RESUMO
The wandering spider, Phoneutria depilata, is one of Colombia's most active nocturnal arthropod predators of vertebrates and invertebrates. Its venom has been a relevant subject of study in the last two decades. However, the scarcity of transcriptomic data for the species limits our knowledge of the distinct components present in its venom for linking the mainly neurotoxic effects of the spider venom to a particular molecular target. The transcriptome of the P. depilata venom gland was analyzed to understand the effect of different diets or sex and the impact of these variables on the composition of the venom. We sequenced venom glands obtained from ten males and ten females from three diet treatments: (i) invertebrate: Tenebrio molitor, (ii) vertebrate: Hemidactylus frenatus, and (iii) mixed (T. molitor + H. frenatus). Of 17,354 assembled transcripts from all samples, 65 transcripts relating to venom production differed between males and females. Among them, 36 were classified as neurotoxins, 14 as serine endopeptidases, 11 as other proteins related to venom production, three as metalloprotease toxins, and one as a venom potentiator. There were no differences in transcripts across the analyzed diets, but when considering the effect of diets on differences between the sexes, 59 transcripts were differentially expressed. Our findings provide essential information on toxins differentially expressed that can be related to sex and the plasticity of the diet of P. depilata and thus can be used as a reference for venomics of other wandering spider species.
Assuntos
Venenos de Aranha , Aranhas , Toxinas Biológicas , Animais , Feminino , Masculino , Perfilação da Expressão Gênica , Vertebrados , Transcriptoma , Aranhas/genética , Aranhas/metabolismo , Peçonhas , Venenos de Aranha/genética , Venenos de Aranha/toxicidade , Venenos de Aranha/metabolismoRESUMO
Acanthoscurria juruenicola is an Amazonian spider described for the first time almost a century ago. However, little is known about their venom composition. Here, we present a multiomics characterization of A. juruenicola venom by a combination of transcriptomics, proteomics, and peptidomics approaches. Transcriptomics of female venom glands resulted in 93,979 unique assembled mRNA transcript encoding proteins. A total of 92 proteins were identified in the venom by mass spectrometry, including 14 mature cysteine-rich peptides (CRPs). Quantitative analysis showed that CRPs, cysteine-rich secretory proteins, metalloproteases, carbonic anhydrases, and hyaluronidase comprise >90% of the venom proteome. Relative quantification of venom toxins was performed by DIA and DDA, revealing converging profiles of female and male specimens by both methods. Biochemical assays confirmed the presence of active hyaluronidases, phospholipases, and proteases in the venom. Moreover, the venom promoted in vivo paralytic activities in crickets, consistent with the high concentration of CRPs. Overall, we report a comprehensive analysis of the arsenal of toxins of A. juruenicola and highlight their potential biotechnological and pharmacological applications. Mass spectrometry data were deposited to the ProteomeXchange Consortium via the PRIDE repository with the dataset identifier PXD013149 and via the MassIVE repository with the dataset identifier MSV000087777.
Assuntos
Venenos de Aranha , Aranhas , Animais , Masculino , Feminino , Aranhas/genética , Aranhas/metabolismo , Venenos de Aranha/genética , Venenos de Aranha/química , Venenos de Aranha/metabolismo , Cisteína/metabolismo , Proteômica/métodos , Espectrometria de Massas/métodos , Proteoma/genética , Proteoma/metabolismo , Peptídeos/análiseRESUMO
The transcriptome of the venom glands of the Phoneutria depilata spider was analyzed using RNA-seq with an Illumina protocol, which yielded 86,424 assembled transcripts. A total of 682 transcripts were identified as potentially coding for venom components. Most of the transcripts found were neurotoxins (156) that commonly act on sodium and calcium channels. Nevertheless, transcripts coding for some enzymes (239), growth factors (48), clotting factors (6), and a diuretic hormone (1) were found, which have not been described in this spider genus. Furthermore, an enzymatic characterization of the venom of P. depilata was performed, and the proteomic analysis showed a correlation between active protein bands and protein sequences found in the transcriptome. The transcriptomic analysis of P. depilata venom glands show a deeper description of its protein components, allowing the identification of novel molecules that could lead to the treatment of human diseases, or could be models for developing bioinsecticides.
