The Experts below are selected from a list of 1692 Experts worldwide ranked by ideXlab platform
Bruno Lomonte - One of the best experts on this subject based on the ideXlab platform.
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Effects of bothrops asper snake venom on lymphatic vessels: Insights into a hidden aspect of envenomation. PLoS Negl
2016Co-Authors: Javier Mora, Rodrigo Mora, Bruno LomonteAbstract:Background: Envenomations by the snake Bothrops asper represent a serious medical problem in Central America and parts of South America. These envenomations concur with drastic local tissue pathology, including a prominent edema. Since lymph flow plays a role in the maintenance of tissue fluid balance, the effect of B. asper venom on collecting lymphatic vessels was studied. Methodology/Principal Findings: B. asper venom was applied to mouse mesentery, and the effects were studied using an intravital microscopy methodology coupled with an image analysis program. B. asper venom induced a dose-dependent contraction of collecting lymphatic vessels, resulting in a reduction of their lumen and in a halting of lymph flow. The effect was reproduced by a myotoxic phospholipase A2 (PLA2) homologue isolated from this venom, but not by a hemorrhagic metalloproteinase or a coagulant thrombin-like serine proteinase. In agreement with this, treatment of the venom with fucoidan, a Myotoxin inhibitor, abrogated the effect, whereas no inhibition was observed after incubation with the peptidomimetic metalloproteinase inhibitor Batimastat. Moreover, fucoidan significantly reduced venom-induced footpad edema. The myotoxic PLA2 homologue, known to induce skeletal muscle necrosis, was able to induce cytotoxicity in smooth muscle cells in culture and to promote an increment in the permeability to propidium iodide in these cells. Conclusions/Significance: Our observations indicate that B. asper venom affects collecting lymphatic vessels through th
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a structure based proposal for a comprehensive myotoxic mechanism of phospholipase a2 like proteins from viperid snake venoms
Biochimica et Biophysica Acta, 2014Co-Authors: Carlos A H Fernandes, Bruno Lomonte, Rafael J Borges, Marcos R M FontesAbstract:Envenomation via snakebites is an important public health problem in many tropical and subtropical countries that, in addition to mortality, can result in permanent sequelae as a consequence of local tissue damage, which represents a major challenge to antivenom therapy. Venom phospholipases A2 (PLA2s) and PLA2-like proteins play a leading role in the complex pathogenesis of skeletal muscle necrosis, nevertheless their precise mechanism of action is only partially understood. Recently, detailed structural information has been obtained for more than twenty different members of the PLA2-like Myotoxin subfamily. In this review, we integrate the available structural, biochemical and functional data on these toxins and present a comprehensive hypothesis for their myotoxic mechanism. This process involves an allosteric transition and the participation of two independent interaction sites for docking and disruption of the target membrane, respectively, leading to a five-step mechanism of action. Furthermore, recent functional and structural studies of these toxins complexed with ligands reveal diverse neutralization mechanisms that can be classified into at least three different groups. Therefore, the data summarized here for the PLA2-like Myotoxins could provide a useful molecular basis for the search for novel neutralizing strategies to improve the treatment of envenomation by viperid snakes.
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Synergism between basic Asp49 and Lys49 phospholipase A2 Myotoxins of viperid snake venom in vitro and in vivo.
PloS one, 2014Co-Authors: Diana Mora-obando, Jose Maria Gutierrez, Julián Fernández, Cesare Montecucco, Bruno LomonteAbstract:Two subtypes of phospholipases A2 (PLA2s) with the ability to induce myonecrosis, ‘Asp49’ and ‘Lys49’ Myotoxins, often coexist in viperid snake venoms. Since the latter lack catalytic activity, two different mechanisms are involved in their myotoxicity. A synergism between Asp49 and Lys49 Myotoxins from Bothrops asper was previously observed in vitro, enhancing Ca2+ entry and cell death when acting together upon C2C12 myotubes. These observations are extended for the first time in vivo, by demonstrating a clear enhancement of myonecrosis by the combined action of these two toxins in mice. In addition, novel aspects of their synergism were revealed using myotubes. Proportions of Asp49 Myotoxin as low as 0.1% of the Lys49 Myotoxin are sufficient to enhance cytotoxicity of the latter, but not the opposite. Sublytic amounts of Asp49 Myotoxin also enhanced cytotoxicity of a synthetic peptide encompassing the toxic region of Lys49 Myotoxin. Asp49 Myotoxin rendered myotubes more susceptible to osmotic lysis, whereas Lys49 Myotoxin did not. In contrast to myotoxic Asp49 PLA2, an acidic non-toxic PLA2 from the same venom did not markedly synergize with Lys49 Myotoxin, revealing a functional difference between basic and acidic PLA2 enzymes. It is suggested that Asp49 Myotoxins synergize with Lys49 Myotoxins by virtue of their PLA2 activity. In addition to the membrane-destabilizing effect of this activity, Asp49 Myotoxins may generate anionic patches of hydrolytic reaction products, facilitating electrostatic interactions with Lys49 Myotoxins. These data provide new evidence for the evolutionary adaptive value of the two subtypes of PLA2 Myotoxins acting synergistically in viperid venoms.
