- Email: [email protected]
90. Proteomic Analysis of the Molecular Targets of a Peptide from Wasp Venom Through Developing of an Analytical Platform
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
complement pathogenesis including paroxysmal nocturnal hemoglobinuria (PNH) (2008), myocardial ischemia reperfusion injury (2009), age-related macular degeneration (AMD) (2010), and myasthenia gravis (2011), with no adverse effects observed, including in primates (2010). Keywords: cobra venom factor, complement 10.1016/j.toxicon.2012.04.090
F. Insects 90. Proteomic Analysis of the Molecular Targets of a Peptide from Wasp Venom Through Developing of an Analytical Platform Lucilene Delazari dos Santos 1, 3, José Roberto Aparecido dos Santos Pinto 2, 3, Anally Ribeiro da Silva Menegasso 2, 3, Ana Maria Garcia Caviquioli 2, 3, Daniel Menezes Saidemberg 2, 3, Mario Sergio Palma 2, 3 1 Center of Studies of Venom and Animal Venomous (CEVAP), University of São Paulo State (UNESP), Botucatu, SP; Brazil 2 Institute of Biosciences/Department of Biology, Center of the Study of Social Insects, University of São Paulo State (UNESP), Rio Claro, SP; Brazil 3 Instituto Nacional de Ciência e Tecnologia (INCT) em Imunologia / iii, Brazil E-mail address: [email protected] (L. Delazari dos Santos).
Background: The organisms’ answers to changes in their environment or even in their development generate intracellular signals which are translated, amplified and converted to physiological/ pharmacological answers toward the signals transduction system from plasma membrane. Gproteins are responsible for detection of specific and temporal characteristics of the majority of signal transduction mechanisms. Currently, more than 50% of medicines utilized in the world, act directly or indirectly, by activating or blocking the G-protein coupled receptors (GPCRs). Therefore, the subject of this study was the development of an affinity chromatography platform by using the mastoparan peptide Protopolybia-MP III, previously reported as specific ligand of GPCRs from rat mast cells. Methods: The peptide Protopolybia MP-III (INWLKLGKAVIDAL-NH2) was synthesized by using F-moc strategy and then, coupled to the chromatographic resin Sepharose 4B, in order to set up an affinity chromatography protocol; the column (12 x 2 cm, 15 mL) was built in TRIS-HCl buffer pH 8.0. Rat peritoneal mast cells were collected and lysed with NaCl 1M; the membrane extract was reconstituted into proteoliposomes of 300 nm of diameter. The elution of proteoliposomes suspension was carried-out at a flow rate of 0.5 mL/min and monitored at 280 nm; fractions of 1 mL were collected. The liganded proteins (under proteoliposome form) were removed under salt gradient from 0 to 1 M NaCl in the same equilibration buffer. The proteins eluted in each proteoliposome fraction were extracted with a solution of 0.02% (w/v) SDS and submitted to 1-D SDS-PAGE, stained with 0.025% (w/v) Coomassie Brilliant Blue and submitted to proteomic analysis. Protein profile showed five protein bands with molecular weights of 18 to 66 kDa, which were excised from gel, processed and sequenced by ESI-IT-MS/MS. Results and Discussion: This experimental approach, associated to SDS-PAGE, in gel trypsin digestion and
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proteomic analysis, permitted the identification of five endosomal proteins: Rho GTPases Cdc 42 and the exocyst complex component 7- as components of the Caþ2 independent FcεRI-mediated exocytosis pathway; synaptosomal-associated protein 29 and the GTP-binding protein Rab-3D as components of the Caþ2 dependent FcεRI-mediated exocytosis pathway, which are related to cell signal transduction. Conclusion: This novel analytical strategy permitted the identification of some GPCRs, promoting a better understanding of the mast cell activation by peptides from wasp venom. Keywords: mastoparan; wasp venom; exocytosis; proteoliposomes; proteomics; mass spectrometry; affinity chromatography 10.1016/j.toxicon.2012.04.091
