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Sergio Marangoni - One of the best experts on this subject based on the ideXlab platform.

  • Neuromuscular effects of venoms and Crotoxin-like proteins from Crotalus durissus ruruima and Crotalus durissus cumanensis.
    Toxicon : official journal of the International Society on Toxinology, 2015
    Co-Authors: Walter L.g. Cavalcante, Luis Alberto Ponce-soto, Sergio Marangoni, Márcia Gallacci
    Abstract:

    A myographic study was performed to compare the neuromuscular effects of venoms and Crotoxin-like proteins from Crotalus durissus ruruima and Crotalus durissus cumanensis in mice phrenic-diaphragm preparation. It was concluded that both venoms present neurotoxic activity as a consequence of their Crotoxin content. Furthermore, Crotoxin from C.d. cumanensis is more potent than that from C.d. ruruima venom. At the concentration range in which both venoms express neurotoxic activity, only C.d. cumanensis venom also manifest a direct myotoxic effect that probably involves the synergic participation of other components than Crotoxin.

  • ability of rabbit antiserum against crotapotin to neutralize the neurotoxic myotoxic and phospholipase a2 activities of Crotoxin from crotalus durissus cascavella snake venom
    Toxicology in Vitro, 2008
    Co-Authors: Daniela G Beghini, Daniela C S Damico, Maria Alice Da Cruzhofling, Lea Rodriguessimioni, Maria Carolina Delatorre, Stephen Hyslop, Sergio Marangoni
    Abstract:

    The toxicity of Crotoxin, the major toxin of Crotalus durissus terrificus (South American rattlesnake) venom, is mediated by its basic phospholipase A2 (PLA2) subunit. This PLA2 is non-covalently associated with crotapotin, an acidic, enzymatically inactive subunit of the Crotoxin complex. In this work, rabbit antiserum raised against crotapotin purified from Crotalus durissus cascavella venom was tested for its ability to neutralize the neurotoxicity of this venom and its Crotoxin in vitro. The ability of this antiserum to inhibit the enzymatic activity of the Crotoxin complex and PLA2 alone was also assessed, and its potency in preventing myotoxicity was compared with that of antisera raised against Crotoxin and PLA2. Antiserum to crotapotin partially neutralized the neuromuscular blockade caused by venom and Crotoxin in electrically stimulated mouse phrenic nerve-hemidiaphragm preparations and prevented the venom-induced myotoxicity, but did not inhibit the enzymatic activity of Crotoxin and purified PLA2. In contrast, previous findings showed that antisera against Crotoxin and PLA2 from C. d. cascavella effectively neutralized the neuromuscular blockade and PLA2 activity of this venom and its Crotoxin. The partial neutralization of Crotoxin-mediated neurotoxicity by antiserum to crotapotin probably reduced the binding of Crotoxin to its receptor following interaction of the antiserum with the crotapotin moiety of the complex.

  • systemic and local myotoxicity induced by snake venom group ii phospholipases a2 comparison between Crotoxin Crotoxin b and a lys49 pla2 homologue
    Toxicon, 2008
    Co-Authors: Jose Maria Gutierrez, Sergio Marangoni, Luis Alberto Poncesoto, Bruno Lomonte
    Abstract:

    The patterns of myotoxicity induced in mice by Crotoxin, Crotoxin B and a Lys49 phospholipase A2 (PLA2) homologue were compared. Lys49 PLA2-induced local myotoxicity is reflected by creatine kinase (CK) loss in injected gastrocnemius muscle, and by a profile of CK increase in plasma characterized by a rapid increment and drop after intramuscular injection, and by a lack of CK increase in plasma after intravenous injection. In contrast, Crotoxin and Crotoxin B, which induce local and systemic myotoxicity, provoked a more prolonged increment in plasma CK activity upon intramuscular injection, and induced increments in plasma CK after intravenous injection. The three toxins promoted a similar extent of local myotoxicity, assessed by the loss of CK in injected gastrocnemius. A method for the quantitative assessment of the ability of toxins to induce systemic myotoxicity is proposed, based on the estimation of the ratio between the area under the curve in the plasma CK activity (total myotoxicity) to the loss of CK in injected gastrocnemius (local myotoxicity). The highest ratio corresponded to Crotoxin, and the lowest corresponded to Lys49 PLA2, the former being a systemic myotoxin and the latter a local myotoxin. Neutralization by antivenoms also differed between the toxins: a drastic reduction in plasma CK, with very poor neutralization of local CK loss, was achieved in the case of Crotoxin B when antivenom was injected intravenously, whereas no neutralization was achieved in the case of Lys49 PLA2. When tested in undifferentiated myoblasts in culture, Lys49 PLA2 induced cytotoxicity, whereas Crotoxin and Crotoxin B did not, evidencing that the latter are devoid of widespread cytolytic activity. Molecular modeling analysis showed that Lys49 PLA2 has a conspicuous cationic face, which is likely to interact with diverse membranes. In contrast, Crotoxin B, despite its overall basic pI, has a lower density of positively charged residues at this molecular region. It is suggested that Lys49 PLA2s homologues interact, through this cationic face, with many different cell types, thus lacking specificity for muscle cells. In contrast, Crotoxin B has a more selective interaction with targets in the muscle cell membrane. This selectivity might be the basis for the ability of Crotoxin and Crotoxin B to induce systemic myotoxicity.

