RESUMEN

Variability in snake venom composition is well-documented and crucial for understanding snake ecology and predicting snakebites. In this study, we characterize the venom composition and biological activities of newborn female and male Bothrops moojeni and their mother. Our results reveal significant differences between the venom of newborn females and males, demonstrating a broad and diverse range of proteins. The venoms of newborn females showed higher serine protease effects, increased hemorrhagic activity, and greater lethality compared to the venom of newborn males. However, no differences were observed in phospholipase A2 and coagulant activity. The differences in protein composition and toxic activities between maternal and neonatal venom, as well as between the venoms of newborn females and males, contribute to understanding the diverse outcomes of snakebites. These results underscore the importance of considering sex and ontogeny in understanding venom composition in snakes.

Asunto(s)

Animales Recién Nacidos , Bothrops , Venenos de Crotálidos , Animales , Bothrops/clasificación , Bothrops/fisiología , Femenino , Masculino , Factores Sexuales

RESUMEN

Hemorrhage is one of the most striking effects of bites by viper snakes resulting in fast bleeding and ischemia in affected tissues. Snake venom metalloproteinases (SVMPs) are responsible for hemorrhagic activity, but the mechanisms involved in SVMP-induced hemorrhage are not entirely understood and the study of such mechanisms greatly depends on in vivo experiments. In vivo, hemorrhagic SVMPs accumulate on basement membrane (BM) of venules and capillary vessels allowing the hydrolysis of collagen IV with consequent weakness and rupture of capillary walls. These effects are not reproducible in vitro with conventional endothelial cell cultures. In this study we used two-dimension (2D) or three-dimension (3D) cultures of HUVECs on matrigel and observed the same characteristics as in ex vivo experiments: only the hemorrhagic toxin was able to localize on surfaces or internalize endothelial cells in 2D cultures or in the surface of tubules formed on 3D cultures. The contribution of matrigel, fibronectin and collagen matrices in jararhagin-induced endothelial cell damage was then analyzed. Collagen and matrigel substrates enhanced the endothelial cell damage induced by jararhagin allowing toxin binding to focal adhesions, disruption of stress fibers, detachment and apoptosis. The higher affinity of jararhagin to collagen than to fibronectin explains the localization of the toxin within BM. Moreover, once located in BM, interactions of jararhagin with α2ß1 integrin would favor its localization on focal adhesions, as observed in our study. The accumulation of toxin in focal adhesions, observed only in cells grown in collagen matrices, would explain the enhancement of cell damage in these matrices and reflects the actual interaction among toxin, endothelial cells and BM components that occurs in vivo and results in the hemorrhagic lesions induced by viper venoms.

Asunto(s)

Colágeno/efectos de los fármacos , Venenos de Crotálidos/farmacología , Fibronectinas/efectos de los fármacos , Metaloendopeptidasas/farmacología , Apoptosis/efectos de los fármacos , Membrana Basal/efectos de los fármacos , Técnicas de Cultivo de Célula , Uniones Célula-Matriz/efectos de los fármacos , Venenos de Crotálidos/análisis , Fragmentación del ADN/efectos de los fármacos , Combinación de Medicamentos , Células Endoteliales/efectos de los fármacos , Citometría de Flujo , Adhesiones Focales/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Laminina , Metaloendopeptidasas/análisis , Modelos Biológicos , Proteoglicanos , Veneno de Bothrops Jararaca

RESUMEN

Disintegrins are cysteine-rich toxins containing the RGD motif exposed in a loop that binds integrins such as αIIbß3, α5ß1 and αvß3. The flexibility of the RGD loop, controlled by the profile of the cysteine pairs and the residues flanking the RGD sequence, are key structural features for the functional activity of these molecules. Recently, our group reported a transcript in the venom gland of Bothrops neuwiedi corresponding to a new P-II SVMP precursor, BnMPIIx, in which the RGD-binding loop includes many substituted residues and unique cysteine residues at the C-terminal. In this paper, we obtained the recombinant disintegrin domain of BnMPIIx, Neuwiedin, which inhibited ADP-induced platelet aggregation, endothelial cell adhesion to fibrinogen and tube formation in Matrigel with no particular selectivity to αIIbß3 or endothelial cell integrins. This value was also comparable to the inhibition observed with other recombinant disintegrins with conserved cysteine positions and residues in RGD loop. In this regard, Neuwiedin is an important component to understand the functional relevance of the diversity generated by accelerated evolution of venom toxins as well as to find out eventual new disintegrin-dependent targets that may be approached with disintegrins.

