Snake Venomics of Crotalus tigris: The Minimalist Toxin Arsenalof the Deadliest Neartic Rattlesnake Venom. EvolutionaryClues for Generating a Pan-Specific Antivenom against CrotalidType II VenomsJuan J. Calvete,*,†,‡ Alicia Perez,‡ Bruno Lomonte,§ Elda E. Sanchez,¶ and Libia Sanz‡

†Departamento de Biotecnología, Universidad Politecnica de Valencia, Valencia, Spain‡Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas (CSIC), Jaime Roig 11, 46010 Valencia, Spain§Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San Jose, Costa Rica¶National Natural Toxins Research Center, Department of Chemistry, Texas A&M University-Kingsville, MSC 158, 975 WestAvenue B, Kingsville, Texas 78363, United States

*S Supporting Information

ABSTRACT: We report the proteomic and antivenomic characterization of Crotalustigris venom. This venom exhibits the highest lethality for mice among rattlesnakes andthe simplest toxin proteome reported to date. The venom proteome of C. tigris com-prises 7−8 gene products from 6 toxin families; the presynaptic β-neurotoxic heterodimericPLA2, Mojave toxin, and two serine proteinases comprise, respectively, 66 and 27% ofthe C. tigris toxin arsenal, whereas a VEGF-like protein, a CRISP molecule, a medium-sized disintegrin, and 1−2 PIII-SVMPs each represent 0.1−5% of the total venomproteome. This toxin profile really explains the systemic neuro- and myotoxic effectsobserved in envenomated animals. In addition, we found that venom lethality of C. tigrisand other North American rattlesnake type II venoms correlates with the concentrationof Mojave toxin A-subunit, supporting the view that the neurotoxic venom phenotype ofcrotalid type II venoms may be described as a single-allele adaptation. Our data suggestthat the evolutionary trend toward neurotoxicity, which has been also reported for theSouth American rattlesnakes, may have resulted by pedomorphism. The ability of an experimental antivenom to effectivelyimmunodeplete proteins from the type II venoms of C. tigris, Crotalus horridus, Crotalus oreganus helleri, Crotalus scutulatusscutulatus, and Sistrurus catenatus catenatus indicated the feasibility of generating a pan-American anti-Crotalus type II antivenom,suggested by the identification of shared evolutionary trends among South and North American Crotalus species.

KEYWORDS: North American rattlesnake, Crotalus tigris, snake venomics, snake venom neurotoxicity, antivenomics

■ INTRODUCTIONCrotalus tigris, the tiger rattlesnake, is a ground-dwelling, medium-sized pitviper (the largest specimen on record measured 88.5 cm).1

The monotypic C. tigris is found in isolated populations in rockyhabitats or in mesquite grasslands at elevations of near sea level toabout 1400 m, in the southwestern United States (central, south-central, and extreme southeastern Arizona), extending southwardinto northwestern Mexico (Sonoran Desertscrub, Chihuahuan Des-ertscrub, Interior Chaparral, and Madrean Evergreen Woodland),and on Isla Tiburon in the Gulf of California.1−3 Thirty-five to52 irregular, gray or brownish diffuse dorsal crossbands (“tigerbands”) and the pink to lavender tinge of its body distinguish thetiger rattlesnake from other species of rattlesnakes that occurwithin some areas of its range, Crotalus atrox, Crotalus cerastes,Crotalus mitchellii, Crotalus molossus, and Crotalus scutulatus.3

C. tigris has a very small triangular head relative to the size of thebody and a large rattle, which can make a lot of sound, reachinga loudness of about 77 dB4 (http://www.californiaherps.com/noncal/southwest/swsnakes/pages/c.tigris.html).