Assuntos
Venenos de Aranha , Aranhas , Animais , Colômbia , Proteômica , Venenos de Aranha/genética , Venenos de Aranha/metabolismo , Aranhas/genética , TranscriptomaRESUMO
Several classes of toxins are present in the venom of Brown spiders (Loxosceles genus), some of them are highly expressed and others are less expressed. In this work, we aimed to clone the sequence of a little expressed novel toxin from Loxosceles venom identified as a serine protease inhibitor (serpin), as well as to express and characterize its biochemical and biological properties. It was named LSPILT, derived from Loxoscelesserine protease inhibitor-like toxin. Multiple alignment analysis revealed high identity between LSPILT and other serpin molecules from spiders and crab. LSPILT was produced in baculovirus-infected insect cells, resulting in a 46-kDa protein fused to a His-tag. Immunological assays showed epitopes in LSPILT that resemble native venom toxins of Loxosceles spiders. The inhibitory activity of LSPILT on trypsin was found both by reverse zymography and fluorescent gelatin-degradation assay. Additionally, LSPILT inhibited the complement-dependent lysis of Trypanosoma cruzi epimastigotes, reduced thrombin-dependent clotting and suppressed B16-F10 melanoma cells migration. Results described herein prove the existence of conserved serpin-like toxins in Loxosceles venoms. The availability of a recombinant serpin enabled the determination of its biological and biochemical properties and indicates potential applications in future studies regarding the pathophysiology of the envenoming or for biotechnological purposes.
Assuntos
Antineoplásicos/farmacologia , Fibrinolíticos/farmacologia , Serpinas/genética , Serpinas/metabolismo , Aranhas/metabolismo , Trypanosoma cruzi/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Baculoviridae , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Clonagem Molecular , Camundongos , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Coelhos , Células Sf9 , Venenos de Aranha/genética , Venenos de Aranha/metabolismo , Aranhas/genética , TripsinaRESUMO
Loxoscelism is a recognized public health problem in Brazil, but the venom from Loxosceles similis, which is widespread in Brazil due to its adaptability to the urban environment, remains poorly characterized. Loxtox is a family of phospholipase D enzymes (PLDs), which are the major components of Loxosceles venom and are responsible for the clinical effects of loxoscelism. Loxtox toxins correspond to 15% of L. similis venom gland transcripts, but the Loxtox family of L. similis has yet to be fully described. In this study, we cloned and functionally characterized recLoxtox s1A and recLoxtox s11A. These recombinant toxins exhibited different in vitro activities depending on pH, and recLoxtox s1A had more intense effects on rabbit skin than did recLoxtox s11A in vivo. Both recombinant toxins were used in immunization protocols, and mapping of their epitopes revealed different immunological reactions for the produced immune serums. Additionally, polyclonal antibodies raised against recLoxtox s1A had greater capacity to significantly reduce the in vitro and in vivo effects of L. similis venom. In summary, we obtained and characterized two novel Loxtox isoforms from L. similis venom, which may be valuable biotechnological and immunological tools against loxoscelism.
