Purification and characterization of the first gamma-phospholipase inhibitor (gamma PLI) from Bothrops jararaca snake serum

Autor(es)

Serino-Silva, Caroline ^{[1, 2]} ; Morais-Zani, Karen ^[1] ; Toyama, Marcos Hikari ^[3] ; Toyama, Daniela de Oliveira ^[3] ; Gaeta, Henrique Hesse ^[3] ; Bittencourt Rodrigues, Caroline Fabri ^{[1, 2]} ; Aguiar, Weslei da Silva ^[2] ; Tashima, Alexandre Keiji ^[4] ; Grego, Kathleen Fernandes ^[1] ; Tanaka-Azevedo, Anita Mitico ^[1]

Afiliação do(s) autor(es)

^[1] Inst Butantan, Lab Herpetol, Sao Paulo, SP - Brazil

^[2] Univ Sao Paulo, Inst Pesquisas Tecnol, Inst Butantan, Interunidades Biotecnol, Sao Paulo, SP - Brazil

^[3] Univ Estadual Paulista, Inst Biociencias Litoral Paulista, Sao Vicente, SP - Brazil

^[4] Univ Fed Sao Paulo, Dept Bioquim, Sao Paulo, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: PLoS One; v. 13, n. 3 MAR 5 2018.
• Citações Web of Science: 1

Resumo

Phospholipases A(2) (PLA(2)) 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 gamma PLI from Bothrops jararaca serum, named gamma BjPLI. PLA(2) 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 PLA(2) 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 gamma BjPLI suggests a role of this inhibitor for protection of these snakes against self-envenomation. (AU)

• Processo FAPESP: 13/05357-4 - Estudo comparativo do veneno botrópico de referência em relação ao veneno das serpentes Bothrops jararaca nascidas em cativeiro no Laboratório de Herpetologia do Instituto Butantan
• Beneficiário: Anita Mitico Tanaka-Azevedo
• Linha de fomento: Auxílio à Pesquisa - Regular
• Processo FAPESP: 17/16908-2 - Caracterização proteômica e das atividades enzimáticas e fisiopatológicas do veneno das espécies de serpentes que compõem o grupo Bothrops neuwiedi
• Beneficiário: Karen de Morais Zani
• Linha de fomento: Auxílio à Pesquisa - Regular
• Processo FAPESP: 13/12826-0 - Identificação das proteínas responsáveis pela proteção contra o auto-envenenamento no plasma da serpente Bothrops jararaca
• Beneficiário: Caroline Serino Silva
• Linha de fomento: Bolsas no Brasil - Iniciação Científica
• Processo FAPESP: 17/01890-0 - Estudo comparativo da composição do veneno das serpentes Crotalus durissus terrificus nascidas em cativeiro no Laboratório de Herpetologia do Instituto Butantan e do veneno crotálico de referência nacional
• Beneficiário: Anita Mitico Tanaka-Azevedo
• Linha de fomento: Auxílio à Pesquisa - Regular
• Processo FAPESP: 14/11108-0 - Estudo comparativo da composição das biomoléculas dos venenos das serpentes Bothrops jararaca e Crotalus durissus terrificus nascidas e mantidas no Instituto Butantan e recém-chegadas da natureza
• Beneficiário: Karen de Morais Zani
• Linha de fomento: Auxílio à Pesquisa - Regular
• Processo FAPESP: 12/19321-9 - Caracterização proteômica e peptidômica dos venenos de aranhas brasileiras por espectrometria de massas
• Beneficiário: Alexandre Keiji Tashima
• Linha de fomento: Auxílio à Pesquisa - Regular