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First crotoxin-like phospholipase A2complex from a New World non-rattlesnake species: Nigroviriditoxin, from the arboreal Neotropicalsnake Bothriechis nigroviridis

Bruno Lomonte a , *, Diana Mora-Obandoa, Julian Fernandeza, Libia Sanz b , Davinia Pla b,Jose Mara Gutierrez a , Juan J. Calvete b

a Instituto Clodomiro Picado, Facultad de Microbiologa, Universidad de Costa Rica, San Jose 11501, Costa Ricab Instituto de Biomedicina de Valencia, CSIC, Jaume Roig 11, 46010 Valencia, Spain

a r t i c l e i n f o

Article history:

Received 19 September 2014Received in revised form21 November 2014Accepted 27 November 2014Available online 28 November 2014

Keywords:

Snake venomViperidaePhospholipase A2CrotoxinBothriechis nigroviridis

a b s t r a c t

Bothriechis nigroviridis is an arboreal Neotropical pitviper found in Costa Rica and Panama. A previousproteomic proling of its venom revealed the presence of proteins with homology to the A and B sub-units of crotoxin/Mojave toxin, a heterodimeric phospholipase A2 (PLA2) complex only described inrattlesnake venoms (genera Crotalus and Sistrurus). The native crotoxin-like heterodimer, namednigroviriditoxin, and its A and B subunits were isolated in the present work, and the complete amino acidsequence of the B subunit was determined. The puried A and B components were demonstrated to forma complex when reconstituted under native conditions. Nigroviriditoxin presents features similar tocrotoxin, albeit displaying lower toxicity: the A component decreases the PLA2 activity of the Bcomponent, and increases its lethal potency in mice. Also in similarity to crotoxin B, nigroviriditoxin Binduces myonecrosis. Its 122 amino acid sequence presents 81% identity with crotoxin B. Accordingly,nigroviriditoxin B was cross-recognized by equine antibodies from a Crotalus durissus terricus anti-venom. Phylogenetic analysis shows that the novel PLA2fromB. nigroviridisvenom is basal to the branch

including all the homologous PLA2enzymes described in rattlesnakes, and more distant from PLA2s fromBothriechis species. Nigroviriditoxin is the rst heterodimeric PLA2complex found in a non-rattlesnake,Neotropical viperid venom, which displays structural, functional, and immunochemical similarities tocrotoxin. The present ndings are compatible with the existence of the particular structural trait ofcrotoxin-like molecules in New World pitvipers before the split of the Meso-South American and theNearctic clades.

2014 Elsevier Ltd. All rights reserved.

1. Introduction

The black-speckled palm snake, Bothriechis nigroviridis, is an

arboreal Neotropical pit viper that inhabits subtropical rainforestsand temperate forests at medium to high elevations(1150e3000 m) on the Cordillera de Tilaran (highlands of Mon-teverde) and Cordillera Volcanica Central in Costa Rica, southeast-ward through the Cordillera de Talamanca to the Chiriqu provincein Panama(Savage, 2002; Campbell and Lamar, 2004; Solorzano,2004). Its Latin name originates from its distinctive speckled co-lor pattern combining black (nigro) and emerald green (viridis)

spots. Adults have been described to prey on small rodents, lizards,frogs, and occasionally small birds (Solorzano, 2004). Owing to itsconnement to undisturbed habitats with little human contact,

envenomings by this pitviper species appear to be extremely un-common, as no documented cases could be found in a literaturesearch.

In a previous study, the venom ofB. nigroviridiswas analyzed bya combined proteomic and toxicological approach (Fernandez et al.,2010), revealing remarkable differences with the venoms of threeother Bothriechisspecies found in Costa Rica: Bothriechis lateralis,Bothriechis schlegelii(Lomonte et al., 2008) and Bothriechis supra-ciliaris(Lomonte et al., 2012). Surprisingly,B. nigroviridisvenom isdevoid of metalloproteinases (Fernandez et al., 2010), a widespreadand often predominant protein family in viperid venoms (Calveteet al., 2007; Lomonte et al., 2014). In agreement with its lack of

* Corresponding author.E-mail address:[email protected](B. Lomonte).