Assuntos
Diester Fosfórico Hidrolases/metabolismo , Venenos de Aranha/metabolismo , Aranhas/metabolismo , Animais , Clonagem Molecular , Epitopos/química , Feminino , Concentração de Íons de Hidrogênio , Soros Imunes/imunologia , Testes de Neutralização , Fosfolipase D/metabolismo , Diester Fosfórico Hidrolases/genética , Isoformas de Proteínas , Coelhos , Proteínas Recombinantes/metabolismo , Pele/efeitos dos fármacos , Esfingomielina Fosfodiesterase/metabolismo , Venenos de Aranha/genéticaRESUMO
The mechanosensitivity of cells depends on the lipid-protein interactions of the plasma membrane. Affectations in the lipid region of the plasma membrane affect the transduction of mechanical forces, and any molecule that modifies the biophysical integrity of the lipid bilayer can alter the mechanical activity of the proteins inside the membrane. To understand whether inhibitors of mechanically activated ion channels affect the mechanical properties of the plasma membrane, we evaluated the rigidity of the membrane of sensory neurons of the DRG of mice using a variant of the scanning ion conductance microscopy method, which allows us to calculate the Young's modulus of individual cells before and after the perfusion of different doses of Gd3+, ruthenium red and GsMTx-4. Our results suggest that these molecules compromise the membrane by increasing the Young's modulus value, which indicates that the membrane becomes more rigid; these compounds act through different mechanisms and by a non-specific manner, each one shows a certain preference for specific cell subpopulations, depending on their cell size and their reactivity to isolectin B4. Our results support the idea that the biophysical properties that result from the interactions that arise in the membranes are part of the mechanotransduction process.
Assuntos
Membrana Celular/metabolismo , Moduladores de Transporte de Membrana/metabolismo , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/ultraestrutura , Animais , Cádmio/metabolismo , Linhagem Celular , Células Cultivadas , Módulo de Elasticidade , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Mecanotransdução Celular , Camundongos , Rutênio Vermelho/metabolismo , Transdução de Sinais , Venenos de Aranha/metabolismoRESUMO
Human accidents with spiders of the genus Loxosceles are an important health problem affecting thousands of people worldwide. Patients evolve to severe local injuries and, in many cases, to systemic disturbances as acute renal failure, in which cases antivenoms are considered to be the most effective treatment. However, for antivenom production, the extraction of the venom used in the immunization process is laborious and the yield is very low. Thus, many groups have been exploring the use of recombinant Loxosceles toxins, particularly phospholipases D (PLDs), to produce the antivenom. Nonetheless, some important venom activities are not neutralized by anti-PLD antibodies. Astacin-like metalloproteases (ALMPs) are the second most expressed toxin acting on the extracellular matrix, indicating the importance of its inclusion in the antigen's formulation to provide a better antivenom. Here we show the construction of a hybrid recombinant immunogen, called LgRec1ALP1, composed of hydrophilic regions of the PLD and the ALMP toxins from Loxosceles gaucho. Although the LgRec1ALP1 was expressed as inclusion bodies, it resulted in good yields and it was effective to produce neutralizing antibodies in mice. The antiserum neutralized fibrinogenolytic, platelet aggregation and dermonecrotic activities elicited by L. gaucho, L. laeta, and L. intermedia venoms, indicating that the hybrid recombinant antigen may be a valuable source for the production of protective antibodies against Loxosceles ssp. venoms. In addition, the hybrid recombinant toxin approach may enrich and expand the alternative antigens for antisera production for other venoms.
