RESUMO
In the Americas and specially in Brazil, the Loxosceles intermedia, Loxosceles gaucho and Loxosceles laeta are the three most medically relevant brown spider species, and whose bites can lead to the condition known as loxoscelism. Here, we report the development of a tool capable of identifying a common epitope amongst Loxosceles sp. venom's toxins. A murine monoclonal antibody (LmAb12) and its recombinant fragments (scFv12P and diabody12P) have been produced and characterized. This antibody and its recombinant constructs were able to recognize proteins of Loxosceles spider venoms with specificity. The scFv12P variant was also able to detect low concentrations of Loxosceles venom in a competitive ELISA assay, displaying potential as a venom identification tool. The primary antigenic target of LmAb12 is a knottin, a venom neurotoxin, that has a shared identity of 100 % between the L. intermedia and L. gaucho species and high similarity to L. laeta. Furthermore, we observed LmAb12 was able to partially inhibit in vitro hemolysis, a cellular event typically induced by the Loxosceles sp. venoms. Such behavior might be due to LmAb12 cross-reactivity between the antigenic target of LmAb12 and the venom's dermonecrotic toxins, the PLDs, or even the existence of synergism between these two toxins.
Assuntos
Venenos de Aranha , Aranhas , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Antígenos/química , Antivenenos/química , Reações Cruzadas , Miniproteínas Nó de Cistina/química , Fosfolipase D/química , Venenos de Aranha/química , Aranhas/química , Epitopos/químicaRESUMO
Loxosceles spider envenomation results in dermonecrosis, principally due to phospholipases D (PLDs) present in the venom. These enzymes have a strongly conserved sequence, 273ATXXDNPW280, in the C-terminal region (SMD-tail) that make contact with ß-sheets of the TIM barrel, in which the amino acids Asp277 and Trp280 establish the energetically strongest contacts. The SMD-tail is conserved in PLDs from different species but absent in the non-toxic PLD ancestral glycerophosphodiester phosphodiesterases (GDPDs). This work aims to understand the role of the C-terminal region in the structural stability and/or function of phospholipases D. Through site-directed mutagenesis of the rLiD1 protein (recombinant Loxosceles intermedia dermonecrotic protein 1), we produced two mutants: rLiD1D277A and rLiD1W280A (both with sphingomyelinase activity), in which Asp277 and Trp280 were replaced by alanine. rLiD1D277A showed similar sphingomyelinase activity but at least 2 times more dermonecrotic activity than rLiD1 (wild-type protein). Conversely, while the rLiD1W280A displayed a slight increase in sphingomyelinase activity, its biological activity was similar or lower compared to rLiD1, potentially due to its decreased thermostability and formation of amyloid aggregates. In conclusion, these new findings provide evidence that SMD-tail mutants impact the structure and function of these proteins and point out that residues outside the active site can even increase the function of these enzymes.
Assuntos
Fosfolipase D , Venenos de Aranha , Aranhas , Animais , Fosfolipase D/genética , Fosfolipase D/química , Fosfolipase D/metabolismo , Domínio Catalítico , Esfingomielina Fosfodiesterase , Diester Fosfórico Hidrolases/genética , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Aranhas/genética , Venenos de Aranha/genética , Venenos de Aranha/químicaRESUMO
Bites of Loxosceles spiders can lead to a set of clinical manifestations called loxoscelism, and are considered a public health problem in many regions. The signs and symptoms of loxoscelism are divided into cutaneous and systemic forms. The former is more frequent and includes signs of envenoming at the bite site or neighboring regions. Systemic loxoscelism, although much less frequent, is associated with complications, and can even lead to death. It may include intravascular hemolysis, acute renal failure, and thrombocytopenia. Loxosceles venoms are enriched with phospholipases D (PLDs), which are a family of isoforms found at intra-species and inter-species levels. Under experimental conditions, these enzymes reproduce the main clinical signs of loxoscelism, including an exacerbated inflammatory response at the bite site and dermonecrosis, as well as thrombocytopenia, intravascular hemolysis, and acute renal failure. The role of PLDs in cutaneous loxoscelism was described over forty years ago, when studies identified and purified toxins featured as sphingomyelinase D. More recently, the production of recombinant PLDs and discoveries about their structure and mechanism has enabled a deeper characterization of these enzymes. In this review, we describe these biochemical and functional features of Loxosceles PLDs that determine their involvement in systemic loxoscelism.
