Biochemical characterization of venom from Pseudoboa neuwiedii (Neuwied’s false boa; Xenodontinae; Pseudoboini)

Kristian A. Torres-Bonillaa, Débora Andrade-Silvab, Solange M.T. Serranob, Stephen Hyslopa,⁎

Abstract

Snake venom metalloproteinase (SVMP) In this work, we examined the proteolytic and phospholipase A2 (PLA2) activities of venom from the opisthoglyphous colubrid Pseudoboa neuwiedii. Proteolytic activity (3 and 10 μg of venom) was comparable to that of Bothrops neuwiedii venom but less than Bothrops atrox. This activity was inhibited by EDTA and 1,10-phenanthroline but only slightly affected (≤30% inhibition) by PMSF and AEBSF, indicating it was mediated by snake venom metalloproteinases (SVMPs). The pH and temperature optima for proteolytic activity were 8.0 and 37 °C, respectively. The venom had no esterase activity, whereas PLA2 activity was similar to B. atrox, greater than B. neuwiedii but less than B. jararacussu. SDS-PAGE revealed venom proteins >100 kDa, 45–70 kDa, 21–24 kDa and ~15 kDa, and mass spectrometry of protein bands revealed SVMPs, cysteine-rich secretory proteins (CRISPs) and PLA2, but no serine proteinases. In gelatin zymography, the most active bands occurred at 65–68 kDa (seen with 0.05–0.25 μg of venom). Caseinolytic activity occurred at 50–66 kDa and was generally weaker than gelatinolytic activity. RP-HPLC of venom yielded 15 peaks, five of which showed gelatinolytic activity; peak 7 was the most active and apparently contained a P-III class SVMP. The venom showed α-fibrinogenase activity, without affecting the β and γ chains; this activity was inhibited by EDTA and 1,10-phenanthroline. The venom did not clot rat citrated plasma but reduced the rate and extent of coagulation after plasma recalcification. In conclusion, P. neuwiedii venom is highly proteolytic and could potentially affect coagulation in vivo by degrading fibrinogen via SVMPs.

Keywords:
Back-fanged colubrid
Caseinolytic
Esterase
Fibrinogenase
Phospholipase A2
Proteolytic
Pseudoboa neuwiedii

1. Introduction

Colubrid snakes belonging to the tribe Pseudoboini (family Dipsadidae, subfamily Xenodontinae) are considered to be a monophyletic group (Zaher et al., 2009; Vidal et al., 2010; Grazziotin et al., 2012) consisting of 11 genera (Boiruna, Clelia, Drepanoides, Mussurana, Oxyrhopus, Paraphimophis, Phimophis, Pseudoboa, Rhachidelus, Rodriguesophis and Siphlophis) and 47 species of moderate-size snakes with terrestrial, semi-arboreal and semi-fossorial habits (Pizzatto and Marques, 2002; Pizzatto, 2005; Bernarde and Abe, 2006; Scott et al., 2006). Most pseudoboine species inhabit forests and savannas and feed mainly on lizards, small mammals and occasionally other snakes (Alencar et al., 2013; Gaiarsa et al., 2013).
Human envenomation by pseudoboines has been reported for several species (Prado-Franceschi and Hyslop, 2002; Weinstein et al., 2011, 2013), including Boiruna maculata (Santos-Costa et al., 2000), Clelia plumbea and Clelia clelia (Pinto et al., 1991; Salomão et al., 2003) and Oxyrhopus spp. (often mistaken for truly venomous coralsnakes, Micrurus spp., because of their similar coloration patterns) (Salomão et al., 2003). This envenomation is characterized by a combination of edema, erythema, ecchymosis/local hemorrhage, ‘stinging’ pain, cyanosis around the wound and lymphadenopathy of varying degrees of intensity; systemic envenomation is generally absent.
Pseudoboa neuwiedii (Neuwied’s false boa; ratonel) is a back-fanged nocturnal terrestrial species with a wide distribution throughout most of northern South America (Colombia, Guyana, French Guiana, Surinam, Venezuela and Peru) and much of the Brazilian Amazon. This species can reach up to 105 cm in length and dorsally is a uniform reddish brown or faded red (often with scattered black spots), except for the head and neck, which are black/brown with a variable single yellowish collar band in the region of the temples; the underside is a yellowish straw color (Fig. 1). Pseudoboa neuwiedii feeds on vertebrates, e.g., anurans, reptiles (including other snakes), and mice, killing them by constriction (Martins and Oliveira, 1998; Boos, 2001).
As a back-fanged species, P. neuwiedii produces venom, but little is known of it composition or actions, and there are no reports of human envenomation by this species. This lack of reports on envenomation probably reflects a combination of (1) the docile, non-aggressive behavior of this species, even when handled by humans, (2) the fact that this species is most abundant in regions such as the Amazon basin where the human population density is generally very low, and (3) possible under-reporting of bites since envenomation by pseudoboines usually results in only mild local manifestations. We have recently shown that P. neuwiedii venom contains considerable proteolytic (caseinolytic) activity, low phospholipase A2 (PLA2) and negligible esterase activity; the venom also adversely affects neurotransmission in chick biventer cervicis neuromuscular preparations in vitro by producing moderate blockade and attenuating the muscle contractures to exogenously added acetylcholine and potassium chloride, in addition to producing mild muscle damage (Torres-Bonilla et al., 2017). In this report, we provide additional information on the biochemical characterization of P. neuwiedii venom, particularly in relation to its proteolytic activity and principal toxin families.