91. Proteomic analyses of the Venom from the Giant Ant Dinoponera quadriceps: A Comparative Study and Characterization of the Major Components of the Venom Derived from 4 Different Areas of Brazil Camila T. Cologna 1, Jaqueline Cardoso 2, Michel Degueldre 1, Ana P.T. Uetanabaro 3, Eraldo M.C. Neto 3, Edwin de Pauw 1, Loic Quinton 1 1
Laboratory of Mass Spectrometry , Université de Liège, Liège, Belgium Laboratory of Animal Studies, Universidade do Estado da Bahia, Bahia, Brazil 3 Laboratory of Quality Control, Department of Biological Sciences, Universidade Estadual de Feira de Santana, Bahia,Brazil E-mail address: [email protected] (C.T. Cologna). 2
Background: The order Hymenoptera, which embraces wasps, bees and ants, constitutes the largest group of venomous animals. Within the 120,000 already described species, the group of ants represents around 15,000 species. The Ponerinae subfamily is symbolized by the world’s biggest ants (3-4 cm) and is found in subtropical zones of South America. In this group of ants, the genus Paraponera e Dinoponera reveals particularly remarkable venom describing medical interest. Their sting may produce acute pain, cold sweating, nausea, a vomiting episode, malaise and tachycardia. Moreover, peptides isolated from the genus Paraponera have been described as ion channel modifiers and antimicrobial toxins. Despite those reports, the information about the biological properties and composition of their venom is still very limited. Objectives: To study the venom of the giant neotropical Dinoponera quadriceps ant collected in 4 geographically different regions of Brazil. By using combinatorial mass spectrometric approaches, we aim to: (i) characterizing the venom composition of these ants and (ii) establishing a comparative analysis of the venom from 4 geographically different regions in Brazil. Methods: The ants were captured in the surroundings of Contendas, Manoel Vitorino, Caetite and Feira de Santana (Bahia, Brazil). Venoms were extracted by mechanical stimulation and then dried. An aliquot of each was analyzed by MALDI-TOF/TOF (Ultraflex II, Bruker Daltonics, Bremen, Germany) and ESI-Q-TOF (Synapt-G1, Waters, Manchester, UK) in direct infusion or with a liquid nanochromatographic separation (nanoACQUITY, Waters, Manchester, UK) step before the MS analysis. In
complement pathogenesis including paroxysmal nocturnal hemoglobinuria (PNH) (2008), myocardial ischemia reperfusion injury (2009), age-related macular degeneration (AMD) (2010), and myasthenia gravis (2011), with no adverse effects observed, including in primates (2010). Keywords: cobra venom factor, complement 10.1016/j.toxicon.2012.04.090
F. Insects 90. Proteomic Analysis of the Molecular Targets of a Peptide from Wasp Venom Through Developing of an Analytical Platform Lucilene Delazari dos Santos 1, 3, José Roberto Aparecido dos Santos Pinto 2, 3, Anally Ribeiro da Silva Menegasso 2, 3, Ana Maria Garcia Caviquioli 2, 3, Daniel Menezes Saidemberg 2, 3, Mario Sergio Palma 2, 3 1 Center of Studies of Venom and Animal Venomous (CEVAP), University of São Paulo State (UNESP), Botucatu, SP; Brazil 2 Institute of Biosciences/Department of Biology, Center of the Study of Social Insects, University of São Paulo State (UNESP), Rio Claro, SP; Brazil 3 Instituto Nacional de Ciência e Tecnologia (INCT) em Imunologia / iii, Brazil E-mail address: [email protected] (L. Delazari dos Santos).