Contents lists available at ScienceDirect

Toxicon

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c om / l o c a t e / t o x i c o n

http://dx.doi.org/10.1016/j.toxicon.2014.11.235

0041-0101/

2014 Elsevier Ltd. All rights reserved.

Toxicon 93 (2015) 144e154
mailto:[email protected]://www.sciencedirect.com/science/journal/00410101http://www.elsevier.com/locate/toxiconhttp://dx.doi.org/10.1016/j.toxicon.2014.11.235http://dx.doi.org/10.1016/j.toxicon.2014.11.235http://dx.doi.org/10.1016/j.toxicon.2014.11.235http://dx.doi.org/10.1016/j.toxicon.2014.11.235http://dx.doi.org/10.1016/j.toxicon.2014.11.235http://dx.doi.org/10.1016/j.toxicon.2014.11.235http://www.elsevier.com/locate/toxiconhttp://www.sciencedirect.com/science/journal/00410101http://crossmark.crossref.org/dialog/?doi=10.1016/j.toxicon.2014.11.235&domain=pdfmailto:[email protected]

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metalloproteinases,B. nigroviridisvenom does not induce hemor-rhage in the mouse skin assay (Fernandez et al., 2010). Anotherstriking feature revealed by the proteomic study of this venom wasthe identication of a major phospholipase A2 (PLA2; EC 3.1.1.4)component which showed signicant amino acid sequence ho-mology to crotoxin B from rattlesnakes (Fernandez et al., 2010). Inaddition, this venom presented a protein with homology with theacidic subunit of crotoxin, i.e. crotoxin A (Fernandez et al., 2010).Crotoxin is a well characterized heterodimeric PLA2complex (Airdet al.,1986; Faure et al.,1994; Marchi-Salvador et al., 2008; Sampaioet al., 2010) thus far only found in the venom of rattlesnakes(generaCrotalusand Sistrurus). The unusual presence of crotoxin-like components outside rattlesnake venoms prompted us toisolate the native crotoxin-like complex, and characterize itsfunctional properties, the amino acid sequence of the PLA2component, and its phylogenetic relationships to homologousproteins found in other crotalid snake venoms.

2. Materials and methods

2.1. Venom

Venom was collected and pooled from three adult specimens ofB. nigroviridiskept at the serpentarium of Instituto Clodomiro Pic-ado, University of Costa Rica. After centrifugation to removeinsoluble matter, the venom was lyophilized and stored at 20 Cuntil use.

2.2. Isolation and characterization of native nigroviriditoxin

heterodimer

To demonstrate the existence of the nigroviriditoxin AB heter-odimer, 50 mg of whole venom in 100 mL of 0.1 M ammonium ac-

etate, pH 6.9, were fractionated using an ETTAN-LC chromatographand a Bio-Sil SEC 250 (300 7.8 mm) size-exclusion chromato-graphic column (Bio-Rad) equilibrated in the same buffer. Theeluate was monitored at 215 nm and the fractions, collectedmanually, were analyzed by LC-MS using a nano-Acquity Ultra-Performance LC (UPLC) using a BEH130 C18(100mm 100 mm,1.7mm particle size) column in-line with a Waters SYNAPT G2 HighDenition Mass Spectrometry System. The ow rate was set to0.6ml/min and the column was developed with a linear gradient of0.1% formic acid in water (solution A) and 0.1% formic acid inacetonitrile (solution B), isocratically 1% B for 1 min, followed by1e12% B for 1 min, 12e40% B for 15 min, and 40e85% B for 2 min.

2.3. Isolation of nigroviriditoxin A and B subunits

Venom aliquots of 2.0e2.5 mg were dissolved in 220 mL of so-lution A (0.1% triuoroacetic acid in water) and centrifuged at3000 g for 5 min. The clear supernatant was fractionated byreverse-phase HPLC, using an Agilent Model 1200 chromatograph.Two-hundred microliters of venom were injected to a C 18column(Teknochroma; 250 4.6 mm; 5 mm particle size) equilibrated withsolution A, and elution was then carried out at a ow rate of 1 mL/min by applying the following gradient toward solution B (0.1% TFAin acetonitrile): 0% B for5 min, 0e15% B over 10 min,15e45% B over60 min, 45e70% B over 10 min, and 70% B over 9 min. The eluantwas monitored at 215 nm, and the fractions of interest werecollected manually, dried by vacuum centrifugation at 45 C, and

stored at 20

C.

