Biochemical properties of a new PI SVMP from Bothrops pauloensis: Inhibition of cell adhesion and angiogenesis

International Journal of Biological Macromolecules 72 (2015) 445–453

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Biochemical properties of a new PI SVMP from Bothrops pauloensis: Inhibition of cell adhesion and angiogenesis David Collares Achê a , Mário Sérgio R. Gomes b , Dayane Lorena Naves de Souza a , Makswell Almeida Silva a , Maria Inês Homsi Brandeburgo a , Kelly Aparecida Geraldo Yoneyama a , Renata Santos Rodrigues a , Márcia Helena Borges c , Daiana Silva Lopes a,∗ , Veridiana de Melo Rodrigues a,∗ a

Institute of Genetics and Biochemistry, Federal University of Uberlandia, UFU, 38400-902 Uberlandia, MG, Brazil Department of Chemical and Physical, State University of Southwest Bahia (UESB), 45506-210 Jequie, BA, Brazil c Ezequiel Dias Foundation, FUNED, 30510-010 Belo Horizonte, MG, Brazil b

a r t i c l e

i n f o

Article history: Received 9 May 2014 Received in revised form 27 August 2014 Accepted 28 August 2014 Available online 6 September 2014 Keywords: Angiogenesis inhibition Bothrops pauloensis Endothelial cells Matrigel Metalloproteinase Snake venom

a b s t r a c t In the present work, we demonstrate some biochemical and functional properties of a new PI snake venom metalloproteinase (SVMP) isolated from Bothrops pauloensis snake venom (BpMP-II), in addition we evaluated its capacity to inhibit endothelial cell adhesion and in vitro angiogenesis. BpMP-II was purified after a combination of three chromatography steps and showed molecular mass of 23,000 Da determined by MALDI-TOF, an isoelectric point of 6.1 and the sequence of some fragments obtained by MS/MS (MALDI TOF\TOF) presented high structural similarity with other PI-SVMPs. BpMP-II showed proteolytic activity against azocasein, was able to degrade bovine fibrinogen and was inhibited by EDTA, 1.10 phenantroline and ␤-mercaptoethanol. BpMP-II did not induce local hemorrhage in the dorsal region of mice even at high doses and did not affect plasma creatine kinase (CK) levels when administered intramuscularly into the gastrocnemius muscle of mice. Moreover, this metalloproteinase decreased tEnd cells viability at concentrations higher than 20 ␮g/mL. With sub-toxic doses this metalloproteinase affected tEnd cell adhesion and was also able to inhibit in vitro angiogenesis. BpMP-II showed very important functional properties suggesting considerable therapeutic potential for this class of protein. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Metalloproteinases or metalloendopeptidases (MP) are hydrolytic enzymes classified as endopeptidases (E.C.3.4.24) dependent on metal binding, usually zinc, for the catalytic site to be activated. They are important in physiological and pathological processes such as cellular adhesion, fertilization, signal transduction, intoxication by venomous animals, etc. These enzymes occur widely in all five kingdoms of life [1,2], the most studied of which are present in snake venom (SVMPs – Snake Venom Metalloproteinases). The group of SVMPs is characterized by a large molecular mass spectrum (20 a 110 kDa) according to the number of

∗ Corresponding authors at: Institute of Genetics and Biochemistry, Federal University of Uberlandia, UFU, 38400-902 Uberlandia, MG, Brazil. Tel.: +55 34 32182203x22; fax: +55 34 32182203x24. E-mail addresses: [email protected], [email protected] (D.S. Lopes), [email protected] (V.d.M. Rodrigues). http://dx.doi.org/10.1016/j.ijbiomac.2014.08.050 0141-8130/© 2014 Elsevier B.V. All rights reserved.

structural domains present in the structure. These domains have been characterized due to their specific functions and are named as catalytic domain, disintegrin or disintegrin-like domain, cysteinerich domain and lectin-like domain. Fox and Serrano (2008) [3] classified SVMPs into 11 subclasses (PIa, PIIa, PIIb, PIIc, PIId, PIIe, D-I, PIIIa, PIIIb, PIIIc and PIIId) based on studies of SVMP precursors and products of post-translational modifications. The structural complexity of SVMPs gives them the capacity to interfere in various physiological processes that resulting in several physiopathological changes such as hemorrhage, myonecrosis, inflammation and inhibition of platelet aggregation [4,5]. The main effect is related to their capacity to degrade extracellular matrix (ECM) proteins, cellular membrane proteins and plasma proteins [4,6–9]. Beyond those effects, metalloproteinases are also capable of interacting with specific receptors such as integrins on the surface of platelets [10,11], fibroblasts [12] and endothelial cells [13,14] thereby activating or inhibiting cellular responses. SVMPs are reported to induce apoptosis and disrupting the angiogenic process of endothelial cells [15–20]. Angiogenesis is important in the pathogenesis of a broad range of disorders such as

