Andrea Senff-Ribeiro

Recent advances in the understanding of brown spider venoms: From the biology of spiders to the molecular mechanisms of toxins.

The Loxosceles genus spiders (the brown spiders) are encountered in all the continents, and the clinical manifestations following spider bites include skin necrosis with gravitational lesion spreading and occasional systemic manifestations, such as intravascular hemolysis, thrombocytopenia and acute renal failure. Brown spider venoms are complex mixtures of toxins especially enriched in three molecular families: the phospholipases D, astacin-like metalloproteases and Inhibitor Cystine Knot (ICK) peptides. Other toxins with low level of expression also present in the venom include the serine proteases, serine protease inhibitors, hyaluronidases, allergen factors and translationally controlled tumor protein (TCTP). The mechanisms by which the Loxosceles venoms act and exert their noxious effects are not fully understood. Except for the brown spider venom phospholipase D, which causes dermonecrosis, hemolysis, thrombocytopenia and renal failure, the pathological activities of the other venom toxins remain unclear. The objective of the present review is to provide insights into the brown spider venoms and loxoscelism based on recent results. These insights include the biology of brown spiders, the clinical features of loxoscelism and the diagnosis and therapy of brown spider bites. Regarding the brown spider venom, this review includes a description of the novel toxins revealed by molecular biology and proteomics techniques, the data regarding three-dimensional toxin structures, and the mechanism of action of these molecules. Finally, the biotechnological applications of the venom components, especially for those toxins reported as recombinant molecules, and the challenges for future study are discussed.

Brown Spider (Loxosceles genus) Venom Toxins: Tools for Biological Purposes

Venomous animals use their venoms as tools for defense or predation. These venoms are complex mixtures, mainly enriched of proteic toxins or peptides with several, and different, biological activities. In general, spider venom is rich in biologically active molecules that are useful in experimental protocols for pharmacology, biochemistry, cell biology and immunology, as well as putative tools for biotechnology and industries. Spider venoms have recently garnered much attention from several research groups worldwide. Brown spider (Loxosceles genus) venom is enriched in low molecular mass proteins (5–40 kDa). Although their venom is produced in minute volumes (a few microliters), and contain only tens of micrograms of protein, the use of techniques based on molecular biology and proteomic analysis has afforded rational projects in the area and permitted the discovery and identification of a great number of novel toxins. The brown spider phospholipase-D family is undoubtedly the most investigated and characterized, although other important toxins, such as low molecular mass insecticidal peptides, metalloproteases and hyaluronidases have also been identified and featured in literature. The molecular pathways of the action of these toxins have been reported and brought new insights in the field of biotechnology. Herein, we shall see how recent reports describing discoveries in the area of brown spider venom have expanded biotechnological uses of molecules identified in these venoms, with special emphasis on the construction of a cDNA library for venom glands, transcriptome analysis, proteomic projects, recombinant expression of different proteic toxins, and finally structural descriptions based on crystallography of toxins.

Biotechnological applications of brown spider (Loxosceles genus) venom toxins

Loxoscelism (the term used to define accidents by the bite of brown spiders) has been reported worldwide. Clinical manifestations following brown spider bites are frequently associated with skin degeneration, a massive inflammatory response at the injured region, intravascular hemolysis, platelet aggregation causing thrombocytopenia and renal disturbances. The mechanisms by which the venom exerts its noxious effects are currently under investigation. The whole venom is a complex mixture of toxins enriched with low molecular mass proteins in the range of 5-40 kDa. Toxins including alkaline phosphatase, hyaluronidase, metalloproteases (astacin-like proteases), low molecular mass (5.6-7.9 kDa) insecticidal peptides and phospholipases-D (dermonecrotic toxins) have been identified in the venom. The purpose of the present review is to describe biotechnological applications of whole venom or some toxins, with especial emphasis upon molecular biology findings obtained in the last years.

Antimelanoma activity of 1,3,4-thiadiazolium mesoionics: a structure-activity relationship study.

