title: Insights on the structure of native CNF, an endogenous phospholipase A2 inhibitor from Crotalus durissus terrificus, the South American rattlesnake.
abstract: Several snake species possess endogenous phospholipase A2 inhibitors (sbPLIs) in their blood plasma, the primary role of which is protection against an eventual presence of toxic phospholipase A2 (PLA2) from their venom glands in the circulation. These inhibitors have an oligomeric structure of, at least, three subunits and have been categorized into three classes (α, β and γ) based on their structural features. SbγPLIs have been further subdivided into two subclasses according to their hetero or homomeric nature, respectively. Despite the considerable number of sbγPLIs described, their structures and mechanisms of action are still not fully understood. In the present study, we focused on the native structure of CNF, a homomeric sbγPLI from Crotalus durissus terrificus, the South American rattlesnake. Based on the results of different biochemical and biophysical experiments, we concluded that, while the native inhibitor occurs as a mixture of oligomers, tetrameric arrangement appears to be the predominant quaternary structure. The inhibitory activity of CNF is most likely associated with this oligomeric conformation. In addition, we suggest that the CNF tetramer has a spherical shape and that tyrosinyl residues could play an important role in the oligomerization. The carbohydrate moiety, which is present in most sbγPLIs, is not essential for the inhibitory activity, oligomerization or complex formation of the CNF with the target PLA2. A minor component, comprising no more than 16% of the sample, was identified in the CNF preparations. The amino-terminal sequence of that component is similar to the B subunits of the heteromeric sbγPLIs; however, the role played by such molecule in the functionality of the CNF, if any, remains to be determined.
authors.affiliation: Diretoria de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias (FUNED), Rua Conde Pereira Carneiro 80, CEP 30510-010, Belo Horizonte, MG, Brazil. Electronic address: consuelo.latorre@funed.mg.gov.br.
authors.forename: Consuelo Latorre
authors.initials: CL
authors.lastname: Fortes-Dias
authors.affiliation: Diretoria de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias (FUNED), Rua Conde Pereira Carneiro 80, CEP 30510-010, Belo Horizonte, MG, Brazil.
authors.forename: Paula Ladeira
authors.initials: PL
authors.lastname: Ortolani
authors.affiliation: Departamento de Física e Biofísica, Instituto de Biociências, Universidade Estadual Paulista, UNESP, Botucatu, SP, Brazil.
authors.forename: Carlos Alexandre H
authors.initials: CA
authors.lastname: Fernandes
authors.affiliation: Diretoria de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias (FUNED), Rua Conde Pereira Carneiro 80, CEP 30510-010, Belo Horizonte, MG, Brazil.
authors.forename: Kelli Roberta
authors.initials: KR
authors.lastname: Lobo
authors.affiliation: Diretoria de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias (FUNED), Rua Conde Pereira Carneiro 80, CEP 30510-010, Belo Horizonte, MG, Brazil.
authors.forename: Lutiana
authors.initials: L
authors.lastname: Amaral de Melo
authors.affiliation: Diretoria de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias (FUNED), Rua Conde Pereira Carneiro 80, CEP 30510-010, Belo Horizonte, MG, Brazil.
authors.forename: Márcia Helena
authors.initials: MH
authors.lastname: Borges
authors.affiliation: Departamento de Física e Matemática, Faculdade de Filosofia Ciências e Letras, USP, Ribeirão Preto, SP, Brazil.
authors.forename: Wallance Moreira
authors.initials: WM
authors.lastname: Pazin
authors.affiliation: Departamento de Física e Biofísica, Instituto de Biociências, Universidade Estadual Paulista, UNESP, Botucatu, SP, Brazil.
authors.forename: Mário de Oliveira
authors.initials: Mde O
authors.lastname: Neto
authors.affiliation: Departamento de Física e Biofísica, Instituto de Biociências, Universidade Estadual Paulista, UNESP, Botucatu, SP, Brazil.
authors.forename: Roberto Morato
authors.initials: RM
authors.lastname: Fernandez
authors.affiliation: Departamento de Física e Biofísica, Instituto de Biociências, Universidade Estadual Paulista, UNESP, Botucatu, SP, Brazil. Electronic address: fontes@ibb.unesp.br.
authors.forename: Marcos Roberto M
authors.initials: MR
authors.lastname: Fontes
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D017839:Crotalus / Q000502:physiology; D003439:Crotoxin / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D064801:Phospholipase A2 Inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D054467:Phospholipases A2 / Q000737:chemistry* / Q000302:isolation & purification; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D030162:Reptilian Proteins / Q000737:chemistry* / Q000302:isolation & purification; D053838:Scattering, Small Angle; D017386:Sequence Homology, Amino Acid; D013020:South America; D014443:Tyrosine / Q000737:chemistry; D014961:X-Ray Diffraction
chemical_list: D006023:Glycoproteins; D064801:Phospholipase A2 Inhibitors; D030162:Reptilian Proteins; C087438:phospholipase A2 inhibitor protein, Crotalus; D014443:Tyrosine; D003439:Crotoxin; D054467:Phospholipases A2
keywords: Crotalus; PLA(2); PLI; Phospholipase A(2); Phospholipase A(2) inhibitor; Quaternary structure