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.     

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.     

Identification,cloning and functional characterization of a novel dermonecrotic toxin (phospholipase D) from brown spider (Loxosceles intermedia) venom

Brown spider bites are associated with lesions including dermonecrosis, gravitational spreading and a massive inflammatory response, along with systemic problems that may include hematological disturbances and renal failure. The mechanisms by which the venom exerts its noxious effects are currently under investigation. It is known that the venom contains a major toxin (dermonecrotic toxin, biochemically a phospholipase D) that can experimentally induce dermonecrosis, inflammatory response, animal mortality and platelet aggregation. Herein, we describe cloning, heterologous expression, purification and functionality of a novel isoform of the 33 kDa dermonecrotic toxin. Circular dichroism analysis evidenced correct folding for the toxin. The recombinant toxin was recognized by whole venom serum antibodies and by a specific antibody to a previously described dermonecrotic toxin. The identified toxin was found to display phospholipase activity and dermonecrotic properties. Additionally, the toxin caused a massive inflammatory response in rabbit skin dermis, evoked platelet aggregation, increased vascular permeability, caused edema and death in mice. These characteristics in combination with functional studies for other dermonecrotic toxins illustrate that a family of dermonecrotic toxins exists, and includes a novel member with high activity that may be useful for future structural and functional studies.     

Identification, cloning and functional characterization of a novel dermonecrotic toxin (phospholipase D) from brown spider (Loxosceles intermedia) venom

Brown spider bites are associated with lesions including dermonecrosis, gravitational spreading and a massive inflammatory response, along with systemic problems that may include hematological disturbances and renal failure. The mechanisms by which the venom exerts its noxious effects are currently under investigation. It is known that the venom contains a major toxin (dermonecrotic toxin, biochemically a phospholipase D) that can experimentally induce dermonecrosis, inflammatory response, animal mortality and platelet aggregation. Herein, we describe cloning, heterologous expression, purification and functionality of a novel isoform of the 33 kDa dermonecrotic toxin. Circular dichroism analysis evidenced correct folding for the toxin. The recombinant toxin was recognized by whole venom serum antibodies and by a specific antibody to a previously described dermonecrotic toxin. The identified toxin was found to display phospholipase activity and dermonecrotic properties. Additionally, the toxin caused a massive inflammatory response in rabbit skin dermis, evoked platelet aggregation, increased vascular permeability, caused edema and death in mice. These characteristics in combination with functional studies for other dermonecrotic toxins illustrate that a family of dermonecrotic toxins exists, and includes a novel member with high activity that may be useful for future structural and functional studies.     

Identification, cloning, expression and functional characterization of an astacin-like metalloprotease toxin from Loxosceles intermedia (brown spider) venom

Injuries caused by brown spiders (Loxosceles genus) are associated with dermonecrotic lesions with gravitational spreading and systemic manifestations. The venom has a complex composition containing many different toxins, of which metalloproteases have been described in many different species of this genus. These toxins may degrade extracellular matrix constituents acting as a spreading factor. By using a cDNA library from an Loxosceles intermedia venom gland, we cloned and expressed a 900 bp cDNA, which encoded a signal peptide and a propeptide, which corresponded to a 30 kDa metalloprotease, now named LALP (Loxosceles astacin-like protease). Recombinant LALP was refolded and used to produce a polyclonal antiserum, which showed cross-reactivity with a 29 kDa native venom protein. CD analysis provided evidence that the recombinant LALP toxin was folded correctly, was still in a native conformation and had not aggregated. LALP addition to endothelial cell cultures resulted in de-adhesion of the cells, and also in the degradation of fibronectin and fibrinogen (this could be inhibited by the presence of the bivalent chelator 1,10-phenanthroline) and of gelatin in vitro. Sequence comparison (nucleotide and deduced amino acid), phylogenetic analysis and analysis of the functional recombinant toxin revealed that LALP is related in both structure and function to the astacin family of metalloproteases. This suggests that an astacin-like toxin is present in a animal venom secretion and indicates that recombinant LALP will be a useful tool for future structural and functional studies on venom and the astacin family.     

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.     

Molecular cloning and expression of a functional dermonecrotic and haemolytic factor from Loxosceles laeta venom

The bite of spiders of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and complement-dependent haemolysis. The aim of this study was to generate recombinant proteins from the Loxosceles spider gland to facilitate structural and functional studies in the mechanisms of loxoscelism. Using "Expressed Sequencing Tag" strategy of aleatory clones from, L. laeta venom gland cDNA library we have identified clones containing inserts coding for proteins with significant similarity with previously obtained N-terminus of sphingomyelinases from Loxosceles intermedia venom [1]. Clone H17 was expressed as a fusion protein containing a 6x His-tag at its N-terminus and yielded a 33kDa protein. The recombinant protein was endowed with all biological properties ascribed to the whole L. laeta venom and sphingomyelinases from L. intermedia, including dermonecrotic and complement-dependent haemolytic activities. Antiserum raised against the recombinant protein recognised a 32-kDa protein in crude L. laeta venom and was able to block the dermonecrotic reaction caused by whole L. laeta venom. This study demonstrates conclusively that the sphingomyelinase activity in the whole venom is responsible for the major pathological effects of Loxosceles spider envenomation.     