Assuntos
Anticorpos Neutralizantes/farmacologia , Antivenenos/farmacologia , Diester Fosfórico Hidrolases/toxicidade , Venenos de Aranha/toxicidade , Animais , Antivenenos/metabolismo , Edema/induzido quimicamente , Edema/tratamento farmacológico , Humanos , Masculino , Metaloproteases/metabolismo , Camundongos Endogâmicos BALB C , Necrose/induzido quimicamente , Necrose/tratamento farmacológico , Diester Fosfórico Hidrolases/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Coelhos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Venenos de Aranha/metabolismo , AranhasRESUMO
Phoneutria nigriventer is one of the largest existing true spiders and one of the few considered medically relevant. Its venom contains several neurotoxic peptides that act on different ion channels and chemical receptors of vertebrates and invertebrates. Some of these venom toxins have been shown as promising models for pharmaceutical or biotechnological use. However, the large diversity and the predominance of low molecular weight toxins in this venom have hampered the identification and deep investigation of the less abundant toxins and the proteins with high molecular weight. Here, we combined conventional and next-generation cDNA sequencing with Multidimensional Protein Identification Technology (MudPIT), to obtain an in-depth panorama of the composition of P. nigriventer spider venom. The results from these three approaches showed that cysteine-rich peptide toxins are the most abundant components in this venom and most of them contain the Inhibitor Cysteine Knot (ICK) structural motif. Ninety-eight sequences corresponding to cysteine-rich peptide toxins were identified by the three methodologies and many of them were considered as putative novel toxins, due to the low similarity to previously described toxins. Furthermore, using next-generation sequencing we identified families of several other classes of toxins, including CAPs (Cysteine Rich Secretory Protein-CRiSP, antigen 5 and Pathogenesis-Related 1-PR-1), serine proteinases, TCTPs (translationally controlled tumor proteins), proteinase inhibitors, metalloproteinases and hyaluronidases, which have been poorly described for this venom. This study provides an overview of the molecular diversity of P. nigriventer venom, revealing several novel components and providing a better basis to understand its toxicity and pharmacological activities.
Assuntos
Proteômica , Venenos de Aranha/metabolismo , Aranhas/metabolismo , Transcriptoma , Sequência de Aminoácidos , Animais , Biomarcadores Tumorais/química , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Peptídeos/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Aranhas/genética , Toxinas Biológicas/genética , Toxinas Biológicas/metabolismo , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
Spider venoms are composed of hundreds of proteins and peptides. Several of these venom toxins are cysteine-rich peptides in the mass range of 3-9 kDa. Small peptides (<3 kDa) can be fully characterized by mass spectrometry analysis, while proteins are generally identified by the bottom-up approach in which proteins are first digested with trypsin to generate shorter peptides for MS/MS characterization. In general, it is sufficient for protein identification to sequence two or more peptides, but for venom peptidomics it is desirable to completely elucidate peptide sequences and the number of disulfide bonds in the molecules. In this chapter we describe a methodology to completely sequence and determine the number of disulfide bonds of spider venom peptides in the mass range of 3-9 kDa by multiple enzyme digestion, mass spectrometry of native and digested peptides, de novo analysis, and sequence overlap alignment.
Assuntos
Espectrometria de Massas/métodos , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/metabolismo , Proteômica/métodos , Venenos de Aranha/metabolismo , Aranhas/metabolismo , AnimaisRESUMO
Loxosceles spiders' venom comprises a complex mixture of biologically active toxins, mostly consisting of low molecular mass components (2-40 kDa). Amongst, isoforms of astacin-like metalloproteases were identified through transcriptome and proteome analyses. Only LALP1 (Loxosceles Astacin-Like protease 1) has been characterized. Herein, we characterized LALP3 as a novel recombinant astacin-like metalloprotease isoform from Loxosceles intermedia venom. LALP3 cDNA was cloned in pET-SUMO vector, and its soluble heterologous expression was performed using a SUMO tag added to LALP3 to achieve solubility in Escherichia coli SHuffle T7 Express LysY cells, which express the disulfide bond isomerase DsbC. Protein purification was conducted by Ni-NTA Agarose resin and assayed for purity by SDS-PAGE under reducing conditions. Immunoblotting analyses were performed with specific antibodies recognizing LALP1 and whole venom. Western blotting showed linear epitopes from recombinant LALP3 that cross-reacted with LALP1, and dot blotting revealed conformational epitopes with native venom astacins. Mass spectrometry analysis revealed that the recombinant expressed protein is an astacin-like metalloprotease from L. intermedia venom. Furthermore, molecular modeling of LALP3 revealed that this isoform contains the zinc binding and Met-turn motifs, forming the active site, as has been observed in astacins. These data confirmed that LALP3, which was successfully obtained by heterologous expression using a prokaryote system, is a new astacin-like metalloprotease isoform present in L. intermedia venom.