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phospholipases a2 from viperidae snake venoms how do they induce skeletal muscle damage
Acta Chimica Slovenica, 2011Co-Authors: Bruno Lomonte, Jose Maria GutierrezAbstract:Phospholipases A 2 (PLA 2 s) are abundant components in snake venoms, which play important toxic roles. This review focuses on group II PLA2s endowed with myotoxic effects, present in Viperidae venoms. These PLA2s are subdivided into catalytically-active (Asp49) PLA 2 s, and catalytically-inactive PLA 2 homologues, the latter most frequently presenting the Lys49 substitution. Both protein subgroups induce skeletal muscle necrosis, although by different mechanisms. Current evidence indicates that phospholipid hydrolysis plays a central role in the necrotizing action of Asp49 Myotoxins, whereas PLA 2 homologues rely on the direct membrane-destabilizing actions of their cationic C-terminal region to achieve such effect, in the absence of catalysis. Both mechanisms converge in sarcolemmal permeabilization, triggering a series of intracellular events that lead to necrosis. Most viperid PLA 2 Myotoxins act only locally, but those forming heterodimeric complexes such as crotoxin spread to distant muscles and induce rhabdomyolysis. This divergence between local and systemic myotoxicity might be related to differences in binding specificity to cell targets. Nevertheless, the identity of molecular targets recognized by viperid PLA 2 Myotoxins remains elusive. Identification of their membrane target(s), and a deeper understanding of the catalytic-dependent and -independent mechanisms that result in membrane destabilization, are two crucial, but still unclarified aspects of their myotoxic action.
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Membrane cholesterol modulates the cytolytic mechanism of Myotoxin II, a Lys49 phospholipase A2 homologue from the venom of Bothrops asper
Cell biochemistry and function, 2011Co-Authors: José Rangel, Jose Maria Gutierrez, Yamileth Angulo, Orestes Quesada, Bruno LomonteAbstract:Lys49 phospholipase A2 (PLA2) homologues present in crotalid snake venoms lack enzymatic activity, yet they induce skeletal muscle necrosis by a membrane permeabilizing mechanism whose details are only partially understood. The present study evaluated the effect of altering the membrane cholesterol content on the cytolytic activity of Myotoxin II, a Lys49 PLA2 isolated from the venom of Bothrops asper, using the myogenic cell line C2C12 as a model target. Cell membrane cholesterol depletion by methyl-β-cyclodextrin (MβCD) treatment enhanced the cytolytic action of Myotoxin II, as well as of its bioactive C-terminal synthetic peptide p115-129. Conversely, cell membrane cholesterol enrichment by preformed cholesterol-MβCD complexes reduced the cytolytic effect of Myotoxin II. The toxic actions of Myotoxin I, a catalytically active PLA2 from the same venom, as well as of the cytolytic peptide melittin from bee venom, also increased in cholesterol-depleted cells. Although physical and functional changes resulting from variations in membrane cholesterol are complex, these findings suggest that membrane fluidity could be a relevant parameter to explain the observed modulation of the cytolytic mechanism of Myotoxin II, possibly influencing bilayer penetration. In concordance, the cytolytic effect of Myotoxin II decreased in direct proportion to lower temperature, a physical factor that affects membrane fluidity. In conclusion, physicochemical properties that depend on membrane cholesterol content significantly influence the cytolytic mechanism of Myotoxin II, reinforcing the concept that the primary site of action of Lys49 PLA2 Myotoxins is the plasma membrane. Copyright © 2011 John Wiley & Sons, Ltd.
Jose Maria Gutierrez - One of the best experts on this subject based on the ideXlab platform.
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Synergism between basic Asp49 and Lys49 phospholipase A2 Myotoxins of viperid snake venom in vitro and in vivo.
PloS one, 2014Co-Authors: Diana Mora-obando, Jose Maria Gutierrez, Julián Fernández, Cesare Montecucco, Bruno LomonteAbstract:Two subtypes of phospholipases A2 (PLA2s) with the ability to induce myonecrosis, ‘Asp49’ and ‘Lys49’ Myotoxins, often coexist in viperid snake venoms. Since the latter lack catalytic activity, two different mechanisms are involved in their myotoxicity. A synergism between Asp49 and Lys49 Myotoxins from Bothrops asper was previously observed in vitro, enhancing Ca2+ entry and cell death when acting together upon C2C12 myotubes. These observations are extended for the first time in vivo, by demonstrating a clear enhancement of myonecrosis by the combined action of these two toxins in mice. In addition, novel aspects of their synergism were revealed using myotubes. Proportions of Asp49 Myotoxin as low as 0.1% of the Lys49 Myotoxin are sufficient to enhance cytotoxicity of the latter, but not the opposite. Sublytic amounts of Asp49 Myotoxin also enhanced cytotoxicity of a synthetic peptide encompassing the toxic region of Lys49 Myotoxin. Asp49 Myotoxin rendered myotubes more susceptible to osmotic lysis, whereas Lys49 Myotoxin did not. In contrast to myotoxic Asp49 PLA2, an acidic non-toxic PLA2 from the same venom did not markedly synergize with Lys49 Myotoxin, revealing a functional difference between basic and acidic PLA2 enzymes. It is suggested that Asp49 Myotoxins synergize with Lys49 Myotoxins by virtue of their PLA2 activity. In addition to the membrane-destabilizing effect of this activity, Asp49 Myotoxins may generate anionic patches of hydrolytic reaction products, facilitating electrostatic interactions with Lys49 Myotoxins. These data provide new evidence for the evolutionary adaptive value of the two subtypes of PLA2 Myotoxins acting synergistically in viperid venoms.