  • Biological and Structural Characterization of Crotoxin and New Isoform of Crotoxin B PLA_2 (F6a) from Crotalus durissus collilineatus Snake Venom
    The Protein Journal, 2007
    Co-Authors: Luis Alberto Ponce-soto, Bruno Lomonte, Lea Rodrigues-simioni, José Camillo Novello, Sergio Marangoni
    Abstract:

    A new Crotoxin B isoform PLA_2 (F6a), from Crotalus durissus collilineatus was purified from by one step reverse phase HPLC chromatography using μ-Bondapack C-18 column analytic. The new Crotoxin B isoform PLA_2 (F6a), complex Crotoxin, the catalytic subunit Crotoxin B isoform PLA_2 (F6a) and two crotapotin isoforms (F3 and F4), were isolated from the venom of Crotalus durissus collilineatus . The crotapotins isoforms F3 and F4 had similar chemical properties, the two proteins different in their ability to inhibit of isoforms of PLA_2 (F6 and F6a). The molecular masses estimated by MALDI-TOF mass spectrometry were: Crotoxin B: 14,943.14 Da, crotapotin F3: 8,693.24 Da, and crotapotin F4: 9 314.56 Da. The new Crotoxin B isoform PLA_2 (F6a) contained 122 amino acid residues and a pI of 8.58. Its amino acid sequence presents high identity with those of other PLA_2s, particularly in the calcium binding loop and active site helix 3. It also presents similarities in the C-terminal region with other myotoxic PLA_2s. The new Crotoxin B isoform PLA_2 (F6a) contained 122 amino acid residues, with a primary structure of HLLQFNKMIK FETRRNAIPP YAFYGCYCGW GGRGRPKDAT DRCCFVHDCC YGKLAKCNTK WDFYRYSLKS GYITCGKGTW CEEQICECDR VAAECLRRSL STYRYGYMIY PDSRCRGPSE TC. A neuromuscular blocking activity was induced by Crotoxin and new Crotoxin B isoform PLA_2 (F6a) in the isolated mouse phrenic nerve diaphragm and the biventer cervicis chick nerve-muscle preparation. Whole Crotoxin was devoid of cytolytic activity upon myoblasts and myotubes in vitro , whereas new Crotoxin B isoform PLA_2 (F6a) was clearly cytotoxic to these cells.

  • crystallization and preliminary x ray crystallographic analysis of the heterodimeric Crotoxin complex and the isolated subunits crotapotin and phospholipase a2
    Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2007
    Co-Authors: K F Santos, M.h. Toyama, Sergio Marangoni, Adelia Cristina Oliveira Cintra, Mario T Murakami, V P Forrer, J Brandao R Neto, Igor Polikarpov, Raghuvir K Arni
    Abstract:

    Crotoxin, a potent neurotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus, exists as a heterodimer formed between a phospholipase A2 and a catalytically inactive acidic phospholipase A2 analogue (crotapotin). Large single crystals of the Crotoxin complex and of the isolated subunits have been obtained. The Crotoxin complex crystal belongs to the orthorhombic space group P21212, with unit-cell parameters a = 38.2, b = 68.7, c = 84.2 A, and diffracted to 1.75 A resolution. The crystal of the phospholipase A2 domain belongs to the hexagonal space group P6122 (or its enantiomorph P6522), with unit-cell parameters a = b = 38.7, c = 286.7 A, and diffracted to 2.6 A resolution. The crotapotin crystal diffracted to 2.3 A resolution; however, the highly diffuse diffraction pattern did not permit unambiguous assignment of the unit-cell parameters.

Cassian Bon - One of the best experts on this subject based on the ideXlab platform.

  • Crotoxin acceptor protein isolated from torpedo electric organ binding properties to Crotoxin by surface plasmon resonance
    Toxicon, 2003
    Co-Authors: Grazyna Faure, Cassian Bon, Alenka Copic, Sabine Le Porrier, Franc Gubensek, Igor Križaj
    Abstract:

    Abstract Crotoxin, a potent neurotoxin from the South American rattlesnake Crotalus durissus terrificus, is a heterodimeric phospholipase A2 (EC 3.1.1.4), which blocks the release of acetylcholine from peripheral neurons. We previously have suggested the existence of a 48 kDa Crotoxin-binding protein in the presynaptic membranes of the electric organ of Torpedo marmorata. Here, we report the purification and characterization of this protein that we called the Crotoxin acceptor protein from Torpedo (CAPT). The membranes of electric organs from Torpedo were solubilized with a detergent (4% (w/v) Triton X-100) and CAPT was isolated by affinity chromatography on a Crotoxin column. SDS–PAGE showed that the purified protein was homogeneous and cross-linking studies with radioiodinated Crotoxin confirmed that it had retained its toxin-binding properties. The purified CAPT has similar molecular mass as crocalbin, a Crotoxin-binding protein isolated from porcine brains, yet anti-crocalbin antiserum failed to recognize CAPT. Surface plasmon resonance biosensor technology was used to measure the specific interaction between Crotoxin and solubilized CAPT. Using this method, it was possible to follow CAPT throughout the purification procedure. As well, an apparent dissociation constant (Kdapp) of 3.4 nM was calculated for the interaction of pure CAPT and Crotoxin from the dissociation rate constant (koff=1.2×10−2 s−1) and the association rate constant (kon=3.5×106 M−1s−1).

