RESUMO
The spider family Sicariidae includes three genera, Hexophthalma, Sicarius and Loxosceles. The three genera share a common characteristic in their venoms: the presence of Sphingomyelinases D (SMase D). SMases D are considered the toxins that cause the main pathological effects of the Loxosceles venom, that is, those responsible for the development of loxoscelism. Some studies have shown that Sicarius spiders have less or undetectable SMase D activity in their venoms, when compared to Hexophthalma. In contrast, our group has shown that Sicarius ornatus, a Brazilian species, has active SMase D and toxic potential to envenomation. However, few species of Sicarius have been characterized for their toxic potential. In order to contribute to a better understanding about the toxicity of Sicarius venoms, the aim of this study was to characterize the toxic properties of male and female venoms from Sicarius tropicus and compare them with that from Loxosceles laeta, one of the most toxic Loxosceles venoms. We show here that S. tropicus venom presents active SMases D. However, regarding hemolysis development, it seems that these toxins in this species present different molecular mechanisms of action than that described for Loxosceles venoms, whereas it is similar to those present in bacteria containing SMase D. Besides, our results also suggest that, in addition to the interspecific differences, intraspecific variations in the venoms' composition may play a role in the toxic potential of venoms from Sicarius species.
Assuntos
Evolução Molecular , Hemólise/efeitos dos fármacos , Diester Fosfórico Hidrolases/toxicidade , Venenos de Aranha/toxicidade , Aranhas/enzimologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Feminino , Células HaCaT , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/patologia , Masculino , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Fatores Sexuais , Especificidade da Espécie , Venenos de Aranha/enzimologia , Venenos de Aranha/genética , Aranhas/classificação , Aranhas/genéticaRESUMO
The spiders of the Loxosceles genus (called brown or violin spiders) are of medical relevance in several countries due to the many human envenomation cases reported. The main component of Loxosceles venom is the enzyme sphingomyelinase D (SMase D), which is responsible for the local and systemic effects induced by the whole venom. Here, we investigated the cytotoxic and genotoxic effects caused by Loxosceles laeta venom and SMase D on human keratinocytes to better understand the dermonecrosis development mechanism. Our findings indicate that whole venom, as well as SMase D, increases intracellular superoxide levels, leading to DNA damage. These effects appear to be dependent on the binding of SMase D to the cell surface, although the complete pathway triggered as a result of the binding still needs to be elucidated. Moreover, after SMase D treatment, we observed the presence of histone γH2AX, suggesting that the cells are undergoing DNA repair. Moreover, when ATR kinase was inhibited, the cell viability of human keratinocytes was decreased. Together, our findings strongly suggest that L. laeta venom, as well as SMase D, increases intracellular superoxide levels, leading to DNA damage in human keratinocytes. Additionally, the induced DNA damage is repaired through the activation of an apparent ATR-mediated DNA-damage response. This knowledge may contribute to a better understanding of the behaviour of human keratinocytes during cutaneous loxoscelism, a condition that affects thousands of people around the world.
Assuntos
Dano ao DNA/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Diester Fosfórico Hidrolases/toxicidade , Venenos de Aranha/toxicidade , Superóxidos/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sobrevivência Celular , Células HaCaT , Histonas/metabolismo , Humanos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/metabolismo , Aranhas/enzimologia , Superóxidos/análiseRESUMO
α-l-Fucosidases are widely occurring enzymes that remove fucose residues from N- and O-fucosylated glycoproteins. Comparison of amino acid sequences of fucosidases reveals that although the nucleophile is conserved among all α-l-fucosidases, the position of the acid/base residue is quite variable. Although several site-directed mutation studies have previously been performed on bacterial fucosidases, the only eukaryotic fucosidase so studied was the human fucosidase. Recent alignments indicate that human and Arthropoda α-l-fucosidases share at least 50% identity and the acid/base residue seems to be conserved among them suggesting a common acid/base residue in Metazoa. Here we describe the cloning and expression in Pichia pastoris of a very active α-l-fucosidase from the spider Nephilingis cruentata (NcFuc) with a Km value for pNPFuc of 0.4 mM. NcFuc hydrolyzed fucoidan, 2´fucosyllactose and also lacto-N-difucohexaose II. Mutants modified at the conserved residues D214N, E209A, E59A were expressed and characterized. The 500-fold lower kcat of D214N than the wild type was consistent with a role in catalysis, as was the 8000-fold lower kcat value of E59A. This was supported by the 57-fold increase in the kcat of E59A upon addition of azide. A complex pH/rate profile was seen for the wild-type and mutant forms of NcFuc, similar to those measured previously for the Sulfolobus fucosidase. The non-conservative catalytic structure and distinct active site organization reinforce the necessity of structural studies of new fucosidases.
