg/mL). Controls were performed in the presence of catalase (0.1 mg/mL). Results are expressed in percentage of cell lysis (mean  SD; n ¼ 3).

under Gramts No 479873/2009-7 and No São Paulo (FAPESP), Brazil, under Grant No 2005/54855-0 and Instituto Nacional de Ciência e Tecnologia de Toxinas (INCTTox, Fapesp/CNPq). Database Model data are available in the PMDB (Protein Model Data Base) under accession number PM0077706 (http://mi. caspur.it/PMDB/user/search.php). The amino acid sequence data are available in the DDBJ/EMBL/GenBank database under the accession number JX171244 (http://www.ebi.ac. uk/ena/data/view/JX171244) and Nucleotide sequence data are available in the DDBJ/EMBL/GenBank databases under the accession number LMUT0069C (http://www.ebi.ac.uk/ ena/data/view/JX171244).

Acknowledgment We are grateful to Dra Elizabeth Abrahams, Departamento de Parasitología, Facultad de Microbiología, Universidad de Costa Rica, for her contribution with some of Trypanosoma strains tested in cytotoxicity experiments.

Thanks are also due to Karla de Castro Figueredo Bordon and Aarón Gómez Argüello for technical assistance. The work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil, under Grants No 479873/2009-7 and No 142711/2007-1 (Ph.D. scholarship), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil, under Grant No 2005/ 54855-0 and Instituto Nacional de Ciência e Tecnologia de Toxinas (INCTTox, Fapesp/CNPq). Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.toxicon.2012.08.008. Conflicts of interest statement The authors declare that there are no conflicts of interest. References Ahn, M.Y., Lee, B.M., Kim, Y.S., 1997. Characterization and cytotoxicity of Lamino acid oxidase from the venom of king cobra (Ophiophagus hannah). Int. J. Biochem. Cell. Biol. 29, 911–919.

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