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Juan J. Calvete - One of the best experts on this subject based on the ideXlab platform.
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PCR amplification of cDNA constructed from venom gland and venom mRNA.
2012Co-Authors: Rachel B. Currier, Paul D. Rowley, Robert A. Harrison, Libia Sanz, Juan J. Calvete, Simon C. WagstaffAbstract:Qualitatively similar PCR products were amplified from cDNA from venom gland (A) or venom (B), using primers complementary to Bitis Arietans venom metalloproteinases (SVMP), phospholipase A2 (PLA2), serine protease (SP), C-type lectins (CTL), vascular endothelial growth factor (VEGF), L-amino acid oxidase (LAO), Kunitz inhibitors (KTI), protein disulphide isomerase (PDI) and QKW inhibitory peptides (QKW). Molecular weight markers (M) are shown to the left.
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exploring the venom proteome of the african puff adder Bitis Arietans using a combinatorial peptide ligand library approach at different phs
Journal of Proteomics, 2010Co-Authors: Elisa Fasoli, Simon C. Wagstaff, Robert A. Harrison, Libia Sanz, Pier Giorgio Righetti, Juan J. CalveteAbstract:We report the 2DE-based proteomic characterization of the venom of the medically important African puff adder, Bitis Arietans, after prefractionation by incubation with a solid-phase combinatorial hexapeptide ligand library (CPLL) at three different pH values. This approach yielded partially overlapping yet clearly distinct sets of proteins. The B. Arietans venom proteome, merged from the four sets of proteins comprises at least 43 distinct proteins from 9 toxin families. In line with a previous reverse-phase HPLC-based venomic characterization on the same species, SVMPs, serine proteinases, C-type lectin-like proteins, and to a minor extent PLA(2), disintegrin Bitistatin, and cystatin, comprise the major toxins in the venom of B. Arietans. However, the 2D-CPLL approach employed here identified both a significantly higher (about double) number of proteins than a previous venomic approach, and many very minor components barely, or not at all, detectable in the 2DE separation of whole venom. 30 proteins from the CPLL-merged venom proteome matched some of the 63 toxin clusters generated by sequencing one thousand randomly selected venom gland cDNA library clones of the same species. The low (47%) concordance between transcriptome and proteome may be interpreted in terms of intraspecific venom variation. Comparison of the reverse-phase HPLC separations of the venom proteins of B. Arietans from Ghana and Nigeria supports this view.
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Molecular Cloning of Disintegrin-like Transcript BA-5A from a Bitis Arietans Venom Gland cDNA Library: A Putative Intermediate in the Evolution of the Long-Chain Disintegrin Bitistatin
Journal of Molecular Evolution, 2006Co-Authors: Paula Juárez, Simon C. Wagstaff, Robert A. Harrison, Jenny Oliver, Libia Sanz, Juan J. CalveteAbstract:We report the cloning and sequence analysis of BA-5A from a venom gland cDNA library of the puff adder, Bitis Arietans , that encodes a novel ECD-disintegrin-like domain. BA-5A is a unique PII disintegrin. It contains the 16 cysteine residues that are conserved in all known disintegrin-like domains of ADAM proteins and snake venom metalloproteinases but lacks the cysteine-rich domain. These features suggest that BA-5A may represent an intermediate in the evolutionary pathway of the long disintegrin Bitistatin and that removal of the cysteine-rich domain and loss of the PIII-specific disulfide bond were separate events along the structural diversification pathway of disintegrins, the former predating the latter. The protein family composition of the Bitis Arietans venom, as determined by combination of reversed-phase HPLC and proteomic analysis, was as follows: Zn^2+-metalloproteinase (38.5%), serine proteinase (19.5%), disintegrin (17.8%), C-type lectin-like (13.2%), PLA_2 (4.3%), Kunitz-type inhibitor (4.1%), cystatin (1.7%), and unknown (0.9%). BA-5A could not be detected in the venom proteome of Bitis Arietans . The occurrence of this very low-abundance (< 0.05%) or nonexpressed disintegrin transcript indicates a hitherto unrecognized structural diversity of this protein family. Whether BA-5A plays a physiological role or represents an orphan protein which could eventually evolve a role in the adaptation of snakes to changing ecological niches and prey habits deserves further investigation.
