Characterization of a novel acidic phospholipase A 2 isolated from the venom of Bothrops mattogrossensis: From purification to structural modeling.

Autor: de Melo Cordeiro Eulálio M; Laboratory of Cellular Immunology Applied to Health, Oswaldo Cruz Foundation, FIOCRUZ Rondônia, Porto Velho, RO, Brazil; Laboratory of Protein Biotechnology and Bioactive Compounds (LABIOPROT), Oswaldo Cruz Foundation, FIOCRUZ Rondônia, Porto Velho, RO, Brazil., de Lima AM; Laboratory of Protein Biotechnology and Bioactive Compounds (LABIOPROT), Oswaldo Cruz Foundation, FIOCRUZ Rondônia, Porto Velho, RO, Brazil., Brant RSC; Carlos Chagas Institute (ICC), Mass Spectrometry Facility of FIOCRUZ Paraná, Curitiba, PR, Brazil., Francisco AF; Laboratory of Protein Biotechnology and Bioactive Compounds (LABIOPROT), Oswaldo Cruz Foundation, FIOCRUZ Rondônia, Porto Velho, RO, Brazil., Santana HM; Laboratory of Cellular Immunology Applied to Health, Oswaldo Cruz Foundation, FIOCRUZ Rondônia, Porto Velho, RO, Brazil., Paloschi MV; Laboratory of Cellular Immunology Applied to Health, Oswaldo Cruz Foundation, FIOCRUZ Rondônia, Porto Velho, RO, Brazil., da Silva Setúbal S; Laboratory of Cellular Immunology Applied to Health, Oswaldo Cruz Foundation, FIOCRUZ Rondônia, Porto Velho, RO, Brazil., da Silva CP; Laboratory of Cellular Immunology Applied to Health, Oswaldo Cruz Foundation, FIOCRUZ Rondônia, Porto Velho, RO, Brazil., Silva MDS; Laboratory of Cellular Immunology Applied to Health, Oswaldo Cruz Foundation, FIOCRUZ Rondônia, Porto Velho, RO, Brazil., Boeno CN; Laboratory of Cellular Immunology Applied to Health, Oswaldo Cruz Foundation, FIOCRUZ Rondônia, Porto Velho, RO, Brazil., Kayano AM; Laboratory of Protein Biotechnology and Bioactive Compounds (LABIOPROT), Oswaldo Cruz Foundation, FIOCRUZ Rondônia, Porto Velho, RO, Brazil; Center for Research in Tropical Medicine (CEPEM/SESAU-RO), Porto Velho, RO, Brazil., Rita PHS; Bioterium of Dom Bosco Catholic University (UCDB), Campo Grande, MS, Brazil., de Azevedo Calderon L; Center for the Study of Biomolecules Applied to Health (CEBio), Oswaldo Cruz Foundation, Fiocruz Rondônia, Porto Velho, RO, Brazil., Soares AM; Laboratory of Protein Biotechnology and Bioactive Compounds (LABIOPROT), Oswaldo Cruz Foundation, FIOCRUZ Rondônia, Porto Velho, RO, Brazil; National Institute of Science and Technology of Epidemiology of Western Amazon, INCT-EpiAmO, Brazil., Salvador DPM; Toxic Protein Crystallography Laboratory (CPrTox-Lab), Federal University of Paraíba (UFPB), João Pessoa, PB, Brazil., Zuliani JP; Laboratory of Cellular Immunology Applied to Health, Oswaldo Cruz Foundation, FIOCRUZ Rondônia, Porto Velho, RO, Brazil; Department of Medicine, Federal University of Rondonia (UNIR), Porto Velho, RO, Brazil. Electronic address: juliana.zuliani@fiocruz.br.
Jazyk: angličtina
Zdroj: International journal of biological macromolecules [Int J Biol Macromol] 2024 Dec 26; Vol. 292, pp. 139217. Date of Electronic Publication: 2024 Dec 26.
DOI: 10.1016/j.ijbiomac.2024.139217
Abstrakt: Phospholipases A 2 (PLA 2 s) are highly prevalent in Bothrops snake venom and play a crucial role in inflammatory responses and immune cell activation during envenomation. Despite their significance, the specific role of PLA 2 s from Bothrops mattogrossensis venom (BmV) in inflammation is not fully understood. This study sought to isolate and characterize a novel acidic PLA 2 from BmV, designated BmPLA 2 -A, and to evaluate its effects on human umbilical vein endothelial cells (HUVECs), with a specific focus on cytotoxicity, adhesion, and detachment. BmPLA 2 -A was isolated through a multi-step chromatographic procedure, involving cation exchange (CM-Sepharose), hydrophobic interaction (n-butyl-Sepharose-HP), and reversed-phase (C-18) chromatography. SDS-PAGE analysis revealed a single protein band of approximately 15 kDa. The primary structure of BmPLA 2 -A was determined by LC-MS/MS, while its tertiary structure was modeled using AlphaFold. Enzymatic activity was verified with the synthetic substrate 4N3OBA. Molecular dynamics simulations were conducted to further investigate the catalytic mechanism of BmPLA 2 -A at the molecular level. In vitro assays on HUVECs revealed that BmPLA 2 -A neither induce cytokine release (IL-6, IL-8, IL-1β, TNF) nor affected cell viability, adhesion, or detachment. The characteristics of BmPLA 2 -A are consistent with those of acidic Asp-49 PLA 2 enzymes, highlighting its potential involvement in the cytotoxic and inflammatory effects of the venom.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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