Assuntos
Biocatálise , Aranhas/enzimologia , alfa-L-Fucosidase/química , alfa-L-Fucosidase/metabolismo , Animais , Domínio Catalítico , Concentração de Íons de Hidrogênio , Mutação , alfa-L-Fucosidase/genética , alfa-L-Fucosidase/isolamento & purificaçãoRESUMO
l-fucose is a constituent of glycoconjugates in different organisms. Fucosidases catalyze the removal of fucose residues, and have been correlated to different physiological and pathological processes, such as fertilization, cancer, fucosidosis, and digestion in molluscs and ticks. An α-l-fucosidase sequence was identified from the transcriptome and proteome from the midgut diverticula of the synanthropic spider Nephilingis cruentata. In this article, we describe the isolation of this α-l-fucosidase and the characterization of its activity using substrates and inhibitors demonstrating different specificities among fucosidases. The enzyme had a Km of 32 and 400 µM for 4-methylumbelliferyl α-l-fucopyranoside and 4-nitrophenyl α-l-fucopyranoside, respectively; and was unable to hydrolyze fucoidan. Nephilingis cruentata α-l-fucosidase was inhibited competitively by fucose and fuconojyrimycin. The fucosidase had two distinct pH optima even in the isolated form, due to oligomerization dependent on pH, as previously described to other fucosidases. Alignment and molecular homology modeling of the protein sequence with other fucosidases indicated that the active sites and catalytic residues were different, including residues involved in acid/base catalysis. Phylogenetic analysis showed, for the first time, gene-duplication events for fucosidases in Arachnida species. All these data reveal that studies on fucosidases in organisms distinct from bacteria, fungi, and humans are important.
Assuntos
Aranhas/enzimologia , alfa-L-Fucosidase/metabolismo , Animais , Feminino , Humanos , Filogenia , Aranhas/genética , Homologia Estrutural de Proteína , alfa-L-Fucosidase/genética , alfa-L-Fucosidase/isolamento & purificaçãoRESUMO
Spider envenomation, from the genus Loxosceles, is frequently reported as a cause of necrotic lesions in humans around the world. Among the many components found in the venom of Loxosceles genus, phospholipases D (PLDs) are the most investigated, since they can cause a massive inflammatory response, dermonecrosis, hemolysis and platelet aggregation, among other effects. Even though the PLDs induce strong platelet aggregation, there are no studies showing how the PLDs interact with platelets to promote this effect. Since many agonists must interact with specific receptors on the platelet membrane to induce aggregation, it is reasonable to expect that the PLDs may, in some way, also interact with platelets, to induce this activity. Therefore, to address this possibility, in this work, a recombinant PLD, called LgRec1, from L. gaucho was fused to enhanced green fluorescent protein (EGFP) and used as a probe to detect the interaction of LgRec1 to platelets, by fluorescence-activated cell sorter (FACS) and confocal microscopy. The preservation of biological activities of this chimera toxin was also analyzed. As a first, the results show that LgRec1 does not require plasma components to bind to platelets, although these components are necessary to LgRec1 to induce platelet aggregation. Also, the attachment of LgRec1 to human platelets' cell membranes suggests that the exposure of phosphatidylserine (PS) may act as a scaffold for coagulation factors. Therefore, the results add new information about the binding of Loxosceles PLDs to platelets, which may help unravel how these toxins promote platelet aggregation.