Assuntos
Reações Cruzadas/imunologia , Metaloendopeptidases/imunologia , Diester Fosfórico Hidrolases/imunologia , Venenos de Aranha/imunologia , Aranhas/imunologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar/genética , Epitopos/imunologia , Epitopos/metabolismo , Immunoblotting , Metaloendopeptidases/classificação , Metaloendopeptidases/genética , Modelos Moleculares , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Filogenia , Domínios Proteicos , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Venenos de Aranha/genética , Venenos de Aranha/metabolismo , Aranhas/genética , Aranhas/metabolismoRESUMO
UNLABELLED: Tarantula spiders, Theraphosidae family, are spread throughout most tropical regions of the world. Despite their size and reputation, there are few reports of accidents. However, like other spiders, their venom is considered a remarkable source of toxins, which have been selected through millions of years of evolution. The present work provides a proteomic overview of the fascinating complexity of the venomous extract of the Grammostola iheringi tarantula, obtained by electrical stimulation of the chelicerae. For analysis a bottom-up proteomic approach Multidimensional Protein Identification Technology (MudPIT) was used. Based on bioinformatics analyses, PepExplorer, a similarity-driven search tool that identifies proteins based on phylogenetically close organisms, a total of 395 proteins were identified in this venomous extract. Most of the identifications (~70%) were classified as predicted (21%), hypothetical (6%) and putative (37%), while a small group (6%) had no predicted function. Identified molecules matched with neurotoxins that act on ions channels; proteases, such as serine proteases, metalloproteinases, cysteine proteinases, aspartic proteinases, carboxypeptidases and cysteine-rich secretory enzymes (CRISP) and some molecules with unknown target. Additionally, non-classical venom proteins were also identified. Up to now, this study represents, to date, the first broad characterization of the composition of G. iheringi venomous extract. Our data provides a tantalizing insight into the diversity of proteins in this venom and their biotechnological potential. SIGNIFICANCE: Animal venoms contain a diversity of molecules able to bind to specific cell targets. Due to their biochemical and physiological properties, these molecules are interesting for medical and biotechnological purposes. In this study, a large number of components of the venomous extract of the spider Grammostola iheringi were identified by the MudPIT technique. It was demonstrated that this approach is a sensitive and adequate method to achieve a broad spectrum of information about animal venoms. Using this bottom-up proteomic method, classical and non-classical venom proteins were identified which stimulate new interest in the systematic research of their protein components.
Assuntos
Proteínas de Artrópodes/metabolismo , Proteômica , Venenos de Aranha/metabolismo , Aranhas/metabolismo , AnimaisRESUMO
Here we present a proteomic characterization of Phoneutria nigriventer venom. A shotgun proteomic approach allowed the identification, for the first time, of O-glycosyl hydrolases (chitinases) in P. nigriventer venom. The electrophoretic profiles under nonreducing and reducing conditions, and protein identification by mass spectrometry, indicated the presence of oligomeric toxin structures in the venom. Complementary proteomic approaches allowed for a qualitative and semi-quantitative profiling of P. nigriventer venom complexity, expanding its known venom proteome diversity.
