RESUMO
Scorpionism is responsible for most accidents involving venomous animals in Brazil, which leads to severe symptoms that can evolve to death. Scorpion venoms consist of complexes cocktails, including peptides, proteins, and non-protein compounds, making separation and purification procedures extremely difficult and time-consuming. Scorpion toxins target different biological systems and can be used in basic science, for clinical, and biotechnological applications. This study is the first to explore the venom content of the unexplored scorpion species Rhopalurus crassicauda, which inhabits exclusively the northernmost state of Brazil, named Roraima, and southern region of Guyana. Here, we pioneer the fractionation of the R. crassicauda venom and isolated and characterized a novel scorpion beta-neurotoxin, designated Rc1, and a monomeric hyaluronidase. R. crassicauda venom and Rc1 (6,882 Da) demonstrated pro-inflammatory activities in vitro and a nociceptive response in vivo. Moreover, Rc1 toxin showed specificity for activating Nav1.4, Nav1.6, and BgNav1 voltage-gated ion channels. This study also represents a new perspective for the treatment of envenomings in Roraima, since the Brazilian scorpion and arachnid antivenoms were not able to recognize R. crassicauda venom and its fractions (with exception of hyaluronidase). Our work provides useful insights for the first understanding of the painful sting and pro-inflammatory effects associated with R. crassicauda envenomings.
Assuntos
Hialuronoglucosaminidase/metabolismo , Mediadores da Inflamação/metabolismo , Peptídeos/metabolismo , Picadas de Escorpião/terapia , Venenos de Escorpião/metabolismo , Animais , Antivenenos/imunologia , Antivenenos/uso terapêutico , Linhagem Celular , Cromatografia Líquida , Reações Cruzadas , Humanos , Hialuronoglucosaminidase/isolamento & purificação , Mediadores da Inflamação/isolamento & purificação , Canais Iônicos/metabolismo , Camundongos , Peptídeos/isolamento & purificação , Venenos de Escorpião/isolamento & purificação , Escorpiões , Análise de Sequência de ProteínaRESUMO
In this study, we characterize the venom of Centruroides edwardsii, one of the most abundant scorpions in urban and rural areas of Costa Rica, in terms of its biochemical constituents and their biological activities. C. edwardsii venom is rich in peptides but also contains some higher molecular weight protein components. No phospholipase A2, hemolytic or fibrinogenolytic activities were found, but the presence of proteolytic and hyaluronidase enzymes was evidenced by zymography. Venom proteomic analysis indicates the presence of a hyaluronidase, several cysteine-rich secretory proteins, metalloproteinases and a peptidylglycine α-hydroxylating monooxygenase like-enzyme. It also includes peptides similar to the K+-channel blocker margatoxin, a dominant toxin in the venom of the related scorpion C. margaritatus. MS and N-terminal sequencing analysis also reveals the presence of Na+-channel-modulating peptides with sequence similarity to orthologs present in other scorpion species of the genera Centruroides and Tityus. We purified the hyaluronidase (which co-eluted with an allergen 5-like CRiSP) and sequenced ~60% of this enzyme. We also sequenced some venom gland transcripts that include other cysteine-containing peptides and a Non-Disulfide Bridged Peptide (NDBP). Our in vivo experiments characterizing the effects on potential predators and prey show that C. edwardsii venom induces paralysis in several species of arthropods and geckos; crickets being the most sensitive and cockroaches and scorpions the most resistant organisms tested. Envenomation signs were also observed in mice, but no lethality was reached by intraperitoneal administration of this venom up to 120⯵g/g body weight.
Assuntos
Venenos de Escorpião/química , Venenos de Escorpião/toxicidade , Escorpiões/química , Animais , Costa Rica , Feminino , Hialuronoglucosaminidase/isolamento & purificação , Insetos , Lagartos , Masculino , Camundongos , Paralisia/induzido quimicamente , Comportamento Predatório , Proteoma , Proteínas de Répteis/química , Venenos de Escorpião/enzimologia , TranscriptomaRESUMO
Snake venoms are complex mixtures mainly composed of proteins and small peptides. Crotoxin is one of the most studied components from Crotalus venoms, but many other components are less known due to their low abundance. The venome of Crotalus durissus terrificus, the most lethal Brazilian snake, was investigated by combining its venom gland transcriptome and proteome to create a holistic database of venom compounds unraveling novel toxins. We constructed a cDNA library from C. d. terrificus venom gland using the Illumina platform and investigated its venom proteome through high resolution liquid chromotography-tandem mass spectrometry. After integrating data from both data sets, more than 30 venom components classes were identified by the transcriptomic analysis and 15 of them were detected in the venom proteome. However, few of them (PLA2, SVMP, SVSP, and VEGF) were relatively abundant. Furthermore, only seven expressed transcripts contributed to â¼82% and â¼73% of the abundance in the transcriptome and proteome, respectively. Additionally, novel venom proteins are reported, and we highlight the importance of using different databases to perform the data integration and discuss the structure of the venom components-related transcripts identified. Concluding, this research paves the way for novel investigations and discovery of future pharmacological agents or targets in the antivenom therapy.
