The Experts below are selected from a list of 309 Experts worldwide ranked by ideXlab platform
Glenn F. King - One of the best experts on this subject based on the ideXlab platform.
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Addition of K22 Converts Spider Venom Peptide Pme2a from an Activator to an Inhibitor of NaV1.7
Biomedicines, 2020Co-Authors: Kathleen Yin, Volker Herzig, Glenn F. King, Jennifer R Deuis, Zoltan Dekan, Ai-hua Jin, Paul F. Alewood, Irina VetterAbstract:Spider Venom is a novel source of disulfide-rich peptides with potent and selective activity at voltage-gated sodium channels (NaV). Here, we describe the discovery of μ-theraphotoxin-Pme1a and μ/δ-theraphotoxin-Pme2a, two novel peptides from the Venom of the Gooty Ornamental tarantula Poecilotheria metallica that modulate NaV channels. Pme1a is a 35 residue peptide that inhibits NaV1.7 peak current (IC50 334 ± 114 nM) and shifts the voltage dependence of activation to more depolarised membrane potentials (V1/2 activation: Δ = +11.6 mV). Pme2a is a 33 residue peptide that delays fast inactivation and inhibits NaV1.7 peak current (EC50 > 10 μM). Synthesis of a [+22K]Pme2a analogue increased potency at NaV1.7 (IC50 5.6 ± 1.1 μM) and removed the effect of the native peptide on fast inactivation, indicating that a lysine at position 22 (Pme2a numbering) is important for inhibitory activity. Results from this study may be used to guide the rational design of Spider Venom-derived peptides with improved potency and selectivity at NaV channels in the future.
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Can we resolve the taxonomic bias in Spider Venom research?
Elsevier, 2019Co-Authors: Volker Herzig, Glenn F. King, Eivind A.b. UndheimAbstract:The rate of discovery of new Spider species greatly exceeds the rate of Spider Venom characterisation, leading to an increasing number of species with unstudied Venoms. However, recent advances in proteomics and genomics that enable the study of Venoms from smaller species has expanded the accessible taxonomic range. Thus, although the number of unstudied Spider Venoms is likely to further increase, future research should focus on the characterisation of Venoms and toxins from previously unstudied Spider families. Keywords: Spider, Venom, Peptide, Diversity, Taxonomic, Transcriptomics, Proteomic
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can we resolve the taxonomic bias in Spider Venom research
Toxicon: X, 2019Co-Authors: Volker Herzig, Glenn F. King, Eivind A.b. UndheimAbstract:The rate of discovery of new Spider species greatly exceeds the rate of Spider Venom characterisation, leading to an increasing number of species with unstudied Venoms. However, recent advances in proteomics and genomics that enable the study of Venoms from smaller species has expanded the accessible taxonomic range. Thus, although the number of unstudied Spider Venoms is likely to further increase, future research should focus on the characterisation of Venoms and toxins from previously unstudied Spider families.
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Efficient enzymatic ligation of inhibitor cystine knot Spider Venom peptides: using sortase a to form double-knottins that probe voltage-gated sodium channel NaV1.7
Bioconjugate chemistry, 2018Co-Authors: Akello J. Agwa, Glenn F. King, Linda V. Blomster, David J. Craik, Christina I. SchroederAbstract:Gating modifier toxins from Spider Venom are disulfide-rich peptides that typically comprise a stabilizing inhibitor cystine knot (ICK). These knottin peptides are being pursued as therapeutic leads for a range of conditions linked to transmembrane proteins. Recently, double-knottin peptides discovered in Spider Venom and produced by recombinant expression have provided insights into the pharmacology of transmembrane channels. Here, we use chemoenzymatic ligation to produce double-knottins to probe the effect of bivalent modulation on the voltage-gated sodium channel subtype 1.7 (NaV1.7), which is implicated in pain signaling. Monovalent knottins were oxidatively folded and then biochemically conjugated using sortase A, to form double-knottins. The structural integrity of the peptides was confirmed using NMR, and fluorescence-based activity assays provided evidence suggesting that coincubated monovalent and bivalent knottins can cooperatively modulate NaV1.7. We anticipate that double-knottins will provid...
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Efficient Enzymatic Ligation of Inhibitor Cystine Knot Spider Venom Peptides: Using Sortase A To Form Double-Knottins That Probe Voltage-Gated Sodium Channel NaV1.7
2018Co-Authors: Akello J. Agwa, Glenn F. King, Linda V. Blomster, David J. Craik, Christina I. SchroederAbstract:Gating modifier toxins from Spider Venom are disulfide-rich peptides that typically comprise a stabilizing inhibitor cystine knot (ICK). These knottin peptides are being pursued as therapeutic leads for a range of conditions linked to transmembrane proteins. Recently, double-knottin peptides discovered in Spider Venom and produced by recombinant expression have provided insights into the pharmacology of transmembrane channels. Here, we use chemoenzymatic ligation to produce double-knottins to probe the effect of bivalent modulation on the voltage-gated sodium channel subtype 1.7 (NaV1.7), which is implicated in pain signaling. Monovalent knottins were oxidatively folded and then biochemically conjugated using sortase A, to form double-knottins. The structural integrity of the peptides was confirmed using NMR, and fluorescence-based activity assays provided evidence suggesting that coincubated monovalent and bivalent knottins can cooperatively modulate NaV1.7. We anticipate that double-knottins will provide novel tools for enhancing our understanding of, and design strategies for, therapeutically relevant voltage-gated ion channels
Volker Herzig - One of the best experts on this subject based on the ideXlab platform.