The effect of a series of 4-phenyl-5-(2′-Y, 4′-X or 4′-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chlorides was evaluated against B16-F10 murine melanoma cells in vitro and against tumors resulting from implanted B16-F10 cells in C57BL/6 mice. These compounds differ from each other only at the cinnamoyl ring substituent (MI-J, X=OH; MI-2,4diF, X=Y=F; MI-4F, X=F and MI-D, X=NO2). The results were compared with those obtained for MI-D, which has already been shown to be a potent and promising drug against melanoma. On exposure of B16-F10 cells to MI-D, MI-2,4diF and MI-4F, all of them at the same micromolar concentration (50 μM) decreased the cell viability to 8, 50 and 22%, respectively, while MI-J did not show any significant effect under the same conditions. However, low doses such as 10 μM MI-D were sufficient to impair cell growth over 72 h, but for MI-2,4diF and MI-4F the effect on B16-F10 proliferation was only observed at a concentration of 25 μM. Furthermore, MI-4F had a slightly better effect than MI-2,4diF in vitro; its effect on tumor growth in vivo was not significant. MI-D inhibited tumor growth by 77%. The greater effectiveness of MI-D compared with MI-2,4diF, MI-4F and MI-J against B16-F10 melanoma cells is probably due to its stronger electron-withdrawing group (NO2), which increases the positive charge on the mesoionic ring and allows extensive conjugation of the side-chain with the exocyclic moiety. This seems to be important for degree of anti-tumor activity of these compounds.

Phospholipase-D activity and inflammatory response induced by brown spider dermonecrotic toxin: endothelial cell membrane phospholipids as targets for toxicity.

Brown spider dermonecrotic toxins (phospholipases-D) are the most well-characterized biochemical constituents of Loxosceles spp. venom. Recombinant forms are capable of reproducing most cutaneous and systemic manifestations such as dermonecrotic lesions, hematological disorders, and renal failure. There is currently no direct confirmation for a relationship between dermonecrosis and inflammation induced by dermonecrotic toxins and their enzymatic activity. We modified a toxin isoform by site-directed mutagenesis to determine if phospholipase-D activity is directly related to these biological effects. The mutated toxin contains an alanine substitution for a histidine residue at position 12 (in the conserved catalytic domain of Loxosceles intermedia Recombinant Dermonecrotic Toxin - LiRecDT1). LiRecDT1H12A sphingomyelinase activity was drastically reduced, despite the fact that circular dichroism analysis demonstrated similar spectra for both toxin isoforms, confirming that the mutation did not change general secondary structures of the molecule or its stability. Antisera against whole venom and LiRecDT1 showed cross-reactivity to both recombinant toxins by ELISA and immunoblotting. Dermonecrosis was abolished by the mutation, and rabbit skin revealed a decreased inflammatory response to LiRecDT1H12A compared to LiRecDT1. Residual phospholipase activity was observed with increasing concentrations of LiRecDT1H12A by dermonecrosis and fluorometric measurement in vitro. Lipid arrays showed that the mutated toxin has an affinity for the same lipids LiRecDT1, and both toxins were detected on RAEC cell surfaces. Data from in vitro choline release and HPTLC analyses of LiRecDT1-treated purified phospholipids and RAEC membrane detergent-extracts corroborate with the morphological changes. These data suggest a phospholipase-D dependent mechanism of toxicity, which has no substrate specificity and thus utilizes a broad range of bioactive lipids.

Cytotoxic effect of a new 1,3,4-thiadiazolium mesoionic compound (MI-D) on cell lines of human melanoma.

