RESUMO
Levels of active Rac1 at epithelial junctions are partially modulated via interaction with Ajuba, an actin binding and scaffolding protein. Here we demonstrate that Ajuba interacts with the Cdc42 GTPase activating protein CdGAP, a GAP for Rac1 and Cdc42, at cell-cell contacts. CdGAP recruitment to junctions does not require Ajuba; rather Ajuba seems to control CdGAP residence at sites of cell-cell adhesion. CdGAP expression potently perturbs junctions and Ajuba binding inhibits CdGAP activity. Ajuba interacts with Rac1 and CdGAP via distinct domains and can potentially bring them in close proximity at junctions to facilitate activity regulation. Functionally, CdGAP-Ajuba interaction maintains junctional integrity in homeostasis and diseases: (i) gain-of-function CdGAP mutants found in Adams-Oliver Syndrome patients strongly destabilize cell-cell contacts and (ii) CdGAP mRNA levels are inversely correlated with E-cadherin protein expression in different cancers. We present conceptual insights on how Ajuba can integrate CdGAP binding and inactivation with the spatio-temporal regulation of Rac1 activity at junctions. Ajuba provides a novel mechanism due to its ability to bind to CdGAP and Rac1 via distinct domains and influence the activation status of both proteins. This functional interplay may contribute towards conserving the epithelial tissue architecture at steady-state and in different pathologies.
Assuntos
Comunicação Celular , Epitélio/metabolismo , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Proteínas com Domínio LIM/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Imunofluorescência , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Junções Intercelulares/metabolismo , Queratinócitos/metabolismo , Proteínas com Domínio LIM/química , Camundongos , Modelos Biológicos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte ProteicoRESUMO
Diagnostic tools are important for clinical management and epidemiological evaluation of Tegumentary (TL) and Visceral (VL) Leishmaniasis. Serology is not frequently used for the diagnosis of the TL form because low antibody titers and cross-reaction with VL. Therefore, it is crucial to identify specific and immunogenic antigens from species associated with the TL form. Here we employed a proteomic approach coupled to an in silico analysis and identified the most abundant and immunogenic proteins from Leishmania amazonensis, Leishmania braziliensis and Leishmania infantum. Of 16 species specific proteins, nine were from the species causative of the TL form (L. amazonensis and L. braziliensis). In silico analysis revealed 18 B-cell epitopes with 0% similarity to Trypanosoma cruzi orthologs and, therefore, less likely to crossreact with sera of patients with Chagas disease. Two proteins reacted exclusively with serum from TL patients and presented several B-cell epitopes without similarity to T. cruzi orthologs: the hypothetical protein GI 134063939 and the metallo-peptidase Clan MA(E)-Family M3. The immunoassay revealed nine peptides with strong reactivity to sera from TL patients. These proteins and peptides may be good candidates to improve the specificity and sensibility of serological tests aiming to diagnose the TL of this neglected human disease. BIOLOGICAL SIGNIFICANCE: As no gold-standard test for tegumentary leishmaniasis (TL) exists, a combination of different diagnostic techniques is often necessary to obtain precise results. Thus, the identification of species-specific, highly immunogenic and abundant proteins that stimulate the humoral immune response in the host should help in the development of serological tests for human TL. Herein we searched for these potential antigens in Leishmania species related to American Leishmaniasis (L. amazonensis, L. braziliensis and L. infantum). To this end, we employed an immunoproteomic approach using proteins from these Leishmania species and sera from TL and Visceral Leishmaniasis (VL) patients. Our study unveils specific proteins and peptides that may represent antigens that will help the efforts to improve the accuracy of serological tests to diagnose the TL form.