Asunto(s)

Bothrops , Venenos de Crotálidos/química , Cisteína/química , Desintegrinas/química , Glándulas Salivales/metabolismo , Secuencia de Aminoácidos , Animales , Adhesión Celular/efectos de los fármacos , Clonación Molecular , Células Endoteliales/efectos de los fármacos , Escherichia coli/genética , Datos de Secuencia Molecular , Agregación Plaquetaria/efectos de los fármacos , Proteínas Recombinantes/química , Alineación de Secuencia , Análisis de Secuencia de ADN

RESUMEN

BaP1 is a P-I class of Snake Venom Metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomations by Bothrops asper, a medically-important species in Central America and parts of South America. Six monoclonal antibodies (MoAb) against BaP1 (MABaP1) were produced and characterized regarding their isotype, dissociation constant (K(d)), specificity and ability to neutralize BaP1-induced hemorrhagic and proteolytic activity. Two MABaP1 are IgM, three are IgG1 and one is IgG2b. The K(d)s of IgG MoAbs were in the nM range. All IgG MoAbs recognized conformational epitopes of BaP1 and B. asper venom components but failed to recognize venoms from 27 species of Viperidae, Colubridae and Elapidae families. Clone 7 cross-reacted with three P-I SVMPs tested (moojeni protease, insularinase and neuwiedase). BaP1-induced hemorrhage was totally neutralized by clones 3, 6 and 8 but not by clone 7. Inhibition of BaP1 enzymatic activity on a synthetic substrate by MABaP1 was totally achieved by clones 3 and 6, and partially by clone 8, but not by clone 7. In conclusion, these neutralizing MoAbs against BaP1 may become important tools to understand structure-function relationships of BaP1 and the role of P-I class SVMP in snakebite envenomation.

Asunto(s)

Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Bothrops/fisiología , Venenos de Crotálidos/enzimología , Metaloendopeptidasas/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Reacciones Cruzadas , Venenos de Crotálidos/antagonistas & inhibidores , Venenos de Crotálidos/toxicidad , Edema/inducido químicamente , Edema/prevención & control , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Hemorragia/inducido químicamente , Hemorragia/prevención & control , Immunoblotting , Inmunoglobulinas , Inyecciones Intraperitoneales , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/toxicidad , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización

RESUMEN

BaP1 is a P-I class of Snake Venom Metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomations by Bothrops asper, a medically-important species in Central America and parts of South America. Six monoclonal antibodies (MoAb) against BaP1 (MABaP1) were produced and characterized regarding their isotype, dissociation constant (Kd), specificity and ability to neutralize BaP1-induced hemorrhagic and proteolytic activity. Two MABaP1 are IgM, three are IgG1 and one is IgG2b. The Kds of IgG MoAbs were in the nM range. All IgG MoAbs recognized conformational epitopes of BaP1 and B. asper venom components but failed to recognize venoms from 27 species of Viperidae, Colubridae and Elapidae families. Clone 7 cross-reacted with three P-I SVMPs tested (moojeni protease, insularinase and neuwiedase). BaP1-induced hemorrhage was totally neutralized by clones 3, 6 and 8 but not by clone 7. Inhibition of BaP1 enzymatic activity on a synthetic substrate by MABaP1 was totally achieved by clones 3 and 6, and partially by clone 8, but not by clone 7. In conclusion, these neutralizing MoAbs against BaP1 may become important tools to understand structure–function relationships of BaP1 and the role of P-I class SVMP in snakebite envenomation.

Asunto(s)

Animales , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/inmunología , Antivenenos/inmunología , Bothrops/clasificación , Metaloproteasas/clasificación , Metaloproteasas/toxicidad , Venenos de Serpiente/inmunología , Anticuerpos Neutralizantes , Colubridae , Elapidae , Viperidae

RESUMEN

Local and systemic hemorrhages are major problems concerning bites by viper snakes. Therefore, accessing venom hemorrhagic activity is an important feature in order to characterize viper venom major toxicities or to assay antivenom efficacy. The methods currently used to access hemorrhagic activity involve animal experiments and according to the general ethical committees, these procedures should be substituted to in vitro assays in order to minimize animal use in research. In this work, we have developed an immunoassay to detect the content of hemorrhagic metalloproteinases in snake venoms using a neutralizing monoclonal antibody anti-jararhagin (MAJar 3). The correlation between the reactivity of this monoclonal antibody and venom-induced hemorrhage was further revealed by a study comparing the hemorrhagic activity of venom samples collected individually from 88 specimens of Bothrops jararacussu with their reactivity with MAJar 3. As a result, a significant correlation (r=0.942) was achieved between samples hemorrhagic activity and their reactivity with MAJar 3, suggesting that this assay can be used as a substitute of the conventional tests performed in vivo to estimate the hemorrhagic activity.