Assuntos
Fosfolipase D , Picada de Aranha , Venenos de Aranha , Aranhas , Trombocitopenia , Animais , Hemólise , Diester Fosfórico Hidrolases/toxicidade , Fosfolipase D/química , Venenos de Aranha/toxicidade , Venenos de Aranha/química , Isoformas de Proteínas , Aranhas/química , Picada de Aranha/complicaçõesRESUMO
Accidents involving Brown spiders are reported throughout the world. In the venom, the major toxins involved in the deleterious effects are phospholipases D (PLDs). In this work, recombinant mutated phospholipases D from three endemic species medically relevant in South America (Loxosceles intermedia, L. laeta and L. gaucho) were tested as antigens in a vaccination protocol. In such isoforms, key amino acid residues involved in catalysis, magnesium-ion coordination, and binding to substrates were replaced by Alanine (H12A-H47A, E32A-D34A and W230A). These mutations eliminated the phospholipase activity and reduced the generation of skin necrosis and edema to residual levels. Molecular modeling of mutated isoforms indicated that the three-dimensional structures, topologies, and surface charges did not undergo significant changes. Mutated isoforms were recognized by sera against the crude venoms. Vaccination protocols in rabbits using mutated isoforms generated a serum that recognized the native PLDs of crude venoms and neutralized dermonecrosis and edema induced by L. intermedia venom. Vaccination of mice prevented the lethal effects of L. intermedia crude venom. Furthermore, vaccination of rabbits prevented the cutaneous lesion triggered by the three venoms. These results indicate a great potential for mutated recombinant PLDs to be employed as antigens in developing protective vaccines for Loxoscelism.
Assuntos
Aranha Marrom Reclusa , Proteínas Mutantes/imunologia , Fosfolipase D/imunologia , Picada de Aranha/imunologia , Picada de Aranha/terapia , Vacinas/imunologia , Acidentes , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Antivenenos/sangue , Antivenenos/imunologia , Biomarcadores , Modelos Animais de Doenças , Imunogenicidade da Vacina , Contagem de Leucócitos , Camundongos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Testes de Neutralização , Fosfolipase D/química , Fosfolipase D/genética , Coelhos , Picada de Aranha/diagnóstico , Picada de Aranha/prevenção & controle , Venenos de Aranha/imunologia , Relação Estrutura-Atividade , Resultado do Tratamento , Vacinação , Vacinas/administração & dosagemRESUMO
Spiders of the genus Loxosceles, popularly known as Brown spiders, are considered a serious public health issue, especially in regions of hot or temperate climates, such as parts of North and South America. Although the venoms of these arachnids are complex in molecular composition, often containing proteins with distinct biochemical characteristics, the literature has primarily described a family of toxins, the Phospholipases-D (PLDs), which are highly conserved in all Loxosceles species. PLDs trigger most of the major clinical symptoms of loxoscelism i.e., dermonecrosis, thrombocytopenia, hemolysis, and acute renal failure. The key role played by PLDs in the symptomatology of loxoscelism was first described 40 years ago, when researches purified a hemolytic toxin that cleaved sphingomyelin and generated choline, and was referred to as a Sphingomyelinase-D, which was subsequently changed to Phospholipase-D when it was demonstrated that the enzyme also cleaved other cellular phospholipids. In this review, we present the information gleaned over the last 40 years about PLDs from Loxosceles venoms especially with regard to the production and characterization of recombinant isoforms. The history of obtaining these toxins is discussed, as well as their molecular organization and mechanisms of interaction with their substrates. We will address cellular biology aspects of these toxins and how they can be used in the development of drugs to address inflammatory processes and loxoscelism. Present and future aspects of loxoscelism diagnosis will be discussed, as well as their biotechnological applications and actions expected for the future in this field.