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phospholipases a2 from viperidae snake venoms how do they induce skeletal muscle damage
Acta Chimica Slovenica, 2011Co-Authors: Bruno Lomonte, Jose Maria GutierrezAbstract:Phospholipases A 2 (PLA 2 s) are abundant components in snake venoms, which play important toxic roles. This review focuses on group II PLA2s endowed with myotoxic effects, present in Viperidae venoms. These PLA2s are subdivided into catalytically-active (Asp49) PLA 2 s, and catalytically-inactive PLA 2 homologues, the latter most frequently presenting the Lys49 substitution. Both protein subgroups induce skeletal muscle necrosis, although by different mechanisms. Current evidence indicates that phospholipid hydrolysis plays a central role in the necrotizing action of Asp49 Myotoxins, whereas PLA 2 homologues rely on the direct membrane-destabilizing actions of their cationic C-terminal region to achieve such effect, in the absence of catalysis. Both mechanisms converge in sarcolemmal permeabilization, triggering a series of intracellular events that lead to necrosis. Most viperid PLA 2 Myotoxins act only locally, but those forming heterodimeric complexes such as crotoxin spread to distant muscles and induce rhabdomyolysis. This divergence between local and systemic myotoxicity might be related to differences in binding specificity to cell targets. Nevertheless, the identity of molecular targets recognized by viperid PLA 2 Myotoxins remains elusive. Identification of their membrane target(s), and a deeper understanding of the catalytic-dependent and -independent mechanisms that result in membrane destabilization, are two crucial, but still unclarified aspects of their myotoxic action.
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Membrane cholesterol modulates the cytolytic mechanism of Myotoxin II, a Lys49 phospholipase A2 homologue from the venom of Bothrops asper
Cell biochemistry and function, 2011Co-Authors: José Rangel, Jose Maria Gutierrez, Yamileth Angulo, Orestes Quesada, Bruno LomonteAbstract:Lys49 phospholipase A2 (PLA2) homologues present in crotalid snake venoms lack enzymatic activity, yet they induce skeletal muscle necrosis by a membrane permeabilizing mechanism whose details are only partially understood. The present study evaluated the effect of altering the membrane cholesterol content on the cytolytic activity of Myotoxin II, a Lys49 PLA2 isolated from the venom of Bothrops asper, using the myogenic cell line C2C12 as a model target. Cell membrane cholesterol depletion by methyl-β-cyclodextrin (MβCD) treatment enhanced the cytolytic action of Myotoxin II, as well as of its bioactive C-terminal synthetic peptide p115-129. Conversely, cell membrane cholesterol enrichment by preformed cholesterol-MβCD complexes reduced the cytolytic effect of Myotoxin II. The toxic actions of Myotoxin I, a catalytically active PLA2 from the same venom, as well as of the cytolytic peptide melittin from bee venom, also increased in cholesterol-depleted cells. Although physical and functional changes resulting from variations in membrane cholesterol are complex, these findings suggest that membrane fluidity could be a relevant parameter to explain the observed modulation of the cytolytic mechanism of Myotoxin II, possibly influencing bilayer penetration. In concordance, the cytolytic effect of Myotoxin II decreased in direct proportion to lower temperature, a physical factor that affects membrane fluidity. In conclusion, physicochemical properties that depend on membrane cholesterol content significantly influence the cytolytic mechanism of Myotoxin II, reinforcing the concept that the primary site of action of Lys49 PLA2 Myotoxins is the plasma membrane. Copyright © 2011 John Wiley & Sons, Ltd.
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Effect of calcineurin inhibitors on myotoxic activity of crotoxin and Bothrops asper phospholipase A2 Myotoxins in vivo and in vitro.
Comparative biochemistry and physiology. Toxicology & pharmacology : CBP, 2006Co-Authors: Elen Haruka Miyabara, Jose Maria Gutierrez, Bruno Lomonte, Igor L. Baptista, Heloisa S. Selistre-de-araujo, Anselmo Sigari MoriscotAbstract:Abstract Previous studies have shown that calcineurin activity plays a critical role in the myotoxic activity induced by crotoxin (CTX), a group II phospholipase A2 (PLA2) with neurotoxic and myotoxic actions. In order to address whether calcineurin is also important for the activity of non-neurotoxic group II PLA2 Myotoxins we have compared the effects of calcineurin inhibition on the myotoxic capacity of CTX and the non-neurotoxic PLA2s, Myotoxin II (Mt II) and Myotoxin III (Mt III) from Bothrops asper venom. Rats were treated with cyclosporin A (CsA) or FK506, calcineurin inhibitors, and received an intramuscular injection of either CTX, Mt II or Mt III into the tibialis anterior. Animals were killed 24 h after injection of toxins. Tibialis anterior was removed and stored in liquid nitrogen. Myofibers in culture were also treated with CsA or FK506 and exposed to CTX, Mt II and Mt III. It was observed that, in contrast to CTX, CsA and FK506 do not attenuate myotoxic effects induced by both Mt II and Mt III in vivo and in vitro. The results of the present study suggest that calcineurin is not essential for the myotoxic activity of Mt II and Mt III, indicating that distinct intracellular pathways might be involved in myonecrosis induced by neurotoxic CTX and non-neurotoxic Bothrops sp. PLA2 Myotoxins. Alternatively, calcineurin dependent fast fiber type shift might render the muscle resistant to the action of CTX, without affecting its susceptibility to Bothrops sp. Myotoxins.