2.4. SDS-PAGE and nESI-mass spectrometry

Peaks corresponding to the A and B subunits of nigroviriditoxinobtained from RP-HPLC were redissolved in water, and their proteinconcentration was estimated by measuring the absorbance at280 nm in a NanoDrop 2000c instrument (Thermo Scientic).Samples were then analyzed by SDS-PAGE using pre-cast gradientgels (4e20%; Bio-Rad) under reducing or non-reducing conditions.Proteins were visualized by Coomassie blue R-250 staining, andrecorded using a ChemiDoc gel imager with ImageLab software(Bio-Rad). Molecular mass markers were run in parallel. Theisotope-averaged molecular mass of puried A and B subunits,respectively, was determined by nESI-MS on a QTrap-3200 massspectrometer (Applied Biosystems). The proteins were diluted in50% acetonitrile containing 0.1% formic acid, and directly infusedinto a nanospray source for ionization at 1200 V. Analysis wasperformed in positive enhanced multicharge mode in the range600e1700m/z, aided by the Analyst v.1.5 software (ABSciex).

2.5. Complex formation between nigroviriditoxin A and B subunits

The A and B subunits of nigroviriditoxin were incubated

together or alone for 15 min at room temperature, and then sub-jected to agarose gel electrophoresis under native conditions. Theagarose was dissolved at 1% (w/v) in 0.116 M Tris, 0.3 M glycine, pH8.6 buffer. Electrophoresis was performed at 2 mA/cm for 90 min,and proteins were visualized by Coomassie R-250 staining.

2.6. Amino acid sequencing of nigroviriditoxin B

The N-terminal amino acid sequence of nigroviriditoxin B wasobtained by automated direct Edman degradation in a Procise in-strument (Applied Biosystems) following manufacturer's in-structions (Fernandez et al., 2010). The protein sequence wascompleted by tandem mass spectrometry of peptides obtained af-ter the digestion of the DTT-reduced and iodoacetamide-alkylated

protein with trypsin or chymotrypsin. Peptides were diluted 1:2with a saturated a-cyanohydroxycinnamic acid solution in 50%acetonitrile and 0.1% triuoroacetic acid, spotted onto an Opti-TOF-384 plate, dried, and analyzed by MALDI-TOF-TOF on a Model4800-Plus Proteomics Analyzer (Applied Biosystems). Spectra wereacquired in positive reector mode at 2 kV in the 875e4000m/zrange using a laser intensity of 3000 and 1500 shots (Lomonte et al.,2012). CalMix standards (ABSciex) spotted on the same plate wereused for external calibration. Few peptides were analyzed by nESI-MS/MS on a QTrap2000 mass spectrometer (Applied Biosystems)by direct infusion into a nanospray source. Selected doubly- ortriply-charged ions from spectra obtained in enhanced resolutionmode (250 amu/s) were subjected to fragmentation using theenhanced product ion option with Q0trapping. Settings were: Q1,

unit resolution; collision energy, 25e40 eV; linear ion trap Q3 lltime, 250 ms; and Q3 scan rate, 1000 amu/s (Calvete et al., 2007).All fragmentation spectra obtained by MALDI- or ESI-mass spec-trometry were interpreted manually to derive de novoamino acidsequences.

2.7. Phospholipase A2activity

The PLA2activity of nigroviriditoxin B was determined on themonodisperse synthetic substrate 4-nitro-3-octanoyl-benzoic acid(NOBA) (Holzer and Mackessy,1996). Various amounts of the toxin,dissolved in 25mL of 10 mM Tris, 10 mM CaCl2, 0.1 M NaCl, pH 8.0buffer, were added to 200 mL of this buffer in triplicate wells of amicroplate. After mixing, 25 mL of NOBA (1 mg/mL in acetonitrile)

were added, to achieve a

nal substrate concentration of 0.32 mM.

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The mixtures were incubated for 60 min at 37 C, and absorbancesat 405 nm were recorded. PLA2activity was expressed as the nalchange in absorbance 1000. Forcomparison, puried B subunit ofcrotoxin from Crotalus durissus terricus (kindly provided by Prof.Andreimar Soares, Universidade Federal de Rondonia, Brazil) wasincluded in the assay. In another experiment, the effect of the Acomponent of nigroviriditoxin on the PLA2 activity of the Bcomponent was tested by mixing both at 1.5:1 or 3:1 M ratios (A:B)and then comparing enzyme activity to that of component B alone,as described above.