  • inhibition of Crotoxin phospholipase a2 activity by manoalide associated with inactivation of Crotoxin toxicity and dissociation of the heterodimeric neurotoxic complex
    Biochemical Pharmacology, 2002
    Co-Authors: Frederic Dorandeu, Cassian Bon, Remi Hesters, Fabien Girard, Elise Four, Annie Foquin, Guy Lallement, Grazyna Faure
    Abstract:

    Abstract Crotoxin (CACB complex) is a convulsant heterodimeric neurotoxic phospholipase A2 (PLA2). The role of phospholipid hydrolysis in its epileptogenic properties remains unresolved. We, thus, studied the effect of manoalide (MLD), a PLA2 inhibitor, on the toxin catalytic activity and its central and peripheral toxicity. Incubation of Crotoxin with MLD fully and irreversibly inactivated its enzymatic activity. Interestingly, Crotoxin also lost its central neurotoxicity after intracerebroventricular injection and peripheral toxicity after intravenous administration. MLD-treated Crotoxin prevented the high affinity binding of [125I]-radiolabeled Crotoxin on rat cortex synaptic plasma membranes. Further analysis of MLD-treated Crotoxin by non-denaturing PAGE and surface plasmon resonance indicated that the Crotoxin complex was dissociated after MLD treatment. Although the loss of MLD-treated Crotoxin peripheral neurotoxicity could not be attributed to this dissociation, the presence of free CA subunit might explain the observed competition in binding experiments. In conclusion, the dissociation of the Crotoxin complex by MLD, as demonstrated in this study, did not permit to specify the role of the enzymatic activity in Crotoxin epileptogenic properties. Other approaches would be required to resolve this question.

  • interaction of the neurotoxic and nontoxic secretory phospholipases a2 with the Crotoxin inhibitor from crotalus serum
    FEBS Journal, 2000
    Co-Authors: Grazyna Faure, Jonas Perales, Christina Villela, Cassian Bon
    Abstract:

    Crotalus durissus terrificus snakes possess a protein in their blood, named Crotoxin inhibitor from Crotalus serum (CICS), which protects them against Crotoxin, the main toxin of their venom. CICS neutralizes the lethal potency of Crotoxin and inhibits its phospholipase A2 (PLA2) activity. The aim of the present study is to investigate the specificity of CICS towards snake venom neurotoxic PLA2s (beta-neurotoxins) and nontoxic mammalian PLA2s. This investigation shows that CICS does not affect the enzymatic activity of pancreatic and nonpancreatic PLA2s, bee venom PLA2 and Elapidae beta-neurotoxins but strongly inhibits the PLA2 activity of Viperidae beta-neurotoxins. Surface plasmon resonance and PAGE studies further demonstrated that CICS makes complexes with monomeric and multimeric Viperidae beta-neurotoxins but does not interact with nontoxic PLA2s. In the case of dimeric beta-neurotoxins from Viperidae venoms (Crotoxin, Mojave toxin and CbICbII), which are made by the noncovalent association of a PLA2 with a nonenzymatic subunit, CICS does not react with the noncatalytic subunit, instead it binds tightly to the PLA2 subunit and induces the dissociation of the heterocomplex. In vitro assays performed with Torpedo synaptosomes showed a protective action of CICS against Viperidae beta-neurotoxins but not against other PLA2 neurotoxins, on primary and evoked liberation of acetylcholine. In conclusion, CICS is a specific PLA2 inhibitor of the beta-neurotoxins from the Viperidae family.

  • biologically active human anti Crotoxin scfv isolated from a semi synthetic phage library
    Immunotechnology, 1997
    Co-Authors: Pierre Lafaye, Cassian Bon, Valerie Choumet, Caroline Demangel, Jeanclaude Mazie
    Abstract:

    Abstract Background : The display of repertoires of antibody fragments on the surface of filamentous bacteriophages offers a new way of making antibodies with predefined binding specificities. Objectives : Here we explored the use of this technology to find human antibodies with biological properties. Phage-scFv specific for Crotoxin, the main toxic component of the venom of the South-American rattlesnake Crotalus durissus terrificus , were isolated from a `single pot' repertoire of more than 10 8 clones made in vitro from human V gene segments [1] . The Crotoxin molecule is composed of two noncovalently linked subunits: a basic and weakly toxic phospholipase A 2 (PLA 2 ) called component B (CB) and an acidic, nonenzymatic and nontoxic subunit called component A (CA). CA is able to increase the toxicity as well as the specificity of action of CB simultaneously reducing its enzymatic activity. Study design : Two clones were isolated (4-21 and 5-3-1) which are specific of the basic subunit CB, but of a moderate affinity (about 10 −7 M). Clones 4-21 and 5-3-1 have different amino acid sequences and different effects on CB properties suggesting that they are raised against different CB epitopes. Purely cholinergic synaptosomes isolated from Torpedo electric organs provide a suitable model to study the presynaptic effects of Crotoxin. In this model, CB was shown to induce a larger acetylcholine release than Crotoxin. Results : A dose-dependent increase of acetylcholine release was observed when Crotoxin was incubated with increasing amounts of phage-scFv 4-21. This clone was also shown to increase the enzymatic activity of Crotoxin. These observations suggest that phage-scFv might dissociate the complex CA-CB. It could be therefore a neutralizing antibody since CB is much less toxic than Crotoxin. This shows that `single pot' libraries are capable of providing not only immunochemical reagents of high specificity but also biological reagents of high quality. The use of this library appears to open new possibilities for immune passive therapy.