Asunto(s)

Bothrops/metabolismo , Venenos de Crotálidos/enzimología , Venenos de Crotálidos/toxicidad , Ensayo de Inmunoadsorción Enzimática/métodos , Hemorragia/inducido químicamente , Metaloproteasas/análisis , Animales , Anticuerpos Monoclonales , Ratones , Proteínas/metabolismo , Conejos , Proteínas de Reptiles/metabolismo , Proteínas de Reptiles/toxicidad

RESUMEN

Snake venom metalloproteinases (SVMPs) have been extensively studied and their effects associated with the local bleeding observed in human accidents by viper snakes. Representatives of P-I and P-III classes of SVMPs similarly hydrolyze extracellular matrix proteins or coagulation factors while only P-III SVMPs induce significant hemorrhage in experimental models. In this work, the effects of P-I and P-III SVMPs on plasma proteins and cultures of muscle and endothelial cells were compared in order to enlighten the mechanisms involved in venom-induced hemorrhage. To reach this comparison, BnP1 was isolated from B. neuwiedi venom and used as a weakly hemorrhagic P-I SVMPs and jararhagin was used as a model of potently hemorrhagic P-III SVMP. BnP1 was isolated by size exclusion and anion-exchange chromatographies, showing apparent molecular mass of approximately 24kDa and sequence similarity with other members of SVMPs, which allowed its classification as a group P-I SVMP. The comparison of local effects induced by SVMPs showed that BnP1 was devoid of significant myotoxic and hemorrhagic activities and jararhagin presented only hemorrhagic activity. BnP1 and jararhagin were able to hydrolyze fibrinogen and fibrin, although the latter displayed higher activity in both systems. Using HUVEC primary cultures, we observed that BnP1 induced cell detachment and a decrease in the number of viable endothelial cells in levels comparable to those observed by treatment with jararhagin. Moreover, both BnP1 and jararhagin induced apoptosis in HUVECs while only a small increase in LDH supernatant levels was observed after treatment with jararhagin, suggesting that the major mechanism involved in endothelial cell death is apoptosis. Jararhagin and BnP1 induced little effects on C2C12 muscle cell cultures, characterized by a partial detachment 24h after treatment and a mild necrotic effect as evidenced by a small increase in the supernatants LDH levels. Taken together, our data show that P-I and P-III SVMPs presented comparable effects except for the hemorrhagic activity, suggesting that hydrolysis of coagulation factors or damage to endothelial cells are not sufficient for induction of local bleeding.

Asunto(s)

Bothrops/metabolismo , Venenos de Crotálidos/química , Metaloendopeptidasas/farmacología , Metaloproteasas/farmacología , Secuencia de Aminoácidos , Animales , Benchmarking , Factores de Coagulación Sanguínea , Células Cultivadas , Venenos de Crotálidos/farmacología , Células Endoteliales/efectos de los fármacos , Hemorragia/inducido químicamente , Humanos , Metaloendopeptidasas/química , Metaloproteasas/química , Ratones , Datos de Secuencia Molecular , Veneno de Bothrops Jararaca

RESUMEN

Snake venom metalloproteinases (SVMPs) are multifunctional enzymes involved in several symptoms following snakebite, such as severe local hemorrhage. Multidomain P-III SVMPs are strongly hemorrhagic, whereas single domain P-I SVMPs are not. This indicates that disintegrin-like and cysteine-rich domains allocate motifs that enable catalytic degradation of ECM components leading to disruption of capillary vessels. Interestingly, some P-III SVMPs are completely devoid of hemorrhagic activity despite their highly conserved disintegrin-like and cysteine-rich domains. This observation was approached in the present study by comparing the effects of jararhagin, a hemorrhagic P-III SVMP, and berythractivase, a pro-coagulant and non-hemorrhagic P-III SVMP. Both toxins inhibited collagen-induced platelet aggregation, but only jararhagin was able to bind to collagen I with high affinity. The monoclonal antibody MAJar 3, that neutralizes the hemorrhagic effect of Bothrops venoms and jararhagin binding to collagen, did not react with berythractivase. The three-dimensional structures of jararhagin and berythractivase were compared to explain the differential binding to collagen and MAJar 3. Thereby, we pinpointed a motif within the Da disintegrin subdomain located opposite to the catalytic domain. Jararhagin binds to both collagen I and IV in a triple helix-dependent manner and inhibited in vitro fibrillogenesis. The jararhagin-collagen complex retained the catalytic activity of the toxin as observed by hydrolysis of fibrin. Thus, we suggest that binding of hemorrhagic SVMPs to collagens I and IV occurs through a motif located in the Da subdomain. This allows accumulation of toxin molecules at the site of injection, close to capillary vessels, where their catalytic activity leads to a local hemorrhage. Toxins devoid of this motif would be more available for vascular internalization leading to systemic pro-coagulant effects. This reveals a novel function of the disintegrin domain in hemorrhage formation.

