title: Isolation, structural and functional characterization of a new Lys49 phospholipase A2 homologue from Bothrops neuwiedi urutu with bactericidal potential.
abstract: Snake venom is a complex mixture of active compounds consisting of 80-90% proteins and peptides that exhibit a variety of biological actions that are not completely clarified or identified. Of these, phospholipase A2 is one of the molecules that has shown great biotechnological potential. The objectives of this study were to isolate, biochemically and biologically characterize a Lys49 phospholipase A2 homologue from the venom of Bothrops neuwiedi urutu. The protein was purified after two chromatographic steps, anion exchange and reverse phase. The purity and relative molecular mass were assessed by SDS-PAGE, observing a molecular weight typical of PLA2s, subsequently confirmed by mass spectrometry obtaining a mass of 13,733 Da. As for phospholipase activity, the PLA2 proved to be enzymatically inactive. The analyses by Edman degradation and sequencing of the peptide fragments allowed for the identification of 108 amino acid residues; this sequence showed high identity with other phospholipases A2 from Bothrops snake venoms, and identified this molecule as a novel PLA2 isoform from B. neuwiedi urutu venom, called BnuTX-I. In murine models, both BnuTX-I as well as the venom induced edema and myotoxic responses. The cytotoxic effect of BnuTX-I in murine macrophages was observed at concentrations above 12 μg/mL. BnuTX-I also presented antimicrobial activity against gram-positive and negative bacterial strains, having the greatest inhibitory effect on Pseudomonas aeruginosa. The results allowed for the identification of a new myotoxin isoform with PLA2 structure with promising biotechnological applications.
authors.affiliation: Centro de Estudos de Biomoléculas Aplicadas à Saúde, CEBio, Fundação Oswaldo Cruz, FIOCRUZ, Fiocruz Rondônia, and Departamento de Medicina, Universidade Federal de Rondônia, UNIR, Porto Velho-RO, Brazil; Instituto Federal de Educação, Ciência e Tecnologia de Rondônia, IFRO, Porto Velho-RO, Brazil; Programa de Pós-Graduação em Biodiversidade e Biotecnologia da Rede BIONORTE, Brazil.
authors.forename: Edailson A
authors.initials: EA
authors.lastname: Corrêa
authors.affiliation: Centro de Estudos de Biomoléculas Aplicadas à Saúde, CEBio, Fundação Oswaldo Cruz, FIOCRUZ, Fiocruz Rondônia, and Departamento de Medicina, Universidade Federal de Rondônia, UNIR, Porto Velho-RO, Brazil.
authors.forename: Anderson M
authors.initials: AM
authors.lastname: Kayano
authors.affiliation: Centro de Estudos de Biomoléculas Aplicadas à Saúde, CEBio, Fundação Oswaldo Cruz, FIOCRUZ, Fiocruz Rondônia, and Departamento de Medicina, Universidade Federal de Rondônia, UNIR, Porto Velho-RO, Brazil.
authors.forename: Rafaela
authors.initials: R
authors.lastname: Diniz-Sousa
authors.affiliation: Laboratório de Imunologia Celular Aplicada à Saúde, Fundação Oswaldo Cruz, FIOCRUZ, Fiocruz Rondônia, Porto Velho-RO, Brazil.
authors.forename: Sulamita S
authors.initials: SS
authors.lastname: Setúbal
authors.affiliation: Centro de Estudos de Biomoléculas Aplicadas à Saúde, CEBio, Fundação Oswaldo Cruz, FIOCRUZ, Fiocruz Rondônia, and Departamento de Medicina, Universidade Federal de Rondônia, UNIR, Porto Velho-RO, Brazil; Programa de Pós-Graduação em Biodiversidade e Biotecnologia da Rede BIONORTE, Brazil.
authors.forename: Fernando B
authors.initials: FB
authors.lastname: Zanchi
authors.affiliation: Programa de Pós-Graduação em Biodiversidade e Biotecnologia da Rede BIONORTE, Brazil; Laboratório de Imunologia Celular Aplicada à Saúde, Fundação Oswaldo Cruz, FIOCRUZ, Fiocruz Rondônia, Porto Velho-RO, Brazil.
authors.forename: Juliana P
authors.initials: JP
authors.lastname: Zuliani
authors.affiliation: Programa de Pós-Graduação em Biodiversidade e Biotecnologia da Rede BIONORTE, Brazil; Centro de Pesquisa em Medicina Tropical de Rondônia, CEPEM, and Fundação Oswaldo Cruz, FIOCRUZ, Fiocruz Rondônia, Porto Velho-RO, Brazil.
authors.forename: Najla B
authors.initials: NB
authors.lastname: Matos
authors.affiliation: Departamento de Bioquímica, Universidade Estadual de Campinas, UNICAMP, Campinas-SP, Brazil.
authors.forename: José R
authors.initials: JR
authors.lastname: Almeida
authors.affiliation: Departamento de Bioquímica, Universidade Estadual de Campinas, UNICAMP, Campinas-SP, Brazil.
authors.forename: Letícia M
authors.initials: LM
authors.lastname: Resende
authors.affiliation: Departamento de Bioquímica, Universidade Estadual de Campinas, UNICAMP, Campinas-SP, Brazil.
authors.forename: Sérgio
authors.initials: S
authors.lastname: Marangoni
authors.affiliation: Universidade Regional Amazónica IKIAM, Via Muyuna Km 7, Tena, Napo, Ecuador.
authors.forename: Saulo L
authors.initials: SL
authors.lastname: da Silva
authors.affiliation: Centro de Estudos de Biomoléculas Aplicadas à Saúde, CEBio, Fundação Oswaldo Cruz, FIOCRUZ, Fiocruz Rondônia, and Departamento de Medicina, Universidade Federal de Rondônia, UNIR, Porto Velho-RO, Brazil; Programa de Pós-Graduação em Biodiversidade e Biotecnologia da Rede BIONORTE, Brazil; Faculdade São Lucas, FSL, Porto Velho-RO, Brazil. Electronic address: andreimar@fiocruz.br.
authors.forename: Andreimar M
authors.initials: AM
authors.lastname: Soares
authors.affiliation: Centro de Estudos de Biomoléculas Aplicadas à Saúde, CEBio, Fundação Oswaldo Cruz, FIOCRUZ, Fiocruz Rondônia, and Departamento de Medicina, Universidade Federal de Rondônia, UNIR, Porto Velho-RO, Brazil; Programa de Pós-Graduação em Biodiversidade e Biotecnologia da Rede BIONORTE, Brazil.
authors.forename: Leonardo A
authors.initials: LA
authors.lastname: Calderon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D017837:Bothrops / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D003435:Crotalid Venoms / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D004487:Edema / Q000097:blood / Q000139:chemically induced / Q000473:pathology; D004591:Electrophoresis, Polyacrylamide Gel; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006094:Gram-Positive Bacteria / Q000187:drug effects; D053583:Interleukin-1beta / Q000097:blood; D008297:Male; D051379:Mice; D054467:Phospholipases A2 / Q000737:chemistry / Q000494:pharmacology*; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000187:drug effects; D016415:Sequence Alignment; D014409:Tumor Necrosis Factor-alpha / Q000097:blood
chemical_list: D000900:Anti-Bacterial Agents; D003435:Crotalid Venoms; D053583:Interleukin-1beta; D014409:Tumor Necrosis Factor-alpha; C018932:myotoxin A; D054467:Phospholipases A2
keywords: Bactericidal activity; Bothrops neuwiedi; Phospholipase A(2); Snake venom