Little is known about the natural history of the C. tigris. It ischiefly nocturnal during the hot summer months, diurnal andcrepuscular in the fall, and in hibernation over the cold monthsof late fall and winter in rock crevices or animal burrows.1−3 Inspite of being a ground-dwelling inhabitant of the desert, itsactivity is not restricted to the ground. It swims readily and alsohas been found in bushes 60 cm above the floor.1 The tigerrattlesnake ambushes much of its prey but also actively foragessmall rodents and lizards,2,5 with juveniles relying heavily onlizards and adults depending more on rodents. In addition,these small rattlesnakes have been known to eat fairly largeprey, including kangaroo rats, packrats, and even spiny lizards.6

This is based upon its venom’s high lethality, rated the highestof all rattlesnake venoms (LD50 value for mice is 0.07 mg/kgintraperitoneal, 0.056 mg/kg intravenous, and 0.21 mg/kgsubcutaneous).7−9

Received: October 13, 2011Published: December 19, 2011

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© 2011 American Chemical Society 1382 dx.doi.org/10.1021/pr201021d | J. Proteome Res. 2012, 11, 1382−1390

Approximately 7000−8000 reptile bites are reported to theAmerican Association of Poison Control Centers (AAPCC)each year.10,11 Most bites result from the eastern diamondbackrattlesnake (Crotalus adamanteus), the western diamondbackrattlesnake (C. atrox), the prairie and Pacific rattlesnakes(Crotalus viridis), the timber rattlesnake (Crotalus horridus), andthe pygmy rattlesnake (Sisturus miliarius) when a snake is handledor abused. The eastern and western diamondback rattlesnakesaccount for the most fatalities. Human bites by C. tigris areinfrequent, and literature available on bites by this snake isscarce. The several recorded human envenomations by tigerrattlers produced little local pain, swelling, or other reactionfollowing the bite, and despite the toxicity of its venom, nosignificant systemic symptoms.12,13 The comparatively lowvenom yield (6.4−11 mg dried venom) and short 4.0−4.6 mmfangs1,2 of C. tigris possibly prevent severe envenoming in adulthumans. However, the clinical picture could be very more seriousif the person bitten was a child or a slight build individual. Theearly therapeutic use of antivenom is important if significantenvenomation is suspected. The purpose of this report was tocharacterize the venom toxin proteome of the tiger rattlesnake,establish composition−toxicity correlations, and investigate theimmunoreactivity profile of an experimental and a commercialantivenom.

■ EXPERIMENTAL SECTION

Venoms and Antivenoms

The venoms of adult C. tigris (Tiger rattlesnake), C. horridus(Timber rattlesnake), C. scutulatus scutulatus type A (Mojaverattlesnake), and Crotalus oreganus helleri (Southern Pacificrattlesnake) were extracted from specimens kept in captivity inthe serpentarium of the National Natural Toxins ResearchCenter (Kingsville, TX, http://ntrc.tamuk.edu) by biting on aparafilm-wrapped container.The anticrotalic antivenom used for in vivo neutralization

assay studies was produced at Instituto Butantan (Butantan, SaoPaulo, Brazil, http://www.butantan.gov.br) by hyperimmuniza-tion of horses with a pool of equal amounts of Crotalus durissusterrif icus and C. durissus collilineatus venom collected in South-eastern and Midwestern Brazil, in the states of Sao Paulo, MatoGrosso, and Minas Gerais (Marisa Maria Teixeira da Rocha,Instituto Butantan, personal communication). The antivenomwas composed of purified F(ab′)2 fragments generated by pepsicdigestion of ammonium sulfate-precipitated IgGs. F(ab′)2concentration was determined spectrophotometrically using anextinction coefficient (ε) of 1.4 for a 1 mg/mL concentration at280 nm using a 1 cm light path length cuvette.14

An experimental antiserum was raised in rabbits by sub-cutaneous injections of sublethal amounts of a mixture of venomsfrom C. d. terrif icus, Crotalus simus, and Crotalus lepidus lepidus.The first injection comprised 100 μg of venom in 100 μL of PBS(20 mM phosphate, 135 mM NaCl, pH 7.3) emulsified with anequal volume of Freund’s complete adjuvant. Booster injectionscomprising increasing amounts (200−500 μg) of immunogenemulsified in Freund’s incomplete adjuvant were administeredevery 2 weeks for a period of 2−3 months (depending on the titerof the antiserum determined by a standard ELISA procedure).Terminal cardiac blood collection, done by intracardiac punctureperformed under general anesthesia, was approved by the IBV’sEthics Commission. The IgG fraction was purified by ammoniumsulfate precipitation followed by affinity chromatography on

Sepharose-Protein A (Agarose Bead Technologies, Tampa, FL)following the manufacturer’s instructions.