Assuntos
Proteômica/métodos , Venenos de Aranha/química , Aranhas/química , Sequência de Aminoácidos , Animais , Espectrometria de Massas , Dados de Sequência Molecular , Venenos de Aranha/genética , Venenos de Aranha/metabolismo , Venenos de Aranha/toxicidade , Aranhas/genética , Aranhas/metabolismoRESUMO
Evidence is accumulating that commonly used pesticides are linked to decline of pollinator populations; adverse effects of three neonicotinoids on bees have led to bans on their use across the European Union. Developing insecticides that pose negligible risks to beneficial organisms such as honeybees is desirable and timely. One strategy is to use recombinant fusion proteins containing neuroactive peptides/proteins linked to a 'carrier' protein that confers oral toxicity. Hv1a/GNA (Galanthus nivalis agglutinin), containing an insect-specific spider venom calcium channel blocker (ω-hexatoxin-Hv1a) linked to snowdrop lectin (GNA) as a 'carrier', is an effective oral biopesticide towards various insect pests. Effects of Hv1a/GNA towards a non-target species, Apis mellifera, were assessed through a thorough early-tier risk assessment. Following feeding, honeybees internalized Hv1a/GNA, which reached the brain within 1 h after exposure. However, survival was only slightly affected by ingestion (LD50>100 µg bee(-1)) or injection of fusion protein. Bees fed acute (100 µg bee(-1)) or chronic (0.35 mg ml(-1)) doses of Hv1a/GNA and trained in an olfactory learning task had similar rates of learning and memory to no-pesticide controls. Larvae were unaffected, being able to degrade Hv1a/GNA. These tests suggest that Hv1a/GNA is unlikely to cause detrimental effects on honeybees, indicating that atracotoxins targeting calcium channels are potential alternatives to conventional pesticides.
Assuntos
Abelhas/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/toxicidade , Inseticidas/toxicidade , Lectinas de Ligação a Manose/toxicidade , Lectinas de Plantas/toxicidade , Venenos de Aranha/toxicidade , Animais , Abelhas/crescimento & desenvolvimento , Bloqueadores dos Canais de Cálcio/metabolismo , Galanthus/química , Inseticidas/metabolismo , Larva/efeitos dos fármacos , Aprendizagem/efeitos dos fármacos , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Lectinas de Plantas/genética , Lectinas de Plantas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/toxicidade , Venenos de Aranha/genética , Venenos de Aranha/metabolismoRESUMO
Phoneutria (Ctenidae) is among the most dangerous venomous spiders in Brazil. Its venom is composed of a mixture of pharmacologically active components, some of which have been quite extensively studied due to their potentiality as models for new pharmaceutical drugs. Nevertheless, literature data on the venom-producing glands are very limited. In the present study, we follow the biological development of intra-cocoon stages of Phoneutria nigriventer spiders, mainly regarding the formation of the venomous apparatus and venom production. The results showed that the venom glands of Phoneutria are already present in the early 1st pre-larva stage. The venomous apparatus is completely formed in the larva, a stage that precedes the spider eclosion from the cocoon. At embryo stages, transcripts of a vertebrate-active neurotoxin (PhTx1) were shown to be present, as well as, unidentified venom proteins that were immunolabeled by anti-venom antibodies. It seems that venom toxins play roles in the protection and survival of those early developmental stages of Phoneutria spiders.
Assuntos
Embrião não Mamífero/embriologia , Desenvolvimento Embrionário/fisiologia , Venenos de Aranha/metabolismo , Aranhas/embriologia , Animais , Embrião não Mamífero/fisiologia , Embrião não Mamífero/ultraestrutura , Glândulas Exócrinas/embriologia , Glândulas Exócrinas/metabolismo , Feminino , Expressão Gênica , Larva/crescimento & desenvolvimento , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Aranhas/fisiologiaRESUMO
Spiders of the Loxosceles genus are cosmopolitan, and their venom components possess remarkable biological properties associated with their ability to act upon different molecules and receptors. Accidents with Loxosceles intermedia specimens are recognized as a public health problem in the south of Brazil. To describe the transcriptional profile of the L. intermedia venom gland, we generated a wide cDNA library, and its transcripts were functionally and structurally analyzed. After initial analyses, 1843 expressed sequence tags (ESTs) produced readable sequences that were grouped into 538 clusters, 281 of which were singletons. 985 reads (53% of total ESTs) matched to known proteins. Similarity searches showed that toxin-encoding transcripts account for 43% of the total library and comprise a great number of ESTs. The most frequent toxins were from the LiTx family, which are known for their insecticidal activity. Both phospholipase D and astacin-like metalloproteases toxins account for approximately 9% of total transcripts. Toxins components such as serine proteases, hyaluronidases and venom allergens were also found but with minor representation. Almost 10% of the ESTs encode for proteins involved in cellular processes. These data provide an important overview of the L. intermedia venom gland expression scenario and revealed significant differences from profiles of other spiders from the Loxosceles genus. Furthermore, our results also confirm that this venom constitutes an amazing source of novel compounds with potential agrochemical, industrial and pharmacological applications.
