- Email: [email protected]
226. Processing of SVMPs: Detection of SVMP Zymogens and Pro-Domain in Bothrops jararaca Venom and Venom Glands
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
225. Proteomic Analysis and Pharmacological Activities of the Venom of the Moroccan Cobra Naja haje legionis. Ibtissam Malih 1, 2, 3, Muhamad Rusdi Ahmad Rusmili 2, Ting Yee Tee 2, Rachid Saile 3, Noreddine Ghalim 1, Iekhsan Othman 2 1
Venoms and Toxins Laboratory, Pasteur Institute of Morocco, Casablanca, Morocco 2 Department of Biomedical Sciences, School of Medicine and Health Sciences, Monash University Sunway Campus, Malaysia 3 URAC 34, Hassan II University Mohammedia - Casablanca, Faculty of Science Ben M'sik, Morocco E-mail address: [email protected] (I. Malih).
Background: In Morocco, envenomation by snake bites poses a serious problem to public health. The cobra Naja haje legionis is endemic of the country and one of the most dangerous species known. In this work, we report the proteomic and pharmacological characterizations of biologically active proteins from the venom of this species. Methods: The various proteins in the crude venom were fractionated and analyzed by a combination of chromatographic separations and proteomic analysis techniques such as gel filtration, RP-HPLC, 1D electrophoresis, in-gel digestion, tandem mass spectrometry and protein database search. The pharmacological properties of Naja haje legionis venom were assessed using in vitro preparations using rodents and chicks. Results: Our venomic strategy allowed the identification of 64 proteins and peptides from known database which can classified into 17 families according to their biological activities. We were able to identify cobra venom factor, L-aminoacid oxidases, acetylcholinesterase, metalloproteinase, disintegrin, cysteine-rich secretory proteins, nerve growth factor, phospholipases A2, vespryns, kunitz-type inhibitor, short neurotoxins, long neurotoxins, weak neurotoxins, neurotoxin like proteins, muscarinic toxins, cytotoxins and cardiotoxins. Pharmacological tests showed that Naja haje legionis venom contained neurotoxic activities inducing irreversible blockage of neuromuscular transmission in both rodent and chick nerve-muscle preparations. This venom also exhibit myotoxic and cardiotoxic activities. Conclusions: These results showed the potency and the complexity of the various proteins present in the venom of Naja haje legionis, which present a very similar pattern to other cobra venoms. The contribution of the different components in venom toxicity deserves further investigations. Keywords: Naja haje legionis, characterization, neurotoxins, proteomics, pharmacology 10.1016/j.toxicon.2012.04.226
226. Processing of SVMPs: Detection of SVMP Zymogens and Pro-Domain in Bothrops jararaca Venom and Venom Glands J.A. Portes-Junior 1, G.S. Magalhães 1, S.S. Sant'Anna 2, M.R. Junqueira 3, N. Yamanouye 4, A.M. Moura-da-Silva 1, G.B. Domont 3 1 2
Laboratório de Imunopatologia, Instituto Butantan, Sâo Paulo, SP, Brazil Laboratório de Herpetologia, Instituto Butantan, São Paulo, SP, Brazil
211
3 Unidade Proteômica, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil 4 Lab. de Farmacologia, Instituto Butantan, São Paulo, SP, Brazil E-mail address: [email protected] (G.B. Domont).
