Two novel dermonecrotic toxins LiRecDT4 and LiRecDT5 from brown spider (Loxosceles intermedia) venom: from cloning to functional characterization.

Loxoscelism (the condition produced by the bite of brown spiders) has been reported worldwide, but especially in warmer regions. Clinical manifestations include skin necrosis with gravitational spreading while systemic loxoscelism may include renal failure, hemolysis and thrombocytopenia. The venom contains several toxins, of which the best biochemically and biologically studied is the dermonecrotic toxin, a phospholipase-D. Purified toxin induces cutaneous and systemic loxoscelism, especially necrotic lesions, hematological disturbances and renal failure. Herein, we describe cloning, heterologous expression and purification of two novel dermonecrotic toxins: LiRecDT4 and LiRecDT5. The recombinant proteins stably expressed in Escherichia coli cells were purified from culture supernatants in a single step using Ni(2+)-chelating chromatography producing soluble proteins of 34 kDa (LiRecDT4) and 37 kDa (LiRecDT5). Circular dichroism analysis evidenced correctly folding for toxins but differences in secondary structures. Both proteins were recognized by whole venom serum antibodies and by a specific antibody to dermonecrotic toxin. Also, recombinant toxins with phospholipase activity induced experimental skin lesions and caused a massive inflammatory response in rabbit skin dermis. Nevertheless, toxins displayed different effects upon platelet aggregation, increase in vascular permeability and not caused death in mice. These characteristics in combination with functional studies illustrates that a family of dermonecrotic toxins exists, and includes two novel members that are useful for future structural and functional studies. They will also be useful in biotechnological ends, for example, as inflammatory and platelet aggregating studies, as antigens for serum therapy source and for lipids biochemical research.

Hyaluronidases in Loxosceles intermedia (Brown spider) venom are endo-beta-N-acetyl-d-hexosaminidases hydrolases.

In studying Loxosceles venom, we detected degradation of purified hyaluronic acid (HA) and hydrolysis of purified chondroitin sulphate (CS) while neither dermatan sulphate, heparin or heparan sulphate were affected. In addition, with HA-degrading kinetic assays, we show that a hydrolase enzyme was involved in the HA cleavage. By use of the Reissig colorimetric reaction, we found that venom hyaluronidase is an endo-beta-N-acetyl-d-hexosaminidase that generates terminal N-acetylglucosamine residues upon cleavage of HA. Zymogram analysis of L. intermedia venom showed HA lytic activities at 41 and 43kDa, and, when CS was used as a substrate, zymograph experiments resulted in 41 and 43kDa lytic zones. Thus, these results support the hypothesis that the same molecules are involved in cleaving HA and CS residues. Experiments to compare L. intermedia electrostimulated venom and venom gland extract also demonstrated very similar HA lytic activity, suggesting again that hyaluronidases are self-components of Loxosceles spider venom instead of oral egesta contamination. HA degradation as a function of pH in these hydrolase enzymes showed no apparent activities at low or high pH, with optimal activity at 6.0-8.0 pH. Finally, we confirmed the cleaving action of the venom hyaluronidases on HA in the extracellular matrix of the dermis of rabbit by fluorescence reaction to HA and confocal microscope analysis. Thus, hyaluronidases type hydrolases endo-beta-N-acetyl-d-hexosaminidase are implicated as self-components of Loxosceles spider venom and can be involved in venom effects as spreading factors.

The effect of brown spider venom on endothelial cell morphology and adhesive structures.

