RESUMO
The yellow-bellied sea snake, Pelamis platura, is the most broadly distributed snake species. Despite being endowed with a highly lethal venom, a proteomic analysis of its toxin composition was unavailable. The venoms of specimens collected in Golfo de Papagayo and Golfo Dulce (Costa Rica), where two distinctive color morphs occur, were chromatographically compared. The latter inhabits a fjord-like gulf where the transit of oceanic sea snakes into and from the basin is restricted, thus possibly affecting gene flow. RP-HPLC evidenced a conserved venom protein profile in both populations, despite their divergent color phenotypes. Following a trend observed in other sea snakes, P. platura venom is relatively simple, being composed of proteins of the three-finger toxin (3FTx), phospholipase A2 (PLA2), cysteine-rich secretory protein (CRISP), 5'-nucleotidase, and metalloproteinase families. The first three groups represent 49.9%, 32.9%, and 9.1% of total venom protein, respectively. The most abundant component (~26%) is pelamitoxin (P62388), a short-chain 3FTx, followed by a major basic PLA2 (~20%) and a group of three isoforms of CRISPs (~9%). Whereas isolated pelamitoxin was highly lethal to mice, neither the PLA2 nor the CRISP fraction caused death. However, the PLA2 rapidly increased plasma creatine kinase activity after intramuscular injection, indicating its myotoxic action. Differing from myotoxic PLA2s of viperids, this PLA2 was not cytolytic to murine myogenic cells in vitro, suggesting possible differences in its mechanism of action. The median lethal dose (LD50) estimates for P. platura crude venom in mice and in three species of fishes did not differ significantly. The sea snake antivenom manufactured by CSL Ltd. (Australia), which uses Enhydrina schistosa as immunogen, cross-recognized the three major components of P. platura venom and, accordingly, neutralized the lethal activity of crude venom and pelamitoxin, therefore being of potential usefulness in the treatment of envenomations by this species. BIOLOGICAL SIGNIFICANCE: Integrative analyses of animal venoms that combine the power of proteomics (venomics) with the characterization of their functional and immunological properties are significantly expanding knowledge on these remarkable bioweapons, both from a basic and a medical perspective. Costa Rica harbors a unique population of the yellow-bellied sea snake, Pelamis platura, that is restricted to a fjord-like gulf (Golfo Dulce). This population differs markedly from oceanic populations found elsewhere along the Pacific coast of this country, by presenting a patternless bright yellow coloration, instead of the typical bicolored or tricolored pattern of this species. It has been suggested that the dominance of this yellow-morph in Golfo Dulce might reflect gene flow restrictions, caused by the oceanographic conditions at this location. The present study demonstrates that the remarkable phenotypic variation between the two color morphs inhabiting Golfo Dulce and Golfo de Papagayo, respectively, is not associated with differences in the expression of venom components, as shown by their conserved RP-HPLC profiles. Proteomic analysis revealed the relatively simple toxin composition of P. platura venom, which contains three predominant types of proteins: three-finger toxins (protein abundance: 49.9%), phospholipases A2 (32.9%), and cysteine-rich secretory proteins (9.1%), together with few minor components. Further, the involvement of these most abundant proteins in the toxic effects of the venom, and their cross-recognition and neutralization by a sea snake antivenom produced against the venom of Enhydrina schistosa, were analyzed.
Assuntos
Antivenenos/farmacologia , Venenos Elapídicos/química , Elapidae/genética , Animais , Costa Rica , Venenos Elapídicos/toxicidade , Feminino , Peixes , Dose Letal Mediana , Masculino , Camundongos , Fosfolipases A/toxicidadeRESUMO
A polyspecific antivenom is used in Central America for the treatment of envenomings by viperid snakes. This antivenom is generated in horses hyperimmunized with a mixture of venoms from Bothrops asper, Crotalus simus and Lachesis stenophrys. The present study analyzed the ability of this antivenom to neutralize the venoms of three Central American viperid species of the 'Porthidium group', i.e. Porthidium nasutum, Porthidium ophryomegas and Cerrophidion sasai, formerly classified as Cerrophidion godmani. In addition, the immunorecognition of the components of these venoms was assessed by immunoaffinity antivenomics. The antivenom proved effective in neutralizing the lethal, hemorrhagic, myotoxic, phospholipase A(2) (PLA(2)) and proteinase activities of the three venoms, albeit exhibiting quantitative differences in the values of the Median Effective Doses (ED(50)). Excepting for certain low molecular mass bands corresponding to disintegrins, and some PLA(2)s and PI-metalloproteinases, Western blotting and immunoaffinity chromatography revealed immunorecognition of most Porthidium and Cerrophidion venom proteins. In agreement with in vivo neutralization assays, immobilized antivenom IgGs showed higher immunocapturing activity of toxins from both Porthidium taxa than from C. sasai. Overall our results demonstrate a significant paraspecific protection of the Costa Rican polyspecific antivenom against the three venoms sampled. They also stress the need to search for novel ways to enhance the immune response of horses against several weakly immunogenic venom components.