  • neurotoxic phospholipases a2 ammodytoxin and Crotoxin bind to distinct high affinity protein acceptors in torpedo marmorata electric organ
    Biochemistry, 1997
    Co-Authors: I Krizaj, Grazyna Faure, F Gubensek, Cassian Bon
    Abstract:

    We studied the binding of radioiodinated ammodytoxin C, a monomeric phospholipase A2 neurotoxin from Vipera ammodytes, and of radioiodinated Crotoxin, a dimeric phospholipase A2 neurotoxin from Crotalus durissus terrificus, to presynaptic membranes from the electric organ of Torpedo marmorata. In both cases, two different families of specific binding sites were identified and characterized. The high-affinity binding sites for both toxins have been shown to be proteins. The low-affinity binding sites were not affected by proteinases or heat, suggesting the involvement of certain lipid structures in this type of binding. By affinity-labeling, [125I]ammodytoxin C was shown to be associated predominantly with membrane proteins of apparent molecular masses of 70,000 and 20,000 Da and to a lesser extent with several proteins of apparent molecular masses ranging between 39,000 and 57,000 Da. [125I]Crotoxin, on the other hand bound primarily to a 48,000 Da membrane protein. All phospholipases A2 tested, except beta-bungarotoxin, inhibited the low-affinity specific binding of ammodytoxin C, whereas only neurotoxic phospholipases A2 prevented the high-affinity binding and the cross-linking of ammodytoxin C and Crotoxin. The inhibition profiles of high-affinity binding for [125I]Crotoxin and for [125I]ammodytoxin C were quite different. Ammodytoxin C and Crotoxin did not inhibit each other on their respective high-affinity binding sites. These observations indicate that at least high-affinity binding sites of these two toxins are different. In contrast with Crotoxin, the isolated basic subunit CB of Crotoxin was able to completely inhibit the high-affinity binding of [125I]ammodytoxin C. Therefore, the acidic subunit CA of Crotoxin does not simply act as a chaperone for CB subunit, but it also confers a distinct binding specificity to the Crotoxin.

Grazyna Faure - One of the best experts on this subject based on the ideXlab platform.

  • crystallographic characterization of functional sites of Crotoxin and ammodytoxin potent β neurotoxins from viperidae venom
    Toxicon, 2012
    Co-Authors: Grazyna Faure, F A Saul
    Abstract:

    Abstract This review will focus on a description of the three-dimensional structures of two β-neurotoxins, the monomeric PLA 2 ammodytoxin from Vipera ammodytes ammodytes , and heterodimeric Crotoxin from Crotalus durissus terrificus , and a detailed structural analysis of their multiple functional sites. We have recently determined at high resolution the crystal structures of two natural isoforms of ammodytoxin (AtxA and AtxC) ( Saul et al., 2010 ) which exhibit different toxicity profiles and different anticoagulant properties. Comparative structural analysis of these two PLA 2 isoforms, which differ only by two amino acid residues, allowed us to detect local conformational changes and delineate the role of critical residues in the anticoagulant and neurotoxic functions of these PLA 2 ( Saul et al., 2010 ). We have also determined, at 1.35 A resolution, the crystal structure of heterodimeric Crotoxin ( Faure et al., 2011 ). The three-dimensional structure of Crotoxin revealed details of the binding interface between its acidic (CA) and basic (CB) subunits and allowed us to identify key residues involved in the stability and toxicity of this potent heterodimeric β-neurotoxin ( Faure et al., 2011 ). The precise spatial orientation of the three covalently linked polypeptide chains in the mature CA subunit complexed with CB helps us to understand the role played by critical residues of the CA subunit in the increased toxicity of the Crotoxin complex. Since the CA subunit is a natural inhibitor of the catalytic and anticoagulant activities of CB, identification of the CA–CB binding interface describes residues involved in this inhibition. We propose future research directions based on knowledge of the recently reported 3D structures of Crotoxin and ammodytoxin.

  • crystal structure of Crotoxin reveals key residues involved in the stability and toxicity of this potent heterodimeric β neurotoxin
    Journal of Molecular Biology, 2011
    Co-Authors: Grazyna Faure, F A Saul
    Abstract:

    Abstract The crystal structure of Crotoxin, a potent presynaptic neurotoxin from Crotalus durissus terrificus , was solved at 1.35 A resolution. It shows the architecture of the three disulfide-linked polypeptide chains (α, β, and γ) of the acidic subunit CA noncovalently complexed with the basic phospholipase A 2 (PLA 2 ) subunit CB. The unique structural scaffold of the association of the CA and CB subunits indicates that posttranslational cleavage of the pro-CA precursor is a prerequisite for the assembly of the CA–CB complex. These studies provide novel structural insights to explain the role of the CA subunit in the mechanism of action of Crotoxin. The crystal structure of the highly toxic and stable CA 2 CBb complex crystallized here allows us to identify key amino acid residues responsible for significant differences in the pharmacological activities of the two classes of Crotoxin complexes. In particular, we show that critical residues Trp31 and Trp70 of the CBb subunit establish intermolecular polar contacts with Asp99 and Asp89, respectively, of the β-chain of CA 2 and contribute to the stability and toxicity of the CA 2 CBb complex. These interactions also lead to decreased PLA 2 activity by partially blocking substrate access to the catalytic dyad and by masking several interfacial binding surface residues important for PLA 2 interaction with phospholipids. Identification of the binding interface between the CA subunits and the CB subunits of Crotoxin is important for the structure-based design of antineurotoxic inhibitors. Since Crotoxin displays numerous physiological functions, including antitumoral properties, knowledge of its three-dimensional structure will be useful for the understanding of these diverse effects.