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Addition of K22 Converts Spider Venom Peptide Pme2a from an Activator to an Inhibitor of NaV1.7
Biomedicines, 2020Co-Authors: Kathleen Yin, Volker Herzig, Glenn F. King, Jennifer R Deuis, Zoltan Dekan, Ai-hua Jin, Paul F. Alewood, Irina VetterAbstract:Spider Venom is a novel source of disulfide-rich peptides with potent and selective activity at voltage-gated sodium channels (NaV). Here, we describe the discovery of μ-theraphotoxin-Pme1a and μ/δ-theraphotoxin-Pme2a, two novel peptides from the Venom of the Gooty Ornamental tarantula Poecilotheria metallica that modulate NaV channels. Pme1a is a 35 residue peptide that inhibits NaV1.7 peak current (IC50 334 ± 114 nM) and shifts the voltage dependence of activation to more depolarised membrane potentials (V1/2 activation: Δ = +11.6 mV). Pme2a is a 33 residue peptide that delays fast inactivation and inhibits NaV1.7 peak current (EC50 > 10 μM). Synthesis of a [+22K]Pme2a analogue increased potency at NaV1.7 (IC50 5.6 ± 1.1 μM) and removed the effect of the native peptide on fast inactivation, indicating that a lysine at position 22 (Pme2a numbering) is important for inhibitory activity. Results from this study may be used to guide the rational design of Spider Venom-derived peptides with improved potency and selectivity at NaV channels in the future.
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Can we resolve the taxonomic bias in Spider Venom research?
Elsevier, 2019Co-Authors: Volker Herzig, Glenn F. King, Eivind A.b. UndheimAbstract:The rate of discovery of new Spider species greatly exceeds the rate of Spider Venom characterisation, leading to an increasing number of species with unstudied Venoms. However, recent advances in proteomics and genomics that enable the study of Venoms from smaller species has expanded the accessible taxonomic range. Thus, although the number of unstudied Spider Venoms is likely to further increase, future research should focus on the characterisation of Venoms and toxins from previously unstudied Spider families. Keywords: Spider, Venom, Peptide, Diversity, Taxonomic, Transcriptomics, Proteomic
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can we resolve the taxonomic bias in Spider Venom research
Toxicon: X, 2019Co-Authors: Volker Herzig, Glenn F. King, Eivind A.b. UndheimAbstract:The rate of discovery of new Spider species greatly exceeds the rate of Spider Venom characterisation, leading to an increasing number of species with unstudied Venoms. However, recent advances in proteomics and genomics that enable the study of Venoms from smaller species has expanded the accessible taxonomic range. Thus, although the number of unstudied Spider Venoms is likely to further increase, future research should focus on the characterisation of Venoms and toxins from previously unstudied Spider families.
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Versatile Spider Venom peptides and their medical and agricultural applications.
Toxicon : official journal of the International Society on Toxinology, 2018Co-Authors: Natalie J. Saez, Volker HerzigAbstract:Spiders have been evolving complex and diverse repertoires of peptides in their Venoms with vast pharmacological activities for more than 300 million years. Spiders use their Venoms for prey capture and defense, hence they contain peptides that target both prey (mainly arthropods) and predators (other arthropods or vertebrates). This includes peptides that potently and selectively modulate a range of targets such as ion channels, receptors and signaling pathways involved in physiological processes. The contribution of these targets in particular disease pathophysiologies makes Spider Venoms a valuable source of peptides with potential therapeutic use. In addition, peptides with insecticidal activities, used for prey capture, can be exploited for the development of novel bioinsecticides for agricultural use. Although we have already reviewed potential applications of Spider Venom peptides as therapeutics (in 2010) and as bioinsecticides (in 2012), a considerable number of research articles on both topics have been published since, warranting an updated review. Here we explore the most recent research on the use of Spider Venom peptides for both medical and agricultural applications.
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corrigendum pharmacological characterisation of the highly nav1 7 selective Spider Venom peptide pn3a
Scientific Reports, 2017Co-Authors: Jennifer R Deuis, Volker Herzig, Zoltan Dekan, Joshua S Wingerd, Jennifer J Smith, Nehan R Munasinghe, Rebecca F Bhola, Wendy L Imlach, David A Armstrong, Johan K RosengrenAbstract:Corrigendum: Pharmacological characterisation of the highly Na V 1.7 selective Spider Venom peptide Pn3a
Eugene V Grishin - One of the best experts on this subject based on the ideXlab platform.
