Snake Venomics of the Lesser Antillean Pit Vipers Bothrops caribbaeus and Bothrops lanceolatus: Correlation with Toxicological Activities and Immunoreactivity of a Heterologous Antivenom† Jose´ Marı´a Gutie´rrez,*,‡ Libia Sanz,§ Jose´ Escolano,§ Julia´n Ferna´ndez,‡ Bruno Lomonte,‡ Yamileth Angulo,‡ Alexandra Rucavado,‡ David A. Warrell,| and Juan J. Calvete*,§ Instituto Clodomiro Picado, Facultad de Microbiologı´a, Universidad de Costa Rica, San Jose´, Costa Rica, Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Cientı´ficas (CSIC), Jaume Roig 11, 46010 Valencia, Spain, and Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom Received May 26, 2008
The venom proteomes of the snakes Bothrops caribbaeus and Bothrops lanceolatus, endemic to the Lesser Antillean islands of Saint Lucia and Martinique, respectively, were characterized by reversephase HPLC fractionation, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The venoms contain proteins belonging to seven (B. caribbaeus) and five (B. lanceolatus) types of toxins. B. caribbaeus and B. lanceolatus venoms contain phospholipases A2, serine proteinases, L-amino acid oxidases and zinc-dependent metalloproteinases, whereas a long disintegrin, DC-fragments and a CRISP molecule were present only in the venom of B. caribbaeus, and a C-type lectin-like molecule was characterized in the venom of B. lanceolatus. Compositional differences between venoms among closely related species from different geographic regions may be due to evolutionary environmental pressure acting on isolated populations. The venoms of these two species differed in the composition and the relative abundance of their component toxins, but they exhibited similar toxicological and enzymatic profiles in mice, characterized by lethal, hemorrhagic, edemaforming, phospholipase A2 and proteolytic activities. The venoms of B. caribbaeus and B. lanceolatus are devoid of coagulant and defibrinogenating effects and induce only mild local myotoxicity in mice. The characteristic thrombotic effect described in human envenomings by these species was not reproduced in the mouse model. The toxicological profile observed is consistent with the abundance of metalloproteinases, PLA2s and serine proteinases in the venoms. A polyvalent (Crotalinae) antivenom produced in Costa Rica was able to immunodeplete ∼80% of the proteins from both B. caribbaeus and B. lanceolatus venoms, and was effective in neutralizing the lethal, hemorrhagic, phospholipase A2 and proteolytic activities of these venoms. Keywords: Bothrops caribbaeus • Bothrops lanceolatus; Saint Lucia lancehead viper • Fer-de-lance pitviper • snake venom protein families • proteomics • viperid toxins • snake venomics • immunodepletion • N-terminal sequencing • mass spectrometry • polyvalent (Crotalinae) antivenom
Introduction The genus Bothrops (Serpentes, Viperidae) comprises 37 species of pitvipers,1 commonly referred as lancehead vipers. Bothrops species are diverse in their morphology and natural * Address correspondence to: Juan J. Calvete, Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Cientı´ficas (CSIC), Jaume Roig 11, 46010 Valencia, Spain. Phone, +34-96-339-1778; fax, +34-96-3690800; e-mail,
[email protected] (for the proteomic aspects of the study). Jose´ M. Gutie´rrez, Instituto Clodomiro Picado, Facultad de Microbiologı´a, Universidad de Costa Rica, San Jose´, Costa Rica. Phone, 506-2229-3135; fax, 506-2292-0485; e-mail,
[email protected] (for the toxinological aspects of the study). ‡ Universidad de Costa Rica. § Instituto de Biomedicina de Valencia. | University of Oxford. † This paper is dedicated to the memory of Cassian Bon and Andre´ Me´nez, who left us during 2008. Their research on the structure and function of venom toxins represents a precious legacy to the field of Toxinology.