Assuntos
Antígenos de Protozoários/análise , Leishmaniose Cutânea/diagnóstico , Leishmaniose Visceral/diagnóstico , Testes Sorológicos/métodos , Reações Cruzadas , Diagnóstico Diferencial , Humanos , Leishmaniose Cutânea/imunologia , Leishmaniose Visceral/imunologia , Doenças Negligenciadas/diagnóstico , Proteômica/métodos , Proteínas de Protozoários/análise , Sensibilidade e Especificidade , Testes Sorológicos/normas , Especificidade da EspécieRESUMO
Atroxlysin-I (Atr-I) is a hemorrhagic snake venom metalloproteinase (SVMP) from Bothrops atrox venom, the snake responsible for the majority of bites in the north region of South America. SVMPs like Atr-I produce toxic effects in victims including hemorrhage, inflammation, necrosis and blood coagulation deficiency. Mapping of B-cell epitopes in SVMPs might result in the identification of non-toxic molecules capable of inducing neutralizing antibodies and improving the anti-venom therapy. Here, using the SPOT-synthesis technique we identified two epitopes located in the N-ter region of Atr-I (AtrEp1-(22)YNGNSDKIRRRIHQM(36); and AtrEp2-(55)GVEIWSNKDLINVQ(68)). Based on the sequence of AtrEp1 and AtrEp2 a third peptide named Atr-I biepitope (AtrBiEp) was designed and synthesized ((23)NGNSDKIRRRIH(34)GG(55)GVEIWSNKDLINVQ(68)). AtrBiEp was used to immunize BALB/c mice. Anti-AtrBiEp serum cross-reacted against Atr-I in western blot and was able to fully neutralize the hemorrhagic activity of Atr-I. Our results provide a rational basis for the identification of neutralizing epitopes on Atr-I snake venom toxin and show that the use of synthetic peptides could improve the generation of immuno-therapeutics.
Assuntos
Antivenenos/imunologia , Epitopos de Linfócito B/imunologia , Metaloendopeptidases/imunologia , Venenos de Serpentes/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Bothrops , Reações Cruzadas , Mapeamento de Epitopos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Testes de Neutralização , Peptídeos/imunologia , Estrutura Terciária de ProteínaRESUMO
MOTIVATION: Antibodies are an important class of biological drugs, but with limitations, such as inadequate pharmacokinetics, adverse immunogenicity and high production costs. Synthetic peptides for the desired target represent an important alternative to antibodies. However, no computational tool exists to guide the design of these peptides. RESULTS: To identify the interacting residues in a given antibody-antigen (Ab-Ag) interface we used Interface Interacting Residue (I2R), a selection method based on computed molecular interactions. The aggregation of all the molecular interactions between epitope and paratope residues allowed us to transform the 3D Ab-Ag complex structures into interface graphs. Based on these data and the probability of molecular interaction we developed EPI-Peptide Designer tool that uses predicted paratope residues for an epitope of interest to generate targeted peptide ligand libraries. EPI-Peptide Designer successfully predicted 301 peptides able to bind to LiD1 target protein (65% of the experimentally tested peptides), an enrichment of 22% compared to randomly generated peptides. This tool should enable the development of a new generation of synthetic interacting peptides that could be very useful in the biosensor, diagnostic and therapeutic fields. AVAILABILITY AND IMPLEMENTATION: All software developed in this work are available at http://www.biocomp.icb.ufmg.br/biocomp/ CONTACT: liza@icb.ufmg.br SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Epitopos , Sítios de Ligação de Anticorpos , Ligantes , Biblioteca de Peptídeos , PeptídeosRESUMO
