Characterization of the gene encoding component C3 of the complement system from the spider Loxosceles laeta venom glands: Phylogenetic implications.

A transcriptome analysis of the venom glands of the spider Loxosceles laeta, performed by our group, in a previous study (Fernandes-Pedrosa et al., 2008), revealed a transcript with a sequence similar to the human complement component C3. Here we present the analysis of this transcript. cDNA fragments encoding the C3 homologue (Lox-C3) were amplified from total RNA isolated from the venom glands of L. laeta by RACE-PCR. Lox-C3 is a 5178 bps cDNA sequence encoding a 190kDa protein, with a domain configuration similar to human C3. Multiple alignments of C3-like proteins revealed two processing sites, suggesting that Lox-C3 is composed of three chains. Furthermore, the amino acids consensus sequences for the thioester was found, in addition to putative sequences responsible for FB binding. The phylogenetic analysis showed that Lox-C3 belongs to the same group as two C3 isoforms from the spider Hasarius adansoni (Family Salcitidae), showing 53% homology with these. This is the first characterization of a Loxosceles cDNA sequence encoding a human C3 homologue, and this finding, together with our previous finding of the expression of a FB-like molecule, suggests that this spider species also has a complement system. This work will help to improve our understanding of the innate immune system in these spiders and the ancestral structure of C3.

Characterization of anti-crotalic antibodies.

Crotalus durissus terrificus, C. d. collilineatus, C. d. cascavella and C. d. marajoensis are responsible minor but severe snake bites in Brazil. The venoms of these snakes share the presence of crotoxin, a neurotoxin comprising of two associated components, crotapotin and phospholipase A2 (PLA2). Treatment of the victims with specific antiserum is the unique effective therapeutic measure. The ability of anti-Crotalus antisera produced by the routine using crude venom to immunize horses or purified crotoxin and PLA2 as individual immunogens was compared. Antisera obtained from horses immunized with C. durissus terrificus crude venom were able to recognize and neutralize not only the toxins presents in C. durissus terrificus, but also the ones present in the venoms from C. d. collilineatus, C. d. cascavella and C. d. marajoensis. Antisera from horses immunized with individual crotoxin or PLA2, although in lesser titers, were also able of recognizing the toxins in all four Crotalus species and neutralize the lethality of the C. d. terrificus venom.

A new anti-loxoscelic serum produced against recombinant sphingomyelinase D Results of preclinical trials

Envenomation by Loxosceles species (brown spider) can lead to local dermonecrosis and to serious systemic effects. The main toxic component in the venom of these spiders is sphingomyelinase D (SMase D) and various isoforms of this toxin are present in Loxosceles venoms. We have produced a new anti-loxoscelic serum by immunizing horses with recombinant SMase D. In the present study, we compared the neutralization efficacy of the new anti-loxoscelic serum and anti-arachnidic serum (the latter serum is used for therapy for loxoscelism in Brazil) against the toxic effects of venoms from spiders of the genus Loxosceles. Neutralization tests showed that anti-SMase D serum has a higher activity against toxic effects of L. intermedia and L. laeta venoms and similar or slightly weaker activity against toxic effects of L. gaucho than that of Arachnidic serum. These results demonstrate that recombinant SMase D can replace venom for anti-venom production and therapy.

Transcriptome analysis of Loxosceles laeta (Araneae, Sicariidae) spider venomous gland using expressed sequence tags

