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Isolation and characterization of the 35 kDa protein of Loxosceles gaucho spider
534
Abstracts/Toxicon 38 (2000) 48~595
Characterization of the dermonecrotic component (35 kDa) of Loxosceles gaucho spider venom using monoclonal antibodies. P. Guilherme , D. Muramatsu, I. Fernandes, K.C. Barbaro (Laborat6rio de Imunopatologia, ]nstituto Butantan, Av. Vital Brazil, 1500, 05503-900, S~o Paulo, SP, Brazil). Loxoscelism has been recognized as a health problem in Brazil since 1957 and nowdays it is considered the most important form of araneism in the country. L. gaucho, L. laeta and L. intermedia are the main species found in the south of Brazil. The most common finding after the spider's bite is a dermonecrotic lesion. A definite therapy for the pathology caused by the Loxosceles venom has not been established yet. Toxins with molecular mass of 32-35 kDa are the principal components responsible for the dermonecrotic activity of the venom (J. Toxicol.Toxins Reviews 14, 40l, 1995). Objective: The aim of this study was to characterize the component responsible for the dermonecrotic activity of the L. gaucho venom (involved in the large majority of accidents in the state of S~o Paulo) using monoclonal antibodies (MoAb) against 35 kDa protein of this venom. Methods and results: To generate MoAbs to the 35 kDa protein, popliteal lymph node cells from Balb/c mice immunized with L. gaucho venom were fused with SP2-O cells. Supernatants from the hibridomas were screened by ELISA using plates coated with the 35 kDa protein or whole L. gaucho venom. ELISA's titers of the MoAbs (purified from ascitic fluid by affinity chromatography Protein A-Sepharose) were similar using both whole venom or 35 kDa purified component. By immunoblotting it was observed that the 4 MoAbs of IgG1 isotype, named MoALgl to Lg4, were able to recognize mainly the 35 kDa protein of L. gaucho venom, but detected weakly the 35 kDa protein of L. intermedia and failed to detect any component of L. laeta venom. On the other side, MoALg5 (IgM isotype) recognize all components of L. gaucho, L. laeta and L. intermedia venoms. Only MoALgl was able to neutralize the dermonecrotic activity of the L. gaucho venom. All MoAbs obtained failed in neutralize the dermonecrotic activity of the heterologous venoms. Conclusion: Loxosceles venoms presented crossreactivity when species-specific antisera were used (Toxicon 32, 113, 1994). Besides, they have similarity in the N-terminal sequence of their dermonecrotic components (J. Protein. Chem. 15, 337, 1996). In despite of these, MoALgl neutralized only the dermonecrotic activity of the homologous venom but not the one of L. intermedia venom although it was able to recognize it by immunoblotting. These results suggest that different epitopes are present in the protein responsible for the dermonecrotic activity of Loxosceles venoms. Furthermore, since this MoAb neutralized the dermonecrotic activity, it probably recognizes the active "toxic" site of the 35 kDa protein of L. gaucho venom. This observation may explain the differences of toxicity between L. gaucho, L. laeta and L. intermedia venoms. Further studies are in progress to investigate this question. Acknowledgements: Supported by FAPESP (97/14188-7, 97/09681-6).
Isolation and characterization of the 35 kDa protein oj Loxosceles gaucho spider.