Asunto(s)

Colágeno/efectos de los fármacos , Venenos de Crotálidos/toxicidad , Metaloendopeptidasas/toxicidad , Secuencia de Aminoácidos , Animales , Sitios de Unión , Colágeno/química , Colágeno/metabolismo , Venenos de Crotálidos/química , Venenos de Crotálidos/metabolismo , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/metabolismo , Veneno de Bothrops Jararaca

RESUMEN

Snake venom metalloproteinases (SVMPs) are widely distributed in snake venoms and play important roles in hemostatic disorders and local tissue damage that follows snakebite. The impact of SVMPs on hemostasis has been extensively studied showing diverse effects both on soluble factors and cellular components. The action of SVMPs involves catalytic and anti-adhesive properties, as well as direct cellular activation and/or the release of endogenous bioactive components. The purpose of this review is to overview the action of SVMPs on the inhibition of platelet functions; angiogenesis, particularly inducing apoptosis of endothelial cells; and regarding the pro-inflammatory reaction that follows snakebite. We discuss the structural features of the molecules that may be involved in such activities. The versatility and availability of SVMPs make them important tools for cell biology research into the mechanisms of action of endogenous metalloproteinases, for insights into cellular-matrix interactions and for clinical investigations into the treatment of snakebites.

Asunto(s)

Plaquetas/enzimología , Células Endoteliales/enzimología , Mediadores de Inflamación/fisiología , Metaloproteasas/fisiología , Venenos de Serpiente/enzimología , Animales , Plaquetas/patología , Células Endoteliales/patología , Humanos , Mediadores de Inflamación/química , Metaloproteasas/química , Venenos de Serpiente/química

RESUMEN

Jararhagin is a multi-domain SVMP from Bothrops jararaca venom comprising catalytic, disintegrin-like and cysteine-rich domains, which cause a local reaction manifested by hemorrhage, edema, cytokine release and inflammatory cell recruitment. In this study, the importance of disintegrin-like/cysteine-rich domains of jararhagin was addressed by analyzing the effects of jararhagin-C, which lacks the catalytic domain, in induction of leukocyte rolling and release of pro-inflammatory cytokines. Jararhagin-C was isolated from B. jararaca venom conserving the same ability of complete jararhagin molecule in inhibiting collagen-induced platelet-aggregation. Treatment of trans-illuminated cremaster muscle in vivo with jararhagin-C increased number of rolling leukocytes (approximately 250%) in post-capillary venules in all periods analyzed, without interfering with microvasculature haemodynamic, like vessel diameter, the erythrocyte speed or the blood flow rate. The release of pro-inflammatory cytokines TNF-alpha, IL-1beta and IL-6 was significantly enhanced in the local of jararhagin-C injection, showing the maximum levels in periods between 2 and 4 h after treatment. Besides the action of jararhagin-C, the presence of the inactivated catalytic domain in o-phenanthrolin-treated jararhagin was related to a higher increase in the number of rolling leukocytes. Moreover, the levels of IL-6 and IL-1beta induced by catalytically active jararhagin were higher than those induced by jararhagin-C. In conclusion, our findings suggest that the disintegrin-like/cysteine-rich domains of jararhagin are sufficient to locally activate the early events of an acute inflammatory response as leukocyte rolling and pro-inflammatory cytokine release and this action may add to the effect of catalysis, which enhances the primary cell activation.