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arthritis and cancer. The microvascular endothelial cell recruited by a tumor is an important target in cancer therapy and treating both the cancer cell and the endothelial cell in a tumor may be more effective than treating the cancer cell alone [21,22]. Currently, anticarcinogenic activities of snake crude venoms and their components have been recognized [23]. Thus the biodiversity of venoms and toxins makes them a unique source from which novel therapeutics may be developed. Bothrops pauloensis venom toxins have been isolated in recent years and their functional and structural characteristics have been demonstrated [24–26]. Additionally, the transcriptomics and proteomics studies of the Bothrops pauloensis venom gland revealed the presence of many peptides and proteins that have not yet been isolated and characterized in this venom, such as metalloproteinases [27]. Due the great diversity of metalloproteinases present in B. pauloensis venom [27] and their great therapeutic potential [28], the present work aimed to isolate and to evaluate the biochemical and enzymatic properties of a new PI-metalloproteinase from B. pauloensis, as well as to evaluate its capacity to inhibit endothelial cell adhesion and in vitro angiogenesis. 2. Materials and methods 2.1. Venom and animals Bothrops pauloensis venom was obtained from specimens kept at Serpentarium Bioagents, LTDA, Batatais-SP, Brazil. This serpentarium is registered in the Brazilian Institute of the Environment and Renewable Natural Resources (no. 471301). Male BALB/c mice were obtained from CBEA (Centro de Bioterismo e Experimentac¸ão Animal) of Federal University of Uberlandia and maintained under standard conditions (12 h light/dark cycle, temperature 22 ± 1 ◦ C, diet and water ad libitum). The procedures protocol was approved by the Committee of Ethics for Use of Animals of the Federal University of Uberlandia, Minas Gerais, Brazil (protocol number 046/09), and was in agreement with the ethical principles of animal experimentation according to the Brazilian Society of Science for laboratory animals (COBEA/SBCAL). 2.2. Reagents CM-Sepharose, Sephacryl S300, HiTrap Capto-Q and Reverse phase C18 (Whatman® ) chromatographic columns were obtained from Amersham Bioscience of Brazil. Acrylamide, N,N -methylenebisacrylamide, N,N,N ,N , tetramethylethylenediamine (TEMED), ammonium persulfate (PSA), sodium dodecyl sulfate (SDS), bromophenol blue, bovine fibrinogen, ethylenediaminetetraacetic acid (EDTA), bovine serum albumin (BSA), aprotinin, benzamidine, ␤-mercaptoethanol, 1,10-phenanthroline and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium) bromide were obtained from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA). Molecular weight markers were purchased from GE Healthcare Bio-Sciences (Pittsburgh, USA). 2.3. BpMP-II Isolation Metalloproteinase BpMP-II was purified from B. pauloensis snake venom according to methods previously described by our research group [26,29] with some modifications. Initially, desiccated crude venom (200 mg) was dissolved in 2 mL of 50 mM ammonium bicarbonate (AMBIC) buffer (pH 7.8) and centrifuged at 3000 × g for 10 min at 4 ◦ C. The supernatant was recovered (137 mg) and submitted to a CM-Sepharose column previously equilibrated with the same buffer. Fractions of 1 mL/tube were collected in a linear gradient from 0.05 M to 0.5 M AMBIC buffer pH 7.8 at a flow rate

of 6.5 mL/h and monitored by an Ultrospec 1000 Spectrophotometer (Amersham Pharmacia Biotech) with an absorbance of 280 nm. The fractions were lyophilized and stored at −20 ◦ C. Fraction CM1 (40 mg) obtained from chromatography using the CM-Sepharose column was then dissolved in 0.05 M AMBIC and applied onto a HiPrep 26/60 Sephacryl S300 column previously equilibrated with AMBIC buffer (0.05 M, pH 7.8). Elution was performed using the same buffer with a flow rate of 0.2 mL/min on the Akta Prime Plus (Amersham Biosciences) system. Fractions were collected, lyophilized and stored at −20 ◦ C. The fraction CM1S3 obtained from the previous step was then submitted to anion exchange chromatography using a Capto-Q column previously equilibrated with 0.05 M AMBIC buffer. Fractions of 1 mL/tube were collected in a linear gradient with 0.5 M AMBIC buffer at a flow rate of 0.4 mL/min. The main peak from the previous step, named BpMP-II, was diluted in 1 mL of trifluoracetic acid (TFA, 0.1%, (v/v)) and submitted to an HPLC system using a reverse phase column (Partisphere C18 Whatman® ) (4.6 mm × 125 mm). Sample elution was initially made with 0.1% trifluoroacetic acid followed by a linear gradient of 80% acetonitrile at a flow rate of 1 mL/min at room temperature. The peak was isolated and lyophilized for further structural and chemical characterization. The protein concentration was determined according to Bradford (1976) [30]. In this method, each sample was dissolved in 100 ␮L of deionized water and 3 mL of Bradford (Sigma–Aldrich Chemical Co.) reagent was added. The concentration was determined spectrophotometrically at 595 nm in triplicate. 2.4. Biochemical characterization 2.4.1. Determination of molecular Mr BpMP-II Mr was estimated by 12.5% (w/v) SDS-PAGE [31]. Samples of protein (10 ␮g) were heated at 100 ◦ C for 5 min and the run was performed under reducing conditions. Afterwards, the gel was stained with Coomassie Brilliant Blue R-250. The molecular mass marker used contained bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), ␤lactoglobulin (18 kDa) and ␣-lactalbumin (14.4 kDa) (Amersham Biosciences). Protein molecular mass was estimated by interpolation from a linear logarithmic plot of the relative molecular mass versus the distance of migration. 2.4.2. Molecular mass determination by MALDI-TOF BpMP-II molecular mass was also determined by matrixassisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Analyses were made by MALDI double TOF mass spectrometry (MALDI-TOF/TOF, AutoFlex III Bruker Daltonics, Bremen, Germany). Protein (80 ␮g) was resuspended in 10 ␮L of 0.1% TFA. Afterwards, 0.5 ␮L of this solution were mixture with 0.5 ␮L of sinapinic acid (0.1% TFA, 50% ACN) and added to the MALDI target plate (Bruker Daltonics) where they were homogenized and dried at room temperature. According to their mass ranges, protein was recorded in linear positive mode. 2.4.3. Isoelectric focusing Isoelectric focusing was performed according to the manufacturer’s instructions (GE Healthcare). Twenty-five micrograms of protein were dissolved in rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS (w/v), 10 mM DTT, 0.002% bromophenol blue) also containing IPG buffer (GE Healthcare). Polyacrylamide strips with immobilized pH (pH 3 to 10) were rehydrated in the IPGbox for 16 h. Afterwards, isoelectric focusing was performed on strips in the Ettan IPGphor 3 system using the following program: 200 V/1 h, 3000 V/1 h, 4000 V/1 h, 1250 V/1 h and 50 V/1 h. Between the first and second dimensions, focused strips were equilibrated in 10 mL of 1.5 M Tris–HCl pH 8.8, 6 M urea, 30%

