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

Author(s)

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]

Affiliation

^[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

• Document type: Journal article
• Source: PLoS One; v. 13, n. 3 MAR 5 2018.
• Web of Science Citations: 1

Abstract

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)

• FAPESP's process: 14/11108-0 - Comparative study of biomolecules composition of Bothrops jararaca and Crotalus durissus terrificus venom born and maintained at the Butantan Institute and newly arrived from nature
• Grantee: Karen de Morais Zani
• Support Opportunities: Regular Research Grants
• FAPESP's process: 13/12826-0 - Identification of Bothrops jararaca snake plasma proteins responsible for its protection against self-envenomation
• Grantee: Caroline Serino Silva
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• FAPESP's process: 17/01890-0 - Comparative study of Crotalus durissus terrificus snake venoms born in captivity in the Laboratory of Herpetology of Butantan Institute and the national crotalic reference venom
• Grantee: Anita Mitico Tanaka-Azevedo
• Support Opportunities: Regular Research Grants
• FAPESP's process: 17/16908-2 - Proteomic characterization and enzymatic and pathophysiological activities of the venom of the snake species that compose the Bothrops neuwiedi group
• Grantee: Karen de Morais Zani
• Support Opportunities: Regular Research Grants
• FAPESP's process: 12/19321-9 - Proteomic and peptidomic characterization of Brazilian spider venoms by mass spectrometry
• Grantee: Alexandre Keiji Tashima
• Support Opportunities: Regular Research Grants
• FAPESP's process: 13/05357-4 - Comparative study of reference Bothrops venom in relation to the venom of Bothrops jararaca born in captivity in herpetology laboratory of the Instituto Butantan
• Grantee: Anita Mitico Tanaka-Azevedo
• Support Opportunities: Regular Research Grants