Comparative proteomic profiling and functional characterization of venom pooled from captive Crotalus durissus terrificus specimens and the Brazilian crotalic reference venom

10 págs, 5 figuras. Información suplementaria en: https://doi.org/10.1016/j.toxicon.2020.07.001The South American rattlesnake Crotalus durissus spp has a wide geographic distribution in Brazil. Although responsible for only a low proportion of ophidian accidents, it is considered one of the most medically important species of venomous snakes due to the high mortality rate (1.87%). Snake venom is a complex phenotype commonly subjected to individual intraspecific, ontogenetic and geographic variability. Compositional differences in pooled venom used in the immunization process may impact the efficacy of the antivenom. In order to assure standardized high-quality antivenom, the potency of each Brazilian crotalic antivenom batch is determined against the 'Brazilian Crotalic Reference Venom' (BCRV). BCRV is produced by Instituto Butantan using venom obtained from the first milking of recently wild-caught C. d. terrificus specimens brought to the Institute. The decrease in the number of snake donations experienced in recent years can become a threat to the production of future batches of BCRV. To evaluate the feasibility of using venom from long-term captive animals in the formulation of BCRV, we have compared the proteomic, biochemical and biological profiles of C. d. terrificus venom pooled from captive specimens (CVP- captive venom pool) and BCRV. Electrophoretic and venomics analyses revealed a very similar venom composition profile, but also certain differences in toxins abundance, with some low abundant protein families found only in BCRV. Enzymatic (L-amino acid oxidase, phospholipase A2 and proteolytic) and biological (myotoxic and coagulant) activities showed higher values in CVP than in BCRV. CVP also possessed slightly higher lethal effect, although the Instituto Butantan crotalic antivenom showed equivalent potency neutralizing BCRV and CVP. Our results strongly suggest that venom from long-term captive C. d. terrificus might be a valid alternative to generate an immunization mixture of equivalent quality to the currently in use reference venom.This work was financially supported by Fundaç~ao de Amparo �a Pesquisa do Estado de S~ao Paulo – FAPESP (grant numbers: 2017/ 01890–0, 2018/25786–0; 2018/25899–0, 2017/26533–6 and 2017/ 16908–2), Conselho Nacional de Desenvolvimento Científico e Tec-nol�ogico (CNPq) (405399/2018–9) and partly funded by grant BFU2017-89103-P (Ministerio de Ciencia, Innovación y Universidades, Madrid, Spain).Peer reviewe

Purification and characterization of the first gamma-type phospholipase A2 inhibitor present in Bothrops jararaca snake serum.

As Fosfolipases A2 (PLA2) são enzimas que atuam desconstruindo membranas celulares, resultando em ácidos graxos e lisofosfolipidios, causando inflamação tecidual. Evidências indicam que serpentes possuem uma resistência natural devido a propriedades presentes no sangue, que inibem ações de proteínas presentes no veneno. Portanto, no presente trabalho foi isolado e caracterizado bioquimicamente e biologicamente o primeiro inibidor de PLA2 do tipo gama (γPLI) do soro da serpente B. jararaca, denominado PLI_BJ. O inibidor de PLA2 foi isolado utilizando dois passos cromatográficos. O PLI_BJ mostrou, por SDS-PAGE, uma massa molecular aparente de 25 000 e 20 000 em condições redutoras e não redutoras, respectivamente. A sequência de aminoácidos parcial de PLI_BJ foi determinada por espectrometria de massa e corresponde a 72% e 68% de cobertura da sequência de aminoácidos de duas proteínas já descritas como PLI. O PLI_BJ mostrou também atividade inibitória satisfatória nos três testes realizados sugerindo um papel deste inibidor nos efeitos de envenenamento da serpente.Phospholipases A2 (PLA2) are enzymes that act on cell membrane phospholipids resulting in fatty acids and lysophospholipids, deconstructing the cell wall causing tissue inflammation. Evidence indicates that snakes have natural resistance due to protective properties of blood that inhibits the action of proteins present in the venom. This study aimed to purify and characterize PLA2 inhibitors (PLI) from serum of the Bothrops jararaca snakes. PLA2 inhibitor was isolated using two chromatographic steps, and was named PLI_BJ. The purity of the PLI_BJ was confirmed by HPLC and SEC. The PLI_BJ showed, by SDS-PAGE, an molecular mass of 25,000 and 20,000 under reducing and non-reducing conditions, respectively. The partial amino acid sequence of PLI_BJ was determined by mass spectrometry and it corresponds to 72% and 68% of coverage of the amino acid sequence of two proteins already described as PLI. The PLI_BJ also showed satisfactory inhibitory activity in the three tests performed suggesting a role of this inhibitor in snake envenomation effects

Ontogenetic study of the composition of snake venom inhibitors and evaluation of the neutralizing activity of the γ-PLI present in the plasma of <i>Bothrops jararaca</i> snake.