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Molecular Cloning of Disintegrin-like Transcript BA-5A from a Bitis Arietans Venom Gland cDNA Library: A Putative Intermediate in the Evolution of the Long-Chain Disintegrin Bitistatin
Journal of Molecular Evolution, 2006Co-Authors: Paula Juárez, Simon C. Wagstaff, Robert A. Harrison, Jenny Oliver, Libia Sanz, Juan J. CalveteAbstract:We report the cloning and sequence analysis of BA-5A from a venom gland cDNA library of the puff adder, Bitis Arietans , that encodes a novel ECD-disintegrin-like domain. BA-5A is a unique PII disintegrin. It contains the 16 cysteine residues that are conserved in all known disintegrin-like domains of ADAM proteins and snake venom metalloproteinases but lacks the cysteine-rich domain. These features suggest that BA-5A may represent an intermediate in the evolutionary pathway of the long disintegrin Bitistatin and that removal of the cysteine-rich domain and loss of the PIII-specific disulfide bond were separate events along the structural diversification pathway of disintegrins, the former predating the latter. The protein family composition of the Bitis Arietans venom, as determined by combination of reversed-phase HPLC and proteomic analysis, was as follows: Zn^2+-metalloproteinase (38.5%), serine proteinase (19.5%), disintegrin (17.8%), C-type lectin-like (13.2%), PLA_2 (4.3%), Kunitz-type inhibitor (4.1%), cystatin (1.7%), and unknown (0.9%). BA-5A could not be detected in the venom proteome of Bitis Arietans . The occurrence of this very low-abundance (< 0.05%) or nonexpressed disintegrin transcript indicates a hitherto unrecognized structural diversity of this protein family. Whether BA-5A plays a physiological role or represents an orphan protein which could eventually evolve a role in the adaptation of snakes to changing ecological niches and prey habits deserves further investigation.
Simon C. Wagstaff - One of the best experts on this subject based on the ideXlab platform.
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Bitis Arietans primers.
2016Co-Authors: Gareth Whiteley, Rhiannon A. E. Logan, Kam-yin D. Leung, Fiona J. Newberry, Paul D. Rowley, John P. Dunbar, Simon C. Wagstaff, Nicholas R. Casewell, Robert A. HarrisonAbstract:Bitis Arietans primers.
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PCR amplification of cDNA constructed from venom gland and venom mRNA.
2012Co-Authors: Rachel B. Currier, Paul D. Rowley, Robert A. Harrison, Libia Sanz, Juan J. Calvete, Simon C. WagstaffAbstract:Qualitatively similar PCR products were amplified from cDNA from venom gland (A) or venom (B), using primers complementary to Bitis Arietans venom metalloproteinases (SVMP), phospholipase A2 (PLA2), serine protease (SP), C-type lectins (CTL), vascular endothelial growth factor (VEGF), L-amino acid oxidase (LAO), Kunitz inhibitors (KTI), protein disulphide isomerase (PDI) and QKW inhibitory peptides (QKW). Molecular weight markers (M) are shown to the left.