Background Envenoming by coral snakes (Elapidae: Micrurus), although not abundant, represent a serious health threat in the Americas, especially because antivenoms are scarce. The development of adequate amounts of antielapidic serum for the treatment of accidents caused by snakes like Micrurus corallinus is a challenging task due to characteristics such as low venom yield, fossorial habit, relatively small sizes and ophiophagous diet. These features make it difficult to capture and keep these snakes in captivity for venom collection. Furthermore, there are reports of antivenom scarcity in USA, leading to an increase in morbidity and mortality, with patients needing to be intubated and ventilated while the toxin wears off. The development of an alternative method for the production of an antielapidic serum, with no need for snake collection and maintenance in captivity, would be a plausible solution for the antielapidic serum shortage. Methods and Findings In this work we describe the mapping, by the SPOT-synthesis technique, of potential B-cell epitopes from five putative toxins from M. corallinus, which were used to design two multiepitope DNA strings for the genetic immunisation of female BALB/c mice. Results demonstrate that sera obtained from animals that were genetically immunised with these multiepitope constructs, followed by booster doses of recombinant proteins lead to a 60% survival in a lethal dose neutralisation assay. Conclusion Here we describe that the genetic immunisation with a synthetic multiepitope gene followed by booster doses with recombinant protein is a promising approach to develop an alternative antielapidic serum against M. corallinus venom without the need of collection and the very challenging maintenance of these snakes in captivity
Assuntos
Toxicologia , Alergia e Imunologia , FarmacologiaRESUMO
This work used eleven Peruvian snake venoms (Bothrops andianus, Bothrops atrox, Bothrops barnetti, Bothrops castelnaudi, Bothriopsis chloromelas, Bothrocophias microphthalmus, Bothrops neuwiedi, Bothriopsis oligolepis, Bothriopsis peruviana, Bothrops pictus and Bothriopsis taeniata) to perform in vitro experimentation and determine its main characteristics. Hyaluronidase (HYAL), phospholipase A2 (PLA2), snake venom metalloproteinase (SVMP), snake venom serine protease (SVSP) and L-amino acid oxidase (LAAO) activities; toxicity by cell viability assays using MGSO3, VERO and HeLa cell lineages; and crossed immunoreactivity with Peruvian (PAV) and Brazilian (BAV) antibothropic polyvalent antivenoms, through ELISA and Western Blotting assays, were determined. Results show that the activities tested in this study were not similar amongst the venoms and each species present their own peculiarities, highlighting the diversity within Bothrops complex. All venoms were capable of reducing cell viability of all tested lineages. It was also demonstrated the crossed recognition of all tested venoms by both antivenoms.
Assuntos
Antivenenos/farmacologia , Bothrops , Venenos de Crotalídeos/toxicidade , Animais , Western Blotting , Brasil , Linhagem Celular , Chlorocebus aethiops , Venenos de Crotalídeos/enzimologia , Venenos de Crotalídeos/imunologia , Ensaio de Imunoadsorção Enzimática , Células HeLa , Humanos , Hialuronoglucosaminidase/metabolismo , L-Aminoácido Oxidase/metabolismo , Metaloproteases/metabolismo , Peru , Fosfolipases A2/metabolismo , Serina Proteases/metabolismo , Células VeroRESUMO
Toxic effects of Peruvian Hadruroides lunatus scorpion venom on different biochemical and enzymatic parameters in blood serum of Wistar rats and Swiss mice were determined after experimental envenomation. An increase in enzymatic activities of Aspartate Aminotransferase (AST), Lactate Dehydrogenase (LDH) and levels of serum protein and albumin were observed while a decrease in creatinine level in serum was perceived after 30 min of envenomation. No alterations in urea levels and in kidney histology were detected in the envenomed rats. The global leukocytes count was diminished, with decrease in lymphocytes, eosinophils and neutrophils levels in the bloodstream, while no alterations were found in hematological parameters of red series in rats injected with H. lunatus venom. IL-2, IL-4, IL-6, INF-γ, TNF, IL-17A and IL-10 levels were evaluated 0.5, 3 and 6 h after experimental envenomation of mice with H. lunatus venom. From all the analyzed cytokines, only IL-6 showed an increase in serum levels. Taken together, these results point out that envenomation by H. lunatus can impair hematological and immunological parameters and therefore might be monitored in accidents involving this species.
