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available at www.sciencedirect.com
www.elsevier.com/locate/jprot
Snake venomics and antivenomics of Bothrops atrox venoms from Colombia and the Amazon regions of Brazil, Perú and Ecuador suggest the occurrence of geographic variation of venom phenotype by a trend towards paedomorphism Vitelbina Núñez a,b , Pedro Cid c , Libia Sanz c , Pilar De La Torre c , Yamileth Angulo d , Bruno Lomonte d , José María Gutiérrez d , Juan J. Calvete c,⁎ a
Programa de Ofidismo/Escorpionismo, Universidad de Antioquia, Medellín 1226, Colombia Escuela de Microbiología, Universidad de Antioquia, Medellín 1226, Colombia c Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain d Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica b
AR TIC LE I N FO
ABS TR ACT
Article history:
The venom proteomes of Bothrops atrox from Colombia, Brazil, Ecuador, and Perú were
Received 11 June 2009
characterized using venomic and antivenomic strategies. Our results evidence the existence of
Accepted 29 July 2009
two geographically differentiated venom phenotypes. The venom from Colombia comprises at least 26 different proteins belonging to 9 different groups of toxins. PI-metalloproteinases and
Keywords:
K49-PLA2 molecules represent the most abundant toxins. On the other hand, the venoms from
Bothrops atrox
Brazilian, Ecuadorian, and Peruvian B. atrox contain predominantly PIII-metalloproteinases.
Common lancehead
These toxin profiles correlate with the venom phenotypes of adult and juvenile B. asper from
Snake venomics
Costa Rica, respectively, suggesting that paedomorphism represented a selective trend during the
Antivenomics
trans-Amazonian southward expansion of B. atrox through the Andean Corridor. The high degree
Venom proteome
of crossreactivity of a Costa Rican polyvalent (Bothrops asper, Lachesis stenophrys, Crotalus simus)
Viperid toxins
antivenom against B. atrox venoms further evidenced the close evolutionary kinship between B.
N-terminal sequencing
asper and B. atrox. This antivenom was more efficient immunodepleting proteins from the
Mass spectrometry
venoms of B. atrox from Brazil, Ecuador, and Perú than from Colombia. Such behaviour may be
Geographic venom
rationalized taking into account the lower content of poorly immunogenic toxins, such as PLA2
phenotype variation
molecules and PI-SVMPs in the paedomorphic venoms. The immunological profile of the Costa
Paedomorphism
Rican antivenom strongly suggests the possibility of using this antivenom for the management of snakebites by B. atrox in Colombia and the Amazon regions of Ecuador, Perú and Brazil. © 2009 Elsevier B.V. All rights reserved.
1.
Introduction
Neartic pitvipers appear to have dispersed into the New World as a single lineage from Asia during the late Oligocene or the
early Miocene [1–3], between 16 and 19 million year ago (Mya), at a time when eastern North America and Eurasia were widely separated across the Atlantic, whereas northeastern Asia and Alaska remained connected via the Behring land
⁎ Corresponding author. Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas (CSIC), Jaume Roig 11, 46010 Valencia, Spain. Tel.: +34 96 339 1778; fax: +34 96 369 0800. E-mail address: jcalvete@ibv.csic.es (J.J. Calvete). 1874-3919/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2009.07.013
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bridge. Initial colonization of the New World was followed by rapid stepwise adaptive radiation progressing across Middle and South America throughout the middle-late Miocene and early Pliocene (16–3.6 Mya) giving rise to most of the approximately 126 currently recognized species included in the 12 genera of New World pitvipers [1–5]. Speciation may have occurred as the consequence of a combination of mountain uplift and fluctuations in climate during Pleistocene glacial periods. The genus Bothrops (subfamily Crotalinae of Viperidae) comprises 32 (http://www.reptile-database.org) or 37 species [6] of neotropical pitvipers, commonly referred as lanceheads, which are widely distributed in tropical Latin America, from the Atlantic lowlands of northeastern Mexico to Argentina, and the southern parts of the lower Caribbean islands, Saint Lucia and Martinique [6]. The common ancestor of all Bothrops was the first viperid to colonize South America, sometime during the late Miocene, 10–15 Mya [2]. Rapid dispersal and diversification of Bothrops across South America may have occurred through adaptive radiations into habitats devoid of viperid competitors [2]. A single species, the ancestor of B. asper, reinvaded Central America, where it remains the only widespread species of Bothrops [2]. Comparative studies of the venom composition of Costa Rican B. asper and Venezuelan B. colombiensis populations point
at the ancestor of B. colombiensis as the founding Middle American B. asper ancestor species [7]. Arrival of the botropoid colonist species may have preceded the uplift of the mountains of lower Central America (8–5 Mya), which culminated with the closure of the Isthmus of Panama 3.5 Mya [8] and fragmented the original homogeneous lowland Costa Rican herpetofauna into allopatric Caribbean and Pacific populations [9]. The phylogeny of Bothrops has undergone several taxonomic revisions but it remains still incompletely understood [3,6,10]. Bothrops (sensu lato) is a paraphyletic clade composed of at least five separate lineages [3,6,10]. The Bothrops asper–atrox complex represents a monophyletic clade of medium to large-sized pitvipers widely spread throughout the tropical parts of Central and South America northward from the Amazon Basin [3,6] (Fig. 1). B. atrox is found in the tropical lowlands of South America east of the Andes, including southeastern Colombia, southern and eastern Venezuela, Guyana, Suriname, French Guiana, eastern Ecuador, eastern Peru, northern Bolivia and the northern half of Brazil. It also occurs on the island of Trinidad [6]. The status and phylogenetic alliances of many of the conventionally recognised species within the asper–atrox group (B. asper, B. atrox, B. colombiensis, B. isabelae, B. leucurus, B. marajoensis, B. moojeni, and B. pradoi) are still the subject of vivid debates [3,10,11].
Fig. 1 – Distribution of snake species of the Bothrops asper–atrox complex in Central and South America. Physical map of Central and northern South America highlighting the geographic distribution of B. asper (closed circles), B. atrox (gray), and B. colombiensis (white and yellow). B. asper is found in the Atlantic lowlands of eastern Mexico and Central America, including Guatemala, Belize, Honduras, Nicaragua, Costa Rica and Panama. In northern South America it is found in the Pacific versant of the Colombian and Ecuadorian Andes and the adjacent coastal lowlands, and in northern Venezuela. B. atrox inhabits tropical lowlands of northern South America east of the Andes, including southeastern Colombia, southern Venezuela, Guyana, Suriname, French Guiana, eastern Ecuador, eastern Peru, northern Bolivia and the northern half of Brazil. The range of B. atrox partly encircles the purported range of B. colombiensis (western, central and north-eastern Venezuela and north of the Orinoco River), although these species are not sympatric. The picture shown at the lower left corner corresponds to an adult specimen of Colombian B. atrox photographed by Alejandro Ramírez (Programa de Ofidismo/ Escorpionismo, Universidad de Medellín, Colombia) in the Department of Meta (filled orange triangle). The common lancehead, Bothrops atrox, is a moderately heavy-bodied terrestrial pitviper. Adults usually attain a total length of 75–125 cm. The Andean Corridor connecting the Colombian and Venezuelan llanos with the southern block of savannas through the Andean slopes, which is hypothesized to have served in the trans-Amazonian southward expansion of B. atrox, is represented by broken arrows.
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Among the B. asper–atrox complex, B. asper, B. atrox, and B. colombiensis cause most of the snakebite accidents occurring within their habitats in Central and northern South America (Fig. 1). B. asper is the leading cause of snakebite mortality and morbidity in Central America [12–16]; B. colombiensis has been reported to account for over 36% of the more than 5000 accidents caused yearly by snake bites in Venezuela [17,18]; and B. atrox is responsible for more human fatalities in South America than any other American reptile [14]. B. asper and B. atrox inhabit the tropical rainforest up to 1200 m east (both species) and north and west (B. asper) of the Colombian Andes [6], and inflict 70–90% of the 3000 bites reported every year in Colombia [19–21]. B. atrox is implicated in most of the approximately 3500 human snakebites registered in the Brazilian Amazon region [22,23]. The estimated mortality among rubber tappers and indigenous people reaches 400/100,000 population in some areas of the rain forest [24]. Each year in Ecuador, 1200–1400 cases of snake bite are reported in 19 of the 21 provinces. East of the Andes, the principal venomous species are B. atrox (58% of bites) and the two-striped forest pit viper, Bothriopsis bilineata smaragdina (36%) [25]. Snakebite envenomations represent also a public health problem in Perú. The vast majority of snakebites in Perú are inflicted by species of the genus Bothrops, including B. barnetti and B. pictus in the western dry coastal regions, and B. atrox, B. brazili, and B. bilineatus in the tropical rainforests located in the eastern part of the country [26]. The principal clinical effects of envenoming by botropoid venoms are local tissue damage (myonecrosis, hemorrhage and edema), life threatening bleeding and blood coagulation disorders, shock, and renal failure. Necrosis and bacterial infection at the site of the bite may cause permanent physical handicap [12–14]. B. asper and B. atrox are highly adaptable and widely distributed species (Fig. 1), and ontogenetic and geographical variability in their venom composition and pharmacological profile have been reported [9,21,27–29]. Intraspecific venom variation represents a well documented phenomenon since more than 70 years [30,31], and is particularly notorious among species that have a wide distribution range, highlighting the concept that these species should be considered as a group of metapopulations. Many researchers have described differences in symptomatology after ophidian envenomation involving specimens from the same species in different geographical locations [32]. Intraspecific variability resulting in clinical variability of envenomation deserves utmost consideration since bites by specific populations may require different treatments. A robust knowledge of venom composition and of the onset of geographic variability may have also an impact in the interpretation of toxinological data and in the selection of specimens for the generation of improved antidotes [33]. In addition, typification of geographic-associated venom phenotypes may render valuable molecular markers for taxonomical as well as medical purposes [7,34–36]. Here, we present a comparative analysis of the proteomes and the immunoreactivity profile against a polyvalent antivenom of the venoms of B. atrox specimens from Colombia, Brazil, Perú, and Ecuador. The results evidence the existence of two geographically (northern and southern) differentiated venom phenotypes and suggest the occurrence of paedomorphism in the diversification of B. atrox venoms.
2.