Assuntos
Plaquetas/efeitos dos fármacos , Fosfatidilserinas/metabolismo , Fosfolipase D/farmacologia , Aranhas/enzimologia , Animais , Plaquetas/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/farmacologia , Hemólise/efeitos dos fármacos , Humanos , L-Lactato Desidrogenase/metabolismo , Fosfolipase D/genética , Agregação Plaquetária/efeitos dos fármacos , Plasma Rico em Plaquetas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Sphingomyelinases D have only been identified in arachnid venoms, Corynebacteria, Arcanobacterium, Photobacterium and in the fungi Aspergillus and Coccidioides. The arachnid and bacterial enzymes share very low sequence identity and do not contain the HKD sequence motif characteristic of the phospholipase D superfamily, however, molecular modeling and circular dichroism of SMases D from Loxosceles intermedia and Corynebacterium pseudotuberculosis indicate similar folds. The phospholipase, hemolytic and necrotic activities and mice vessel permeabilities were compared and both enzymes possess the ability to hydrolyze phospholipids and also promote similar pathological reactions in the host suggesting the existence of a common underlying mechanism in tissue disruption. J. Cell. Biochem. 118:2053-2063, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Proteínas de Artrópodes/toxicidade , Proteínas de Bactérias/toxicidade , Permeabilidade Capilar/efeitos dos fármacos , Corynebacterium pseudotuberculosis/química , Diester Fosfórico Hidrolases/toxicidade , Aranhas/química , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Corynebacterium pseudotuberculosis/enzimologia , Corynebacterium pseudotuberculosis/patogenicidade , Eritrócitos/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Hemólise/efeitos dos fármacos , Cavalos , Humanos , Camundongos , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Carneiro Doméstico , Pele/efeitos dos fármacos , Pele/patologia , Aranhas/enzimologia , Aranhas/patogenicidadeRESUMO
Envenomation by Loxosceles spider is characterized by the development of dermonecrosis. In previous studies, we have demonstrated that increased expression/secretion of matrix metalloproteinases 2 and 9, induced by Loxosceles intermedia venom Class 2 SMases D (the main toxin in the spider venom), contribute to the development of cutaneous loxoscelism. In the present study we show that the more potent venom containing the Class 1 SMase D from Loxosceles laeta, in addition to increasing the expression/secretion of MMP2 and MMP9, also stimulates the expression of MMP7 (Matrilysin-1), which was associated with keratinocyte cell death. Tetracycline, a matrix metalloproteinase inhibitor, prevented cell death and reduced MMPs expression. Considering that L. laeta venom is more potent at inducing dermonecrosis than L. intermedia venom, our results suggest that MMP7 may play an important role in the severity of dermonecrosis induced by L. laeta spider venom SMase D. In addition, the inhibition of MMPs by e.g. tetracyclines may be considered for the treatment of the cutaneous loxoscelism.
Assuntos
Proteínas de Artrópodes/farmacologia , Queratinócitos/efeitos dos fármacos , Diester Fosfórico Hidrolases/farmacologia , Venenos de Aranha/farmacologia , Aranhas/enzimologia , Animais , Antibacterianos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Queratinócitos/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 7 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Necrose/prevenção & controle , Coelhos , Pele/efeitos dos fármacos , Pele/patologia , Venenos de Aranha/enzimologia , Tetraciclina/farmacologia , Fatores de TempoRESUMO
BACKGROUND: Sphingomyelinase D is the main toxin present in the venom of Loxosceles spiders. Several isoforms present in these venoms can be structurally classified in two groups. Class I Sphingomyelinase D contains a single disulphide bridge and variable loop. Class II Sphingomyelinase D presents an additional intrachain disulphide bridge that links a flexible loop with a catalytic loop. These classes exhibit differences in their toxic potential. In this paper we address the distribution of the structural classes of SMase D within and among species of spiders and also their evolutionary origin by means of phylogenetic analyses. We also conducted tests to assess the action of natural selection in their evolution combined to structural modelling of the affected sites. RESULTS: The majority of the Class I enzymes belong to the same clade, which indicates a recent evolution from a single common ancestor. Positively selected sites are located on the catalytic interface, which contributes to a distinct surface charge distribution between the classes. Sites that may prevent the formation of an additional bridge were found in Class I enzymes. CONCLUSIONS: The evolution of Sphingomyelinase D has been driven by natural selection toward an increase in noxiousness, and this might help explain the toxic variation between classes.