2. Material and methods

2.1. Reagents

The reagents for electrophoresis were obtained from Sigma Chemical Co. (St. Louis, MO, USA) or GE LifeSciences (Piscataway, NJ, USA). Molecular mass markers were from BioRad Laboratories (Hercules, CA, USA). 4-(2-Aminoethyl)benzenesulfonyl fluoride (AEBSF), azocasein, N-benzoyl-L-arginine p-nitroanilide (BApNA), casein, ethylenediaminetetraacetic acid (EDTA), gelatin, glycine, 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 4-nitro-3-(octanoyloxy) benzoic acid, 1,10-phenanthroline, phenylmethylsulfonyl fluoride (PMSF), plasminogen-free bovine fibrinogen, Tris hydrochloride, Triton X-100 and trypsin were from Sigma. Isoflurane was from Cristália (Itapira, SP, Brazil). The other reagents were of analytical grade obtained from GE LifeSciences, Mallinckrodt/J.T. Baker (Mexico City, DF, Mexico) and Merck S.A. (Rio de Janeiro, RJ, Brazil).

2.2. Venoms

Pseudoboa neuwiedii venom was a pool collected from two adult snakes (1M, 1F) maintained at the Laboratorio de Herpetología, EcoFisiología y Etología of the Universidad del Tolima (Ibagué, capital city of Tolima, Colombia) under Environmental License No. 2046 (June 13, 2012) provided by Corporación Autónoma Regional del Tolima (CORTOLIMA). The venom was collected using micropipettes placed over the enlarged rear fangs and then lyophilized and stored at −20 °C (Ferlan et al., 1983; Assakura et al., 1992). The mean volume and yield of venom per extraction was 200 μl and 2 mg of dried venom, respectively, based on two extractions from each of the specimens used here. When required, the venom was freshly dissolved in saline (0.9% NaCl) solution prior to use. Venoms from adult Bothrops atrox, Bothrops jararacussu and Bothrops neuwiedii snakes of both sexes were obtained from the Centro de Extração de Toxinas Animais (CETA, Morungaba, SP, Brazil) and stored lyophilized at −20 °C.

2.3. Animals

Male Wistar rats (300–400 g) obtained from the Multidisciplinary Center for Biological Investigation (CEMIB) at UNICAMP were housed in ventilated racks (Alesco®) in standard plastic cages with a wood shaving substrate (5 rats/cage) at 23 °C on a 12 h light/dark cycle (lights on at 6 a.m.) and had free access to standard rodent chow (Nuvital®) and water. The animal protocols were approved by an institutional Committee for Ethics in Animal Use (CEUA/UNICAMP, protocol no. 4479-1/2017) and the experiments were done according to the general ethical guidelines for animal use established by the Brazilian Society of Laboratory Animal Science (SBCAL; http://www. sbcal.org.br/conteudo/view?ID_CONTEUDO=65) and Brazilian legislation (Federal Law no. 11,794, of October 8, 2008), in conjunction with the guidelines for animal experiments established by the Brazilian National Council for Animal Experimentation (CONCEA, http://www. mct.gov.br/index.php/content/view/310553.html; http://www.mct. gov.br/upd_blob/0234/234054.pdf) and EU Directive 2010/63/EU for the Protection of Animals Used for Scientific Purposes.

2.4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymography

SDS-PAGE was done in a discontinuous system with a 4% polyacrylamide stacking gel prepared in 0.5 M Tris-HCl, pH 6.8, containing 0.4% SDS, and a 10% running gel prepared in 1.5 M Tris-HCl, pH 8.8, containing 0.4% SDS (Laemmli, 1970). The samples were diluted in sample buffer containing 4% bromophenol blue, 0.06 M Tris-HCl, 2% SDS and 10% glycerol and boiled for 5 min prior to loading onto the stacking gel. The gels (10 cm × 12.5 cm) were run in a mini-VE 206E system (GE Life Sciences) at constant voltage (100 mV), with 0.625 M Tris-HCl, 1.92 M glycine and 1% SDS, pH 6.8, as the running buffer. Molecular mass markers were included in all runs. After electrophoresis, the gels were stained with Coomassie brilliant blue G250 and documented.
Zymography was done as previously described (Miyazaki et al., 1990). Briefly, samples were mixed with SDS sample buffer in the absence of a reducing agent, incubated for 20 min at 37 °C and separated on a 10% polyacrylamide gel containing gelatin (final concentration: 0.1%) or casein (final concentration: 0.8%). After electrophoresis, gels were soaked in 2.5% Triton X-100 for 40 min then digested by incubating the gel in 50 mM Tris-HCl, pH 7.4, containing 0.1 mM NaCl, 1 M dehydrated CaCl2 and 0.02% NaN3 at 37 °C for 16 h. The gels were stained with 0.1% Coomassie brilliant blue G250 and proteolytic activity was detected as clear bands against a blue background.

2.5. Reverse-phase high performance liquid chromatography (RP-HPLC)

Pseudoboa neuwiedii venom (1 mg) was dissolved in 0.1% trifluoroacetic acid (TFA) and centrifuged (13,000 rpm, 10 min, 4 °C). The sample (100 μg) was loaded onto a reverse-phase C18 HPLC column (Discovery C18 HPLC column, 25 cm × 4.6 mm; Sigma) and elution was performed with 0.1% TFA and a gradient (0–100%) of buffer B (100% acetonitrile in 0.1% TFA) over 215 min at a flow rate of 1 ml/ min. Protein fractions were collected manually, lyophilized and stored at −80 °C.