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inhibition of myotoxic activity of bothrops asper Myotoxin ii by the anti trypanosomal drug suramin
Journal of Molecular Biology, 2005Co-Authors: M T Murakami, Paulo A. Melo, Jose Maria Gutierrez, Bruno Lomonte, Emerson Z Arruda, Ana B Martinez, Sabrina Calilelias, Marcelo A Tomaz, R K ArniAbstract:Suramin, a synthetic polysulfonated compound, developed initially for the treatment of African trypanosomiasis and onchocerciasis, is currently used for the treatment of several medically relevant disorders. Suramin, heparin, and other polyanions inhibit the myotoxic activity of Lys49 phospholipase A2 analogues both in vitro and in vivo, and are thus of potential importance as therapeutic agents in the treatment of viperid snake bites. Due to its conformational flexibility around the single bonds that link the central phenyl rings to the secondary amide backbone, the symmetrical suramin molecule binds by an induced-fit mechanism complementing the hydrophobic surfaces of the dimer and adopts a novel conformation that lacks C2 symmetry in the dimeric crystal structure of the suramin–Bothrops asper Myotoxin II complex. The simultaneous binding of suramin at the surfaces of the two monomers partially restricts access to the nominal active sites and significantly changes the overall charge of the interfacial recognition face of the protein, resulting in the inhibition of myotoxicity.
Yamileth Angulo - One of the best experts on this subject based on the ideXlab platform.
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Membrane cholesterol modulates the cytolytic mechanism of Myotoxin II, a Lys49 phospholipase A2 homologue from the venom of Bothrops asper
Cell biochemistry and function, 2011Co-Authors: José Rangel, Jose Maria Gutierrez, Yamileth Angulo, Orestes Quesada, Bruno LomonteAbstract:Lys49 phospholipase A2 (PLA2) homologues present in crotalid snake venoms lack enzymatic activity, yet they induce skeletal muscle necrosis by a membrane permeabilizing mechanism whose details are only partially understood. The present study evaluated the effect of altering the membrane cholesterol content on the cytolytic activity of Myotoxin II, a Lys49 PLA2 isolated from the venom of Bothrops asper, using the myogenic cell line C2C12 as a model target. Cell membrane cholesterol depletion by methyl-β-cyclodextrin (MβCD) treatment enhanced the cytolytic action of Myotoxin II, as well as of its bioactive C-terminal synthetic peptide p115-129. Conversely, cell membrane cholesterol enrichment by preformed cholesterol-MβCD complexes reduced the cytolytic effect of Myotoxin II. The toxic actions of Myotoxin I, a catalytically active PLA2 from the same venom, as well as of the cytolytic peptide melittin from bee venom, also increased in cholesterol-depleted cells. Although physical and functional changes resulting from variations in membrane cholesterol are complex, these findings suggest that membrane fluidity could be a relevant parameter to explain the observed modulation of the cytolytic mechanism of Myotoxin II, possibly influencing bilayer penetration. In concordance, the cytolytic effect of Myotoxin II decreased in direct proportion to lower temperature, a physical factor that affects membrane fluidity. In conclusion, physicochemical properties that depend on membrane cholesterol content significantly influence the cytolytic mechanism of Myotoxin II, reinforcing the concept that the primary site of action of Lys49 PLA2 Myotoxins is the plasma membrane. Copyright © 2011 John Wiley & Sons, Ltd.
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The C-terminal region of a Lys49 Myotoxin mediates Ca2+ influx in C2C12 myotubes.
Toxicon : official journal of the International Society on Toxinology, 2009Co-Authors: Mariana Cintra-francischinelli, Yamileth Angulo, Cesare Montecucco, Paola Pizzo, José M Gutiérrez, Bruno LomonteAbstract:Myotoxins are abundant components of snake venoms, being a significant public health problem worldwide. Among them, Lys49 phospholipase A(2) homologue Myotoxins cause extensive necrosis in skeletal muscle tissue. Their mechanisms of action are still poorly understood, but there is evidence that the C-terminal region is involved in membrane damage leading to myotoxicity. To investigate the effect of the C-terminal peptide 115-129 of Agkistrodon contortrix laticinctus Myotoxin on the plasma membrane of myoblasts and myotubes, the entry of Ca(2+) was monitored by fluorescence imaging, and the ensuing cytotoxicity was determined. The Myotoxin synthetic peptide was found to act selectively on myotubes, which were rapidly overloaded with Ca(2+) with ensuing necrosis. The profile of intracellular Ca(2+) increase induced by the C-terminal peptide, but not by its scrambled version control, reproduces the second, prominent wave of the biphasic response documented in previous studies using whole Lys49 Myotoxins. These observations provide relevant insights into the mechanism of action of this family of toxins, with implications for the understanding of their structure-function relationships.