The bite of spiders belonging to the genus Loxosceles can induce a variety of clinical symptoms, including dermonecrosis, thrombosis, vascular leakage, haemolysis, and persistent inflammation. In order to examine the transcripts expressed in venom gland of Loxosceles laeta spider and to unveil the potential of its products on cellular structure and functional aspects, we generated 3,008 expressed sequence tags (ESTs) from a cDNA library. Results: All ESTs were clustered into 1,357 clusters, of which 16.4% of the total ESTs belong to recognized toxin-coding sequences, being the Sphingomyelinases D the most abundant transcript; 14.5% include "possible toxins", whose transcripts correspond to metalloproteinases, serinoproteinases, hyaluronidases, lipases, C-lectins, cystein peptidases and inhibitors. Thirty three percent of the ESTs are similar to cellular transcripts, being the major part represented by molecules involved in gene and protein expression, reflecting the specialization of this tissue for protein synthesis. In addition, a considerable number of sequences, 25%, has no significant similarity to any known sequence. Conclusion: This study provides a first global view of the gene expression scenario of the venom gland of L. laeta described so far, indicating the molecular bases of its venom composition.

Loxosceles spider venom induces the release of thrombomodulin and endothelial protein C receptor: implications for the pathogenesis of intravascular coagulation as observed in loxoscelism.

BACKGROUND: The venom of the spider Loxosceles can cause both local and systemic effects including disseminated intravascular coagulation. AIM: The aim of this study was to investigate the effects of the venom of Loxosceles intermedia (L. intermedia) and the purified Sphingomyelinase D (SMaseD) toxin upon the Protein C (PC) natural anticoagulant pathway. RESULTS: Both the venom and e purified SMaseD reduced the cell surface expression of thrombomodulin (TM) and Endothelial PC Receptor on endothelial cells in culture. The reduction of cell surface expression was caused by cleavage from the cell surface mediated by activation of an endogenous metalloproteinase. Reduction of TM and Endothelial PC Receptor on the surface of these cells resulted in an impaired ability of the cells to assist in the thrombin-induced activation of PC. CONCLUSION: This novel observation gives further insight into the mechanisms of the pathology induced by venom from Loxosceles spiders and may aid the development of a suitable therapy.

Sphingomyelinases D induce direct association of C1q to the erythrocyte membrane causing complement mediated autologous haemolysis.

Bites by Loxosceles spiders can induce severe clinical symptoms, including dermonecrosis, thrombosis, vascular leakage, haemolysis and persistent inflammation. The causative toxin is a sphingomyelinase D (SMase D) that cleaves sphingomyelin into choline and ceramide-1-phosphate. A similar enzyme, showing comparable bioactivity, is secreted by certain pathogenic corynebacteria and acts as a potent virulence factor. We have previously found that SMase D toxins led to an increased susceptibility of human erythrocytes (E) to activation of complement (C) via the classical pathway (CP) in the absence of antibodies. In the present study we have investigated the CP initiating components involved in the haemolysis induced by SMases from Corynebacterium pseudotuberculosis (PLD) and from Loxosceles intermedia venom (P1). When P1 or PLD treated E were incubated with C8-depleted human serum, an increase in C1q, serum amyloid protein (SAP) and C-reactive protein (CRP) binding was observed. While purified C1q, SAP and CRP were found to bind to P1 or PLD treated E, depletion of SAP or CRP from human serum did not prevent C-mediated lysis, suggesting that pentraxins are not involved in the initiation of C-activation. However depletion of C1 lead to a greatly reduced haemolysis, demonstrating that the activation of the CP is caused by direct binding of C1q to the SMase treated cells. Binding of fluid phase C-regulators C4b-binding protein and factor H was also observed, however these C-regulators in conjunction with the membrane bound C-regulators were unable to prevent haemolysis, demonstrating the potency of SMase D facilitated binding of C1 and activation of C.

Genetic selection for resistance or susceptibility to oral tolerance imparts correlation to both Immunoglobulin E level and mast cell number phenotypes with a profound impact on the atopic potential of the individual.