  • Crotoxin acceptor protein isolated from torpedo electric organ binding properties to Crotoxin by surface plasmon resonance
    Toxicon, 2003
    Co-Authors: Grazyna Faure, Cassian Bon, Alenka Copic, Sabine Le Porrier, Franc Gubensek, Igor Križaj
    Abstract:

    Abstract Crotoxin, a potent neurotoxin from the South American rattlesnake Crotalus durissus terrificus, is a heterodimeric phospholipase A2 (EC 3.1.1.4), which blocks the release of acetylcholine from peripheral neurons. We previously have suggested the existence of a 48 kDa Crotoxin-binding protein in the presynaptic membranes of the electric organ of Torpedo marmorata. Here, we report the purification and characterization of this protein that we called the Crotoxin acceptor protein from Torpedo (CAPT). The membranes of electric organs from Torpedo were solubilized with a detergent (4% (w/v) Triton X-100) and CAPT was isolated by affinity chromatography on a Crotoxin column. SDS–PAGE showed that the purified protein was homogeneous and cross-linking studies with radioiodinated Crotoxin confirmed that it had retained its toxin-binding properties. The purified CAPT has similar molecular mass as crocalbin, a Crotoxin-binding protein isolated from porcine brains, yet anti-crocalbin antiserum failed to recognize CAPT. Surface plasmon resonance biosensor technology was used to measure the specific interaction between Crotoxin and solubilized CAPT. Using this method, it was possible to follow CAPT throughout the purification procedure. As well, an apparent dissociation constant (Kdapp) of 3.4 nM was calculated for the interaction of pure CAPT and Crotoxin from the dissociation rate constant (koff=1.2×10−2 s−1) and the association rate constant (kon=3.5×106 M−1s−1).

  • inhibition of Crotoxin phospholipase a2 activity by manoalide associated with inactivation of Crotoxin toxicity and dissociation of the heterodimeric neurotoxic complex
    Biochemical Pharmacology, 2002
    Co-Authors: Frederic Dorandeu, Cassian Bon, Remi Hesters, Fabien Girard, Elise Four, Annie Foquin, Guy Lallement, Grazyna Faure
    Abstract:

    Abstract Crotoxin (CACB complex) is a convulsant heterodimeric neurotoxic phospholipase A2 (PLA2). The role of phospholipid hydrolysis in its epileptogenic properties remains unresolved. We, thus, studied the effect of manoalide (MLD), a PLA2 inhibitor, on the toxin catalytic activity and its central and peripheral toxicity. Incubation of Crotoxin with MLD fully and irreversibly inactivated its enzymatic activity. Interestingly, Crotoxin also lost its central neurotoxicity after intracerebroventricular injection and peripheral toxicity after intravenous administration. MLD-treated Crotoxin prevented the high affinity binding of [125I]-radiolabeled Crotoxin on rat cortex synaptic plasma membranes. Further analysis of MLD-treated Crotoxin by non-denaturing PAGE and surface plasmon resonance indicated that the Crotoxin complex was dissociated after MLD treatment. Although the loss of MLD-treated Crotoxin peripheral neurotoxicity could not be attributed to this dissociation, the presence of free CA subunit might explain the observed competition in binding experiments. In conclusion, the dissociation of the Crotoxin complex by MLD, as demonstrated in this study, did not permit to specify the role of the enzymatic activity in Crotoxin epileptogenic properties. Other approaches would be required to resolve this question.

  • interaction of the neurotoxic and nontoxic secretory phospholipases a2 with the Crotoxin inhibitor from crotalus serum
    FEBS Journal, 2000
    Co-Authors: Grazyna Faure, Jonas Perales, Christina Villela, Cassian Bon
    Abstract:

    Crotalus durissus terrificus snakes possess a protein in their blood, named Crotoxin inhibitor from Crotalus serum (CICS), which protects them against Crotoxin, the main toxin of their venom. CICS neutralizes the lethal potency of Crotoxin and inhibits its phospholipase A2 (PLA2) activity. The aim of the present study is to investigate the specificity of CICS towards snake venom neurotoxic PLA2s (beta-neurotoxins) and nontoxic mammalian PLA2s. This investigation shows that CICS does not affect the enzymatic activity of pancreatic and nonpancreatic PLA2s, bee venom PLA2 and Elapidae beta-neurotoxins but strongly inhibits the PLA2 activity of Viperidae beta-neurotoxins. Surface plasmon resonance and PAGE studies further demonstrated that CICS makes complexes with monomeric and multimeric Viperidae beta-neurotoxins but does not interact with nontoxic PLA2s. In the case of dimeric beta-neurotoxins from Viperidae venoms (Crotoxin, Mojave toxin and CbICbII), which are made by the noncovalent association of a PLA2 with a nonenzymatic subunit, CICS does not react with the noncatalytic subunit, instead it binds tightly to the PLA2 subunit and induces the dissociation of the heterocomplex. In vitro assays performed with Torpedo synaptosomes showed a protective action of CICS against Viperidae beta-neurotoxins but not against other PLA2 neurotoxins, on primary and evoked liberation of acetylcholine. In conclusion, CICS is a specific PLA2 inhibitor of the beta-neurotoxins from the Viperidae family.

Daniela G Beghini - One of the best experts on this subject based on the ideXlab platform.

