Comparative structural studies on Lys49-phospholipases A(2) from Bothrops genus reveal their myotoxic site

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Insights into the role of oligomeric state on the biological activities of crotoxin: crystal structure of a tetrameric phospholipase A(2) formed by two isoforms of crotoxin B from Cratalus durissus terrificus venom

Crotoxin B (CB or Cdt PLA(2)) is a basic Asp49-PLA(2) found in the venom of Crotalus durissus terrificus and it is one of the subunits that constitute the crotoxin (Cro). This heterodimeric toxin, main component of the C. d. terrificus venom, is completed by an acidic, nontoxic, and nonenzymatic component (crotoxin A, CA or crotapotin), and it is related to important envenomation effects such as neurological disorders, myotoxicity, and renal failure. Although Cro has been crystallized since 1938, no crystal structure of this toxin or its subunits is currently available. In this work, the authors present the crystal structure of novel tetrameric complex formed by two dimers of crotoxin B isoforms (CB1 and CB2). The results suggest that these assemblies are stable in solution and show that Ser1 and Glu92 of CB1 and CB2, respectively, play an important role in the oligomerization. The tetrameric and dimeric conformations resulting from the association of the isoforms may increase the neurotoxicity of the toxin CB by the creation of new binding sites, which could improve the affinity of the molecular complexes to the presynaptic membrane.

Crystallization and preliminary X-ray diffraction analysis of crotoxin B from Crotalus durissus collilineatus venom

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Kinetics of venom and antivenom serum levels, clinical evaluation and therapeutic effectiveness in dogs inoculated with Crotalus durissus terrificus venom

This work evaluated the clinical and therapeutic aspects as well as serum levels of venom and antivenom IgG by enzyme-linked immunosorbent assay (ELISA) in experimental envenomation of dogs with Crotalus durissus terrificus venom. Twenty-eight mixed breed adult dogs were divided into four groups of seven animals each, Group I: only venom; Group II, venom + 50 ml of anti-bothropic-crotalic serum (50mg) + fluid therapy; Group III, venom + 50 ml of anti-bothropic-crotalic serum + fluid therapy + urine alkalination; Group IV, 50 ml of anti-bothropic-crotalic serum. The lyophilized venom of Crotalus durissus terrificus was reconstituted in saline solution and subcutaneously inoculated at the dose of 1mg/kg body weight. The dogs presented clinical signs of local pain, weakness, mandibular ptosis, mydriasis, emesis and salivation. The venom levels detected by ELISA ranged from 0 to 90ng/ml, according to the severity of the clinical signs. Serum antivenom ranged from 0 to 3ug/ml and was detected for up to 138h after treatment. ELISA results showed the effectiveness of the serum therapy for the venom neutralization.

Mass spectrometric characterization of two novel inflammatory peptides from the venom of the social wasp Polybia paulista

The social wasp P. paulista is relatively common in southeast Brazil causing many medically important stinging incidents. The seriousness of these incidents is dependent on the amount of venom inoculated by the wasps into the victims, and the characteristic envenomation symptoms are strongly dependent on the types of peptides present in the venom. In order to identify some of these naturally occurring peptides available in very low amounts, an analytical protocol was developed that uses a combination of reversed-phase and normal-phase high-performance liquid chromatography (HPLC) of wasp venom for peptide purification, with matrix-assisted laser desorption/ionization time-of-flight post-source decay mass spectrometry (MALDI-Tof-PSD-MS) and low-energy collision-induced dissociation (CID) in a quadrupole time-of-flight tandem mass spectrometry (QTof-MS/MS) instrument for peptide sequencing at the sub-picomole level. The distinction between Leu and Ile was achieved both by observing d-type fragment ions obtained under CID conditions and by comparison of retention times of the natural peptides and their synthetic counterparts (with different combinations of I and/or L at N- and C-terminal positions). To distinguish the isobaric residues K and Q, acetylation of peptides was followed by Q-Tof-MS analysis. The primary sequences obtained were INWLKLGKMVIDAL-NH2 (MW 1611.98Da) and IDWLKLGKMVMDVL-NH2 (MW 1658.98Da). Micro-scale bioassay protocols characterized both peptides as presenting potent hemolytic action, mast cell degranulation, and chemotaxis of poly-morphonucleated leukocyte (PMNL) cells. The primary sequences and the bioassay results suggest that these toxins constitute members of a new sub-class of mastoparan toxins, directly involved in the occurrence of inflammatory processes after wasp stinging. Copyright (C) 2004 John Wiley Sons, Ltd.

