What’s in a mass

This short essay pretends to make the reader reflect on the concept of biological mass and on the added value that the determination of this molecular property of a protein brings to the interpretation of evolutionary and translational snake venomics research. Starting from the premise that the amino acid sequence is the most distinctive primary molecular characteristics of any protein, the thesis underlying the first part of this essay is that the isotopic distribution of a protein's molecular mass serves to unambiguously differentiate it from any other of an organism's proteome. In the second part of the essay, we discuss examples of collaborative projects among our laboratories, where mass profiling of snake venom PLA2 across conspecific populations played a key role revealing dispersal routes that determined the current phylogeographic pattern of the species.Ministerio de Ciencia, Innovación y Universidades/[BFU2017-89103-P]/MICIU/EspañaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Differential transcript profile of inhibitors with potential anti-venom role in the liver of juvenile and adult Bothrops jararaca snake

Background. Snakes belonging to the Bothrops genus are vastly distributed in Central and South America and are responsible for most cases of reported snake bites in Latin America. The clinical manifestations of the envenomation caused by this genus are due to three major activities-proteolytic, hemorrhagic and coagulant-mediated by metalloproteinases, serine proteinases, phospholipases A(2) and other toxic compounds present in snake venom. Interestingly, it was observed that snakes are resistant to the toxic effects of its own and other snake's venoms. This natural immunity may occur due the absence of toxin target or the presence of molecules in the snake plasma able to neutralize such toxins. Methods. In order to identify anti-venom molecules, we construct a cDNA library from the liver of B. jararaca snakes. Moreover, we analyzed the expression profile of four molecules-the already known anti-hemorrhagic factor Bj46a, one gamma-phospholipase A(2) inhibitor, one inter-alpha inhibitor and one C1 plasma protease inhibitor-in the liver of juvenile and adult snakes by qPCR. Results. The results revealed a 30-fold increase of gamma-phospholipase A(2) inhibitor and a minor increase of the inter-alpha inhibitor (5-fold) and of the C1 inhibitor (3-fold) in adults. However, the Bj46a factor seems to be equally transcribed in adults and juveniles. Discussion. The results suggest the up-regulation of different inhibitors observed in the adult snakes might be a physiological adaptation to the recurrent contact with their own and even other snake's venoms throughout its lifespan. This is the first comparative analysis of ontogenetic variation of expression profiles of plasmatic proteins with potential anti-venom activities of the venomous snake B. jararaca. Furthermore, the present data contributes to the understanding of the natural resistance described in these snakes.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Tecnologico (CNPq)INCT - Entomologia MolecularInst Butantan, Lab Herpetol, Sao Paulo, BrazilUniv Sao Paulo, Interunidades Biotecnol, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Bioquim, Sao Paulo, BrazilUniv Fed Rio de Janeiro, Dept Bioquim, Rio De Janeiro, BrazilInst Nacl Ciencia & Tecnol Entomol Mol, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Bioquim, Escola Paulista Med, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Bioquim, Sao Paulo, BrazilFAPESP: 2010/10266-5FAPESP: 2012/03657-8FAPESP: 2013/05357-4FAPESP: 2014/11108-0CNPq: 308780/2013-2Web of Scienc

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

Study of anti-inflammatory activity of Bothrops jararaca native antithrombin. Antithrombin cloning.

A antitrombina de B. jararaca foi isolada por meio de cromatografia de afinidade em coluna HiTrap Heparin HP (GE Healthcare). A análise da interação da antitrombina humana ou de B. jararaca com a heparina em sistema BIAcoreT200 demonstrou que a antitrombina de B. jararaca apresenta maior afinidade pela heparina que a antitrombina humana. Com relação à sua atividade anti-inflamatória, os resultados obtidos evidenciaram o efeito anti-inflamatório do pré e do pós-tratamento com a antitrombina de B. jararaca na resposta inflamatória aguda. A análise proteômica do exsudato inflamatório de camundongos identificou algumas proteínas possivelmente relacionadas ao mecanismo de inibição da antitrombina, como a enzima C3 do sistema complemento, a sorotransferrina, a a1-antitripsina, a apolipoproteína AI, o fibrinogênio, o cininogênio e a albumina. O processo de clonagem permitiu a obtenção da sequência completa da antitrombina de B. jararaca e apesar da longa distância evolutiva entre serpentes e humanos, diversas características da antitrombina encontram-se conservadas.B. jararaca antithrombin was isolated by affinity chromatography using HiTrap Heparin HP column (GE Healthcare). The interaction analysis of human or B. jararaca antithrombin with heparin using a BIAcoreT200 system (GE Healthcare) demonstrated that the affinity of B. jararaca antithrombin for heparin is higher than human antithrombin. Regarding the anti-inflammatory activity of B. jararaca antithrombin, the results showed the anti-inflammatory effect of pre- and post-treatment with this molecule in acute inflammation. The proteomic analysis of inflammatory exudate of mice identified some proteins possibly related to the mechanism of inhibition of antithrombin, such as C3 complement, serum transferrin, a1-antitrypsin, apolipoprotein AI, fibrinogen, albumin and kininogen. The molecular cloning process allowed the determination of the complete sequence of B. jararaca antithrombin and despite the evolutionary distance between snakes and human, a number of characteristics are preserved in antithrombin molecule

A novel trypsin Kazal-type inhibitor from Aedes aegypti with thrombin coagulant inhibitory activity

Kazal-type inhibitors play several important roles in invertebrates, such as anticoagulant, vasodilator and antimicrobial activities. Putative Kazal-type inhibitors were described in several insect transcriptomes. In this paper we characterized for the first time a Kazal unique domain trypsin inhibitor from the Aedes aegypti mosquito. Previously, analyses of sialotranscriptome of A. aegypti showed the potential presence of a Kazal-type serine protease inhibitor, in female salivary glands, carcass and also in whole male, which we named AaTI (A. aegypti trypsin inhibitor). AaTI sequence showed amino acid sequence similarity with insect thrombin inhibitors, serine protease inhibitor from Litopenaeus vannamei hemocytes and tryptase inhibitor from leech Hirudo medicinalis (LDTI). In this work we expressed, purified and characterized the recombinant AaTI (rAaTI). Molecular weight of purified rAaTI was 7 kDa rAaTI presented dissociation constant (K(i)) of 0.15 and 3.8 nM toward trypsin and plasmin, respectively, and it weakly inhibited thrombin amidolytic activity. The rAaTI was also able to prolong prothrombin time, activated partial thromboplastin time and thrombin time. AaTI transcription was confirmed in A. aegypti female salivary gland and gut 3 h and 24 h after blood feeding, suggesting that this molecule can act as anticoagulant during the feeding and digestive processes. Its transcription in larvae and pupae suggested that AaTI may also play other functions during the mosquito`s development. (C) 2010 Elsevier Masson SAS. All rights reserved.FAPESP Fundacao de Amparo a Pesquisa do Estado de Sao Paulo[05/03514-9]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CNPq Conselho Nacional de Desenvolvimento Cientifico e Tecnologico[470070/2004-8]Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CNPq Conselho Nacional de Desenvolvimento Cientifico e Tecnologico[575829/2008-7]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP[07/56614-6