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Latarcins: versatile Spider Venom peptides
Cellular and Molecular Life Sciences, 2015Co-Authors: Peter V. Dubovskii, Eugene V Grishin, Alexey V. Feofanov, Alexander A Vassilevski, Sergey A Kozlov, Roman G. EfremovAbstract:Arthropod Venoms feature the presence of cytolytic peptides believed to act synergetically with neurotoxins to paralyze prey or deter aggressors. Many of them are linear, i.e., lack disulfide bonds. When isolated from the Venom, or obtained by other means, these peptides exhibit common properties. They are cationic; being mostly disordered in aqueous solution, assume amphiphilic α-helical structure in contact with lipid membranes; and exhibit general cytotoxicity, including antifungal, antimicrobial, hemolytic, and anticancer activities. To suit the pharmacological needs, the activity spectrum of these peptides should be modified by rational engineering. As an example, we provide a detailed review on latarcins (Ltc), linear cytolytic peptides from Lachesana tarabaevi Spider Venom. Diverse experimental and computational techniques were used to investigate the spatial structure of Ltc in membrane-mimicking environments and their effects on model lipid bilayers. The antibacterial activity of Ltc was studied against a panel of Gram-negative and Gram-positive bacteria. In addition, the action of Ltc on erythrocytes and cancer cells was investigated in detail with confocal laser scanning microscopy. In the present review, we give a critical account of the progress in the research of Ltc. We explore the relationship between Ltc structure and their biological activity and derive molecular characteristics, which can be used for optimization of other linear peptides. Current applications of Ltc and prospective use of similar membrane-active peptides are outlined.
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bacterial production of latarcin 2a a potent antimicrobial peptide from Spider Venom
Protein Expression and Purification, 2008Co-Authors: Yuri M Shlyapnikov, Sergey A Kozlov, Alexander A Vassilevski, Yaroslav A Andreev, Eugene V GrishinAbstract:Natural Venoms are promising sources of candidate therapeutics including antibiotics. A recently described potent antimicrobial peptide latarcin 2a (Ltc 2a) from Lachesana tarabaevi Spider Venom shows a broad-spectrum antibacterial activity. This peptide consists of 26 amino acid residues and therefore its production using chemical synthesis, although trivial, is costly. We describe an easy approach to Ltc 2a production in Escherichia coli using the conventional fusion partner thioredoxin. Latarcin 2a synthetic gene was cloned into the expression vector pET-32b, which was then used to transform E. coli BL21(DE3) strain. His-tagged fusion purification was achieved using metal-chelate affinity chromatography. Since no methionine residues are present in the latarcin 2a sequence, cyanogen bromide could be effectively utilized to separate the target product from the carrier protein. Reverse-phase HPLC was used as the final step of purification; the final yield was ∼3 mg/L of bacterial culture. To increase the yields, we attempted incorporation of Ltc 2a tandem repeats into the fusion protein; however, production rates greatly decreased due to enhanced fusion toxicity. Moreover, we probed constructs to produce an Ltc 2a dimer and the Ltc 2a propeptide to study their functional properties. Recombinant peptides were produced at appreciable yields and biological tests to determine their activities were performed. Latarcin 2a is the first linear peptide from Spider Venom and one of the first membrane-active peptides from Venomous animals to be biosynthetically produced.
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bacterial production of latarcin 2a a potent antimicrobial peptide from Spider Venom
Protein Expression and Purification, 2008Co-Authors: Yuri M Shlyapnikov, Sergey A Kozlov, Alexander A Vassilevski, Yaroslav A Andreev, Eugene V GrishinAbstract:Natural Venoms are promising sources of candidate therapeutics including antibiotics. A recently described potent antimicrobial peptide latarcin 2a (Ltc 2a) from Lachesana tarabaevi Spider Venom shows a broad-spectrum antibacterial activity. This peptide consists of 26 amino acid residues and therefore its production using chemical synthesis, although trivial, is costly. We describe an easy approach to Ltc 2a production in Escherichia coli using the conventional fusion partner thioredoxin. Latarcin 2a synthetic gene was cloned into the expression vector pET-32b, which was then used to transform E. coli BL21(DE3) strain. His-tagged fusion purification was achieved using metal-chelate affinity chromatography. Since no methionine residues are present in the latarcin 2a sequence, cyanogen bromide could be effectively utilized to separate the target product from the carrier protein. Reverse-phase HPLC was used as the final step of purification; the final yield was approximately 3 mg/L of bacterial culture. To increase the yields, we attempted incorporation of Ltc 2a tandem repeats into the fusion protein; however, production rates greatly decreased due to enhanced fusion toxicity. Moreover, we probed constructs to produce an Ltc 2a dimer and the Ltc 2a propeptide to study their functional properties. Recombinant peptides were produced at appreciable yields and biological tests to determine their activities were performed. Latarcin 2a is the first linear peptide from Spider Venom and one of the first membrane-active peptides from Venomous animals to be biosynthetically produced.