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exploring the venom proteome of the african puff adder Bitis Arietans using a combinatorial peptide ligand library approach at different phs
Journal of Proteomics, 2010Co-Authors: Elisa Fasoli, Simon C. Wagstaff, Robert A. Harrison, Libia Sanz, Pier Giorgio Righetti, Juan J. CalveteAbstract:We report the 2DE-based proteomic characterization of the venom of the medically important African puff adder, Bitis Arietans, after prefractionation by incubation with a solid-phase combinatorial hexapeptide ligand library (CPLL) at three different pH values. This approach yielded partially overlapping yet clearly distinct sets of proteins. The B. Arietans venom proteome, merged from the four sets of proteins comprises at least 43 distinct proteins from 9 toxin families. In line with a previous reverse-phase HPLC-based venomic characterization on the same species, SVMPs, serine proteinases, C-type lectin-like proteins, and to a minor extent PLA(2), disintegrin Bitistatin, and cystatin, comprise the major toxins in the venom of B. Arietans. However, the 2D-CPLL approach employed here identified both a significantly higher (about double) number of proteins than a previous venomic approach, and many very minor components barely, or not at all, detectable in the 2DE separation of whole venom. 30 proteins from the CPLL-merged venom proteome matched some of the 63 toxin clusters generated by sequencing one thousand randomly selected venom gland cDNA library clones of the same species. The low (47%) concordance between transcriptome and proteome may be interpreted in terms of intraspecific venom variation. Comparison of the reverse-phase HPLC separations of the venom proteins of B. Arietans from Ghana and Nigeria supports this view.
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Intra-specific variation in venom of the African Puff Adder (Bitis Arietans): Differential expression and activity of snake venom metalloproteinases (SVMPs).
Toxicon, 2010Co-Authors: Rachel B. Currier, Paul D. Rowley, Robert A. Harrison, Gavin D. Laing, Simon C. WagstaffAbstract:Bitis Arietans is considered one of the most medically significant snakes in Africa, primarily due to a combination of its extensive geographical distribution, common occurrence and highly potent haemorrhagic and cytotoxic venom. Our investigation has revealed a remarkable degree of intra-species variation between pooled venom samples from different geographical origins across sub-Saharan Africa and Arabia, and within a group of individual specimens from the same origin in Nigeria as determined by a combination of immunological, biochemical and proteomic assays. We demonstrate significant quantitative and qualitative differences between B. Arietans venom in terms of protein expression, immunogenicity and activity of snake venom metalloproteinases (SVMPs); toxins with a primary role in the haemorrhagic and tissue-necrotic pathologies suffered by envenomed victims. Specifically, we have identified a processed PII SVMP that exhibits striking inter-specimen variability.
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Molecular Cloning of Disintegrin-like Transcript BA-5A from a Bitis Arietans Venom Gland cDNA Library: A Putative Intermediate in the Evolution of the Long-Chain Disintegrin Bitistatin
Journal of Molecular Evolution, 2006Co-Authors: Paula Juárez, Simon C. Wagstaff, Robert A. Harrison, Jenny Oliver, Libia Sanz, Juan J. CalveteAbstract:We report the cloning and sequence analysis of BA-5A from a venom gland cDNA library of the puff adder, Bitis Arietans , that encodes a novel ECD-disintegrin-like domain. BA-5A is a unique PII disintegrin. It contains the 16 cysteine residues that are conserved in all known disintegrin-like domains of ADAM proteins and snake venom metalloproteinases but lacks the cysteine-rich domain. These features suggest that BA-5A may represent an intermediate in the evolutionary pathway of the long disintegrin Bitistatin and that removal of the cysteine-rich domain and loss of the PIII-specific disulfide bond were separate events along the structural diversification pathway of disintegrins, the former predating the latter. The protein family composition of the Bitis Arietans venom, as determined by combination of reversed-phase HPLC and proteomic analysis, was as follows: Zn^2+-metalloproteinase (38.5%), serine proteinase (19.5%), disintegrin (17.8%), C-type lectin-like (13.2%), PLA_2 (4.3%), Kunitz-type inhibitor (4.1%), cystatin (1.7%), and unknown (0.9%). BA-5A could not be detected in the venom proteome of Bitis Arietans . The occurrence of this very low-abundance (< 0.05%) or nonexpressed disintegrin transcript indicates a hitherto unrecognized structural diversity of this protein family. Whether BA-5A plays a physiological role or represents an orphan protein which could eventually evolve a role in the adaptation of snakes to changing ecological niches and prey habits deserves further investigation.
Robert A. Harrison - One of the best experts on this subject based on the ideXlab platform.
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Bitis Arietans primers.
