Snake Venomics and Antivenomics of Bothrops diporus, a Medically Important Pitviper in Northeastern Argentina

Snake species within genus Bothrops are responsible for more than 80% of the snakebites occurring in South America. The species that cause most envenomings in Argentina, B. diporus, is widely distributed throughout the country, but principally found in the Northeast, the region with the highest rates of snakebites. The venom proteome of this medically relevant snake was unveiled using a venomic approach. It comprises toxins belonging to fourteen protein families, being dominated by PI- and PIII-SVMPs, PLA2 molecules, BPP-like peptides, L-amino acid oxidase and serine proteinases. This toxin profile largely explains the characteristic pathophysiological effects of bothropic snakebites observed in patients envenomed by B. diporus. Antivenomic analysis of the SAB antivenom (Instituto Vital Brazil) against the venom of B. diporus showed that this pentabothropic antivenom efficiently recognized all the venom proteins and exhibited poor affinity towards the small peptide (BPPs and tripeptide inhibitors of PIII-SVMPs) components of the venom.


Introduction
Bothrops diporus [1], commonly known as "yarará chica", is a venomous terrestrial lancehead pit viper endemic to South America. B. diporus was initially classified as one of the twelve subspecies of the Bothrops neuwiedi complex [2]. Revisions of the systematics within the neuwiedi group elevated five subspecies, including B. n. diporus, to the species level [3]. Recently, Fenwick and co-workers have proposed an alternative taxonomic arrangement [4]. These authors proposed a new genus, Bothropoides (derived from the Greek bothros and ops referring to the facial pit, and the term oides meaning "similar to") to include B. alcatraz, B. diporus, B. erythromelas, B. insularis, B. jararaca, B. lutzi, B. mattogrossensis, B. neuwiedi, B. pubescens, and B. pauloensis. Taxonomical revisions may have an important impact in the medical area because a correct identification of the species responsible for snakebite accidents in a given area is of applied importance for the production and efficient use of the antivenoms. In addition, an accurate taxonomy will enlighten research in comparative biology, trait evolution, historical biogeography, and other fields. In this respect, Carrasco et al. [5] highlighted the incongruence between their analyses using morphological, ecological and molecular information of all species of the B. neuwiedi group, and the classification proposed by Fenwick and co-workers [4]. The demonstration that B. neuwiedi is a highly supported monophyletic  PIII-SVMP and PI-SVMP, snake venom metalloproteinases of class PIII and PI, respectively; SVMPi, snake venom metalloproteinase tripeptide inhibitors; PLB, phospholipase B; QC, glutaminyl cyclase; SVSP, snake venom serine proteinase; 5′NT, 5′ nucleotidase; PLA2, phospholipase A2; VAP (BPP, bradykinin-potentiating peptide and BPP-like peptides); CRISP, cysteine-rich secretory protein; CTL, C-type lectin-like protein; DC, disintegrin-like/cysteine-rich domains; PDE, phosphodiesterase; PLB, phospholipase B; LAO, L-amino acid oxidase.