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cyto insectotoxins a novel class of cytolytic and insecticidal peptides from Spider Venom
Biochemical Journal, 2008Co-Authors: Alexander A Vassilevski, Sergey A Kozlov, Olga V Samsonova, Natalya S Egorova, Dmitry V Karpunin, K A Pluzhnikov, Alexei V Feofanov, Eugene V GrishinAbstract:Eight linear cationic peptides with cytolytic and insecticidal activity, designated cyto-insectotoxins (CITs), were identified in Lachesana tarabaevi Spider Venom. The peptides showed antibiotic activity towards Gram-positive and Gram-negative bacteria at micromolar concentrations as well as toxicity to insects. The primary structures of the toxins were established by direct Edman sequencing in combination with enzymatic and chemical polypeptide degradation and MS. CITs represent a novel class of cytolytic molecules and Spider Venom toxins. They are the first example of molecules showing equally potent antimicrobial and insecticidal effects. Analysis of L. tarabaevi Venom gland expressed sequence tag database revealed the primary structures of the protein precursors; eight peptides homologous with the purified toxins were additionally predicted. CIT precursors share a conventional prepropeptide structure with an acidic prosequence and a processing motif common to most Spider toxin precursors. The most abundant peptide, CIT 1a, was chemically synthesized, and its lytic activity on different bacterial strains, human erythrocytes and lymphocytes, insect cells, planar lipid bilayers and lipid vesicles was characterized. The Spider L. tarabaevi is suggested to have evolved to rely on a unique set of linear cytolytic toxins, as opposed to the more common disulfide-containing Spider neurotoxins.
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vitamin b1 thiazole derivative reduces transmembrane current through ionic channels formed by toxins from black widow Spider Venom and sea anemone in planar phospholipid membranes
Biochimica et Biophysica Acta, 2007Co-Authors: Oleg Ya Shatursky, Tatyana M Volkova, Olexander V Romanenko, N H Himmelreich, Eugene V GrishinAbstract:The vitamin B 1 (thiamine) structural analogue 3-decyloxycarbonylmethyl-4-methyl-5-(β-hydroxyethyl) thiazole chloride (DMHT) (0.1 mM) reversibly reduced transmembrane currents in CaCl 2 and KC1 solutions via ionic channels produced by latrotoxins (a-latrotoxin (a-LT) and α-latroinsectotoxin (a-LIT)) from black widow Spider Venom and sea anemone toxin (RTX) in the bilayer lipid membranes (BLMs). Introduction of DMHT from the cis-side of BLM bathed in 10 mM CaCl 2 inhibited transmembrane current by 31.6±3% and by 61.8±3% from the trans-side of BLM for α-LT channels. Application of DMHT in the solution of 10 mM CaCl 2 to the cis-side of BLM decreased the current through the α-LIT and RTX channels by 52±4% and 50±5%, respectively. Addition of Cd 2+ (1 mM) to the cis- or trans-side of the membrane after the DMHT-induced depression of Ca 2+ -current across the α-LT channels caused its further decrease by 85 ±5% that coincides favorably with the intensity of Cd 2+ blocking in control experiments without DMHT. These data suggest that DMHT inhibiting is not specific for latrotoxin channels only and DMHT may exert its action on α-LT channels without considerable influence on the ionogenic groups of Ca 2+ -selective site inside the channel cavity. The binding kinetics of DMHT with the α-LT channel shows no cooperativity and allows to expect that the DMHT binding site of the toxin is formed by one ionogenic group as the slopes of inhibition rate determined in log-log coordinates are 1.25 on the trans-side and 0.68 on the cis-side. Similar pK of binding (5.4 on the trans-side and 5.7 on the cis-side) also suggest that DMHT may interact with the same high affinity site of α-LT channel on either side of the BLM. The comparative analysis of effective radii measured for α-LT, α-LIT and RTX channels on the cis-side (0.9 nm, 0.53 nm and 0.55 nm, correspondingly) and for α-LT channel on the trans-side (0.28±0.18 nm) with the intensity of DMHT inhibitory action obtained on these channels allowed to conclude that the potency of DMHT inhibition increased on toxin pores of smaller lumen. © 2006 Elsevier B.V. All rights reserved.
Waldemiro Gremski - One of the best experts on this subject based on the ideXlab platform.