Asunto(s)

Venenos de Crotálidos/química , Venenos de Crotálidos/toxicidad , Cisteína/química , Desintegrinas/química , Inflamación/inducido químicamente , Metaloendopeptidasas/química , Metaloendopeptidasas/toxicidad , Animales , Bothrops/metabolismo , Dominio Catalítico , Citocinas , Endotelio Vascular/metabolismo , Inflamación/patología , Interleucina-6/metabolismo , Rodamiento de Leucocito/efectos de los fármacos , Ratones , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/citología , Inhibidores de Agregación Plaquetaria , Factores de Tiempo , Vénulas , Veneno de Bothrops Jararaca

RESUMEN

Thalassophryne nattereri (niquim) is a venomous fish found on the northern and northeastern coasts of Brazil. Every year, hundreds of humans are affected by the poison, which causes excruciating local pain, edema, and necrosis, and can lead to permanent disabilities. In experimental models, T. nattereri venom induces edema and nociception, which are correlated to human symptoms and dependent on venom kininogenase activity; myotoxicity; impairment of blood flow; platelet lysis and cytotoxicity on endothelial cells. These effects were observed with minute amounts of venom. To characterize the primary structure of T. nattereri venom toxins, a list of transcripts within the venom gland was made using the expressed sequence tag (EST) strategy. Here we report the analysis of 775 ESTs that were obtained from a directional cDNA library of T. nattereri venom gland. Of these ESTs, 527 (68%) were related to sequences previously described. These were categorized into 10 groups according to their biological functions. Sequences involved in gene and protein expression accounted for 14.3% of the ESTs, reflecting the important role of protein synthesis in this gland. Other groups included proteins engaged in the assembly of disulfide bonds (0.5%), chaperones involved in the folding of nascent proteins (1.4%), and sequences related to clusterin (1.5%), as well as transcripts related to calcium binding proteins (1.0%). We detected a large cluster (1.3%) related to cocaine- and amphetamine-regulated transcript (CART), a peptide involved in the regulation of food intake. Surprisingly, several retrotransposon-like sequences (1.0%) were found in the library. It may be that their presence accounts for some of the variation in venom toxins. The toxin category (18.8%) included natterins (18%), which are a new group of kininogenases recently described by our group, and a group of C-type lectins (0.8%). In addition, a considerable number of sequences (32%) was not related to sequences in the databases, which indicates that a great number of new toxins and proteins are still to be discovered from this fish venom gland.

Asunto(s)

Etiquetas de Secuencia Expresada , Venenos de los Peces/genética , Peces Venenosos/genética , Perfilación de la Expresión Génica , Transcripción Genética/genética , Secuencia de Aminoácidos , Animales , Proteínas de Unión al Calcio , ADN Complementario/genética , Proteínas de Peces/química , Proteínas de Peces/genética , Venenos de los Peces/química , Humanos , Lectinas Tipo C , Chaperonas Moleculares , Datos de Secuencia Molecular , Análisis de Secuencia de ADN

RESUMEN

There is an increasing interest of obtaining venom by other ways than from extracting it from snakes captured in the wild. A readily available source of this venom will be useful for all pharmacological and biotechnological studies, as well as providing an improved avenue for treatments of snakebites. Here, we show that secretory cells of venom gland can be a good in vitro apparatus to produce venom. We have maintained and morphologically characterized the secretory cells of the Bothrops jararaca venom gland cultured up to 21 days. The isolated cells assemble into acini that growth in size up to 21st day, instead of adhering to the substrate. Bothropasin, a venom metalloprotease, was localized in secretory vesicles by immunoelectron microscopy and venom was also detected in culture medium in a concentration as high as 63 microg/ml. These data show that the acini formed in culture are functionally viable; they can produce and secrete venom.

Asunto(s)

Bothrops , Venenos de Crotálidos/metabolismo , Glándulas Exocrinas/citología , Metaloendopeptidasas/metabolismo , Ponzoñas/biosíntesis , Animales , Western Blotting , Células Cultivadas , Venenos de Crotálidos/análisis , Medios de Cultivo , Glándulas Exocrinas/ultraestructura , Metaloendopeptidasas/análisis , Microscopía Inmunoelectrónica , Factores de Tiempo

RESUMEN

Thalassophryne nattereri (niquim) is a venomous fish found on the northern and northeastern coasts of Brazil. Every year, hundreds of humans are affected by the poison, which causes excruciating local pain, edema, and necrosis, and can lead to permanent disabilities. In experimental models, T. nattereri venom induces edema and nociception, which are correlated to human symptoms and dependent on venom kininogenase activity; myotoxicity; impairment of blood flow; platelet lysis and cytotoxicity on endothelial cells. These effects were observed with minute amounts of venom. To characterize the primary structure of T. nattereri venom toxins, a list of transcripts within the venom gland was made using the expressed sequence tag (EST) strategy. Here we report the analysis of 775 ESTs that were obtained from a directional cDNA library of T. nattereri venom gland.