Correlations between Major Venom Proteins and Venom Toxicity
The venom of B. diporus is highly proteolytic, hemorrhagic and myotoxic [10,16,20,[23][24][25]. Its toxin profile here reported potentially explains the local and systemic effects observed in envenomings by this species, which, as in most accidents due to species of the genus Bothrops [26,27], are associated with major local effects (rapid edema formation, pain, inflammation, ecchymosis, hemorrhage, and local myonecrosis, dermonecrosis and blistering) and, in moderate to severe cases, systemic manifestations (blood clotting perturbations, hypotensive shock, and kidney failure) may also develop [23,28].
When injected at relatively low doses, B. diporus venom induces desfibrinogenation in mice [21]. PI-SVMPs are potent proteolytic and alpha > β-fibrino(geno)lytic enzymes [34,35]. PI-SVMPs may thus contribute to the depletion of circulating clottable fibrinogen and can, in conjunction with the action of thrombin-like serine proteinases targeting coagulation factors [36][37][38], synergistically potentiate the activity of hemorrhagic PIII-SVMPs, resulting in increased incidence of systemic bleeding. C-type lectin-like molecules also interact with components of the human hemostatic system, affecting the blood coagulation cascade and platelet aggregation, further contributing to blood clotting perturbations caused by B. diporus venom [39,40].
L-amino acid oxidases, flavoenzymes that catalyze oxidative deamination of L-amino acids to form corresponding α-keto acids, hydrogen peroxide and ammonia, are widely distributed in viperid venoms [41]. Certain L-amino acid oxidases, such as B. pauloensis B5AR80, have been reported to induce platelet aggregation in platelet-rich plasma [42]. However, the contribution to the envenoming process of the B. diporus LAO eluted in RP-HPLC fraction 30, representing 7.5% of the total venom proteins, remains elusive.
The high abundance of putative vasoactive peptides (15.9% of the venom proteome) (Figure 2) is remarkable. BPP + BPP-like peptides, commonly found in the venom of many Bothrops and other snake species [43], include inhibitors of the angiotensin I-converting enzyme that enhance the hypotensive effect of circulating bradykinin, causing a vascular shock in the snake's prey or victim [44][45][46]. However, BPP-like peptides lacking the C-terminal sequence (PXIPP) caused no potentiation of bradykinin hypotensive effect [47,48] and, despite being a major venom component of Lachesis spp. venoms, do not represent a serious clinical concern in the treatment of envenomings by species of this genus [49]. This evidence suggests that the PXIPP motif is crucial for the proper expression of the pharmacological activities of BPPs. Peptides ZARPPHPPIPP (RP-HPLC-4), (254.3)VNAPXNPSIPP and (237.2)ADPRAPNIPP (RP-HPLC-10), and ZGGQPTPQIPP (RP-HPLC-11), representing, respectively, 1.7%, 1.3%, and 7.1% of the venom fractions, bear this C-terminal sequence motif (Table S1). A related peptide, ZGGWPRPGPEIPP, isolated from "B. neuwiedi" venom showed bradykinin-potentiating action on isolated guinea-pig ileum and a relevant angiotensin-converting enzyme competitive inhibiting activity [50]. The pharmacological activity of B. diporus venom BPPs deserves future detailed investigation. BPPs typically contain an N-terminal 5 1 -oxoprolinyl (pyroglutaminyl) residue. Snake venom QC may catalyze the cyclization of N-terminal glutamic acid and glutamine residues [51] of BPPs and other venom components, such as PIII-SVMPs and their endogenous tripeptide inhibitors (SVMPi), ZNW, ZQW, and ZKW [52,53]. Present at millimolar concentrations [54], these low-affinity antagonists maintain SVMPs catalytically inactive in the lumen of the venom gland, and their inhibition is instantly disengaged when venom is injected into tissue of the prey or victim [54].

Minor Venom Proteins
Cysteine-rich secretory proteins (CRISP) represent a widely distributed protein family in viperid and elapid snake venoms [55,56]. Reported activities of some purified CRISPs include inhibition of cyclic nucleotide-gated ion channels and smooth muscle contraction.
The occurrence of PLB in snake venoms was initially reported by Doery and Pearson [57], and have been characterized as responsible for the high direct hemolytic activity of Australian elapid venoms by Takasaki and Tamiya [58] and Bernheimer and co-workers [59,60]. A recent shotgun proteomic analysis of the venoms of B. atrox, B. jararacussu, B. jararaca, B. neuwiedi, B. alternatus, and B. cotiara, identified PLB molecules in these six species [23], strongly suggesting that this class of toxins may be more widely distributed in Viperidae than previously thought. However, as in the case of CRISPs, the participation of this class of proteins in envenoming requires future detailed studies. 5 1 -nucleotidase (5 1 NT) was first reported by Gullan and Jackson in 1938 [61]. Since then, 5 1 -nucleotidase has been found in a number of snake venoms [62]. However, the pharmacological activities of this hydrolytic enzyme has not been unambiguously defined. Purines appear to be the most primitive and widespread chemical messengers in the animal and plant kingdoms [63]. The identification of free purines as endogenous constituent of venoms has further supported the role of purinergic signaling in envenomation [64].
Purines are known to potentiate venom-induced hypotension and paralysis via purine receptors. Thus, it has been proposed that, following cell disruption brought about by the venom proteinases, hemorrhagins, myotoxins and cytotoxins, purines nucleosides generated from endogenous precursors in the prey by the action of nucleotidases (5 1 NT, ATPase, ADPase), nucleases (phosphodiesterase (PDE), DNases, and RNases), and phosphatases (acid and alkaline phosphomonoesterases), may play role in prey immobilization [64]. In addition, ATP released from skeletal muscle by the myotoxic action of PLA 2 s acts as a "danger signal" stimulating purinergic receptors to enhance and spread the muscle damage caused by the myotoxins and pain [65,66].

Antivenomics
The degree of cross-immunorecognition of the venom toxins of B. diporus by the pentabothropic antivenom (SAB) produced by Instituto Vital Brazil was assessed by immunoaffinity antivenomics [67]. The results displayed in Figure 3 clearly show that the SAB antivenom column efficiently immunoretained all the venom components eluting in RP-HPLC fractions 12-38 and poorly those recovered in chromatographic fractions 1-11. These fractions contain endogenous tripeptide inhibitors of SVMPs (SVMPi), BPP and BPP-like peptides, and a previous investigation [49] indicated that they may not represent a serious clinical concern. On the other hand, while comparing the levels of immune recognition gathered from in antivenomics with the in vivo neutralization efficacy of an antivenom is not straightforward, since both experiments involve radically different protocols, in our experience, even a moderate immunocapturing capability of~25% correlates with a good outcome in the in vivo neutralization tests [68]. Hence, we conclude that the SAB antivenom may represent a therapeutic alternative for treating envenomings by B. diporus.