Venomics

Venom proteins were separated by reverse-phase HPLC using aTeknokroma Europa C18 (0.4 cm × 25 cm, 5 μm particle size,300 Å pore size) column as described.15 Protein detection wasat 215 nm and peaks were collected manually and dried in aSpeed-Vac (Savant). The relative abundances (% of the totalvenom proteins) of the different protein families in the venomswere estimated from the relation of the sum of the areas of thereverse-phase chromatographic peaks containing proteins fromthe same family to the total area of venom protein peaks. In astrict sense, and according to the Lambert−Beer law, the cal-culated relative amounts correspond to the % of total pep-tide bonds in the sample, which is a good estimate of the %by weight (g/100 g) of a particular venom component. Therelative contributions of different proteins eluting in the samechromatographic fraction were estimated by densitometry afterSDS-PAGE separation.HPLC fractions were analyzed by SDS-PAGE (using 15%

polyacrylamide gels) and N-terminal sequencing (using a Prociseinstrument, Applied Biosystems, Foster City, CA). Amino acidsequence similarity searches were performed against the availabledatabanks using the BLAST program16 implemented in the WU-BLAST2 search engine at http://www.bork.embl-heidelberg.de.In some cases, this information is sufficient to assign a venomtoxin to a protein family represented in the databases. Molecularmass determination was performed by MALDI-TOF massspectrometry (using an Applied Biosystems Voyager-DE Proinstrument) and electrospray ionization (ESI) mass spectrom-etry (using an Applied Biosystems QTrap 2000 mass spectrom-eter). Protein bands of interest were excised from Coomassiebrilliant blue-stained SDS-PAGE gels and subjected to auto-mated reduction, alkylation, and in-gel digestion with sequencing-grade porcine pancreatic trypsin (Promega). The tryptic peptidemixtures were dried in a SpeedVac and dissolved in 5 mL of50% ACN and 0.1% TFA. Digest (0.85 mL) was spotted ontoa MALDI-TOF sample holder, mixed with an equal volumeof a 1:10 (v/v) dilution of a saturated solution of α-cyano-4-hydroxycinnamic acid (Sigma) in 50% ACN containing 0.1%TFA, dried, and analyzed with an Applied Biosystems Voyager-DE Pro MALDI-TOF mass spectrometer, operated in delayedextraction and reflector modes. For peptide ion sequencing, theprotein digest mixtures were loaded in nanospray capillaries(http://www.proxeon.com) and submitted to electrospray ioniza-tion mass spectrometric analysis using an Applied Biosystem'sQTrap 2000 mass spectrometer. Enhanced multiply charged modewas run at 250 amu/s across the entire mass range to determinethe charge state of the ions. Monoisotopic doubly or triply chargedprecursor ions were selected (within a window of ±0.5 m/z) andsequenced by CID-MS/MS using the enhanced product ion modewith Q0 trapping option; Q1 was operated at unit resolution, theQ1-to-Q2 collision energy was set to 30 (for m/z ≤ 700) or 35 eV(for m/z > 700), the Q3 entry barrier was 8 V, the linear iontrap (LIT) Q3 fill time was 250 ms, and the scan rate in Q3 was1000 amu/s. CID spectra were interpreted manually (i.e., de novosequenced) or using MASCOT as a seach engine, either throughits publicly available Web site (http://www.matrixscience.com) orusing a licensed version (2.0) of the MASCOT program. Searcheswere done against the default nonredundant datababes or a privatedatabase containing 1763 viperid protein sequences depositedin the SwissProt/TrEMBL database (http://www.uniprot.org/)

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plus the previously assigned peptide ion sequences from snakevenomics projects carried out in our laboratories.15,17 MS/MSmass tolerance was set to ±0.6 Da. Carbamidomethyl cysteineand oxidation of methionine were fixed and variable modifica-tions, respectively.

Neutralization of Venom Lethality

To assess the ability of the anticrotalic antivenom produced atInstituto Butantan to neutralize the lethal activity of C. tigrisvenom, five mice received an intraperitoneal (i.p.) injection of avenom challenge dose of 5 μg (∼4 LD50 for mice of 16−18 gbody weight) in 250 μL of phosphate-buffered saline (0.12 MNaCl, 0.04 M sodium phosphate, pH 7.2). An i.p. median lethaldose (LD50) of 0.07 μg/g body weight was assumed.9 Anothergroup of five mice received an identical injection of venom thathad been preincubated with the antivenom, at a ratio of 4 μL ofantivenom/μg of venom, for 30 min at room temperature.Deaths were scored after a period of 48 h.