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molecular cloning heterologous expression and functional characterization of a novel translationally controlled tumor protein tctp family member from loxosceles intermedia brown Spider Venom
The International Journal of Biochemistry & Cell Biology, 2012Co-Authors: Youssef Bacila Sade, Rafael Bertoni Da Silveira, Olga Meiri Chaim, Waldemiro Gremski, Andrea Senffribeiro, Marianna Boiaferreira, Luiza Helena Gremski, Silvio Sanches VeigaAbstract:EnVenoming with brown Spiders (Loxosceles genus) is common throughout the world. Cutaneous symptoms following Spider bite accidents include dermonecrosis, erythema, itching and pain. In some cases, accidents can cause hypersensibility or even allergic reactions. These responses could be associated with histaminergic events, such as an increase in vascular permeability and vasodilatation. A protein that may be related to the effects of Spider Venom was identified from a previously obtained cDNA library of the L. intermedia Venom gland. The amino acid sequence of this protein is homologous to proteins from the TCTP (translationally-controlled tumor protein) family, which are extracellular histamine-releasing factors (HRF) that are associated with the allergic reactions to parasites. Herein, we described the cloning, heterologous expression, purification and functional characterization of a novel member of the TCTP family from the Loxosceles intermedia Venom gland. This recombinant protein, named LiRecTCTP, causes edema, enhances vascular permeability and is likely related to the inflammatory activity of the Venom. Moreover, LiRecTCTP presents an immunological relationship with mammalian TCTPs.
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the relationship between calcium and the metabolism of plasma membrane phospholipids in hemolysis induced by brown Spider Venom phospholipase d toxin
Journal of Cellular Biochemistry, 2011Co-Authors: Daniele Chavesmoreira, Olga Meiri Chaim, Oldemir C Mangili, Waldemiro Gremski, Fernanda Souza, Rosalvo T H Fogaca, Andrea Senffribeiro, Silvio Sanches VeigaAbstract:Brown Spider Venom phospholipase-D belongs to a family of toxins characterized as potent bioactive agents. These toxins have been involved in numerous aspects of cell pathophysiology including inflammatory response, platelet aggregation, endothelial cell hyperactivation, renal disorders, and hemolysis. The molecular mechanism by which these toxins cause hemolysis is under investigation; literature data have suggested that enzyme catalysis is necessary for the biological activities triggered by the toxin. However, the way by which phospholipase-D activity is directly related with human hemolysis has not been determined. To evaluate how brown Spider Venom phospholipase-D activity causes hemolysis, we examined the impact of recombinant phospholipase-D on human red blood cells. Using six different purified recombinant phospholipase-D molecules obtained from a cDNA Venom gland library, we demonstrated that there is a correlation of hemolytic effect and phospholipase-D activity. Studying LiRecDT1, a potent hemolytic and phospholipase-D recombinant toxin, we determined that the toxin degrades synthetic sphingomyelin, lysophosphatidylcholine, and lyso-platelet-activating factor. Additionally, we determined that the toxin degrades phospholipids in a detergent extract of human erythrocytes, as well as phospholipids from ghosts of human red blood cells. The products of the degradation of synthetic sphingomyelin and lysophosphatidylcholine following LiRecDT1 treatments caused hemolysis of human erythrocytes. This hemolysis, dependent on products of metabolism of phospholipids, is also dependent on Calcium ion concentration because the percentage of hemolysis increased with an increase in the dose of Calcium in the medium. Recombinant phospholipase-D treatment of human erythrocytes stimulated an influx of Calcium into the cells that was detected by a Calcium-sensitive fluorescent probe (Fluo-4). This Calcium influx was shown to be channel-mediated rather than leak-promoted because the influx was inhibited by L-type Calcium channel inhibitors but not by a T-type Calcium channel blocker, Sodium channel inhibitor or a specific inhibitor of Calcium activated Potassium channels. Finally, this inhibition of hemolysis following recombinant phospholipase-D treatment occurred in a concentration-dependent manner in the presence of L-type Calcium channel blockers such as Nifedipine and Verapamil. The data provided herein, suggest that the brown Spider Venom phospholipase-D-induced hemolysis of human erythrocytes is dependent on the metabolism of membrane phospholipids, such as sphingomyelin and lysophosphatidylcholine, generating bioactive products that stimulate a Calcium influx into red blood cells mediated by the L-type channel. J. Cell. Biochem. © 2011 Wiley-Liss, Inc. Language: en
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hyaluronidases in loxosceles intermedia brown Spider Venom are endo β n acetyl d hexosaminidases hydrolases
Toxicon, 2007Co-Authors: Rafael Bertoni Da Silveira, Olga Meiri Chaim, Oldemir C Mangili, Waldemiro Gremski, Carl P Dietrich, Helena B Nader, Silvio Sanches VeigaAbstract:In studying Loxosceles Venom, we detected degradation of purified hyaluronic acid (HA) and hydrolysis of purified chondroitin sulphate (CS) while neither dermatan sulphate, heparin or heparan sulphate were affected. In addition, with HA-degrading kinetic assays, we show that a hydrolase enzyme was involved in the HA cleavage. By use of the Reissig colorimetric reaction, we found that Venom hyaluronidase is an endo-beta-N-acetyl-d-hexosaminidase that generates terminal N-acetylglucosamine residues upon cleavage of HA. Zymogram analysis of L. intermedia Venom showed HA lytic activities at 41 and 43kDa, and, when CS was used as a substrate, zymograph experiments resulted in 41 and 43kDa lytic zones. Thus, these results support the hypothesis that the same molecules are involved in cleaving HA and CS residues. Experiments to compare L. intermedia electrostimulated Venom and Venom gland extract also demonstrated very similar HA lytic activity, suggesting again that hyaluronidases are self-components of Loxosceles Spider Venom instead of oral egesta contamination. HA degradation as a function of pH in these hydrolase enzymes showed no apparent activities at low or high pH, with optimal activity at 6.0-8.0 pH. Finally, we confirmed the cleaving action of the Venom hyaluronidases on HA in the extracellular matrix of the dermis of rabbit by fluorescence reaction to HA and confocal microscope analysis. Thus, hyaluronidases type hydrolases endo-beta-N-acetyl-d-hexosaminidase are implicated as self-components of Loxosceles Spider Venom and can be involved in Venom effects as spreading factors.