BACKGROUND: Immunological oral tolerance is being studied with great interest due to its therapeutic potential in allergy and autoimmunity processes, although the cellular and molecular mechanisms linking these different phenomena remain elusive. In the present study, two mouse lines with extreme phenotypes for susceptibility [TS Line] or resistance [TR Line] to oral tolerance and their [TS x TR]F2 segregants were used in order to evaluate the impact of these traits on the atopic potential of the individuals. OBJECTIVE: Demonstrate whether the tr and ts genes, cumulated during 18 generations of bidirectional genetic selection, influence expression of two important immunobiological traits (IgE and mast cell) critical to allergic response. METHODS: Mice with extreme phenotypes for oral tolerance to ovalbumin (OVA), produced by assortative mating (TS and TR Line), and their (TS x TR)F2 segregating were used. Serum IgE levels assayed by ELISA, and mastocytes counted with toluidine blue staining were evaluated in naïve mice. Anaphylaxis was induced by intravenous injection of OVA, intestinal inflammation by oral administration of OVA 7 days after immunization, and pulmonary inflammation by intranasal and nebulization OVA challenges. Specific IgE was dosed by passive cutaneous anaphylaxis. RESULTS: The naïve TS mice have a 20-fold lower serum IgE level and two- to threefold diminished mast cell numbers in mucosal sites, when compared with TR-mice, which were highly susceptible to allergic inflammation and anaphylactic shock. The associations of oral tolerance, serum IgE levels and mast cell numbers in naïve animals were confirmed analysing the simultaneous presence of these traits in individuals of a [TS x TR]F2 -segregating population. CONCLUSION: The results suggest that the complex of genes controlling TS and TR phenotypes play a main role in the regulation of the atopic potential of the individual. The studies of these traits in interline F2 segregants demonstrated a co-segregation of TS and TR phenotypes with IgE responsiveness and mast cell numbers. Thus, the opposite capacity of the genetically modified mice may be involved in co-adaptative mechanisms reflecting a dynamic relation between gene frequencies in a natural population. These correlations give circumstantial evidence to support clinical applications of oral tolerance in allergic and autoimmune diseases.

Ordered mesoporous silica SBA-15 A new effective adjuvant to induce antibody response

The applicability of SBA-15 mesostructure as an adjuvant and evaluation of its efficiency to induce antibody response, was discussed. It was observed that better encapsulation of biomolecules of variable shape and size can be achieved using a antigen to SBA-15 weight ratio of 1: 2.5. Efficient antibody generation could be achieved because SBA-15 was able to attract antigens effectively due to its high surface area and proper mesopore size. The results show that SBA-15 and related silica mesostructures are promising nanosystems for vaccine delivery.

The improvement of the therapeutic anti-Lachesis muta serum production in horses.

The main features associated with pit viper envenomations include the intense local lesions such as oedema, necrosis, acute renal failure and other effects. The severity of these reactions to snakebite depends on the degree of envenomation. Lachesis muta venom (LMV) has weak lethal activity, but due to the large amount often inoculated, the effects are extremely severe and demand anti-venom with a high neutralizing capacity. LMV had the lowest neutralizing antibody induction capacity in horses when compared with that of other venoms. For example, Bothrops anti-venom serum neutralizes 180 times the equivalent LD(50) to Bothrops venom; Crotalus anti-venom neutralizes 250 LD(50) of this venom, while Lachesis anti-venom neutralizes only five LD(50) of the Lachesis toxins. To examine the reasons for this low antibody induction, the H(GP) mouse line, genetically selected for high antibody production received, at different times during immunization with sheep erythrocytes (SE), whole LMV and isolated venom fractions I-VI eluted by gel-filtration chromatography on Superdex75. The specific antibody responsiveness showed a partial, but significant suppression of the anti-SE antibody responses during the kinetics of the primary and even the secondary immunizations, after 50-100 microg of fractions IV and V administration 72-48 h before the first antigen injections. Fraction IV was then applied in a Superose 12 column and three samples were obtained. The peak IVA containing a component of Mr 27 kDa was liable with the immunosuppressive effect as made evident by its effect on the H mice anti-SE responses. Horses receiving the LMV exempt of fractions IV and V produce highly significant anti-Lachesis sera with a 45 LD(50) neutralizing activity, providing, for the first time, an efficient specific therapeutic heterologous serum for human use.