Assuntos
Picadas de Escorpião/patologia , Venenos de Escorpião/toxicidade , Escorpiões/metabolismo , Animais , Aspartato Aminotransferases/sangue , Creatinina/sangue , Interferon gama/sangue , Interleucina-10/sangue , Interleucina-17/sangue , Interleucina-2/sangue , Interleucina-4/sangue , Interleucina-6/sangue , L-Lactato Desidrogenase/sangue , Masculino , Camundongos , Ratos , Ratos Wistar , Albumina Sérica/metabolismoRESUMO
This communication describes the general characteristics of the venom from the Brazilian scorpion Tityus fasciolatus, which is an endemic species found in the central Brazil (States of Goiás and Minas Gerais), being responsible for sting accidents in this area. The soluble venom obtained from this scorpion is toxic to mice being the LD50 is 2.984 mg/kg (subcutaneally). SDS-PAGE of the soluble venom resulted in 10 fractions ranged in size from 6 to 10-80 kDa. Sheep were employed for anti-T. fasciolatus venom serum production. Western blotting analysis showed that most of these venom proteins are immunogenic. T. fasciolatus anti-venom revealed consistent cross-reactivity with venom antigens from Tityus serrulatus. Using known primers for T. serrulatus toxins, we have identified three toxins sequences from T. fasciolatus venom. Linear epitopes of these toxins were localized and fifty-five overlapping pentadecapeptides covering complete amino acid sequence of the three toxins were synthesized in cellulose membrane (spot-synthesis technique). The epitopes were located on the 3D structures and some important residues for structure/function were identified.
Assuntos
Antivenenos/análise , Proteínas de Artrópodes/toxicidade , Modelos Moleculares , Picadas de Escorpião/imunologia , Venenos de Escorpião/toxicidade , Escorpiões/imunologia , Sequência de Aminoácidos , Animais , Antivenenos/metabolismo , Antivenenos/uso terapêutico , Proteínas de Artrópodes/antagonistas & inibidores , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Brasil , Reações Cruzadas , Bases de Dados de Proteínas , Mapeamento de Epitopos , Dose Letal Mediana , Masculino , Camundongos , Dados de Sequência Molecular , Peso Molecular , Conformação Proteica , Picadas de Escorpião/sangue , Venenos de Escorpião/antagonistas & inibidores , Venenos de Escorpião/química , Venenos de Escorpião/metabolismo , Escorpiões/fisiologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , OvinosRESUMO
Scorpionism represents a serious public health problem resulting in the death of children and debilitated individuals. Scorpion sting treatment employs various strategies including the use of specific medicines such as antiserum, especially for patients with severe symptoms. In 1909 Charles Todd described the production of an antiserum against the venom of the scorpion Buthus quinquestriatus. Based on Todd's work, researchers worldwide began producing antiserum using the same approach i.e., immunization of horses with crude venom as antigen. Despite achieving satisfactory results using this approach, researchers in this field have developed alternative approaches for the production of scorpion antivenom serum. In this review, we describe the work published by experts in toxinology to the development of scorpion venom antiserum. Methods and results describing the use of specific antigens, detoxified venom or toxins, purified toxins and or venom fractions, native toxoids, recombinant toxins, synthetic peptides, monoclonal and recombinant antibodies, and alternative animal models are presented.
Assuntos
Antivenenos/biossíntese , Imunização/métodos , Modelos Animais , Picadas de Escorpião/tratamento farmacológico , Picadas de Escorpião/epidemiologia , Venenos de Escorpião/antagonistas & inibidores , Anticorpos Monoclonais/uso terapêutico , Antivenenos/história , Antivenenos/uso terapêutico , História do Século XIX , História do Século XX , História do Século XXI , Humanos , Proteínas Recombinantes/uso terapêutico , Venenos de Escorpião/química , Venenos de Escorpião/toxicidade , Especificidade da EspécieRESUMO
In this work, an anti-loxoscelic serum was produced by immunizing horses with a recombinant dermonecrotic protein from Loxosceles intermedia (rLiD1). Anti-rLiD1 antibodies were able to recognize different species of Loxosceles venoms by Western Blot and ELISA. The efficacy of anti-rLiD1 serum against the toxic effects of Loxosceles laeta (Peru) venom was tested, showing that anti-rLiD1 serum can neutralize those effects. This study confirms that recombinant proteins can be good candidates to replace crude venoms for antivenom production.