Experimental section
2.1.
Venoms and antivenom
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The venom of B. atrox (Colombia) was pooled from in-captivity born adult offspring of snakes captured in the Colombian Department of Meta (Fig. 1A) and maintained at the Serpentarium of the University of Antioquia, Medellín, Colombia. The venom of B. atrox (Brazilian Amazon) was pooled from adult specimens and purchased from Latoxan (Valence, France). Venoms pooled from at least 5–6 adult Peruvian and Ecuadorian B. atrox specimens were generously donated by the Instituto Nacional de Salud (Lima, Perú) and the Instituto Nacional de Higiene y Medicina Tropical ‘Leopoldo Izquieta Pérez’ (Guayaquil, Ecuador), respectively. The polyvalent (Crotalinae) antivenom (batch 4201007POLQ, expiry date: October 2010) manufactured at the Instituto Clodomiro Picado (ICP), Universidad de Costa Rica, was produced by immunizing horses with a mixture of equal amounts of the venoms of Bothrops asper, Crotalus simus, and Lachesis stenophrys obtained from adult specimens kept in captivity at the ICP serpentarium [37]. Whole immunoglobulins were purified by caprylic acid precipitation [38]. IgG concentration was determined spectrophotometrically using an extinction coefficient (ε) of 1.4 for a 1 mg/ml IgG concentration at 280 nm using a 1 cm light pathlength cuvette [39].
2.2.
Isolation of venom proteins
For reverse-phase HPLC separations, 2–5 mg of crude, lyophilized venom was dissolved in 100 µL of 0.05% trifluoroacetic acid (TFA) and 5% acetonitrile, and insoluble material was removed by centrifugation in an Eppendorff centrifuge at 13,000 × g for 10 min at room temperature. Proteins in the soluble material were separated using an ETTAN™ LC HPLC system (Amersham Biosciences) and a Lichrosphere RP100 C18 column (250× 4 mm, 5 µm particle size) eluted at 1 mL/min with a linear gradient of 0.1% TFA in water (solution A) and acetonitrile (solution B) (5%B for 10 min, followed by 5–15%B over 20 min, 15–45%B over 120 min, and 45–70%B over 20 min). Protein detection was at 215 nm and peaks were collected manually and dried in a SpeedVac (Savant). Given that the wavelength of absorbance for a peptide bond is 190–230 nm, protein detection at 215 nm allows to estimate the relative abundances (expressed as percentage of the total venom proteins) of the different protein families from the relation of the sum of the areas of the reverse-phase chromatographic peaks containing proteins from the same family to the total area of venom protein peaks in the reversephase chromatogram. In a strict sense, and according to the Lambert–Beer law, the calculated relative amounts correspond to the “% of total peptide bonds in the sample”, which is a good estimate of the % by weight (gr/100gr) of a particular venom component. In those cases where an HPLC fraction shows the presence of several co-eluting proteins, the relative abundance of each component was estimated by densitometry.
2.3.
Characterization of HPLC-isolated proteins
Isolated protein fractions were subjected to N-terminal sequence analysis (using a Procise instrument, Applied
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Biosystems, Foster City, CA, USA) following the manufacturer's instructions. Amino acid sequence similarity searches were performed against the available databanks using the BLAST program [40] implemented in the WU-BLAST2 search engine at http://www.bork.embl-heidelberg.de. The molecular masses of the purified proteins were determined by SDS-PAGE (on 12 or 15% polyacrylamide gels), MALDI-TOF mass spectrometry (using an Applied Biosystems Voyager-DE Pro™ instrument, and by electrospray ionization (ESI) mass spectrometry using an Applied Biosystems QTrap™ 2000 mass spectrometer [41] operated in Enhanced Multiple Charge mode in the range m/z 600-1700.
2.4.
In-gel enzymatic digestion and mass fingerprinting
Protein bands of interest were excised from Coomassie Brilliant Blue-stained SDS-PAGE gels and subjected to automated reduction with DTT and alkylation with iodoacetamide, and in-gel digestion with sequencing grade porcine pancreas trypsin (Promega) using a ProGest digestor (Genomic Solutions) following the manufacturer's instructions. 0.65 µL of the tryptic peptide mixtures (from a total volume of ∼20 µL) were spotted onto a MALDI-TOF sample holder, mixed with an equal volume of a saturated solution of α-cyano-4hydroxycinnamic acid (Sigma) in 70% acetonitrile containing 0.1% TFA, air-dried, and analyzed with an Applied Biosystems Voyager-DE Pro MALDI-TOF mass spectrometer, operated in delayed extraction and reflector modes. A tryptic peptide mixture of Cratylia floribunda seed lectin (SwissProt accession code P81517) prepared and previously characterized in our laboratory was used as mass calibration standard (mass range, 450–3300 Da).
2.5. Collision-induced dissociation tandem mass spectrometry (CID-MS/MS) For peptide sequencing, the protein digest mixture was loaded in a nanospray capillary column and subjected to electrospray ionization (ESI) mass spectrometric analysis using a QTrap 2000 mass spectrometer (Applied Biosystems) [41] equipped with a nanospray source (Protana, Denmark). Doubly- or triplycharged ions of selected peptides from the MALDI-TOF mass fingerprint spectra were analyzed in Enhanced Resolution MS mode and the monoisotopic ions were fragmented using the Enhanced Product Ion tool with Q0 trapping. Enhanced Resolution was performed at 250 amu/s across the entire mass range. Settings for MS/MS experiments were as follows: Q1 — unit resolution; Q1-to-Q2 collision energy — 30–40 eV; Q3 entry barrier — 8 V; LIT (linear ion trap) Q3 fill time — 250 ms; and Q3 scan rate — 1000 amu/s. CID spectra were interpreted manually or using a licensed version of the MASCOT program (http://www.matrixscience.com) against a private database containing 1083 viperid protein sequences deposited in the SwissProt/TrEMBL database (UniProtKB/Swiss-Prot Release 56.7 of 20-Jan-2009; http://us.expasy.org/sprot/) plus the previously assigned peptide ion sequences from snake venomics projects carried out in our laboratory [7,9,33,42–49]. MS/MS mass tolerance was set to ± 0.6 Da. Carbamidomethyl cysteine and oxidation of methionine were fixed and variable modifications, respectively.
2.6. Antivenomics: immunodepletion of venom proteins by a Costa Rican polyvalent antivenom We have coined the term “antivenomics” for the identification of venom proteins bearing epitopes recognized by an antivenom using proteomic techniques [7,33,43,44]. Briefly, 2 mg of whole venom were dissolved in 70 µL of 20 mM phosphate buffer, pH 7.0, mixed with 4 mg of purified polyvalent antivenom IgGs, and incubated with gentle stirring for 1 h at 37 °C. IgG concentration was determined spectrophotometrically using an extinction coefficient (ε) of 1.4 for a 1 mg/ml IgG concentration at 280 nm using a 1 cm light pathlength cuvette [39]. Thereafter, 6 mg of rabbit anti-horse IgG antiserum (Sigma) in 350 µL of 20 mM phosphate buffer, pH 7.0, were added, and the mixture was incubated for another 1 h at 37 °C. Immunocomplexes were precipitated by centrifugation at 13,000 rpm for 30 min in an Eppendorf centrifuge and the supernatant was submitted to reverse-phase separation as described for the isolation of venom proteins. HPLC-fractions were characterized as described above. Control samples were subjected to the same procedure except that antivenom IgGs were not included in the reaction mixture.
3.
Results and discussion
3.1. Distinct venom proteomes in B. atrox from Colombia and Brazil Intraspecific venom variation resulting in clinical variability of envenomation appears to be particularly notorious among species that, like B. atrox, have a wide distribution range (Fig. 1) [30,31]. Intraspecific diversity resulting in clinical variability of envenomation deserves utmost consideration since bites by specific populations may require different treatments. However, despite its recognized medical importance, literature on the toxin composition and geographical variation of the venom of B. atrox is scarce [27,28]. To address this open question, the protein compositions of pooled venoms from B. atrox specimens from Colombia and from Brazil were investigated using the proteomic protocols previously applied to characterize the venoms of a number of viperid species from different genera [7,9,33,35,36,42–49]. Briefly, the crude venoms were fractionated by reverse-phase HPLC (Figs. 2A and 3A), followed by analysis of each chromatographic fraction by SDS-PAGE (Figs. 2B and 3B), N-terminal sequencing, and molecular mass determination by MALDI-TOF and ESI mass spectrometry (Table 1). Protein fractions showing single electrophoretic band, molecular mass, and N-terminal sequence were straightforwardly assigned by BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST) to a known protein family. Protein fractions showing heterogeneous or blocked N-termini were analyzed by SDS-PAGE and the bands of interest were excised and subjected to automated reduction, carbamidomethylation, and in-gel tryptic digestion. The resulting tryptic peptides were then analyzed by MALDI-TOF mass fingerprinting followed by amino acid sequence determination of selected doubly- and triply-charged peptide ions by collision-induced dissociation tandem mass spectrometry. Product ion spectra were submitted through the on-line form
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Fig. 2 – Characterization of the venom proteome of B. atrox from Colombia. Panel A displays a reverse-phase HPLC separation of the venom proteins. Two milligrams of total venom proteins were applied to a Lichrosphere RP100 C18 column, which was then developed with the following chromatographic conditions: isocratically (5% B) for 10 min, followed by 5–15% B for 20 min, 15–45% B for 120 min, and 45–70% B for 20 min. Fractions were collected manually and submitted to N-terminal sequencing and molecular determination by SDS-PAGE, ESI and MALDI mass spectrometry. The results are shown in Table 1. (B) SDS-PAGE showing the protein composition of the reverse-phase HPLC separated venom protein fractions displayed in panel A and run under non-reduced (upper panels) and reduced (lower panels) conditions. Molecular mass markers (in kDa) are indicated at the side of each gel. Protein bands were excised and characterized by mass fingerprinting and CID-MS/MS of selected doubly- or triply-charged peptide ions. The results are shown in Table 1.