Assuntos
Fosfolipase D/história , Diester Fosfórico Hidrolases/história , Venenos de Aranha/história , Animais , Catálise , História do Século XX , História do Século XXI , Humanos , Fosfolipase D/química , Fosfolipase D/farmacologia , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/farmacologia , Proteômica , Proteínas Recombinantes/farmacologia , Picada de Aranha/diagnóstico , Picada de Aranha/tratamento farmacológico , Picada de Aranha/enzimologia , Venenos de Aranha/química , Venenos de Aranha/farmacologiaRESUMO
Loxoscelism refers to the clinical symptoms that develop after brown spider bites. Brown spider venoms contain several phospholipase-D isoforms, which are the main toxins responsible for both the cutaneous and systemic effects of loxoscelism. Understanding of the phospholipase-D catalytic mechanism is crucial for the development of specific treatment that could reverse the toxic effects caused by the spider bite. Based on enzymatic, biological, structural, and thermodynamic tests, we show some features suitable for designing drugs against loxoscelism. Firstly, through molecular docking and molecular dynamics predictions, we found three different molecules (Suramin, Vu0155056, and Vu0359595) that were able to bind the enzyme's catalytic site and interact with catalytically important residues (His12 or His47) and with the Mg2+ co-factor. The binding promoted a decrease in the recombinant brown spider venom phospholipase-D (LiRecDT1) enzymatic activity. Furthermore, the presence of the inhibitors reduced the hemolytic, dermonecrotic, and inflammatory activities of the venom toxin in biological assays. Altogether, these results indicate the mode of action of three different LiRecDT1 inhibitors, which were able to prevent the venom toxic effects. This strengthen the idea of the importance of designing a specific drug to treat the serious clinical symptoms caused by the brown spider bite, a public health problem in several parts of the world, and until now without specific treatment. J. Cell. Biochem. 118: 726-738, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Proteínas de Artrópodes/antagonistas & inibidores , Aranha Marrom Reclusa/enzimologia , Desenho de Fármacos , Fosfolipase D/antagonistas & inibidores , Venenos de Aranha/antagonistas & inibidores , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Benzimidazóis/farmacologia , Aranha Marrom Reclusa/genética , Aranha Marrom Reclusa/patogenicidade , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Hemólise/efeitos dos fármacos , Humanos , Cinética , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Necrose , Fosfolipase D/química , Fosfolipase D/genética , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Piperidinas/farmacologia , Coelhos , Proteínas Recombinantes/genética , Pele/efeitos dos fármacos , Pele/patologia , Picada de Aranha/tratamento farmacológico , Picada de Aranha/enzimologia , Venenos de Aranha/química , Venenos de Aranha/genética , Suramina/farmacologiaRESUMO
Brown spider phospholipases D from Loxosceles venoms are among the most widely studied toxins since they induce dermonecrosis, triggering inflammatory responses, increase vascular permeability, cause hemolysis, and renal failure. The catalytic (H12 and H47) and metal-ion binding (E32 and D34) residues in Loxosceles intermedia phospholipase D (LiRecDT1) were mutated to understand their roles in the observed activities. All mutants were identified using whole venom serum antibodies and a specific antibody to wild-type LiRecDT1, they were also analyzed by circular dichroism (CD) and differential scanning calorimetry (DSC). The phospholipase D activities of H12A, H47A, H12A-H47A, E32, D34 and E32A-D34A, such as vascular permeability, dermonecrosis, and hemolytic effects were inhibited. The mutant Y228A was equally detrimental to biochemical and biological effects of phospholipase D, suggesting an essential role of this residue in substrate recognition and binding. On the other hand, the mutant C53A-C201A reduced the enzyme's ability to hydrolyze phospholipids and promote dermonecrosis, hemolytic, and vascular effects. These results provide the basis understanding the importance of specific residues in the observed activities and contribute to the design of synthetic and specific inhibitors for Brown spider venom phospholipases D.