Crystallization and preliminary crystallographic analysis of SMase I, a sphingomyelinase from Loxosceles laeta spider venom.

SMase I, a 32 kDa sphingomyelinase found in Loxosceles laeta venom, is responsible for the major pathological effects of spider envenomation. This toxin has been cloned and functionally expressed as a fusion protein containing a 6 x His tag at its N-terminus to yield a 33 kDa protein [Fernandes-Pedrosa et al. (2002), Biochem. Biophys. Res. Commun. 298, 638-645]. The recombinant protein possesses all the biological properties ascribed to the whole L. laeta venom, including dermonecrotic and complement-dependent haemolytic activities. Dynamic light-scattering experiments conducted at 291 K demonstrate that the sample possesses a monomodal distribution, with a hydrodynamic radius of 3.57 nm. L. laeta SMase I was crystallized by the hanging-drop vapour-diffusion technique using the sparse-matrix method. Single crystals were obtained using a buffer solution consisting of 0.08 M HEPES and 0.9 M trisodium citrate, which was titrated to pH 7.5 using 0.25 M sodium hydroxide. Complete three-dimensional diffraction data were collected to 1.8 angstroms at the Laboratório Nacional de Luz Síncrotron (LNLS, Campinas, Brazil). The crystals belong to the hexagonal system (space group P6(1) or P6(5)), with unit-cell parameters a = b = 140.6, c = 113.6 angstroms. A search for heavy-atom derivatives has been initiated and elucidation of the crystal structure is currently in progress.

Mechanism of induction of complement susceptibility of erythrocytes by spider and bacterial sphingomyelinases

We have recently shown that the sphingomyelinase toxins P1 and P2 from the venom of the spider Loxosceles intermedia induce complement (C)-dependent lysis of autologous erythrocytes by induction of the cleavage of cell surface glycophorins through activation of an endogenous metalloproteinase facilitating the activation of the alternative pathway of C. Phospholipase D (PLD) from Corynebacterium pseudotuberculosis shows some degree of homology with the spider sphingomyelinases and can induce similar clinical symptoms to those observed after spider envenomation. The aim of this study was to investigate if the bacterial PLD-induced haemolysis of human erythrocytes was C dependent and if cleavage of glycophorins occurred. We show here that haemolysis of both PLD- and P1-treated human erythrocytes was C dependent, but while PLD-mediated haemolysis was dependent on activation of the classical pathway of C, P1 induced lysis via both the classical and alternative pathways. P1, but not PLD, induced cleavage of glycophorins and no change in expression of complement regulators was induced by either of the toxins. In both cases, annexin V binding sites were exposed, suggesting that the membrane asymmetry had been disturbed causing exposure of phosphatidylserine to the cell surface. Our results suggest that C susceptibility induced by L. intermedia and C. pseudotuberculosis PLD is a result of exposure of phosphatidylserine, and the higher potency of P1 toxin can be explained by its additional effect of cleavage of glycophorins.

[Presence of Loxosceles similis Moenkhaus, 1898 (Araneae, Sicariidae) in Serra da Bodoquena, State of Mato Grosso do Sul, Brazil]. / Presença de Loxosceles similis Moenkhaus, 1898 (Araneae, Sicariidae) na Serra da Bodoquena, Estado de Mato Grosso do Sul, Brasil.