2016Co-Authors: Gareth Whiteley, Rhiannon A. E. Logan, Kam-yin D. Leung, Fiona J. Newberry, Paul D. Rowley, John P. Dunbar, Simon C. Wagstaff, Nicholas R. Casewell, Robert A. HarrisonAbstract:Bitis Arietans primers.
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PCR amplification of cDNA constructed from venom gland and venom mRNA.
2012Co-Authors: Rachel B. Currier, Paul D. Rowley, Robert A. Harrison, Libia Sanz, Juan J. Calvete, Simon C. WagstaffAbstract:Qualitatively similar PCR products were amplified from cDNA from venom gland (A) or venom (B), using primers complementary to Bitis Arietans venom metalloproteinases (SVMP), phospholipase A2 (PLA2), serine protease (SP), C-type lectins (CTL), vascular endothelial growth factor (VEGF), L-amino acid oxidase (LAO), Kunitz inhibitors (KTI), protein disulphide isomerase (PDI) and QKW inhibitory peptides (QKW). Molecular weight markers (M) are shown to the left.
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exploring the venom proteome of the african puff adder Bitis Arietans using a combinatorial peptide ligand library approach at different phs
Journal of Proteomics, 2010Co-Authors: Elisa Fasoli, Simon C. Wagstaff, Robert A. Harrison, Libia Sanz, Pier Giorgio Righetti, Juan J. CalveteAbstract:We report the 2DE-based proteomic characterization of the venom of the medically important African puff adder, Bitis Arietans, after prefractionation by incubation with a solid-phase combinatorial hexapeptide ligand library (CPLL) at three different pH values. This approach yielded partially overlapping yet clearly distinct sets of proteins. The B. Arietans venom proteome, merged from the four sets of proteins comprises at least 43 distinct proteins from 9 toxin families. In line with a previous reverse-phase HPLC-based venomic characterization on the same species, SVMPs, serine proteinases, C-type lectin-like proteins, and to a minor extent PLA(2), disintegrin Bitistatin, and cystatin, comprise the major toxins in the venom of B. Arietans. However, the 2D-CPLL approach employed here identified both a significantly higher (about double) number of proteins than a previous venomic approach, and many very minor components barely, or not at all, detectable in the 2DE separation of whole venom. 30 proteins from the CPLL-merged venom proteome matched some of the 63 toxin clusters generated by sequencing one thousand randomly selected venom gland cDNA library clones of the same species. The low (47%) concordance between transcriptome and proteome may be interpreted in terms of intraspecific venom variation. Comparison of the reverse-phase HPLC separations of the venom proteins of B. Arietans from Ghana and Nigeria supports this view.
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Intra-specific variation in venom of the African Puff Adder (Bitis Arietans): Differential expression and activity of snake venom metalloproteinases (SVMPs).
Toxicon, 2010Co-Authors: Rachel B. Currier, Paul D. Rowley, Robert A. Harrison, Gavin D. Laing, Simon C. WagstaffAbstract:Bitis Arietans is considered one of the most medically significant snakes in Africa, primarily due to a combination of its extensive geographical distribution, common occurrence and highly potent haemorrhagic and cytotoxic venom. Our investigation has revealed a remarkable degree of intra-species variation between pooled venom samples from different geographical origins across sub-Saharan Africa and Arabia, and within a group of individual specimens from the same origin in Nigeria as determined by a combination of immunological, biochemical and proteomic assays. We demonstrate significant quantitative and qualitative differences between B. Arietans venom in terms of protein expression, immunogenicity and activity of snake venom metalloproteinases (SVMPs); toxins with a primary role in the haemorrhagic and tissue-necrotic pathologies suffered by envenomed victims. Specifically, we have identified a processed PII SVMP that exhibits striking inter-specimen variability.