Assuntos
Domínio Catalítico/genética , Fosfolipase D/química , Fosfolipídeos/química , Venenos de Aranha/enzimologia , Animais , Aranha Marrom Reclusa/química , Aranha Marrom Reclusa/enzimologia , Permeabilidade Capilar , Dicroísmo Circular , Hemólise , Mutação , Fosfolipase D/metabolismo , Fosfolipídeos/metabolismo , Diester Fosfórico Hidrolases/química , Venenos de Aranha/químicaRESUMO
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
UNLABELLED: Brown spider (Loxosceles genus) bites have been reported worldwide. The venom contains a complex composition of several toxins, including phospholipases-D. Native or recombinant phospholipase-D toxins induce cutaneous and systemic loxoscelism, particularly necrotic lesions, inflammatory response, renal failure, and hematological disturbances. Herein, we describe the cloning, heterologous expression and purification of a novel phospholipase-D toxin, LiRecDT7 in reference to six other previously described in phospholipase-D toxin family. The complete cDNA sequence of this novel brown spider phospholipase-D isoform was obtained and the calculated molecular mass of the predicted mature protein is 34.4 kDa. Similarity analyses revealed that LiRecDT7 is homologous to the other dermonecrotic toxin family members particularly to LiRecDT6, sharing 71% sequence identity. LiRecDT7 possesses the conserved amino acid residues involved in catalysis except for a conservative mutation (D233E) in the catalytic site. Purified LiRecDT7 was detected as a soluble 36 kDa protein using anti-whole venom and anti-LiRecDT1 sera, indicating immunological cross-reactivity and evidencing sequence-epitopes identities similar to those of other phospholipase-D family members. Also, LiRecDT7 exhibits sphingomyelinase activity in a concentration dependent-manner and induces experimental skin lesions with swelling, erythema and dermonecrosis. In addition, LiRecDT7 induced a massive inflammatory response in rabbit skin dermis, which is a hallmark of brown spider venom phospholipase-D toxins. Moreover, LiRecDT7 induced in vitro hemolysis in human erythrocytes and increased blood vessel permeability. These features suggest that this novel member of the brown spider venom phospholipase-D family, which naturally contains a mutation (D233E) in the catalytic site, could be useful for future structural and functional studies concerning loxoscelism and lipid biochemistry. HIGHLIGHTS: 1- Novel brown spider phospholipase-D recombinant toxin contains a conservative mutation (D233E) on the catalytic site. 2-LiRecDT7 shares high identity level with isoforms of Loxosceles genus. 3-LiRecDT7 is a recombinant protein immunodetected by specific antibodies to native and recombinant phospholipase-D toxins. 4-LiRecDT7 shows sphingomyelinase-D activity in a concentration-dependent manner, but less intense than other isoforms. 5-LiRecDT7 induces dermonecrosis and inflammatory response in rabbit skin. 6-LiRecDT7 increases vascular permeability in mice. 7-LiRecDT7 triggers direct complement-independent hemolysis in erythrocytes.
Assuntos
Fosfolipase D/química , Isoformas de Proteínas/química , Animais , Domínio Catalítico , Biologia Computacional , Hemólise/efeitos dos fármacos , Humanos , Camundongos , Dados de Sequência Molecular , Mutação/genética , Fosfolipase D/genética , Fosfolipase D/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/farmacologia , CoelhosRESUMO
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
Brown spider venom phospholipase-D belongs to a family of toxins characterized as potent bioactive agents. These toxins have been involved in numerous aspects of cell pathophysiology including inflammatory response, platelet aggregation, endothelial cell hyperactivation, renal disorders, and hemolysis. The molecular mechanism by which these toxins cause hemolysis is under investigation; literature data have suggested that enzyme catalysis is necessary for the biological activities triggered by the toxin. However, the way by which phospholipase-D activity is directly related with human hemolysis has not been determined. To evaluate how brown spider venom phospholipase-D activity causes hemolysis, we examined the impact of recombinant phospholipase-D on human red blood cells. Using six different purified recombinant phospholipase-D molecules obtained from a cDNA venom gland library, we demonstrated that there is a correlation of hemolytic effect and phospholipase-D activity. Studying recombinant phospholipase-D, a potent hemolytic and phospholipase-D recombinant toxin (LiRecDT1), we determined that the toxin degrades synthetic sphingomyelin (SM), lysophosphatidylcholine (LPC), and lyso-platelet-activating factor. Additionally, we determined that the toxin degrades phospholipids in a detergent extract of human erythrocytes, as well as phospholipids from ghosts of human red blood cells. The products of the degradation of synthetic SM and LPC following recombinant phospholipase-D treatments caused hemolysis of human erythrocytes. This hemolysis, dependent on products of metabolism of phospholipids, is also dependent on calcium ion concentration because the percentage of hemolysis increased with an increase in the dose of calcium in the medium. Recombinant phospholipase-D treatment of human erythrocytes stimulated an influx of calcium into the cells that was detected by a calcium-sensitive fluorescent probe (Fluo-4). This calcium influx was shown to be channel-mediated rather than leak-promoted because the influx was inhibited by L-type calcium channel inhibitors but not by a T-type calcium channel blocker, sodium channel inhibitor or a specific inhibitor of calcium activated potassium channels. Finally, this inhibition of hemolysis following recombinant phospholipase-D treatment occurred in a concentration-dependent manner in the presence of L-type calcium channel blockers such as nifedipine and verapamil. The data provided herein, suggest that the brown spider venom phospholipase-D-induced hemolysis of human erythrocytes is dependent on the metabolism of membrane phospholipids, such as SM and LPC, generating bioactive products that stimulate a calcium influx into red blood cells mediated by the L-type channel.