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Ability of fucoidan to prevent muscle necrosis induced by snake venom Myotoxins: comparison of high- and low-molecular weight fractions.
Toxicon : official journal of the International Society on Toxinology, 2007Co-Authors: Karol Azofeifa, Yamileth Angulo, Bruno LomonteAbstract:Abstract Fucoidan, a natural polysaccharide extracted from brown seaweed, inhibits the myotoxic phospholipases A 2 present in the venoms of crotalid snakes. This study evaluated the influence of molecular weight on the ability of fucoidan to prevent muscle necrosis when rapidly administered after injection of a purified Myotoxin or crude venom of Bothrops asper , in a mouse model. It was hypothesized that smaller fucoidan fragments, being of higher diffusibility to tissues, might have a better neutralizing efficiency in vivo . Fucoidan was subjected to acid hydrolysis to obtain low-molecular weight fragments ( F L ), or to gel filtration to isolate its high-molecular weight fraction ( F H ). These two preparations were standardized to the same neutralizing potency by preincubation assays, and subsequently tested in vivo , by independent administration assays. Local i.m. administration of either F H or F L , immediately after i.m. injection of Myotoxin II, prevented nearly 50% of muscle necrosis, albeit with no difference between the two preparations. Muscle necrosis was not reduced when either F H or F L was administered by i.v. route, immediately after i.m. toxin injection. When tested against crude venom, which contains several Myotoxin isoforms, the immediate in situ i.m. injection of F H still inhibited myonecrosis by nearly one-half of the effect recorded in the untreated group, whereas F L was ineffective. It is concluded that, in this model, and in contrast to expectations, the use of smaller fucoidan fragments to prevent muscle damage induced by snake venom Myotoxins is not advantageous, when compared with larger fucoidan molecules.
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isolation characterization and molecular cloning of anmip a new α type phospholipase a2 Myotoxin inhibitor from the plasma of the snake atropoides nummifer viperidae crotalinae
Comparative Biochemistry and Physiology B, 2007Co-Authors: Steve Quiros, Alberto Alapegiron, Yamileth Angulo, Bruno LomonteAbstract:A new phospholipase A2 (PLA2)-inhibitory protein was isolated from the plasma of Atropoides nummifer, a crotaline snake from Central America. This inhibitor was named AnMIP, given its ability to neutralize the activity of basic PLA2 Myotoxins of its own and related venoms. The cDNA of AnMIP was cloned and sequenced, showing that it belongs to the α group of phospholipase A2 inhibitors (PLIs). AnMIP appears as a homotrimer in the native state, held together by non-covalent forces, with a subunit molecular mass of 22,247–22,301 and an isoelectric point of 4.1–4.7. This trimeric structure is the first observed in a PLIα from American crotaline snakes, previously reported only in Asian species. Sequencing, mass spectrometry, and analytical isoelectrofocusing indicated the existence of isoforms, as reported for other PLIαs isolated from snake plasma. The inhibitory profile of AnMIP showed specificity towards group II PLA2s, either belonging to the catalytically-active (D49) or -inactive (K49) subtypes, exemplified in this study by Bothrops asper Myotoxin I and A. nummifer Myotoxin II, respectively. By phylogenetic analysis it was shown that AnMIP is closely related to CgMIP-II, previously isolated from the plasma of Cerrophidion godmani, showing 93% amino acid sequence identity.
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structure of Myotoxin ii a catalytically inactive lys49 phospholipase a2 homologue from atropoides nummifer venom
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2006Co-Authors: Mario T Murakami, Bruno Lomonte, Yamileth Angulo, Cristiane C Melo, Raghuvir K. ArniAbstract:Lys49 snake-venom phospholipase A2 (PLA2) homologues are highly myotoxic proteins which, although lacking catalytic activity, possess the ability to disrupt biological membranes, inducing significant muscle-tissue loss and permanent disability in severely envenomed patients. Since the structural basis for their toxic activity is still only partially understood, the structure of Myotoxin II, a monomeric Lys49 PLA2 homologue from Atropoides nummifer, has been determined at 2.08 A resolution and the anion-binding site has been characterized.
Charlotte L Ownby - One of the best experts on this subject based on the ideXlab platform.