2.6. In-gel digestion and LC-MS/MS analysis

2.6.1. In-gel digestion

Venom protein bands were excised from the Coomassie blue-stained SDS-PAGE gel and in-gel digested with trypsin as described by Shevchenko et al. (1996).

2.6.2. Analysis by liquid chromatography coupled to mass spectrometry (LC-MS/MS)

The peptide-containing buffer from the overnight digestion was dried in a vacuum centrifuge (Christ) and the peptides then dissolved in 20 μl of 0.1% formic acid prior to analysis by mass spectrometry using an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific) coupled to a nanoLC Easy II (Proxeon) system. For analysis, a 4 μl sample was injected onto a C18 pre-column (100 μm × 40 mm × 5 μm; internal diameter × length × particle size) and the peptides were separated on an analytical column packed with Aqua® C18 beads (Phenomenex; 75 μm × 150 mm × 3 μm) that was eluted with a linear gradient composed of water (solution A) and acetonitrile (solution B) containing 0.1% formic acid at a flow rate of 200 nl/min for 15 min. The gradient started with a concentration of 4% B and finished with 35% B. The MS1 spectra were obtained with the FTMS analyzer at a resolution of 30,000 in an m/z range of 300–2000. The fragmentation method chosen was CID and only the ten most intense ions from each MS1 and those with two or more charges were selected for fragmentation. The collision energy used to obtain the fragments (MS/MS spectra) was 35 eV and the fragments were analyzed in the ion trap mode. The source voltage was fixed at 2 kV and the dynamic exclusion time was adjusted for 90 s; a list containing 500 ions was used to minimize the repeated acquisition of the same m/z value.

2.6.3. Database searches

The resulting file from the LC-MS/MS analysis was converted to MGF format by RawConverter software (He et al., 2015) and was used for database searches with the program Mascot (version 2.4.1). The database used consisted of the sequences stored in Uniprot for snakes (up to 21/07/2017; http://www.uniprot.org/; 70,087 sequences) and 79 complete sequences of toxins obtained from B. jararaca gland venom transcripts (Junqueira-de-Azevedo et al., 2015). The oxidation of methionine residues and deamidation of asparagine and glutamine residues were considered as variable modifications, whereas the carbamidomethylation of cysteine residues was considered a fixed modification. The tolerance values for the observed masses were 10 ppm for the precursors selected in MS1 and 0.5 Da for fragments analyzed in the ion trap. The enzyme selected was trypsin, with a maximum tolerance of one lost cleavage site. The criteria applied to the analysis of the identified proteins were a false discovery rate < 1% and at least two peptides identified, at least one of which was unique. The ion score cut-off was set at < 0.05.

2.7. Enzymatic activities

2.7.1. Proteolytic activity

Caseinolytic activity was measured colorimetrically using azocasein as substrate, as described elsewhere (Wang and Huang, 2002) and modified as follows. The standard assay mixture contained 90 μl of azocasein (5 mg/ml) in reaction buffer (0.05 M Tris-HCl, pH 8.0, 1 mM CaCl2) and 10 μl of P. neuwiedii venom (0.1, 0.3, 1, 3 and 10 μg) in a final volume of 100 μl that was incubated at 37 °C for 90 min. The reaction was stopped by adding 200 μl of 5% trichloroacetic acid followed by incubation at room temperature for 5 min. After centrifugation (8000g, 5 min), 150 μl of the supernatant was mixed with an equal volume of 0.5 M NaOH and the absorbance was measured at 440 nm using a SpectraMax 340 multiwell plate reader (Molecular Devices, Sunnyvale, CA, USA). One unit of activity was defined as an increase in absorbance (A440nm) of 0.001/min. Bothrops atrox and B. neuwiedii venoms were used as positive controls.
The effect of pH, temperature and inhibitors on P. neuwiedii venom proteolytic activity was tested using the caseinolytic assay described above. The optimum pH for proteolytic activity was determined over a pH range of 5.0–11.0, using acetate (pH 5.0), HEPES (pH 6.0 and 7.0), Tris-HCl (pH 8.0 and 9.0) and glycine (pH 10.0 and 11.0) buffers (all 50 mM and containing 1 mM CaCl2). The temperature at which the venom (10 μg) showed maximum activity was examined by incubating the reaction mixture at the optimum pH and at 25, 30, 37, 55 or 80 °C for 90 min. The effect of the inhibitors EDTA and 1.10-phenanthroline (metalloproteinase inhibitors) and PMSF and AEBSF (serine protease inhibitors) was examined by incubating P. neuwiedii venom (10 μg) with these compounds (10 μM to 10 mM) for 30 min at 37 °C prior to assaying residual enzymatic activity.

2.7.2. Esterase activity

Esterase activity was assayed using N-benzoyl-L-arginine p-nitroanilide (BApNA) as substrate. Activity towards BApNA was assayed in 96-well plates. The standard assay mixture contained 50 μl of buffer (10 mM Tris-HCl, pH 8.0, 10 mM CaCl2 and 100 mM NaCl), 200 μl of substrate solution (1 mM), 15 μl of water and 10 μl of P. neuwiedii venom (0.1, 0.3, 1, 3 and 10 μg) in a final volume of 270 μl. The reactions were run in a SpectraMax 340 plate reader for 30 min at 37 °C, with the absorbance being read at 410 nm. The results were expressed as the initial velocity of reaction calculated based on the amount of pnitroanilide formed (Erlanger et al., 1961). Bothrops atrox and B. neuwiedii venoms were used as positive controls.