4396 Journal of Proteome Research 2008, 7, 4396–4408 Published on Web 09/12/2008
history, and are widely distributed in tropical Latin America, from northeastern Mexico to Argentina, and to a few of the lower Caribbean islands.1 The species of this genus are responsible for the vast majority of snakebites in Central and South America.2 Within this genus, Bothrops caribbaeus and Bothrops lanceolatus are endemic to the Lesser Antillean islands of Saint Lucia and Martinique, respectively.1,3 Morphological and molecular data suggest that B. caribbaeus and B. lanceolatus originated as a result of long-distance dispersal of mainland species of the Bothrops atrox-asper complex.3 The two species form a monophyletic group, which in turn forms the sister clade to the Bothrops atrox-asper complex.3 The timing of the split between the Lesser Antillean Bothrops clade and the B. atroxasper clade may have occurred during the late Miocene or early Pliocene, 4.2-8.9 million years ago (Mya).3 On the other hand, molecular and quantitative morphological data indicate that 10.1021/pr8003826 CCC: $40.75
2008 American Chemical Society
Venomics of B. caribbaeus and B. lanceolatus
Figure 1. Reverse-phase HPLC separation of the venom proteins from B. caribbaeus. Fractions were collected manually and characterized by N-terminal sequencing, ESI mass spectrometry, and SDS-PAGE. The results are shown in Table 1. Insets, right, picture of B. caribbaeus from Western Saint Lucia (copyright D. A. Warrell); left, SDS-PAGE showing the protein composition of the reverse-phase HPLC separated venom protein fractions run under nonreduced (upper panel) and reduced (lower panel) conditions. Molecular mass markers (in kDa) are indicated at the left of each gel. Protein bands were excised and characterized by mass fingerprinting and CID-MS/MS. The results are shown in Table 1.
the divergence between B. caribbaeus and B. lanceolatus can be dated to a 3.1-6.5 Mya stepping-stone colonization, including initial dispersal of the B. caribbaeus ancestor from the mainland to Saint Lucia, followed by a further dispersal event to Martinique.3 B. caribbaeus, commonly known as Saint Lucia lancehead pitviper and locally as ‘the serpent’ (Figure 1, inset), is endemic to this island where it is restricted to the coastal lowlands of the middle two-thirds of the island. It inhabits coastal tropical forests and riverine areas below 180 m, being abundant in coconut plantations. B. caribbaeus is a terrestrial to semiarboreal species whose total length (average 100 cm) can reach 213 cm.1 B. lanceolatus, known as Fer-de-lance or Martinique serpent (Figure 2, inset), is endemic to the island of Martinique, where it is distributed in two isolated populations in the humid upland regions in the northern and southern portions of the island, from sea level to approximately 1300 m. It is also semiarboreal, and its average total length is 150 cm, but specimens approaching 300 cm are reported.1 These two species predate on warm-blooded animals, mostly mammals like mice and rats, but also on birds.1 In Martinique, there is an average of 20 reported bites by B. lanceolatus each year, with occasional fatalities. Without early and adequate treatment with specific antivenom, severe envenoming may result, characterized by local tissue damage and delayed multifocal thrombotic occlusions of small/medium sized arteries, causing cerebral, myocardial, pulmonary and other infarctions.4-7 The inflammatory events induced by injection of B. lanceolatus venom in rodents have been investigated,8-10 and various toxic and enzymatic activities have been described for the crude venom.11,12 In Saint Lucia, B. caribbaeus causes about 12 bites each year with identical clinical effects.6,13 The pathogenesis of the arterial thromboses caused by B. lanceolatus cannot be attributed to direct coagu-
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Figure 2. Reverse-phase HPLC separation of the venom proteins from B. lanceolatus. Fractions were collected manually and characterized by N-terminal sequencing, ESI mass spectrometry, and SDS-PAGE.. The results are shown in Table 2. Insets, right, picture of B. lanceolatus from South-Eastern Martinique (copyright D. A. Warrell); left, SDS-PAGE showing the protein composition of the reverse-phase HPLC separated venom protein fractions run under nonreduced (upper panel) and reduced (lower panel) conditions. Molecular mass markers (in kDa) are indicated at the left of each gel. Protein bands were excised and characterized by mass fingerprinting and CID-MS/MS. The results are shown in Table 2.