Antivenomics

For antivenomics,17 1 mg of crude C. tigris venom in 300 μL of0.2 M phosphate, pH 7.0, was incubated overnight at roomtemperature and with gentle stirring with 10 mg of rabbit IgGantibodies affinity-purified from the antiserum raised against amixture of venoms from C. d. terrif icus, C. simus, and C. lepiduslepidus. IgG-antigen immunocomplexes were pulled down withAgarose-Protein-A (ABT) beads capable of retaining 25 mg ofIgG molecules. After centrifugation at 13 000 rpm for 3 minin an Eppendorf centrifuge, the supernatant containing thenonbound venom proteins was submitted to reverse-phaseseparation as described above. Control samples were subjected

to the same procedure except that (i) preimmune rabbit serumIgGs were employed or (ii) antivenom IgGs were not includedin the reaction mixture.

■ RESULTS AND DISCUSSION

The Minimalist Venom Proteome of C. tigris Suggests ThatNeurotoxicity Represents a Paedomorphic Trend in NearticType II Venoms

Rattlesnake venoms belong to one of two distinct phenotypes,which broadly correspond to type I (high levels of SVMPs andlow toxicity, LD50 > 1 mg/g of mouse body weight) and type II(low metalloproteinase activity and high toxicity, LD50 < 1 mg/gof mouse body weight) defined by Mackessy.18,19 The hightoxicity of type II venoms and the characteristic systemic neuro-and myotoxic effects observed in envenomations appear to bedirectly related to the high concentration of the presynapticβ-neurotoxic heterodimeric PLA2 molecules in these venoms.The venom proteome of C. tigris appears to be composed of only7−8 gene products from six different toxin families (Table 1,Figures 1 and 2). In particular, the low metalloproteinasecontent, the high concentration of Mojave toxin subunits (66%of the total venom proteins) (Table 2), and its high toxicity,LD50 0.05 (i.v)−0.07 (i.p.) mg/g of mouse body weight, whichis the highest known for any rattlesnake venom,7−9 placeC. tigris venom into the type II class defined by Mackessy.18,19

This is by far the simplest viperid snake venom toxin pro-teome reported to date. Hence, most snake venoms of familyViperidae (vipers and pitvipers) analyzed by state-of-the-artproteomic techniques comprise several tens to some hundredsof molecules.17,20−26 Cerberus rynchops venom represents

Table 1. Assignment of the Reverse-Phase Fractions from the Venoms of C. tigris, Isolated as in Figure 1, to Protein Families byN-Terminal Edman Sequencing, Mass Spectrometry, and Collision-Induced Fragmentation by nESI-MS/MS of Selected PeptideIons from In-Gel Digested Protein Bands Separated by SDS-PAGE (Inset in Figure 1)a

peptide ion

HPLCfraction N-terminal sequence

molecularmass m/z z MS/MS-derived sequence

Mowsescore protein family

1 AGEECDCGSPANP 7320 Da disintegrin2 SPEXCQG 9 kDa Mojave toxin acid chain [P18998]

SYGCYCG3 HLLQFNKMIKFETRK 14187 Da 649.8 2 YGYMFYPDSR 75 Mojave toxin basic chain [P62023]

871.8 2 GTWCEEQICECDR 752158.9 1 NAIPFYAFYGCYCGWGGRb

1978.0 1 SLSTYKYGYMFYPDSRb

4 ND 36 kDa 692.3 2 ZAMPFMEVYER de novo svVEGF5 SVDFDSESPRKPEIQ 24 kDa 569.6 2 SVDFDSESPR 36 CRISP

769.3 2 MEWYPEAAANAER 686 VVGGDECNINEHR 31 kDa 749.8 2 VVGGDECDINEHR 60 serine proteinase

657.4 2 XGNWGSXTPXTR de novo552.6 2 TXCAGXVQGGK 88

7,10 VIGGDECNINEHRFL 31 kDa 756.8 2 VIGGDECNINEHR 42 serine proteinase [∼ ABY65929]1321.8 1 HILIYVGVHDRb