Venom
B. diporus venom was collected from 6 adult snakes kept in the Serpentarium of Corrientes, a province in Northeast Argentina. For venom extraction, the snakes were anesthetized with CO2. Venoms were pooled, lyophilized, and stored at −20 °C until used.

Isolation and Characterization of Venom Proteins
Two milligrams of crude, lyophilized venom were dissolved in 200 μL of 5% acetonitrile in water containing 0.1% trifluoroacetic acid (TFA), centrifuged to remove debris, and separated by reverse-phase HPLC using a Teknokroma Europa Protein 300 C18 (0.4 cm × 25 cm, 5 mm particle size, 300 Å pore

Venom
B. diporus venom was collected from 6 adult snakes kept in the Serpentarium of Corrientes, a province in Northeast Argentina. For venom extraction, the snakes were anesthetized with CO 2 . Venoms were pooled, lyophilized, and stored at´20˝C until used.

Isolation and Characterization of Venom Proteins
Two milligrams of crude, lyophilized venom were dissolved in 200 µL of 5% acetonitrile in water containing 0.1% trifluoroacetic acid (TFA), centrifuged to remove debris, and separated by reverse-phase HPLC using a Teknokroma Europa Protein 300 C18 (0.4 cmˆ25 cm, 5 mm particle size, 300 Å pore size) column and an LC 1100 High Pressure Gradient System (Agilent Technologies, Santa Clara, CA, USA) equipped with DAD detector and micro-Auto-sampler. The flow-rate was set to 1 mL/min and the column was developed with a linear gradient of 0.1% TFA in water (solution A) and acetonitrile (solution B) using the following column elution conditions: isocratically (5% B) for 5 min, followed by 5%-25% B for 10 min, 25%-45% B for 60 min, and 45%-70% for 10 min. Protein detection was carried out at 215 nm with a reference wavelength of 400 nm. Fractions were collected manually, dried in a vacuum centrifuge (Savant), and redissolved in water, and submitted to molecular mass determination using a QTrap™ 2000 mass spectrometer (ABSciex, Concord, ON, Canada) equipped with a nanospray source (Protana, Denmark), and SDS-PAGE analysis in 12% polyacrylamide gels, under reducing and non-reducing conditions. Gels were stained with Coomassie Brilliant Blue R-250 (Sigma-Aldrich, St. Louis, MO, USA).

