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Structural, functional, and bioinformatics studies reveal a new snake venom homologue phospholipase A2 class

Authors

  • Juliana I. dos Santos,

    1. Departamento de Física e Biofísica, Instituto de Biociências, UNESP – Univ Estadual Paulista, Botucatu-SP and Instituto Nacional de Ciência e Tecnologia em Toxinas, CNPq, Brazil
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    • The authors Juliana I. dos Santos and Mariana Cintra-Francischinelli contributed equally to this work.

  • Mariana Cintra-Francischinelli,

    1. Dipartimento di Scienze Biomediche, Università di Padova, Padova, Italy
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    • Rafael J. Borges,

      1. Departamento de Física e Biofísica, Instituto de Biociências, UNESP – Univ Estadual Paulista, Botucatu-SP and Instituto Nacional de Ciência e Tecnologia em Toxinas, CNPq, Brazil
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    • Carlos A. H. Fernandes,

      1. Departamento de Física e Biofísica, Instituto de Biociências, UNESP – Univ Estadual Paulista, Botucatu-SP and Instituto Nacional de Ciência e Tecnologia em Toxinas, CNPq, Brazil
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    • Paola Pizzo,

      1. Dipartimento di Scienze Biomediche, Università di Padova, Padova, Italy
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    • Adélia C. O. Cintra,

      1. Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, FCFRP, USP, Ribeirão Preto-SP, Brazil
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    • Antonio S. K. Braz,

      1. Departamento de Física e Biofísica, Instituto de Biociências, UNESP – Univ Estadual Paulista, Botucatu-SP and Instituto Nacional de Ciência e Tecnologia em Toxinas, CNPq, Brazil
      Current affiliation:
      1. Univ Federal do ABC, CCNH, Santo André-SP, Brazil
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    • Andreimar M. Soares,

      1. Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, FCFRP, USP, Ribeirão Preto-SP, Brazil
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    • Marcos R. M. Fontes

      Corresponding author
      1. Departamento de Física e Biofísica, Instituto de Biociências, UNESP – Univ Estadual Paulista, Botucatu-SP and Instituto Nacional de Ciência e Tecnologia em Toxinas, CNPq, Brazil
      • Departamento de Física e Biofísica, Instituto de Biociências, UNESP – Univ Estadual Paulista, Botucatu-SP and Instituto Nacional de Ciência e Tecnologia em Toxinas, CNPq, Brazil
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    Abstract

    Phospholipases A2 (PLA2s) are enzymes responsible for membrane disruption through Ca2+-dependent hydrolysis of phospholipids. Lys49-PLA2s are well-characterized homologue PLA2s that do not show catalytic activity but can exert a pronounced local myotoxic effect. These homologue PLA2s were first believed to present residual catalytic activity but experiments with a recombinant toxin show they are incapable of catalysis. Herein, we present a new homologue Asp49-PLA2 (BthTX-II) that is also able to exert muscle damage. This toxin was isolated in 1992 and characterized as presenting very low catalytic activity. Interestingly, this myotoxic homologue Asp49-PLA2 conserves all the residues responsible for Ca2+ coordination and of the catalytic network, features thought to be fundamental for PLA2 enzymatic activity. Previous crystallographic studies of apo BthTX-II suggested this toxin could be catalytically inactive since a distortion in the calcium binding loop was observed. In this article, we show BthTX-II is not catalytic based on an in vitro cell viability assay and time-lapse experiments on C2C12 myotube cell cultures, X-ray crystallography and phylogenetic studies. Cell culture experiments show that BthTX-II is devoid of catalytic activity, as already observed for Lys49-PLA2s. Crystallographic studies of the complex BthTX-II/Ca2+ show that the distortion of the calcium binding loop is still present and impairs ion coordination even though Ca2+ are found interacting with other regions of the protein. Phylogenetic studies demonstrate that BthTX-II is more phylogenetically related to Lys49-PLA2s than to other Asp49-PLA2s, thus allowing Crotalinae subfamily PLA2s to be classified into two main branches: a catalytic and a myotoxic one. Proteins 2010. © 2010 Wiley-Liss, Inc.

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