lant activity of the venom, since it does not clot human plasma.11 Human victims show negligible or no evidence of coagulopathy but may be thrombocytopenic.6 The biochemistry and toxicology of B. caribbaeus venom have not been addressed in the literature. A highly effective antivenom (Sanofi-Pasteur ‘Bothrofav’) for the treatment of envenomings by B. lanceolatus has been developed.5,14 There exhibits an excellent preclinical profile of neutralization11 and its timely administration prevents the development of the most serious effects of envenoming, including thrombosis.5,14 However, the restricted availability of the antivenom in the neighboring island of Saint Lucia and in zoos and herpetariums where these species may be kept is a matter of concern. Hence, there is a need to test other antivenoms for their efficacy against the effects of B. caribbaeus and B. lanceolatus venoms for the treatment of snakebite envenomings in Santa Lucia and Martinique. A thorough characterization of the venom proteomes of these medically important Caribbean Bothrops species might contribute to a deeper understanding of the biology, ecology and pathophysiology of envenomings by these snakes, and would also serve as a starting point for studying structure-function correlations of individual toxins. In addition, knowledge of the relative contributions of different venom toxin families to the composition of the venoms might be relevant for generating immunization protocols that elicit the production of toxin-specific antibodies showing greater specificity and effectiveness than conventional antivenoms raised by immunizing horses with whole venom. Here, we report detailed proteomic studies of the venoms of B. caribbaeus and B. lanceolatus using snake venomic and immunodepletion approaches,15-17 with the aims of (i) correlating venom composition and evolutionary trends associated Journal of Proteome Research • Vol. 7, No. 10, 2008 4397
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Table 1. Assignment of the Reverse-Phase Fractions of B. caribbeus Venom, Isolated as in Figure 2, 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 (Separated by SDS-PAGE as in Figure 2)a HPLC fraction Bcar-
N-terminal sequence
1-3 4 5 6 7 8 9 10 11 12
n.p. GRPPR SPPVCGNYYRELGEDCDCGPPANCQNPCCD SPPVCGNYFVEVGEE SPPVCGNYFVEVGEE SVDFDSESPRKPEIQ DLWQFGKMISIVMRKNVVFLYFSYGCYCG V(V/I)GGDECDINEHRSL VIGGDECDINEHRSL Blocked
26 kDa9/1 28 kDa9/1 24921 13958 27 kDa9/1 24 kDa9/1 52 kDa9/1
Blocked ADDRNPLEECFRETD
58 kDa9/1 54 kDa9/1
Blocked TPEQQRYVELFIVVDHGMFMKYNGDSDKIR N.D.
110 kDa9/56 kDa1 23 kDa9/1 56 kDa9/1 812.8 48 kDa9/1
13
14 15 16
m M
peptide ion molecular mass
m/z
z
MS/MS-derived sequence
8802
36 kDa9/1
812.8 526.1 615.2 626.9 594.2 532.3 646.6 676.4 743.6 638.8 757.6 526.3
2 2 2 2 2 2 2 2 2 3 3 2
XYEXVNTVNEXYR GNYYGYCR DNSPGQNNPCK MFYSSNDEHK GNQDXYYCR NPLEECFR EGWYANLGPMR SAGQLYEESLQK ETDYEEFLEIAK ETLSVTADYVIVCTTSR IYFAGEYTAQAHGWIDSTIK GNYYGYCR
526.3
2 2 2 2 3
GNYYGYCR XYEXVNTVNEXYR YXEXXXGQHR (201.1)XSADDTQTAFGEWK (715.3)SQCILNEPLR
671.3 884.8 651.8
protein family unknown Long disintegrin DC-fragment DC-fragment CRISP PLA2 Serine proteinase Serine proteinase PIII-metalloproteinase
PIII-metalloprotease acid oxidase
L-amino
PIII-metalloproteinase PI-metalloproteinase PIII-metalloproteinase PIII-metalloproteinase PIII-metalloproteinase
a X, Ile or Leu. Unless other stated, for MS/MS analyses, cysteine residues were carbamidomethylated; molecular masses of native proteins were determined by electrospray-ionization ((0.02%) or MALDI-TOF ((0.2%) mass spectrometry. Apparent molecular mass determined by SDS-PAGE of non-reduced (9) and reduced (1) samples; n.p., non-peptidic material found. M and m, denote, respectively, major and minor products within the same HPLC fraction.
with allopatric speciation in geographically isolated Lesser Antillean islands; (ii) characterizing the toxicological profile of the venom of B. caribbaeus and comparing it with the previously reported profiles of the venoms of B. lanceolatus and its sister clade species B. asper and B. atrox; and (iii) investigating the ability of a heterologous polyvalent antivenom produced in Costa Rica to immunorecognize and neutralize the toxic activities of components from the venoms of the two Caribbean Bothrops species.