Assuntos
Estruturas Animais/metabolismo , Perfilação da Expressão Gênica , Venenos de Aranha/genética , Aranhas/anatomia & histologia , Aranhas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Eletroforese em Gel de Poliacrilamida , Etiquetas de Sequências Expressas , Regulação da Expressão Gênica , Biblioteca Gênica , Dados de Sequência Molecular , Peptídeos/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Venenos de Aranha/química , Venenos de Aranha/isolamento & purificação , Venenos de Aranha/metabolismoRESUMO
Loxosceles intermedia spider venom was subjected to proteomic analysis through a MudPIT shot-gun approach to identify the protein composition. Were identified 39 proteins which seem to responsible by the lesion of different types of tissues, to some physiopathological actions and by the prevention of structural damage to the toxin structures.
Assuntos
Diester Fosfórico Hidrolases/análise , Proteínas/análise , Venenos de Aranha/análise , Aranhas , Animais , Humanos , Espectrometria de Massas , Diester Fosfórico Hidrolases/metabolismo , Diester Fosfórico Hidrolases/toxicidade , Proteínas/metabolismo , Proteínas/toxicidade , Proteoma/análise , Mordeduras de Serpentes/fisiopatologia , Venenos de Aranha/metabolismo , Venenos de Aranha/toxicidadeRESUMO
Tarantula venoms are a cocktail of proteins and peptides that have been increasingly studied in recent years. In contrast, less attention has been given to analyzing the structure of the paired cephalic glands that produce the venom. We have used light, electron, and confocal microscopy to study the organization and structure of the venom gland of the Brazilian tarantula Vitalius dubius. The chelicerae are hairy chitinous structures, each with a single curved hollow fang that opens via an orifice on the anterior surface. Internally, each chelicera contains striated muscle fiber bundles that control fang extension and retraction, and a cylindrical conical venom gland surrounded by a thick well-developed layer of obliquely arranged muscle fibers. Light microscopy of longitudinal and transverse sections showed that the gland secretory epithelium consists of a sponge-like network of slender epithelial cell processes with numerous bridges and interconnections that form lacunae containing secretion. This secretory epithelium is supported by a basement membrane containing elastic fibers. The entire epithelial structure of the venom-secreting cells is reinforced by a dense network of F-actin intermediate filaments, as shown by staining with phalloidin. Neural elements (axons and acetylcholinesterase activity) are also associated with the venom gland. Transmission electron microscopy of the epithelium revealed an ultrastructure typical of secretory cells, including abundant rough and smooth endoplasmic reticulum, an extensive Golgi apparatus, and numerous mitochondria.