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molecular cloning and functional characterization of two isoforms of dermonecrotic toxin from loxosceles intermedia brown Spider Venom gland
Biochimie, 2006Co-Authors: Rafael Bertoni Da Silveira, Olga Meiri Chaim, Oldemir C Mangili, Waldemiro Gremski, Marcia Helena Appel, Juliana L Dreyfuss, Romine Bachmann Pigozzo, Leny TomaAbstract:Abstract Brown Spider (Genus Loxosceles) bites are normally associated with necrotic skin degeneration, gravitational spreading, massive inflammatory response at injured region, platelet aggregation causing thrombocytopenia and renal disturbances. Brown Spider Venom has a complex composition containing many different toxins, of which a well-studied component is the dermonecrotic toxin. This toxin alone may produce necrotic lesions, inflammatory response and platelet aggregation. Biochemically, dermonecrotic toxin belongs to a family of toxins with 30–35 kDa characterized as sphingomyelinase-D. Here, employing a cDNA library of Loxosceles intermedia Venom gland, we cloned and expressed two recombinant isoforms of the dermonecrotic toxin LiRecDT2 (1062 bp cDNA) and LiRecDT3 (1007 bp cDNA) that encode for signal peptides and complete mature proteins. Phylogenetic tree analysis revealed a structural relationship for these toxins compared to other members of family. Recombinant molecules were expressed as N-terminal His-tag fusion proteins in Escherichia coli and were purified to homogeneity from cell lysates by Ni2+ chelating chromatography, resulting in proteins of 33.8 kDa for LiRecDT2 and 34.0 kDa for LiRecDT3. Additional evidence for related toxins containing sequence/epitopes identity comes from antigenic cross-reactivity using antibodies against crude Venom toxins and antibodies raised with a purified dermonecrotic toxin. Recombinant toxins showed differential functionality in rabbits: LiRecDT2 caused a macroscopic lesion with gravitational spreading upon intradermal injection, while LiRecDT3 evoked transient swelling and erythema upon injection site. Light microscopic analysis of skin biopsies revealed edema, a collection of inflammatory cells in and around blood vessels and a proteinaceous network at the dermis. Moreover, differential functionality for recombinant toxins was also demonstrated by a high sphingomyelinase activity for LiRecDT2 and low activity for LiRecDT3 as well as greater in vitro platelet aggregation and blood vessel permeability induced by LiRecDT2 and residual activity for LiRecDT3. Cloning and expression of two recombinant dermonecrotic toxins demonstrate an intraspecific family of homologous toxins that act in synergism for deleterious activities of the Venom and open possibilities for biotechnological applications for recombinant toxins as research tools for understanding the inflammatory response, vascular integrity and platelet aggregation modulators.
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histopathological findings in rabbits after experimental acute exposure to the loxosceles intermedia brown Spider Venom
International Journal of Experimental Pathology, 2003Co-Authors: Katia Zoghbi Ospedal, Oldemir C Mangili, Silvio Sanches Veiga, Marcia Helena Appel, Jose Fillus Neto, Waldemiro GremskiAbstract:Loxoscelism, the term used to describe enVenomation with brown Spiders, is characterized by a dermonecrotic lesion at the bite site. In the present investigation we submitted albino rabbits to an acute experimental enVenomation protocol using Loxosceles intermedia (brown Spider) Venom, with in order to determine the pathogenesic features of the lesion induced by this Spider, which is the cause of several accidents throughout the world. Rabbits received intradermal injections of the Venom and were monitored over the first 4 h, and then at 12 h and 1, 2 and 5 days after enVenomation. Histological specimens from 3 rabbits per time point were collected from euthanized animals and processed for histological examination by light microscopy. Major findings observed during the first 4 h were oedema, haemorrhage, degeneration of blood vessel walls, plasma exudation, thrombosis, neutrophil accumulation in and around blood vessels with an intensive diapedesis, a diffuse collection of inflammatory cells (polymorphonuclear leucocytes) in the dermis, and subcutaneous muscular oedema. Over the following hours and up to 5 days after enVenomation the changes progressed to massive neutrophil infiltration (with no other leucocytes) into the dermis and even into subcutaneous muscle tissue, destruction of blood vessels, thrombosis, haemorrhage, myonecrosis, and coagulative necrosis on the 5th day.