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glycerol (v/v), 2% SDS (m/v) and bromophenol blue solution, containing 10 mg/mL DTT for 15 min. A second process using the same solution with 25 mg/mL iodoacetamide was performed. After equilibration, the strips were submitted to 12.5% SDS-PAGE using a Mini VE system (GE Healthcare) and the gel was stained with 2% (w/v) Coomassie Fast Blue R-350. The gel was scanned using Image Master LabScanTM v. 5.0. Analyses were performed with Platinum 2D Master imaging software (GE Healthcare).

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Similarly, this activity was performed by incubating 5 ␮g of enzyme with 50 ␮L of fibrinogen for different time intervals (5 min, 10 min, 15 min, 30 min, 1 h, 2 h and 24 h) at 37 ◦ C. The effect of various protease inhibitors was also evaluated. The enzyme (5 ␮g) was preincubated separately with 10 ␮L solutions containing 10 mM of ␤-mercaptoethanol, EDTA, 1,10-phenanthroline, benzamidine and aprotinin for 1 h at 37 ◦ C. Afterwards, 50 ␮L of bovine fibrinogen (1 mg/mL in PBS pH 7.8) were added. The products of the reactions were analyzed by 12.5% SDS-PAGE.

2.5. Partial sequence determination BpMP-II (100 ␮g) was dispersed in 100 mM of NH4 HCO3 and 8 M urea. It was reduced with 10 mM DTT in 50 mM NH4 HCO3 at 37 ◦ C for 60 min. Alkylation was performed with 50 mM iodoacetamide in 50 mM NH4 HCO3 at 37 ◦ C for 30 min in the dark. An aliquot of Trypsin Gold (Promega, Madison, WI, EUA) 0.5 ␮g/2.5 ␮L was added to the sample and the reaction was allowed to take place overnight at 37 ◦ C. The reaction was interrupted with the addition of 1% TFA (v/v) and the mixture was concentrated using a SpeedVac. The digest products were desalted and separated on a Sep-Pak C18 (Millipore) column (10 mm × 10 mm) using a linear gradient of 0–70% acetonitrile in 0.1% TFA (v/v). The sample was added to 3 ␮L of ␣-cyano-4-hydroxycinnamic acid (␣-CHCA) matrix, applied to a plate (Bruker Daltonics, Bremen, Germany) and dried at room temperature. Monoisotopic peptide masses were determined on external calibration mode. The peptide spectrum (MS/MS) was obtained by LIFT fragmentation. Mass spectra were analyzed using Flex Analysis software (Bruker Daltonics) and protein identification was performed using Biotools and Mascot softwares [32]. The partial sequence of BpMP-II was compared to related proteins in the NCBI database using the FASTA 3 and BLAST programs. For the obtained sequences, alignment with database sequences was done using the ClustalW program [33]. 2.6. Functional characterization 2.6.1. Azocaseinolytic and fibrinogenolytic activities Enzymatic characterization of BpMP-II was initially determined by using azocasein as substrate according to Gomes et al. [34] with modifications. Samples containing 100 ␮L of azocasein (1 mg/mL) in 0.05 M Tris–HCl and 0.15 M NaCl were incubated for 30 min at 37 ◦ C with 45 ␮L of BpMP-II at different concentrations (0.5 ␮g, 1 ␮g, 2 ␮g, 5 ␮g, 10 ␮g and 20 ␮g) in the 96-well polystyrene plates (NUNC MaxiSorp). After, 45 ␮L of trichloroacetic acid 20% (m/v) were added to each sample. The plate was incubated at room temperature for 30 min, and then centrifuged at 3000 × g for 20 min. The absorbance of the supernatant at 366 nm was determined by an EL800 plate reader (BioTek, Washington DC, USA). One unit (U) of azocaseinolytic activity was defined as an increase of 0.01 absorbance units at 366 nm under the standard assay conditions. All assays were performed in triplicate. The effect of different pH values and temperatures upon azocaseinolytic activity was evaluated by incubating 10 ␮g of BpMP-II for 1 h with different buffer solutions at various pH intervals (2.5, 5.0, 7.5 and 10.5) and temperatures (4 ◦ C, 25 ◦ C, 37 ◦ C, 60 ◦ C and 100 ◦ C). The enzymatic assay was performed as described above. Fibrinogenolytic activity was performed as described by Rodrigues et al. [35] with some modifications. Briefly, 50 ␮L samples of bovine fibrinogen (1 mg/mL in PBS pH 7.8) were incubated with different enzyme concentrations (1 ␮g, 2 ␮g, 5 ␮g, 10 ␮g and 20 ␮g) for 1 h at 37 ◦ C. The reaction was stopped with 25 ␮L of 0.06 M Tris–HCl, pH 6.8, containing 10% (v/v) glycerol, 10% (v/v) ␤mercaptoethanol, 2% (w/v) SDS and 0.05% (w/v) bromophenol blue. The samples were then heated at 100 ◦ C for 4 min and analyzed by 12.5% (w/v) SDS-PAGE.