1770.9 1 ILCAGVLEGGIDTCHRb

8,9 ND 46 kDa 578.3 3 MYDXVNVXTPXYHR 42 PIII-SVMP (∼ Q9DGB9)1155.6 1 EGNHYGYCRb

1283.8 1 EGNHYGYCRKb

604.6 3 QGAQCAEGLCCDQCR 35aIn MS/MS-derived sequences, X = Ile or Leu; Z, pyrrolidone carboxylic acid; B, Lys or Gln. Unless otherwise stated, for MS/MS analyses, cysteineresidues were carbamidomethylated. Molecular masses of native proteins were determined by electrospray-ionization (±0.02%) or MALDI-TOF(±0.2%) mass spectrometry. Apparent molecular masses were determined by SDS-PAGE of reduced samples. “De novo” indicates a peptidesequence determined by manual interpretation of MS/MS spectra that did not produce any hit by MASCOT search. bPeptide sequences from theMS/MS-based assigned proteins matching ions present in the tryptic peptide mass fingerprint spectra.

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another very low complexity proteome. It appears to contain atotal of five major proteins, one isoform each of metalloproteinase,CRISP and C-type lectin, and two major isoforms of ryncolins.27

C. rynchops (dog-faced water snake) belongs to Homalopsidae of

Colubroidea (rear-fanged snakes). The pharmacological profileof C. rynchops venom remains elusive, but given the centralrole that diet has played in the adaptive radiation ofsnakes,28−30 venom may represent a key trophic adaptativetrait.31 In the frame of this view, the relative composition ofC. tigris venom (Table 2) suggests that the pharmacologicalrelevance of its toxins may vary widely: the two subunitsof Mojave toxin and two serine proteinases comprise,respectively, 66 and 27% of the C. tigris toxin arsenal,whereas a VEGF-like protein, a CRISP molecule, a medium-sized disintegrin, and 1−2 PIII-SVMPs each represent 0.1−5%of the total venom proteome (Table 2).The toxin profile of C. tigris venom may explain the effects

observed in envenomated animals.8,32 Mice injected subcutaneously(s.c.) with C. tigris venom characteristically showed circling move-ments, ataxia, and flaccid paralysis. Local subcutaneous hemorrhagewas not observed except with doses about ten times the LD50. Inaddition, although the venom exhibits low but significant proteaseactivity, it does not seem to cause any hemolytic activity. Thesesystemic neuro- and myotoxic effects appear to be directly relatedto the concentration of the presynaptic β-neurotoxic heterodimericPLA2 molecules, Mojave toxin (in Neartic rattlesnakes),33 crotoxin(in Central and South American rattlesnake venoms),34−36

and sistruxin (in Sistrurus catenatus catenatus and S. catenatustergeminus venoms).37,38 The occurrence of a high concen-tration of a toxin immunologically related to Mojave toxin inC. tigris venom had been reported by Weinstein et al.,39 andsubsequently the presence of Mojave toxin subunits A and B inC. tigris was verified using DNA analysis of blood and toxin-specific immunological analysis of venom.40 Hawgood41 com-piled a review of pathophysiological effects of Mojave toxin,Castinolia et al.42 demonstrated inhibition of neuromusculartransmission, and Ho and Lee43 and Gopalakrishnakone et al.44

found the site of action to be presynaptic and also describedthe toxin as myonecrotic and capable of causing pulmonaryhemorrhage. Mice injected with the isolated toxin at a dosage ofabout the LD50 level of crude venom displayed ataxia and ashort period of hyperexcitability, which were followed by pros-tration and tachypnea. Extensive respiratory distress occurredrapidly and led to death within 15 min postinjection.39 Thispattern of pharmacological activities resembles the symptomsobserved in envenomings by South American rattlesnakes(C. durissus sp.), characterized by severe systemic effects associatedwith neurotoxicity and systemic myotoxicity.45,46