Background: Snake Venom Metalloproteinases (SVMPs) responsible for Bothrops envenoming cause severe local and systemic complications in humans. They are multidomain enzymes synthesized as zymogens and the enzyme activation is regulated by hydrolysis of the propeptide also known as pro-domain. Methods: In this work we aim to trace the SVMP processing route by detecting the zymogen molecule or cleaved pro-domain in Bothrops jararaca venom glands using antipro-domain antibodies and mass spectrometry. The prodomain was obtained by expression of its cDNA extracted from venom glands in pAE vector/E. coli. The recombinant protein (PD-Jar) was used to immunize mice to obtain polyclonal antibodies. The presence of pro-domains in zymogen or processed forms was then evaluated by Western Blotting in venom samples and venom gland extracts collected at 0, 4, 7, 10, 14 and 21 days after milking. Results: The antiserum was able to recognize bands of 22 and 47 kDa in venom samples collected 7 and 10 days after milking, which contained pro-domain peptides detected by MS/MS. In the extracts of venom glands, antiserum was able to recognize bands of 47 and 76 kDa in all samples. The reactive bands were extracted by pulldown using anti-pro-domain antibodies and subjected to characterization by mass spectrometry. Discussion and Conclusions: Our results indicate that SVMPs are mainly found as zymogens within the venomsecreting cells and processing is very likely to occur in the lumen of the venom gland, in a time-dependent manner. Supported by: FAPESP, CNPq and CAPES Keywords: zymogen, SVMPs, Bothrops jararaca, processing 10.1016/j.toxicon.2012.04.227
227. Proteopeptidome Determination of Bothrops jararaca Venom: an Innovative Approach in Snake Venomics Carolina A. Nicolau 1, 3, 4, André Teixeira-Ferreira 1, 3, 4, Paulo C. Carvalho 1, 3, Magno Junqueira 2, 3, 4, Jonas Perales 1, 3, 4, Ana Gisele -Ferreira C. Neves 1, 3, 4, Richard H. Valente 1, 3, 4 1
Oswaldo Cruz Foundation, RJ, Brazil Federal University of Brasilia, DF, Brazil 3 Proteomic Network, RJ, Brazil 4 INCTTOX/ CNPq, Brazil E-mail address: [email protected] (C.A. Nicolau). 2
Background: Aiming for a breakthrough regarding the actual limitations of data volume generated in snake venomics analyses, our group implemented the use of isoelectrofocusing technology (OFFGEL) followed by reversedphase nanochromatography coupled to high resolution mass spectrometry, in order to simultaneously analyze the proteome and peptidome (PROTEOPEPTIDOME) of Bothrops jararaca venom.
225. Proteomic Analysis and Pharmacological Activities of the Venom of the Moroccan Cobra Naja haje legionis. Ibtissam Malih 1, 2, 3, Muhamad Rusdi Ahmad Rusmili 2, Ting Yee Tee 2, Rachid Saile 3, Noreddine Ghalim 1, Iekhsan Othman 2 1
Venoms and Toxins Laboratory, Pasteur Institute of Morocco, Casablanca, Morocco 2 Department of Biomedical Sciences, School of Medicine and Health Sciences, Monash University Sunway Campus, Malaysia 3 URAC 34, Hassan II University Mohammedia - Casablanca, Faculty of Science Ben M'sik, Morocco E-mail address: [email protected] (I. Malih).