Spiders of the Loxosceles genus have been responsible for severe clinical cases of envenomation worldwide. Accidents involving brown spiders can cause dermonecrotic injury, hemorrhage, hemolysis, platelet aggregation and renal failure. Histological findings of animals treated by venom have shown subendothelial blebs, vacuoles and endothelial cell membrane degeneration of blood vessel walls, as well as fibrin and thrombus formation. The mechanisms by which the venom causes these disorders are poorly understood. In this work, with an endothelial cell line derived from rabbit aorta, we were able to demonstrate that venom binds to the cell surface and the extracellular matrix. Moreover, we observed that the venom also induced morphological alterations, such as cell retraction, homophilic disadhesion and an increasing in filopodia projections. We also demonstrated that toxins present in the venom disorganized focal adhesion points and actin microfilaments of endothelial cells. Nevertheless, endothelial cell viability showed no alterations compared to controls. Additionally, venom treatment changed the fibronectin matrix profile synthesized by these cells as well as cell adhesion to fibronectin. These results suggest that the deleterious effects of venom on blood vessel walls could be a consequence of the direct effect on the endothelial cell surface and adhesive structures involved in blood vessel stability. These effects indirectly lead to leukocyte and platelet activation, disseminated intravascular coagulation and an increase in vessel permeability.

Molecular cloning and functional characterization of two isoforms of dermonecrotic toxin from Loxosceles intermedia (brown spider) venom gland.

Brown spider (Genus Loxosceles) bites are normally associated with necrotic skin degeneration, gravitational spreading, massive inflammatory response at injured region, platelet aggregation causing thrombocytopenia and renal disturbances. Brown spider venom has a complex composition containing many different toxins, of which a well-studied component is the dermonecrotic toxin. This toxin alone may produce necrotic lesions, inflammatory response and platelet aggregation. Biochemically, dermonecrotic toxin belongs to a family of toxins with 30-35 kDa characterized as sphingomyelinase-D. Here, employing a cDNA library of Loxosceles intermedia venom gland, we cloned and expressed two recombinant isoforms of the dermonecrotic toxin LiRecDT2 (1062 bp cDNA) and LiRecDT3 (1007 bp cDNA) that encode for signal peptides and complete mature proteins. Phylogenetic tree analysis revealed a structural relationship for these toxins compared to other members of family. Recombinant molecules were expressed as N-terminal His-tag fusion proteins in Escherichia coli and were purified to homogeneity from cell lysates by Ni(2+) chelating chromatography, resulting in proteins of 33.8 kDa for LiRecDT2 and 34.0 kDa for LiRecDT3. Additional evidence for related toxins containing sequence/epitopes identity comes from antigenic cross-reactivity using antibodies against crude venom toxins and antibodies raised with a purified dermonecrotic toxin. Recombinant toxins showed differential functionality in rabbits: LiRecDT2 caused a macroscopic lesion with gravitational spreading upon intradermal injection, while LiRecDT3 evoked transient swelling and erythema upon injection site. Light microscopic analysis of skin biopsies revealed edema, a collection of inflammatory cells in and around blood vessels and a proteinaceous network at the dermis. Moreover, differential functionality for recombinant toxins was also demonstrated by a high sphingomyelinase activity for LiRecDT2 and low activity for LiRecDT3 as well as greater in vitro platelet aggregation and blood vessel permeability induced by LiRecDT2 and residual activity for LiRecDT3. Cloning and expression of two recombinant dermonecrotic toxins demonstrate an intraspecific family of homologous toxins that act in synergism for deleterious activities of the venom and open possibilities for biotechnological applications for recombinant toxins as research tools for understanding the inflammatory response, vascular integrity and platelet aggregation modulators.

Brown spider dermonecrotic toxin directly induces nephrotoxicity.