Experimental Procedures Venoms and Antivenom. The venom of B. caribbaeus was pooled from four F1 adult specimens born in the Kentucky Reptile Zoo (KY) from snakes collected in different locations of the island of Saint Lucia, and was kindly donated by Kristen Wiley and James Harrison. Venom of B. lanceolatus was purchased from Latoxan (Valence, France) and was pooled from >12 specimens captured from different localities of Martinique during the last 5-10 years and maintained since then in captivity. These B. caribbaeus and B. lanceolatus snakes eat mice and small rats. In some experiments, the venom of adult specimens of B. asper, collected in the Pacific region of Costa Rica and kept at the serpentarium of Instituto Clodomiro Picado (ICP), was used for comparative purposes. Venoms were lyophilized and stored at -20 °C. The polyvalent (Crotalinae) antivenom (batch 4201007POLQ, expiry date: October 2010) produced at ICP, Universidad de Costa Rica, was used in the antivenomic studies and in neutralization assays. It is produced by immunizing horses with a mixture of equal amounts of the venoms of B. asper, Crotalus durissus durissus, and Lachesis stenophrys obtained from adult specimens kept in captivity at the ICP serpentarium.18 Whole immunoglobulins were purified by caprylic acid precipitation.19 4398
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Isolation of Venom Proteins. For reverse-phase HPLC separations, 2-5 mg of crude, lyophilized venoms of B. caribbaeus and B. lanceolatus 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 000g 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 Speed-Vac (Savant). The relative abundances (% of the total venom proteins) of the different protein families in the venoms were estimated 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. Characterization of HPLC-Isolated Proteins. Isolated protein fractions were subjected to N-terminal sequence analysis (using a Procise instrument, Applied Biosystems, Foster City, CA) following the manufacturer’s instructions. Amino acid sequence similarity searches were performed against the available databanks using the BLAST program20 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) and by electrospray ionization (ESI) mass spectrometry using an Applied Biosystems QTrap 2000 mass spectrometer21 operated in Enhanced Multiple Charge mode in the range m/z 600-1700.
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Venomics of B. caribbaeus and B. lanceolatus
Table 2. Assignment of the Reverse-Phase Fractions of B. lanceolatus Venom, Isolated as in Figure 3, 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 (Separated by SDS-PAGE as in Figure 3) HPLC fraction Blan-
1-3 4 5 6
peptide ion N-terminal sequence
molecular mass
m/z
z
MS/MS-derived sequence
protein family
n.p GRPPR DLWQFRKMISVVMRK VVGGDECNINEHR(F/S)L QLW(A/K)FRKMISVVMRK VIGGDECNINEHRSL TLCAFIMEGGKSMC VIGGDECNINEHRSL QLW(A/K)FRKMISVVMRK VIGGDECNINEHRSL Blocked
13965 32, 38 kDa9 18 kDa9 25, 33 kDa9
unknown PLA2 Serine proteinase PLA2 2-chain serine proteinase
28, 33, 36 kDa9 18 kDa9 54, 27 kDa9/1 52 kDa9/1
Serine proteinase PLA2 Serine proteinase PIII-metalloproteinase
13
DCPSDWSS QAYNPYRYVEFXIVV VIGGDECNINEHRSL N.D. VIGGDECNINEHRSL ADDRNPLEECFRETD
27 52 26 52 26 54
14 15 16
VIGGDECNINEHRSL DCPSDWSSYEGHCYK Blocked TPEQQRYVELFIVVD Blocked
26 kDa9 26 kDa9 110 kDa9/54 kDa1 23 kDa9/1 52 kDa9/1 48 kDa9/1
7 8 9 10 10-13 11 12
TPEQQRYVELFIVVD
kDa9/14 kDa1 kDa9/1 kDa9 kDa9/1 kDa9 kDa9/1
36 kDa9/1 23 kDa9/1
526.8 813.1
2 2
GNYYGYCR XYEXVNTVNEXYR
884.8
2
(201.1)XSADDTQTAFGEWK
884.8
2
(201.1)XSADDTQTAFGEWK
532.3 646.6 676.4 743.6 638.8 757.6
2 2 2 2 3 3
NPLEECFR EGWYANLGPMR SAGQLYEESLQK ETDYEEFLEIAK ETLSVTADYVIVCTTSR IYFAGEYTAQAHGWIDSTIK
526.3
2
GNYYGYCR
671.3 671.3 884.8 651.8
2 2 2 3
YXEXXXGQHR YXEXXXGQHR (201.1)XSADDTQTAFGEWK (715.3)SQCILNEPLR
C-type lectin-like PIII-metalloproteinase Serine proteinase PIII-metalloproteinase Serine proteinase L-amino acid oxidase
Serine proteinase C-type lectin-like PIII-metalloproteinase PI-metalloproteinase PIII-metalloproteinase PIII-metalloproteinase PIII-metalloproteinase PI-metalloproteinase
X, Ile or Leu. Unless other stated, for MS/MS analyses, cysteine residues were carbamidomethylated; molecular masses of native proteins were determined by electrospray-ionization ((0.02%) or MALDI-TOF ((0.2%) mass spectrometry. Apparent molecular mass determined by SDS-PAGE of non-reduced (9) and reduced (1) samples; n.p., non-peptidic material found.
