title: Snake venom serine proteinases specificity mapping by proteomic identification of cleavage sites.
abstract: Many snake venom toxins are serine proteases but their specific in vivo targets are mostly unknown. Various act on components of the coagulation cascade, and fibrinolytic and kallikrein-kinin systems to trigger various pathological effects observed in the envenomation. Despite showing high similarity in terms of primary structure snake venom serine proteinases (SVSPs) show exquisite specificity towards macromolecular substrates. Therefore, the characterization of their peptide bond specificity is important for understanding the active site preference associated with effective proteolysis as well as for the design of peptide substrates and inhibitors. Bothrops jararaca contains various SVSPs among which Bothrops protease A is a specific fibrinogenolytic agent and PA-BJ is a platelet-activating enzyme. In this study we used proteome derived peptide libraries in the Proteomic Identification of protease Cleavage Sites (PICS) approach to explore the peptide bond specificity of Bothrops protease A and PA-BJ in order to determine their individual peptide cleavage sequences. A total of 371 cleavage sites (208 for Bothrops protease A and 163 for PA-BJ) were detected and both proteinases displayed a clear preference for arginine at the P1 position. Moreover, the analysis of the specificity profiles of Bothrops protease A and PA-BJ revealed subtle differences in the preferences along P6-P6', despite a common yet unusual preference for Pro at P2. Taken together, these results map the subsite specificity of both SVSPs and shed light in the functional differences between these proteinases.
  
  
  CONCLUSIONS
  Proteolysis is key to various pathological effects observed upon envenomation by viperid snakes. The use of the Proteomic Identification of protease Cleavage Sites (PICS) approach for the easy mapping of proteinase subsite preferences at both the prime- and non-prime sides concurrently gives rise to a fresh understanding of the interaction of the snake venom serine proteinases with peptide and macromolecular substrates and indicates that their hydrolytic activity is influenced by the amino acid sequences adjacent to the scissile bond.
authors.affiliation: Laboratório Especial de Toxinologia Aplicada, Center of Toxins, Immune-response and Cell Signaling-CeTICS, Instituto Butantan, Brazil.
authors.forename: André
authors.initials: A
authors.lastname: Zelanis
authors.affiliation: Centre for Blood Research, University of British Columbia, Vancouver, Canada; Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, Canada; Central Institute for Engineering, Electronics and Analytics (ZEA-3), Forschungszentrum Jülich, Jülich, Germany.
authors.forename: Pitter F
authors.initials: PF
authors.lastname: Huesgen
authors.affiliation: Laboratório Especial de Toxinologia Aplicada, Center of Toxins, Immune-response and Cell Signaling-CeTICS, Instituto Butantan, Brazil; Instituto de Química, Departamento de Bioquímica, Universidade de São Paulo, Brazil.
authors.forename: Ana Karina
authors.initials: AK
authors.lastname: Oliveira
authors.affiliation: Departamento de Bioquímica, Universidade Federal de São Paulo, Brazil.
authors.forename: Alexandre K
authors.initials: AK
authors.lastname: Tashima
authors.affiliation: Laboratório Especial de Toxinologia Aplicada, Center of Toxins, Immune-response and Cell Signaling-CeTICS, Instituto Butantan, Brazil. Electronic address: solange.serrano@butantan.gov.br.
authors.forename: Solange M T
authors.initials: SM
authors.lastname: Serrano
authors.affiliation: Centre for Blood Research, University of British Columbia, Vancouver, Canada; Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, Canada; Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada. Electronic address: chris.overall@ubc.ca.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Overall
mesh_terms: D000818:Animals; D017837:Bothrops; D003435:Crotalid Venoms / Q000201:enzymology*; D019151:Peptide Library; D010972:Platelet Activating Factor / Q000737:chemistry* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D003435:Crotalid Venoms; D019151:Peptide Library; D010972:Platelet Activating Factor; C038929:Bothrops jararaca protease A; D012697:Serine Endopeptidases
keywords: Peptide bond specificity; Proteome derived peptide library; Proteomic Identification of protease Cleavage Sites; Serine proteinase; Snake venom