C. tigris also exhibits a toxin venom profile and lethal mediantoxicity (LD50) closely resembling those of neurotoxic SouthAmerican C. durissus subspecies (terrif icus, cascavella, collilinea-tus)47,48 (compare Figures 1 and 3D; Table 3). Moreover, reverse-phase HPLC profiling of the venoms from C. scutulatus scutulatus(Css, Mojave rattlesnake), C. horridus (Ch, Timber rattlesnake),and the Southern Pacific rattlesnake, C. oreganus helleri (Coh)displayed in Figure 3 highlights the occurrence of Mojave toxinsubunits in these New World rattlesnakes and shows the closeresemblance between North American type II and neurotoxicSouth American crotalid venoms. Mojave toxin moleculesisolated from these taxa exhibit identical N-terminal sequences(A-subunit, SSYGCYCGAGGQGWP + SPENCQGESQPC;B-subunit, HLLQFNKMIKFETRK) and very similar electro-spray-ionization isotope-averaged molecular masses (A-sub-units, 9 kDa; B-subunits: 14186 Da (Css), 14156 Da (Ch),14177 Da (Coh), 14187 Da (Cdc)).Rattlesnakes had their origin ∼20 Mya in the Sierra Madre

Occidental in the north-central Mexican Plateau49 and

Figure 1. Characterization of the venom proteome of C. tigris. Reverse-phase HPLC separation of the venom proteins of C. tigris. Inset: SDS-PAGE of the reverse-phase HPLC separated venom proteins run underreduced conditions. Molecular mass markers (in kDa) are indicated atthe side of each gel. Protein bands were excised and characterized bymass fingerprinting and CID-MS/MS of selected doubly or triplycharged peptide ions (Table 1).

Figure 2. Overall protein composition of the venoms of C. tigris. Relativeoccurrence of proteins from different toxin families in the venoms of adultC. tigris. PIII-SVMP, snake venom Zn2+-metalloproteinase (SVMPs) ofclass III; CRISP, cysteine-rich secretory protein; svVEGF, snake venomvascular endothelial growth factor. For details of the individual proteinscharacterized, consult Table 1. The percentages of the different toxinfamilies in the venoms are listed in Table 2.

Table 2. Overview of the Relative Occurrence of Proteins(in Percentage of the Total HPLC-Separated Proteins of theDifferent Families) in the Venom of C. tigris

protein family % of total venom proteins

Mojave toxin 66.2acid chain 22.1basic chain 44.1

serine proteinase 26.8svVEGF 5.0CRISP 1.9disintegrin 0.2PIII-SVMP 0.1

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dispersed northward into North America and southward intoSouth America.1,3 The gain of neurotoxicity and lethality torodents represents a pedomorphic trend that correlates with anincreased concentration of crotoxin along the axis of Crotalusradiation in South America.47,48 The phylogeny and evolutionof β-neurotoxic PLA2s present in the venoms of rattlesnakeshave been investigated by Werman.49 The distribution of thehighly closely related Mojave toxin resembles a mosaic fromMexico northward.50 The lack of phylogenetic clustering amongrattlesnakes with neurotoxin PLA2 molecules in their venoms(Figure 4) indicates that phylogeny may not be an importantconsideration in the evolution of rattlesnake type II venoms.51

The distribution of Mojave toxin varies not only between speciesbut also between geographic populations within the samespecies.51 Powell and Lieb52 have suggested that the extremelyhigh neurotoxicity exhibited by North American rattlesnakes

Table 3. Correlation between Intraperitoneal Median LethalToxicity (LD50, in mg of Venom/g of Mouse) and RelativeAbundance (in Percentage of the Total HPLC-SeparatedProteins) of Mojave Toxin or Crotoxin Subunits (Figures 1and 3) in the Venom Proteomes of Crotalid Species

A-chain B-chain % ABa LD50

C. oreganus helleri 2 9 22 1.518

S. catenatus catenatus 3.6 12.8 28 0.13−0.4330

C. scutulatus scutulatus (A) 4.5 19 24 0.22−0.4662

C. oreganus concolor 21 0.4618

C. horridus 7 22 32 0.22−1.063

C. durissus collilineatus 17 50 34 0.07−0.164

C. durissus terrif icus 19 40.5 46 0.06−0.0964

C. durissus cascavella 20 52.5 39 0.07−0.164

C. tigris 21 45 46 0.05−0.077−9aEstimated percentage of AB heterodimer.

Figure 3. Comparison of the toxin profiles of type II venoms. The venom proteins of C. scutulatus scutulatus type A (A), C. oreganus helleri (B),C. horridus (C), and C. durissus cascavella (D) were separated by reverse-phase HPLC. Peaks containing the acidic and the basic subunits of neurotoxinMojave toxin or crotoxin are labeled “a” and “b”, respectively. Inset: Electrospray-ionization mass spectra of the neurotoxin B-subunits.