  • Ability of rabbit antiserum against crotapotin to neutralize the neurotoxic, myotoxic and phospholipase A(2) activities of Crotoxin from Crotalus durissus cascavella snake venom
    Inglaterra, 2015
    Co-Authors: Daniela G Beghini, Damico Dcs, Maria Carolina Delatorre
    Abstract:

    The toxicity of Crotoxin, the major toxin of Crotalus durissus terrificus (South American rattlesnake) venom, is mediated by its basic phospholipase A(2) (PLA(2)) subunit. This PLA(2) is non-covalently associated with crotapotin, an acidic, enzymatically inactive subunit of the Crotoxin complex. In this work, rabbit antiserum raised against crotapotin purified from Crotalus durissus eascavella venom was tested for its ability to neutralize the neurotoxicity of this venom and its Crotoxin in vitro. The ability of this antiserum to inhibit the enzymatic activity of the Crotoxin complex and PLA(2) alone was also assessed, and its potency in preventing myotoxicity was compared with that of antisera raised against Crotoxin and PLA(2). Antiserum to crotapotin partially neutralized the neuromuscular blockade caused by venom and Crotoxin in electrically stimulated mouse phrenic nerve-hemidiaphragm preparations and prevented the venom-induced myotoxicity, but did not inhibit the enzymatic activity of Crotoxin and purified PLA(2). In contrast, previous findings showed that antisera against Crotoxin and PLA(2) from C. d. cascavella effectively neutralized the neuromuscular blockade and PLA(2) activity of this venom and its Crotoxin. The partial neutralization of Crotoxin-mediated neurotoxicity by antiserum to crotapotin probably reduced the binding of Crotoxin to its receptor following interaction of the antiserum with the crotapotin moiety of the complex. (c) 2007 Elsevier Ltd. All rights reserved.22124024

  • Cross-neutralization of the neurotoxicity of Crotalus durissus terrificus and Bothrops jararacussu venoms by antisera against Crotoxin and phospholipase A(2) from Crotalus durissus cascavella venom
    Inglaterra, 2015
    Co-Authors: Daniela G Beghini, Jc Novello
    Abstract:

    We have previously demonstrated that rabbit antisera raised against Crotoxin from Crotalus durissus cascavella venom (cdc-Crotoxin) and its PLA(2) (cdc-PLA(2)) neutralized the neurotoxicity of this venom and its Crotoxin. In this study, we examined the ability of these antisera to neutralize the neurotoxicity of Crotalus durissus terrificus and Bothrops jararacussu venoms and their major toxins, cdt-Crotoxin and bothropstoxin-1 (BthTX-1), respectively, in mouse isolated phrenic nerve-diaphragm preparations. Immunoblotting showed that antiserum to cdc-Crotoxin recognized cdt-Crotoxin and BthTX-1, while antiserum to cdc-PLA(2) recognized cdt-PLA(2) and BthTX-1. ELISA corroborated this cross-reactivity. Antiserum to cdc-Crotoxin prevented the neuromuscular blockade caused by C d. terrificus venom and its Crotoxin at a venom/Crotoxin: antiserum ratio of 1:3. Antiserum to cdc-PLA(2) also neutralized the neuromuscular blockade caused by C. d. terrificus venom or its Crotoxin at venom or toxin:antiserum ratios of 1:3 and 1: 1, respectively. The neuromuscular blockade caused by B. jararacussu venom and BthTX-1 was also neutralized by the antisera to cdc-Crotoxin and cdc-PLA(2) at a venom/toxin:antiserum ratio of 1: 10 for both. Commercial equine antivenom raised against C.d. terrificus venom was effective in preventing the neuromuscular blockade typical of B. jararacussu venom (venom: anti venom ratio of 1:2), whereas for BthTX-1 the ratio was 1:10. These results show that antiserum produced against PLA(2) the major toxin in C durissus cascavella venom, efficiently neutralized the neurotoxicity of C. d. terrificus and B. jararacussu venoms and their PLA(2) toxins. (c) 2005 Published by Elsevier Ltd.46660461

  • ability of rabbit antiserum against crotapotin to neutralize the neurotoxic myotoxic and phospholipase a2 activities of Crotoxin from crotalus durissus cascavella snake venom
    Toxicology in Vitro, 2008
    Co-Authors: Daniela G Beghini, Daniela C S Damico, Maria Alice Da Cruzhofling, Lea Rodriguessimioni, Maria Carolina Delatorre, Stephen Hyslop, Sergio Marangoni
    Abstract:

    The toxicity of Crotoxin, the major toxin of Crotalus durissus terrificus (South American rattlesnake) venom, is mediated by its basic phospholipase A2 (PLA2) subunit. This PLA2 is non-covalently associated with crotapotin, an acidic, enzymatically inactive subunit of the Crotoxin complex. In this work, rabbit antiserum raised against crotapotin purified from Crotalus durissus cascavella venom was tested for its ability to neutralize the neurotoxicity of this venom and its Crotoxin in vitro. The ability of this antiserum to inhibit the enzymatic activity of the Crotoxin complex and PLA2 alone was also assessed, and its potency in preventing myotoxicity was compared with that of antisera raised against Crotoxin and PLA2. Antiserum to crotapotin partially neutralized the neuromuscular blockade caused by venom and Crotoxin in electrically stimulated mouse phrenic nerve-hemidiaphragm preparations and prevented the venom-induced myotoxicity, but did not inhibit the enzymatic activity of Crotoxin and purified PLA2. In contrast, previous findings showed that antisera against Crotoxin and PLA2 from C. d. cascavella effectively neutralized the neuromuscular blockade and PLA2 activity of this venom and its Crotoxin. The partial neutralization of Crotoxin-mediated neurotoxicity by antiserum to crotapotin probably reduced the binding of Crotoxin to its receptor following interaction of the antiserum with the crotapotin moiety of the complex.