The venom of Loxosceles spiders causes dermonecrotic lesion and induces complement-dependent intravascular haemolysis that characterizes a severe systemic effect. In Brazil, L. gaucho, L. intermedia and L. laeta, present in the anthropic environment, have been pointed out as the most important agents of the loxoscelism. Besides these species there are others that, by predominating in the natural environment, have not been evaluated regarding human health risk, as in the case of Loxosceles similis. The development of a research project in Bodoquena Range, for ecological observation and identification of insects of medical interest, enabled the capture of Loxosceles similis specimens in the "Pitangueiras" cave and "Lago Azul" cave, in Bodoquena Range, municipality of Bonito, State of Mato Grosso do Sul, Brazil. The objectives of this study were to define the parameters for identification, environmental features of the habitat of this species, as well as an update of its geographical distribution.

Presença de Loxosceles similis Moenkhaus, 1898 (Araneae, Sicariidae) na Serra da Bodoquena, Estado de Mato Grosso do Sul, Brasil / Presence of Loxosceles similis Moenkhaus, 1898 (Araneae, Sicariidae) in Serra da Bodoquena, State of Mato Grosso do Sul, Brazil

The venom of Loxosceles spiders causes dermonecrotic lesion and induces complement-dependent intravascular haemolysis that characterizes a severe systemic effect. In Brazil, L. gaucho, L. intermedia and L. laeta, present in the anthropic environment, have been pointed out as the most important agents of the loxoscelism. Besides these species there are others that, by predominating in the natural environment, have not been evaluated regarding human health risk, as in the case of Loxosceles similis. The development of a research project in Bodoquena Range, for ecological observation and identification of insects of medical interest, enabled the capture of Loxosceles similis specimens in the [quot ]Pitangueiras[quot ] cave and [quot ]Lago Azul[quot ] cave, in Bodoquena Range, municipality of Bonito, State of Mato Grosso do Sul, Brazil. The objectives of this study were to define the parameters for identification, environmental features of the habitat of this species, as well as an update of its geographical distribution.

O veneno das aranhas do gênero Loxosceles causa lesão dermonecrótica e induz hemólise intravascular dependente de complemento, configurando um quadro clínico de intensa gravidade. No Brasil, as espécies L. gaucho L. intermedia e L. laeta, presentes no ambiente antrópico, têm sido apontadas como principais agentes do loxoscelismo. Além destas, existem outras espécies, que por predominarem no ambiente natural, não têm sido avaliadas quanto ao risco à saúde do homem, como é o caso de Loxosceles similis. O desenvolvimento de projeto de pesquisa, na Serra da Bodoquena, para observações ecológicas e identificação de insetos de interesse médico, possibilitou a captura de espécimens de Loxosceles similis na Serra da Bodoquena, Município de Bonito, Estado do Mato Grosso do Sul, Brasil, nas grutas Pitangueiras e do Lago Azul. Os parâmetros para identificação, características ambientais do habitat da espécie, bem como atualização de sua distribuição geográfica são objetos deste trabalho.

Molecular and immunochemical evidences demonstrate that endooligopeptidase A is the predominant cytosolic oligopeptidase of rabbit brain.

Oligopeptidases are tissue endopeptidases that do not attack proteins and are likely to be involved in the maturation and degradation of peptide hormones and neuropeptides. The rabbit brain endooligopeptidase A and the rat testes soluble metallopeptidase (EC 3.4.24.15) are thiol-activated oligopeptidases which are able to generate enkephalin from a number of opioid peptides and to inactivate bradykinin and neurotensin by hydrolyzing the same peptide bonds. A monospecific antibody raised against the purified rabbit brain endooligopeptidase A allowed the identification of a 2. 3 kb cDNA coding for a truncated enzyme of 512 amino acids, displaying the same enzymatic features as endooligopeptidase A. In spite of all efforts, employing several strategies, the full-length cDNA could not be cloned until now. The analysis of the deduced amino acid sequence showed no similarity to the rat testes metalloendopeptidase sequence, except for the presence of the typical metalloprotease consensus sequence [HEXXH]. The antibody raised against recombinant endooligopeptidase A specifically inhibited its own activity and reduced the thiol-activated oligopeptidase activity of rabbit brain cytosol to less than 30%. Analysis of the endooligopeptidase A tissue distribution indicated that this enzyme is mainly expressed in the CNS, whereas the soluble metallo EC 3.4.24.15 is mainly expressed in peripheral tissues.