Silvio Sanches Veiga - One of the best experts on this subject based on the ideXlab platform.
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molecular cloning heterologous expression and functional characterization of a novel translationally controlled tumor protein tctp family member from loxosceles intermedia brown Spider Venom
The International Journal of Biochemistry & Cell Biology, 2012Co-Authors: Youssef Bacila Sade, Rafael Bertoni Da Silveira, Olga Meiri Chaim, Waldemiro Gremski, Andrea Senffribeiro, Marianna Boiaferreira, Luiza Helena Gremski, Silvio Sanches VeigaAbstract:EnVenoming with brown Spiders (Loxosceles genus) is common throughout the world. Cutaneous symptoms following Spider bite accidents include dermonecrosis, erythema, itching and pain. In some cases, accidents can cause hypersensibility or even allergic reactions. These responses could be associated with histaminergic events, such as an increase in vascular permeability and vasodilatation. A protein that may be related to the effects of Spider Venom was identified from a previously obtained cDNA library of the L. intermedia Venom gland. The amino acid sequence of this protein is homologous to proteins from the TCTP (translationally-controlled tumor protein) family, which are extracellular histamine-releasing factors (HRF) that are associated with the allergic reactions to parasites. Herein, we described the cloning, heterologous expression, purification and functional characterization of a novel member of the TCTP family from the Loxosceles intermedia Venom gland. This recombinant protein, named LiRecTCTP, causes edema, enhances vascular permeability and is likely related to the inflammatory activity of the Venom. Moreover, LiRecTCTP presents an immunological relationship with mammalian TCTPs.
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the relationship between calcium and the metabolism of plasma membrane phospholipids in hemolysis induced by brown Spider Venom phospholipase d toxin
Journal of Cellular Biochemistry, 2011Co-Authors: Daniele Chavesmoreira, Olga Meiri Chaim, Oldemir C Mangili, Waldemiro Gremski, Fernanda Souza, Rosalvo T H Fogaca, Andrea Senffribeiro, Silvio Sanches VeigaAbstract:Brown Spider Venom phospholipase-D belongs to a family of toxins characterized as potent bioactive agents. These toxins have been involved in numerous aspects of cell pathophysiology including inflammatory response, platelet aggregation, endothelial cell hyperactivation, renal disorders, and hemolysis. The molecular mechanism by which these toxins cause hemolysis is under investigation; literature data have suggested that enzyme catalysis is necessary for the biological activities triggered by the toxin. However, the way by which phospholipase-D activity is directly related with human hemolysis has not been determined. To evaluate how brown Spider Venom phospholipase-D activity causes hemolysis, we examined the impact of recombinant phospholipase-D on human red blood cells. Using six different purified recombinant phospholipase-D molecules obtained from a cDNA Venom gland library, we demonstrated that there is a correlation of hemolytic effect and phospholipase-D activity. Studying LiRecDT1, a potent hemolytic and phospholipase-D recombinant toxin, we determined that the toxin degrades synthetic sphingomyelin, lysophosphatidylcholine, and lyso-platelet-activating factor. Additionally, we determined that the toxin degrades phospholipids in a detergent extract of human erythrocytes, as well as phospholipids from ghosts of human red blood cells. The products of the degradation of synthetic sphingomyelin and lysophosphatidylcholine following LiRecDT1 treatments caused hemolysis of human erythrocytes. This hemolysis, dependent on products of metabolism of phospholipids, is also dependent on Calcium ion concentration because the percentage of hemolysis increased with an increase in the dose of Calcium in the medium. Recombinant phospholipase-D treatment of human erythrocytes stimulated an influx of Calcium into the cells that was detected by a Calcium-sensitive fluorescent probe (Fluo-4). This Calcium influx was shown to be channel-mediated rather than leak-promoted because the influx was inhibited by L-type Calcium channel inhibitors but not by a T-type Calcium channel blocker, Sodium channel inhibitor or a specific inhibitor of Calcium activated Potassium channels. Finally, this inhibition of hemolysis following recombinant phospholipase-D treatment occurred in a concentration-dependent manner in the presence of L-type Calcium channel blockers such as Nifedipine and Verapamil. The data provided herein, suggest that the brown Spider Venom phospholipase-D-induced hemolysis of human erythrocytes is dependent on the metabolism of membrane phospholipids, such as sphingomyelin and lysophosphatidylcholine, generating bioactive products that stimulate a Calcium influx into red blood cells mediated by the L-type channel. J. Cell. Biochem. © 2011 Wiley-Liss, Inc. Language: en
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hyaluronidases in loxosceles intermedia brown Spider Venom are endo β n acetyl d hexosaminidases hydrolases
Toxicon, 2007Co-Authors: Rafael Bertoni Da Silveira, Olga Meiri Chaim, Oldemir C Mangili, Waldemiro Gremski, Carl P Dietrich, Helena B Nader, Silvio Sanches VeigaAbstract:In studying Loxosceles Venom, we detected degradation of purified hyaluronic acid (HA) and hydrolysis of purified chondroitin sulphate (CS) while neither dermatan sulphate, heparin or heparan sulphate were affected. In addition, with HA-degrading kinetic assays, we show that a hydrolase enzyme was involved in the HA cleavage. By use of the Reissig colorimetric reaction, we found that Venom hyaluronidase is an endo-beta-N-acetyl-d-hexosaminidase that generates terminal N-acetylglucosamine residues upon cleavage of HA. Zymogram analysis of L. intermedia Venom showed HA lytic activities at 41 and 43kDa, and, when CS was used as a substrate, zymograph experiments resulted in 41 and 43kDa lytic zones. Thus, these results support the hypothesis that the same molecules are involved in cleaving HA and CS residues. Experiments to compare L. intermedia electrostimulated Venom and Venom gland extract also demonstrated very similar HA lytic activity, suggesting again that hyaluronidases are self-components of Loxosceles Spider Venom instead of oral egesta contamination. HA degradation as a function of pH in these hydrolase enzymes showed no apparent activities at low or high pH, with optimal activity at 6.0-8.0 pH. Finally, we confirmed the cleaving action of the Venom hyaluronidases on HA in the extracellular matrix of the dermis of rabbit by fluorescence reaction to HA and confocal microscope analysis. Thus, hyaluronidases type hydrolases endo-beta-N-acetyl-d-hexosaminidase are implicated as self-components of Loxosceles Spider Venom and can be involved in Venom effects as spreading factors.