2.6.2. Hemorrhagic activity Hemorrhagic activity was determined as described by Nikai et al. [36], with some modifications. Samples containing 10 ␮g to 100 ␮g of BpMP-II were dissolved in 50 ␮L of PBS and injected intradermally (i.d.) into the dorsal skin of male Balb-C mice (20–25 g, n = 4) to induce the hemorrhagic process. Control mice received only PBS. After 3 h, the animals were sacrificed, the dorsal skin removed and the extent of hemorrhagic spots measured using a low pressure caliper rule. 2.6.3. Myotoxic activity Male Balb-C mice (20–25 g, n = 3) were injected intramuscularly (i.m.) in the gastrocnemius muscle with 20 ␮g of BpMP-II or 10 ␮g of crude venom dissolved in 50 ␮L of PBS. The control group received only 50 ␮L of PBS. After 3 h, the animals were anesthetized with ketamine® 10% (0.05 mL/kg) and xylazine® 2% (0.025 mL/kg), bled by cardiac puncture and the sera were assayed for creatinekinase activity with a commercial kit (Biotécnica). 2.6.4. Cellular viability Murine endothelial cells tEnd (thymic endothelium) were used as established by Bussolino et al. [37]. Cells were maintained in RPMI 1640 medium supplemented with 10% bovine fetal serum, 2 mM l-glutamine, 2 mM sodium pyruvate, 1 mM non-essentials amino acids, 100 U/mL penicillin and 100 ␮g/mL streptomycin. Cells were incubated at 37 ◦ C and 5% CO2. The viability of cultured tEnd cells treated with BpMP-II was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method. To determine cell viability, 1.5 × 104 cells/well were seeded on a 96-well plate. After adhesion, the medium was changed and the cells were treated with different concentrations of BpMP-II or culture medium (control group) for 24 h at 37 ◦ C and 5% CO2. After 24 h, cells were incubated with 5 mg/mL MTT for 3 h at 37 ◦ C. Formazan crystals resulting from MTT reduction were dissolved by the addition of 100 ␮L of PBS containing 10% SDS and 0.01 M HCl (18 h, 37 ◦ C and 5% CO2 ). The reaction intensity was measured by optical density at 570 nm using an ELISA reader (BioTek Elx50). 2.6.5. Adhesion assay to extracellular matrix proteins For adhesion assay, tEnd cells were preincubated for 30 min at room temperature with BpMP-II (10 and 40 ␮g/mL) or culture medium (control group) and then added to 24-well plate previously coated with Matrigel 1 mg/mL (BD Bioscience) for 1 h at 37 ◦ C. After 18 h, cells were photographed under a microscope at ×20 magnification. 2.6.6. Tube formation inhibition assay The influence of BpMP-II on endothelial cell tube formation was evaluated by matrigel tube formation assays. The experiments were performed as described by Yeh et al. [38] with modifications. Briefly, 24-well plates were coated with 250 ␮L of Matrigel 5.25 mg/mL (BD Bioscience) and incubated for 60 min at 37 ◦ C. tEnds cells (2 × 105 cells/well) were preincubated with 10 and 40 ␮g/mL of BpMP-II or culture medium (control group) for 30 min at room temperature and plated on Matrigel in a total volume of

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Fig. 1. Purification of BpMP-II. (A) Chromatographic profile of crude venom (137 mg) on CM-Sepharose Fast Flow equilibrated with ammonium bicarbonate buffer 0.05 M pH 7.8. A convex gradient was applied to the column using ammonium bicarbonate buffer 0.5 M pH 7.8. Fractions (1 mL/tube) were collected in a 6 mL/h flow rate at room temperature. (B) Chromatographic profile of CM1 (40 mg) fraction on Sephacryl S300 gel filtration column previously equilibrated with ammonium bicarbonate buffer 0.05 M pH 7.8. Fractions (1 mL/tube) were collected in a 0.2 mL/min flow rate at room temperature, then lyophilized and stored as −20 ◦ C. (C) Chromatographic profile of fraction CM1S3 (11 mg) on Capto-Q ion-exchange column. Column was previously equilibrated with ammonium bicarbonate buffer 0.05 M pH 7.8 and gradient made with the same buffer 0.5 M and pH 7.8. (D) SDS-PAGE 12.5% (w/v): Molecular weight marker (MW) and BpMP-II (10 ␮g) reduced with ␤-mercaptoethanol. (E) BpMP-II on (RP-HPLC) C18. Linear gradient with acetonitrile 80%. Absorbance at 280 nm (blue line) and 214 nm (red line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

200 ␮L of culture medium supplemented with bFGF (30 ng/mL) for 18 h. The cells were photographed under a microscope at ×20 magnification. 2.7. Statistical analysis Results were presented as means ± standard deviation (S.D.). The statistical significance of the results was evaluated using Student’s t-test and the Graphpad Prism 5 Project program. A value of p > 0.05 was considered significant.

Table 1 Protein recovery and enzymatic activities of fractions and BpMP-II. Purification steps

Amount (mg/total)*

Protein yield (%)

Azocaseinolytic activity (U/mg)**

Crude venom CM1 CM1S3 BpMP-II

137 40 11 0.55

100 29.0 8.0 0.4

1700 1240 1010 1110

*

According to Bradford (1974). 1 U represents the increase of 0.01 in absorbance (366 nm) in which the enzyme hydrolyses the substrate (azocasein). **