  • anti sera raised in rabbits against Crotoxin and phospholipase a2 from crotalus durissus cascavella venom neutralize the neurotoxicity of the venom and Crotoxin
    Toxicon, 2004
    Co-Authors: Daniela G Beghini, Lea Rodriguessimioni, Stephen Hyslop, Jc Novello, Saraguaci Hernandezoliveira, Sergio Marangoni
    Abstract:

    Crotoxin, the principal neurotoxin in venom of the South American rattlesnakes Crotalus durissus terrificus and Crotalus durissus cascavella, contains a basic phospholipase A2 (PLA2) and an acidic protein, crotapotin. In this work, we examined the ability of rabbit anti-sera against Crotoxin and its PLA2 subunit to neutralize the neurotoxicity of venom and Crotoxin from C. d. cascavella in mouse phrenic nerve-diaphragm and chick biventer cervicis preparations. Immunoblotting showed that the anti-sera recognized C. d. cascavella Crotoxin and PLA2. This was confirmed by ELISA, with both anti-sera having end-point dilutions of 3 x 10(-6). Anti-Crotoxin serum neutralized the neuromuscular blockade in phrenic nerve-diaphragm muscle preparations at venom or Crotoxin:anti-serum ratios of 1:2 and 1:3, respectively. Anti-PLA2 serum also neutralized this neuromuscular activity at a venom or Crotoxin:anti-serum ratio of 1:1. In biventer cervicis preparations, the corresponding ratio for anti-Crotoxin serum was 1:3 for venom and Crotoxin, and 1:1 and 1:2 for anti-PLA2 serum. The neutralizing capacity of the sera in mouse preparations was comparable to that of commercial anti-serum raised against C. d. terrificus venom. These results show that anti-sera against Crotoxin and PLA2 from C. d. cascavella venom neutralized the neuromuscular blockade induced by venom and Crotoxin in both nerve-muscle preparations, with the anti-serum against Crotoxin being slightly less potent than that against Crotoxin.

  • neurotoxic and myotoxic actions of Crotoxin like and crotalus durissus cascavella whole venom in the chick biventer cervicis preparation
    Toxicon, 2004
    Co-Authors: Daniela G Beghini, Maria Alice Da Cruzhofling, Lea Rodriguessimioni, M.h. Toyama, Jc Novello, Sergio Marangoni
    Abstract:

    Crotoxin from Crotalus durissus cascavella venom was purified by a combination of molecular exclusion chromatography (Superdex 75 column) and HPLC molecular exclusion (Protein Pack 300SW column). Neurotoxic and myotoxic effects from C. durissus cascavella whole venom and its main fraction, the Crotoxin-like, were studied in the chick biventer cervicis (CBC) nerve–muscle preparation. Both venom and its Crotoxin showed significant (p<0.05) blockade of neuromuscular transmission at concentrations as low as 0.2–1, 5 and 25 μg/ml, but no significant effect has been shown with a concentration of 0.04 μg/ml (n=5 each). The time required to produce 50% neuromuscular blockade with the venom and its Crotoxin was 53.6±8.2 and 65.9±4.9 min (0.2 μg/ml), 29.7±1.9 and 34.3±1.9 min (1 μg/ml), 24.8±1.6 and 21.1±1.5 min (5 μg/ml), 20.9±3.7 and 20.1±1.4 min (25 μg/ml), respectively. The addition to the incubation bath of acetylcholine (55 and 110 μM) or KCl (20.1 mM), either before or after the venom or the Crotoxin induced contracture in the presence of a total blockade, in all the concentrations used. Morphological analysis showed that the damage caused by C. durissus cascavella venom is stronger than that caused by Crotoxin. The myonecrotic picture was more marked at higher venom and Crotoxin doses (1, 5 or 25 μg/ml). Only at 25 μg/ml concentrations of the venom and Crotoxin, marked muscle fiber changes were detected. We concluded that the Crotoxin-like and the whole venom from C. durissus cascavella possess a preponderant and quite potent neurotoxic action in this preparation, and a myotoxic action which is observed only at higher doses.

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  • Crotoxin the major toxin from the rattlesnake crotalus durissus terrificus inhibits 3h choline uptake in guinea pig ileum
    Brazilian Journal of Medical and Biological Research, 2000
    Co-Authors: L S Kattah, C R Diniz, Marcelo M Santoro, M E De Lima
    Abstract:

    We examined the effect of Crotoxin, the neurotoxic complex from the venom of the South American rattlesnake Crotalus durissus terrificus, on the uptake of 3H-choline in minces of smooth muscle myenteric plexus from guinea pig ileum. In the concentration range used (0.03-1 µM) and up to 10 min of treatment, Crotoxin decreased 3H-choline uptake by 50-75% compared to control. This inhibition was time dependent and did not seem to be associated with the disruption of the neuronal membrane, because at least for the first 20 min of tissue exposure to the toxin (up to 1 µM) the levels of lactate dehydrogenase (LDH) released into the supernatant were similar to those of controls. Higher concentrations of Crotoxin or more extensive incubation times with this toxin resulted in elevation of LDH activity detected in the assay supernatant. The inhibitory effect of Crotoxin on 3H-choline uptake seems to be associated with its phospholipase activity since the equimolar substitution of Sr2+ for Ca2+ in the incubation medium or the modification of the toxin with p-bromophenacyl bromide substantially decreased this effect. Our results show that Crotoxin inhibits 3H-choline uptake with high affinity (EC25 = 10 ± 5 nM). We suggest that this inhibition could explain, at least in part, the blocking effect of Crotoxin on neurotransmission.