2.7.3. Phospholipase A2 (PLA2) activity

Venom PLA2 activity was assayed in 96-well plates using 4-nitro-3(octanoyloxy) benzoic acid in 0.1 M Tris-HCl, pH 8, containing 0.01 M Ca2+ for 30 min at 37 °C (Ponce-Soto et al., 2002). The standard assay mixture contained 200 μl of buffer, 20 μl of substrate and 20 μl of P. neuwiedii venom (0.1, 0.3, 1, 3, 10 and 20 μg) in a final volume of 240 μl. The reactions were run in a SpectraMax 340 plate reader for 30 min at 37 °C, with the change in absorbance being read at 425 nm. One unit of activity was defined as an increase in absorbance (A425nm) of 0.001/min. Bothrops atrox, B. neuwiedii and B. jararacussu venoms were used as positive controls.

2.8. Pro- and anticoagulant activities

2.8.1. Fibrinogenolytic activity

Fibrinogenolytic activity was assayed by incubating 100 μl of plasminogen-free bovine fibrinogen (5 mg/ml; dissolved in 0.1 M Tris-HCl, pH 7.5, containing 0.1 M NaCl) with 100 μl of venom (10 μg) at 37 °C. At predetermined times (10 and 30 min, and 1, 2, 4 and 6 h) after venom addition, 20 μl aliquots were withdrawn from the incubation mixture and transferred to polypropylene Eppendorf tubes containing 20 μl of 2% β-mercaptoethanol and 2% SDS. After mixing, the samples were loaded onto 12% polyacrylamide gels and the proteins separated by SDS-PAGE; the gels were subsequently stained with Coomassie brilliant blue R250 (Cavinato et al., 1998). Undegraded fibrinogen and molecular mass markers were included in the runs to allow assessment of fibrinogen chain digestion. In some assays, the venom was treated with protease inhibitors (EDTA, 1,10-phenanthroline, PMSF or AEBSF, 10 mM each) for 30 min at 37 °C prior to incubation with fibrinogen for 1 h. The remainder of the assay was done as described above.

2.8.2. Coagulant activity

The coagulant activity of P. neuwiedii venom was examined using rat citrated platelet-poor plasma (PPP). Rats were anesthetized with isoflurane and arterial blood was collected into polypropylene tubes containing 3.8% sodium citrate (anticoagulant:blood ratio 1:9, v/v), after which the animals were killed with an overdose of anesthetic. Platelet-rich plasma (PRP) was obtained by centrifugation (400g, 15 min, room temperature) and further centrifugation of PRP (800g, 13 min, room temperature) was used to obtain platelet-poor plasma (PPP) for the coagulation test. For the assay, 190 μl of PPP was incubated at 37 °C with 10 μl of sample (P. neuwiedii venom: 10 μg, B. atrox venom: 10 μg as a positive control) and the time required for clot formation was recorded.

2.8.3. Plasma recalcification time

The influence of P. neuwiedii venom on the plasma recalcification time was assayed as described elsewhere (Berger et al., 2008; da Silva et al., 2012). Briefly, PPP was obtained from rat citrated blood by centrifugation (3000g, 15 min, room temperature) and 80 μl aliquots were transferred to a 96-well plate and incubated with 50 mM Tris-HCl, pH 7.4 (control, 10 μl) or P. neuwiedii venom (5 or 10 μg in 10 μl of TrisHCl buffer) at 37 °C. After a 5 min incubation with venom, 10 μl of 250 mM CaCl2 was added to each well (total reaction volume: 100 μl) and clot formation was monitored for 30 min at 650 nm in a SpectraMax 340 plate reader.

2.9. Statistical analysis

The results were expressed as the mean ± SD. Statistical comparisons were done using one-way or two-way ANOVA followed by the Tukey-Kramer post-hoc test. A value of p < 0.05 indicated significance.