3. Results and discussion 3.1. Isolation and biochemical characterization Fractionation of Bothrops pauloensis venom was performed initially as described by [26]. Desiccated crude venom (200 mg) was dissolved in 0.05 AMBIC buffer (pH 7.8) and after centrifugation the protein recovery was 137 mg. This low protein recovery can be due to the presence of un-dissolvable materials and degraded toxins, since that B. pauloensis venom possesses a great diversity of biologically active compounds represented mainly by proteases [27]. Total protein (137 mg) was submitted to an ion-exchange chromatographic column (CM-Sepharose) resulted in six fractions named CM1, CM2, CM3 CM4, CM5a and CM5b (Fig. 1A). The CM1 fraction was shown to be very heterogeneous with high proteolytic activity against azocasein (Table 1). Fraction CM1 was applied to a gel filtration Sephacryl S300 column according to Ferreira et al. [29] and seven peaks were obtained

at 280 nm (CM1S1, CM1S2, CM1S3, CM1S4, CM1S5, CM1S6 and CM1S7) (Fig. 1B). CM1S3 also showed azocaseinolytic activity (Table 1) and was then submitted to an ion-exchange column (Capto-Q) previously equilibrated with 0.05 M AMBIC buffer at pH 7.8 (Fig. 1C). The main peak (absorbance 280 nm) containing the protein of interest (BpMP-II) was visualized by 12.5% SDS-PAGE under reducing conditions and showed Mr of 23,800 (Fig. 1D). BpMP-II showed proteolytic activity upon azocasein and represented approximately 0.4% of the crude venom (Table 1). Although SVMPs represent one of the greatest fractions in bothropic venom, when isolated, they can present low recovery [35]. BpMP-II was also applied to a reverse phase column (C18/RP-HPLC) (Fig. 1E) and the unique peak was submitted to analyzes of MALDI-TOF, isoelectric focusing and MS/MS. The protein presented molecular mass of 23,000 Da by MALDI-TOF (Fig. 2) and pI of 6.1 (data not shown). The partial sequence of BpMP-II was determined by mass spectrometry. One hundred micrograms of the protein were

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Fig. 2. Molecular mass of BpMP-II by MALDI-TOF-MS using an AutoFlex III MALDITOF-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). The software Flex Analysis 3.0 (Bruker Daltonics, Bremen, Germany) was used for analysis of the mass spectrometric data.

submitted to hydrolysis with trypsin and the Ms spectrum of digested fragments are showed in Fig. 3A. To identify the protein, peptides with masses of 1096.509, 1549.669 and 1822.743 Da (Fig. 3A) were fragmented by the LIFT method (Bruker Daltonics) (data not shown). MS/MS spectrum were analyzed by Flex Analysis 3.0 (Bruker Daltonics) and submitted to Biotools (Bruker Daltonics) and Matrix Science (Mascot Search) software for protein identification. Results showed that BpMP-II had match with other

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Fig. 3. (A) Ms1 spectrum of mass profile from peptides obtained by trypsin fragmentation of BpMP-II. Spectrum analysis was made by the Biotools software (Bruker Daltonics, USA) and values shown in Da. (B) Histogram showing the highly significant score above 50 (p < 0,05), indicating reliability on the results obtained from MS/MSM fragmentation.

snake venom metaloproteinases (score of 110, significance > 50 and p < 0,05) (Fig. 3B). BpMP-II fragments 1096.509 Da (KTLTSFGEWRE), 1549.669 Da (RVHEMVNTVNGFFRS) and 1822.743 Da (RTRVHEMVNTVNGFFRS) showed similarity with other PI SVMP sequences, such as BpMPI from Bothrops pauloensis [26], MP-I [39] and BaP1from B. asper [40] (Fig. 4). BpMP-II can be considered a new isoform PI

Fig. 4. BpMP-II partial sequence alignment with some PI metalloproteinase homologous sequences obtained from BLASTP (PubMed – Medline) and Uniprot protein data bank. Access codes: BpMP-I from Bothrops pauloensis [26]; MP-I from Bothrops neuwiedi (E3UJL3) [39]; BaP1 from Bothrops asper (P83512) [40]. The symbol (*) represents conserved amino acid residues when compared to the BpMP-I sequence. The symbols (:) and (.) represent amino acid residues with same and different chemical characteristics, respectively.

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Fig. 5. Proteolytic activity of BpMP-II upon azocasein. (A) Enzyme at different concentration (0.5 ␮g; 1.0 ␮g; 2 ␮g; 5 ␮g; 10 ␮g; 20 ␮g) was incubated with 100 ␮L of azocasein (1 mg/mL) for 1 h at 37 ◦ C (see Section 2). BpMP-II (5 ␮g) was also previously incubating for 1 h at 37 ◦ C incubated with 100 ␮L azocasein (1 mg/mL) at different (B) temperatures or (C) pH solutions. The azocaseinolytic activity was assayed at 366 nm. Azocasein solutions without enzyme were used as control of reaction. Results are shown as mean ± SD (n = 3). * Differences statistically significant (p < 0.05).

Fig. 6. Fibrinogenolytic activity of BpMP-II. SDS-PAGE at 12.5% (w/v) of bovine fibrinogen digest. Samples of 50 ␮L fibrinogen (1 mg/mL) were incubated with BpMP-II at 37 ◦ C in different conditions: (A) Enzyme concentration (incubation time, 1 h, pH 7.8); (B) Incubation time (enzyme concentration, 5 ␮g, pH 8.0); (C) Presence of inhibitors (enzyme concentration, 5 ␮g, pH 7.8 for 1 h).