Asunto(s)

Animales , Etiquetas de Secuencia Expresada , Peces Venenosos/genética , Proteínas de Peces/genética , Proteínas de Peces/química , Secuencia de Aminoácidos/genética , Venenos de los Peces/genética , Venenos de los Peces/química , Análisis de Secuencia de ADN , Perfilación de la Expresión Génica , Proteínas de Unión al Calcio

RESUMEN

Jararhagin is a snake venom metalloproteinase (SVMP) from Bothrops jararaca involved in several hemostatic and inflammatory disorders that occur in human envenomings. In this study, we evaluated the effect of jararhagin on endothelial cells (tEnd). The exposure of tEnd to jararhagin (20 and 40microg/ml) resulted in apoptosis with activation of pro-caspase-3 and alterations in the ratio between Bax/Bcl-xL. We observed that apoptosis was followed by decrease of cell viability and the loss of cell adhesion. Jararhagin induced changes in cell shape with a decrease in cell spreading, rounding up and detachment. This was accompanied by a rearrangement of actin network and a decrease in FAK association to actin and in tyrosine phosphorylated proteins. Morphological alterations and apoptosis were abolished when jararhagin catalytic activity was inhibited, indicating the importance of catalysis. Treatment of murine peritoneal adherent cells or fibroblasts with jararhagin did not result in apoptosis. The data indicate that the pro-apoptotic effect of jararhagin is selective to endothelial cells, interfering with the adhesion mechanisms and inducing anoikis. The present model might be useful for the study of the relationships between the architectural changes in the cytoskeleton and the complex phenomenon named anoikis.

Asunto(s)

Anoicis/efectos de los fármacos , Venenos de Crotálidos/farmacología , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Metaloendopeptidasas/farmacología , Metaloproteasas/farmacología , Venenos de Serpiente/enzimología , Actinas/metabolismo , Animales , Bothrops , Caspasa 3/metabolismo , Adhesión Celular/efectos de los fármacos , Línea Celular , Línea Celular Transformada , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Cinética , Masculino , Ratones , Ratones Endogámicos BALB C , Fosforilación/efectos de los fármacos , Proteína X Asociada a bcl-2/metabolismo , Proteína bcl-X/metabolismo , Veneno de Bothrops Jararaca

RESUMEN

A novel family of proteins with kininogenase activity and unique primary structure was characterized using combined pharmacological, proteomic and transcriptomic approaches of Thalassophryne nattereri fish venom. The major venom components were isolated and submitted to bioassays corresponding to its main effects: nociception and edema. These activities were mostly located in one fraction (MS3), which was further fractionated. The isolated protein, named natterin, was able to induce edema, nociception and cleave human kininogen and kininogen-derived synthetic peptides, releasing kallidin (Lys-bradykinin). The enzymatic digestion was inhibited by kallikrein inhibitors as Trasylol and TKI. Natterin N-terminal peptide showed no similarity with already known proteins present in databanks. Primary structure of natterin was obtained by a transcriptomic approach using a representative cDNA library constructed from T. nattereri venom glands. Several expressed sequence tags (ESTs) were obtained and processed by bioinformatics revealing a major group (18%) of related sequences unknown to gene or protein sequence databases. This group included sequences showing the N-terminus of isolated natterin and was named Natterin family. Analysis of this family allowed us to identify five related sequences, which we called natterin 1-4 and P. Natterin 1 and 2 sequences include the N-terminus of the isolated natterin. Furthermore, internal peptides of natterin 1-3 were found in major spots of whole venom submitted to mass spectrometry/2DGE. Similarly to the ESTs, the complete sequences of natterins did not show any significant similarity with already described tissue kallikreins, kininogenases or any proteinase, all being entirely new. These data present a new task for the knowledge of the action of kininogenases and may help in understanding the mechanisms of T. nattereri fish envenoming, which is an important medical problem in North and Northeast of Brazil.

Asunto(s)

Batrachoidiformes/metabolismo , Venenos de los Peces/aislamiento & purificación , Calicreínas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromatografía en Gel , Electroforesis en Gel Bidimensional , Venenos de los Peces/química , Peces Venenosos , Biblioteca de Genes , Calicreínas/química , Datos de Secuencia Molecular , Alineación de Secuencia