Abstracts/Toxzcon 38 (2000) 487--595
535
D. Muramatsu, P. Guilherme, K.C. Barbaro (Laborat6rio de Imunopatologia, Instituto Butantan, Av. Vital Brazil, 1500, 05503-900, Silo Paulo, SP, Brazil), Spiders of Loxosceles genus are represented by more than thirty species in South America. In Brazil there are seven species, three of them, L. gaucho, L. laeta and L. intermedia are usually found in south and south-east regions. Envenomations by Loxosceles spiders are an important medical problem characterized by dermonecrotic lesion in the bitten site and occasionally by systemic effects that may be lethal. Previous results showed that the dermonecrotic and lethal activities of the L. gaucho venom are due to a fraction of the venom that contains mainly components with 35 kDa. (J. Protein. Chem. 15, 337, 1996). Objective: The aim of this project was to study some properties of L. gaucho venom (responsible for the large majority of accidents in the state of Silo Paulo), attempting to characterize the components responsible for its dermonecrotic activity. Methods and results: Loxosceles gaucho venom proteins were fractionated by cation exchange chromatography (Mono S-HR 5/5) using a FPLC system. Proteins bound to the column were eluted with a linear gradient of NaC1 (0-1 M) in 50 mM acetate buffer, pH 5.0. Fractionation of the dialised venom resulted in 14 peaks. Analysis by SDS-PAGE showed that the major protein of the L. gaucho venom has approximately 35 kDa. Isoeletric focusing of the L. gaucho venom showed acid and basic proteins. Two major basic bands were detected with pI of 8.2 and 7.1 and three major acid bands with pI of 4.5; 3.9 and 3.7. Dermonecrotic activity was assayed by i.d. injections, into a shaved area of the rabbit dorsum of 0.2 ml containing 3 ~tg of venom or its fractions (0.5 pg) in 0.15 M NaC1. The dermonecrotic activity was presented mainly in fraction 7, that contained the component of 35 kDa and pI of 8.2. Conclusion: The major protein in the L. gaucho venom has approximately 35 kDa. It is a basic protein with pI of 8.2 and it is the main component responsible for the dermonecrotic activity. This is in agreement with previous results obtained with L. reclusa spider venom (Am. J. Med. Sci., 304, 261-267, 1992). It was observed that Loxosceles venoms presented cross-reactivity when species specific antisera were used (Toxicon 32, 113, 1994) and has similarity in the N-terminal sequence of dermonecrotic components from different species (J. Protein. Chem. 15, 337, 1996). Despite of these differences of toxicity between L. gaucho, L. laeta and L. intermedia venoms were also detected (Braz. J. Med. Biol. Res. 29, 1491-1497, 1996). The isolation and characterization of other proteins present in the venoms are in progress to investigate this question. Acknowledgements: Supported by FAPESP (97/14187-0, 97/09681-6).
Presence and biological role of oligosaccharides in Loxosceles intermedia (brown spider) venom proteins. J.L. Dreyfuss a, A.M. Pereira a, V.L.P. Santos a, S.S. Veiga a, O.C. Mangili b, H.B. Nader c, R.R. Brentani d, W. Gremski a (aDepartment of Cell Biology, Federal University of Parana, Curitiba, PR, Brazil; bDepartment of Physiology, Federal University of Parana, Curitiba, PR, Brazil; °Department of Biochemistry, Federal University of Silo Paulo, SP, Brazil; dLudwig Institute for Cancer Research, SP, Brazil).
Abstracts/Toxicon 38 (2000) 48~595
Characterization of the dermonecrotic component (35 kDa) of Loxosceles gaucho spider venom using monoclonal antibodies. P. Guilherme , D. Muramatsu, I. Fernandes, K.C. Barbaro (Laborat6rio de Imunopatologia, ]nstituto Butantan, Av. Vital Brazil, 1500, 05503-900, S~o Paulo, SP, Brazil). Loxoscelism has been recognized as a health problem in Brazil since 1957 and nowdays it is considered the most important form of araneism in the country. L. gaucho, L. laeta and L. intermedia are the main species found in the south of Brazil. The most common finding after the spider's bite is a dermonecrotic lesion. A definite therapy for the pathology caused by the Loxosceles venom has not been established yet. Toxins with molecular mass of 32-35 kDa are the principal components responsible for the dermonecrotic activity of the venom (J. Toxicol.Toxins Reviews 14, 40l, 1995). Objective: The aim of this study was to characterize the component responsible for the dermonecrotic activity of the L. gaucho venom (involved in the large majority of accidents in the state of S~o Paulo) using monoclonal antibodies (MoAb) against 35 kDa protein of this venom. Methods and results: To generate MoAbs to the 35 kDa protein, popliteal lymph node cells from Balb/c mice immunized with L. gaucho venom were fused with SP2-O cells. Supernatants from the hibridomas were screened by ELISA using plates coated with the 35 kDa protein or whole L. gaucho venom. ELISA's titers of the MoAbs (purified from ascitic fluid by affinity chromatography Protein A-Sepharose) were similar using both whole venom or 35 kDa purified component. By immunoblotting it was observed that the 4 MoAbs of IgG1 isotype, named MoALgl to Lg4, were able to recognize mainly the 35 kDa protein of L. gaucho venom, but detected weakly the 35 kDa protein of L. intermedia and failed to detect any component of L. laeta venom. On the other side, MoALg5 (IgM isotype) recognize all components of L. gaucho, L. laeta and L. intermedia venoms. Only MoALgl was able to neutralize the dermonecrotic activity of the L. gaucho venom. All MoAbs obtained failed in neutralize the dermonecrotic activity of the heterologous venoms. Conclusion: Loxosceles venoms presented crossreactivity when species-specific antisera were used (Toxicon 32, 113, 1994). Besides, they have similarity in the N-terminal sequence of their dermonecrotic components (J. Protein. Chem. 15, 337, 1996). In despite of these, MoALgl neutralized only the dermonecrotic activity of the homologous venom but not the one of L. intermedia venom although it was able to recognize it by immunoblotting. These results suggest that different epitopes are present in the protein responsible for the dermonecrotic activity of Loxosceles venoms. Furthermore, since this MoAb neutralized the dermonecrotic activity, it probably recognizes the active "toxic" site of the 35 kDa protein of L. gaucho venom. This observation may explain the differences of toxicity between L. gaucho, L. laeta and L. intermedia venoms. Further studies are in progress to investigate this question. Acknowledgements: Supported by FAPESP (97/14188-7, 97/09681-6).