Os inibidores naturais de venenos de serpentes têm sido estudados há mais de um século. Entre eles, estão as proteínas de plasma de alguns mamíferos e de serpentes não peçonhentas e peçonhentas, que possuem a capacidade de promover uma resistência ao veneno. Estas proteínas têm despertado o interesse biotecnológico, já que são uma ótima alternativa para o desenvolvimento de novos fármacos. As principais proteínas encontradas no plasma desses animais, com capacidade de neutralização do veneno, são os inibidores de metaloproteinases e os inibidores de PLA2 (PLIs). A PLA2 atua no efeito local do envenenamento, causando inflamação do tecido na área afetada. Nesse contexto, recentemente foi isolado o γBjPLI, que é um PLI presente no sangue da serpente peçonhenta <i>Bothrops jararaca</i>. Esta proteína pertence à classe gama (γ), com base em domínios estruturais característicos. O γBjPLI possui aproximadamente 22 kDa e inibe as atividades enzimáticas, edematogênicas e mionecróticas das PLA2, sugerindo um papel desse inibidor na proteção dessas serpentes contra o autoenvenenamento. Baseado nessas informações, neste trabalho, foi estudado de forma comparativa a composição proteica do plasma de neonatos, jovens e adultos, fêmeas e machos de <i>B jararaca</i>, por análises de SDS-PAGE, <i>Western blotting</i>, cromatografia de afinidade, ELISA e espectrometria de massa. Tais análises permitiram identificar proteínas do tipo PLI (αPLI, βPLI e γPLI) em todas as fases da vida, sendo que αPLI e βPLI foram identificados pela primeira vez no plasma da serpente <i>B. jararaca</i>. Também foi realizada a avaliação da interação e neutralização da atividade anticoagulante da crotoxina por tromboelastometria, em que o γBjPLI foi capaz de inibir até 43% da atividade dessa toxina. Ademais, mostrou uma maior afinidade pela PLA2 Asp-49 em comparação à PLA2 Lys-49, tanto por apresentar uma alteração em espectros obtidos por dicroísmo circular, quanto pela inibição das atividades miotóxicas e edematogênicas, com valores de 19,6% e 38,5% para a PLA2 Asp-49, superiores aos 16% e 19,6% para a PLA2 Lys-49, respectivamente. Além disso, para estabelecer novos métodos de obtenção destes inibidores com maiores rendimentos mantendo a estabilidade e atividade, o γBjPLI foi clonado e expresso utilizando o vetor pET28a e a cepa de expressão <i>SHuffle</i> da espécie de bactéria <i>Escherichia coli.</i>.Snake venom inhibitors have been studied for over a century. Among them are the plasma proteins of some mammals and of non-venomous and venomous snakes, which have the ability to promote resistance to the venom. These proteins have aroused biotechnological interest, as they are an excellent alternative for the development of new drugs. The main proteins found in animal plasma, capable of neutralizing venom, are metalloproteinase inhibitors and PLA2 inhibitors (PLIs). The PLA2 acts in the local effect of envenomation, causing tissue inflammation in the affected area. Based on this information, BjPLI was recently isolated, which is a PLI present in the blood of the venomous snake <i>Bothrops jararaca</i>. γBjPLI belongs to the gamma (γ) class, based on its characteristic domains. γBjPLI has approximately 22 kDa and inhibits the enzymatic, edematogenic and myonecrotic activities of PLA2, suggesting a role of this inhibitor in the protection of these snakes against self-envenomation. Knowing that, in this work it is presented a comparative study of the protein composition of the plasma of newborns, young and adults, females and males of <i>B. jararaca</i>, by analysis of SDS-PAGE, Western blotting, affinity chromatography, ELISA and mass spectrometry. Such analysis allowed to identify PLI-like proteins (αPLI, βPLI and γPLI) at all stages of life, with αPLI and βPLI were being found for the first time in the <i>B. jararaca</i> snake plasma. An evaluation of the interaction and neutralization of crotoxin anticoagulant activity by thromboelastometry was also performed, and γBjPLI was able to inhibit up to 43% of the activity. In addition, it showed a greater affinity for PLA2 Asp-49 compared to PLA2 Lys- 49, evidenced by an alteration in spectra obtained by circular dichroism, and by the inhibition of myotoxic and edematogenic activities, with values of 19.6% and 38. 5% for PLA2 Asp-49, higher than 16% and 19.6% for PLA2 Lys-49, respectively. Furthermore, to establish new methods for obtaining these inhibitors with greater yield, stability, and activity, γBjPLI was cloned and expressed using the pET28a vector and a SHuffle expression strain of the bacterium species <i>Escherichia coli.</i&gt