RESUMEN

A novel family of proteins with kininogenase activity and unique primary structure was characterized using combined pharmacological, proteomic and transcriptomic approaches of Thalassophryne nattereri fish venom. The major venom components were isolated and submitted to bioassays corresponding to its main effects: nociception and edema. These activities were mostly located in one fraction (MS3), which was further fractionated. The isolated protein, named natterin, was able to induce edema, nociception and cleave human kininogen and kininogen-derived synthetic peptides, releasing kallidin (Lys-bradykinin). The enzymatic digestion was inhibited by kallikrein inhibitors as Trasylol and TKI. Natterin N-terminal peptide showed no similarity with already known proteins present in databanks. Primary structure of natterin was obtained by a transcriptomic approach using a representative cDNA library constructed from T. nattereri venom glands. Several expressed sequence tags (ESTs) were obtained and processed by bioinformatics revealing a major group (18%) of related sequences unknown to gene or protein sequence databases. This group included sequences showing the N-terminus of isolated natterin and was named Natterin family. Analysis of this family allowed us to identify five related sequences, which we called natterin 1-4 and P. Natterin 1 and 2 sequences include the N-terminus of the isolated natterin. Furthermore, internal peptides of natterin 1-3 were found in major spots of whole venom submitted to mass spectrometry/2DGE. Similarly to the ESTs, the complete sequences of natterins did not show any significant similarity with already described tissue kallikreins, kininogenases or any proteinase, all being entirely new. These data present a new task for the knowledge of the action of kininogenases and may help in understanding the mechanisms of T. nattereri fish envenoming, which is an important medical problem in North and Northeast of Brazil.

Asunto(s)

Animales , Batrachoidiformes/metabolismo , Calicreínas/aislamiento & purificación , Calicreínas/química , Peces Venenosos/clasificación , Venenos de los Peces/aislamiento & purificación , Venenos de los Peces/química , Biblioteca de Genes , Brasil , Cromatografía en Gel , Datos de Secuencia Molecular , Electroforesis en Gel Bidimensional , Proteínas , Secuencia de Aminoácidos

RESUMEN

The reprolysin subfamily of metalloproteinases includes snake venom metalloproteinases (SVMP) and mammalian disintegrin/metalloproteinase. These proteins are synthesized as zymogens and undergo proteolytic processing resulting in a variety of multifunctional proteins. Jararhagin is a P-III SVMP isolated from the venom of Bothrops jararaca. In crude venom, two forms of jararhagin are typically found, full-length jararhagin and jararhagin-C, a proteolytically processed form of jararhagin that is composed of the disintegrin-like and cysteine-rich domains of jararhagin. To better understand the structural and mechanistic bases for these forms of jararhagin in the venom of B. jararaca and the source of venom complexity in general, we have examined the jararhagin forms isolated from venom and the autolysis of isolated jararhagin under the conditions of varying pH, calcium ion concentration, and reducing agents. From our results, jararhagin isolated from venom appears as two forms: a predominant form that is stable to in vitro autolysis and a minor form that is susceptible to autolysis under a variety of conditions including alkaline pH, low calcium ion concentrations, or reducing agent. The autolysis site for production of jararhagin-C from isolated jararhagin was different from that observed for jararhagin-C as isolated from crude venom. Taken together, these data lead us to the conclusion that during the biosynthesis of jararhagin in the venom gland at least three forms are present: one form which is rapidly processed to give rise to jararhagin-C, one form which is resistant to processing in the venom and autolysis in vitro, and one minor form which is susceptible to autolysis under conditions that promote destabilization of its structure. The presence of these different forms of jararhagin contributes to greater structural and functional complexity of the venom and may be a common feature among all snake venoms. The biological and biochemical features in the venom gland responsible for these jararhagin isoforms are currently under investigation.

Asunto(s)

Bothrops , Venenos de Crotálidos/genética , Variación Genética , Metaloendopeptidasas/genética , Secuencia de Aminoácidos , Animales , Calcio/farmacología , Cromatografía Liquida , Venenos de Crotálidos/química , Venenos de Crotálidos/aislamiento & purificación , Cisteína/química , Desintegrinas/química , Concentración de Iones de Hidrógeno , Espectrometría de Masas , Metaloendopeptidasas/química , Metaloendopeptidasas/efectos de los fármacos , Metaloendopeptidasas/aislamiento & purificación , Peso Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Veneno de Bothrops Jararaca

RESUMEN

The inflammatory action of jararhagin, a hemorrhagic metalloproteinase from Bothrops jararaca venom, was studied in mice using dorsal air pouches. The injection of the toxin in 6-day-old air pouches resulted in a leukocyte accumulation comparable to that induced by LPS and whole venom. Polymorphonuclear and mononuclear cells were present in this infiltrate, with a predominance of neutrophils. Treatment of jararhagin with 1,10-phenantroline abolished its proteolytic activity and reduced the pro-inflammatory effect in approximately 50%. Cell influx was not observed when jararhagin was injected into 1-hr air pouches devoid of macrophages, except when it was injected together with 10(6) syngeneic peritoneal macrophages. Supernatants of macrophages stimulated in vitro with jararhagin did not induce leukocyte influx in 1-hr air pouches; the influx occurred after injection of the pellets of stimulated cultures. In summary, jararhagin is an important pro-inflammatory component of B. jararaca venom, and its activity is dependent upon the proteolytic activity and the presence of macrophages.