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ability of wedelolactone heparin and para bromophenacyl bromide to antagonize the myotoxic effects of two crotaline venoms and their pla2 Myotoxins
Toxicon, 1999Co-Authors: Paulo A. Melo, Charlotte L OwnbyAbstract:We examined the ability of wedelolactone, heparin and para-bromophenacyl bromide to antagonize the myotoxic activity in mice of venoms from Crotalus viridis viridis and Agkistrodon contortrix laticinctus and two phospholipase A2 Myotoxins, CVV Myotoxin and ACL Myotoxin, isolated from them. Myotoxicity was measured by the increase in plasma creatine kinase (CK) activity at two hours and histological changes in extensor digitorum longus muscle (EDL) at three hours after injection of the test solution. Both heparin and wedelolactone independently reduced the myotoxic effect of both crude venoms and both Myotoxins, but wedelolactone was more effective. Wedelolactone plus heparin reduced the myotoxic effect of CVV Myotoxin more than either antagonist alone. The PLA2 inhibitor, para-bromophenacyl bromide (pBPB), reduced the myotoxic effect of both Myotoxins more than either wedelolactone or heparin. On the other hand, the myotoxic effect of polylysine was not reduced by either wedelolactone or para-bromophenacyl bromide, but it was reduced by heparin. These results indicate that wedelolactone, para-bromophenacyl bromide and heparin are antagonists of these two phospholipase A2 Myotoxins, and that antagonism by the first two compounds may be due to a more specific interaction with these proteins than that by the latter.
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Structure, Function and Biophysical Aspects of the Myotoxins from Snake Venoms
Journal of Toxicology: Toxin Reviews, 1998Co-Authors: Charlotte L OwnbyAbstract:AbstractSnake venom Myotoxins can be categorized into three types: small, basic polypeptides such as Myotoxin a and crotamine; cardiotoxins from cobra venoms; and phospholipase A2, toxins such as crotoxin and notexin. All three types of Myotoxins induce depolarization and contraction of skeletal muscle cells. However, the myonecrosis induced by the small, basic polypeptide Myotoxins is different from that induced by the cardiotoxins and phospholipase A2 Myotoxins in that the former do not appear to lyse the sarcolemma whereas the latter two types cause lysis of the sarcolemma which is of rapid onset. Molecular properties of the toxins are similar in that they are all highly basic proteins, and a large portion of their surface charge is positive Also, they all have considerable β-sheet structure which may be involved in interaction with the membrane The purpose of this review is to describe the structure and function of these Myotoxins and to evaluate features they might share which could shed light on the...
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preliminary x ray crystallographic data for a Myotoxin from agkistrodon contortrix laticinctus broad banded copperhead venom
Toxicon, 1997Co-Authors: A C Treharne, Charlotte L Ownby, Steven P. White, H Selistre S De Araujo, S I FoundlingAbstract:A Type II phospholipase A2 Myotoxin from Agkistrodon contortrix laticinctus was purified to homogeneity and crystallized. The protein had only myotoxic activity. X-ray diffraction quality crystals were obtained by the hanging drop vapour diffusion method from a crystallization solution containing 2.0 M ammonium sulphate. X-ray data were collected to a resolution of 2.3 A, and the crystals are fully characterized.
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Isolation, characterization and crystallization of a phospholipase A2 Myotoxin from the venom of the prairie rattlesnake (Crotalus viridis viridis).
Toxicon : official journal of the International Society on Toxinology, 1997Co-Authors: Charlotte L Ownby, Terry R. Colberg, Steven P. WhiteAbstract:Abstract A Myotoxin with phospholipase A 2 (PLA 2 ) activity was isolated from the venom of the prairie rattlesnake ( Crotalus viridis viridis , CVV) by cation-exchange chromatography. The toxin contains 123 amino acids and has an estimated mol. wt of 14,000. It is basic, with a p I above 9. Comparison of the N-terminal 33 residues of this Myotoxin with other PLA 2 proteins from snake venoms showed that CVV Myotoxin has highest homology (91%) to one isoform of the B component of crotoxin from Crotalus durissus terrificus venom, and less homology (73–75%) to mojave toxin from Crotalus scutulatus scutulatus venom and agkistrotoxin from Agkistrodon halys Pallas venom. It has the least homology (40–43%) to PLA 2 s from venom of two other snakes in the Crotalus genus which are neither neurotoxic nor myotoxic. CVV Myotoxin induces the type of myonecrosis typical of snake venom Myotoxins with the PLA 2 structure, i.e. rapid disruption of the plasma membrane as indicated by the presence of delta lesions, hypercontraction and clumping of the myofilaments, and necrosis of affected skeletal muscle cells. Inhibition of the phospholipase activity of the toxin with p -bromophenacyl bromide inhibits the myotoxic activity, indicating that for some Myotoxins with the PLA 2 structure, the catalytic activity is important for myotoxic activity. This is the first report of the isolation of a non-neurotoxic, single-chain PLA 2 Myotoxin from the venom of a snake from the Crotalus genus.