Increments in serum cytokine and nitric oxide levels in mice injected with Bothrops asper and Bothrops jararaca snake venoms.

Changes in serum levels of several cytokines and nitric oxide were studied in BALB/c mice injected intraperitoneally with one median lethal dose (LD(50)) of the venoms of Bothrops asper and Bothrops jararaca, two of the medically most important poisonous snakes of Latin America. Despite differences observed in the time-course of cytokine increments and in serum cytokine levels, both venoms induced prominent elevations of TNF-alpha, IL-1, IL-6, IL-10 and IFN-gamma. There was an early increase in TNF-alpha and IL-1, followed by a more pronounced increment by 18 h. IL-6 levels peaked between 4 and 6 h, and this cytokine probably modulates the secretion of TNF-alpha and IL-1 and the synthesis of acute-phase proteins. Both venoms induced an early increment in serum IL-10, whereas IFN-gamma levels reached higher values in mice injected with B. jararaca venom than in those receiving B. asper venom. Serum nitric oxide concentration increased in mice injected with both venoms rapidly after envenomation, remaining elevated for 24 h. It is concluded that a complex pattern of cytokine and nitric oxide synthesis and secretion occurs in severe experimental envenomation by B. asper and B. jararaca venoms. Furthermore, it is suggested that some of these mediators, particularly TNF-alpha, IL-1 and nitric oxide, might play a relevant role in the pathophysiology of systemic alterations induced by these venoms.

Loxosceles intermedia spider envenomation induces activation of an endogenous metalloproteinase, resulting in cleavage of glycophorins from the erythrocyte surface and facilitating complement-mediated lysis.

Loxosceles is the most venomous spider in Brazil, and envenomation causes dermonecrosis and complement (C)-dependent intravascular hemolysis. The authors studied the mechanism of induction of C-induced hemolysis. Purified Loxosceles toxins rendered human erythrocytes susceptible to lysis by human C but did not have an effect on the E-bound C-regulators DAF, CR1, or CD59. However, incubation with venom toxins caused cleavage of glycophorin from the erythrocyte (E) surface, facilitating C activation and hemolysis. The results suggest that glycophorin is an important factor in the protection of E against homologous C. Cleavage of glycophorin (GP) A, GPB, and GPC occurred at sites close to the membrane but could not be accomplished using purified GPA and purified toxins, demonstrating that cleavage was not an effect of a direct proteolytic action of the Loxosceles toxins on the glycophorins. Inhibition of the cleavage of glycophorins induced by Loxosceles venom was achieved with 1,10-phenanthroline. The authors propose that the sphingomyelinase activity of the toxins induces activation of an endogenous metalloproteinase, which then cleaves glycophorins. They observed the transfer of C-dependent hemolysis to other cells, suggesting that the Loxosceles toxins can act on multiple cells. This observation can explain the extent of hemolysis observed in patients after envenomation. Identification of the mechanism of induction of susceptibility to C-mediated lysis after Loxosceles envenomation opens up the possibility of the development of an effective therapeutic strategy. (Blood. 2000;95:683-691)

Bothrops asper snake venom and its metalloproteinase BaP-1 activate the complement system. Role in leucocyte recruitment.