Purification and characterization of the first γ-phospholipase inhibitor (γPLI) from Bothrops jararaca snake serum.

Phospholipases A2 (PLA2) are enzymes acting on the cell membrane phospholipids resulting in fatty acids and lysophospholipids and deconstructing the cell membrane. This protein is commonly found in snake venoms, causing tissue inflammation in the affected area. Evidence indicates that snakes have natural resistance to their own venom due to protective properties in plasma, that inhibit the action of proteins present in their venom. Given that, this study aimed to purify and characterize a γPLI from Bothrops jararaca serum, named γBjPLI. PLA2 inhibitor was isolated using two chromatographic steps: an ion exchange column (DEAE), followed by an affinity column (crotoxin coupled to a CNBr-activated Sepharose resin). The purity and biochemical characterization of the isolated protein were analyzed by RP-HPLC, SEC, SDS-PAGE, circular dichroism and mass spectrometry. The ability to inhibit PLA2 was determined by enzymatic activity, neutralization of paw edema and myonecrosis. The protein purity was confirmed by RP-HPLC and SEC, whilst an apparent molecular mass of 25 kDa and 20 kDa was obtained by SDS-PAGE, under reducing and non-reducing conditions, respectively. According to mass spectrometry analysis, this protein showed 72% and 68% of coverage when aligned to amino acid sequences of two proteins already described as PLIs. Thus, the inhibitory activity of enzymatic, edema and myonecrotic activities by γBjPLI suggests a role of this inhibitor for protection of these snakes against self-envenomation

Comparative compositional and functional analyses of Bothrops moojeni specimens reveal several individual variations.

Snake venoms are complex protein mixtures with different biological activities that can act in both their preys and human victims. Many of these proteins play a role in prey capture and in the digestive process of these animals. It is known that some snakes are resistant to the toxicity of their own venom by mechanisms not yet fully elucidated. However, it was observed in the Laboratory of Herpetology of Instituto Butantan that some Bothrops moojeni individuals injured by the same snake species showed mortalities caused by envenoming effects. This study analyzed the biochemical composition of 13 venom and plasma samples from Bothrops moojeni specimens to assess differences in their protein composition. Application of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed distinct venom protein profiles, but very homogeneous plasma profiles. Western Blotting (WB) was performed with plasma samples, which were submitted to incubation with the respective venom. Some individuals showed an immunorecognized band zone around 25 kDa, indicating interaction between the same individual plasma and venom proteins. Crossed-WB assay using non-self-plasma and venom showed that this variability is due to venom protein composition instead of plasma composition. These venoms presented higher caseinolytic, collagenolytic and coagulant activities than the venoms without these regions recognized by WB. Mass spectrometry analyses performed on two individuals revealed that these individuals present, in addition to higher protein concentrations, other exclusive proteins in their composition. When these same two samples were tested in vivo, the results also showed higher lethality in these venoms, but lower hemorrhagic activity than in the venoms without these regions recognized by WB. In conclusion, some Bothrops moojeni specimens differ in venom composition, which may have implications in envenomation. Moreover, the high individual venom variability found in this species demonstrates the importance to work with individual analyses in studies involving intraspecific venom variability and venom evolution

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

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 βPLI 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