Assuntos
Evolução Molecular , Diester Fosfórico Hidrolases/genética , Venenos de Aranha/enzimologia , Aranhas/classificação , Aranhas/genética , Animais , Modelos Moleculares , Diester Fosfórico Hidrolases/química , Filogenia , Seleção Genética , Venenos de Aranha/genética , Aranhas/enzimologiaRESUMO
Hemocyanin of the spider Polybetes pythagoricus, in addition to its typical role as an oxygen transporter, also exhibits a phenoloxidase activity induced by micellar concentrations of SDS. In the present work, we found the kinetic parameters Km and Vmax of Polybetes pythagoricus hemocyanin (PpHc) PO activity to be 0.407 mM and 0.081 µmolmin(-1) mg protein(-1) , respectively. Dopamine was used as the substrate with SDS at a final concentration of 10 mM and a 30-min incubation at 25°C. Conformational changes in Hc associated with the SDS treatment were analyzed using far-UV circular dichroism, intrinsic fluorescence and absorption spectroscopy. The secondary and tertiary structural changes of PpHc induced by SDS led to increases in α-helical content and tryptophan fluorescence intensity. A reduction in the absorption spectrum at 340 nm in the presence of SDS was also observed. These results suggest that the SDS-induced PO activity of PpHc can be ascribed to conformational changes in the local environment of the typer-3 copper active site.
Assuntos
Hemocianinas/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Aranhas/enzimologia , Animais , CinéticaRESUMO
Cysteine cathepsins are widely spread on living organisms associated to protein degradation in lysosomes, but some groups of Arthropoda (Heteroptera, Coleoptera, Crustacea and Acari) present these enzymes related to digestion of the meal proteins. Although spiders combine a mechanism of extra-oral with intracellular digestion, the sporadic studies on this subject were mainly concerned with the digestive fluid (DF) analysis. Thus, a more complete scenario of the digestive process in spiders is still lacking in the literature. In this paper we describe the identification and characterization of cysteine cathepsins in the midgut diverticula (MD) and DF of the spider Nephilengys cruentata by using enzymological assays. Furthermore, qualitative and quantitative data from transcriptomic followed by proteomic experiments were used together with biochemical assays for results interpretation. Five cathepsins L, one cathepsin F and one cathepsin B were identified by mass spectrometry, with cathepsins L1 (NcCTSL1) and 2 (NcCTSL2) as the most abundant enzymes. The native cysteine cathepsins presented acidic characteristics such as pH optima of 5.5, pH stability in acidic range and zymogen conversion to the mature form after in vitro acidification. NcCTSL1 seems to be a lysosomal enzyme with its recombinant form displaying acidic characteristics as the native ones and being inhibited by pepstatin. Evolutionarily, arachnid cathepsin L may have acquired different roles but its use for digestion is a common feature to studied taxa. Now a more elucidative picture of the digestive process in spiders can be depicted, with trypsins and astacins acting extra-orally under alkaline conditions whereas cysteine cathepsins will act in an acidic environment, likely in the digestive vacuoles or lysosome-like vesicles.