3. Results and discussion

Colubrid venoms contain a variety of toxins such as snake venom metalloproteinases (SVMPs), snake venom serine proteases (SVSPs), Ctype lectins, cysteine-rich secretory proteins (CRISPs), three-finger toxins (3FTx) and other proteins, many of which have counterparts in the venoms of highly venomous crotalids, viperids and elapids (Junqueira-de-Azevedo et al., 2016). SVMPs are very common in colubrid venoms (Zelanis et al., 2010; Peichoto et al., 2012; Sanchez et al., 2014; Junqueira-de-Azevedo et al., 2016) and some of them have been purified and characterized, e.g., the metallofibrinogenases PofibC1, C2, C3 and H from Philodryas olfersii (Assakura et al., 1994), patagonfibrase from Philodryas patagoniensis (Peichoto et al., 2007, 2010, 2011; Peichoto and Santoro, 2016), and alsophinase from Alsophis portoricensis (Weldon and Mackessy, 2012). SVMPs are classified into three classes (P-I, P-II and P-III) that vary in structure and biological activities (Gutiérrez and Rucavado, 2000; Gutiérrez et al., 2005; Fox and Serrano, 2008, 2009; Markland and Swenson, 2013). P-III SVMPs are among the best studied because of their potent hemorrhagic activity, with P-I and P-II SVMPs being less studied, although they also have important physiological effects in envenoming (Escalante et al., 2011). SVMP activity in vitro is frequently monitored based on proteolytic activity, typically the cleavage of casein or azocasein. We have previously shown that P. neuwiedii venom has proteolytic (caseinolytic) activity (TorresBonilla et al., 2017), and we examined this activity in more detail in this work.
Pseudoboa neuwiedii venom had similar caseinolytic activity to B. neuwiedii venom when tested over the same range of doses, but considerably less activity than B. atrox at the two highest doses (Fig. 2A).
This caseinolytic activity was mediated by metalloproteinases, most probably SVMPs, since it was strongly inhibited by EDTA and 1,10phenanthroline (10 mM each) that chelate metal ions, including Zn2+ that is essential for SVMP activity. In contrast, PMSF and AEBSF (serine proteinase inhibitors) at the highest concentration tested (10 mM) caused ≤30% inhibition that was more marked with PMSF (Fig. 2B). The optimum pH for the caseinolytic activity of P. neuwiedii venom was pH 8.0, with a rapid decrease in activity at pH < 6.0 or > 9.0 (Fig. 2C), while the ideal temperature for activity was 37 °C (Fig. 2D).
The majority of caseinolytic proteases in colubrid venoms are metalloenzymes (Mackessy, 2002; Junqueira-de-Azevedo et al., 2016) that may have a role in prey digestion (Hill and Mackessy, 2000; Mackessy, 2002). The SVMPs of South American colubrids studied so far have an important role in the pathophysiology of envenoming, and contribute to hemorrhage through the degradation of specific basement membrane components and the disruption of capillary walls (Assakura et al., 1992; Acosta et al., 2003; Lemoine and Rodríguez-Acosta, 2003; Lemoine et al., 2004a,b; Weldon and Mackessy, 2010; Sanchez et al., 2014). These proteins can also contribute to inflammation (Acosta de Perez et al., 2003; Peichoto et al., 2011) and myotoxicity (Peichoto et al., 2004; Sanchez et al., 2014).
Although the proteolytic activity of P. neuwiedii venom was slightly inhibited by PMSF and AEBSF at the highest concentration tested, this may have been a non-specific effect since the venom showed virtually no activity towards the esterase substrate BapNA, even at the highest amount tested (10 μg) compared with the Bothrops venoms used as positive controls (Fig. 2E). This finding indicates that P. neuwiedii venom lacks SVSPs. Indeed, few reports have detected SVSP activity in colubrid venoms (Assakura et al., 1994; Mackessy, 2002; Weldon and Mackessy, 2010; Junqueira-de-Azevedo et al., 2016) and, where present, this activity is generally much lower than in front-fanged snakes (Mackessy, 1993).
In contrast to many other colubrid venoms, P. neuwiedii venom showed PLA2 activity comparable to that of B. atrox venom and somewhat greater than B. neuwiedii, but lower than B. jararacussu at the highest amount tested (20 μg) (Fig. 2F). Compared to its wide distribution in the venoms of front-fanged snakes, PLA2 activity is absent in many rear-fanged species (Junqueira-de-Azevedo et al., 2016) but has been detected in venoms of other South American dipsadids, including Erythrolamprus bizona (Torres-Bonilla et al., 2017), Leptodeira annulata (Mebs, 1968; Torres-Bonilla et al., 2016), Thamnodynastes strigatus (Hill and Mackessy, 2000; Mackessy, 2002), Tomodon dorsatus and Trimorphodon biscutatus (Zelanis et al., 2010; Peichoto et al., 2012). The greater PLA2 activity found in this work compared to a previous report (Torres-Bonilla et al., 2017) most probably reflects the different sensitivities of the assays used in these studies since, in the previous report, a pH-sensitive dye assay (Price, 2007) was used that was less sensitive and more complicated to run than the colorimetric assay based on 4-nitro-3-(octanoyloxy) benzoic acid used here.
Since proteolytic activity was the highest of the three enzymatic activities detected in P. neuwiedii venom, we investigated this in more detail. SDS-PAGE of P. neuwiedii venom (30 μg) showed several bands with molecular masses > 100 kDa, 45–70 kDa, 21–24 kDa and ~15 kDa (Fig. 3A, lane 2). This electrophoretic profile was considerably simpler than that of two Bothrops venoms (B. atrox and B. neuwiedii; 30 μg each) run under the same conditions; the components of the latter two venoms ranged from 15 kDa to ~100 kDa (Fig. 3A, lanes 3 and 4, respectively). The high molecular mass bands of P. neuwiedii venom probably corresponded to P-III SVMPs, a conclusion confirmed by mass spectrometric analysis of the protein bands (see below).