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 bovine pancreas trypsin (Roche) using a ProGest digestor (Genomic Solutions) following the manufacturer’s instructions. A total of 0.65 µL of the tryptic peptide mixtures (total volume of ∼ 20 µL) was spotted onto a MALDI-TOF sample holder, mixed with an equal volume of a saturated solution of R-cyano-4-hydroxycinnamic acid (Sigma) in 50% acetonitrile containing 0.1% TFA, 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 (Swiss-Prot accession code P81517) prepared and previously characterized in our laboratory was used as mass calibration standard (mass range, 450-3300 Da). 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 mass spectrometer (Applied Biosystems)21 equipped with a nanospray source (Protana, Denmark). Doubly- or triply charged 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 927 viperid protein sequences deposited in the Swiss-Prot/TrEMBL database (Knowledgebase Release 12 of July 2007; http://us.expasy.org/sprot/; 212 in Swiss-Prot, 715 in TrEMBL) plus the previously assigned peptide ion sequences from snake venomics projects carried out in our laboratory.15,16,22-27 MS/MS mass tolerance was set to (0.6 Da. Carbamidomethyl cysteine and oxidation of methionine were fixed and variable modifications, respectively. Immunodepletion of Venom Proteins by a Costa Rican Polyvalent antivenom. For the identification of venom proteins bearing epitopes recognized by an antivenom using proteomic techniques,17 2 mg of whole venom was 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 Journal of Proteome Research • Vol. 7, No. 10, 2008 4399
research articles for 1 h at 37 °C. Thereafter, 6 mg of rabbit anti-horse IgG antiserum (Sigma) in 350 µL of 20 mM phosphate buffer, pH 7.0, was 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. The control sample was subjected to the same procedure except that antivenom IgGs were not included in the reaction mixture. Western Blot. The occurrence within the polyvalent antivenom of antibodies directed against antigenic determinants exhibited by B. caribbaeus and B. lanceolatus venom proteins, which may or may not be immunoprecipitated by the immunodepletion approach described above, was investigated by Western blot analysis.17 To this end, the reverse-phase HPLC chromatographic fractions were electrophoresed in SDS-(10%) polyacrylamide gels under nonreduced conditions followed by electrotransfer to nitrocellulose membranes as described28 using a Bio-Rad minitransfer cell operated at 150 mA during 90 min. To assess transfer efficiency, the nitrocellulose membranes were previsualized by reversible Ponceau-S Red stain. Then, the membranes were incubated in 1% bovine serum albumin in phosphate-buffered saline (PBS; 0.12 M NaCl, 0.04 M sodium phosphate, pH 7.2) for 30 min at room temperature to block nonspecific binding sites, and the membranes were subsequently incubated overnight at 4 °C with either ICP polyvalent antivenom or normal equine serum, diluted 1:1000 in PBS. After washing 4 times with PBS containing 0.1% albumin and 0.05% Tween-20, the membranes were incubated with an antihorse IgG-alkaline phospatase conjugate (Sigma), diluted 1:1000, during 2 h at room temperature. Finally, membranes were washed 4 times as above, and color development was performed by adding BCIP/NBT (Chemicon) substrate. Toxicological and Enzymatic Activities of Venoms. Toxic effects were studied in CD-1 mice (18-20 g body weight), unless otherwise stated. Lyophilized venom was dissolved in PBS prior to each test. All experimental protocols involving the use of mice were approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUA) of the University of Costa Rica. Lethality. Various amounts of venom, dissolved in 200 µL of PBS, were injected intravenously (iv), in the caudal vein, to groups of four mice. For some experiments, mice were injected venom intraperitoneally (ip) in a volume of 0.5 mL. Control animals received the same volume of PBS alone. Deaths occurring within 24 h were recorded and the Median Lethal Dose (LD50) was estimated by probits. Hemorrhagic Activity. Various amounts of venom, dissolved in 100 µL of PBS, were injected