Assuntos
Proteínas de Artrópodes/metabolismo , Catepsinas/metabolismo , Digestão , Aranhas/enzimologia , Animais , Proteínas de Artrópodes/genética , Catepsinas/genética , Feminino , Trato Gastrointestinal/enzimologia , Espectrometria de Massas , Filogenia , Aranhas/genéticaRESUMO
We report the production of a neutralizing monoclonal antibody able to recognize the venoms of three major medically important species of Loxosceles spiders in Brazil. The mAb was produced by immunization of mice with a toxic recombinant L. intermedia sphingomyelinase D {SMases D isoform (rLiD1)} [1] and screened by enzyme-linked immunosorbent assay (ELISA) using L. intermedia, L. laeta and L. gaucho venoms as antigens. One clone (LiD1mAb16) out of seventeen anti-rLiD1 hybridomas was cross-reactive with the three whole Loxosceles venoms. 2D Western blot analysis indicated that LiD1mAb16 was capable of interacting with 34 proteins of 29-36kDa in L. intermedia, 33 in L. gaucho and 27 in L. laeta venoms. The results of immunoassays with cellulose-bound peptides revealed that the LiD1mAb16 recognizes a highly conserved linear epitope localized in the catalytic region of SMases D toxins. The selected mAb displayed in vivo protective activity in rabbits after challenge with rLiD1. These results show the potential usefulness of monoclonal antibodies for future therapeutic approaches and also opens up the perspective of utilization of these antibodies for immunodiagnostic assays in loxoscelism.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Diester Fosfórico Hidrolases/imunologia , Venenos de Aranha/enzimologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Reações Cruzadas , Mapeamento de Epitopos , Hibridomas , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência Molecular , Testes de Neutralização , Coelhos , Proteínas Recombinantes/imunologia , Venenos de Aranha/imunologia , Aranhas/enzimologiaRESUMO
Sphingomyelinases D (SMases D) or dermonecrotic toxins are well characterized in Loxosceles spider venoms and have been described in some strains of pathogenic microorganisms, such as Corynebacterium sp. After spider bites, the SMase D molecules cause skin necrosis and occasional severe systemic manifestations, such as acute renal failure. In this paper, we identified new SMase D amino acid sequences from various organisms belonging to 24 distinct genera, of which, 19 are new. These SMases D share a conserved active site and a C-terminal motif. We suggest that the C-terminal tail is responsible for stabilizing the entire internal structure of the SMase D Tim barrel and that it can be considered an SMase D hallmark in combination with the amino acid residues from the active site. Most of these enzyme sequences were discovered from fungi and the SMase D activity was experimentally confirmed in the fungus Aspergillus flavus. Because most of these novel SMases D are from organisms that are endowed with pathogenic properties similar to those evoked by these enzymes alone, they might be associated with their pathogenic mechanisms.
Assuntos
Corynebacterium pseudotuberculosis/enzimologia , Fungos/enzimologia , Ixodes/enzimologia , Diester Fosfórico Hidrolases/metabolismo , Aranhas/enzimologia , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Aspergillus flavus/enzimologia , Aspergillus flavus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Corynebacterium pseudotuberculosis/classificação , Corynebacterium pseudotuberculosis/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos/classificação , Fungos/genética , Ixodes/classificação , Ixodes/genética , Modelos Moleculares , Dados de Sequência Molecular , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Filogenia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Esfingomielinas/química , Esfingomielinas/metabolismo , Aranhas/classificação , Aranhas/genéticaRESUMO
Loxosceles bites have been associated with characteristic dermonecrotic lesions with gravitational spreading and systemic manifestations. Venom primarily comprises peptides and protein molecules (5-40 kDa) with multiple biological activities. Although poorly studied, metalloproteases have been identified in venoms of several Loxosceles species, presenting proteolytic effects on extracellular matrix components. The characterization of an Astacin-like protease (LALP) in Loxosceles intermedia venom was the first report of an Astacin family member as a component of animal venom. Recently, these proteases were described as a gene family in L. intermedia, Loxosceles laeta and Loxosceles gaucho. Herein, the whole venom complexity of these three Loxosceles species was analyzed using two-dimensional electrophoresis (2DE). Subproteomes of LALPs were explored through 2DE immunostaining using anti-LALP1 antibodies and 2DE gelatin zymogram. Proteins presented molecular masses ranging from 24 to 29 kDa and the majority of these molecules had basic or neutral isoelectric points (6.89-9.93). Likewise, the measurement of gelatinolytic effects of Loxosceles venom using fluorescein-gelatin showed that the three venoms have distinct proteolytic activities. The metalloprotease fibrinogenolytic activities were also evaluated. All venoms showed fibrinogenolytic activity with different proteolytic effects on Aα and Bß chains of fibrinogen. The results reported herein suggest that the LALP family is larger than indicated in previously published data and that the complex profile of the gelatinolytic activity reflects their relevance in loxoscelism. Furthermore, our investigation implicates the brown spider venom as a source of Astacin-like proteases for use in loxoscelism studies, cell biology research and biotechnological applications.