Figure 4. Phylogenetic distribution of rattlesnake type II venoms. Taxaconfirmed for the presence of neurotoxic PLA2 complexes (sistruxin,crotoxin, canebrake toxin, Mojave toxin, concolor toxin) in their venomsare boxed in a gray background. The cladogram, based on Castoe andParkinson,65 shows taxonomic relationships among rattlesnakes(Crotalus and Sistrurus) and highlights the lack of phylogenetic clusteringamong species with neurotoxin molecules in their venoms.

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represents a transitory populational phenomenon associatedwith novel prey bases.The evolution of rattlesnakes in the warm deserts of western

North America has been investigated by evaluating mitochondrialDNA (mtDNA) sequences.53 The most recent commonancestor for the rattlesnakes Sistrurus/Crotalus was estimatedat 12.7 Mya (mid-Miocene), the subsequent divergence ofS. miliarius anbd S. catenatus sp. appears to have occurred 10.2Mya, and C. tigris seemingly diverged from C. mitchellii at theLate Miocene/Early Pliocene boundary (5.6 Ma).53 On theother hand, Wuster and co-workers54 have traced the dispersalof C. durissus in South America, revealing a classical pattern ofstepwise colonization progressing from a northern center oforigin in Mexico to northern South America and across theAmazon Basin. The biogeographical data suggested an ancientbasal cladogenesis in the Central American C. simus clade datedback to the late Miocene/early Pliocene (6.4−6.7 Mya), and arelatively recent (1.7−1.1 Mya) basal South American dispersalacross a central trans-Amazonian corridor during the middlePleistocene (1.1−1.0 Mya).54,55 The timing of these cladogeneticevents yielding taxa with type II venoms scattered through thephylogenetic tree of the rattlesnakes (Figure 4) may reflect theconvergence of an evolutionary trend toward neurotoxicity.Assuming that the evolutionary trend reported for the SouthAmerican rattlesnakes47,48 holds true for the North AmericanCrotalus species, the occurrence of Mojave toxin in the venomof adult specimens of certain populations within terminal cladesof recently divergent North American taxa (Figure 4) may alsohave resulted by pedomorphism. Furthermore, and taking forgranted that experimental verification is required (e.g., throughcomparative proteomic analysis of neonate and adult venoms),the reported ontogenetic variation in the venom composition ofNorth American rattlesnakes,55,56 would support this hypoth-esis. Of note is that neonate, juvenile, and adult C. o. concolorvenoms are essentially similar in composition, with respect totoxicity, amount of concolor toxin (PLA2), and low metal-loproteinase activity.55

Venom Lethality in North American Rattlesnake Type IIVenoms Correlates with Concentration of Mojave Toxina-Subunit

The relative abundances of the Mojave toxin (or crotoxin)A- and B-subunits in the proteomes of type II venoms wereestimated from the areas of their reverse-phase chromato-graphic peaks (Table 3). In line with the fact that theseneurotoxic PLA2s are composed of a nontoxic acidic (A-)3-chain subunit, derived from the proteolytic cleavage ofa single PLA2 precursor molecule, and a mildy toxic basic(B-) single-chain PLA2 subunit that associate noncovalentlyinto dimers, increasing thereby the toxicity 10−30times,33−35 there is a clear correlation between hetero-dimeric Mojave toxin (or crotoxin) concentration and thereported venom LD50 (Table 3). In the venoms sampled inthis work, the B-subunit was present in 2.1−4.5-fold excesswith respect to the A-subunit, suggesting that translationinto the venom of the acidic subunit is the limiting factorfor confering enhanced venom neurotoxicity. In line withthis view, the Mojave toxin B-subunit gene is widespreadamong Crotalus species, and its occurrence is independentof the A-subunit gene.51−53 The neurotoxic venom phenotypemay thus be described as a single-allele (A-subunit)adaptation.51,52 This view is supported by previous reportsshowing that Mojave rattlesnakes (C. scutulatus scutulatus)