Pseudoboa neuwiedii venom showed strong proteolytic activity in gelatin zymography, with activity being detected using as little as 0.05 μg of venom (Fig. 3B, lane 8). Analysis of lanes 6–8 in Fig. 3B indicated that P. neuwiedii venom contained various proteins with activity towards this substrate, but that most of the activity was associated with two bands with molecular masses of ~65–68 kDa; with higher amounts of venom (lanes 2–5), additional ‘bands’ were seen at ≥97 kDa that appeared as smears and most likely reflected the elevated proteolytic activity of the venom components during electrophoretic migration. This high gelatinase activity was similar to that reported for the venoms of the colubrids Alsophis portoricensis and Hydrodynastes gigas (Hill and Mackessy, 2000; Weldon and Mackessy, 2010) and more prominent than the activity in the venoms of the Asian Amphiesma stolatum and Rhabdophis tigrinus (Hill and Mackessy, 2000) and the South American Philodryas spp. (P. olfersii, P. patagonensis, P. nattereri), Thamnodynastes strigatus and Tomodon dorsatus (Hill and Mackessy, 2000; Zelanis et al., 2010). For comparison with P. neuwiedii, under similar conditions, the gelatinolytic activity of B. atrox and B. neuwiedii venoms was detectable with ≥3 μg of venom (Fig. 3D, lanes 2–5 and 6–9, respectively).
The venom also showed proteolytic activity towards casein in zymography, but this was less intense than towards gelatin, with activity being observed in the range of 5–40 μg (Fig. 3C). In addition, some of the proteins involved apparently differed from those for gelatinolytic activity since the molecular mass range was lower than for the latter activity, e.g., ~50–66 kDa compared to ~65–68 kDa for gelatinolytic activity; the greatest caseinolytic activity was seen with a band of ~50 kDa (Fig. 3C).
To explore the composition of P. neuwiedii venom further, venom protein bands were excised from the SDS-PAGE gel, digested with trypsin and analyzed by LC-MS/MS. This analysis revealed the presence of a variety of components, including cysteine-rich secretory proteins (CRISPs), PLA2, SVMPs, snake venom matrix metalloproteinases (svMMPs) and less abundant components such as C-type lectin-like protein (CLP), phospholipase B (PLB) and vascular endothelial growth factor (VEGF); significantly, no serine proteinases (SVSPs) were identified (Table S1, Supplementary material). The intensity of the protein bands and their corresponding identified toxins suggested that CRISPs and SVMPs were the major venom components, with CLP and PLA2 being minor components (Fig. 4); these proteins have also been identified in the venoms or venom glands of other colubrids (Junqueira-deAzevedo et al., 2016).
SVMPs are widespread in crotalid and viperid venoms, where they are responsible for a variety of effects, including hemorrhage, inflammation, and interference with coagulation and platelet aggregation (Gutiérrez and Rucavado, 2000; Gutiérrez et al., 2005; Fox and Serrano, 2008; Escalante et al., 2011). SVMPs are also the most widespread of the major enzymatic components in colubrid venoms and are regularly detected in transcriptomic and proteomic analyses (Kamiguti et al., 2000; Campos et al., 2016; Junqueira-de-Azevedo et al., 2016). Colubrid venom SVMPs are P-III SVMPs and our findings generally agree with this since the P. neuwiedii SVMP fragments showed homology with P-III SVMPs from colubrid and crotalid (Bothrops spp.) venoms. Two high molecular mass bands (> 100 kDa) yielded fragments with homology to SVMPs, but it is unclear whether these proteins represent novel or incompletely processed SVMPs or simply proteins with anomalous migratory behavior in SDS-PAGE. In addition, SVMP-related fragments were detected in a band of ~21 kDa that is within the molecular mass range of P-I class SVMPs although, as indicated above, colubrid SVMPs are generally P-III proteins.
The two best studied colubrid SVMPs so far, alsophinase from Alsophis (Borikenophis) portoricensis (Weldon and Mackessy, 2012) and patagonfibrase from Philodryas patagoniensis (Peichoto et al., 2007, 2010, 2011; Peichoto and Santoro, 2016) are fibrinogenolytic (α-fibrinogenases), pro-inflammatory, hemorrhagic and necrotic. As indicated below, P. neuwiedii venom also showed α-fibrinogenolytic activity (with no effect on the β and γ chains) that was inhibited by EDTA and 1,10-phenanthroline, but not by PMSF and AEBSF, indicating that SVMPs were involved. This finding, together with the fragment homologies, indicates that P-III SVMPs are responsible for this activity in P. neuwiedii venom.
Cysteine-rich secretory proteins (CRISPs) are widespread in snake venoms and exert a variety of activities, including the modulation of cyclic nucleotide-gated ion channels and voltage-gated Ca2+ (but not K+) channels, attenuation of K+-induced contractions of arterial smooth muscle, pro-inflammatory effects and human complement activation; however, these activities are not consistently found among these proteins (Yamazaki et al., 2002; Jin et al., 2003; Yamazaki et al., 2003; Fry et al., 2008; Suzuki et al., 2008; Lodovicho et al., 2017). CRISPs have also been detected to varying degrees in transcriptomic and proteomic analyses of most colubrid venoms examined so far (Junqueira-de-Azevedo et al., 2016). The fragments detected in P. neuwiedii venom (particularly MEWYPEAAANAER that is generally well-conserved among snake venoms) showed homology with a variety of colubrid and crotalid CRISPs (Fig. 4, Table S1). Among colubrids, homology was detected with helicopsin purified from Helicops angulatus (Estrella et al., 2011), CRISPs cloned from P. chamissonis (Urra et al., 2015), and a protein from Erythrolamprus poecilogyrus.
Despite their widespread distribution, few CRISPs have been purified from colubrid venoms and their precise role in envenomation remains unclear. The best characterized colubrid CRISPs are helicopsin from H. angulatus (Estrella et al., 2011) and patagonin from Philodryas patagoniensis (Peichoto et al., 2009). Helicopsin is not proteolytic or hemorrhagic but is neurotoxic and lethal to mice (minimum lethal dose: 0.4 mg/kg, i.p.), with death occurring within a few minutes through respiratory paralysis (Estrella et al., 2011). Patagonin is also devoid of proteolytic, edematogenic and hemorrhagic activities, does not stimulate platelet aggregation or inhibit platelet aggregation induced by a variety of agonists, and has no effect on the basal tension or K+-induced contraction of rat endothelium-denuded aortic rings; however, this protein causes myonecrosis and an inflammatory infiltrate in mouse gastrocnemius muscle (Peichoto et al., 2009). Since P. neuwiedii venom causes myonecrosis in chick biventer cervicis muscle in vitro (TorresBonilla et al., 2017), this damage could be partially mediated by CRISPs.