Assuntos
Metaloproteases/metabolismo , Venenos de Aranha/enzimologia , Aranhas/enzimologia , Animais , Eletroforese em Gel Bidimensional , Feminino , Masculino , Metaloproteases/química , Proteoma , Especificidade da EspécieRESUMO
An important step in the development of therapeutic antivenoms is the pre-clinical testing using in vivo methods to assess their neutralizing potency. For spider antivenoms (Loxosceles species), horse serum potency against the necrotizing activities of Loxosceles intermedia crude venom is currently tested in rabbits. These procedures are time consuming and involve a large number of animals. The aim of this study was to develop an in vitro method to assess the neutralizing potency of anti-Loxosceles sera. We first demonstrated that it was not possible to establish a correlation between the ELISA antibody reactivity of horse anti-Loxosceles serum and their neutralizing potency. We then showed that the antivenoms recognized several peptide epitopes from different regions of SMase-D proteins, which are toxic antigens from Loxosceles venoms. The recognition of some peptides was observed only when high neutralizing potency sera was used. Based on these results, three peptides (peptide 1, DNRRPIWNLAHMVNA and peptide 3, DFSGPYLPSLPTLDA corresponding to residues 2-16 and 164-178, respectively, of SMase-1 protein from Loxosceles laeta, and peptide 2, EFVNLGANSIETDVS corresponding to residues 22-36 of A1H - LoxGa protein from Loxosceles gaucho and LiD1 protein from L. intermedia) were selected. The peptides were synthesized, coupled to bovine serum albumin (BSA), and used as antigens in indirect ELISA to test their reactivity with horse anti-Loxosceles serum of varying neutralizing potencies. We found certain assay conditions that discriminated between the high and low neutralizing potency sera. This study introduced an in vitro and peptide-based neutralization assay for anti-Loxosceles antivenoms.
Assuntos
Antivenenos/biossíntese , Antivenenos/farmacologia , Desenho de Fármacos , Testes de Neutralização/métodos , Venenos de Aranha/antagonistas & inibidores , Aranhas/química , Sequência de Aminoácidos , Análise de Variância , Animais , Biologia Computacional , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Epitopos/metabolismo , Cavalos/sangue , Soros Imunes/metabolismo , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Soroalbumina Bovina , Aranhas/enzimologiaRESUMO
The toxicity of Loxosceles spider venom has been attributed to a rare enzyme, sphingomyelinase D, which transforms sphingomyelin to ceramide-1-phosphate. The bases of its inflammatory and dermonecrotic activity, however, remain unclear. In this work the effects of ceramide-1-phosphate on model membranes were studied both by in situ generation of this lipid using a recombinant sphingomyelinase D from the spider Loxosceles laeta and by pre-mixing it with sphingomyelin and cholesterol. The systems of choice were large unilamellar vesicles for bulk studies (enzyme kinetics, fluorescence spectroscopy and dynamic light scattering) and giant unilamellar vesicles for fluorescence microscopy examination using a variety of fluorescent probes. The influence of membrane lateral structure on the kinetics of enzyme activity and the consequences of enzyme activity on the structure of target membranes containing sphingomyelin were examined. The findings indicate that: 1) ceramide-1-phosphate (particularly lauroyl ceramide-1-phosphate) can be incorporated into sphingomyelin bilayers in a concentration-dependent manner and generates coexistence of liquid disordered/solid ordered domains, 2) the activity of sphingomyelinase D is clearly influenced by the supramolecular organization of its substrate in membranes and, 3) in situ ceramide-1-phosphate generation by enzymatic activity profoundly alters the lateral structure and morphology of the target membranes.