Asunto(s)

Inflamación/inducido químicamente , Macrófagos Peritoneales/fisiología , Metaloendopeptidasas/metabolismo , Venenos de Víboras/farmacología , Animales , Bothrops , Quimiotaxis de Leucocito/efectos de los fármacos , Inflamación/patología , Recuento de Leucocitos , Leucocitos/citología , Leucocitos/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Venenos de Víboras/administración & dosificación , Venenos de Víboras/enzimología

RESUMEN

The release of pro-inflammatory cytokines (IL-1beta, IL-6 and TNF-alpha) from murine peritoneal adherent cells (MPAC) was studied after exposure to jararhagin, a metalloproteinase/disintegrin isolated from Bothrops jararaca venom. MPACs were treated with LPS (lipopolysaccharide), jararhagin, or EDTA-inactivated jararhagin for up to 24h. Following incubation, the culture supernatant was assayed by ELISA for the presence of cytokines, while the cells were analysed for viability and cytokine mRNA expression. The cells exposed to native jararhagin released TNF-alpha and IL-1beta after 4 and 24h respectively. When MPACs were exposed to Jararhagin treated with EDTA, TNF-alpha and IL-1beta production was sustained throughout the culture period and IL-6 production was observed. TNF-alpha, IL-6 and IL-1beta mRNA were detected 4h after stimulation with either native or EDTA-treated jararhagin. Addition of jararhagin to LPS stimulated cells resulted in a dramatic decrease in the release of IL-6 and TNF-alpha. RT-PCR showed that this inhibition does not occur at the transcriptional level and further experiments showed that jararhagin degraded soluble cytokines by proteolytic activity. This study suggests that jararhagin induces TNF-alpha, IL-1beta and IL-6 expression, which may be rapidly degraded by its proteolytic activity.

Asunto(s)

Bothrops , Venenos de Crotálidos/antagonistas & inhibidores , Venenos de Crotálidos/farmacología , Citocinas/farmacología , Ácido Edético/farmacología , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/inmunología , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/farmacología , Cavidad Peritoneal/citología , ARN Mensajero/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Venenos de Crotálidos/química , Venenos de Crotálidos/enzimología , Venenos de Crotálidos/toxicidad , Combinación de Medicamentos , Ácido Edético/antagonistas & inhibidores , Ensayo de Inmunoadsorción Enzimática , Inflamación , Quelantes del Hierro , Lipopolisacáridos/antagonistas & inhibidores , Macrófagos Peritoneales/efectos de los fármacos , Metaloendopeptidasas/química , Metaloendopeptidasas/aislamiento & purificación , Metaloendopeptidasas/metabolismo , Ratones , Ratones Endogámicos BALB C , Inhibidores de Agregación Plaquetaria , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Veneno de Bothrops Jararaca

RESUMEN

Jararhagin is a toxic protein, isolated from the venom of the snake Bothrops jararaca, which is composed of a metalloprotease domain coupled to a disintegrin/cysteine-rich domain. It induces local haemorrhage owing to the proteolytic digestion of the basement membrane of capillaries. Jararhagin also cleaves the alpha(2)beta(1) integrin on the surface of platelets, thereby acting as a potent inhibitor of collagen-induced platelet aggregation. Crystals of jararhagin were obtained by the vapour-diffusion technique at 273 K in 200 mM sodium acetate, 100 mM cacodylate buffer pH 6.5 and 30% PEG 8000. Diffraction data have been obtained to a resolution of 2.8 A from a single frozen crystal, which belonged to space group P2(1)2(1)2(1) with unit-cell parameters a = 73.7, b = 100.3, c = 133.4 A. The asymmetric unit contains two jararhagin molecules and has a solvent content of 45%. A molecular-replacement solution has been obtained using a homology-built model based on the crystal structure of acutolysin, a haemorrhagic zinc metalloproteinase from the venom of the snake Agkistrodon acutus; attempts are under way to locate the remaining domains.

Asunto(s)

Bothrops , Venenos de Crotálidos/química , Venenos de Crotálidos/enzimología , Metaloendopeptidasas/química , Animales , Cristalización , Cristalografía por Rayos X , Desintegrinas/química , Modelos Moleculares , Conformación Proteica , Veneno de Bothrops Jararaca