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Fig. 3 – Characterization of the venom proteome of B. atrox from Brazil. Venom proteins were separated by reverse-phase HPLC separation (panel A) as in Fig. 2. Fractions were collected manually and submitted to N-terminal sequencing and molecular determination by SDS-PAGE, ESI and MALDI mass spectrometry. The results are shown in Table 2. (B) SDS-PAGE showing the protein composition of the reverse-phase HPLC separated venom protein fractions displayed in panel A and run under non-reduced (upper panels) and reduced (lower panels) conditions. Molecular mass markers (in kDa) are indicated at the side of each gel. Protein bands were excised and characterized by mass fingerprinting and CID-MS/MS of selected doubly- or triply-charged peptide ions. The results are shown in Table 2.
of the search engine MASCOT (http://www.matrixscience. com) to identify snake venom proteins. The MS/MS data were also manually interpreted for de novo sequencing and the CID-MS/MS-deduced peptide ion sequences (Tables 1 and 2) were submitted to BLAST similarity searches against a nonredundant database. The 33 fractions isolated by reverse-phase HPLC of the venom of B. atrox (Colombia) (Fig. 2A) comprised at least 26
different gene products (Table 1) belonging to 9 different groups of toxins (Fig. 4A, Table 3) distributed into 3 major protein groups (SVMP, PLA2, serine proteinase), which account for about 83.5% of the total venom proteins, and 6 minor protein families (bradykinin-potentiating peptides, disintegrin, DC-fragment, cysteine-rich secretory protein, L-amino acid oxidase, and C-type lectin-like), each representing less than 8% of the total venom proteins. On the other hand,
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at least 35 distinct proteins were identified in the 36 HPLC fractions collected from the venom of B. atrox (Brazil) (Table 2). These proteins belong to the same families found in B. atrox (Colombia), though the Colombian and the Brazilian venoms strongly depart from each other in the relative concentrations and the identity of their toxins (Tables 1–3). Variation in venom protein composition between these taxa was estimated using a Protein Similarity Coefficient [PSCab = [2 × (no. of proteins shared between a and b) / (total number of distinct proteins in a + total number of distinct proteins in b)] × 100] based on bandsharing coefficient used to compare individual genetic profiles using multilocus DNA fingerprints [50]. We judged two proteins (listed in Tables 1 and 2) as being different when they met one or more of these criteria: 1) had different N-terminal sequences and/or distinct internal peptides sequences (derived from MS/MS data) corresponding to homologous regions; 2) had different peptide mass fingerprints; 3) were of different sizes (±0.4% for MALDI-TOF MS derived masses and + 1.4 kDa for SDS-PAGE-determined masses); 4) Eluted in different reverse-phase HPLC peaks. Based on these criteria, Colombian and Brazilian B. atrox venom share just a few toxins, including the medium-sized disintegrin batroxostatin, a bradykinin-potentiating peptide, two K49- and one D49-PLA2 molecules, a CRISP, and L-amino acid oxidase (labelled with the symbol Φ in Tables 1 and 2). On the other hand, each venom shows also distinct proteins, i.e. a galactose-specific lectin in B. atrox from Colombia, and three D49-PLA2s, C-type lectin-like proteins, and a glutaminyl cyclase in B. atrox from Brazil. These proteins, which may represent population markers [36], are identified by the symbol ♦ in Tables 1 and 2. Fig. 5 displays the electrospray ionization mass spectra of the major shared and unique PLA2 molecules of B. atrox from Colombia and Brazil. The complement of serine proteinases and metalloproteinases (both PI and PIII) appear to be also highly variable among the venoms from Brazil and Colombia. Venom is a shared derived trait of the advanced snakes (Caenophidia). Snake venom composition is inherited rather than environmentally induced, being under a strict genetic control [51] and thus venom protein typification may render valuable molecular markers for phylogeographical purposes [7,34–36]. An estimate of the similarity of venom proteins between the Colombian and Brazilian B. atrox populations may be close to 23%. We emphasize that this measure should be regarded as a maximum estimate of the similarity between the venom profiles of the two B. atrox populations; it is suspected that a number of the proteins that we judge to be the same using the above criteria would be found to differ at one or more of these criteria if more complete information were available.
3.2. Geographic variation of the venom phenotype among B. atrox: a case of paedomorphism? Intraspecific compositional variation between venoms among specimens inhabiting different geographic regions may be due to evolutionary environmental pressure acting on isolated populations. Within B. atrox, there is considerable geographic variation in colour pattern, scalation, size and other morphological characteristics [6]. Whether this variation is a reflection of the varied habitats and ecologies of a widespread, highly
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variable species or an indication that this species is a composite of several (sub)species remains controversial. Wüster et al. [52] have provided evidence of incongruence between mtDNA haplotype lineages and morphological variation, as well as between morphology and phylogeography. The authors suggested that this pattern is more consistent with the hypothesis of a single, highly variable species than with the recognition of multiple species within B. atrox. Our results showing prominent geographical variation in the venom composition within this species further complicates the taxonomical status of allopatric B. atrox phylogroups. Though we are aware that inference of the causes underlying the geographical variation requires further molecular studies on a large sample size, the venom of Brazilian B. atrox exhibits a toxin phenotype that may be viewed as paedomorphic within the B. atrox–asper clade. Thus, it contains a large proportion of PIII-metalloproteinases and moderate amounts of PI-SVMPs and D49-PLA2 molecules (Table 3). In contrast, the venom of B. atrox from Colombia comprises predominantly PI-metalloproteinases and K49-PLA2 molecules (Table 3). Such toxin profiles correlate with the venom phenotypes of juvenile and adult B. asper from Costa Rica [9], respectively. Most Bothrops species present ontogenetic variability in their venom composition [9,21,27,53–55]. A shift from PIII to PI metalloproteinases has been reported in venom samples from B. asper from both, the Caribbean and the Pacific versants of Costa Rica [9], as well as in the venoms of B. atrox from the Brazilian Amazon [27] and from Meta (Villavicencio, Colombia) [21]. The ontogenetic change correlates with the biochemical characteristics and pharmacological profile of venoms from newborn and juvenile specimens, which show higher lethal, hemorrhagic, edema forming and coagulant activities than venoms from 3-year old specimens, which exhibit higher indirect hemolytic, i.e. phospholipase A2 activity [21]. This character is corroborated by clinical reports [14]. The high hemorrhagic activity of newborn and juvenile venoms correlates with the abundance of metalloproteinases of the PIII class, which are amongst the most potent hemorrhagic toxins [56,57]. In particular, PIII-SVMPs are more hemorrhagic (determined by their minimal hemorrhagic dose — MHD) than the lower molecular weight metalloproteinases of the PI class [58].
3.3. B. atrox from Ecuador and Perú possess Amazonian venom phenotypes: a phylogeographic pattern of trans-Amazonian dispersal The finding of geographic variation in the venom phenotype among B. atrox from Colombia and Brazil prompted us to sample other Amazonian B. atrox populations. Fig. 6A and B display, respectively, the reverse-phase chromatographic separation of the venom proteins from Ecuadorian and Peruvian B. atrox. A detailed proteomic analysis of these venoms will be published elsewhere. Nevertheless, it is evident that both venoms exhibit a greater similarity with the Brazilian phenotype (Fig. 3A) than with the venom phenotype characterized in B. atrox from Colombia (Fig. 2A). Hence, like the venom of Brazilian B. atrox, the composition of the venoms from Ecuador-Perú is dominated by metalloproteinases of the PIII class. This group of toxins comprise well over 50% of the total proteins of each venom, suggesting that B. atrox from Ecuador,
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Table 1 – Assignment of the reverse-phase isolated fractions of venom pooled from Bothrops atrox from Colombia (Fig. 2A) to protein families by N-terminal Edman sequencing, mass spectrometry, and collision-induced fragmentation by nESI-MS/MS of selected peptide ions from in-gel digested protein bands (Fig. 2B). HPLC fraction
N-terminal sequencing
Isotope-averaged molecular mass
Batx_Col1-4, 6,7 5 8
n.p. N.D. EAGEECDCGTPENPCCD
11
Blocked
12 13 14
N.D. N.D. SLVELGKMILQETGK
7648 7588 7167 7018 6966 6807 7740 7611 1116.6 1146.6 1385.1 999.5 28.5 kDa▼/■ 24.5 kDa▼/■ 13,858
15
SLVELGKMILQETGK
13,826
16
SVDFDSESPRKPEIQ
24,858
17-19
VIGGDECNINEHRSL
33▼/35■ kDa
NNCPQDWLPMNGLCY
28▼/16■ kDa
9 10
GEECDCGTPENPCCDAA EAGEECDCGTPENPCCDA AGEECDCGTPENPCCDAA
MS/MS-derived sequence
m/z
z
639.3 684.3 576.1
2 3 2
SETHYSPDGRK LRPGAQCAEGLCCDQCR CTGQSADCPR
558.8 573.8 693.1 500.3 526.6 534.8 592.3 712.8 634.9 592.3 552.8 614.3 712.8 789.9 634.9 935.0 569.6 768.9 635.6 612.4 532.4 670.8 621.2 637.8 812.0 3277.6
2 2 2 2 2 2 2 2 3 2 2 2 2 2 3 3 2 2 3 2 2 3 2 2 2 1
(222.1)GVVDPNVPP (222.1)GMVDPNVPP ZKWPRPGPEIPP (217.7)QGDPDGPP GNYYGYCR GXCCDQCR NNYXKPFCK DATDRCCYVHK NPLTSYAGAGCNCGVGGR NNYXKPFCK DRYSYSWK GAYGCNCGVGGR DATDRCCYVHK ELCECDKAVAICLR NPLTSYAGAGCNCGVGGR MILQETGKNPLTSYAGAGCNCGVGGR SVDFDSESPR MEWYPEAAANAER KPEIQNEIVDLHNSLR XNXXNHAXCR EKFXCPNR NNCPQDWLPMNGLCYK DFSWEWTDR SCTDYLSWDK EFCVEXVSYTGYR YKPGCHLASIHLYGESPEIAEYISDYHK
Protein/protein family
BaP1 PI-SVMP fragment [P83512] ∼Disintegrin batroxostatin 1-72Φ [P18618] ∼Disintegrin batroxostatin2-72 ∼Disintegrin batroxostatin 3-72 ∼Disintegrin batroxostatin 4-72 ∼Disintegrin batroxostatin 4-69 ∼Disintegrin batroxostatin 4-68 Disintegrin batroxostatin 3-67 Disintegrin batroxostatin 1-72 Disintegrin batroxostatin 2-72 Unknown Unknown Bradykinin-potentiating peptideΦ Unknown DC-fragmentΦ DC-fragment K49-PLA2 [∼Q6JK69]Φ
K49-PLA2 [Q6JK69]Φ
CRISPΦ
Serine proteinase [∼AAQ62580] Galactose-specific lectin [∼P83519]♦
J O U RN A L OF P ROT EO M IC S 7 3 (2 0 0 9) 5 7– 7 8
AGEECDCGTPENPCCDA GEECDCGTPENPCCDAA EECDCGTPENPCCDAAT
1277.5 7770
Peptide ion
VIGGDECNINEHRSL
26▼/30 kDa
18,19
NLMQFETLIMQIAGR
13704
19 20 21
VIGGDECNINEH VIGGDECDINEHPFL VIGGDECDINEHPFL
35 kDa▼ 28 kDa▼/■ 31 kDa▼
22
VIGGDECNINEHR VIGGDECNINEHR
26 kDa▼ 26 kDa▼
23
Blocked
23,296
25-33
N.D. ADDRNPLEECFRETD
110▼/56■ kDa 52 kDa▼
27
N.D. TPEQQRYVDLFIVVD
78 kDa▼ 25469
28
Blocked
33 kDa▼/■
29
N.D.
