Snake Venomics of the Lesser Antillean Pit Vipers Bothrops caribbaeus and Bothrops lanceolatus: Correlation with Toxicological Activities and Immunoreactivity of a Heterologous Antivenom

Abstract

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 reverse-phase 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 A 2, 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, edema-forming, phospholipase A 2 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, PLA 2s and serine proteinases in the venoms. A polyvalent (Crotalinae) antivenom produced in Costa Rica was able to immunodeplete approximately 80% of the proteins from both B. caribbaeus and B. lanceolatus venoms, and was effective in neutralizing the lethal, hemorrhagic, phospholipase A 2 and proteolytic activities of these venoms.

Venoms, venomics, antivenomics

“…Firstly, these messengers may exhibit transient, individual or a temporal expression patterns over the life time of the snake. Alternatively, mRNAs whose predicted polypeptides were not found to be secreted in venom may represent very low abundance toxins that play a hitherto unrecognised physiological function in the venom gland, or may simply represent a hidden repertoire of non-translated orphan molecules which may eventually become functional for the adaptation of snakes to Table 1 Overview of the relative occurrence of proteins (in percentage of the total HPLC-separated proteins) of the toxin families in the venoms of Sistrurus catenatus catenatus (SCC), Sistrurus catenatus tergeminus (SCT), and Sistrurus catenatus edwardsii (SCE) from USA [34]; Sistrurus miliarius barbouri (SMB) from USA [52]; the Tunisian snakes Cerastes cerastes cerastes (CCC), Cerastes vipera (CV) and Macrovipera lebetina transmediterranea (MLT) [48]; African Bitis arietans (BA) [50]; Bitis gabonica gabonica (BGG) [49]; Bitis gabonica rhinoceros (BGR), Bitis nasicornis (BN), and Bitis caudalis (BC) [56]; Echis ocellatus (EO) [51]; Lachesis muta (LM) [53]; Crotalus atrox (CA), and Agkistrodon contortrix contortrix (ACC) from USA (Calvete et al, unpublished); Armenian vipers Macrovipera lebetina obtusa (Mlo), and Vipera raddei (Vr) [54]; Atropoides picadoi (Api), and Atropoides mexicanus (Amex) [57] from Costa Rica; Bothrops asper (Bas) from the Caribbean (C) and the Pacific versants of Costa Rica [60]; Lesser Antillean pitvipers Bothrops caribbaeus (Bcar) (Santa Lucía), and Bothrops lanceolatus (Blan) (Martinique) [58]; Brazilian Bothrops fonsecai (Bfon), and Bothrops cotiara (Bco) [59]; Bothriechis lateralis (Bolat), and Bothriechis schlegelii (Bosch) [55] from Costa Rica; and Lachesis stenophrys (Lste) [53] changing ecological niches and prey habits. These data suggest that the final composition of venom is influenced by transcriptional and post-translational mechanisms that may be more complex than previously appreciated.…”

Antivenomic Assessment of the Immunological Reactivity of EchiTAb-Plus-ICP, an Antivenom for the Treatment of Snakebite Envenoming in Sub-Saharan Africa

“…We have coined the term "antivenomics" to describe our proteomic protocol for identifying venom proteins bearing epitopes reactive with an antivenom. 21,[28][29][30][31][32] Briefly, two milligrams 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 hr at 37°C. The IgG concentration was determined spectrophotometrically using an extinction coefficient (e) of 1.4 for a 1 mg/mL IgG concentration at 280 nm using a 1 cm light pathlength cuvette.…”

“…com ) against a private database containing 1,083 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. 21,[28][29][30]31,[36][37][38][39][40][41][42][43] MS/MS mass tolerance was set to ±0.6 Da. Carbamidomethyl cysteine and oxidation of methionine were fixed and variable modifications, respectively.…”

Snake Venomics, Antivenomics, and Venom Phenotyping: The Ménage à Trois of Proteomic Tools Aimed at Understanding the Biodiversity of Venoms

“…To date, we have explored a number of venom proteomes of medically relevant vipers (Table 4.2) from different regions of different continents (Fig. 4.3) (Alape-Girón et al, 2008;Angulo et al, 2008;Bazaa et al, 2005;Calvete et al, 2007bCalvete et al, , c, 2009bGutiérrez et al, 2008;Juárez et al, 2004Juárez et al, , 2006Lomonte et al, 2008;Núñez et al, 2009;Sanz et al, 2006Sanz et al, , 2008aTashima et al, 2008;Wagstaff et al, 2009). A few other laboratories have also reported qualitative proteomic studies on several venoms, including those from the European vipers, Vipera ammodytes ammodytes and Vipera ammodytes meridionalis (Georgieva et al, 2008), Vipera aspis (Ferquel et al, 2007), Asian Daboia russelli siamensis (Risch et al, 2009), Amazonian Bothrops atrox (Guércio et al, 2006), and Brazilian Bothrops jararaca (Fox et al, 2006) and Bothrops insularis (Valente et al, 2009).…”