Assuntos
Antivenenos/imunologia , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Proteínas Recombinantes/metabolismo , Venenos de Aranha/química , Animais , Antivenenos/farmacologia , Western Blotting , Brasil , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Cavalos , Testes de Neutralização , Peru , Diester Fosfórico Hidrolases/análise , Especificidade da Espécie , Venenos de Aranha/enzimologiaRESUMO
The main goal of this work was to develop a strategy to identify B-cell epitopes on four different three finger toxins (3FTX) and one phospholipase A2 (PLA2) from Micrurus corallinus snake venom. 3FTx and PLA2 are highly abundant components in Elapidic venoms and are the major responsibles for the toxicity observed in envenomation by coral snakes. Overlapping peptides from the sequence of each toxin were prepared by SPOT method and three different anti-elapidic sera were used to map the epitopes. After immunogenicity analysis of the spot-reactive peptides by EPITOPIA, a computational method, nine sequences from the five toxins were chemically synthesized and antigenically and immunogenically characterized. All the peptides were used together as immunogens in rabbits, delivered with Freund's adjuvant for a first cycle of immunization and Montanide in the second. A good antibody response against individual synthetic peptides and M. corallinus venom was achieved. Anti-peptide IgGs were also cross-reactive against Micrurus frontalis and Micrurus lemniscatus crude venoms. In addition, anti-peptide IgGs inhibits the lethal and phospholipasic activities of M. corallinus crude venom. Our results provide a rational basis to the identification of neutralizing epitopes on coral snake toxins and show that their corresponding synthetic peptides could improve the generation of immuno-therapeutics. The use of synthetic peptide for immunization is a reasonable approach, since it enables poly-specificity, low risk of toxic effects and large scale production.
Assuntos
Venenos Elapídicos/química , Elapidae , Epitopos de Linfócito B/genética , Fosfolipases A2/genética , Toxinas Biológicas/genética , Sequência de Aminoácidos , Animais , Formação de Anticorpos , Brasil , Técnicas de Química Sintética , Ensaio de Imunoadsorção Enzimática , Imunoglobulina G/metabolismo , Dados de Sequência Molecular , Testes de Neutralização , Peptídeos/genética , Peptídeos/imunologiaAssuntos
Toxicologia , Toxicologia , Venenos , Intoxicação , Toxinas Biológicas , Toxicologia , Biologia Celular , GenéticaRESUMO
Tityus serrulatus is a Brazilian scorpion species with great medical significance. While the effects of neurotoxins have been extensively studied, little is known about the proteases expressed in the venom gland of this arthropod. In this study, clones from a T. serrulatus (Ts) venom gland cDNA library were selected according to homology to proteases. The sequences were aligned in the database and classified by homology. Similarity and identity analyses of the sequences were carried out, and a phylogenetic tree was constructed with the sequences of other proteases. These cDNA sequences correspond to ten different metalloproteases, named metalloserrulases (TsMS). TsMS 1-9 belong to the metzincin family, which has three domains: signal peptide, propeptide, and metalloprotease domain; while TsMS 10 belongs to the gluzincin family. The proteolytic activity of the venom was inferred from the cleavage of fibrinogen, and the residues recognized by the proteases were determined by cleavage of a tripeptide library using a fluorescence resonance energy transfer assay. The Ts venom showed proteolytic activity on fibrinogen and preferential cleavage close to the basic residues K and R. Its activity could be inhibited by EDTA, indicating that the venom from this scorpion predominantly consists of metalloproteases.
Assuntos
Metaloproteases/genética , Metaloproteases/toxicidade , Venenos de Escorpião/enzimologia , Sequência de Aminoácidos , Animais , DNA Complementar/genética , Fibrinogênio/metabolismo , Metaloproteases/química , Dados de Sequência Molecular , Filogenia , Escorpiões , Homologia de Sequência de AminoácidosRESUMO
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/enzimologiaAssuntos
Toxicologia , Toxicologia , Biodiversidade , Venenos , Intoxicação , Toxinas Biológicas , ToxicologiaRESUMO
Members of the spider genus Lasiodora are widely distributed in Brazil, where they are commonly known as caranguejeiras. Lasiodora spider venom is slightly harmful to humans. The bite of this spider causes local pain, edema and erythema. However, Lasiodora sp. spider venom may be a source of important pharmacological tools. Our research group has described previously that Lasiodora sp. venom produces bradycardia in the isolated rat heart. In the present work, we sought to evaluate the vascular effect of Lasiodora sp. venom and to isolate the vasoactive compounds from the venom. The results showed that Lasiodora spider venom induced a concentration-dependent vasodilation in rat aortic rings, which was dependent on the presence of a functional endothelium and abolished by the nitric oxide synthase (NOS) inhibitor L-NAME. Western blot experiments revealed that the venom also increased endothelial NOS function by increasing phosphorylation of the Ser¹¹77 residue. Assay-directed fractionation isolated a vasoactive fraction from Lasiodora sp. venom. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) assays identified a mixture of two compounds: adenosine diphosphate (ADP, approximately 90%) and adenosine monophosphate (AMP, approximately 10%). The vasodilator effects of Lasiodora sp. whole venom, as well as ADP, were significantly inhibited by suramin, which is a purinergic P2-receptor antagonist. Therefore, the results of the present work indicate that ADP is a main vasodilator component of Lasiodora sp. spider venom.
