Proteomics and life-history variability of Endogenous Phospholipases A2 Inhibitors (PLIs) in Bothrops jararaca plasma.

In Brazil, the genus Bothrops is responsible for most ophidian accidents. Snake venoms have a wide variety of proteins and peptides exhibiting a broad repertoire of pharmacological and toxic effects that elicit systemic injury and characteristic local effects. The snakes' natural resistance to envenomation caused by the presence of inhibitory compounds on their plasma have been extensively studied. However, the presence of these inhibitors in different developmental stages is yet to be further discussed. The aim of this study was to evaluate the ontogeny of Bothrops jararaca plasma inhibitor composition and, to this end, plasma samples of B. jararaca were obtained from different developmental stages (neonates, youngs, and adults) and sexes (female and male). SDS-PAGE, Western blotting, affinity chromatography, and mass spectrometry were performed to analyze the protein profile and interaction between B. jararaca plasma and venom proteins. In addition, the presence of γBjPLI, a PLA2 inhibitor previously identified and characterized in B. jararaca serum, was confirmed by Western blotting. According to our results, 9-17% of plasma proteins were capable of binding to venom proteins in the three developmental stages. The presence of different endogenous inhibitors and, more specifically, different PLA2 inhibitor (PLI) classes and antihemorrhagic factors were confirmed in specimens of B. jararaca from newborn by mass spectrometry. For the first time, the αPLI and ßPLI were detected in B. jararaca plasma, although low or no ontogenetic and sexual correlation were found. The γPLI were more abundant in adult female, than in neonate and young female, but similar to neonate, young and adult male according to the results of mass spectrometry analysis. Our results suggest that there are proteins in the plasma of these animals that can help counteract the effects of self-envenomation from birth.

A comparative study of endogenous phospholipase A2 inhibitors in the serum of Brazilian pit vipers.

This work compared the presence of phospholipase A2 inhibitors (PLIs) in the serum of 19 snake species maintained at Instituto Butantan to better understand the mechanisms of venom resistance in snakes and improve the treatment of snakebite. PLI was isolated from blood of 19 snake species by one-step chromatography and identified in all samples, besides its identity was confirmed through the interaction with both phospholipase A2 and anti-γPLI. These findings highlight the diversity of snake serum PLIs and emphasize the importance of structure-function studies.

Venom complexity of Bothrops atrox (common lancehead) siblings.

BACKGROUND: Variability in snake venoms is a well-studied phenomenon. However, sex-based variation of Bothrops atrox snake venom using siblings is poorly investigated. Bothrops atrox is responsible for the majority of snakebite accidents in the Brazilian Amazon region. Differences in the venom composition of Bothrops genus have been linked to several factors such as ontogeny, geographical distribution, prey preferences and sex. Thus, in the current study, venom samples of Bothrops atrox male and female siblings were analyzed in order to compare their biochemical and biological characteristics. METHODS: Venoms were collected from five females and four males born from a snake captured from the wild in São Bento (Maranhão, Brazil), and kept in the Laboratory of Herpetology of Butantan Intitute. The venoms were analyzed individually and as a pool of each gender. The assays consisted in protein quantification, 1-DE, mass spectrometry, proteolytic, phospholipase A2, L-amino acid oxidase activities, minimum coagulant dose upon plasma, minimum hemorrhagic dose and lethal dose 50%. RESULTS: Electrophoretic profiles of male's and female's venom pools were quite similar, with minor sex-based variation. Male venom showed higher LAAO, PLA2 and hemorrhagic activities, while female venom showed higher coagulant activity. On the other hand, the proteolytic activities did not show statistical differences between pools, although some individual variations were observed. Meanwhile, proteomic profile revealed 112 different protein compounds; of which 105 were common proteins of female's and male's venom pools and seven were unique to females. Despite individual variations, lethality of both pools showed similar values. CONCLUSION: Although differences between female and male venoms were observed, our results show that individual variations are significant even between siblings, highlighting that biological activities of venoms and its composition are influenced by other factors beyond gender.

BoaγPLI: Structural and functional characterization of the gamma phospholipase A2 plasma inhibitor from the non-venomous Brazilian snake Boa constrictor.

Plasma in several organisms has components that promote resistance to envenomation by inhibiting specific proteins from snake venoms, such as phospholipases A2 (PLA2s). The major hypothesis for inhibitor's presence would be the protection against self-envenomation in venomous snakes, but the occurrence of inhibitors in non-venomous snakes and other animals has opened new perspectives for this molecule. Thus, this study showed for the first time the structural and functional characterization of the PLA2 inhibitor from the Boa constrictor serum (BoaγPLI), a non-venomous snake that dwells extensively the Brazilian territory. Therefore, the inhibitor was isolated from B. constrictor serum, with 0.63% of recovery. SDS-PAGE showed a band at ~25 kDa under reducing conditions and ~20 kDa under non-reducing conditions. Chromatographic analyses showed the presence of oligomers formed by BoaγPLI. Primary structure of BoaγPLI suggested an estimated molecular mass of 22 kDa. When BoaγPLI was incubated with Asp-49 and Lys-49 PLA2 there was no severe change in its dichroism spectrum, suggesting a non-covalent interaction. The enzymatic assay showed a dose-dependent inhibition, up to 48.2%, when BoaγPLI was incubated with Asp-49 PLA2, since Lys-49 PLA2 has a lack of enzymatic activity. The edematogenic and myotoxic effects of PLA2s were also inhibited by BoaγPLI. In summary, the present work provides new insights into inhibitors from non-venomous snakes, which possess PLIs in their plasma, although the contact with venom is unlikely.

Venom complexity of Bothrops atrox (common lancehead) siblings

Variability in snake venoms is a well-studied phenomenon. However, sex-based variation of Bothrops atrox snake venom using siblings is poorly investigated. Bothrops atrox is responsible for the majority of snakebite accidents in the Brazilian Amazon region. Differences in the venom composition of Bothrops genus have been linked to several factors such as ontogeny, geographical distribution, prey preferences and sex. Thus, in the current study, venom samples of Bothrops atrox male and female siblings were analyzed in order to compare their biochemical and biological characteristics. Methods: Venoms were collected from five females and four males born from a snake captured from the wild in São Bento (Maranhão, Brazil), and kept in the Laboratory of Herpetology of Butantan Intitute. The venoms were analyzed individually and as a pool of each gender. The assays consisted in protein quantification, 1-DE, mass spectrometry, proteolytic, phospholipase A2, L-amino acid oxidase activities, minimum coagulant dose upon plasma, minimum hemorrhagic dose and lethal dose 50%. Results: Electrophoretic profiles of male's and female's venom pools were quite similar, with minor sex-based variation. Male venom showed higher LAAO, PLA2 and hemorrhagic activities, while female venom showed higher coagulant activity. On the other hand, the proteolytic activities did not show statistical differences between pools, although some individual variations were observed. Meanwhile, proteomic profile revealed 112 different protein compounds; of which 105 were common proteins of female's and male's venom pools and seven were unique to females. Despite individual variations, lethality of both pools showed similar values. Conclusion: Although differences between female and male venoms were observed, our results show that individual variations are significant even between siblings, highlighting that biological activities of venoms and its composition are influenced by other factors beyond gender.(AU)