The structural characteristics of mesoionic compounds, which contain distinct regions of positive and negative charges associated with a poly-heteroatomic system, enable them to cross cellular membranes and interact strongly with biomolecules. Potential biological applications have been described for mesoionic compounds. 1,3,4-Thiadiazolium mesoionic compound (MI-D), a new mesoionic compound, has been demonstrated to be extremely cytotoxic to B16-F10 murine melanoma cells when compared to fotemustine and dacarbazine, drugs of reference in melanoma treatment protocols, describing inhibition of tumours grown in vitro and in vivo. We now evaluate the effects of mesoionic compound MI-D on different human melanoma cell lines. The drug decreased the viability and proliferation of MEL-85, SK-MEL, A2058 and MEWO cell lines in vitro, showing a considerable cytotoxic activity on these human cells. Adhesion of MEL-85 cells was evaluated in the presence of the drug using different extracellular matrix (ECM) constituents. MI-D decreased MEL-85 adhesion to laminin, fibronectin and matrigel. The morphology and actin cytoskeleton organisation of MEL-85 cells were also modified on MI-D treatment. These results on human melanoma cell lines indicate that MI-D is a very encouraging drug against melanoma, a tumour that is extremely resistant to chemotherapy.

A novel hyaluronidase from brown spider (Loxosceles intermedia) venom (Dietrich's Hyaluronidase): from cloning to functional characterization.

Loxoscelism is the designation given to clinical symptoms evoked by Loxosceles spiders bites. Clinical manifestations include skin necrosis with gravitational spreading and systemic disturbs. The venom contains several enzymatic toxins. Herein, we describe the cloning, expression, refolding and biological evaluation of a novel brown spider protein characterized as a hyaluronidase. Employing a venom gland cDNA library, we cloned a hyaluronidase (1200 bp cDNA) that encodes for a signal peptide and a mature protein. Amino acid alignment revealed a structural relationship with members of hyaluronidase family, such as scorpion and snake species. Recombinant hyaluronidase was expressed as N-terminal His-tag fusion protein (∼45 kDa) in inclusion bodies and activity was achieved using refolding. Immunoblot analysis showed that antibodies that recognize the recombinant protein cross-reacted with hyaluronidase from whole venom as well as an anti-venom serum reacted with recombinant protein. Recombinant hyaluronidase was able to degrade purified hyaluronic acid (HA) and chondroitin sulfate (CS), while dermatan sulfate (DS) and heparan sulfate (HS) were not affected. Zymograph experiments resulted in ∼45 kDa lytic zones in hyaluronic acid (HA) and chondroitin sulfate (CS) substrates. Through in vivo experiments of dermonecrosis using rabbit skin, the recombinant hyaluronidase was shown to increase the dermonecrotic effect produced by recombinant dermonecrotic toxin from L. intermedia venom (LiRecDT1). These data support the hypothesis that hyaluronidase is a “spreading factor”. Recombinant hyaluronidase provides a useful tool for biotechnological ends. We propose the name Dietrichs Hyaluronidase for this enzyme, in honor of Professor Carl Peter von Dietrich, who dedicated his life to studying proteoglycans and glycosaminoglycans.

Identification of a direct hemolytic effect dependent on the catalytic activity induced by phospholipase‐D (dermonecrotic toxin) from brown spider venom

Brown spiders have world‐wide distribution and are the cause of health problems known as loxoscelism. Necrotic cutaneous lesions surrounding the bites and less intense systemic signs like renal failure, DIC, and hemolysis were observed. We studied molecular mechanism by which recombinant toxin, biochemically characterized as phospholipase‐D, causes direct hemolysis (complement independent). Human erythrocytes treated with toxin showed direct hemolysis in a dose‐dependent and time‐dependent manner, as well as morphological changes in cell size and shape. Erythrocytes from human, rabbit, and sheep were more susceptible than those from horse. Hemolysis was not dependent on ABO group or Rhesus system. Confocal and FACS analyses using antibodies or GFP‐phospholipase‐D protein showed direct toxin binding to erythrocytes membrane. Moreover, toxin‐treated erythrocytes reacted with annexin‐V and showed alterations in their lipid raft profile. Divalent ion chelators significantly inhibited hemolysis evoked by phospholipase‐D, which has magnesium at the catalytic domain. Chelators were more effective than PMSF (serine‐protease inhibitor) that had no effect on hemolysis. By site‐directed mutation at catalytic domain (histidine 12 by alanine), hemolysis and morphologic changes of erythrocytes (but not the toxins ability of membrane binding) were inhibited, supporting that catalytic activity is involved in hemolysis and cellular alterations but not toxin cell binding. The results provide evidence that L. intermedia venom phospholipase‐D triggers direct human blood cell hemolysis in a catalytic‐dependent manner. J. Cell. Biochem. 107: 655–666, 2009.