lacking the acidic subunit DNA sequence lack Mojave toxin intheir venom.57

Antivenomics of C. tigris and Other Crotalid Type IIVenoms

The evolutionary trend toward neurotoxicity observed in SouthAmerican and North American rattlesnakes suggested thefeasibility of generating a pan-American anticrotalid type IIvenoms. To check this possibility, we first assessed the abilityof the anticrotalic antivenom produced at Instituto Butantanagainst C. d. terrif icus venom to neutralize the lethal activityof C. tigris venom. This antivenom showed a very higheffectiveness in the neutralization of the lethal, myotoxic, andneurotoxic effects of the crotoxin-rich venoms of C. durissussubspecies and newborn C. simus.58 All five mice receiving anintraperitoneal injection of ∼4 LD50 of venom died within16 h, whereas all mice that received the venom/antivenommixture survived throughout the 48 h observation period,demonstrating that the Butantan anti-C. d. terrif icus anti-venom is also able to neutralize the lethal action of C. tigrisvenom. Next, we applied our antivenomics protocol17,59,60 toassess the ability of an experimental (C. simus, C. l. lepidus,C. d. terrif icus) antivenom to immunodeplete proteinsfrom the venoms of C. tigris, C. horridus, C. oreganus helleri,C. scutulatus scutulatus, and S. catenatus catenatus. The results,illustrated in Figure 5, clearly showed that the trivalentantivenom was very effective targeting the toxins of these type IIvenoms.

Concluding Remarks and Perspectives

The characterization of the venom of C. tigris and finding oflargely conserved toxin profile in the venoms of otherneurotoxic North American rattlesnakes provides a proteomicframework to interpret previous biochemical, immunochemical,and pharmacological investigations on crotalid type II venoms.Of particular relevance is the correlation between thetranslational level of Mojave toxin A-subunit and venom lethalactivity. In addition, the crystal structure of crotoxin from C. d.terrif icus, recently solved at 1.35 Å resolution, indicates thatposttranslational cleavage of the acidic subunit precursor is aprerequisite for the assembly of the heterodimeric β-neuro-toxin.61 However, the identity of the protease responsible forthe proteolytic processing of the acidic subunit precursor ofneurotoxic PLA2 complexes, Mojave toxin and crotoxin,remains elusive. Whether any of the two serine proteinasespresent in C. tigris venom bears this activity deserves furtherinvestigation.Our antivenomic results and the lack of phylogenetic

clustering among rattlesnakes with neurotoxin PLA2 moleculesin their venoms (a pedomorphic trend?) strongly indicate thatproteomic-guided identification of evolutionary and immuno-logical trends among venoms may aid replacing the traditionalgeographic- and phylogenetic-driven hypotheses for antivenomproduction strategies by a more rational approach based onvenom proteome phenotyping and immunological profilesimilarities. In this respect, we predict that the neurotoxicvenoms of C. lepidus klauberi and C. mitchelli mitchelli mayexhibit the proteomic and evolutionary trends outlined here fortype II Crotalus venoms. The identification of sharedevolutionary trends among South American and NorthAmerican Crotalus reported here may impact the choice ofvenoms for immunization to produce an effective pan-Americananti-Crotalus antivenom.

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■ ASSOCIATED CONTENT

*S Supporting Information

Mass spectra from this study. This material is available free ofcharge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author

*Phone: +34 96 339 1778. Fax: +34 96 369 0800. E-mail:jcalvete@ibv.csic.es.

Figure 5. Immunodepletion of venom proteins by an experimental antivenom. Reverse-phase separations of the venom proteins (upperchromatograms) from C. tigris (A), C. horridus (B), C. oreganus helleri (C), C. scutulatus scutulatus type A (D), and S. catenatus catenatus (E), and thetoxins recovered after incubation of the crude venoms with the experimental trivalent antivenom against C. simus, C. l. lepidus, and C. d. terrificus(lower chromatograms), followed by immunoprecipitation with Agarose-Protein A.

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■ ACKNOWLEDGMENTSThis work has been financed by Grants BFU2010-17373(from the Ministerio de Ciencia e Innovacion, Madrid, Spain),CRUSA-CSIC (Project 2009CR0021), PROMETEO/2010/005 from the Generalitat Valenciana (Valencia, Spain), NIH/VIPER Resource Grant (#5 P40 RR018300-09), and TexasA&M University-Kingsville.

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