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Fig. 7. Effect of BpMP-II on endothelial cell viability. tEnd cells were incubated with different concentrations of BpMP-II or culture medium (control group) for 24 h and viability were evaluated by MTT as described in experimental procedures. Samples were analyzed in triplicate and (*) p < 0.05 compared to control.

metalloproteinase from B. pauloensis venom, once that its primary sequence presented some different amino acid residues when compared to BpMP-I from this same venom (Fig. 4). 3.2. Functional characterization The proteolytic activity of BpMP-II was assayed initially by degradation of azocasein, which showed be dose dependent (Fig. 5A). According to these results, 5 ␮g of BpMP-II were used to assay protein stability at different pH solutions and temperatures. BpMP-II showed best activity at 4 ◦ C when compared to the others tested temperatures and lost its activity at 60 ◦ C and 100 ◦ C (Fig. 5B). The protein showed optimal activity at neutral pHs (Fig. 5C). BpMPII acted like other SVMPs that present optimum activity close to neutral pH and low temperatures (4 ◦ C) [26,35,36,41]. The loss or

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decrease of proteolytic activity is related to a decrease in the polarization of the Glu177 residue (essential to catalysis) and to the protonation state of His residues present in the active site. The protonation of residues related to metalloproteinase catalysis modifies the affinity for Zn2+ , thus interfering with zinc-dependent activities [42]. Hemostasis interference may be an important systemic complication caused by bothropic envenomation. This action is caused by enzymes that are able to degrade fibrinogen and other plasma components. BpMP-II was able to degrade bovine fibrinogen (Fig. 6A) in a dose and time dependent manner. BpMP-II (5 ␮g) induced a rapid degradation of A␣ chain (15 min) and a slower degradation of B␤ chain (24 h). The ␥ chains were unaffected (Fig. 6B). This activity was completely inhibited by EDTA, 1,10-phenantroline and by ␤-mercaptoethanol and was not inhibited by benzamidine and aprotinin, showing that this enzyme is a Zn2+ metalloproteinase (Fig. 6C). This enzyme preferentially degraded A␣ chain of fibrinogen like others PI SVMPs [26,34,43–45]. BpMP-II did not cause hemorrhage when injected intradermally and was not able to induce myotoxicity in mouse gastrocnemius muscle as indicated by plasma levels of creatine-Kinase (data not shown). These data are consistent with some results described in the literature. A PI metalloproteinase isolated from Bothrops neuwiedi venom named BnP1 was described as nonhemorrhagic and non-myotoxic metalloproteinase [46]. Although PI SVMPs show low or absent hemorrhagic activity, some of them can cause myotoxicity [26,47,48]. Baldo et al. [49] demonstrated that neuwiedase, a non-hemorrhagic snake venom metalloproteinase from PI class, was able to cause skeletal muscle damage, probably due to its proteolytic action on the connective tissue surrounding muscle cells, followed by proteolysis of myofibrillar components. Low or absent of hemorrhagic activity is a characteristic of SVMPs belonging to the PI class, showing that myotoxicity is not always related to ischemia from hemorrhagic effects, but may be the result of the direct action of a metalloproteinase on muscle cells.

Fig. 8. Effect of BpMP-II on adhesion of endothelial cells in matrigel. (A) tEnd cells preincubated with medium (Control). Samples of 10 ␮g/mL (B) or 40 ␮g/mL of BpMP-II (C) were preincubated with tEnd cells for 30 min at room temperature. Images were registered under magnification of ×20. Three experiments were conducted independently (n = 3).

Fig. 9. The inhibitory effect of BpMP-II on tEnd tube formation in Matrigel assay (see Section 2). (A) tEnd cells preincubated with medium (Control). Cells arranged capillary-like form. (B) tEnd cells treated with BpMP-II (10 ␮g/mL) (C) tEnd cells treated with BpMP-II (40 ␮g/mL). Images were registered under magnification of ×20. Three experiments were conducted independently (n = 3).

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BpMP-II was toxic on tEnd cells at doses higher than 20 ␮g/mL (Fig. 7) and showed a LC50 of 40 ␮g/mL. This protein at 10 ␮g/mL or 40 ␮g/mL inhibited the adhesion of tEnd cells to extracellular matrix proteins (Matrigel) (Fig. 8) and was also able to inhibit capillary formation in Matrigel when compared to control (Fig. 9). Endothelial cells are targets of SVMPs by interfering with viability, adhesion and angiogenesis. They can interfere in these processes by interacting with cell surface integrins [13,50–52] or by degrading extracellular matrix proteins as fibronectin, vitronectin, collagen I and collagen IV or plasma proteins [5,9,16,35,49]. Cell surface integrins are targets of disintegrins and disintegrinlike/cysteine rich domains of SVMPs, which act as antagonists of integrins, interfering in adhesion, migration, angiogenesis and cell communication. These effects has been demonstrated by jararhagin from Bothrops jararaca, atrolysin-A from Bothrops atrox and alternagin from Bothrops alternatus, which were able to interfere with ␣2␤1 integrin function [13,51–53]. Besides, the catalytic domain can be involved in disruption of endothelial cell integrity and inhibition of angiogenesis. Tanjoni et al. [15] and Baldo et al. [46] showed that jararhagin and BnP1, respectively were able to reduce viability of endothelial cell by interfering with cell attachment inducing apoptosis and that these effects are completely dependent on their catalytic activities. In addition, jararhagin promoted proteolytic cleavage of plasminogen generating the angiostatin peptide, an anti-angiogenic factor that inhibits endothelial cell proliferation in vitro [17,54]. In according to our results of inhibition of cell adhesion and in vitro angiogenesis of tEnd cells, we suggest that BpMP-II interferes in these processes probably by a proteolytic mechanism, however new studies must reveal more details about its mechanisms of action on endothelial cells. 4. Conclusions BpMP-II is a new non-hemorrhagic fibrinogenolytic SVMP isolated from B. pauloensis that showed low toxicity in vitro and in vivo, exhibiting adhesion cell and angiogenesis inhibition. These results open perspectives for future investigations into the use of BpMP-II as a model for a therapeutic agent. Conflicts of interest The authors declared that there are no conflicts of interest. Acknowledgments The authors gratefully acknowledge the financial support by Fundac¸ão de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Universidade Federal de Uberlândia (UFU), Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES), National Institute of Science and Technology (INCT) in Nano-Biopharmaceutical, Brazil and the technical support of Fundac¸ão Ezequiel Dias (Belo Horizonte, Minas Gerais, Brazil). References [1] F.X. Gomis-Rüth, Mol. Biotechnol. 24 (2003) 157–202. [2] F.X. Gomis-Rüth, T.O. Botelho, W. Bode, Biochim. Biophys. Acta 1824 (2012) 157–163. [3] J.W. Fox, S.M.T. Serrano, FEBS Lett. 275 (2008) 3016–3030. [4] J.J. Gutierrez, A. Rucavado, T. Escalante, C. Diaz, Toxicon 45 (2005) 997–1011. [5] F.S. Markland, S. Swenson, Toxicon 62 (2013) 3–18. [6] A.S. Kamiguti, P. Gallagher, C. Marcinkiewicz, R.D. Theakston, M. Zuzel, J.W. Fox, FEBS Lett. 549 (2003) 129–134. [7] M.B. Silva, M. Schattner, C.R. Ramos, I.L. Junqueira-de-azevedo, M.C. Guarnieri, M.A. Lazzari, C.A. Sampaio, R.G. Pozner, J.S. Ventura, P.L. Ho, A.M. ChudzinskiTavassi, Biochem. J. 369 (2003) 129–139.