82 kDa▼
32
N.D.
44 kDa▼/■
33
N.D.
55–58 kDa▼/■
756.9 570.6 552.9 826.4 769.1 644.8 490.3 630.6 753.6 882.9 809.6
716.1 773.5 862.2 910.4 756.9 756.9 574.9 547.3 611.8 838.1 682.8 532.6 647.3 757.9 498.3 533.9 555.8 759.6 548.6 539.8 577.3 526.8 464.3 555.3 739.9 526.8
2 2 2 3 3 2 2 2 2 2 2
2 3 3 3 2 2 2 2 2 2 3 2 2 2 2 2 3 2 2 2 2 2 2 2 2
VIGGDECNINEHR VSDYTEWIK VLNEDEQTR VSNSEHIAPLSLPSSPPSVGSVCR AAYPWQPVSSTTLCAGILQGGK NFQMQLGVHSK QXCECDR DTYDNKYWR CCFVHDCCYGK NLMQFETLIMQIAGR DNKDTYDNKYWR
NVITDKDIMLIR NSEHIAPLSLPSNPPSVGSVCR YFCGMTLINQEWVLTAAHCNR VIGGDECDINEHPFLAFMYYSPR VIGGDECNINEHR VIGGDECNINEHR VAGWGSXSVGR YNSNLNTIR HSVGVVRDHSK YIELAVVADHGIFTK ITDQAXPSVTXDXFGNWR NPLEECFR EGWYANLGPMR ETDYEEFLEIAR XYYGYCR YNGNXDQXR TXDSFGEWR DMXNVQPAAPKTXDSFGEWR TXTSFGEWR VSTSFGEWR TGXTXFGEWR GNYYGYCR (199.1) FYFPR GDEYFYCR (240.1)PQCILNEPLR GNYYGYCR
Thrombin-like serine proteinase [Q072L6]
D49-PLA2 [∼ P81480]Φ
Serine proteinase Serine proteinase Serine proteinase [P04971]
Serine proteinase Serine proteinase PI-SVMP BaP1 [∼P83512]
PIII-SVMP L-amino acid oxidase [∼Q6TGQ9]Φ
PIII-SVMP PI-SVMP [∼Q8QG89]
PI-SVMP [∼Q5XUW8] PI-SVMP [∼Q072L5] PI-SVMP PIII-SVMP
J O U RN A L OF P ROT EO MI CS 7 3 (2 0 0 9) 5 7– 7 8
17
PIII-SVMP [∼Q3HTN2] PIII-SVMP
X, Ile or Leu. Unless other stated, for N-terminal sequencing and MS/MS analyses, cysteine residues were pyridylethylated and carbamidomethylated, respectively; molecular masses of the native proteins were determined by electrospray-ionization mass spectrometry, MALDI-TOF (⁎) or by SDS-PAGE before (▼) or after (■) sample reduction with β-mercaptoethanol; n.p., non peptidic material found. N.D., not determined. ♦, protein class uniquely found in B. atrox from Colombia; Φ, proteins found also in B. atrox from Brazil (see Table 2).
65
66 Table 2 – Assignment of the reverse-phase isolated fractions of venom pooled from Bothrops atrox from Brazil (Fig. 3A) to protein families by N-terminal Edman sequencing, mass spectrometry, and collision-induced fragmentation by nESI-MS/MS of selected peptide ions from in-gel digested protein bands (Fig. 3B). HPLC fraction
N-terminal sequencing
Isotope-averaged molecular mass
Batx_Bra 1 2-5 6
7281 7413 7212 7598 7560 7767 7567 1385.1 28–31 kDa■ 13,860 13,826
15
SLIEFANMILEETKK
13,875
16
IIGGDECNINEHR
31 kDa■
SVDFDSESPRKPEIQ
24,858
17
N.D. HLVQFEKLIQIIAGR
36 kDa▼/■ 13,783
18
NLMQFETLIMQIAGR
13,704
19
VVGGDECNINEHR
31 kDa■
7 8
MS/MS-derived sequence
m/z
z
639.5
2
693.1 526.3 738.4 552.6 416.9 562.9 738.4 766.9 648.6 731.4 958.6 752.9 653.9 523.9 584.6 763.6 925.6 601.3 569.6 768.9 635.6 869.6 639.9 554.6 752.9 490.3 630.6 752.9 809.6 882.9 714.9 749.6
2 2 2 2 2 2 2 2 3 2 3 2 2 2 2 2 2 2 2 2 3 2 2 2 2 2 2 2 2 2 2 2
YAAVGGGGGGGGGGGAR
ZKWPRPGPEIPP GNYYGYCR CCYVHKCCYK DRYSYSWK YSYSWK ENLDTYNKK CCYVHKCCYK SYGAYGCNCGVLGR NPVTSYGAYGCNCGVLGR ITVCDENNPCLK MILQETGKNPVTSYGAYGCNCGVLGR CCFVHDCCYGK YMAYPDLLFCK TDRYSYSR VSDYTEWXR IIGGDECNINEHR XAPXSXPSNPP(525.9)CR (199.1)DKDXMXXR SVDFDSESPR MEWYPEAAANAER KPEIQNEXVDXHNSXR TNCPASCFCHNEXK AAYPEXPAEYR KQXCECDR CCFVHDCCYGK QXCECDR DTYDNKYWR CCFVHDCCYGK DNKDTYDNKYWR NLMQFETLIMQIAGR SXPSSPPSVGSVCR VVGGDECNINEHR
Protein/protein family
Fragment from BPP/CNP precursor Batroxostatin [P18618]1-69 Batroxostatin [P18618]3-71 Batroxostatin [P18618]5-71 Batroxostatin [P18618]1-71 Batroxostatin [P18618]3-72 ∼Disintegrin batroxostatin 1-72Φ ∼Disintegrin batroxostatin 3-72 Bradykinin-potentiating peptide DC-fragmentsΦ K49-PLA2 [∼Q6JK69]Φ K49-PLA2 [∼Q6JK69]Φ
D49-PLA2 [∼ P20474 B.asper]♦
Serine proteinase
CRISPΦ
Serine proteinase D49-PLA♦2 D49-PLA2 [∼P81480]Φ
Serine proteinase
J O U RN A L OF P ROT EO M IC S 7 3 (2 0 0 9) 5 7– 7 8
9 10-12 13 14
N.D. n.p. EAGEECDCGTPENP GEECDCGTPENPCCD ECDCGTPENPCCDAT EAGEECDCGTPENP GEECDCGTPENPCCD AGEECDCGTPENPCCD GEECDCGTPENPCCDA Blocked SPPVCGNELLEVGEE SLVELGKMILQETGK SLVELGKMILQETGK
Peptide ion
13,856
20
VIGGDECDINEHPFL
36 kDa▼/■
20, 21
N.D.
27 kDa■
21
V(V/F)GGDECNINEHR
31 + 32 kDa■
21
N.D.
24 kDa▼/■
22
N.D.
33 kDa▼/■
23,24,26
DCPSDWSSYEGHCYK
16 kDa■
DCPPDWSSYEGHCY
14 kDa■
24 25,27,30
Blocked ADPRNPLEECFRETD
110▼/54■ kDa 56 kDa▼
26
Blocked
110▼/■ kDa
28,29 30
TPEQQRYVDLFIVVD N.D.
22,919 55 kDa▼/■
Blocked
29 kDa▼/■
639.9 752.9 490.3 486.5 558.9 752.9 715.6 574.6 756.3 773.8 749.6 639.9 618.6 714.9 716.1 609.1 770.8 542.1 471.6 595.6 535.1 710.9 818.9 838.1 849.6 441.6 592.1 532.6 647.3 676.4 638.9 569.3 704.9 744.3 715.9 560.9 692.9 2 615.4 555.8 721.6 577.3 769.2 832.9 617.8 737.9 861.4 548.6 610.9
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
AAYPEXPAEYR CCFVHDCCYGK QICECDR XYCGXHTR SVANDDEVXR SVANDDEVXRYPK NVITDKSIMLIR VMGWGSXSSPK VIGGDECNINEHR VFGGDECNINEHR VVGGDECNINEHR AAYPEXPAEYR (223.4)PEXXPEYR XSPSSPPSVVGSCR VSXTNXEXWSNR AVVFNENVXGR (582.8)AXXXTAVVFNENVXGR FXAFXYPGR XYXGXHAR (198.8)MNWADAER YYVWXGXR (314.2)FECPSDWSTHR (402.4)DDVNWDDTVR EEADFVVSXTSADFR TSNDQWWSFPCTR XFWCTR ZSNXTPEQQR NPLEECFR EGWYANLGPMR SAGQLYEESLQK ETLSVTADYVIVCTTSR HDDIFAYEK LPTSMYQAIQEK ETDYEEFLEIAK EEIQAICRPSMIQR NPFYTPSPAK XTPFAHNXPYGR FYMYEGPAPR TXDSFGEWR LIFFDGEEAFVR YPGSPGSYAVR NPVFPVYFLNTAR MWQNDLHPILIER WSPSDSLYGSR HPVEDDHIPFLR NLNDLGLLNNYSSER TXTSFGEWR HSVGVVRDHSK
D49-PLA♦2 Serine proteinase [∼P04971]
B. atrox [AAA48553] Serine proteinase Serine proteinase Serine proteinase
PIII-SVMP domain
Serine proteinase C-lectin α-subunit♦
C-lectin β-subunit♦
PIII-SVMP L-amino acid oxidase [∼Q6TGQ9]Φ
J O U RN A L OF P ROT EO MI CS 7 3 (2 0 0 9) 5 7– 7 8
SGVQFETLIMKIAGR
Unknown
PI-SVMP atroxlysin-1 [P85420] Glutaminyl cyclase [Q90YA8]♦
PI-SVMP
67
(continued nextpage) page) (continued ononnext
68
Table 2 (continued) HPLC fraction
N-terminal sequencing
Isotope-averaged molecular mass
Batx_Bra TPEQQRYVELLIVVD
22,828
32,33
Blocked
55 kDa▼/■
Blocked
110▼/55■ kDa
Blocked
42 kDa■
Blocked
58 kDa▼/■
N.D. N.D.