2.8. Myotoxic activity

The ability of nigroviriditoxin B to induce skeletal muscle ne-crosis was evaluated in groups ofveCD-1 mice of 18e20 g of bodyweight, following protocols approved by the Institutional Com-mittee for the Use and Care of Animals (CICUA) at University ofCosta Rica. Twentymg of the toxin were injected intramuscularly inthe gastrocnemius, in a volume of 100 mL of PBS (0.12 M NaCl,0.04 M sodium phosphate buffer, pH 7.2). A control group receivedan identical injection of PBS alone. After 3 h, a blood sample was

obtained from the tail of each animal into a heparinized capillarytube, and after centrifugation, 4 mL of plasma were used to deter-mine the creatine kinase (CK) activity using a UV-kinetic assay (CK-Nac, Biocon Diagnostik). CK activity was expressed in units/L. Forcomparison, 20mg of crotoxin B was injected into another group ofmice and the plasma CK activity was assessed as described.

2.9. Lethal activity of nigroviriditoxin B and potentiation by

nigroviriditoxin A

Variable doses of nigroviriditoxin B (10e80 mg) were injectedintravenously (i.v.) in the tail vein in groups of four mice of 16e18 gof body weight, in 100mL of PBS. Deaths were recorded after 48 hand the median lethal dose (LD50 S.E.) was calculated by probits

(Finney, 1971) using the BioStat v.2009 software (AnalystSoft). Asimilar assay was performed for the isolated subunit A component,using doses up to 70 mg. For evaluation of synergy, the naturalproportion of the A and B components of nigroviriditoxin in thevenom was estimated by integrating their corresponding chro-matographic peak areas in the RP-HPLC signal at 215 nm, roughlycorresponding to peptide bondabundance. The molar proportion ofA:B components was estimated to be approximately 1:2. On thisbasis, the lethality of mixtures of both components was tested inmice at 1:2 M ratio, and compared to the results obtained wheneach component was assayed independently.

2.10. Immunochemical cross-recognition of nigroviriditoxin B by

antivenom against C. d. terricus

Puried nigroviriditoxin A subunit, B subunit, or crotoxin B,respectively, were adsorbed onto wells of an ELISA plate (NuncMaxisorp) by incubating 0.2 mg/well of the proteins in 100 mL of0.1 M Tris, 0.15 M NaCl, pH 9.0 buffer overnight at 4 C. Afterwashing ve times with PBS, free sites were blocked with PBScontaining 1% bovine serum albumin (BSA) for 1 h. Then, serialdilutions of equine antivenom againstC. d. terricusvenom (Insti-tuto Butantan, batch 0309-38), or normal equine serum as anegative control, were added and incubated 1 h. Plates were thenwashed with 0.05 M Tris, 0.15 M NaCl, 20 mM ZnCl2, 1 mM MgCl2(pH 7.4), and bound antibodies were detected by incubation withan anti-horse total IgG-alkaline phosphatase conjugate (1:3000;Sigma) for 1 h, followed by washing and color development withp-

nitrophenylphosphate (1 mg/mL) in diethanolamine buffer (pH

9.8). Absorbances were recorded on a microplate reader (Labsys-tems) at 405 nm. Assays were performed in triplicate wells.

2.11. Phylogenetic analysis of nigroviriditoxin B

Proteins showing amino acid sequence identity values of at least75% and a score of at least 191 in comparison to nigroviriditoxin B

were retrieved after a BLAST search (http://blast.ncbi.nlm.gov).These proteins, together with three available sequences corre-sponding to Asp49-PLA2s characterized from the genus Bothriechis(Q6EER4, C0HJC1, A8E2V4), were aligned with the program MUS-CLE (Edgar, 2004) using the MEGA6 software (Tamura et al., 2013).The evolutionary history was inferred with MEGA6, by using themaximum likelihood method based on the JTT matrix-based model(Jones et al., 1992). Three sequences corresponding to PLA2s fromelapid snake species (P81167 from Micrurus nigrocinctus, P00605from Naja nigricollis and P00614 from Oxyuranus scutellatus scu-tellatus) wereused as an outgroup in this analysis, which involved atotal of 22 amino acid sequences.