PLA2 are ubiquitous in elapid, crotalid and viperid venoms, but colubrid venoms are generally devoid of this enzyme (Junqueira-deAzevedo et al., 2016); hence, the detection of PLA2 activity in P. neuwiedii venom and the identification of PLA2 fragments in the mass spectrometric analysis is particularly interesting. The fragments identified showed 100% identity with regions of myotoxin II, a basic, noncatalytic Lys49 PLA2 homolog (UniProtKB/Swiss-Prot accession no.: Q9I834) from venom of the South American lancehead pitviper Bothrops moojeni (de Azevedo et al., 1997; Soares et al., 1998), as indicated below (the underlined regions correspond to the fragments identified in P. neuwiedi venom mapped onto the myotoxin II sequence): SLFELGKMILQETGKNPAKSYGVYGCNCGVGGRGKPKDATDRCCYVHKCCYKKLTGCDPKKDRYSYSWKDKTIVCGENNSCLKELCECDKAVAICLRENLDTYNKKYRYNYLKPFCKKADPC.
Additional identity was also observed between P. neuwiedii fragments and PLA2 of other Bothrops spp. (B. atrox, B. brazili) and with the basic PLA2 subunit of crotoxin, a dimeric PLA2 from venom of the South American rattlesnake, Crotalus durissus terrificus; there was no homology with elapid PLA2. These findings indicate that P. neuwiedii PLA2s belong to group IIA PLA2 that contains crotalid and viperid enzymes, rather than to group IA that contains elapid PLA2. To date, only one report has described the purification and characterization of a PLA2 from colubrid venom, i.e., trimorphin from the venom Trimorphodon biscutatus lambda (Huang and Mackessy, 2004). The N-terminal sequence of the first 50 amino acids of this protein indicated greatest homology with hydrophine (sea snakes and Australian elapids) PLA2, indicating it was a group IA PLA2; the enzyme contained Asp49 and His48 in the active site that accounted for its enzymatic activity. The catalytic activity showed a broad pH optimum (7–9) and was abolished by EDTA, which suggested a Ca2+-dependence similar to other catalytically active snake venom PLA2; no further characterization of the biological activities was reported. In contrast to these findings of PLA2 related to crotalid/viperid and elapid venoms, transcriptomic analyses have detected type IIE PLA2 in the venom glands of Dispholidus typus (boomslang; Colubridae) and Leioheterodon madagascariensis (Madagascan giant hognose snake; Lamprophiidae) (Fry et al., 2012), Oxyrhopus guibei (false coralsnake; Dipsadidae) (Junqueira-de-Azevedo et al., 2016), Pantherophis guttatus (corn snake; Colubridae) and Opheodrys aestivus (rough green snake; Colubridae) (Hargreaves et al., 2014). While the role of type IIE PLA2 in colubrid envenomation is unknown, the type IA and IIA PLA2 are probably involved in local effects such as pain, edema (enhanced vascular permeability), inflammatory responses and myonecrosis, in a manner analogous to that of PLA2 from crotalid venoms (Gutiérrez and Ownby, 2003; Teixeira et al., 2003; Zambelli et al., 2017). In this context, the necrosis and muscle fiber edema caused by P. neuwiedii venom in chick biventer cervicis muscle in vitro may be partially mediated by PLA2 (TorresBonilla et al., 2017).
C-Type lectin proteins (CLPs) have been detected in various colubrid venom gland transcriptomes and venom proteomes (Junqueira-deAzevedo et al., 2016) but to-date none has been purified or characterized. The fragment GGHLASLGSIEEGKFVGK detected here showed homology with the α-subunit of a CLP cloned from Philodryas chamissonis venom gland and, by extension, with the corresponding subunit of a CLP from Philodryas olfersii with which the P. chamissonis protein shares 96% sequence identity (Urra et al., 2015). In elapid, crotalid and viperid venoms, CLP can interfere with blood coagulation and platelet aggregation (Morita, 2005; Clemetson, 2010; Arlinghaus and Eble, 2012) and similar activities could potentially be exerted by P. neuwiedii venom, but this remains to be determined.
In contrast to SVMPs that are abundant in colubrid venoms, snake venom matrix metalloproteinases (svMMPs), which appear to be colubrid-specific venom components, are less abundant and have been identified in several venoms primarily through transcriptomic and proteomic analyses (Ching et al., 2012; Junqueira-de-Azevedo et al., 2016). Pseudoboa neuwiedii venom showed several peptide matches with svMMP from the colubrid Phalotris mertensi and with matrix metalloproteinase-9 from Rhabdophis tigrinus (structurally similar to svMMP; Table S1). The function of svMMPs in colubrid venoms is unclear, but they could potentially act synergistically with SVMPs during envenomation (Junqueira-de-Azevedo et al., 2016). In this regard, an svMMP purified from an extract of macerated R. tigrinus Duvernoy’s gland showed strong proteolytic (gelatinolytic) activity that was inhibited by EDTA and 1,10-phenanthroline (Komori et al., 2006).
Overall, the LC-MS/MS findings indicating the presence of SVMPs and PLA2s agreed with the enzymatic assays that detected proteolytic and PLA2 activities in P. neuwiedii venom. Similarly, the lack of sequences corresponding to SVSPs agreed with the absence of amidolytic activity in the venom. The latter finding also agreed with the widespread absence of SVSPs and esterase activity in colubrid venoms (Mackessy, 2002; Zelanis et al., 2010; Junqueira-de-Azevedo et al., 2016). In addition to well-known venom components, the LC-MS/MS analysis also revealed the presence of a variety of cellular proteins (Table S1, Supplementary material), such as glutathione peroxidase, serpin, annexin, leucocyte elastase inhibitor and cyclotransferase; these proteins from venom gland epithelial cells may or may not have relevant contributions to the biological actions of the venom.