Crystallization and preliminary crystallographic analysis of SMase I, a sphingomyelinase from Loxosceles laeta spider venom

SMase I, a 32 kDa sphingomyelinase found in Loxosceles laeta venom, is responsible for the major pathological effects of spider envenomation. This toxin has been cloned and functionally expressed as a fusion protein containing a 6 x His tag at its N-terminus to yield a 33 kDa protein [Fernandes-Pedrosa et al. (2002), Biochem. Biophys. Res. Commun. 298, 638 - 645]. The recombinant protein possesses all the biological properties ascribed to the whole L. laeta venom, including dermonecrotic and complement-dependent haemolytic activities. Dynamic light-scattering experiments conducted at 291 K demonstrate that the sample possesses a monomodal distribution, with a hydrodynamic radius of 3.57 nm. L. laeta SMase I was crystallized by the hanging-drop vapour-diffusion technique using the sparse-matrix method. Single crystals were obtained using a buffer solution consisting of 0.08 M HEPES and 0.9 M trisodium citrate, which was titrated to pH 7.5 using 0.25 M sodium hydroxide. Complete three-dimensional diffraction data were collected to 1.8 Angstrom at the Laboratorio Nacional de Luz Sincrotron (LNLS, Campinas, Brazil). The crystals belong to the hexagonal system ( space group P6(1) or P6(5)), with unit-cell parameters a = b = 140.6, c = 113.6 Angstrom. A search for heavy-atom derivatives has been initiated and elucidation of the crystal structure is currently in progress.

Crystal structure of a dimeric Ser49 PLA(2)-like myotoxic component of the Vipera ammodytes meridionalis venomics reveals determinants of myotoxicity and membrane damaging activity

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

The Venomics of Bothrops alternatus is a Pool of Acidic Proteins with Predominant Hemorrhagic and Coagulopathic Activities

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Acidentes por lepidópteros (larvas e adultos de mariposas): estudo dos aspectos epidemiológicos, clínicos e terapêuticos

Os autores apresentam e discutem aspectos dos acidentes causados por larvas de lepidópteros (mariposas), enfatizando as manifestações dermatológicas e a dor intensa que caracterizam estes agravos. Além disso, são apresentadas larvas de mariposas que causam manifestações extracutâneas, tais como severos distúrbios de coagulação e artropatias anquilosantes, e ainda dermatites provocadas por insetos adultos. Os principais grupos de lepidópteros causadores de acidentes em humanos são demonstrados, e as medidas terapêuticas atualizadas são discutidas. O lepidopterismo e o erucismo são acidentes comuns, e é importante que o dermatologista saiba reconhecer e tratar esse tipo de envenenamento.

Sea urchin granuloma

Os acidentes por animais aquáticos traumatizantes e venenosos podem provocar morbidez importante em humanos. Equinodermos marinhos incluem mais de 6000 espécies de estrelas-do-mar, ouriços-do-mar, bolachas-de-praia e pepinos-do-mar. Vários equinodermos têm sido responsabilizados por acidentes em humanos. Granulomas por ouriço-do-mar são lesões de caráter granulomatoso, crônicas, causada por acidentes com espículas de ouriço-do-mar. Os autores relatam um caso típico de granulomas por ouriço-do-mar ocorrido em um pescador e enfatizam as implicações terapêuticas aplicadas.

Venomous mollusks: the risks of human accidents by conus snails (gastropoda: conidae) in Brazil

Os moluscos do gênero Conus apresentam um aparato venenoso composto de uma rádula quitinosa ligada a glândulas de peçonha, causando envenenamentos humanos graves e mesmo óbitos pela ação neurotóxica indutora do bloqueio de vários receptores e paralisia muscular. Não há casos descritos de envenenamento no país, mas determinadas populações correm risco de acidentes.

Purification, sequencing and structural characterization of the phospholipase A(1) from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae)

The biochemical and functional characterization of wasp venom toxins is an important prerequisite for the development of new tools both for the therapy of the toxic reactions due to envenomation caused by multiple stinging accidents and also for the diagnosis and therapy of allergic reactions caused by this type of venom. PLA(1) was purified from the venom of the neotropical social wasp Polybia paulista by using molecular exclusion and cation exchange chromatographies; its amino acid sequence was determined by using automated Edman degradation and compared to the sequences of other vespid venom PLA(1)'s. The enzyme exists as a 33,961.40 da protein, which was identified as a lipase of the GX class, liprotein lipase superfamily, pancreatic lipases (ab20.3) homologous family and RP2 sub-group of phospholipase. P. paulista PLA(1) is 53-82% identical to the phospholipases from wasp species from Northern Hemisphere. The use restrained-based modeling permitted to describe the 3-D structure of the enzyme, revealing that its molecule presents 23% alpha-helix, 28% beta-sheet and 49% coil. The protein structure has the alpha/beta fold common to many lipases; the core consists of a tightly packed beta-sheet constituted of six-stranded parallel and one anti-parallel beta-strand, surrounded by four alpha-helices. P. paulista PLA(1) exhibits direct hemolytic action against washed red blood cells with activity similar to the Cobra cardiotoxin from Naja naja atra. In addition to this, PLA(1) was immunoreactive to specific IgE from the sera of P. paulista-sensitive patients. (c) 2007 Elsevier Ltd. All rights reserved.