Assuntos
Ceramidas/química , Ceramidas/metabolismo , Membranas Artificiais , Diester Fosfórico Hidrolases/metabolismo , 2-Naftilamina/análogos & derivados , 2-Naftilamina/metabolismo , Animais , Varredura Diferencial de Calorimetria , Colesterol/metabolismo , Transferência Ressonante de Energia de Fluorescência , Cinética , Lauratos/metabolismo , Luz , Microscopia de Fluorescência , Fosfatidilcolinas , Espalhamento de Radiação , Esfingomielinas/metabolismo , Aranhas/enzimologia , Temperatura , Lipossomas Unilamelares/metabolismoRESUMO
Phospholipases D (PLDs) are principally responsible for the local and systemic effects of Loxosceles envenomation including dermonecrosis and hemolysis. Despite their clinical relevance in loxoscelism, to date, only the SMase I from Loxosceles laeta, a class I member, has been structurally characterized. The crystal structure of a class II member from Loxosceles intermedia venom has been determined at 1.7Å resolution. Structural comparison to the class I member showed that the presence of an additional disulphide bridge which links the catalytic loop to the flexible loop significantly changes the volume and shape of the catalytic cleft. An examination of the crystal structures of PLD homologues in the presence of low molecular weight compounds at their active sites suggests the existence of a ligand-dependent rotamer conformation of the highly conserved residue Trp230 (equivalent to Trp192 in the glycerophosphodiester phosphodiesterase from Thermus thermophofilus, PDB code: 1VD6) indicating its role in substrate binding in both enzymes. Sequence and structural analyses suggest that the reduced sphingomyelinase activity observed in some class IIb PLDs is probably due to point mutations which lead to a different substrate preference.
Assuntos
Fosfolipase D/química , Fosfolipase D/classificação , Venenos de Aranha/enzimologia , Aranhas/enzimologia , Sequência de Aminoácidos , Animais , Domínio Catalítico , Cristalografia por Raios X , Cisteína/química , Dados de Sequência MolecularRESUMO
Phospholipases D are the major dermonecrotic component of Loxosceles venom and catalyze the hydrolysis of phospholipids, resulting in the formation of lipid mediators such as ceramide-1-phosphate and lysophosphatidic acid which can induce pathological and biological responses. Phospholipases D can be classified into two classes depending on their catalytic efficiency and the presence of an additional disulfide bridge. In this work, both wild-type and H12A-mutant forms of the class II phospholipase D from L. intermedia venom were crystallized. Wild-type and H12A-mutant crystals were grown under very similar conditions using PEG 200 as a precipitant and belonged to space group P12(1)1, with unit-cell parameters a = 50.1, b = 49.5, c = 56.5â Å, ß = 105.9°. Wild-type and H12A-mutant crystals diffracted to maximum resolutions of 1.95 and 1.60â Å, respectively.
Assuntos
Fosfolipase D/química , Fosfolipase D/classificação , Venenos de Aranha/enzimologia , Aranhas/enzimologia , Sequência de Aminoácidos , Animais , Cristalização , Cristalografia por Raios X/métodos , Difusão , Dissulfetos/química , Escherichia coli/genética , Histidina/química , Temperatura Alta , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Peso Molecular , Mutação , Fosfolipase D/genética , Fosfolipase D/isolamento & purificação , Diester Fosfórico Hidrolases , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/classificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Homologia de Sequência de Aminoácidos , Transformação Bacteriana , Difração de Raios XRESUMO
Brown spiders have a worldwide distribution, and their venom has a complex composition containing many different molecules. Herein, we report the existence of a family of astacin-like metalloprotease toxins in Loxosceles intermedia venom, as well as in the venom of different species of Loxosceles. Using a cDNA library from the L. intermedia venom gland, we cloned two novel cDNAs encoding astacin-like metalloprotease toxins, LALP2 and LALP3. Using an anti-serum against the previously described astacin-like toxin in L. intermedia venom (LALP1), we detected the presence of immunologically-related toxins in the venoms of L. intermedia, Loxosceles laeta, and Loxosceles gaucho. Zymographic experiments showed gelatinolytic activity of crude venoms of L. intermedia, L. laeta, and L. gaucho (which could be inhibited by the divalent metal chelator 1,10-phenanthroline) at electrophoretic mobilities identical to those reported for immunological cross-reactivity. Moreover, mRNAs extracted from L. laeta and L. gaucho venom glands were screened for astacin-like metalloproteases, and cDNAs obtained using LALP1-specific primers were sequenced, and their deduced amino acid sequences confirmed they were members of the astacin family with the family signatures (HEXXHXXGXXHE and MXY), LALP4 and LALP5, respectively. Sequence comparison of deduced amino acid sequences revealed that LALP2, LALP3, LALP4, and LALP5 are related to the astacin family. This study identified the existence of gene family of astacin-like toxins in the venoms of brown spiders and raises the possibility that these molecules are involved in the deleterious effects triggered by the venom.