2.12. Homology modeling of nigroviriditoxin B

The Swiss-Model automated homology modeler (http://swissmodel.expasy.org/; Biasini et al., 2014; Arnold et al., 2006)was used to predict the three-dimensional structure of nigrovir-iditoxin B. The modeler selected crotoxin B (PDB access code 3R0L;Faure et al., 2011) as the best template, with a resolution of 1.35 and 80.5% sequence identity with the target. Swiss-PDB viewerv.4.1 was used to superimpose the obtained model to the template,and to calculate r.m.s.d values for main chain a-carbons and back-bones. Electrostatic surface potential of the proteins were repre-sented with the DS ViewerPro v.6.0 software (Accelrys).

3. Results

Fractionation of B. nigroviridis venom by RP-HPLC on C18 isshown inFig. 1.The peaks eluting at ~42 and ~51 min had beenpreviously identied to have homology to the acidic (A) and basic(B) subunits of crotoxin, respectively, in a proteomic study on thisvenom (Fernandez et al., 2010). Therefore, we explored if these twoproteins could form a crotoxin-like complex, hereby namednigroviriditoxin. By SDS-PAGE, the PLA2 B subunit migrated as aband of ~15 kDa under reducing conditions, whereas the A-chainmigrated as a main band of ~9 kDa (Fig. 2). Under non-reducingconditions, the A chain was observed at ~15 kDa, suggesting apossible dimerization or an anomalous migration. Similarly, the Bchain appeared to aggregate, as evidenced by a continuous smear inthe 15e25 kDa range (Fig. 2), commonly observed under non-

Fig. 1. Isolation of the A and B subunits of nigroviriditoxin from the venom of

Bothriechis nigroviridis. Crude venom (~2 mg) was fractionated by RP-HPLC on a C 18column (4.6 250 mm) and monitored at 215 nm. Protein elution was performed with

an acetonitrile gradient (dashed line) as described in Materials and methods. The

peaks corresponding to A (acidic chain) and B (PLA 2) subunits are labeled.

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reducing conditions in group II PLA2s of viperid snake venoms(Soares et al., 2000; Angulo et al., 2000; N~nez et al., 2004),

including crotoxin (Faure et al., 1994). By ESI-MS analysis, the Asubunit presented an isotope-averaged molecular mass of 9607 Da(1), whereas the B subunit showed a main molecular mass of14,083 Da, and an additional mass of 14,113 Da (2) (Fig. 3). In thelatter case, the observed mass heterogeneity likely correspond tothe presence of PLA2isoforms, a frequent nding in snake venoms(Doley et al., 2010) and in crotoxin preparations, due to the complexmultigene natureof this protein family (Faure et al.,1994; Faure andBon, 1988).

We explored the capability of the two proteins to associate intoa crotoxin-like complex using two different approaches. On the onehand, B. nigroviridis venom was fractionated by size-exclusionchromatography and the composition of the resulting fractionswas analyzed by mass spectrometry. A major peak eluted at 10.1 mL(Fig. 4A), corresponding to the position expected for a 22.9 kDastandard protein (Fig. 4B), and contained components of 9605.6 Da(A1), 9421.5 Da (A2), and 14,081.3 Da (B) (Fig. 4C, upper panel).Reverse-phase chromatography prior to MS analysis separated the14 kDa (Fig. 4C, middle panel) from the 9 kDa (Fig. 4C, lower panel)proteins. As a whole, these data clearly indicate that the crotoxin-like proteins ofB. nigroviridis associate into heterodimers, herebynamed nigroviriditoxin A1B and A2B.

The capability of the RP-HPLC-dissociated and isolated A and Bcomponents of nigroviriditoxin to reconstitute the heterodimer(s)was also analyzed by agarose electrophoresis under native condi-tions. The free A component of nigroviriditoxin migrated rapidlytowards the anode, indicating its acidic character, whereas the B

Fig. 2. Electrophoretic mobility of the A and B subunits of nigroviriditoxin by SDS-

PAGE (4e20% gradient gel). Samples were analyzed after reducing (r) or non-

reducing (nr) conditions) and visualized by Coomassie blue R-250 staining. Values

for the molecular mass markers (m) are shown to the right, in kDa. (For interpretation

of the references to color in this gure legend, the reader is referred to the web versionof this article.)

Fig. 3. Molecular mass of the A and B subunits of nigroviriditoxin as determined by nano-electrospray ionization mass spectrometry (ESI-MS). The multiply-charged ion series for

the A(A) and B (C) subunits are deconvoluted in panels (B) and (D), respectively.