Assuntos
Venenos de Crotalídeos/química , Crotalus/fisiologia , Proteoma/isolamento & purificação , Transcriptoma , Sequência de Aminoácidos , Animais , Carboxipeptidases/genética , Carboxipeptidases/isolamento & purificação , Carboxipeptidases/metabolismo , Cromatografia Líquida/métodos , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/isolamento & purificação , Fatores de Crescimento de Fibroblastos/metabolismo , Expressão Gênica , Biblioteca Gênica , Ontologia Genética , Hialuronoglucosaminidase/genética , Hialuronoglucosaminidase/isolamento & purificação , Hialuronoglucosaminidase/metabolismo , Anotação de Sequência Molecular , Proteoma/genética , Proteoma/metabolismo , Alinhamento de Sequência , Análise de Sequência de RNA , Espectrometria de Massas em Tandem/métodosRESUMO
Theraphosid spider venoms are extremely complex mixtures, composed mainly by low molecular compounds, peptides, and enzymes. The large size of these spiders and their ability to breed in captivity permits access to rather large amounts of venom and an easier venom extraction. In the present study, we conducted a comparative investigation about the content of hyaluronidase-like enzymes in the venoms from several theraphosid spiders, with a special focus on the Poecilotheria species, which are considered as underestimated theraphosids of medical importance. The following species were analyzed: Poecilotheria regalis, Poecilotheria ornata, Poecilotheria rufilata, Poecilotheria vittata, Bonnetina papalutlensis, Aphonopelma sp., Brachypelma smithi, Brachypelma epicureanum, Brachypelma boehmei, Grammostola porteri, Lasiodora klugi, Ceratogyrus darlingi, and Nhandu chromatus. The presence of hyaluronidase-like enzymes was evidenced in all venoms by a turbidimetric method and zymography. Several isoforms of acid-active hyaluronidase-like enzymes were detected in the venoms from Poecilotheria species. These results provide some biochemical characteristics of the high molecular mass proteins of the theraphosid venoms.
Assuntos
Proteínas de Artrópodes/isolamento & purificação , Hialuronoglucosaminidase/isolamento & purificação , Venenos de Aranha/enzimologia , Aranhas/química , Animais , Especificidade da EspécieRESUMO
The Loxosceles genus spiders (the brown spiders) are encountered in all the continents, and the clinical manifestations following spider bites include skin necrosis with gravitational lesion spreading and occasional systemic manifestations, such as intravascular hemolysis, thrombocytopenia and acute renal failure. Brown spider venoms are complex mixtures of toxins especially enriched in three molecular families: the phospholipases D, astacin-like metalloproteases and Inhibitor Cystine Knot (ICK) peptides. Other toxins with low level of expression also present in the venom include the serine proteases, serine protease inhibitors, hyaluronidases, allergen factors and translationally controlled tumor protein (TCTP). The mechanisms by which the Loxosceles venoms act and exert their noxious effects are not fully understood. Except for the brown spider venom phospholipase D, which causes dermonecrosis, hemolysis, thrombocytopenia and renal failure, the pathological activities of the other venom toxins remain unclear. The objective of the present review is to provide insights into the brown spider venoms and loxoscelism based on recent results. These insights include the biology of brown spiders, the clinical features of loxoscelism and the diagnosis and therapy of brown spider bites. Regarding the brown spider venom, this review includes a description of the novel toxins revealed by molecular biology and proteomics techniques, the data regarding three-dimensional toxin structures, and the mechanism of action of these molecules. Finally, the biotechnological applications of the venom components, especially for those toxins reported as recombinant molecules, and the challenges for future study are discussed.