[8] W.J. Wang, C.H. Shih, T.F. Huang, Biochimie 87 (2005) 1065–1077. [9] C.P. Bernardes, D.L. Menaldo, E. Camacho, J.C. Rosa, T. Escalante, A. Rucavado, B. Lomont, J.M. Gutiérrez, S.V. Sampaio, J. Proteomics 80 (2013) 250–267. [10] M. De Luca, L.C. Dunlop, R.K. Andrews, J.V. Flannery Jr., R. Ettling, D.A. Cumming, G.M. Veldman, M.C. Berndt, J. Biol. Chem. 270 (1995) 26734–26737. [11] W.K. You, H.J. Seo, K.H. Chung, D.S. Kim, J. Biochem. 134 (2003) 739–749. [12] P. Zigrino, A.S. Kamiguti, J. Eble, C. Drescher, R. Nischt, J.W. Fox, C. Mauch, J. Biol. Chem. 227 (2002) 40528–40535. [13] D.H. Souza, M.R. Iemma, L.L. Ferreira, J.P. Faria, M.L. Oliva, R.B. Zingali, S. Iewiarowski, H.S. Selistre-de-araujo, Arch. Biochem. Biophys. 384 (2000) 341–350. [14] M.R. Cominetti, C.H. Terruggi, O.H. Ramos, J.W. Fox, A. Mariano-Oliveira, M.S. de Freitas, C.C. Figueiredo, V. Morandi, H.S. Selistre-de-araujo, J. Biol. Chem. 279 (2004) 18247–18255. [15] I. Tanjoni, R. Weinlich, M.S. Della-Casa, P.B. Clissa, R.F. Saldanha-Gama, M.S. de Freitas, C. Barja-Fidalgo, G.P. Amarante-Mendes, A.M. Moura-da-Silva, Apoptosis 10 (2005) 851–861. [16] A.M. Moura-da-Silva, D. Butera, I. Tanjoni, Curr. Pharm. Des. 13 (2007) 2893–2905. [17] P.L. Ho, S.M.T. Serrano, A.M. Chudzinski-Tavassi, A.M. Moura-da-Silva, R. Mentele, C. Caldas, M.L.V. Oliva, C. BIF, L.S. OM, Biochem. Biophys. Res. Commun. 294 (2002) 879–885. [18] K. Trummal, K. Tonismagi, E. Siigur, A. Aaspollu, A. Lopp, T. Sillat, et al., Toxicon 46 (2005) 46–61. [19] C.H. Yeh, H.C. Peng, T.F. Huang, Blood 92 (1998) 3268–3276. [20] T.F. Huang, C.H. Yeh, W.B. Wu, Haemostasis 31 (2001) 192–206. [21] J. Folkman, Nat. Rev. Drug Discov. 6 (2007) 273–286. [22] G.C. Alghisi, C. Ruegg, Endothelium 13 (2006) 113–135. [23] L.A. Calderon, J.C. Sobrinho, K.D. Zaqueo, A.A. de Moura, A.N. Grabner, M.V. Mazzi, S.M.A. Nomizo, C.F.C. Fernandes, J.P. Zuliani, B.A. Carvalho, S.L. da Silva, R.G. Stábeli, A.M. Soares, BioMed. Res. Int. 2014 (2014) 19. [24] R.S. Rodrigues, L.F.M. Izidoro, A. Fuly, M.I. Homsi-Brandeburgo, A. Hamaguchi, H.S. Selistre de Araujo, J.R. Giglio, A.M. Soares, V.M. Rodrigues, Toxicon 50 (2007) 153–165. [25] F.L. Costa, R.S. Rodrigues, L.F.M. Izidoro, D.L. Menaldo, A. Hamaguchi, M.I. Homsi-Brandeburgo, A.L. Fuly, S.G. Soares, H.S. Selistre-deAraújo, B. Barraviera, A.M. Soares, V.M. Rodrigues, Toxicon 54 (2009) 725–735. [26] D.L.S. Naves, M.S.R. Gomes, F.B. Ferreira, R.S. Rodrigues, D.C. Achê, M. Richardson, M.H. Borges, V.M. Rodrigues, Comp. Biochem. Physiol. B 161 (2012) 102–109. [27] R.S. Rodrigues, J. Boldrini-Franc¸a, F.P.P. Fonseca, P. de La Torre, F. HenriqueSilva, L. Sanz, J.J. Calvete, V.M. Rodrigues, J. Proteomics 75 (2012) 2707–2720. [28] P.A. Kenny, M.J. Bissell, J. Clin. Invest. 117 (2007) 337–345. [29] F.B. Ferreira, M.S. Gomes, D.L. de Souza, S.N. Gimenes, L.E. Castanheira, M.H. Borges, R.S. Rodrigues, K.A. Yoneyama, M.I. Brandeburgo, V.M. Rodrigues, Toxins (Basel) 5 (2013) 2403–2419. [30] M.M. Bradford, Anal. Biochem. 72 (1976) 248–254. [31] U.K. Laemmli, Nature 227 (1970) 680–685. [32] D.N. Perkins, D.J. Pappin, D.M. Creasy, J.S. Cottrell, Electrophoresis 20 (1992) 3551–3567. [33] M.A. Larkin, G. Blackshields, N.P. Brown, R. Chenna, P.A. McGettigan, H. McWilliam, F. Valentin, I.M. Wallace, A. Wilm, R. Lopez, J.D. Thompson, T.J. Gibson, D.G. Higgins, Bioinformatics 23 (2007) 2947–2948. [34] M.S.R. Gomes, M.R. Queiroz, C.C.N. Mamede, M.M. Mendes, A. Hamaguchi, M.I. Homsi-Brandeburgo, M.V. Sousa, E.N. Aquino, M.S. Castro, F. Oliveira, V.M. Rodrigues, Comp. Biochem. Physiol. C 153 (2011) 290–300. [35] V.M. Rodrigues, A.M. Soares, R. Guerra-Sa, V. Rodrigues, M.R.M. Fontes, J.R. Giglio, Arch. Biochem. Biophys. 381 (2000) 213–224. [36] T. Nikai, N. Mori, M. Kishida, H. Sugihara, A.T. Tu, Arch. Biochem. Biophys. 231 (1984) 309–319. [37] F. Bussolino, M. Ziche, J.M. Wang, D. Alessi, L. Morbidelli, O. Cremona, A. Bosia, P.C. Marchisio, A. Mantovani, J. Clin. Invest. 87 (1991) 986–995. [38] C.H. Yeh, H.C. Peng, R.S. Yang, T.F. Huang, Mol. Pharmacol. 59 (2001) 1333–1342. [39] A.M. Moura-da-Silva, M.S. Furlan, M.C. Caporrino, K.F. Grego, J.A. Portes-Junior, P.B. Clissa, R.H. Valente, G.S. Magalhães, BMC Genet. 12 (12) (2011) 94. [40] L. Watanabe, J.D. Shannon, R.H. Valente, A. Rucavado, A. Alape-Giron, A.S. Kamiguti, R.D.G. Theakston, J.W. Fox, J.M. Gutierrez, R.K. Arni, Protein Sci. 12 (2003) 2273–2281. [41] X.L. Xu, X.H.H. Liu, B. Wu, Y. Liu, W.Q. Liu, Y.S. Xie, L.Q. Liu, Biopolymers 74 (2004) 336–344. [42] X.Y. Zhu, Z.L. Zhu, W.M. Gong, M.K. Teng, L.W. Niu, Sheng Wu Hua Yu Sheng Wu Wu LI Xue Bao (Shanghai) 29 (1997) 163–169. [43] O.H.P. Ramos, H.S. Selistre-de-Araujo, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 142 (2006) 328–346. [44] S. Swenson, C.F. Toombs, L. Pena, J. Johansson, F.S. Markland JR., Curr. Drug Targets Cardiovasc. Haematol. Disord. 4 (2004) 417–435. [45] M. Berger, A.F. Pinto, J.A. Guimaraes, Toxicon 51 (2008) 488–501. [46] C. Baldo, I. Tanjoni, I.R. Leon, I.F.C. Batista, M.S. Della-Casa, P.B. Clissa, R. Weinlich, M. Lopes-Ferreira, I. Lebrun, G.P. Amarante-Mendes, V.M. Rodrigues, J. Perales, R.H. Valente, A.M. Moura-da-Silva, Toxicon 51 (2008) 54–65. [47] E.F. Sanchez, F.S. Schneider, A. Yarleque, M.H. Borges, M. Richardson, S.G. Figueiredo, K.S. Evangelista, J.A. Eble, Arch. Biochem. Biophys. 49 (2010) 9–20.