29 kDa▼ 26 kDa▼/■
N.D.
24 kDa▼
N.D.
110▼/55■ kDa
34,35
36
MS/MS-derived sequence
m/z
z
555.8 752.6 526.6 718.3 526.6 718.3 502.3 718.3 685.6 748.1 583.6 522.6 835.6 535.2 555.8 752.6 838.3 589.6 2 604.6 862.9 685.6 450.1
2 3 2 3 2 2 2 3 2 2 2 2 2 2 2 3 2 2 2 2 2 2
TXDSFGEWR DLINVQPAAPQTLDSFGEWR GNYYGYCR ITNXPDVDYTXNSFAEWR GNYYGYCR ITNXPDVDYTXNSFAEWR XXVNEXFR ITNXPDVDYTXNSFAEWR XAXVGXEXWSNR (256.8)PQCDEPEXPR GEECDCGSPR MPGNXDEXR XYEXVNFXNEXFR YYVWXXGR TXDSFGEWR DLINVQPAAPQTLDSFGEWR EEADFVVSXTSADHR GSCYNFV(351.8) XAYVVCEAQR (143.4)SDGSSVSYENVVER XAXVGXEXWSNR YWDXFR
Protein/protein family
PI-SVMP ∼ atroxlysin-1 PIII-SVMP Dimeric PIII-SVMP PIII-SVMP PIII-SVMP
C-type lectin-like♦ PI-SVMP ∼ atroxlysin-1 C-type lectin-like♦
Dimeric PIII-SVMP
X, Ile or Leu. Z, pyrrolidone carboxylic acid. unless other stated, for N-terminal sequencing and MS/MS analyses, cysteine residues were pyridylethylated and carbamidomethylated, respectively; molecular masses of the native proteins were determined by electrospray-ionization mass spectrometry, MALDI-TOF (⁎) or by SDS-PAGE before (▼) or after (■) sample reduction with β-mercaptoethanol; n.p., non peptidic material found. N.D., not determined. ♦, protein class uniquely found in B. atrox from Brazil; Φ, proteins found also in B. atrox from Colombia (see Table 1).
J O U RN A L OF P ROT EO M IC S 7 3 (2 0 0 9) 5 7– 7 8
31
Peptide ion
69
J O U RN A L OF P ROT EO MI CS 7 3 (2 0 0 9) 5 7– 7 8
Table 3 – Overview of the relative occurrence of proteins from different families in the venoms of Bothrops atrox from Colombia and Brazil. Protein family
BPP/C-NP Medium disintegrin DC-fragment CRISP PLA2 ● K49 ● D49
f
Serine proteinase L-amino acid oxidase C-type lectin ● Gal-specific lectin ● αβ C-type lectin-like
f
Zn2+-metalloproteinase ● PIII ● PI
f
Other proteins ● Glutaminyl cyclase
Fig. 4 – Overall protein composition of B. atrox venoms. Panels A and B display, respectively, the relative occurrence of proteins from different toxin families in the venoms of Bothrops atrox from Colombia and Brazil. BPP, bradykinin-potentiating peptide; DC-fragments, disintegrin/cysteine-rich fragment from PIII snake venom Zn2+-metalloproteinase (SVMPs); LAO, L-amino acid oxidase; PLA2, phospholipase A2; CRISP, cysteine-rich secretory protein; Gal-lectin, galactose-specific lectin; C-lectin, C-type lectin-like molecule. Details of the individual proteins characterized are shown in Table 1, and the percentages of the different toxin families in the venom are listed in Table 3.
Perú share the paedomorphic character described in this work for the Brazilian B. atrox venom phenotype. However, comparison of their chromatographic and SDS-PAGE patterns revealed geographic venom composition variation among the B. atrox populations sampled. Strikingly, Ecuadorian, Peruvian, Brazilian and Colombian B. atrox venoms contain an identical K49-PLA2 molecule of isotope-averaged molecular mass 13,826 Da (Fig. 5A) though each venom also possesses unique PLA2 proteins (Fig. 5B–F). These data strongly argue for selective pressure acting to preserve the function of the common PLA2 molecule. K49, and other PLA2 homologues devoid of enzymatic activity, diverged from ancestral group II myotoxic D49 PLA2s [59]. Their proposed adaptive roles in Viperidae venoms have been recently discussed [60,61].
% of total venom proteins Colombia
Brazil
0.3 1.7 < 0.1 2.6 24.1 18.5 5.6 10.9 4.7 7.1
< 0.1 < 0.1 0.9 1.8 14.3 2.5 11.8 4.6 2.1 0.7
7.1 – 48.5
– 0.7 72.1
3.1 45.4
49.1 23.0
–
0.2
Although some studies have pointed to an a link between the presence of K49-PLA2 homologues in the venom and a rodent-rich diet [62–64], this association may not be a general trend [61]. On the other hand, variable, taxa-specific PLA2 proteins may serve as taxonomical markers [36] and may provide clues to trace the divergence of the group.
3.4. Antivenomic analysis of the crossreactivity of B. atrox venoms against a Costa Rican polyvalent (Crotalinae) antivenom Accidental envenomation by Bothrops species constitutes a relevant public health issue in Central and South America [12– 14,65]. Adequate treatment of snake envenoming is critically dependent on the ability of antivenoms to reverse venominduced coagulopathy, haemorrhage, hypotensive shock and other signs of systemic envenoming. Several antivenoms are produced in Latin America using different venoms in the immunization schemes [66,67]. Each of these antivenoms is effective against envenomations by snake venoms not included in the immunization protocol, demonstrating the high degree of immunological cross reactivity between Central and South American crotaline snake venoms. A practical consequence of this fortunate circumstance is the possibility of using heterologous antivenoms to circumvent the restricted availability of species-specific antivenoms in some regions. However, before testing in clinical trials, antivenoms need to be evaluated experimentally by assessing their neutralizing ability against the most relevant toxic and enzymatic activities of snake venoms. Simple laboratory tests have been adapted for the evaluation of antivenoms [68 and references cited]. Our antivenomics approach [33,43] is simple and easy to implement in a protein chemistry laboratory, and may thus represent another useful protocol for investigating the immunoreactivity, and thus the potential therapeutic usefulness, of
70
J O U RN A L OF P ROT EO M IC S 7 3 (2 0 0 9) 5 7– 7 8
Fig. 5 – PLA2 molecules as taxonomical markers of allopatric B. atrox phylogroups. Panel A display the electrospray-ionization mass spectrum (ESI-MS) of the major K49-PLA2 molecule, isotope-averaged molecular mass 13,826 Da, shared by B. atrox venoms from Colombia (Col-15, Fig. 2A, Table 1), Brazil (Bra-14, Fig. 3A, Table 2), Ecuador (Ecu-4, Fig. 6A), and Perú (P-10, Fig. 6B). Panel B, ESI-MS of the D49-PLA2 molecule (13,704 Da) present in the venoms of B. atrox from Colombia (Col-19, Fig. 2A, Table 1) and Brazil (Bra-18, Fig. 3A, Table 2). Panels C and D show electrospray ionization mass spectra of D49-PLA2 proteins (isotope-averaged molecular masses of 13,875 Da (Bra-15) and 13,783 Da (Bra-17) (Fig. 3A, Table 2), respectively) uniquely found in the venom of B. atrox from Brazil. Panels E and F, ESI-MS of taxa-specific PLA2 proteins isolated from the venoms of Ecuadorian and Peruvian B. atrox (calculated isotope-averaged molecular masses of 13,799 Da and 13,738 Da, respectively) (RP-HPLC peaks 6 and 11 of Fig. 6A and B, respectively).
J O U RN A L OF P ROT EO MI CS 7 3 (2 0 0 9) 5 7â&#x20AC;&#x201C; 7 8
71
Fig. 5 (continued).
antivenoms towards homologous and heterologous venoms [7,44,48]. Following recommendations of international workshops pointing to the need of strengthening antivenom production
and distribution in Central and South America, and in other regions of the world, [69â&#x20AC;&#x201C;71], a number of studies have been performed to assess the cross-reactivity of different antivenoms manufactured in different countries. In the case of Latin
72
J O U RN A L OF P ROT EO M IC S 7 3 (2 0 0 9) 5 7– 7 8
Fig. 6 – Reverse-phase HPLC separation of the venom proteins of B. atrox from Perú (A) and Ecuador (B). Chromatographic conditions were as in Fig. 2. Inserts, SDS-PAGE showing the protein composition of selected reverse-phase HPLC fractions run under non-reduced (upper panels) and reduced (lower panels) conditions. Molecular mass markers (in kDa) are indicated at the side of each gel.