RP-HPLC of P. neuwiedii venom yielded 15 peaks with retention times of 130–190 min (Fig. 5A). SDS-PAGE of these peaks revealed protein bands (most of which showed weak silver staining) in peaks 3 (~200 kDa), 4–6 (~15–20 kDa region) and 7–9 (~37–75 kDa region) (Fig. 5B, C); these bands generally corresponded to the major bands seen in the electrophoretic profile of the venom (Fig. 3A). Screening of all peaks for gelatinolytic activity indicated that only peaks 7–11 were active (Fig. 5D, E), with three protein bands of ~50–66 kDa being detected; these bands corresponded to the main protein bands seen in gelatin zymography of the venom (Fig. 3B). Although peaks 7–11 showed proteolytic activity in gelatin zymography, no protein bands were seen in SDS-PAGE for peaks 10 and 11, probably because of the low amount of venom (100 μg) used for RP-HPLC (the total amount of venom available for this work was very limited). This finding nevertheless indicated very potent activity of the proteins involved (gelatinolytic activity was seen with nanogram quantities of protein). Indeed, serial dilutions of peak 7 corresponding to 250 ng to 32.5 ng of protein confirmed that this peak had very potent activity in gelatin zymography that was completely inhibited by 10 mM EDTA (Fig. 6A, B) indicating the presence of SVMPs (probably belonging to the P-III class based on their molecular mass and sensitivity to EDTA). The components of this peak are likely to have an important role in mediating the biological activities of this venom.
Pseudoboa neuwiedii venom degraded the Aα-chain of fibrinogen, whereas the Bβ-chain and γ-chain were not affected. Digestion of the Aα-chain resulted in the appearance of degradation products with molecular masses of 21–50 kDa (Fig. 7A). The inhibition assays showed that EDTA and 1,10-phenanthroline abolished the fibrinogenolytic activity of P. neuwiedii venom, whereas PMSF and AEBSF had no effect on this activity (Fig. 7B). These results indicate that degradation of the Aαchain was mediated by SVMPs. The venom (10 μg) did not clot rat citrated plasma even after 5 min at 37 °C (n = 6) whereas the positive control (B. atrox venom, 10 μg) clotted citrated plasma in 49 ± 12 s (n = 6). These results indicate that the venom did not contain thrombin-like and/or procoagulant enzymes (that are typically SVSPs), in agreement with the lack of esterase activity noted above. However, P. neuwiedii venom (10 μg) delayed the initiation of coagulation by ~9 min in response to plasma recalcification and reduced the extent of coagulation by ~80%; a lower amount of venom (5 μg) had a less marked effect on the clotting of recalcified plasma (Fig. 7C).
This delay in coagulation agreed with findings for other colubrids. The venoms of the colubrids P. olfersii, P. patagonensis, P. baroni and A. portoricensis interfere with blood coagulation by causing hypofibrinogenemia through the action of fibrinogenases that likely contribute to the bleeding and prolonged clotting times associated with bites by these snakes (Assakura et al., 1992; Peichoto et al., 2005; Weldon and Mackessy, 2010; Sanchez et al., 2014). The strong fibrinogenolytic activity of P. neuwiedii venom that adversely affected the rate and extent of recalcification was most likely mediated by P-III SVMPs since (a) this venom does not contain SVSPs, (b) studies with this class of SVMPs from other colubrid snake venoms indicate that they have potent fibrinogenolytic activity (Assakura et al., 1994; Peichoto et al., 2007) and (c) this class of SVMPs is generally resistant (partly because of their high molecular mass) to inhibition by plasma α2macroglobulin that usually inhibits P-I SVMPs (Markland, 1998; Escalante et al., 2011). In addition to P-III SVMPs, it is possible that the venom PLA2 could also contribute to the delay in plasma recalcification since these enzymes can inhibit formation of the prothrombinase complex that is necessary for coagulation by degrading phospholipids involved in this complex (Markland, 1998; Kini, 2005); this possibility was not investigated here because of the limited amount of venom available for detailed studies.
Envenomation by pseudoboines is characterized by a variety of local effects summarized in Table 1. Although detailed biochemical studies to identify the toxins responsible for these effects have not been undertaken for this group of snakes, comparison with similar responses seen in envenomation by the much better characterized Bothrops pitvipers suggests that the venom components most probably involved in these manifestations are SVMPs (Gutiérrez and Rucavado, 2000; Gutiérrez et al., 2005; Escalante et al., 2011) and PLA2 (Teixeira et al., 2003, 2009). This conclusion agrees with our demonstration here of high proteolytic and moderate PLA2 activity in P. neuwiedii venom and suggests that envenomation by this species may well result in the local effects already described for other pseudoboines. Although P. neuwiedii venom is apparently devoid of thrombin-like activity, its ability to degrade fibrinogen and delay plasma recalcification suggests that this venom could affect coagulation locally and possibly systemically. This activity, in conjunction with potent SVMP action, could potentially contribute to marked local, and possibly systemic, hemorrhage after envenomation. The limited amount of material available for this investigation meant that it was not possible to examine the local and systemic biological activities of this venom.
In conclusion, the results described here show that P. neuwiedii venom contains primarily SVMPs, CRISPs and PLA2. Enzymatically, the venom is highly proteolytic and this activity could potentially affect coagulation in vivo through its ability to degrade fibrinogen via SVMPs. The purification and characterization of SVMPs, CRISPs and PLA2 from this venom would shed light on their ability to interfere with coagulation and platelet aggregation and their potential role in the local and systemic effects of envenoming by this species.

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