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Fig. 4. Panel A, fractionation ofB. nigroviridisvenom (green trace) by size-exclusion chromatography. The column was calibrated with a mixture of dextran blue (2000 kDa), bovine

serum albumin (66.4 kDa), equine cytochromec(12.3 kDa), and vitamin B12 (1.35 kDa), and used for estimating the apparent molecular mass of nigroviriditoxin (peak labeled with

an asterisk) as 22.9 kDa (panel B). The heterodimeric association of nigroviriditoxin A and B subunits was demonstrated by mass spectrometry (panel C). The major peak eluting at

10.1 mL contained components of 9605.6 Da (A1), 9421.5 Da (A2) and 14,081.3 Da (B) (upper panel). Reverse-phase chromatographic separation prior to MS analysis separated the

14 kDa B-subunit (middle panel) from the 9 kDa A1 and A2 subunits (lower panel). (For interpretation of the references to color in this gure legend, the reader is referred to the

web version of this article.)


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component migrated toward the cathode, indicating its basic pI(Fig. 5). When these two components were mixed, a new bandappeared at half-way the migration distance between both freecomponents (Fig. 5), clearly indicating the formation of an A Bcomplex. Accordingly, an evident reduction in the intensity of the Asubunit was observed, and no free B subunit was detected. Furtherevidence for the similarity of the A component of nigroviriditoxinto crotoxin A wasobtained in this nativegel assay, by observing thata mixture of the former with crotoxin B also results in the formationof a complex, concomitant with the disappearance of free crotoxinB(Fig. 5).

Due to the limited availability of B. nigroviridis venom, which

yielded only small amounts of isolated nigroviriditoxin A (Fig. 1),and considering its expected structural complexity (assumingsimilarity to crotoxin A, formed by three covalently-linked chainsgenerated by proteolytic processing at the same polypeptide bondsas crotoxin; Fernandez et al., 2010), the determination of the aminoacid sequence of this component could not be attempted. On theother hand, the complete sequence of nigroviriditoxin B wasdetermined by the combination of N-terminal Edman degradationand tandem MS of proteolytic peptides (Fig. 6). Only the C-terminal

cysteine residue of this sequence could not be directly observed inthe tryptic peptide ion ofm/z946.13 due to the preceding lysine,but was inferred on the basis of itsabsolute conservation in groupIIPLA2s from snake venoms (Arni and Ward, 1996), and the isotope-averaged molecular mass determined for the native protein. Thesequence of nigroviriditoxin B consists of 122 amino acid residues,with all canonical cysteine positions of group II PLA2s beingconserved. This sequence will appear in UniProt (http://www.uniprot.org/) under the accession number C0HJL8. The theoreticalmolecular mass predicted by the sequence is 14,112 Da, whichpresents a difference of ~29e30 Da with the most abundant form(14,083 Da) or coincides, within instrumental error, with the sec-ond form observed (14,113 Da) by ESI-MS analysis (Fig. 3). Ac-cording to its sequence, the theoretically expected pI of this proteinwould be 8.5, in agreement with its observed cathodic migration inthe native agarose gel system (Fig. 5).

Nigroviriditoxin B presents sequence homology to crotoxin Bisoforms described in the venom of C. d. terricus (Fig. 7), withidentity values ranging from 77 to 80%. The highest identity (81%)was observed in comparison to a PLA2 from Sistrurus catenatustergeminus(Q6EER2;Chen et al., 2004). By multiple alignment ofnigroviriditoxin B with PLA2s having 75% identity, and with three

Asp49-PLA2s described in the genus Bothriechis, a phylogenetic treewas inferred (Fig. 8). Elapid PLA2s served as an outgroup, as theybelong to the group I classication of this enzyme superfamily.Noteworthy, nigroviriditoxin B appears basal to the group con-taining all the different crotoxin B variants described in rattle-snakes, i.e. Crotalus and Sistrurus species, and does not clustertogether with other PLA2s characterized from venoms of species ofthe genus Bothriechis (Fig. 8). The branch of nigroviriditoxin B stemsfrom acidic PLA2s of Old-world crotalid species (Gloydius halys,Ovophis monticola) and three acidic PLA2s isolated from B. lateralisandB. schlegelii(Fig. 8).