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CDNA CLONING AND SEQUENCE ANALYSIS OF A LYSINE-49 PHOSPHOLIPASE A2 Myotoxin FROM AGKISTRODON CONTORTRIX LATICINCTUS SNAKE VENOM
Archives of biochemistry and biophysics, 1996Co-Authors: Heloísa S. Selistre De Araújo, Steven P. White, Charlotte L OwnbyAbstract:A cDNA clone (ACLPREMT1) for a K49 phospholipase A2(PLA2) Myotoxin fromAgkistrodon contortrix laticinctussnake venom was isolated from a venom gland library and sequenced. The ACLPREMT1 cDNA is 734 bp in length and has an open reading frame of 414 bp. It codes for a K49 phospholipase A2with 121 amino acid residues. The sequence of the first 20 amino acid residues of the predicted mature protein matches exactly with the N-terminal sequence of the purified Myotoxin. Comparison of the ACLPREMT1 cDNA sequence with PLA2cDNAs from Viperidae snakes shows that it has a similar organization: highly conserved 5′ and 3′ untranslated regions, a sequence encoding a 16-amino acid signal peptide, and the mature protein coding region. Comparison of the predicted sequence of ACL Myotoxin and other K49 and D49 PLA2Myotoxins shows that, despite the homology (85–97%) at the nucleotide level, K49 PLA2Myotoxins are distinct from the D49 PLA2s and form a highly conserved protein family. In addition to the substitution of D49K, K49 Myotoxins have several invariant residues not found in the D49 group, including K7, K78, K80, K115, and K116. There are also some conserved residues (E12, T13, K16, and N17) in all myotoxic proteins, including some neurotoxic and myotoxic PLA2s. Molecular modeling of ACL Myotoxin shows that these residues are close together on the surface of one side of the molecule which suggests a potential site for binding to membranes and/or induction of toxicity.
Leonel Calderon - One of the best experts on this subject based on the ideXlab platform.
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an overview of lysine 49 phospholipase a2 Myotoxins from crotalid snake venoms and their structural determinants of myotoxic action
Toxicon, 2003Co-Authors: Bruno Lomonte, Yamileth Angulo, Leonel CalderonAbstract:In 1984, the first venom phospholipase A2 (PLA2) with a lysine substituting for the highly conserved aspartate 49 was discovered, in the North American crotalid snake Agkistrodon p. piscivorus [J. Biol. Chem. 259 (1984) 13839]. Ten years later, the first mapping of a ‘toxic region’ on a Lys49 PLA2 was reported, in Bothrops asper Myotoxin II [J. Biol. Chem. 269 (1994) 29867]. After a further decade of research on the Lys49 PLA2s, a better understanding of their structural determinants of toxicity and mode of action is rapidly emerging, with myotoxic effector sites identified at the C-terminal region in at least four proteins: B. asper Myotoxin II, A. p. piscivorus K49 PLA2, A. c. laticinctus ACL Myotoxin, and B. jararacussu bothropstoxin I. Although important features still remain to be established, their toxic mode of action has now been understood in its more general concepts, and a consistent working hypothesis can be experimentally supported. It is proposed that all the toxic activities of Lys49 PLA2s are related to their ability to destabilize natural (eukaryotic and prokaryotic) and artificial membranes, using a cationic/hydrophobic effector site located at their C-terminal loop. This review summarizes the general properties of the Lys49 PLA2 Myotoxins, emphasizing the development of current concepts and hypotheses concerning the molecular basis of their toxic activities.
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Inhibition of the myotoxic activity of Bothrops asper Myotoxin II in mice by immunization with its synthetic 13-mer peptide 115–129
Toxicon : official journal of the International Society on Toxinology, 1999Co-Authors: Leonel Calderon, Bruno LomonteAbstract:The region comprising amino acid residues 115-129 of Myotoxin II, a Lys49 phospholipase A2 from the venom of Bothrops asper, was previously shown to constitute a heparin binding site, and to be associated with its toxic activities. The corresponding synthetic peptide, KKYRYYLKPLCKK, was coupled to diphtheria toxoid as a carrier, and utilized as an immunogen in mice, to explore the possible protection from the myotoxic activity induced by Myotoxin II in vivo. Mice receiving peptide-carrier injections produced antibodies to peptide 115-129, which cross-reacted to Myotoxin II, as determined by enzyme-immunoassay. In contrast, no antibodies against peptide 115-129 were detected in mice immunized with Myotoxin II, despite the strong antibody response to the whole antigen. Thus, region 115-129 of Myotoxin II is not an immunodominant B-cell epitope in the mouse. After immunization with conjugated peptide or Myotoxin II, mice were challenged with Myotoxin II, and the extent of myonecrosis was estimated by determining their plasma creatine kinase activity, in comparison to non-immunized mice. After the challenge, both the group immunized with Myotoxin II, and the group immunized with peptide 115-129, had a significant reduction of myonecrosis. These results demonstrate that region 115-129 of Myotoxin II constitutes a neutralizing epitope, and provide further evidence for the relevance of this region in its myotoxic effect in vivo.