Assuntos
Antivenenos/uso terapêutico , Venenos de Crotalídeos/toxicidade , Viperidae/metabolismo , Animais , Antivenenos/análise , Antivenenos/imunologia , Cromatografia de Afinidade/métodos , Venenos de Crotalídeos/química , Avaliação Pré-Clínica de Medicamentos , Hemorragia/induzido quimicamente , Hemorragia/patologia , Hemorragia/prevenção & controle , Cavalos/imunologia , Injeções Intraperitoneais , Dose Letal Mediana , Longevidade/efeitos dos fármacos , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Doenças Musculares/induzido quimicamente , Doenças Musculares/patologia , Doenças Musculares/prevenção & controle , Testes de Neutralização , Inibidores de Fosfolipase A2 , Proteômica/métodos , Mordeduras de Serpentes/tratamento farmacológico , Mordeduras de Serpentes/imunologia , Viperidae/imunologiaRESUMO
The genus Bothriechis comprises a lineage of nine species of Neotropical pitvipers distributed mainly in highlands across Middle America, all adapted to arboreal habitats. Bothriechis supraciliaris is a relatively recently described species that inhabits the Pacific southwest of Costa Rica, whose venom had never been studied. A proteomic and toxicological profiling of its venom is here reported. Proteins or peptides that belong to eleven families were found, with a predominance of bradykinin-potentiating peptides (21.9%), followed by serine proteinases (15.2%) and phospholipases A(2) (13.4%). A group of short polyglycine peptides, resembling the poly-His/poly-Gly metalloproteinase inhibitors described in Atheris and Echis snake venoms, was observed for the first time in a Bothriechis venom. Comparison of the venom proteome of B. supraciliaris with those of Bothriechis schlegelii, Bothriechis lateralis, and Bothriechis nigroviridis, confirms the highly diverse toxicological strategies evolved by these arboreal snakes in each case, as possible alternative solutions to the same trophic purpose. Toxicological profiling of B. supraciliaris venom revealed a potent hemorrhagic action, moderate myotoxicity, and very weak procoagulant activity. Importantly from the medical perspective, the lethal activity of its venom (mouse intraperitoneal LD(50): 7.1 µg/g) was efficiently neutralized by a polyvalent (Viperidae) antivenom of therapeutic use in Central America.
Assuntos
Venenos de Crotalídeos/análise , Venenos de Crotalídeos/toxicidade , Proteômica , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Costa Rica , Feminino , Dose Letal Mediana , Masculino , Camundongos , Dados de Sequência Molecular , Filogenia , Viperidae/classificaçãoRESUMO
Within the Neotropical pit vipers, a lineage of primarily Middle American snake species referred to as the "Porthidium group" includes the genera Atropoides, Cerrophidion, and Porthidium. In this study, the venom proteomes of Porthidium nasutum, P. ophryomegas, and Cerrophidion godmani from Costa Rica were analyzed, and correlated to their toxic and enzymatic activities. Their HPLC profiles revealed a higher similarity between the two Porthidium species than between these and C. godmani. Proteins belonging to nine (P. nasutum), eight (P. ophryomegas), and nine (C. godmani) families were identified by mass spectrometry or N-terminal sequencing. Final cataloging of proteins and their relative abundances confirmed the close relationship between venoms of P. nasutum and P. ophryomegas, departing from that of C. godmani. Since the latter species had been taxonomically classified as Porthidium godmani previously, our venomic analyses agree with its current generic status. Venoms of P. nasutum and P. ophryomegas, despite containing abundant metalloproteinases and serine proteinases, lack procoagulant activity on human plasma, in contrast to venom of C. godmani. The latter induced strong myotoxicity in mice, which correlates with its high proportion of phospholipases A(2), whereas venoms from the two Porthidium species, containing lower amounts of these enzymes, induced only mild muscle damage.