Background: The organisms’ answers to changes in their environment or even in their development generate intracellular signals which are translated, amplified and converted to physiological/ pharmacological answers toward the signals transduction system from plasma membrane. Gproteins are responsible for detection of specific and temporal characteristics of the majority of signal transduction mechanisms. Currently, more than 50% of medicines utilized in the world, act directly or indirectly, by activating or blocking the G-protein coupled receptors (GPCRs). Therefore, the subject of this study was the development of an affinity chromatography platform by using the mastoparan peptide Protopolybia-MP III, previously reported as specific ligand of GPCRs from rat mast cells. Methods: The peptide Protopolybia MP-III (INWLKLGKAVIDAL-NH2) was synthesized by using F-moc strategy and then, coupled to the chromatographic resin Sepharose 4B, in order to set up an affinity chromatography protocol; the column (12 x 2 cm, 15 mL) was built in TRIS-HCl buffer pH 8.0. Rat peritoneal mast cells were collected and lysed with NaCl 1M; the membrane extract was reconstituted into proteoliposomes of 300 nm of diameter. The elution of proteoliposomes suspension was carried-out at a flow rate of 0.5 mL/min and monitored at 280 nm; fractions of 1 mL were collected. The liganded proteins (under proteoliposome form) were removed under salt gradient from 0 to 1 M NaCl in the same equilibration buffer. The proteins eluted in each proteoliposome fraction were extracted with a solution of 0.02% (w/v) SDS and submitted to 1-D SDS-PAGE, stained with 0.025% (w/v) Coomassie Brilliant Blue and submitted to proteomic analysis. Protein profile showed five protein bands with molecular weights of 18 to 66 kDa, which were excised from gel, processed and sequenced by ESI-IT-MS/MS. Results and Discussion: This experimental approach, associated to SDS-PAGE, in gel trypsin digestion and
141
proteomic analysis, permitted the identification of five endosomal proteins: Rho GTPases Cdc 42 and the exocyst complex component 7- as components of the Caþ2 independent FcεRI-mediated exocytosis pathway; synaptosomal-associated protein 29 and the GTP-binding protein Rab-3D as components of the Caþ2 dependent FcεRI-mediated exocytosis pathway, which are related to cell signal transduction. Conclusion: This novel analytical strategy permitted the identification of some GPCRs, promoting a better understanding of the mast cell activation by peptides from wasp venom. Keywords: mastoparan; wasp venom; exocytosis; proteoliposomes; proteomics; mass spectrometry; affinity chromatography 10.1016/j.toxicon.2012.04.091
91. Proteomic analyses of the Venom from the Giant Ant Dinoponera quadriceps: A Comparative Study and Characterization of the Major Components of the Venom Derived from 4 Different Areas of Brazil Camila T. Cologna 1, Jaqueline Cardoso 2, Michel Degueldre 1, Ana P.T. Uetanabaro 3, Eraldo M.C. Neto 3, Edwin de Pauw 1, Loic Quinton 1 1
Laboratory of Mass Spectrometry , Université de Liège, Liège, Belgium Laboratory of Animal Studies, Universidade do Estado da Bahia, Bahia, Brazil 3 Laboratory of Quality Control, Department of Biological Sciences, Universidade Estadual de Feira de Santana, Bahia,Brazil E-mail address: [email protected] (C.T. Cologna). 2
Background: The order Hymenoptera, which embraces wasps, bees and ants, constitutes the largest group of venomous animals. Within the 120,000 already described species, the group of ants represents around 15,000 species. The Ponerinae subfamily is symbolized by the world’s biggest ants (3-4 cm) and is found in subtropical zones of South America. In this group of ants, the genus Paraponera e Dinoponera reveals particularly remarkable venom describing medical interest. Their sting may produce acute pain, cold sweating, nausea, a vomiting episode, malaise and tachycardia. Moreover, peptides isolated from the genus Paraponera have been described as ion channel modifiers and antimicrobial toxins. Despite those reports, the information about the biological properties and composition of their venom is still very limited. Objectives: To study the venom of the giant neotropical Dinoponera quadriceps ant collected in 4 geographically different regions of Brazil. By using combinatorial mass spectrometric approaches, we aim to: (i) characterizing the venom composition of these ants and (ii) establishing a comparative analysis of the venom from 4 geographically different regions in Brazil. Methods: The ants were captured in the surroundings of Contendas, Manoel Vitorino, Caetite and Feira de Santana (Bahia, Brazil). Venoms were extracted by mechanical stimulation and then dried. An aliquot of each was analyzed by MALDI-TOF/TOF (Ultraflex II, Bruker Daltonics, Bremen, Germany) and ESI-Q-TOF (Synapt-G1, Waters, Manchester, UK) in direct infusion or with a liquid nanochromatographic separation (nanoACQUITY, Waters, Manchester, UK) step before the MS analysis. In