Biological and biochemical properties of the Brazilian Potamotrygon stingrays : Potamotrygon cf. scobina and Potamotrygon gr. orbignyi

Stingrays of the family Potamotrygonidae are widespread throughout river systems of South America that drain into the Atlantic Ocean. Some species are endemic to the most extreme freshwater environment of the Brazil and cause frequent accidents to humans. The envenomation causes immediate, local, and intense pain, soft tissue edema, and a variable extent of bleeding. The present study was carried out in order to describe the principal biological and some biochemical properties of the Brazilian Potamotrygon fish venoms (Potamotrygon cf. scobina and P. gr. orbignyi). Both stingray venoms induced significant edematogenic and nociceptive responses in mice. Edematogenic and nociceptive responses were reduced when the venom was incubated at 37 or 56 degrees C. The results showed striking augments of leukocytes rolling and adherent cells to the endothelium of cremaster mice induced by both venoms. The data also presented that injection of both venoms induced necrosis, low level of proteolytic activity, without inducing haemorrhage. But when the venoms of both stingray species were injected together with their mucus secretion, the necrotizing activity was more vigorous. The present study provided in vivo evidence of toxic effects for P. cf. scobina and P. gr. orbignyi venoms. (c) 2006 Elsevier Ltd. All fights reserved.

A new class of neurotoxin from wasp venom slows inactivation of sodium current

The effects of alpha-pompilidotoxin (alpha-PMTX), a new neurotoxin isolated from the venom of a solitary wasp, were studied on the neuromuscular synapses in lobster walking leg and the rat trigeminal ganglion (TG) neurons. Paired intracellular recordings from the presynaptic axon terminals and the innervating lobster leg muscles revealed that alpha-PMTX induced long bursts of action potentials in the presynaptic axon, which resulted in facilitated excitatory and inhibitory synaptic transmission. The action or alpha-PMTX was distinct from that of other known facilitatory presynaptic toxins, including sea anemone toxins and alpha-scorpion toxins, which modify the fast inactivation of Na+ current. We further characterized the action of alpha-PMTX on Na+ channels by whole-cell recordings from rat trigeminal neurons. We found that alpha-PMTX stowed the Na+ channels inactivation process without changing the peak current-voltage relationship or the activation time course of tetrodotoxin (TTX)-sensitive Na+ currents, and that alpha-PMTX had voltage-dependent effects on the rate of recovery from Na+ current inactivation and deactivating tail currents. The results suggest that alpha-PMTX slows or blocks conformational changes required for fast inactivation of the Na+ channels on the extracellular surface. The simple structure of alpha-PMTX, consisting of 13 amino acids, would be advantageous for understanding the functional architecture of Na+ channel protein.

Kinetic and mechanistic characterization of the Sphingomyelinases D from Loxosceles intermedia spider venom

Envenomation by arachnids of the genus Loxosceles leads to local dermonecrosis and serious systemic toxicity mainly induced by sphingomyelinases D (SMase D). These enzymes catalyze the hydrolysis of sphingomyelin resulting in the formation of ceramide-phosphate and choline as well as the cleavage of lysophosphatidyl choline generating the lipid mediator lysophosphatidic acid. We have, previously, cloned and expressed two functional SMase D isoforms, named P1 and P2, from Loxosceles intertnedia venom and comparative protein sequence analysis revealed that they are highly homologous to SMase I from Loxosceles laeta which folds to form an (alpha/beta)(8) barrel. In order to further characterize these proteins, pH dependence kinetic experiments and chemical modification of the two active SMases D isoforms were performed. We show here that the amino acids involved in catalysis and in the metal ion binding sites are strictly conserved in the SMase D isoforms from L. intermedia. However, the kinetic studies indicate that SMase P1 hydrolyzes sphingomyelin less efficiently than P2, which can be attributed to a substitution at position 203 (Pro-Leu) and local amino acid substitutions in the hydrophobic channel that could probably play a role in the substrate recognition and binding. (c) 2005 Elsevier Ltd. All rights reserved.