Background: In Morocco, envenomation by snake bites poses a serious problem to public health. The cobra Naja haje legionis is endemic of the country and one of the most dangerous species known. In this work, we report the proteomic and pharmacological characterizations of biologically active proteins from the venom of this species. Methods: The various proteins in the crude venom were fractionated and analyzed by a combination of chromatographic separations and proteomic analysis techniques such as gel filtration, RP-HPLC, 1D electrophoresis, in-gel digestion, tandem mass spectrometry and protein database search. The pharmacological properties of Naja haje legionis venom were assessed using in vitro preparations using rodents and chicks. Results: Our venomic strategy allowed the identification of 64 proteins and peptides from known database which can classified into 17 families according to their biological activities. We were able to identify cobra venom factor, L-aminoacid oxidases, acetylcholinesterase, metalloproteinase, disintegrin, cysteine-rich secretory proteins, nerve growth factor, phospholipases A2, vespryns, kunitz-type inhibitor, short neurotoxins, long neurotoxins, weak neurotoxins, neurotoxin like proteins, muscarinic toxins, cytotoxins and cardiotoxins. Pharmacological tests showed that Naja haje legionis venom contained neurotoxic activities inducing irreversible blockage of neuromuscular transmission in both rodent and chick nerve-muscle preparations. This venom also exhibit myotoxic and cardiotoxic activities. Conclusions: These results showed the potency and the complexity of the various proteins present in the venom of Naja haje legionis, which present a very similar pattern to other cobra venoms. The contribution of the different components in venom toxicity deserves further investigations. Keywords: Naja haje legionis, characterization, neurotoxins, proteomics, pharmacology 10.1016/j.toxicon.2012.04.226
226. Processing of SVMPs: Detection of SVMP Zymogens and Pro-Domain in Bothrops jararaca Venom and Venom Glands J.A. Portes-Junior 1, G.S. Magalhães 1, S.S. Sant'Anna 2, M.R. Junqueira 3, N. Yamanouye 4, A.M. Moura-da-Silva 1, G.B. Domont 3 1 2
Laboratório de Imunopatologia, Instituto Butantan, Sâo Paulo, SP, Brazil Laboratório de Herpetologia, Instituto Butantan, São Paulo, SP, Brazil
211
3 Unidade Proteômica, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil 4 Lab. de Farmacologia, Instituto Butantan, São Paulo, SP, Brazil E-mail address: [email protected] (G.B. Domont).
Background: Snake Venom Metalloproteinases (SVMPs) responsible for Bothrops envenoming cause severe local and systemic complications in humans. They are multidomain enzymes synthesized as zymogens and the enzyme activation is regulated by hydrolysis of the propeptide also known as pro-domain. Methods: In this work we aim to trace the SVMP processing route by detecting the zymogen molecule or cleaved pro-domain in Bothrops jararaca venom glands using antipro-domain antibodies and mass spectrometry. The prodomain was obtained by expression of its cDNA extracted from venom glands in pAE vector/E. coli. The recombinant protein (PD-Jar) was used to immunize mice to obtain polyclonal antibodies. The presence of pro-domains in zymogen or processed forms was then evaluated by Western Blotting in venom samples and venom gland extracts collected at 0, 4, 7, 10, 14 and 21 days after milking. Results: The antiserum was able to recognize bands of 22 and 47 kDa in venom samples collected 7 and 10 days after milking, which contained pro-domain peptides detected by MS/MS. In the extracts of venom glands, antiserum was able to recognize bands of 47 and 76 kDa in all samples. The reactive bands were extracted by pulldown using anti-pro-domain antibodies and subjected to characterization by mass spectrometry. Discussion and Conclusions: Our results indicate that SVMPs are mainly found as zymogens within the venomsecreting cells and processing is very likely to occur in the lumen of the venom gland, in a time-dependent manner. Supported by: FAPESP, CNPq and CAPES Keywords: zymogen, SVMPs, Bothrops jararaca, processing 10.1016/j.toxicon.2012.04.227
227. Proteopeptidome Determination of Bothrops jararaca Venom: an Innovative Approach in Snake Venomics Carolina A. Nicolau 1, 3, 4, André Teixeira-Ferreira 1, 3, 4, Paulo C. Carvalho 1, 3, Magno Junqueira 2, 3, 4, Jonas Perales 1, 3, 4, Ana Gisele -Ferreira C. Neves 1, 3, 4, Richard H. Valente 1, 3, 4 1
Oswaldo Cruz Foundation, RJ, Brazil Federal University of Brasilia, DF, Brazil 3 Proteomic Network, RJ, Brazil 4 INCTTOX/ CNPq, Brazil E-mail address: [email protected] (C.A. Nicolau). 2
Background: Aiming for a breakthrough regarding the actual limitations of data volume generated in snake venomics analyses, our group implemented the use of isoelectrofocusing technology (OFFGEL) followed by reversedphase nanochromatography coupled to high resolution mass spectrometry, in order to simultaneously analyze the proteome and peptidome (PROTEOPEPTIDOME) of Bothrops jararaca venom.