America, the investigations have revealed a high-degree of crossprotection between several antivenoms generated against Bothrops sp venoms [33 and references cited]. Here, we have investigated the spectrum of recognition of B. atrox toxins by a equine polyvalent antivenom generated against a mixture of the venoms of B. asper, C. simus, and L. stenophrys. Fig. 7 displays
reverse-phase separations of the venom proteins of the four B. atrox phylogroups investigated in this work recovered in the soluble fraction after incubation of venom with the polyvalent antivenom followed by immunoprecipitation with rabbit antihorse IgG antiserum. The antivenom showed essentially the same immunoreactivity against B. atrox from Colombia (Fig. 7A)
J O U RN A L OF P ROT EO MI CS 7 3 (2 0 0 9) 5 7– 7 8
73
Fig. 7 – Immunodepletion of venom proteins by the polyvalent antivenom. Panels A and B show, respectively, reverse-phase separations of the venom proteins from B. atrox from Colombia and Brazil recovered after incubation of the crude venom with the polyvalent (Crotalinae) Costa Rican antivenom, followed by rabbit anti-horse IgG antiserum and immunoprecipitation. The inserts shows SDS-PAGE analyses of β-mercaptoethanol-reduced fractions labelled as in the chromatograms (Figs. 2A and 3A). Protein fraction numbering is as in Tables 1 (B. atrox from Colombia) and 2 (B. atrox from Brazil). Panels C and D show, respectively, reverse-phase chromatographic separations of the non-immunodepleted proteins after incubation of the crude venoms of B. atrox from Ecuador and Perú with the polyvalent (Crotalinae) Costa Rican antivenom, followed by rabbit anti-horse IgG antiserum. Peak labelling as in Fig. 6A and B. The insert in panel D shows SDS-PAGE analyses of β-mercaptoethanol-reduced fractions labelled as in the chromatogram. In A–D, peaks labeled with letters (a–d) correspond to fragments of rabbit IgG heavy-chain constant domain [GenBank accession code ABB21727]. Major fragments “b” and “d” have N-terminal sequence LLGGPSVFIIPPKDTLMISR.
74
J O U RN A L OF P ROT EO M IC S 7 3 (2 0 0 9) 5 7– 7 8
Fig. 7 (continued).
as previously determined for B. colombiensis [7]: it essentially immunoprecipitated medium-sized disintegrins, PIII-SVMPs, C-type lectin-like proteins, CRISP, L-amino acid oxidase, and to a minor extent serine proteinases and DC-fragments, but displayed limited immunoreactivity towards PLA2 molecules and PI-SVMPs. We estimate that 45–50% of the latter toxins were only partially immunodepleted from B. atrox (Colombia) venom by the antivenom. On the other hand, more efficient immunoprecipitation profiles were evidenced when the venoms from Brazil, Ecuador, and Perú were investigated (Fig. 7B–D). The antivenom was particularly efficient towards the Ecuadorian venom, immuno-
depleting 100% of the toxins (Fig. 7C). In the cases of B. atrox from Brazil and Perú, more than 90% of all proteins were depleted from the venoms. The non-immunoprecipitated proteins were a DCfragment, PLA2 molecules and some PIII-SVMPs (both venoms, Fig. 7B and D), and serine proteinases and a C-type lectin-like molecule from B. atrox (Brazil) (Fig. 7B). The observed pattern of crossreactivity of the polyvalent antivenom against B. atrox venoms further evidences the close evolutionary kinship between B. asper and B. atrox. The higher immunodepletion efficiency of proteins from the paedomorphic venoms from the Amazon regions of Ecuador, Perú, and
J O U RN A L OF P ROT EO MI CS 7 3 (2 0 0 9) 5 7– 7 8
Brazil may be rationalized taking into account the lower content of poorly immunogenic toxins, such as PLA2 molecules and PI-SVMPs. The immunological profile of the Costa Rican ICP polyvalent antivenom strongly suggest the possibility of using this antivenom for the management of snakebites by B. atrox in Colombia, Ecuador, Perú and Brazil (i.e. if antivenom availability becomes compromised). This conclusion is in line with previous studies showing that the Costa Rican polyvalent antivenom effectively recognized all the major proteins of B. atrox (Colombia) by Western blot analysis [21] and neutralized the toxic effects of B. atrox venoms from Colombia [72], Brazil [73], and Perú [74]. However, the neutralization ability of the Costa Rican antivenom was assessed using venom pooled from 40 specimens classified as B. atrox asper and collected in four regions of Antioquia (Urabá, Magdalena Medio, Bajo Cauca, Nordeste) and Chocó (Pacific Coast), in northwestern Colombia. The species inhabiting this geographical range has undergone taxonomic revision and is currently regarded as B. asper. Hence, although our results show a similar immunodepletion activity of the antivenom towards B. asper and B. atrox venoms, clinical trials are needed to secure its therapeutic use in envenomations by B. atrox.
4.
Concluding remarks
The occurrence of intraspecies variability in the biochemical composition and symptomatology after envenomation by snakes from different geographical locations and of different ages has long been appreciated by herpetologist and toxinologists, and appears to be a general feature of venoms [32]. Certain variability in venom composition is under genetic control [75], and gene regulation effects on adaptive variation, mainly through mutations in the cis-regulatory regions of genes, are increasingly documented [76]. High interpopulational variance in gene frequencies is characteristic of genetic drift in conspecific populations [77]. Venom compositional differences might be explained in terms of population fragmentation due to the dynamics of rainforest during the Quaternary or by isolation by distance among populations within each region. However, the adaptive role of the observed ontogenetic variations in venom composition and actions remains elusive at present. B. atrox is considered a diet generalist. Its main diet includes small mammals and birds, but also frogs and lizards [6,78]. Most species of Bothrops feed largely on ectothermic prey as juveniles but shift to endothermic prey when it reaches a size sufficient to swallow rodents, marsupials, birds, and other bulky prey items [6]. Shifts in venom lethality for certain kinds of prey follow this general trend [79]. Increased procoagulant activity of B. alcatraz venom, mainly due to metalloproteinase and serine proteinase activities, has been hypothesized to represent a paedomorphic character [79]. B. alcatraz has been isolated in the Alcatrazes Archipielago, 35 km off the Atlantic coast of São Paulo, southeastern Brazil, since approximately 10,000 years ago [80]. Retention of juvenile characters in B. alcatraz appears to be associated with a diet based on ectotherms (mainly centipedes), owing to the absence of small mammalian prey on the islands [79]. Paedomorphosis was first proposed by Walter Garstang in 1922 [81]. The driving force behind paedomorphism is often competition or predation
75
pressure. At this respect, it has been documented that venoms from neonate Crotalus snakes are more toxic to lizards than adult venoms [82]. However, the notion that evolutionary interactions between snakes and their prey may be responsible for variation in venom composition [51,83] has been questioned [84,85]. We envisioned two alternative scenarios that may potentially account for the selective advantages of venom paedomorphism among B. atrox. Specimens that colonized the Amazonian region southwards may have comprised a relatively small population of snakes under selective pressure to maintain a paedomorphic trait to adapt to the new, prey-restricted, ecosystem. On the other hand, lack of prey pressure may have allowed a rapid evolution of the colonizing B. atrox population without the need to shift to the increased venom complexity that characterizes adult snakes that feed on a wide variety of prey (generalist predators). In both scenarios, achieving sexual maturity while maintaining increased hemotoxic and lethal venom activities may have conferred evolutionary fitness to the ancestors of Amazonian B. atrox, thus fueling the paedomorphic trend. The South American populations of the B. atrox species complex (B. atrox, B. colombiensis, B. isabelae, B. leucurus, B. marajoensis, B. moojeni, and B. pradoi) show low levels of mtDNA sequence divergence, which is consistent with the recent origin of the group [3,11]. The northern Venezuelan (B. colombiensis) haplotype was consistently placed as the sister group to all other South American mainland haplotypes [3,11]. The taxonomical status of B. colombiensis has been a matter of continuous debate [6,7 and references cited]. A recent study has revealed that the venoms of B. colombiensis and B. asper (Costa Rica) share approximately 65–70% of their venom proteomes [7], evidencing the close kinship of these two species. These data, and our present finding of two well-supported B. atrox clades characterized by an “adult asper-like” venom phenotype in Colombia and a distinct (“neonate asper-like”) venom profile in the southern population inhabiting the Amazon regions of Ecuador, Perú, and Brazil, support the view that the ancestor of B. colombiensis underwent southward dispersal contributing thereby to the present-day cladogenesis of the B. atrox complex. Historical events leave genetic patterns that can be interpreted with appropriate tools. The geographical distribution of paedomorphic B. atrox in Ecuador, Perú, and Brazil revealed by our proteomic analyses suggests a pattern of allopatric fragmentation along a trans-Amazonian southward expansion through the Andean Corridor connecting the Colombian and Venezuelan llanos with the southern block of savannas through the Andean slopes [86]. However, B. atrox is a widespread species east of the Andes (Fig. 1) and a single corridor scenario may not fully explain its distributional pattern. In addition to the Andean Corridor, two other corridors connecting northern and southern savanna regions have been proposed [86–88]: the Central Amazonian Corridor connects savannas north and south of the Amazon basin, and the Coastal Corridor, which is located close to the Atlantic coast and connects northern and southern blocks of South American open formations. Quijada-Mascareñas et al. [89] have presented phylogeographical and tree topology data congruent with a continuous distribution of the Neotropical rattlesnake (Crotalus durissus complex) along a central Amazonian corridor during the middle Pleistocene. Here, we vindicate the use of the venom signature as a tool to investigate the phylogeography of B. atrox. Proteomic studies on a large sample
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size of B. atrox venoms from Bolivia, on the one hand, and from Venezuela, Guyana, Suriname, French Guyana, and different localities within the northern half of Brazil, on the other hand, are required to establish a coherent scenario for the dispersal and range expansion patterns of B. atrox.
Acknowledgements This study has been financed by grant BFU2007-61563 from the Ministerio de Educación y Ciencia, Madrid, projects from the Vicerrectoría de Investigación, Universidad de Costa Rica (741A7-611), CRUSA-CSIC (2007CR0004), and CYTED (206AC0281). Travelling between Spain and Costa Rica was financed by Acciones Integradas 2006CR0010 between CSIC and the University of Costa Rica (UCR). Alejandro Ramírez (Programa de Ofidismo/Escorpionismo, Universidad de Antioquia, Colombia) is gratefully acknowledged for allowing us reproduction of the picture of B. atrox shown in Fig. 1.