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Molecular Cloning of Disintegrin-like Transcript BA-5A from a Bitis Arietans Venom Gland cDNA Library: A Putative Intermediate in the Evolution of the Long-Chain Disintegrin Bitistatin
Journal of Molecular Evolution, 2006Co-Authors: Paula Juárez, Simon C. Wagstaff, Robert A. Harrison, Jenny Oliver, Libia Sanz, Juan J. CalveteAbstract:We report the cloning and sequence analysis of BA-5A from a venom gland cDNA library of the puff adder, Bitis Arietans , that encodes a novel ECD-disintegrin-like domain. BA-5A is a unique PII disintegrin. It contains the 16 cysteine residues that are conserved in all known disintegrin-like domains of ADAM proteins and snake venom metalloproteinases but lacks the cysteine-rich domain. These features suggest that BA-5A may represent an intermediate in the evolutionary pathway of the long disintegrin Bitistatin and that removal of the cysteine-rich domain and loss of the PIII-specific disulfide bond were separate events along the structural diversification pathway of disintegrins, the former predating the latter. The protein family composition of the Bitis Arietans venom, as determined by combination of reversed-phase HPLC and proteomic analysis, was as follows: Zn^2+-metalloproteinase (38.5%), serine proteinase (19.5%), disintegrin (17.8%), C-type lectin-like (13.2%), PLA_2 (4.3%), Kunitz-type inhibitor (4.1%), cystatin (1.7%), and unknown (0.9%). BA-5A could not be detected in the venom proteome of Bitis Arietans . The occurrence of this very low-abundance (< 0.05%) or nonexpressed disintegrin transcript indicates a hitherto unrecognized structural diversity of this protein family. Whether BA-5A plays a physiological role or represents an orphan protein which could eventually evolve a role in the adaptation of snakes to changing ecological niches and prey habits deserves further investigation.
Libia Sanz - One of the best experts on this subject based on the ideXlab platform.
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PCR amplification of cDNA constructed from venom gland and venom mRNA.
2012Co-Authors: Rachel B. Currier, Paul D. Rowley, Robert A. Harrison, Libia Sanz, Juan J. Calvete, Simon C. WagstaffAbstract:Qualitatively similar PCR products were amplified from cDNA from venom gland (A) or venom (B), using primers complementary to Bitis Arietans venom metalloproteinases (SVMP), phospholipase A2 (PLA2), serine protease (SP), C-type lectins (CTL), vascular endothelial growth factor (VEGF), L-amino acid oxidase (LAO), Kunitz inhibitors (KTI), protein disulphide isomerase (PDI) and QKW inhibitory peptides (QKW). Molecular weight markers (M) are shown to the left.
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exploring the venom proteome of the african puff adder Bitis Arietans using a combinatorial peptide ligand library approach at different phs
Journal of Proteomics, 2010Co-Authors: Elisa Fasoli, Simon C. Wagstaff, Robert A. Harrison, Libia Sanz, Pier Giorgio Righetti, Juan J. CalveteAbstract:We report the 2DE-based proteomic characterization of the venom of the medically important African puff adder, Bitis Arietans, after prefractionation by incubation with a solid-phase combinatorial hexapeptide ligand library (CPLL) at three different pH values. This approach yielded partially overlapping yet clearly distinct sets of proteins. The B. Arietans venom proteome, merged from the four sets of proteins comprises at least 43 distinct proteins from 9 toxin families. In line with a previous reverse-phase HPLC-based venomic characterization on the same species, SVMPs, serine proteinases, C-type lectin-like proteins, and to a minor extent PLA(2), disintegrin Bitistatin, and cystatin, comprise the major toxins in the venom of B. Arietans. However, the 2D-CPLL approach employed here identified both a significantly higher (about double) number of proteins than a previous venomic approach, and many very minor components barely, or not at all, detectable in the 2DE separation of whole venom. 30 proteins from the CPLL-merged venom proteome matched some of the 63 toxin clusters generated by sequencing one thousand randomly selected venom gland cDNA library clones of the same species. The low (47%) concordance between transcriptome and proteome may be interpreted in terms of intraspecific venom variation. Comparison of the reverse-phase HPLC separations of the venom proteins of B. Arietans from Ghana and Nigeria supports this view.