intradermally (id), in the ventral abdominal region, to groups of four mice. Controls received 100 µL of PBS alone. After 2 h, mice were sacrificed by CO2 inhalation, their skins were removed, and the hemorrhagic area in the inner side of the skin was measured to determine the diameter of hemorrhagic spots.29 The Minimum Hemorrhagic Dose (MHD) was estimated as the lowest venom dose inducing a hemorrhagic area of 10 mm diameter.29 Edema-Forming Activity. Various amounts of venom, dissolved in 50 µL of PBS, were injected subcutaneously (sc) in the foot pads of four mice. Control animals received 50 µL of PBS alone. One hour after the injection, the thickness of the injected foot pad was measured by a low-pressure spring caliper.30 Edema was expressed as the percentage increase in 4400
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Gutie´rrez et al. thickness of the foot pad injected with venom, as compared to the pad injected with PBS. The Minimum Edema-forming Dose (MED) corresponded to the dose of venom that induced 30% edema.31 Myotoxicity. Groups of four mice were injected intramuscularly (im), in the right gastrocnemius muscle, with 50 µg of lyophilized venom reconstituted in 50 µL of PBS. Control animals received 50 µL of PBS alone. Three hours after injection, mice were bled, plasma was separated, and the creatine kinase (CK) activity in plasma was determined using the CK-NAc kit (Biocon Diagnostik, Germany), and expressed as Units/L. For comparative purposes, the venoms of B. lanceolatus and B. asper were tested in addition to the venom of B. caribbaeus. The presence of basic PLA2 components was also assessed by PAGE at pH 4.3. 32,33 Coagulant Activity. Various amounts of venom, dissolved in 100 µL of PBS, were added to 200 µL of citrated human plasma that had been previously incubated for 5 min at 37 °C in a water bath. Controls included the addition of 100 µL of PBS to plasma samples. Clotting times were recorded and the Minimum Coagulant Dose (MCD), defined as the lowest venom dose that induced clotting in 60 s, was determined.34,35 Defibrinogenating Activity. Various amounts of venom, dissolved in 200 µL of PBS, were injected iv, into the caudal vein, to groups of four mice. Controls included mice injected with PBS alone. One hour after injection, mice were bled by cardiac puncture, under anesthesia, and blood was placed in dry glass tubes and left undisturbed for 2 h at 22-25 °C. Thereafter, the tubes were gently tilted and the presence, or absence, of clots was recorded. The Minimum Defibrinogenating Dose (MDD) corresponded to the minimum dose of venom that rendered blood unclottable in all mice tested.35 Inhibition of Platelet Aggregation. Platelet-rich plasma (PRP) was obtained by centrifugation of citrated blood from a healthy human volunteer.36 Platelet concentration was adjusted to 200 000 platelets/µL. Various doses of venom, dissolved in 100 µL of PBS, were added to 500 µL of PRP, at 37 °C, and the effect of venom on platelet aggregation was assessed in an aggregometer for 5 min (Chronolog Corp.). For collageninduced platelet aggregation, 2 µL of a 1 mg/mL solution of collagen (Chronolog) was added to the platelet suspension, and the time-dependent aggregation was recorded. Controls consisted of PRP incubated with collagen alone, and PRP incubated with PBS alone. Histopathological Alterations. Groups of four mice were injected iv with various amounts of venom dissolved in 200 µL of PBS. Control animals were injected with PBS alone. Mice were observed, and in the event of death, tissue samples were immediately collected from lungs, heart and brain. Mice that survived 24 h were sacrificed and tissue samples collected. In other experiments, mice were injected im, in the right gastrocnemius, with 50 µg of venom, in 50 µL of PBS. Three hours after injection, mice were sacrificed, the injected muscle was dissected out, and a sample of muscle tissue was collected. Tissue samples were immediately added to 10% formalin in PBS and processed routinely for embedding in paraffin. Eightmicrometers sections were obtained and stained with hematoxylin-eosin for microscopic observation. Phospholipase A2 Activity. The phospholipase A2 activitity of the venoms was determined on micellar phosphatidylcholine according to the phenol red-based colorimetric method.37 Briefly, aliquots of 5 µL, containing varying amounts of venom, were added to 1 mL of substrate in a thermoregulated cuvette