Characterization of the Venom Peptidome and Proteome
Electrophoretic protein bands were excised from a Coomassie Brilliant Blue-stained SDS-PAGE gel and subjected to in-gel reduction (10 mM dithiothreitol) and alkylation (50 mM iodoacetamide), followed by overnight sequencing-grade trypsin digestion (66 ng/µL in 25 mM ammonium bicarbonate, 10% acetonitrile; 0.25 µg/sample) in an automated processor (ProGest Protein Digestion Workstation, Genomic Solution Ltd., Cambridgeshire, UK) following the manufacturer's instructions. Tryptic digests were dried in a SpeedVac (Savant™, Thermo Scientific Inc., West Palm Beach, FL, USA), redissolved in 15 µL of 0.1% formic acid in water, and submitted to LC-MS/MS. To this end, tryptic peptides were separated by nano-Acquity UltraPerformance LC (UPLC ) using BEH130 C18 (100 µmˆ100 mm, 1.7 µm particle size) column in-line with a SYNAPT G2 High Definition Mass Spectrometry System (Waters Corp. Milford Massachusetts, USA). The flow rate was set to 0.6 µL/min and the column was developed with a linear gradient of 0.1% formic acid in water (solution A) and 0.1% formic acid in acetonitrile (solution B), isocratically 1% B for 1 min, followed by 1%-12% B for 1 min, 12%-40% B for 15 min, 40%-85% B for 2 min. Doubly and triply charged ions were selected for collision-induced dissociation (CID) MS/MS. Fragmentation spectra were interpreted (a) manually (de novo sequencing); (b) using the on-line form of the MASCOT program at http://www.matrixscience.com against the NCBI non-redundant database; and (c) processed in Waters Corporation's ProteinLynx Global SERVER 2013 version 2.5.2. (with Expression version 2.0) against the species-specific venom gland cDNA-derived toxin sequences. MS/MS mass tolerance was set to˘0.6 Da. Carbamidomethyl cysteine and oxidation of methionine were selected as fixed and variable modifications, respectively. Amino acid sequence similarity searches were performed against the available databanks using the BLAST program implemented in the WU-BLAST2 search engine at http://www.bork.embl-heidelberg.de.
The relative abundances (expressed as percentage of the total venom proteins) of the different protein families were calculated as the ratio of the sum of the areas of the reverse-phase chromatographic peaks containing proteins from the same family to the total area of venom protein peaks in the reverse-phase chromatogram [22]. When more than one protein band was present in a reverse-phase fraction, their proportions were estimated by densitometry of Coomassie-stained SDS-polyacrylamide gels using ImageJ version 1.47 (http://rsbweb.nih.gov/ij). Conversely, the relative abundances of different proteins contained in the same SDS-PAGE band were estimated based on the relative ion intensities of the three more abundant peptide ions associated with each protein by MS/MS analysis. Finally, protein family abundances were estimated as the percentages of the total venom proteome.
The final formulation consists of purified F(ab 1 ) 2 fragments generated by digestion with pepsin of ammonium sulphate-precipitated IgG molecules [84]. A vial of SAB (10 mL, 18.7 mg F(ab 1 ) 2 /mL) neutralizes 65 mg of B. jararaca reference venom. For preparation of the antivenom affinity column, 300 µL of CNBr-activated Sepharose 4B (GE Healthcare) matrix were packed in a Pierce centrifuge column and washed extensively with 10 matrix volumes of cold 1 mM HCl followed by two matrix volumes of coupling buffer (0.2 M NaHCO 3 , 0.5 M NaCl, pH 8.3) to adjust the pH of the column to 7.0-8.0. 10 mg of antivenom were dissolved in ½ matrix volume of coupling buffer and incubated with the matrix for 4 h at room temperature. Coupling yield was 6.6 mg, as estimated by measuring the pre-coupled and post-coupled antivenom solution by UV-absorbance, using an extinction coefficient at 280 nm of 1.36 for 1 mg/mL [85]. Non-reacted matrix groups were then blocked by washing the column with 300 µL of 0.1M Tris-HCl, pH 8.0, at 4˝C overnight, using an orbital shaker. The affinity column was then washed alternately at high and low pH, with three column volumes of 0.1 M acetate containing 0.5 M NaCl, pH 4.0-5.0, and three column volumes of 0.1 M Tris-HCl, pH 8.5; this treatment was repeated 6 times. The column was then equilibrated with 5 matrix volumes of working buffer solution (20 mM phosphate buffer, 135 mM NaCl, pH 7.4; PBS). For the immunoaffinity assay, 300 µg of adult B. diporus venom were dissolved in ½ matrix volumes of PBS and incubated with the affinity matrix for 1 h at room temperature (25˝C) using an orbital shaker. This corresponded to an venom:antivenom molar ratio of about 1:22. As specificity controls, 300 µL of Sepharose 4 Fast Flow matrix, without or with 7 mg of immobilized control (naive) IgGs, were incubated with venom and the column developed in parallel to the immunoaffinity experiment. Following elution of the non-retained fractions with 2.5 volumes of PBS, the immunocaptured proteins were eluted with 5 matrix volumes of elution buffer (0.1 M glycine-HCl, pH 2.0) and neutralized with 1 M Tris-HCl, pH 9.0. The crude venom and the non-retained and the immunocaptured venom fractions were fractionated by reverse-phase HPLC using a Discovery BIO Wide Pore C 18 (15 cmˆ2.1 mm, 3 µm particle size, 300 Å pore size) column (Sigma-Aldrich, St. Louis, MO, USA) and an LC 1100 High Pressure Gradient System (Agilent Technologies, Santa Clara, CA, USA) equipped with a DAD detector. The flow rate was set to 0.4 mL/min and the column was developed with a linear gradient of 0.1% TFA in water (solution A) and 0.1% TFA in acetonitrile (solution B): isocratically (5% B) for 1 min, followed by 5%-25% B for 5 min, 25%-45% B for 35 min, and 45%-70% solution B for 5 min. Protein detection was carried out at 214 nm with a reference wavelength of 400 nm.
Supplementary Materials: The following are available online at www.mdpi.com/2072-6651/8/1/9/s1. Acknowledgments: This work was supported by Grant BFU2013-42833-P from the Ministerio de Economía y Competitividad, Madrid (Spain). The authors thank the Serpentarium CEPSAN of the local Zoo in Corrientes, Argentina and, especially to Lic. Laura Rey, for generously providing the venom of Bothrops diporus. Carolina Gay was supported by a Partial Financing Program of Short Stays Abroad for Postdoctoral Fellows from CONICET (Consejo Nacional de Investigaciones Científicas y Tecnológicas), Argentina.

Conflicts of Interest:
The authors declare no conflict of interest.