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Immunochemical Characterization and Role in Toxic Activities of Region 115–129 of Myotoxin II, a Lys49 Phospholipase A2fromBothrops asperSnake Venom
Archives of biochemistry and biophysics, 1998Co-Authors: Leonel Calderon, Bruno LomonteAbstract:The region 115–129 of Myotoxin II, a catalytically inactive Lys49 phospholipase A2, was previously shown to constitute a heparin-binding site and to be involved in its cytolytic actionin vitro.An immunochemical approach was utilized to further explore the role of this region in the toxic activities of Myotoxin II. By using a carrier-linked 13-mer synthetic peptide as immunogen, rabbit polyclonal antibodies against region 115–129 were obtained. These antibodies were able to bind to the native protein and to inhibit its myotoxic and cytolytic effects in preincubation-type neutralization experiments. Antibodies to peptide 115–129 formed precipitating macromolecular complexes in gel immunodiffusion, demonstrating the oligomeric state of Myotoxin II not only in its crystalline structure (dimeric), but also in solution. Analyses of the antibody response to carrier-linked peptide 115–129 and native Myotoxin II suggest that region 115–129, although potentially immunogenic, is not an immunodominant B-cell epitope of this protein, failing to elicit significant antibody responses in animals immunized with the native toxin. Antibodies to peptide 115–129 cross-reacted with 15 purified class II myotoxic phospholipases A2found in snake venoms of the generaBothrops, Agkistrodon, Trimeresurus,andVipera,but not with the recombinant human class II phospholipase A2, for which no toxic actions have been described. Myotoxic phospholipases of the class I (notexin) and class III (bee venom) groups were not recognized by antibodies to p115–129. These results demonstrate that the overall antigenic structure of region 115–129 is conserved among class II myotoxic phospholipases A2, despite differences in their corresponding amino acid sequences. Based on the accumulated experimental evidence, a model of the myotoxic region of Myotoxin II, and possibly of related class II Lys49 phospholipase A2Myotoxins, is proposed.
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immunochemical characterization and role in toxic activities of region 115 129 of Myotoxin ii a lys49 phospholipase a2frombothrops aspersnake venom
Archives of Biochemistry and Biophysics, 1998Co-Authors: Leonel Calderon, Bruno LomonteAbstract:The region 115–129 of Myotoxin II, a catalytically inactive Lys49 phospholipase A2, was previously shown to constitute a heparin-binding site and to be involved in its cytolytic actionin vitro.An immunochemical approach was utilized to further explore the role of this region in the toxic activities of Myotoxin II. By using a carrier-linked 13-mer synthetic peptide as immunogen, rabbit polyclonal antibodies against region 115–129 were obtained. These antibodies were able to bind to the native protein and to inhibit its myotoxic and cytolytic effects in preincubation-type neutralization experiments. Antibodies to peptide 115–129 formed precipitating macromolecular complexes in gel immunodiffusion, demonstrating the oligomeric state of Myotoxin II not only in its crystalline structure (dimeric), but also in solution. Analyses of the antibody response to carrier-linked peptide 115–129 and native Myotoxin II suggest that region 115–129, although potentially immunogenic, is not an immunodominant B-cell epitope of this protein, failing to elicit significant antibody responses in animals immunized with the native toxin. Antibodies to peptide 115–129 cross-reacted with 15 purified class II myotoxic phospholipases A2found in snake venoms of the generaBothrops, Agkistrodon, Trimeresurus,andVipera,but not with the recombinant human class II phospholipase A2, for which no toxic actions have been described. Myotoxic phospholipases of the class I (notexin) and class III (bee venom) groups were not recognized by antibodies to p115–129. These results demonstrate that the overall antigenic structure of region 115–129 is conserved among class II myotoxic phospholipases A2, despite differences in their corresponding amino acid sequences. Based on the accumulated experimental evidence, a model of the myotoxic region of Myotoxin II, and possibly of related class II Lys49 phospholipase A2Myotoxins, is proposed.
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Quantitation by enzyme-immunoassay of antibodies against Bothrops Myotoxins in four commercially-available antivenoms.
Toxicon : official journal of the International Society on Toxinology, 1991Co-Authors: Bruno Lomonte, Jose Maria Gutierrez, Gustavo Rojas, Leonel CalderonAbstract:B. Lomonte, J. M. Gutierrez, G. Rojas and L. Calderon. Quantitation by enzyme-immunoassay of antibodies against Bothrops Myotoxins in four commercially-available antivenoms. Toxicon29, 695–702, 1991.—An enzyme-immunoassay (EIA) for the quantitation of antibodies against Myotoxins present in the venoms of Bothrops asper (Costa Rica), B. atrox (Colombia) and B. moojeni (Brazil), was developed. This EIA was utilized for the evaluation of four antivenoms produced in Mexico (Laboratorios Myn; MYN), Costa Rica (Instituto Clodomiro Picado; ICP), Colombia (Instituto Nacional de Salud; INS) and Brazil (Instituto Butantan; IB). Antivenoms ICP, IB and INS showed a high titer of antibodies against the three Myotoxins tested, with only slight differences between them, depending on the antigen utilized. In contrast, MYN antivenom had very low levels of antibodies to the three Myotoxins. Seventeen batches of ICP antivenom were analyzed by EIA, using B. asper Myotoxin II as antigen. Although all batches had high anti-Myotoxin titers, these varied significantly. Batches produced after 1988 had, in general, higher titers than older (1986–1987) ones. Antivenom stored for one year at 37°C had a slight, but significant (P < 0.002) decrease of anti-Myotoxin activity, compared to antivenom stored at 4°C or 23°C. No correlation was found between anti-Myotoxin concentration and lethality-neutralizing ability of the ICP antivenom.