Assuntos
Venenos de Aranha/toxicidade , Aranhas/química , Animais , Antivenenos/química , Proteínas de Artrópodes/química , Proteínas de Artrópodes/isolamento & purificação , Proteínas de Artrópodes/toxicidade , Biomarcadores Tumorais/química , Biomarcadores Tumorais/isolamento & purificação , Feminino , Humanos , Hialuronoglucosaminidase/química , Hialuronoglucosaminidase/isolamento & purificação , Hialuronoglucosaminidase/toxicidade , Masculino , Modelos Moleculares , Fosfolipase D/química , Fosfolipase D/isolamento & purificação , Fosfolipase D/toxicidade , Proteômica , Serina Proteases/química , Serina Proteases/isolamento & purificação , Serina Proteases/toxicidade , Picada de Aranha/patologia , Venenos de Aranha/química , Venenos de Aranha/imunologia , Aranhas/anatomia & histologia , Aranhas/fisiologia , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
Loxoscelism is the designation given to clinical symptoms evoked by Loxosceles spider's bites. Clinical manifestations include skin necrosis with gravitational spreading and systemic disturbs. The venom contains several enzymatic toxins. Herein, we describe the cloning, expression, refolding and biological evaluation of a novel brown spider protein characterized as a hyaluronidase. Employing a venom gland cDNA library, we cloned a hyaluronidase (1200 bp cDNA) that encodes for a signal peptide and a mature protein. Amino acid alignment revealed a structural relationship with members of hyaluronidase family, such as scorpion and snake species. Recombinant hyaluronidase was expressed as N-terminal His-tag fusion protein (â¼45 kDa) in inclusion bodies and activity was achieved using refolding. Immunoblot analysis showed that antibodies that recognize the recombinant protein cross-reacted with hyaluronidase from whole venom as well as an anti-venom serum reacted with recombinant protein. Recombinant hyaluronidase was able to degrade purified hyaluronic acid (HA) and chondroitin sulfate (CS), while dermatan sulfate (DS) and heparan sulfate (HS) were not affected. Zymograph experiments resulted in â¼45 kDa lytic zones in hyaluronic acid (HA) and chondroitin sulfate (CS) substrates. Through in vivo experiments of dermonecrosis using rabbit skin, the recombinant hyaluronidase was shown to increase the dermonecrotic effect produced by recombinant dermonecrotic toxin from L. intermedia venom (LiRecDT1). These data support the hypothesis that hyaluronidase is a "spreading factor". Recombinant hyaluronidase provides a useful tool for biotechnological ends. We propose the name Dietrich's Hyaluronidase for this enzyme, in honor of Professor Carl Peter von Dietrich, who dedicated his life to studying proteoglycans and glycosaminoglycans.
Assuntos
Aracnídeos/enzimologia , Hialuronoglucosaminidase/genética , Hialuronoglucosaminidase/isolamento & purificação , Peçonhas/enzimologia , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/isolamento & purificação , Proteínas de Artrópodes/metabolismo , Sulfatos de Condroitina/metabolismo , Clonagem Molecular , Modelos Animais de Doenças , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/química , Hialuronoglucosaminidase/metabolismo , Mordeduras e Picadas de Insetos/patologia , Dados de Sequência Molecular , Peso Molecular , Filogenia , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
A hyaluronidase (CdtHya1) from Crotalus durissus terrificus snake venom (CdtV) was isolated and showed to exhibit a high activity on hyaluronan cleavage. However, surveys on this enzyme are still limited. This study aimed at its isolation, functional/structural characterization and the evaluation of its effect on the spreading of crotoxin and phospholipase A(2) (PLA(2)). The enzyme was purified through cation exchange, gel filtration and hydrophobic chromatography. After that, it was submitted to a reverse-phase fast protein liquid chromatography (RP-FPLC) and Edman degradation sequencing, which showed the first N-terminal 44 amino acid residues whose sequence evidenced identity with other snake venom hyaluronidases. CdtHya1 is a monomeric glycoprotein of 64.5 kDa estimated by SDS-PAGE under reducing conditions. It exhibited maximum activity in the presence of 0.2 M NaCl, at 37 °C, pH 5.5 and a specificity to hyaluronan higher than that to chondroitin-4-sulphate, chondroitin-6-sulphate or dermatan. Divalent cations (Ca(2+) and Mg(2+)) and 1 M NaCl significantly reduced the enzyme activity. The specific activity of CdtHya1 was 5066 turbidity reducing units (TRU)/mg, against 145 TRU/mg for the soluble venom, representing a 34.9-fold purification. The pure enzyme increased the diffusion of crotoxin and PLA(2) through mice tissues. CdtHya1 (32 TRU/40 µL) potentiated crotoxin action, as evidenced by mice death, and it decreased the oedema caused by subplantar injections of buffer, crotoxin or PLA(2), thus evidencing the relevance of hyaluronidase in the crotalic envenoming. This work yielded a highly active antiedematogenic hyaluronidase from CdtV, the first one isolated from rattlesnake venoms.