The homology of nigroviriditoxin B to the various crotoxin Bisoforms found in the venoms of rattlesnakes is in agreement withits functional characteristics. As shown in Fig. 9A, isolated nigro-

viriditoxin B displays functional PLA2activity, only slightly lower incomparison to crotoxin B. Also in similarity with the latter, nigro-viriditoxin B induced myonecrosis when injected intramuscularlyin mice, although this effect was markedly lower in comparison tocrotoxin B (Fig. 9B). Regarding lethal activity, nigroviriditoxin Balone showed an LD50 of 50 (44.9e55.6) mg/mouse (i.e., 2.9 mg/gbody weight) when injected intravenously. Mice showed signs ofrespiratory paralysis before death. When nigroviriditoxin A wasadded to the B component, at 1:2 (A:B) molar ratio, the potency of

Fig. 5. Complex formation between the A and B subunits of nigroviriditoxin evaluated

by native agarose gel electrophoresis at pH 8.6. Lane 1: A subunit; lane2: B subunit;

lane3: mixture of A and B subunits, at 1.5:1 (A:B) molar ratio; lane 4: crotoxin B; lane

5: mixture of nigroviriditoxin A subunit and crotoxin B, at 1.5:1 (A:B) molar ratio.

Anode () and cathode () positions are indicated. Proteins were loaded at 6 mg/lane

and visualized by Coomassie R-250 staining after electrophoresis.

Fig. 6. Amino acid sequence of nigroviriditoxin B. Peptides generated by the digestion of the protein with trypsin (T) or chymotrypsin (C) were de novo sequenced by mass

spectrometry. The

rst 60 amino acid residues were determined by N-terminal Edman degradation.

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this effect increased by 60%, to an LD50of 31 (27.5e33.9)mg/mouse(2.2 mg/g body weight). Higher amounts of A subunit could not betested due to limitations in venom availability. The isolated Asubunit was not lethal per se, and did not cause any evident alter-ations, when injected at doses up to 70 mg/mouse (4.1 mg/g bodyweight).

The addition of the acidic A component to nigroviriditoxin Bcaused a signicant reduction of its PLA2activityin vitro, nearly by

half (Fig. 10). On the other hand, in ELISA experiments nigrovir-iditoxin B was clearly immunorecognized by an equine antivenomraised against the venom ofC. d. terricus(Fig. 11A), with a signalnearly as intense as that generated by puried crotoxin B (Fig. 11C).Recognition of nigroviriditoxin A by this antivenom was negligible(Fig. 11B), although in this case the unavailability of crotoxin A didnot allow to control for the level of antibodies against the homol-ogous antigen in the antivenom.

The three-dimensional structure of nigroviriditoxin B wasmodeled using the crystal structure of crotoxin B as a template. Theobtained model appears virtually superimposable to the latterstructure, with r.m.s.d. values for a-carbon and backbone atoms of0.06 and 0.08 , respectively (Fig. 12). Both proteins differ at 24out of their 122 amino acid residues, and the corresponding side

chains of these are represented in Fig.12B. Out of the 22 amino acidresidues of crotoxin B proposed to interact with crotoxin A in thecrystallized complex (Faure et al., 2011), nigroviriditoxin B con-serves 18. The only four amino acid residues that change fromcrotoxin B to nigroviriditoxin B in this particular set include His1/Asn, Lys14/Arg, Trp61/Ser, and Pro111/Leu. The high structural simi-larity between nigroviriditoxin B and crotoxin B is also reected bytheir conserved surface charge distributions, compared in Fig. 12C

and D).

4. Discussion

A previous proteomic study on the venom of B. nigroviridisrevealed several unusual characteristics (Fernandez et al., 2010).Among them, the unexpected occurrence of a predominant PLA2with similarity to crotoxin B, together with a protein resemblingthe acidic A subunit of crotoxin, prompted us to investigatewhether this venom would contain a crotoxin-like complex, to dateonly found in the venoms of rattlesnakes (Crotalusand Sistrurus)among New World pitvipers. Crotoxin, rst isolated from the SouthAmerican rattlesnake C. d. terricus, is the major toxic component in

the venom of several rattlesnake species. It is a heterodimeric

Fig. 7. Multiple alignment of the amino acid sequence of nigroviriditoxin B with crotalid venom phospholipases A2selected on the basis of sequence identity values of at least 75%and a BLAST score of at least 191. Three available sequences of phospholipases A2fromBothriechisvenoms were additionally included. Sequences were aligned using MUSCLE (Edgar,

