title: Screening for target toxins of the antiophidic protein DM64 through a gel-based interactomics approach.
abstract: DM64 is a glycosylated protein with antivenom activity isolated from the serum of the opossum Didelphis aurita. It binds non-covalently to myotoxins I (Asp49) and II (Lys49) from Bothrops asper venom and inhibits their myotoxic effect. In this study, an affinity column with immobilized DM64 as bait was used to fish potential target toxins. All ten isolated myotoxins tested were able to effectively bind to the DM64 column. To better access the specificity of the inhibitor, crude venoms from Bothrops (8 species), Crotalus (2 species) and Naja naja atra were submitted to the affinity purification. Venom fractions bound and nonbound to the DM64 column were analyzed by two-dimensional gel electrophoresis and MALDI-TOF/TOF MS. Although venom fractions bound to the column were mainly composed of basic PLA2, a few spots corresponding to acidic PLA2were also observed. Some unexpected protein spots were also identified: C-type lectins and CRISP may represent putative new targets for DM64, whereas the presence of serine peptidases in the venom bound fraction is likely a consequence of nonspecific binding to the column matrix. The present results contribute to better delineate the inhibitory potential of DM64, providing a framework for the development of more specific antivenom therapies.
  
  
  
  Local tissue damage induced by myotoxic PLA2remains a serious consequence of snake envenomation, since it is only partially neutralized by traditional antivenom serotherapy. Myotoxin inhibition by highly specific molecules offers great promise in the treatment of snakebites, a health problem largely neglected by governments and pharmaceutical industries. Bioactive compounds such as DM64 can represent a valuable source of scaffolds for drug development in this area. The present study has systematically profiled the binding specificity of DM64 toward a variety of snake venom toxin classes and therefore can lead to a better understanding of the structure-function relationship of this important antivenom protein.
authors.affiliation: Laboratório de Toxinologia, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ, Brazil; Instituto Nacional de Ciência e Tecnologia em Toxinas, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil.
authors.forename: Surza L G
authors.initials: SL
authors.lastname: Rocha
authors.affiliation: Laboratório de Toxinologia, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ, Brazil; Instituto Nacional de Ciência e Tecnologia em Toxinas, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil. Electronic address: anag@ioc.fiocruz.br.
authors.forename: Ana G C
authors.initials: AG
authors.lastname: Neves-Ferreira
authors.affiliation: Laboratório de Toxinologia, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ, Brazil; Instituto Nacional de Ciência e Tecnologia em Toxinas, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil.
authors.forename: Monique R O
authors.initials: MR
authors.lastname: Trugilho
authors.affiliation: Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.
authors.forename: Yamileth
authors.initials: Y
authors.lastname: Angulo
authors.affiliation: Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.
authors.forename: Bruno
authors.initials: B
authors.lastname: Lomonte
authors.affiliation: Laboratório de Toxinologia, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ, Brazil; Instituto Nacional de Ciência e Tecnologia em Toxinas, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil.
authors.forename: Richard H
authors.initials: RH
authors.lastname: Valente
authors.affiliation: Laboratório de Química de Proteínas/Unidade Proteômica, Dept. Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; Instituto Nacional de Ciência e Tecnologia em Toxinas, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil.
authors.forename: Gilberto B
authors.initials: GB
authors.lastname: Domont
authors.affiliation: Laboratório de Toxinologia, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ, Brazil; Instituto Nacional de Ciência e Tecnologia em Toxinas, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil. Electronic address: jperales@ioc.fiocruz.br.
authors.forename: Jonas
authors.initials: J
authors.lastname: Perales
mesh_terms: D000818:Animals; D001798:Blood Proteins / Q000378:metabolism* / Q000627:therapeutic use; D002846:Chromatography, Affinity; D003435:Crotalid Venoms / Q000037:antagonists & inhibitors*; D015180:Electrophoresis, Gel, Two-Dimensional; D010741:Phospholipases A / Q000032:analysis / Q000037:antagonists & inhibitors; D011485:Protein Binding; D040901:Proteomics / Q000379:methods; D013045:Species Specificity; D053719:Tandem Mass Spectrometry; D014118:Toxins, Biological / Q000032:analysis / Q000037:antagonists & inhibitors
chemical_list: D001798:Blood Proteins; D003435:Crotalid Venoms; C471417:DM64 protein, Didelphis marsupialis; D014118:Toxins, Biological; D010741:Phospholipases A
keywords: Didelphis aurita; Interactomics; Myotoxin; Myotoxin inhibitor; Proteomics; Snake venoms