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Molecular Cloning of Disintegrin-like Transcript BA-5A from a Bitis Arietans Venom Gland cDNA Library: A Putative Intermediate in the Evolution of the Long-Chain Disintegrin Bitistatin
Journal of Molecular Evolution, 2006Co-Authors: Paula Juárez, Simon C. Wagstaff, Robert A. Harrison, Jenny Oliver, Libia Sanz, Juan J. CalveteAbstract:We report the cloning and sequence analysis of BA-5A from a venom gland cDNA library of the puff adder, Bitis Arietans , that encodes a novel ECD-disintegrin-like domain. BA-5A is a unique PII disintegrin. It contains the 16 cysteine residues that are conserved in all known disintegrin-like domains of ADAM proteins and snake venom metalloproteinases but lacks the cysteine-rich domain. These features suggest that BA-5A may represent an intermediate in the evolutionary pathway of the long disintegrin Bitistatin and that removal of the cysteine-rich domain and loss of the PIII-specific disulfide bond were separate events along the structural diversification pathway of disintegrins, the former predating the latter. The protein family composition of the Bitis Arietans venom, as determined by combination of reversed-phase HPLC and proteomic analysis, was as follows: Zn^2+-metalloproteinase (38.5%), serine proteinase (19.5%), disintegrin (17.8%), C-type lectin-like (13.2%), PLA_2 (4.3%), Kunitz-type inhibitor (4.1%), cystatin (1.7%), and unknown (0.9%). BA-5A could not be detected in the venom proteome of Bitis Arietans . The occurrence of this very low-abundance (< 0.05%) or nonexpressed disintegrin transcript indicates a hitherto unrecognized structural diversity of this protein family. Whether BA-5A plays a physiological role or represents an orphan protein which could eventually evolve a role in the adaptation of snakes to changing ecological niches and prey habits deserves further investigation.
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Molecular Cloning of Disintegrin-like Transcript BA-5A from a Bitis Arietans Venom Gland cDNA Library: A Putative Intermediate in the Evolution of the Long-Chain Disintegrin Bitistatin
Journal of Molecular Evolution, 2006Co-Authors: Paula Juárez, Simon C. Wagstaff, Robert A. Harrison, Jenny Oliver, Libia Sanz, Juan J. CalveteAbstract:We report the cloning and sequence analysis of BA-5A from a venom gland cDNA library of the puff adder, Bitis Arietans , that encodes a novel ECD-disintegrin-like domain. BA-5A is a unique PII disintegrin. It contains the 16 cysteine residues that are conserved in all known disintegrin-like domains of ADAM proteins and snake venom metalloproteinases but lacks the cysteine-rich domain. These features suggest that BA-5A may represent an intermediate in the evolutionary pathway of the long disintegrin Bitistatin and that removal of the cysteine-rich domain and loss of the PIII-specific disulfide bond were separate events along the structural diversification pathway of disintegrins, the former predating the latter. The protein family composition of the Bitis Arietans venom, as determined by combination of reversed-phase HPLC and proteomic analysis, was as follows: Zn^2+-metalloproteinase (38.5%), serine proteinase (19.5%), disintegrin (17.8%), C-type lectin-like (13.2%), PLA_2 (4.3%), Kunitz-type inhibitor (4.1%), cystatin (1.7%), and unknown (0.9%). BA-5A could not be detected in the venom proteome of Bitis Arietans . The occurrence of this very low-abundance (< 0.05%) or nonexpressed disintegrin transcript indicates a hitherto unrecognized structural diversity of this protein family. Whether BA-5A plays a physiological role or represents an orphan protein which could eventually evolve a role in the adaptation of snakes to changing ecological niches and prey habits deserves further investigation.
Yuri N. Utkin - One of the best experts on this subject based on the ideXlab platform.