Proteome analysis of brown spider venom: identification of loxnecrogin isoforms in Loxosceles gaucho venom

Brown spiders of the Loxosceles genus are distributed worldwide. In Brazil, eight species are found in Southern states, where the envenomation by Loxosceles venom (loxoscelism) is a health problem. The mechanism of the dermonecrotic action of Loxosceles venom is not totally understood. Two isoforms of dermonecrotic toxins (loxnecrogins) from L. gaucho venom have been previously purified, and showed sequence similarities to sphingomyelinase. Herein we employed a proteomic approach to obtain a global view of the venom proteome, with a particular interest in the loxnecrogin isoforms' pattern. Proteomic two-dimensional gel electrophoresis maps for L. gaucho, L. intermedia, and L. laeta venoms showed a major protein region (30-35 kDa, pI 3-10), where at least eight loxnecrogin isoforms could be separated and identified. Their characterization used a combined approach composed of Edman chemical sequencing, matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and electrospray ionization-quadropole-time of flight tandem mass spectrometry leading to the identification of sphingomyelinases D. The venom was also pre-fractionated by gel filtration on a Superose 12 fast protein liqiud chromatography column, followed by capillary liquid chromatography-mass spectrometry. Eleven possible loxnecrogin isoforms around 30-32 kDa were detected. The identification of dermonecrotic toxin isoforms in L. gaucho venom is an important step towards understanding the physiopathology of the envenomation, leading to improvements in the immunotherapy of loxoscelism.     

Cloning,expression and characterization of a phospholipase D from Loxosceles gaucho venom gland

Loxosceles venom comprises a mixture of diverse toxins that induces intense local inflammatory reaction, dermonecrotic injury, platelet aggregation, hemolytic anemia and acute renal failure. Among several toxins in the venom, phospholipases D (PLDs), also called dermonecrotic toxins, are the most important and best studied, since they account for the main effects observed in loxoscelism. Despite their importance, biological analysis of PLDs is hampered by the minute amounts normally purified from the venom, and therefore many efforts have been made to clone those toxins. However, to date, no PLD from Loxosceles gaucho has been obtained in a heterologous system. Thus, in this work we show the cloning of a PLD from L. gaucho venom gland, named LgRec1, which was successfully expressed in a bacterial system. LgRec1 evoked local reaction (edema, erythema, ecchymosis, and paleness), dermonecrosis and hemolysis. It was also able to hydrolyze sphingomyelin and promote platelet aggregation. ELISA and Western blot analysis showed that LgRec1 was recognized by an anti-L. gaucho venom serum, a commercial arachnidic antivenom as well as a monoclonal antibody raised against the dermonecrotic fraction of L. gaucho venom. In addition, LgRec1 demonstrated to be highly immunogenic and antibodies raised against this recombinant toxin inhibited local reaction (∼65%) and dermonecrosis (∼100%) elicited by L. gaucho whole venom. Since PLDs are considered the major components accounting for the local and systemic envenomation effects caused by spiders from genus Loxosceles, the information provided here may help to understand the mechanisms behind clinical symptomatology.     

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.     

Purification and characterization of loxnecrogin,a dermonecrotic toxin from Loxosceles gaucho brown spider venom

The most common manifestation of Loxosceles spider envenoming is a dermonecrotic lesion at the bite site. Dermonecrotic toxins from Loxosceles gaucho venom were purified and characterized by mass spectrometry (capillary liquid chromatography followed by mass spectrometry detection). Two components were purified: a major one of 31,444 Da, called loxnecrogin A, and a minor one of 31,626 Da, called loxnecrogin B, being probably two isoforms of the toxin. The N-terminal sequence of loxnecrogin A showed similarity with N termini of other sphingomyelinolytic dermonecrotic toxins isolated from venoms of different Loxosceles species. The internal sequences did not present any statistically significant hits in sequence databases searches. However, loxnecrogin A partial sequence showed high similarity to regions of L. intermedia LiD1 recombinant protein sequence, recently described in the literature but not yet deposited in databanks.     

Experimental evidence for a direct cytotoxicity of Loxosceles intermedia (brown spider) venom in renal tissue.