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Venomics of B. caribbaeus and B. lanceolatus at 30 °C, and after a lag period of 20 s, the decrease in absorbance at 558 nm was continuously monitored for 2 min. One unit of PLA2 activity was defined as a change of 0.001 in absorbance per minute. Proteinase Activity. Proteinase activity of the venoms was determined using an azocasein substrate.38 To this end, 20 µL aliquots containing varying amounts of venom were added to 100 µL of substrate (10 mg/mL azocasein dissolved in 25 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.4), followed by incubation at 37 °C for 90 min. After stopping the reaction with 200 µL of 5% trichloroacetic acid, followed by centrifugation, 100 µL of supernatant was mixed with 100 µL of 0.5 M NaOH, and the absorbance at 450 nm was recorded. One unit of proteolytic activity was defined as a change of 0.2 in absorbance per minute. Neutralization of Venoms by Antivenom. The neutralizing ability of the ICP polyvalent antivenom was investigated following previously described protocols.31,39,40 Briefly, a constant dose of venom, known as the ‘challenge dose’, was incubated, at 37 °C for 30 min, with various dilutions of antivenom, using PBS as diluent. Controls consisted of venom incubated with PBS alone and antivenom incubated with PBS alone. Then, aliquots of the mixtures, containing a ‘challenge dose’ of venom, were tested in the corresponding experimental system described above. In the case of lethal activity, a protocol based on intraperitoneal (ip) injection was also used.31 The ‘challenge dose’ of venom corresponded to 4 LD50 (lethality), 10 MHD (hemorrhagic activity), 1 or 2 µg for B. caribbaeus or B. lanceolatus, respectively (phospholipase A2 activity) and 20 µg (proteinase activity). The doses of B. lanceolatus venom were established on the basis of previous results.11 Statistical Analyses. The significance of the differences between the mean values of two experimental groups was assessed by the Student’s t test.
Results and Discussion Characterization of the Venom Proteomes of B. caribbaeus and B. lanceolatus. The protein composition of the venoms of B. caribbaeus and B. lanceolatus was investigated using our snake venomics approach,15 including fractionation of the crude venoms by reverse-phase HPLC and analysis of each chromatographic fraction by SDS-PAGE (Figures 1 and 2), N-terminal sequencing, and tryptic peptide MALDI-TOF mass fingerprinting (Tables 1 and 2). 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 previously reported protein or to a known protein family. Fractions showing heterogeneous or a blocked N-terminal sequence were analyzed by SDS-PAGE and the bands of interest were subjected to automated reduction, carbamidomethylation, and in-gel tryptic digestion followed by sequencing of selected doublyand triply charged tryptic peptide ions by collision-induced dissociation tandem mass spectrometry (Tables 1 and 2). Although several components have been isolated from B. lanceolatus venom, such as a hemorrhagic metalloproteinase,41 a fibrinogenolytic serine proteinase42 and a phospholipase A2,43 no venom protein sequences from B. caribbaeus and B. lanceolatus are available in the Swiss-Prot/TrEMBL database (UniProtKB/Swiss-Prot Release 54.6 of 04-Dec-2007; UniProtKB/TrEMBL Release 37.6 of 04-Dec-2007). This fact, along with the rapid amino acid sequence divergence of venom proteins evolving under accelerated evolution,44-49 may account for the
Table 3. Overview of the Relative Occurrence of Proteins (in Percentage of the Total HPLC-Separated Proteins) of the Different Families in the Venoms of B. caribbaeus and B. lanceolatus % of total venom proteins protein family
B. caribbaeus
B. lanceolatus
Long disintegrin DC-fragments CRISP PLA2 Serine proteinase L-amino acid oxidase C-type lectin-like Zn2+-metalloproteinase (- PI-SVMPs (- PIII-SVMPs
1.5