Brown spider (Loxosceles genus) venom can induce dermonecrotic lesions at the bite site and systemic manifestations including fever, vomiting, convulsions, disseminated intravascular coagulation, hemolytic anemia and acute renal failure. The venom is composed of a mixture of proteins with several molecules biochemically and biologically well characterized. The mechanism by which the venom induces renal damage is unknown. By using mice exposed to Loxosceles intermedia recombinant dermonecrotic toxin (LiRecDT), we showed direct induction of renal injuries. Microscopic analysis of renal biopsies from dermonecrotic toxin-treated mice showed histological alterations including glomerular edema and tubular necrosis. Hyalinization of tubules with deposition of proteinaceous material in the tubule lumen, tubule epithelial cell vacuoles, tubular edema and epithelial cell lysis was also observed. Leukocytic infiltration was neither observed in the glomerulus nor the tubules. Renal vessels showed no sign of inflammatory response. Additionally, biochemical analyses showed such toxin-induced changes in renal function as urine alkalinization, hematuria and azotemia with elevation of blood urea nitrogen levels. Immunofluorescence with dermonecrotic toxin antibodies and confocal microscopy analysis showed deposition and direct binding of this toxin to renal intrinsic structures. By immunoblotting with a hyperimmune dermonecrotic toxin antiserum on renal lysates from toxin-treated mice, we detected a positive signal at the region of 33-35 kDa, which strengthens the idea that renal failure is directly induced by dermonecrotic toxin. Immunofluorescence reaction with dermonecrotic toxin antibodies revealed deposition and binding of this toxin directly in MDCK epithelial cells in culture. Similarly, dermonecrotic toxin treatment caused morphological alterations of MDCK cells including cytoplasmic vacuoles, blebs, evoked impaired spreading and detached cells from each other and from culture substratum. In addition, dermonecrotic toxin treatment of MDCK cells changed their viability evaluated by XTT and Neutral-Red Uptake methodologies. The present results point to brown spider dermonecrotic toxin cytotoxicity upon renal structures in vivo and renal cells in vitro and provide experimental evidence that this brown spider toxin is directly involved in nephrotoxicity evoked during Loxosceles spider venom accidents.

Concentration of hyaluronic acid in primary open-angle glaucoma aqueous humor.

We compared the concentration of hyaluronic acid in the aqueous humor of primary open-angle glaucoma (POAG) patients and non-glaucomatous patients. Aqueous humor samples were obtained from 22 patients just before trabeculectomy for clinically uncontrolled POAG (POAG group). Aqueous humor (0.1 mL) was aspirated by inserting a 26-gauge needle into the anterior chamber. The same procedure was performed for 22 non-glaucomatous patients just before cataract surgery (control group). Immediately after collection, the aqueous humor was stored at -20 degrees C. The concentration of hyaluronic acid was determined by a sensitive, noncompetitive and nonisotopic fluoroassay. The median (range) concentrations of hyaluronic acid of the POAG and control groups were 298.4 microg L(-1) (99.0-743.7 microg L(-1)) and 545.1 microg L(-1) (145.0-2366.0 microg L(-1)), respectively. The difference in concentrations of hyaluronic acid between the groups was statistically significant (P<0.001). In conclusion, the concentration of hyaluronic acid in the aqueous humor in POAG patients is lower than in non-glaucomatous patients. Further studies are necessary to determine the role of hyaluronic acid in the pathophysiology of POAG.

Glycosaminoglycans affect the action of human plasma kallikrein on kininogen hydrolysis and inflammation.

Human plasma kallikrein (huPK) is a serine proteinase involved in many biological processes including those of the kallikrein-kinin system. The action of huPK on kininogen results in bradykinin (BK) release, a potent mediator of inflammatory responses. BK generation may be influenced by several agents, and the aim of this work was to investigate the effect of glycosaminoglycans (GAGs) on human high-molecular-weight kininogen (HK) hydrolysis by huPK and on inflammation. huPK was pre-incubated in the absence and presence of different GAGs, followed by the addition of kininogen. Bradykinin released at different times was measured by radioimmunoassay, and KM and kcat were calculated. Tuna and bovine dermatan sulfates, the most potent GAGs studied, reduced by 80% and 68%, respectively, the catalytic efficiency of huPK (control = 4. x 10(4) M(-1) s(-1) in BK release. The effect of bovine dermatan sulfate (BDS) on inflammatory response was studied in rat paw edema induced by carrageenin and hourly determined (1-4 h) by plethysmography. BDS significantly reduced the inflammatory response in the first and second hours of measurements (24% and 28%, respectively), p < 0.05. GAGs were shown to reduce bradykinin release "in vitro" and in an inflammation model. This reduction may play a role in the control or maintenance of some pathological and physiological processes.