The venom of the snake Bothrops asper, the most important poisonous snake in Central America, evokes an inflammatory response, the mechanisms of which are not well characterized. The objectives of this study were to investigate whether B. asper venom and its purified toxins--phospholipases and metalloproteinase--activate the complement system and the contribution of the effect on leucocyte recruitment. In vitro chemotaxis assays were performed using Boyden's chamber model to investigate the ability of serum incubated with venom and its purified toxins to induce neutrophil migration. The complement consumption by the venom was evaluated using an in vitro haemolytic assay. The importance of complement activation by the venom on neutrophil migration was investigated in vivo by injecting the venom into the peritoneal cavity of C5-deficient mice. Data obtained demonstrated that serum incubated with crude venom and its purified metalloproteinase BaP-1 are able to induce rat neutrophil chemotaxis, probably mediated by agent(s) derived from the complement system. This hypothesis was corroborated by the capacity of the venom to activate this system in vitro. The involvement of C5a in neutrophil chemotaxis induced by venom-activated serum was demonstrated by abolishing migration when neutrophils were pre-incubated with antirat C5a receptor antibody. The relevance of the complement system in in vivo leucocyte mobilization was further demonstrated by the drastic decrease of this response in C5-deficient mice. Pre-incubation of serum with the soluble human recombinant complement receptor type 1 (sCR 1) did not prevent the response induced by the venom, but abolished the migration evoked by metalloproteinase-activated serum. These data show the role of the complement system in bothropic envenomation and the participation of metalloproteinase in the effect. Also, they suggest that the venom may contain other component(s) which can cause direct activation of C5a.

Ontogenetic development of Loxosceles intermedia spider venom.

Envenomation by Loxosceles spider has become a public health problem in the South region of Brazil, mainly due to high levels of domiciliary infestation by Loxosceles intermedia spiders. The toxic effects of L. intermedia venom are mostly associated with a 35 kDa protein (F35) which presents complement-dependent haemolytic and dermonecrotic activities. The aim of this study was to detect, through biological and immunochemical assays, the appearance of the main toxic component, F35, during the ontogenetic development of L. intermedia spiders. The toxin appeared in its fully active form in venom of third instar spiderlings; from then on its activity increased throughout development until adulthood. On the other hand, F35 was not detected in extracts of either eggs or spiderlings of the first and second instars.

Sex-linked variation of Loxosceles intermedia spider venoms.

In order to investigate intraspecific differences in Loxosceles intermedia spider venom we compared some biological properties of male and female venoms. Females produced higher amounts of venom than males. Furthermore, female venom presented more potent dermonecrotic and complement-dependent activities than male venom. Interestingly, the F35 toxin, a dermonecrotic and complement-dependent haemolytic factor, was also present in greater amounts in female venom, as demonstrated by ELISA. Therefore, the higher production and increased toxicity of venom in female specimens as compared to males may contribute to the variability observed in the severity of envenoming caused by L. intermedia spiders.

Sphingomyelinases in the venom of the spider Loxosceles intermedia are responsible for both dermonecrosis and complement-dependent hemolysis.

The bite of spiders of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and complement (C) dependent haemolysis. The aim of this study was to characterise the toxins in the venom responsible for the different biological effects. We have previously shown that a 35 kDa protein, named F35, purified from Loxosceles intermedia venom, incorporates into the membranes of human erythrocytes and renders them susceptible to the alternative pathway of autologous C. Here we have further purified the F35 protein which was resolved by reversed phase chromatography into three tightly contiguous peaks termed P1, P2, and P3. P1 and P2 were shown to be homogeneous by SDS-PAGE and N-terminal aminoacid analysis, while P3 consisted of two highly homologous proteins. N-terminal sequencing of all four proteins showed a high degree of homology, which was confirmed by cross-reactivity of antisera raised against the individual purified proteins. Functional characterisation of P1 and P2 indicated the presence of sphingomyelinase activity and either protein in isolation was capable of inducing all the in vivo effects seen with whole spider venom, including C-dependent haemolysis and dermonecrosis. In all assays, P2 was more active than P1, while P3 was completely inactive. These data show that different biological effects of L. intermedia venom can be assigned to the sphingomyelinase activity of two highly homologous proteins, P1 and P2. Identification of these proteins as inducers of the principal pathological effects induced by whole venom will aid studies of the mechanism of action of the venom and the development of a effective therapy.