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histopathological findings in rabbits after experimental acute exposure to the loxosceles intermedia brown Spider Venom
International Journal of Experimental Pathology, 2003Co-Authors: Katia Zoghbi Ospedal, Oldemir C Mangili, Silvio Sanches Veiga, Marcia Helena Appel, Jose Fillus Neto, Waldemiro GremskiAbstract:Loxoscelism, the term used to describe enVenomation with brown Spiders, is characterized by a dermonecrotic lesion at the bite site. In the present investigation we submitted albino rabbits to an acute experimental enVenomation protocol using Loxosceles intermedia (brown Spider) Venom, with in order to determine the pathogenesic features of the lesion induced by this Spider, which is the cause of several accidents throughout the world. Rabbits received intradermal injections of the Venom and were monitored over the first 4 h, and then at 12 h and 1, 2 and 5 days after enVenomation. Histological specimens from 3 rabbits per time point were collected from euthanized animals and processed for histological examination by light microscopy. Major findings observed during the first 4 h were oedema, haemorrhage, degeneration of blood vessel walls, plasma exudation, thrombosis, neutrophil accumulation in and around blood vessels with an intensive diapedesis, a diffuse collection of inflammatory cells (polymorphonuclear leucocytes) in the dermis, and subcutaneous muscular oedema. Over the following hours and up to 5 days after enVenomation the changes progressed to massive neutrophil infiltration (with no other leucocytes) into the dermis and even into subcutaneous muscle tissue, destruction of blood vessels, thrombosis, haemorrhage, myonecrosis, and coagulative necrosis on the 5th day.
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identification of high molecular weight serine proteases in loxosceles intermedia brown Spider Venom
Toxicon, 2000Co-Authors: Silvio Sanches Veiga, Rafael Bertoni Da Silveira, Oldemir C Mangili, Juliana L Dreyfuss, Juliana Haoach, Aline M Pereira, Waldemiro GremskiAbstract:High molecular weight serine-proteases have been identified in Loxosceles intermedia (brown Spider) Venom. The mechanism by which Loxosceles spp Venoms cause dermonecrotic injury (a hallmark of loxoscelism) is currently under investigation, but it seems to be molecularly complex and in some instance proteases might be expected to play a role in this skin lesion. In the present investigation, when we submitted L. intermedia Venom to linear gradient 3–20% SDS-PAGE stained by a monochromatic silver method we detected a heterogeneous protein profile in molecular weight, ranging from 850- to 5-kDa. In an attempt to detect zymogen molecules of proteolytic enzymes, Venom aliquots were treated with several exogenous proteases. Among them, trypsin activated two gelatinolytic molecules of 85- and 95-kDa in the Venom. In experiments of hydrolysis inactivation using different protease inhibitors for four major class of proteases, we detected that only serine-type protease inhibitors were able to inactivate the 85- and 95-kDa enzymes in the Venom. An examination of the 85- and 95-kDa gelatinolytic activities as a function of pH showed that these proteases had no apparent activities at pH below 5.0 and higher than 9.0 and displayed little activity at pH 6.0, with the optimal pH for their activities ranging from 7.0 to 8.0. Evaluation of the functional specificities of the 85- and 95-kDa Venom proteases showed that these proteases efficiently degrade gelatin (denatured collagen) but have no proteolytic activity on hemoglobin, immunoglobulin, albumin, fibrinogen or laminin, suggesting specificity of their proteolytic actions. We describe here two serine-proteases activities in L. intermedia Venom probably involved in the harmful effects of the Venom.