Assuntos
Difosfato de Adenosina/farmacologia , Venenos de Aranha/química , Aranhas/química , Vasodilatadores/farmacologia , Difosfato de Adenosina/química , Monofosfato de Adenosina/química , Animais , Western Blotting , Fracionamento Químico , Endotélio/efeitos dos fármacos , Técnicas In Vitro , Espectrometria de Massas , NG-Nitroarginina Metil Éster/metabolismo , Óxido Nítrico Sintase/metabolismo , Ressonância Magnética Nuclear Biomolecular , Fosforilação/efeitos dos fármacos , Ratos , Suramina/química , Vasodilatação/efeitos dos fármacos , Vasodilatadores/químicaRESUMO
A chimeric protein was constructed expressing three epitopes of LiD1, a dermonecrotic toxin from the venom of Loxosceles intermedia spider. This species is responsible for a large number of accidents involving spiders in Brazil. We demonstrated that the chimeric protein (rCpLi) generated is atoxic and that antibodies previously developed in rabbits against synthetic epitopes reactive with rCpLi in ELISA and immunoblot assays. The antibody response in rabbits against the rCpLi was evaluated by ELISA and we have detected an antibody response in all immunized animals. Overlapping peptides covering the amino acid sequence of the rCpLi were synthesized on a cellulose membrane, and their recognition by rabbit anti-rCpLi serum assessed. Three different antigenic regions were identified. The percentage of inhibition of the dermonecrotic, hemorrhagic and edematogenic activities caused by the recombinant protein LiD1r in naïve rabbits was assessed by pre-incubation with anti-rCpLi antibodies. Anti-rCpLi induced good dermonecrotic and hemorrhagic protection. The levels of protection were similar to the antiboides anti-LiD1r. In summary, we have developed a polyepitope recombinant chimeric protein capable of inducing multiple responses of neutralizing antibodies in a rabbit model. This engineered protein may be a promising candidate for therapeutic serum development or vaccination.
Assuntos
Proteínas Recombinantes de Fusão/química , Venenos de Aranha/química , Animais , Anticorpos Neutralizantes/imunologia , Antivenenos/imunologia , Edema/imunologia , Edema/prevenção & controle , Epitopos de Linfócito B/genética , Hemorragia/imunologia , Hemorragia/prevenção & controle , Necrose/imunologia , Necrose/prevenção & controle , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Pele/imunologia , Pele/patologia , Esfingomielina Fosfodiesterase/química , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/imunologia , Venenos de Aranha/genética , Venenos de Aranha/imunologia , AranhasRESUMO
This manuscript describes the general biochemical properties and immunological characteristics of Peruvian spider Loxosceles laeta venom (PLlv), which is responsible for the largest number of accidents involving venomous animals in Peru. In this work, we observed that the venom of this spider is more lethal to mice when compared with L. laeta venom from Brazil (BLlv). The LD50 of PLlv was 1.213 mg/kg when the venom was intradermally injected. The venom displayed sphingomyelinase activity and produced dermonecrotic, hemorrhagic and edema effects in rabbits. 2-D SDS-PAGE separation of the soluble venoms resulted in a protein profile ranging from 20 to 205 kDa. Anti-PLlv and anti-BLlv sera produced in rabbits and assayed by ELISA showed that rabbit antibodies cross-reacted with PLlv and BLlv and also with other Brazilian Loxosceles venoms. Western blotting analysis showed that bands corresponding to 25-35 kDa are the proteins best recognized in every Loxosceles spp venoms analyzed. The immunized rabbits displayed protective effect after challenge with PLlv and BLlv. In vitro assays with horse anti-loxoscelic antivenoms produced in Brazil and Peru demonstrated that these commercial antivenoms were efficient to inhibit the sphingomyelinase activity of PLlv and BLlv.