The relationship between calcium and the metabolism of plasma membrane phospholipids in hemolysis induced by brown spider venom phospholipase‐D toxin

Brown spider venom phospholipase‐D belongs to a family of toxins characterized as potent bioactive agents. These toxins have been involved in numerous aspects of cell pathophysiology including inflammatory response, platelet aggregation, endothelial cell hyperactivation, renal disorders, and hemolysis. The molecular mechanism by which these toxins cause hemolysis is under investigation; literature data have suggested that enzyme catalysis is necessary for the biological activities triggered by the toxin. However, the way by which phospholipase‐D activity is directly related with human hemolysis has not been determined. To evaluate how brown spider venom phospholipase‐D activity causes hemolysis, we examined the impact of recombinant phospholipase‐D on human red blood cells. Using six different purified recombinant phospholipase‐D molecules obtained from a cDNA venom gland library, we demonstrated that there is a correlation of hemolytic effect and phospholipase‐D activity. Studying recombinant phospholipase‐D, a potent hemolytic and phospholipase‐D recombinant toxin (LiRecDT1), we determined that the toxin degrades synthetic sphingomyelin (SM), lysophosphatidylcholine (LPC), and lyso‐platelet‐activating factor. Additionally, we determined that the toxin degrades phospholipids in a detergent extract of human erythrocytes, as well as phospholipids from ghosts of human red blood cells. The products of the degradation of synthetic SM and LPC following recombinant phospholipase‐D treatments caused hemolysis of human erythrocytes. This hemolysis, dependent on products of metabolism of phospholipids, is also dependent on calcium ion concentration because the percentage of hemolysis increased with an increase in the dose of calcium in the medium. Recombinant phospholipase‐D treatment of human erythrocytes stimulated an influx of calcium into the cells that was detected by a calcium‐sensitive fluorescent probe (Fluo‐4). This calcium influx was shown to be channel‐mediated rather than leak‐promoted because the influx was inhibited by L‐type calcium channel inhibitors but not by a T‐type calcium channel blocker, sodium channel inhibitor or a specific inhibitor of calcium activated potassium channels. Finally, this inhibition of hemolysis following recombinant phospholipase‐D treatment occurred in a concentration‐dependent manner in the presence of L‐type calcium channel blockers such as nifedipine and verapamil. The data provided herein, suggest that the brown spider venom phospholipase‐D‐induced hemolysis of human erythrocytes is dependent on the metabolism of membrane phospholipids, such as SM and LPC, generating bioactive products that stimulate a calcium influx into red blood cells mediated by the L‐type channel. J. Cell. Biochem. 112: 2529–2540, 2011.

Molecular cloning, heterologous expression and functional characterization of a novel translationally-controlled tumor protein (TCTP) family member from Loxosceles intermedia (brown spider) venom

Envenoming with brown spiders (Loxosceles genus) is common throughout the world. Cutaneous symptoms following spider bite accidents include dermonecrosis, erythema, itching and pain. In some cases, accidents can cause hypersensibility or even allergic reactions. These responses could be associated with histaminergic events, such as an increase in vascular permeability and vasodilatation. A protein that may be related to the effects of spider venom was identified from a previously obtained cDNA library of the L. intermedia venom gland. The amino acid sequence of this protein is homologous to proteins from the TCTP (translationally-controlled tumor protein) family, which are extracellular histamine-releasing factors (HRF) that are associated with the allergic reactions to parasites. Herein, we described the cloning, heterologous expression, purification and functional characterization of a novel member of the TCTP family from the Loxosceles intermedia venom gland. This recombinant protein, named LiRecTCTP, causes edema, enhances vascular permeability and is likely related to the inflammatory activity of the venom. Moreover, LiRecTCTP presents an immunological relationship with mammalian TCTPs.