D.C. Achê et al. / International Journal of Biological Macromolecules 72 (2015) 445–453 [48] V.M. Rodrigues, A.M. Soares, S.H. Andrião-Escarso, A.M. Franceschi, A. Rucavado, J.M. Gutiérrez, J.R. Giglio, Biochimie 83 (2001) 471–479. [49] C. Baldo, L.F.M. Izidoro, E.A.V. Ferro, A. Hamaguchi, M.I. Homsi-Brandeburgo, V.M. Rodrigues, J. Venom Anim. Toxins Incl. Trop. Dis. (Online) 16 (2010) 462–469. [50] C. Marcinkiewicz, Curr. Pharm. Des. 11 (2005) 815–827.

453

[51] H.S. Selistre-de-Araujo, M.R. Cominetti, C.H. Terruggi, A. Mariano- Oliveira, M.S. De Freitas, M. Crepin, et al., Braz. J. Med. Biol. Res. 38 (2005) 1505–1511. [52] A.S. Kamiguti, C.R.M. Hay, M. Zuzel, Biochem. J. 320 (1996) 635–641. [53] L.G. Jia, X.M. Wang, J.D. Shannon, J.B. Bjarnason, J.W. Fox, J. Biol. Chem. 272 (1997) 13094–13102. [54] A.M. Moura-da-Silva, C. Baldo, Toxicon 60 (2012) 280–289.