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an unusual phospholipase a2 from puff adder Bitis Arietans venom a novel blocker of nicotinic acetylcholine receptors
Toxicon, 2011Co-Authors: Catherine A. Vulfius, Elena V. Gorbacheva, Vladislav G. Starkov, Alexey V. Osipov, Igor E. Kasheverov, T. V. Andreeva, M. E. Astashev, Victor I. Tsetlin, Yuri N. UtkinAbstract:Abstract The venoms of snakes from Viperidae family mainly influence the function of various blood components. However, the published data indicate that these venoms contain also neuroactive components, the most studied being neurotoxic phospholipases A 2 (PLA 2 s). Earlier we have shown (Gorbacheva et al., 2008) that several Viperidae venoms blocked nicotinic acetylcholine receptors (nAChRs) and voltage-gated Ca 2+ channels in isolated identified neurons of the fresh-water snail Lymnaea stagnalis . In this paper, we report on isolation from puff adder Bitis Arietans venom and characterization of a novel protein bitanarin that reversibly blocks nAChRs. To isolate the protein, the venom of B. Arietans was fractionated by gel-filtration, ion-exchange and reversed phase chromatography and fractions obtained were screened for capability to block nAChRs. The isolated protein competed with [ 125 I]iodinated α-bungarotoxin for binding to human α7 and Torpedo californica nAChRs, as well as to acetylcholine-binding protein from L. stagnalis , the IC 50 being 20 ± 1.5, 4.3 ± 0.2, and 10.6 ± 0.6 μM, respectively. It also blocked reversibly acetylcholine-elicited current in isolated L. stagnalis neurons with IC 50 of 11.4 μM. Mass-spectrometry analysis determined the molecular mass of 27.4 kDa and the presence of 28 cysteine residues forming 14 disulphide bonds. Edman degradation of the protein and tryptic fragments showed its similarity to PLA 2 s from snake venoms. Indeed, the protein possessed high PLA 2 activity, which was 1.95 mmol/min/μmol. Bitanarin is the first described PLA 2 that contains 14 disulphide bonds and the first nAChR blocker possessing PLA 2 activity.
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An unusual phospholipase A2 from puff adder Bitis Arietans venom – a novel blocker of nicotinic acetylcholine receptors
Toxicon, 2011Co-Authors: Catherine A. Vulfius, Elena V. Gorbacheva, Vladislav G. Starkov, Alexey V. Osipov, Igor E. Kasheverov, T. V. Andreeva, M. E. Astashev, Victor I. Tsetlin, Yuri N. UtkinAbstract:Abstract The venoms of snakes from Viperidae family mainly influence the function of various blood components. However, the published data indicate that these venoms contain also neuroactive components, the most studied being neurotoxic phospholipases A 2 (PLA 2 s). Earlier we have shown (Gorbacheva et al., 2008) that several Viperidae venoms blocked nicotinic acetylcholine receptors (nAChRs) and voltage-gated Ca 2+ channels in isolated identified neurons of the fresh-water snail Lymnaea stagnalis . In this paper, we report on isolation from puff adder Bitis Arietans venom and characterization of a novel protein bitanarin that reversibly blocks nAChRs. To isolate the protein, the venom of B. Arietans was fractionated by gel-filtration, ion-exchange and reversed phase chromatography and fractions obtained were screened for capability to block nAChRs. The isolated protein competed with [ 125 I]iodinated α-bungarotoxin for binding to human α7 and Torpedo californica nAChRs, as well as to acetylcholine-binding protein from L. stagnalis , the IC 50 being 20 ± 1.5, 4.3 ± 0.2, and 10.6 ± 0.6 μM, respectively. It also blocked reversibly acetylcholine-elicited current in isolated L. stagnalis neurons with IC 50 of 11.4 μM. Mass-spectrometry analysis determined the molecular mass of 27.4 kDa and the presence of 28 cysteine residues forming 14 disulphide bonds. Edman degradation of the protein and tryptic fragments showed its similarity to PLA 2 s from snake venoms. Indeed, the protein possessed high PLA 2 activity, which was 1.95 mmol/min/μmol. Bitanarin is the first described PLA 2 that contains 14 disulphide bonds and the first nAChR blocker possessing PLA 2 activity.