REFERENCES [1] Parkinson CL, Campbell JA, Chippindale PT. Multigene phylogenetic analysis of pitvipers with comments on their biogeography. In: Schuett GW, Höggren M, Douglas ME, Greene HW, editors. Biology of the Vipers. Salt Lake City: Eagle Mountain Publishing LC; 2002. p. 93–110. [2] Castoe TA, Daza JM, Smith EN, Sasa M, Kuch U, Campbell JA, Chippindale PT, Parkinson CL. Comparative phylogeography of pitvipers suggests a consensus of ancient Middle American highland biogeography. J Biogeogr 2009;36:88–103. [3] Wüster W, Salomão MG, Quijada-Mascareñas JA, Thorpe RS, Duckett GJ, Puorto MG, et al. Origins and evolution of the South American pitviper fauna: evidence from mitochondrial DNA sequence analysis. In: Schuett GW, Höggren M, Douglas ME, Greene HW, editors. Biology of the Vipers. Eagle Mountain Publishing, LC; 2002. p. 111–28. [4] Wüster W, Ferguson JE, Quijada-Mascareñas JA, Pook CE, Salomão MG, Thorpe RS. Tracing an invasion: landbridges refugia and the phylogeography of the Neotropical rattlesnake (Serpentes: Viperidae: Crotalus durissus). Mol Ecol 2005;14:1095–108. [5] Castoe TA, Parkinson CL. Bayesian mixed models and the phylogeny of pitvipers (Viperidae: Serpentes). Mol Phylogenet Evol 2006;39:91–110. [6] Campbell JA, Lamar WW. The Venomous Reptiles of the Western Hemisphere. Ithaca (NY): Comstock Publishing Associates; 2004. [7] Calvete JJ, Borges A, Segura A, Flores-Díaz M, Alape-Girón A, Gutiérrez JM, Diez N, DeSousa L, Kiriakos D, Sánchez E, Faks JG, Escolano J, Sanz L. Snake venomics and antivenomics of Bothrops colombiensisa medically important pitviper of the Bothrops atrox-asper complex endemic to Venezuela: contributing to its taxonomy and snakebite management. J Proteomics 2009;72:227–40. [8] Coates AG, Obando JA. The geologic evolution of the Central American Isthmus. In: Jackson JBC, Budd AF, Coats AG, editors. Evolution and Environment in Tropical America. Chicago: University of Chicago Press; 1996. p. 21–56. [9] Alape-Girón A, Sanz L, Escolano J, Flores-Díaz M, Madrigal M, Sasa M, et al. Snake venomics of the lancehead pitviper Bothrops asper: geographic, individual, and ontogenetic variations. J Proteome Res 2008;7:3556–71.
[10] Castoe TA, Parkinson CL. Bayesian mixed models and the phylogeny of pitvipers (Viperidae: Serpentes). Mol Phylogenet Evol 2006;39:91–110. [11] Wüster W, Salomão MG, Thorpe RS, Puorto G, Furtado MFD, Hoge SA, Theakston RDG, Warrell DA. Systematics of the Bothrops atrox complex: new insights from multivariate analysis and mitochondrial DNA sequence information. In: Thorpe RS, Wüster W, Malhotra A, editors. Venomous Snakes Ecology Evolution and Snakebite. Oxford: Clarendon Press; 1999. p. 99–114. [12] Gutiérrez JM. Clinical toxicology of snakebite in Central America. In: Meier J, White J, editors. Handbook of Clinical Toxicology of Animal Venoms and Poisons, Boca Raton. CRC Press; 1995. p. 645–65. [13] Thorpe RS, Wüster W, Malhotra A, editors. Venomous Snakes. Ecology, Evolution and Snakebite. Oxford: Clarendon Press; 1997. [14] Warrell DA. Snakebites in Central and South America: epidemiology, clinical features, and clinical management. In: Campbell JA, Lamar WW, editors. The Venomous Reptiles of the Western Hemisphere. Ithaca: Comstock Publishing Associates; 2004. p. 709–62. [15] Meier J, White J, editors. Handbook of Clinical Toxicology of Animal Venoms and Poisons. Boca Raton: CRC; 1995. [16] Fan HW, Cardoso JLC. Clinical toxicology of snake bites in South America. In: Meier J, White J, editors. Handbook of Clinical Toxicology of Animal Venoms and Poisons. Florida: CRC Press; 1995. p. 667–88. [17] Lancini AR, Kornacker PM. Die Schlangen von Venezuela. Caracas: Armitano Editores; 1989. [18] Rodríguez-Acosta A, Uzcategui W, Azuaje R, Aguilar I, Girón M. Análisis clínico y epidemiológico de los accidentes por mordeduras de serpientes del género Bothrops en Venezuela. Rev Cubana Med Trop 2000;52:90–4. [19] Silva J. Las serpientes del género Bothrops en la Amazonia colombiana: aspectos biomédicos (epidemiología clínica y biología del ofidismo). Acta Med Colomb 1989;14:148–65. [20] Otero R. Manual de diagnóstico y tratamiento del accidente ofídico. Medellín (Colombia): Editorial Universidad de Antioquia; 1994. p. 1–15. [21] Saldarriaga MM, Otero R, Núñez V, Toro MF, Díaz A, Gutiérrez JM. Ontogenetic variability of Bothrops atrox and Bothrops asper snake venoms from Colombia. Toxicon 2003;42:405–11. [22] Manual de diagnostico e tratamento de acidentes por animais peconhentos, Ministerio da Saude, Fundacao Nacional de Saude, Brasília, Brazil; 1998. [23] Pardal PPO, Souza AM, Monteiro MRCC, Fan HW, Cardoso JLC, França FOS, et al. Clinical trial of two antivenoms for the treatment of Bothrops and Lachesis bites in the northeastern Amazon region of Brazil. Trans R Soc Trop Med Hyg 2004;98:28–42. [24] Pierini SV, Warrell DA, De Paulo A, Theakston RDG. High incidence of bites and stings by snakes and other animals among rubber tappers and Amazonian Indians of the Jurua valley Acre state Brazil. Toxicon 1996;34:225–36. [25] Smalligan R, Cole J, Brito N, Laing GD, Mertz BL, Manock S, et al. Crotaline snake bite in the Ecuadorian Amazon: randomised double blind comparative trial of three South American polyspecific antivenoms. BMJ 2004;329:1129–35. [26] Zavaleta A, Salas M. Ofidismo: envenenamiento por mordedura de serpientes. In: Martínez-Villaverde JR, León-Barúa R, Vidal-Neira L, Losno-García R, editors. Emergencias en Medicina Interna, Lima, Perú; 1996. p. 241–60. [27] Guércio RAP, Shevchenko A, Shevchenko A, López-Lozano JL, Paba J, Sousa MV, Ricart CAO. Ontogenetic variations in the venom proteome of the Amazonian snake Bothrops atrox. Proteome Sci 2006;4:11. [28] Salazar AM, Rodríguez-Acosta A, Girón ME, Aguilar I, Guerrero B. A comparative analysis of the clotting and fibrinolytic
J O U RN A L OF P ROT EO MI CS 7 3 (2 0 0 9) 5 7– 7 8
[29]
[30] [31] [32]
[33]
[34]
[35]
[36]
[37]
[38]
[39] [40]
[41]
[42] [43]
[44]
[45]
[46]
activities of the snake venom (Bothrops atrox) from different geographical areas in Venezuela. Thromb Res 2007;120:95–104. Otero R, Osorio RG, Valderrama R, Giraldo CA. Efectos farmacológicos y enzimáticos de los venenos de serpientes de Antioquia y Chocó (Colombia). Toxicon 1992;30:611–20. Vellard J. Variation gèographique du venin de Bothrox atrox. C R Acad Sci (Paris) 1937;204:1369–71. Vellard J. Variation gèographique du venin de Crotalus terrificus. C R Soc Biol (Paris) 1939;130:463–4. Chippaux JP, Williams V, White J. Snake venom variability: methods of study results and interpretation. Toxicon 1991;29:1279–303. Gutiérrez JM, Lomonte B, León G, Alape-Girón A, Flores-Díaz M, Sanz L, Angulo Y, Calvete JJ. Snake venomics and antivenomics: proteomic tools in the design and control of antivenoms for the treatment of snakebite envenoming. J Proteomics 2009;72:165–82. Souza GHMF, Catharino RR, Ifa DR, Eberlin MN, Hyslop S. Peptide fingerprinting of snake venoms by direct infusion nano-electrospray ionization mass spectrometry: potential use in venom identification and taxonomy. J Mass Spectrom 2008;43:594–9. Calvete JJ, Escolano J, Sanz L. Snake venomics of Bitis species reveals large intragenus venom toxin composition variation: application to taxonomy of congeneric taxa. J Proteome Res 2007;6:2732–45. Tashima AK, Sanz L, Camargo ACM, Serrano SMT, Calvete JJ. Snake venomics of the Brazilian pitvipers Bothrops cotiara and Bothrops fonsecai. Identification of taxonomy markers. J Proteomics 2008;71:473–85. Angulo Y, Estrada R, Gutiérrez JM. Clinical and laboratory alterations in horses during immunization with snake venoms for the production of polyvalent (Crotalinae) antivenom. Toxicon 1997;35:81–90. Rojas G, Jiménez JM, Gutiérrez JM. Caprylic acid fractionation of hyperimmune horse plasma: description of a simple procedure for antivenom production. Toxicon 1994;32:59–67. Fasman DG, editor. Practical Handbook of Biochemistry and Molecular Biology. Boston: CRC Press; 1992. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997;25:3389–402. Le Blanc JC, Hager JW, Ilisiu AM, Hunter C, Zhong F, Chu I. Unique scanning capabilities of a new hybrid linear ion trap mass spectrometer (QTRAP) used for high sensitivity proteomics applications. Proteomics 2003;3:859–69. Calvete JJ, Juárez P, Sanz L. Snake venomics. Strategy and applications. J Mass Spectrom 2007;42:1405–14. Calvete JJ, Sanz L, Angulo Y, Lomonte B, Gutiérrez JM. Venoms, venomics, antivenomics. FEBS Lett 2009;583:1736–43. Lomonte B, Escolano J, Fernández J, Sanz L, Angulo Y, Gutiérrez JM, Calvete JJ. Snake venomics and antivenomics of the arboreal neotropical pitvipers Bothriechis lateralis and Bothriechis schlegelii. J Proteome Res 2008;7:2445–57. Angulo Y, Escolano J, Lomonte B, Gutiérrez JM, Sanz L, Calvete JJ. Snake venomics of Central American pitvipers. Clues for rationalizing the distinct envenomation profiles of Atropoides nummifer and Atropoides picadoi. J Proteome Res 2008;7:708–19. Sanz L, Escolano J, Ferretti M, Biscoglio MJ, Rivera E, Crescenti EJ, Angulo Y, Lomonte B, Gutiérrez JM, Calvete JJ. Snake venomics of the South and Central American Bushmasters. Comparison of the toxin composition of Lachesis muta gathered from proteomic versus transcriptomic analysis. J Proteomics 2008;71:46–60.