Assuntos
Cálcio/química , Membrana Celular/metabolismo , Hemólise/efeitos dos fármacos , Hemolíticos/toxicidade , Proteínas de Insetos/toxicidade , Fosfolipase D/toxicidade , Fosfolipídeos/metabolismo , Venenos de Aranha/enzimologia , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/metabolismo , Membrana Celular/efeitos dos fármacos , Ensaios Enzimáticos , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Hemolíticos/química , Humanos , Proteínas de Insetos/química , Lisofosfatidilcolinas/química , Fosfolipase D/química , Fosfolipídeos/química , Proteínas Recombinantes/química , Esfingomielinas/química , Venenos de Aranha/química , Venenos de Aranha/toxicidade , AranhasRESUMO
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
Bites from brown spiders (Loxosceles genus) have clinical manifestations including skin necrosis with gravitational spreading, and systemic involvement that may include renal failure, hemolysis, and thrombocytopenia. Mice were exposed to recombinant wild-type phospholipase-D, or to an isoform with a mutation in the catalytic domain resulting in no phospholipasic activity. Renal biopsies from mice treated with the wild-type toxin showed glomerular edema, erythrocytes and collapse of Bowman's space, edema and deposition of proteinaceous material within the tubular lumen. Ultrastructural analyses confirmed cytotoxicity by demonstrating disorders of glomerulus at foot processes and at fenestrated endothelium. Tubule alterations include deposits of amorphous material and edema, as well as an increase of epithelial cytoplasmic multivesicular bodies and electron-dense structures. There was an absence of nephrotoxicity in mice treated with the mutated toxin. Analyses of urine and blood showed that wild type toxin induced hematuria and elevation of blood urea, while treatment with mutated toxin caused no changes. Mouse lethality experiments also showed oliguria and mortality after treatment with wild-type toxin, but not following exposure to the mutated toxin. Immunofluorescence using antibodies to phospholipase-D toxin showed deposition of both toxins along the renal tubular structures as detected by confocal microscopy. Immunoblots of urine showed a 30 kDa band in samples from animals treated with wild-type toxin, but no band from mice exposed to mutated toxin. Wild-type toxin treatment caused cytoplasmic vacuolization, impaired spreading, reduction of cellular viability, and cell-cell and cell-substratum detachment in MDCK cells, while treatment with mutated isoform had no effect. Finally, there is a direct correlation between toxin activity on cell membrane phospholipids generating choline and cytotoxicity. We have defined for the first time a molecular mechanism for Loxosceles venom nephrotoxicity that is dependent on the catalytic activity of phospholipase-D toxin.
Assuntos
Túbulos Renais/efeitos dos fármacos , Fosfolipase D/toxicidade , Diester Fosfórico Hidrolases/toxicidade , Insuficiência Renal/induzido quimicamente , Venenos de Aranha/toxicidade , Animais , Domínio Catalítico/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Túbulos Renais/ultraestrutura , Camundongos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Fosfolipase D/química , Fosfolipase D/genética , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/toxicidade , Proteinúria/induzido quimicamente , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidade , Insuficiência Renal/patologia , Venenos de Aranha/química , Venenos de Aranha/genéticaRESUMO
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.