Assuntos
Venenos de Crotalídeos/enzimologia , Crotalus/metabolismo , Edema/prevenção & controle , Hialuronoglucosaminidase/metabolismo , Hialuronoglucosaminidase/farmacologia , Sequência de Aminoácidos , Animais , Biocatálise/efeitos dos fármacos , Cálcio/farmacologia , Cromatografia/métodos , Crotoxina/metabolismo , Difusão/efeitos dos fármacos , Edema/patologia , Eletroforese em Gel de Poliacrilamida , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/isolamento & purificação , Cinética , Magnésio/farmacologia , Masculino , Camundongos , Dados de Sequência Molecular , Fosfolipases A2/metabolismo , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Cloreto de Sódio/farmacologia , Especificidade por SubstratoRESUMO
Venomous animals use their venoms as tools for defense or predation. These venoms are complex mixtures, mainly enriched of proteic toxins or peptides with several, and different, biological activities. In general, spider venom is rich in biologically active molecules that are useful in experimental protocols for pharmacology, biochemistry, cell biology and immunology, as well as putative tools for biotechnology and industries. Spider venoms have recently garnered much attention from several research groups worldwide. Brown spider (Loxosceles genus) venom is enriched in low molecular mass proteins (5-40 kDa). Although their venom is produced in minute volumes (a few microliters), and contain only tens of micrograms of protein, the use of techniques based on molecular biology and proteomic analysis has afforded rational projects in the area and permitted the discovery and identification of a great number of novel toxins. The brown spider phospholipase-D family is undoubtedly the most investigated and characterized, although other important toxins, such as low molecular mass insecticidal peptides, metalloproteases and hyaluronidases have also been identified and featured in literature. The molecular pathways of the action of these toxins have been reported and brought new insights in the field of biotechnology. Herein, we shall see how recent reports describing discoveries in the area of brown spider venom have expanded biotechnological uses of molecules identified in these venoms, with special emphasis on the construction of a cDNA library for venom glands, transcriptome analysis, proteomic projects, recombinant expression of different proteic toxins, and finally structural descriptions based on crystallography of toxins.
Assuntos
Biotecnologia/métodos , Aranha Marrom Reclusa/metabolismo , Venenos de Aranha/química , Toxinas Biológicas/farmacologia , Animais , Biomarcadores Tumorais/isolamento & purificação , Biomarcadores Tumorais/farmacologia , Hialuronoglucosaminidase/isolamento & purificação , Hialuronoglucosaminidase/farmacologia , Metaloproteases/isolamento & purificação , Metaloproteases/farmacologia , Fosfolipase D/isolamento & purificação , Fosfolipase D/farmacologia , Inibidores de Serina Proteinase/isolamento & purificação , Inibidores de Serina Proteinase/farmacologia , Venenos de Aranha/enzimologia , Toxinas Biológicas/isolamento & purificação , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
Freshwater stingrays (Potamotrygon motoro) are known to cause human accidents through a sting located in its tail. In the State of Goiás, this accident happens especially during the fishing season of the Araguaia River. The P. motoro venom extracted from the sting presented hyaluronidase activity. The enzyme was purified by gel filtration on Sephacryl S-100 and ion-exchange chromatography on SP-Sepharose. A typical procedure provided 376.4-fold purification with a 2.94% yield. The molecular weight of the purified enzyme was 79 kDa as estimated by gel filtration on Sephacryl S-100. The K(m) and V(max) values for hyaluronidase, using hyaluronic acid as substrate, were 4.91 microg/ml and 2.02 U/min, respectively. The pH optimum for the enzyme was pH 4.2 and maximum activity was obtained at 40 degrees C. The hyaluronidase from P. motoro was shown to be heat instable, being stabilized by bovine albumin and DTT, and inhibited by Fe(2+), Mn(2+), Cu(2+) and heparin.
Assuntos
Elasmobrânquios , Hialuronoglucosaminidase/isolamento & purificação , Hialuronoglucosaminidase/metabolismo , Peçonhas/enzimologia , Animais , Cromatografia em Gel , Cromatografia por Troca Iônica , Cobre/farmacologia , Ditiotreitol/farmacologia , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Água Doce , Heparina/farmacologia , Temperatura Alta , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/antagonistas & inibidores , Concentração de Íons de Hidrogênio , Ferro/farmacologia , Manganês/farmacologia , Soroalbumina Bovina/farmacologia , Especificidade por SubstratoRESUMO
The purification procedure of a hyaluronidase from Tityus serrulatus scorpion venom is described. It involves basically an ion-exchange chromatography on CM-cellulose at pH 7.8 followed by a rechromatography of the active fraction on the same column at pH 4.7. The optima pH and temperature for maximum activity of the isolated enzyme was 6.0 and 40 degrees C, respectively. Its K(M) was 69.7 microg/ml at 37 degrees C and its specific activity was 19,900+/-1,730 turbidity reducing units (TRU)/mg against 845+/-88TRU/mg for the whole desiccated venom, representing a 23- to 24-fold purification range. The hyaluronidase activity of the purified protein (51kDa) was inhibited by some flavonoid compounds. This article also showed that T. serrulatus hyaluronidase affected on the activity of the venom's major toxin, tityustoxin-I (TsTX-I or Ts1), as reflected by alterations in the serum levels of creatine kinase (CK), lactate dehydrogenase (LD) and aspartate aminotransferase (AST) following injection of TsTX-I, in the presence or absence of hyaluronidase.