2004) with the MEGA6 software (Tamura et al., 2013), as described inMaterials and methods. Protein access codes correspond to the UniProtKB database at the ExPASy Proteomics

Server. Identical amino acid positions are shaded in gray, with cysteine residues in boldface. Isoelectric points (pI) were calculated with the compute pI/MW tool ( http://web.expasy.

org/compute_pi/), and the basic or acidic proteins are represented in blue or red colors, respectively. (For interpretation of the references to color in this gure legend, the reader is

referred to the web version of this article.)

http://web.expasy.org/compute_pi/http://web.expasy.org/compute_pi/http://web.expasy.org/compute_pi/http://web.expasy.org/compute_pi/

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indicate that nigroviriditoxin B displays a similar prole of activitiesas crotoxin B, although with a general lower potency. Finally,nigroviriditoxin B was strongly recognized by equine antibodies

from an antivenom raised against the venom of C. d. terricus,demonstrating its antigenic similarity with crotoxin B, in agree-ment with their high sequence identity.

From a structural point of view, it is noteworthy that the aminoacid sequence differences between nigroviriditoxin B and crotoxinB, consisting of 24 out of 122 positions, do not predict any majordeviations in the modeled three-dimensional structure of theformer. In the crystal structure of crotoxin analyzed byFaure et al.(2011), 22 amino acid residues of the B subunit were found to beengaged in its interaction with the A subunit. Of these, 18 areconserved in nigroviriditoxin B. Given that nigroviriditoxin A wasshown to form a complex with crotoxin B, the high conservation ofamino acid residues between the latter and nigroviriditoxin Bwould also predict a high conservation of key residues in the Asubunits from both snake species. Among the four amino acidresidues relevant for heterodimerization that differ between cro-toxin B and nigroviriditoxin B, the Trp61/Ser substitution (Trp70/Ser

in the numbering system used by Faure et al. (2011)) deservesspecial attention. Trp30 and Trp61 are considered to be critical to thestability and toxicity of the crotoxin complex, by establishingintermolecular contacts with theb-chain of crotoxin A (Faure et al.,2011). Trp30 is conserved in nigroviriditoxin B, but Trp61 is replacedby Ser, and this could inuence either its binding afnity tonigroviriditoxin A, its toxicity, or both. Interestingly, inspection ofthe multiple alignment of PLA2s homologous to nigroviriditoxin B(Fig. 4), shows that Trp61 (Trp70) is exclusively present in theenzymes from rattlesnake species, whereas nigroviriditoxin B andthe two PLA2s from Old World species (positioned basal to therattlesnake group in the phylogenetic tree; Fig. 5), present Ser atthis position. Considering that nigroviriditoxin B showed weakertoxicity than crotoxin B, it is therefore tempting to speculate that

the mutation leading to the emergence of Trp

61

(

Trp

70

) instead of

Fig. 9. (A)Phospholipase A2activity of nigroviriditoxin B (B), compared to crotoxin B

from Crotalus durissus terricus (C), on the synthetic substrate 4-nitro-3-octanoyl-

benzoic acid. Each point represents mean SD of triplicates. The differences between

both enzymes are statistically signicant (p < 0.05; Student'st-test) at 2.5, 5, and 10mg.

(B) Myotoxic activity of nigroviriditoxin B (Ngvtx B) and crotoxin B (Ctx B) after

intramuscular injection of 20 mg in mice. Plasma creatine kinase activity was deter-mined 3 h after injection. Phosphate-buffered saline (PBS) was injected to a control

group of mice. Each bar represents mean SD ofve mice. Differences between all

groups are statistically signicant (p < 0.05; ANOVA, followed by TukeyeKramer test).

Fig. 10. Modulation of the phospholipase A2 activity of nigroviriditoxin B by nigro-

viriditoxin A. Enzyme activity of the B subunit alone, or in the presence of A subunit at

the indicated molar ratios, was assayed on 4-nitro-3-octanoyl-benzoic acid. Each bar

represents mean SD of triplicate assays. The difference in enzymatic activity of

mixtures at both ratios, compared to nigroviriditoxin B alone is statistically signicant

(p

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