Assuntos
Metaloendopeptidases/química , Metaloproteases/química , Metaloproteases/genética , Venenos de Aranha/enzimologia , Aranhas/enzimologia , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Sequência de Bases , Clonagem Molecular , Reações Cruzadas , DNA Complementar/genética , Gelatina/metabolismo , Humanos , Metaloproteases/imunologia , Metaloproteases/metabolismo , Camundongos , Dados de Sequência Molecular , Fenantrolinas/farmacologia , Diester Fosfórico Hidrolases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Venenos de Aranha/genética , Aranhas/genéticaRESUMO
Envenomation by Loxosceles species (brown spider) can lead to local dermonecrosis and to serious systemic effects. The main toxic component in the venom of these spiders is sphingomyelinase D (SMase D) and various isoforms of this toxin are present in Loxosceles venoms. We have produced a new anti-loxoscelic serum by immunizing horses with recombinant SMase D. In the present study, we compared the neutralization efficacy of the new anti-loxoscelic serum and anti-arachnidic serum (the latter serum is used for therapy for loxoscelism in Brazil) against the toxic effects of venoms from spiders of the genus Loxosceles. Neutralization tests showed that anti-SMase D serum has a higher activity against toxic effects of L. intermedia and L. laeta venoms and similar or slightly weaker activity against toxic effects of L. gaucho than that of Arachnidic serum. These results demonstrate that recombinant SMase D can replace venom for anti-venom production and therapy.
Assuntos
Antivenenos/farmacologia , Diester Fosfórico Hidrolases/imunologia , Picada de Aranha/terapia , Venenos de Aranha/antagonistas & inibidores , Animais , Células Cultivadas , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Eritrócitos/efeitos dos fármacos , Cavalos , Humanos , Imunoquímica , Testes de Neutralização , Diester Fosfórico Hidrolases/metabolismo , Coelhos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Venenos de Aranha/enzimologia , Venenos de Aranha/imunologia , Aranhas/enzimologia , Aranhas/metabolismoRESUMO
Brown spider bites are associated with lesions including dermonecrosis, gravitational spreading and a massive inflammatory response, along with systemic problems that may include hematological disturbances and renal failure. The mechanisms by which the venom exerts its noxious effects are currently under investigation. It is known that the venom contains a major toxin (dermonecrotic toxin, biochemically a phospholipase D) that can experimentally induce dermonecrosis, inflammatory response, animal mortality and platelet aggregation. Herein, we describe cloning, heterologous expression, purification and functionality of a novel isoform of the 33 kDa dermonecrotic toxin. Circular dichroism analysis evidenced correct folding for the toxin. The recombinant toxin was recognized by whole venom serum antibodies and by a specific antibody to a previously described dermonecrotic toxin. The identified toxin was found to display phospholipase activity and dermonecrotic properties. Additionally, the toxin caused a massive inflammatory response in rabbit skin dermis, evoked platelet aggregation, increased vascular permeability, caused edema and death in mice. These characteristics in combination with functional studies for other dermonecrotic toxins illustrate that a family of dermonecrotic toxins exists, and includes a novel member with high activity that may be useful for future structural and functional studies.