  • studies on the specificity of cnf a phospholipase a2 inhibitor isolated from the blood plasma of the south american rattlesnake crotalus durissus terrificus i interaction with pla2 from lachesis muta muta snake venom
    Toxicon, 1999
    Co-Authors: Consuelo Latorre Fortesdias, M L D Jannotti, F J L Franco, A Magalhaes, C R Diniz
    Abstract:

    Abstract A phospholipase A 2 inhibitor has been previously purified and cloned from the blood plasma of the South American rattlesnake, Crotalus durissus terrificus . This inhibitor, named CNF for Crotalus neutralizing factor, interacts with Crotoxin, the main neurotoxin from C. d. terrificus venom, abolishing its phospholipase A 2 activity. Crotoxin is a heterodimer of an acidic subunit (CA) and a basic phospholipase A 2 (CB). CNF acts by forming a stable non-toxic complex with CB, replacing CA in the toxic CA–CB of Crotoxin. In the present investigation, we have shown that CNF has a broader specificity. It is able to inhibit the PLA 2 activity of the whole venom from the bushmaster snake ( Lachesis muta muta ), a species evolutionary related to Crotalus . Inhibition experiments have been carried out with four PLA 2 active components isolated from L. m. muta venom, one basic and three acidic ones. CNF inhibition is not restricted to the basic PLA 2 , but extended to the three acidic forms as well.

  • time factor in the detection of circulating whole venom and Crotoxin and efficacy of antivenom therapy in patients envenomed by crotalus durissus
    Toxicon, 1997
    Co-Authors: Carlos Faria Santos Amaral, C R Diniz, Delio Campolina, M B Dias, Claudio M Bueno, Carlos Chavezolortegui, Claudia L Penaforte, Nilton Alves De Rezende
    Abstract:

    Abstract Thirty-seven patients envenomed by Crotalus durissus were classified into three groups according to the interval between the bite and hospital admission (ΔT): group 1 (n = 14, ΔT 4 hr 8 hr). Venous blood from these patients was sampled for biochemical and hematological analysis and for whole venom, Crotoxin and antivenom enzyme-linked immunosorbent assays before antivenom treatment (T0) and at 1 hr (T1), 6 hr (T6), 12 hr (T12) and 24 hr (T24) after the start of antivenom therapy. The patients were treated with 100–200 ml (10–20 ampules) of C. durissus antivenom. Whole venom and Crotoxin were detected in 13 (92.8%) and 11 (78.6%) of 14 group 1 patients, respectively, in 11 (78.6%) and six (42.9%) of 14 group 2 patients, respectively, and in two (22.2%) and one (11.1%) of nine group 3 patients, respectively, before antivenom treatment. Data from this study show that whole venom and Crotoxin were not detected in most of patients when the time elapsed between the bite and hospital admission was greater than 8 hr, and Crotoxin was not detected in most of the patients who were admitted to the hospital at times ranging from 4 to 8 hr after the snakebite. Plasma whole venom, Crotoxin and antivenom levels measured over time in these patients show the efficacy of antivenom treatment, since circulating venom and Crotoxin were no longer detected 1 hr after antivenom therapy and high antivenom titers persisted for at least 24 hr after serotherapy.

  • a phospholipase a2 inhibitor from the plasma of the south american rattlesnake crotalus durissus terrificus protein structure genomic structure and mechanism of action
    Journal of Biological Chemistry, 1994
    Co-Authors: Consuelo Latorre Fortesdias, J Ewell, C R Diniz
    Abstract:

    Abstract The lethal toxicity of the South American rattlesnake (Crotalus durissus terrificus) venom can be attributed mainly to the presence of a pre-synaptic neurotoxin, Crotoxin, with phospholipase A2 activity. Crotoxin is a heterodimer of an acidic protein (CA) and a basic phospholipase A2 (CB). An anti-toxic protein of subunit molecular mass 23.6 kDa that neutralizes both lethal and PLA2 activity of crotalid venom and Crotoxin has been previously purified from the plasma of this snake (Fortes-Dias, C., Fonseca, B. C. B., Kochva, E., and Diniz, C. R. (1991) Toxicon 29, 997-1008). The protein has been named CNF for Crotalus neutralizing factor. In the present study, we have shown that CNF exists as an oligomeric aggregate of (CNF)n, where n = 6-8, and when it interacts with Crotoxin, it replaces the acidic protein CA of Crotoxin to form a stable near stoichiometric complex of CNF.CB. The CNF.CB complex no longer exhibits PLA2 activity and is inert in vivo. Thus, the exchange reaction between CA.CB of Crotoxin and CNF to form CNF.CB and free CA is reminiscent of the interaction of Crotoxin with its target receptor at the neuromuscular transmission site in the presynaptic cells. A cDNA encoding CNF has been isolated from a liver cDNA library using an appropriate nucleotide probe. The nucleotide sequence codes for a 19-residue signal peptide, followed by a 181-residue protein of which 16 are half-cystines. Calculated molecular mass is 20.06 kDa, and there is a putative N-linked carbohydrate site at Asn157.