Assuntos
Antivenenos/farmacologia , Diester Fosfórico Hidrolases/toxicidade , Venenos de Aranha/toxicidade , Aranhas/metabolismo , Animais , Western Blotting , Brasil , Reações Cruzadas , Edema/induzido quimicamente , Edema/patologia , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Cavalos , Imunização , Dose Letal Mediana , Masculino , Camundongos , Testes de Neutralização , Peru , Coelhos , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/metabolismoRESUMO
PnTx3-4 is a toxin isolated from the venom of the spider Phoneutria nigriventer that blocks N-, P/Q-, and R-type voltage-gated calcium channels and has great potential for clinical applications. In this report we used the SUMO system to express large amounts of recombinant PnTx3-4 peptide, which was found in both soluble and insoluble fractions of bacterial extracts. We purified the recombinant toxin from both fractions and showed that the recombinant peptide showed biological activity similar to the native PnTx3-4. In silico analysis of the primary sequence of PnTx3-4 indicated that the peptide conforms to all the criteria of a knottin scaffold. Additionally, circular dichroism spectrum analysis of the recombinant PnTx3-4 predicted that the toxin structure is composed of approximately 53% turns/unordered, 31% α-helix and 16% ß-strand, which is consistent with predicted model of the PnTx3-4 knottin scaffold available at the knottin database (http://knottin.cbs.cnrs.fr). These studies provide the basis for future large scale production and structure-function investigation of PnTx3-4.
Assuntos
Canais de Cálcio/metabolismo , Neuropeptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Venenos de Aranha/metabolismo , Sequência de Aminoácidos , Análise de Variância , Animais , Dicroísmo Circular , Dados de Sequência Molecular , Neuropeptídeos/genética , Neuropeptídeos/isolamento & purificação , Oligonucleotídeos/genética , Plasmídeos/genética , Dobramento de Proteína , Análise de Sequência de DNA , Sinaptossomos/metabolismoRESUMO
Members of the spider genus Loxosceles pose a marked health risk to humans because of the seriousness of the necrotic and systemic effects of their bite, known as loxoscelism. The recent confirmation of Loxosceles similis in residences of Belo Horizonte in Minas Gerais Province, Brazil increases the local potential risk of loxoscelism at higher levels. The first characterization of the venom from this species showed that its main biological effects had a similar intensity as other species (e.g. Loxosceles intermedia, Loxosceles laeta, and Loxosceles gaucho). Therefore, we wished to further analyse the biological activity of the L. similis venom as well as the capacity of anti-L. similis-venom serum to reduce dermonecrotic effects to rabbit skin. Histological analysis of rabbit skin 2, 4 and 8h after intradermal injection of L. similis venom demonstrated a dense inflammatory infiltrate, edema, degeneration and necrosis of the skin muscle, dissociation of collagen fibers, and disruption of reticular fibers. Importantly, pre-incubation of the venom with anti-L. similis-venom serum significantly decreased all of these effects. Anti-L. similis antivenom generated antibodies that were strongly reactive to L. similis venom and capable of neutralizing the dermonecrotic effects in rabbits caused by this venom. Moreover, the antivenom significantly reduced the sphingomyelinase activity of L. similis crude venom. Venoms produced by male and female spiders were equally reactive towards anti-L. similis and anti-L. intermedia antivenoms, but female venom induced larger lesions on rabbits. In contrast, female venom acted as an immunization enhancer and protected animals from L. similis envenomation to a greater degree than male venom. In conclusion, the results shown in this study for L. similis antivenom merits a more in depth study of its properties, which may become a valuable tool against loxoscelism.