Assuntos
Glândulas Exócrinas/anatomia & histologia , Venenos de Aranha/metabolismo , Aranhas/anatomia & histologia , Animais , Glândulas Exócrinas/metabolismo , Glândulas Exócrinas/ultraestrutura , Feminino , Masculino , Microscopia Eletrônica de Transmissão , Aranhas/metabolismo , Aranhas/ultraestruturaRESUMO
The theraphosid spider genus Vitalius contains several species found in southeastern Brazil. In this work, we used electrostimulation to obtain venom from Vitalius dubius and examined its general composition. Male spiders yielded significantly less (p < 0.05) venom (12.5 +/- 0.7 mg of liquid/spider, n = 16; mean +/- S.E.M.) than female spiders (25.5 +/- 2.0 mg of liquid/spider, n = 11). However, when corrected for spider weight, males yielded slightly more venom (2.89 +/- 0.16 mg/g vs. 2.45 +/- 0.76 mg/g for males and females, respectively, p < 0.05). Venom yield correlated linearly with spider weight for spiders weighing up to approximately 12-13 g, but decreased in very heavy females. There was a marked decrease in venom yield after the first milking. The protein concentration of pooled venom was 18.3 +/- 2.4 mg/ml (n = 4) and accounted for 16.6 +/- 4.7% of the dry venom weight. The venom contained high hyaluronidase activity (275 +/- 24 TRU/mg of protein, n = 4), with a molecular mass of approximately 45 kDa estimated by zymography. SDS-PAGE revealed a few proteins with molecular masses >14 kDa but showed two staining bands of peptides <14 kDa. The venom reacted in ELISA with affinity-purified IgG from commercial arachnidic antivenom. Immunoblotting with this IgG detected proteins of 30-140 kDa only. Fractionation of the venom by reverse-phase chromatography resulted in five major and eight minor peaks.
Assuntos
Venenos de Aranha/química , Aranhas/fisiologia , Animais , Cromatografia , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Masculino , Venenos de Aranha/metabolismoRESUMO
Loxoscelism (the term used to define accidents by the bite of brown spiders) has been reported worldwide. Clinical manifestations following brown spider bites are frequently associated with skin degeneration, a massive inflammatory response at the injured region, intravascular hemolysis, platelet aggregation causing thrombocytopenia and renal disturbances. The mechanisms by which the venom exerts its noxious effects are currently under investigation. The whole venom is a complex mixture of toxins enriched with low molecular mass proteins in the range of 5-40 kDa. Toxins including alkaline phosphatase, hyaluronidase, metalloproteases (astacin-like proteases), low molecular mass (5.6-7.9 kDa) insecticidal peptides and phospholipases-D (dermonecrotic toxins) have been identified in the venom. The purpose of the present review is to describe biotechnological applications of whole venom or some toxins, with especial emphasis upon molecular biology findings obtained in the last years.
Assuntos
Biotecnologia , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/toxicidade , Venenos de Aranha/química , Venenos de Aranha/toxicidade , Aranhas/química , Aranhas/classificação , Animais , Previsões , Humanos , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Picada de Aranha/patologia , Picada de Aranha/terapia , Venenos de Aranha/genética , Venenos de Aranha/metabolismoRESUMO
Herein, we describe a confirmed case of Loxosceles spider bite that illustrates the critical complications seen in loxoscelism, including skin necrosis, rhabdomyolysis, hemolysis, coagulopathy, acute kidney failure, and electrolyte disorders. Upon initial assessment, laboratory studies revealed the following: the white blood cell count was 29,400 WBCs/mm(3), hemoglobin was 9.2g/dL, and the platelet count was 218,000 cells/mm(3). Coagulation studies revealed the following: international normalized ratio, 1.83; activated partial-thromboplastin time, 62 s; D-dimer, 600 ng/mL (normal range <500 ng/mL); free protein S, 37% (normal range=64-114%); protein C, negative; and antithrombin III, negative. Various serum levels were abnormal: urea, 110 mg/dL; creatinine, 3.1mg/dL; indirect bilirubin, 3.8 mg/dL; creatine kinase, 1631 U/L; lactate dehydrogenase, 6591 U/L; potassium 6.2 mmol/L. Urine tests were positive for hemoglobin and bilirubin. In addition, concentrations of interleukin-6 and tumor necrosis factor-alpha were notably elevated in the serum. In conclusion, physicians must be alert to the possibility of loxoscelism when a patient presents with the clinical and laboratory findings described above, especially if the patient resides in an endemic area. Advances in our understanding of multiple pathways and mediators that orchestrate the response to Loxosceles venom might reveal new possibilities for the management of loxoscelism.