77
[47] Sanz L, Ayvazyan N, Calvete JJ. Snake venomics of the Armenian mountain vipers Macrovipera lebetina obtusa and Vipera raddei. J Proteomics 2008;71:198–209. [48] Gutierrez J, Sanz L, Escolano J, Fernandez J, Lomonte B, Angulo Y, Rucavado A, Warrell DA, Calvete JJ. Snake venomics of the Lesser Antillean pit vipers Bothrops caribbaeus and Bothrops lanceolatus. Correlation with toxicological activities and immunoreactivity of a heterologous antivenom. J Proteome Res 2008;7:4396–408. [49] Wagstaff SC, Sanz L, Juárez P, Harrison RA, Calvete JJ. Combined snake venomics and venom gland transcriptomic analysis of the ocellated carpet viper, Echis ocellatus. J Proteomics 2009;71:609–23. [50] Wetton JH, Carter RE, Parkin DT, Walters D. Demographic study of a wild house sparrow population by DNA fingerprinting. Nature 1987;327:147–9. [51] Daltry JC, Wüster W, Thorpe RS. Diet and snake venom evolution. Nature 1996;379:537–40. [52] Wüster W, Salomão MG, Duckett GJ, Thorpe RS, Puorto G, Warrell DA, Theakston RDG. Mitochondrial DNA evolution of the Bothrops atrox species complex (Squamata: Serpentes: Viperidae). Kaupia 1999;8:135–44. [53] López-Lozano JL, Valle de Sousa M, Ricart CAO, Chávez-Olortegui C, Flores-Sánchez E, Muniz EG, Bührnheim PF, Morhy L. Ontogenetic variation of metalloproteinases and plasma coagulant activity in venoms of wild Bothrops atrox specimens from Amazonian rain forest. Toxicon 2002;40:997–1006. [54] Gutiérrez JM, Chaves F, Bolaños R. Estudio comparativo de venenos de ejemplares recién nacidos y adultos de Bothrops asper. Rev Biol Trop 1980;28:341–6. [55] Furtado MFD, Maruyama M, Kamiguti AS, Antonio LC. Comparative study of nine Bothrops snake venoms from adult female snakes and their offspring. Toxicon 1991;29:219–26. [56] Fox JW, Serrano SM. Structural considerations of the snake venom metalloproteinases, key members of the M12 reprolysin family of metalloproteinases. Toxicon 2005;45:969–85. [57] Gutiérrez JM, Rucavado A, Escalante T, Díaz C. Hemorrhage induced by snake venom metalloproteinases: biochemical and biophysical mechanisms involved in microvessel damage. Toxicon 2005;45:997–1011. [58] Escalante T, Shannon J, Moura-da-Silva AM, Gutierrez JM, Fox JW. Novel insights into capillary vessel basement membrane damage by snake venom hemorrhagic metalloproteinases: a biochemical and immunohistochemical study. Arch Biochem Biophys 2006;455:144–53. [59] Lynch VJ. Inventing an arsenal: adaptive evolution and neofunctionalization of snake venom phospholipase A2 genes. BMC Evol Biol 2007;7:2. [60] Gibbs HL, Rossiter W. Rapid evolution by positive selection and gene gain and loss: PLA2 venom genes in closely related Sistrurus rattlesnakes with divergent diets. J Mol Evol 2008;66:151–66. [61] Lomonte B, Angulo Y, Sasa M, Gutiérrez JM. The Phospholipase A2 homologues of snake venoms: biological activities and their possible adaptive roles. Protein Pept Lett 2009;16:860–76. [62] Tsai IH, Wang YM, Chen YH, Tsai TS, Tu MC. Venom phospholipases A2 of bamboo viper (Trimeresurus stejnegeri): molecular characterization, geographic variations and evidence of multiple ancestries. Biochem J 2004;377:215–23. [63] Chijiwa T, Deshimaru M, Nobuhisa I, Nakai M, Ogawa T, Oda N, Nakashima KI, Fukumaki Y, Shimohigashi Y, Hattori S, Ohno M. Regional evolution of venom-gland phospholipase A2 isoenzymes of Trimeresurus flavoviridis snakes in the southwestern islands of Japan. Biochem J 2000;347:491–9. [64] Chijiwa T, Yamaguchi Y, Ogawa T, Deshimaru M, Nobuhisa I, Nakashima KI, Oda-Ueda N, Fukumaki Y, Hattori S, Ohno M.
78
[65]
[66]
[67]
[68]
[69]
[70]
[71]
[72]
[73]
[74]
[75]
J O U RN A L OF P ROT EO M IC S 7 3 (2 0 0 9) 5 7– 7 8
Interisland evolution of Trimeresurus flavoviridis venom phospholipase A2 isozymes. J Mol Evol 2003;56:286–93. Gutiérrez JM, Theakston RD, Warrell DA. Confronting the neglected problem of snake bite envenoming: the need for a global partnership. PLoS Med 2006;3:e150–9. Meier J. Commercially available antivenoms (“hyperimmune sera”, “antivenins”, “antisera”) for antivenom therapy. In: Meier J, White J, editors. Handbook of Clinical Toxicology of Animal Venoms and Poisons. Florida: CRC Press; 1995. p. 689–721. Espino-Solís GP, Riaño-Umbarila L, Becerril B, Possani LD. Antidotes against venomous animals: state of the art and prospectives. J Proteomics 2009;72:183–99. Bogarín G, Romero M, Rojas G, Lutsch C, Casadamont M, Lang J, Otero R, Gutiérrez JM. Neutralization, by a monospecific Bothrops lanceolatus antivenom, of toxic activities induced by homologous and heterologous Bothrops snake venoms. Toxicon 1999;37:551–7. Theakston RDG, Warrell DA, Griffiths E. Report of a WHO workshop on standardization and control of antivenoms. Toxicon 2003;41:541–57. World Health Organization. Rabies and envenomings. A neglected public health issue. Report of a Consultative Meeting. Geneva: WHO; 2007. Gutiérrez JM, Higashi HG, Wen FH, Burnouf T. Strengthening antivenom production in Central and South American public laboratories: report of a workshop. Toxicon 2007;49:30–5. Otero R, Núñez V, Osorio RG, Gutiérrez JM, Giraldo CA, Posada LE. Ability of six Latin American antivenoms to neutralize the venom of mapaná equis (Bothrops atrox) from Antioquia and Chocó (Colombia). Toxicon 1995;33:809–15. Bogarín G, Morais JF, Yamaguchi IK, Stephano MA, Marcelino JR, Nishikawa AK, Guidolin R, Rojas G, Higashi HG, Gutiérrez JM. Neutralization of crotaline snake venoms from Central and South America by antivenoms produced in Brazil and Costa Rica. Toxicon 2000;38:1429–41. Rojas E, Quesada L, Arce V, Lomonte B, Rojas G, Gutiérrez JM. Neutralization of four Peruvian Bothrops sp. snake venoms by polyvalent antivenoms produced in Perú and Costa Rica: preclinical assessment. Acta Trop 2005;93:85–95. Gibbs HL, Sanz L, Calvete JJ. Snake population venomics: proteomics-based analyses of individual variation reveals significant gene regulation effects on venom protein expression in Sistrurus rattlesnakes. J Mol Evol 2009;68:113–25.
[76] Whitehead A, Crawford DL. Variation within and among species in gene expression: raw material for evolution. Mol Ecol 2006;15:1197–211. [77] Lande R. Natural selection and random genetic drift in phenotypic evolution. Evolution 1976;30:314–34. [78] Martins M, Marques OAV, Sazima I. Ecological and phylogenetic correlates of feeding habits in Neotropical pitvipers of the genus Bothrops. In: Schuett G, Höggren M, Green HW, editors. Biology of the Vipers. Indiana: Biological Science Press; 2002. p. 307–28. [79] Marques OAV, Martins M, Sazima I. A new insular species of pitviper from Brazil, with comments on evolutionary biology and conservation of the Bothrops jararaca group (Serpentes, Viperidae). Herpetologica 2002;58:303–12. [80] Grazziotin FG, Monzel M, Echeverrigaray S, Bonatto SL. Phylogeography of the Bothrops jararaca complex (Serpentes: Viperidae): past fragmentation and island colonization in the Brazilian Atlantic forest. Mol Ecol 2006;15:3969–82. [81] Garstang W. The theory of recapitulation: a critical re-statement of the biogenetic law. Linn Soc J Zool 1922; XXXV:81–101. [82] Mackessy SP. Venom ontogeny in the Pacific rattlesnakes Crotalus viridis helleri and C.v. oreganus. Copeia 1988;1988:92–101. [83] Mackessy SP, Sixberry NM, Heyborne WH, Fritts T. Venom of the Brown Treesnake, Boiga irregularis: ontogenetic shifts and taxa-specific toxicity. Toxicon 2006;47:537–48. [84] Sasa M. Diet and snake venom evolution: can local selection alone explain intraspecific venom variation? Toxicon 1999;37:249–52. [85] Mebs D. Toxicity in animals. Trends in evolution? Toxicon 2001;39:87–96. [86] Cardoso da Silva JM, Bates JM. Biogeographical patterns and conservation in the South American cerrado: a tropical savanna horspot. BioScience 2002;52:225–33. [87] Webb SD. Ecogeography nd the Great American Interchange. Paleobiology 1991;17:266–80. [88] Haffer J. Alternative models of vertebrate speciation in Amazonia: an overview. Biodivers Conserv 1997;6:451–76. [89] Quijada-Mascareñas JA, Ferguson JE, Pook CE, Salomão MG, Thorpe RS, Wüster W. Phylogeographic patterns of trans-Amazonian vicariants and Amazonian biogeography: the Neotropical rattlesnake (Crotalus durissus complex) as an example. J Biogeogr 2007;34:1296–312.