title: Crotalid snake venom subproteomes unraveled by the antiophidic protein DM43.
abstract: Snake venoms are mixtures of proteins and peptides with different biological activities, many of which are very toxic. Several animals, including the opossum Didelphis aurita, are resistant to snake venoms due to the presence of neutralizing factors in their blood. An antihemorrhagic protein named DM43 was isolated from opossum serum. It inhibits snake venom metalloproteinases through noncovalent complex formation with these enzymes. In this study, we have used DM43 and proteomic techniques to explore snake venom subproteomes. Four crotalid venoms were chromatographed through an affinity column containing immobilized DM43. Bound fractions were analyzed by one- and two-dimensional gel electrophoresis, followed by identification by MALDI-TOF/TOF mass spectrometry. With this approach, we could easily visualize and compare the metalloproteinase compositions of Bothrops atrox, Bothrops jararaca, Bothrops insularis, and Crotalus atrox snake venoms. The important contribution of proteolytic processing to the complexity of this particular subproteome was demonstrated. Fractions not bound to DM43 column were similarly analyzed and were composed mainly of serine proteinases, C-type lectins, C-type lectin-like proteins, l-amino acid oxidases, nerve growth factor, cysteine-rich secretory protein, a few metalloproteinases (and their fragments), and some unidentified spots. Although very few toxin families were represented in the crotalid venoms analyzed, the number of protein spots detected was in the hundreds, indicating an important protein variability in these natural secretions. DM43 affinity chromatography and associated proteomic techniques proved to be useful tools to separate and identify proteins from snake venoms, contributing to a better comprehension of venom heterogeneity.
authors.affiliation: Laboratorio de Toxinologia, Pavilhão Ozório de Almeida, Instituto Oswaldo Cruz, Fiocruz, Av. Brasil, 4365, 21040-900 Rio de Janeiro, RJ, Brazil.
authors.forename: Surza L G
authors.initials: SL
authors.lastname: Rocha
authors.forename: Ana G C
authors.initials: AG
authors.lastname: Neves-Ferreira
authors.forename: Monique R O
authors.initials: MR
authors.lastname: Trugilho
authors.forename: Alex
authors.initials: A
authors.lastname: Chapeaurouge
authors.forename: Ileana R
authors.initials: IR
authors.lastname: León
authors.forename: Richard H
authors.initials: RH
authors.lastname: Valente
authors.forename: Gilberto B
authors.initials: GB
authors.lastname: Domont
authors.forename: Jonas
authors.initials: J
authors.lastname: Perales
mesh_terms: D000818:Animals; D001798:Blood Proteins / Q000378:metabolism* / Q000494:pharmacology; D017837:Bothrops / Q000145:classification / Q000378:metabolism; D002846:Chromatography, Affinity; D003435:Crotalid Venoms / Q000032:analysis* / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001798:Blood Proteins; D003435:Crotalid Venoms; C454462:DM43 protein, Didelphis marsupialis; D020543:Proteome; D045726:Metalloproteases