Assuntos
Derme/efeitos dos fármacos , Fosfolipase D/química , Fosfolipase D/toxicidade , Venenos de Aranha/química , Venenos de Aranha/enzimologia , Venenos de Aranha/toxicidade , Sequência de Aminoácidos , Animais , Permeabilidade Capilar/efeitos dos fármacos , Clonagem Molecular , DNA Complementar/genética , Derme/patologia , Edema/induzido quimicamente , Camundongos , Dados de Sequência Molecular , Necrose/induzido quimicamente , Fosfolipase D/genética , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/toxicidade , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/toxicidade , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidade , Venenos de Aranha/genética , Aranhas/enzimologiaRESUMO
The clinical features of brown spider bites are the appearance of necrotic skin lesions, which can also be accompanied by systemic involvement, including weakness, vomiting, fever, convulsions, disseminated intravascular coagulation, intravascular hemolysis and renal disturbances. Severe systemic loxoscelism is much less common than the cutaneous form, but it may be the cause of clinical complications and even death following envenomation. Here, by using three recombinant dermonecrotic toxins, LiRecDT1, LiRecDT2 and LiRecDT3 (the major toxins found in the venom), we report the biological, immunological and structural differences for these members of this toxin family. Purified toxins evoked similar inflammatory reactions following injections into rabbit skin. Recombinant toxin treatments of MDCK cells with LiRecDT1 and LiRecDT2 changed cell viability, as evaluated by neutral red uptake and assessment of cell morphology through inverted microscopy, whereas LiRecDT3 caused only residual activity. Differences in cell cytotoxicity triggered by recombinant toxins were confirmed through a human red blood lysis assay, during which LiRecDT1 and LiRecDT2 caused a high degree of hemolysis compared to LiRecDT3, which induced only a small hemolytic effect. Additionally, biological differences for recombinant toxins were corroborated through mice lethality experiments, which showed animal mortality after LiRecDT1 and LiRecDT2 treatments, but an absence of lethality following LiRecDT3 exposure. Moreover, in experiments for edema, both the LiRecDT1 and the LiRecDT2 toxins evoked similar results, causing edema following toxin exposure, whereas LiRecDT3 caused only residual effects. Characterization of antigenic cross-reactivity using sera against crude venom toxins by immunoWestern blotting and immunodot blotting with recombinant LiRecDT1, LiRecDT2 and LiRecDT3 compared among themselves pointed to a higher cross-reactivity for LiRecDT1 compared to LiRecDT2 and LiRecDT3, corroborating structural and antigenic differences for these three toxins. Finally, evidence for structural differences among the recombinant toxins was strengthened by circular dichroism spectra, which suggested that the toxins were folded, and not aggregated or denatured proteins.
Assuntos
Fosfolipase D/toxicidade , Venenos de Aranha/toxicidade , Animais , Linhagem Celular , Dicroísmo Circular , Reações Cruzadas , Cães , Edema/induzido quimicamente , Hemólise/efeitos dos fármacos , Masculino , Fosfolipase D/química , Estrutura Secundária de Proteína , Coelhos , Proteínas Recombinantes/toxicidade , Venenos de Aranha/química , AranhasRESUMO
Phospholipase D (E.C. 3.1.4.4.) was detected in isolated bovine rod outer segments (ROS) and its properties determined. The enzyme activity was assayed using either a sonicated microdispersion of 1,2-diacyl-sn-[2(3)H]glycerol-3-phosphocholine (PC), or [14C]ethanol. Using [3H]PC and ethanol as a substrate, we were able to detect the hydrolytic properties as well as the transphosphatidylation reaction catalyzed by phospholipase D (PLD): formation of [3H]phosphatidic acid and phosphatidylethanol [3H]PtdEt; whereas with [14C]ethanol or [3H]glycerol in the absence of exogenous PC, only transphosphatidylation reactions were detected (formation of [14C]PtdEt or [3H]phosphatidylglycerol, respectively). The use of varying concentrations of [3H]PC and 400 mM of ethanol gave an apparent Km value for PC of 0.51 mM and a Vmax value of 111 nmol x h(-1) x (mg protein)(-1). The activity was linear up to 60 min of incubation and up to 0.2 mg of protein. The optimal ethanol concentration was determined to be 400 mM, with an apparent Km of 202 mM and a Vmax value for ethanol of 125 nmol x h(-1) x (mg protein)(-1). A clear pH optimum was observed around 7. PLD activity was increased in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate or sodium deoxycholate and inhibited with Triton X-100. The enzyme activity was also activated in the presence of Ca2+ or Mg2+ (1 mM) although these ions were not required for measuring PLD activity. The high specific activity of PLD found in purified ROS compared to the activity found in other subcellular fractions of the bovine retina suggests that this enzymatic activity is native to ROS. The present report is the first evidence of PLD activity associated with photoreceptor ROS.