Isolation and characterization of the 35 kDa protein oj Loxosceles gaucho spider.
Abstracts/Toxzcon 38 (2000) 487--595
535
D. Muramatsu, P. Guilherme, K.C. Barbaro (Laborat6rio de Imunopatologia, Instituto Butantan, Av. Vital Brazil, 1500, 05503-900, Silo Paulo, SP, Brazil), Spiders of Loxosceles genus are represented by more than thirty species in South America. In Brazil there are seven species, three of them, L. gaucho, L. laeta and L. intermedia are usually found in south and south-east regions. Envenomations by Loxosceles spiders are an important medical problem characterized by dermonecrotic lesion in the bitten site and occasionally by systemic effects that may be lethal. Previous results showed that the dermonecrotic and lethal activities of the L. gaucho venom are due to a fraction of the venom that contains mainly components with 35 kDa. (J. Protein. Chem. 15, 337, 1996). Objective: The aim of this project was to study some properties of L. gaucho venom (responsible for the large majority of accidents in the state of Silo Paulo), attempting to characterize the components responsible for its dermonecrotic activity. Methods and results: Loxosceles gaucho venom proteins were fractionated by cation exchange chromatography (Mono S-HR 5/5) using a FPLC system. Proteins bound to the column were eluted with a linear gradient of NaC1 (0-1 M) in 50 mM acetate buffer, pH 5.0. Fractionation of the dialised venom resulted in 14 peaks. Analysis by SDS-PAGE showed that the major protein of the L. gaucho venom has approximately 35 kDa. Isoeletric focusing of the L. gaucho venom showed acid and basic proteins. Two major basic bands were detected with pI of 8.2 and 7.1 and three major acid bands with pI of 4.5; 3.9 and 3.7. Dermonecrotic activity was assayed by i.d. injections, into a shaved area of the rabbit dorsum of 0.2 ml containing 3 ~tg of venom or its fractions (0.5 pg) in 0.15 M NaC1. The dermonecrotic activity was presented mainly in fraction 7, that contained the component of 35 kDa and pI of 8.2. Conclusion: The major protein in the L. gaucho venom has approximately 35 kDa. It is a basic protein with pI of 8.2 and it is the main component responsible for the dermonecrotic activity. This is in agreement with previous results obtained with L. reclusa spider venom (Am. J. Med. Sci., 304, 261-267, 1992). It was observed that Loxosceles venoms presented cross-reactivity when species specific antisera were used (Toxicon 32, 113, 1994) and has similarity in the N-terminal sequence of dermonecrotic components from different species (J. Protein. Chem. 15, 337, 1996). Despite of these differences of toxicity between L. gaucho, L. laeta and L. intermedia venoms were also detected (Braz. J. Med. Biol. Res. 29, 1491-1497, 1996). The isolation and characterization of other proteins present in the venoms are in progress to investigate this question. Acknowledgements: Supported by FAPESP (97/14187-0, 97/09681-6).
Presence and biological role of oligosaccharides in Loxosceles intermedia (brown spider) venom proteins. J.L. Dreyfuss a, A.M. Pereira a, V.L.P. Santos a, S.S. Veiga a, O.C. Mangili b, H.B. Nader c, R.R. Brentani d, W. Gremski a (aDepartment of Cell Biology, Federal University of Parana, Curitiba, PR, Brazil; bDepartment of Physiology, Federal University of Parana, Curitiba, PR, Brazil; °Department of Biochemistry, Federal University of Silo Paulo, SP, Brazil; dLudwig Institute for Cancer Research, SP, Brazil).