Bothrops jararacussu

A theoretical molecular model of this domain was built through folding recognition (threading) techniques and refined by molecular dynamics simulation. Then, the final BjussuMP-I catalytic
domain model was compared to other SVMPs and Reprolysin family proteins in order to identify eventual structural differences, which could help to understand the biochemical activities of these enzymes. The presence of large hydrophobic areas and some conserved surface charge-positive
residues were identified as important features of the SVMPs and other metalloproteases.

Marchi-Salvador DP, Fernandes CA, Amui SF, Soares AM, Fontes MR

For the first time, a non-catalytic and myotoxic Lys49-PLA2 (BthTX-I from Bothrops jararacussu venom) has been crystallized with BPB inhibitor. X-ray diffraction data were collected and electron-density calculations showed that the ligand is bound to the His48 residue. BthTX-I with His48 chemically modified by BPB shows strongly reduced myotoxic and cytotoxic activities. This suggests a biological correlation between the modification of His48, which is associated with catalytic activity of PLA2s, and other toxicological activities of Lys49-PLA2s.

Roberto PG, Kashima S, Marcussi S, Pereira JO, Astolfi-Filho S, Nomizo A, Giglio JR, Fontes MR, Soares AM, Franca SC.

Cloning and identification of a complete cDNA coding for a bactericidal and antitumoral acidic phospholipase A2 from Bothrops jararacussu venom.

In order to better understand the function of acidic phospholipases A2 (PLA2s) from snake venoms, expressed sequence tags (ESTs) that code for acidic PLA2s were isolated from a cDNA library prepared from the poly(A)+ RNA of venomous glands of Bothrops jararacussu. The complete nucleotide sequence (366 bp), named BOJU-III, encodes the BthA-I-PLA2 precursor, which includes a signal peptide and the mature protein with 16 and 122 amino acid residues, respectively. Multiple comparison of both the nucleotide and respective deduced amino acid sequence with EST and protein sequences from databases revealed that the full-length cDNA identified (BOJU III--AY145836) is related to an acidic PLA2 sharing similarity, within the range 55-81%, with acidic phospholipases from snake venoms. Moreover, phylogenetic analysis of amino acid sequences of acidic PLA2s from several pit viper genera showed close evolutionary relationships among acidic PLA2s from Bothrops, Crotalus, and Trimeresurus. The molecular modeling showed structural similarity with other dimeric class II PLA2s from snake venoms. The native protein BthA-I-PLA2, a nontoxic acidic PLA2 directly isolated from Bothrops jararacussu snake venom, was purified and submitted to various bioassays. BthA-I-PLA2 displayed high catalytic activity and induced Ca2+-dependent liposome disruption. Edema induced by this PLA2 was inhibited by indomethacin and dexamethasone, thus suggesting involvement of the cyclo-oxygenase pathway. BthA-I-PLA2 showed anticoagulant activity upon human plasma and inhibited phospholipid-dependent platelet aggregation induced by collagen or ADP. In addition, it displayed bactericidal activity against Escherichia coli and Staphylococcus aureus and antitumoral effect upon breast adrenocarcinoma as well as upon human leukemia T and Erlich ascitic tumor. Following chemical modification with p-bromophenacyl bromide, total loss of the enzymatic and pharmacological activities were observed. This is the first report on the isolation and identification of a cDNA encoding a complete acidic PLA2 from Bothrops venom, exhibiting bactericidal and antitumoral effects.

Sant`Ana, CD^(1,3); Curtarelli, MB⁽¹⁾; Pereira, ALA⁽¹⁾; Ticli, FK⁽³⁾; Mazzi, MV⁽³⁾; Marcussi, S^(1,2); Magalhães, MR⁽⁴⁾; Giglio, JR⁽²⁾; Sampaio, SV⁽³⁾; Pereira, PS⁽¹⁾; Marins, MA⁽¹⁾; Soares, AM⁽³⁾

Inibição da atividade nucleotídica de venenos de serpentes por extratos de plantas

Venenos de serpentes são compostos principalmente por enzimas das classes PLA2s, proteases, LAAOs, entre outras. Essas proteínas têm sido intensamente estudadas e utilizadas como ferramentas em laboratórios para fins diagnósticos, sendo até mesmo empregadas para o entendimento de patologias e com aplicação na clínica médica. Neste trabalho foi avaliada a inibição da atividade nucleotídica de venenos de serpentes dos gêneros Bothrops e Crotalus, por extratos aquosos de diferentes plantas (Pentacletra macroloba, Mikania glomerata, Casearia sylvestris e Mandevilla velutina). As partes das plantas utilizadas para a elaboração dos extratos foram escolhidas baseando-se em conhecimentos populares de seu uso anti-ofídico, sendo utilizados o caule de P. macroloba, as raízes de M. glomerata, folhas de C. sylvestris e xilopódio de M. velutina. As amostras de venenos e extratos foram previamente incubadas em volume final de 5µL durante 60min a 37ºC (n=3) e posteriormente avaliadas através da técnica de enzimodifusão, sendo utilizado um gel composto por agarose 2%, tampão Tris-HCl 0,05M com MgSO4 0,01M pH 7.4 acrescido de DNA de esperma de salmão como substrato e brometo de etídio para possibilitar a visualização da atividade em UV. Os extratos de P. macroloba e M. glomerata foram eficientes em inibir 100% da atividade nucleotídica induzida pelos venenos de Bothrops jararacussu (50µg), B. neuwiedi (50 e 100µg), C. d. terríficus (50 e 100µg) e C. d. collineatus (50µg), nas proporções de 1:15 e 1:30 (m/m). Os extratos de C. sylvestris e M. velutina inibiram 100% da atividade nucleotídica do veneno de B. jararacussu apenas nas primeiras 6 horas, mostrando o mesmo efeito para C. d. collineatus em 24 horas de experimento, na proporção de 1:12 (m/m). Os resultados apresentados geram dados para a identificação de novos inibidores naturais dos efeitos tóxicos do envenenamento ofídico, além de servirem como uma ferramenta na compreensão do mecanismo de ação das nucleotidases presentes em venenos de serpentes.