Brown spider (Loxosceles genus) venom causes necrotic lesions often accompanied by fever, hemolysis, thrombocytopenia, and acute renal failure. Using mice exposed to Loxosceles intermedia venom, we aimed to show whether the venom directly induces renal damage. The experimental groups were composed of 50 mice as controls and 50 mice that received the venom. Light microscopic analysis of renal biopsy specimens showed alterations including hyalinization of proximal and distal tubules, erythrocytes in Bowman's space, glomerular collapse, tubule epithelial cell blebs and vacuoles, interstitial edema, and deposition of eosinophilic material in the tubule lumen. Electron microscopic findings indicated changes including glomerular epithelial and endothelial cell cytotoxicity as well as disorders of the basement membrane. Tubule alterations include epithelial cell cytotoxicity with cytoplasmic membrane blebs, mitochondrial changes, increase in smooth endoplasmic reticulum, presence of autophagosomes, and deposits of amorphous material in the tubules. We also found that the venom caused azotemia with elevation of blood urea levels but did not decrease C3 complement concentration or cause hemolysis in vivo. Confocal microscopy with antibodies against venom proteins showed direct binding of toxins to renal structures, confirmed by competition assays. Double-staining immunofluorescence reactions with antibodies against type IV collagen or laminin, antibodies to venom toxins, and fluorescent cytochemistry with DAPI revealed deposition of toxins in glomerular and tubule epithelial cells and in renal basement membranes. Two-dimensional electrophoresis showed venom rich in low molecular mass and cationic toxins. By immunoblotting with antibodies to venom toxins on renal extracts from venom-treated mice, we detected a renal binding toxin at 30 kD. The data provide experimental evidence that L. intermedia venom is directly involved in nephrotoxicity.     

Compared chemical properties of dermonecrotic and lethal toxins from spiders of the genusLoxosceles (Araneae)

Loxosceles spider venom usually causes a typical dermonecrotic lesion in bitten patients, but it may also cause systemic effects that may be lethal. Gel filtration on Sephadex G-100 ofLoxosceles gaucho, L. laeta, orL. intermedia spider venoms resulted in three fractions (A, containing higher molecular mass components, B containing intermediate molecular mass components, and C with lower molecular mass components). The dermonecrotic and lethal activities were detected exclusively in fraction A of all three species. Analysis by SDS-PAGE showed that the major protein contained in fraction A has molecular weight approximately 35 kDa inL. gaucho andL. intermedia, but 32 kDa inL. laeta venom. These toxins were isolated from venoms ofL. gaucho, L. laeta, andL. intermedia by SDS-PAGE followed by blotting to PVDF membrane and sequencing. A database search showed a high level of identity between each toxin and a fragment of theL. reclusa (North American spider) toxin. A multiple sequence alignment of theLoxosceles toxins showed many common identical residues in their N-terminal sequences. Identities ranged from 50.0% (L. gaucho andL. reclusa) to 61.1% (L. intermedia andL. reclusa). The purified toxins were also submitted to capillary electrophoresis peptide mapping afterin situ partial hydrolysis of the blotted samples. The results obtained suggest thatL. intermedia protein is more similar toL. laeta toxin thanL. gaucho toxin and revealed a smaller homology betweenL. intermedia andL gaucho. Altogether these findings suggest that the toxins responsible for most important activities of venoms ofLoxosceles species have a molecular mass of 32–35 kDa and are probably homologous proteins.     

Compared chemical properties of dermonecrotic and lethal toxins from spiders of the genusLoxosceles (Araneae)

Loxosceles spider venom usually causes a typical dermonecrotic lesion in bitten patients, but it may also cause systemic effects that may be lethal. Gel filtration on Sephadex G-100 ofLoxosceles gaucho, L. laeta, orL. intermedia spider venoms resulted in three fractions (A, containing higher molecular mass components, B containing intermediate molecular mass components, and C with lower molecular mass components). The dermonecrotic and lethal activities were detected exclusively in fraction A of all three species. Analysis by SDS-PAGE showed that the major protein contained in fraction A has molecular weight approximately 35 kDa inL. gaucho andL. intermedia, but 32 kDa inL. laeta venom. These toxins were isolated from venoms ofL. gaucho, L. laeta, andL. intermedia by SDS-PAGE followed by blotting to PVDF membrane and sequencing. A database search showed a high level of identity between each toxin and a fragment of theL. reclusa (North American spider) toxin. A multiple sequence alignment of theLoxosceles toxins showed many common identical residues in their N-terminal sequences. Identities ranged from 50.0% (L. gaucho andL. reclusa) to 61.1% (L. intermedia andL. reclusa). The purified toxins were also submitted to capillary electrophoresis peptide mapping afterin situ partial hydrolysis of the blotted samples. The results obtained suggest thatL. intermedia protein is more similar toL. laeta toxin thanL. gaucho toxin and revealed a smaller homology betweenL. intermedia andL gaucho. Altogether these findings suggest that the toxins responsible for most important activities of venoms ofLoxosceles species have a molecular mass of 32–35 kDa and are probably homologous proteins.     

The phylogenetic distribution of sphingomyelinase D activity in venoms of Haplogyne spiders

The venoms of Loxosceles spiders cause severe dermonecrotic lesions in human tissues. The venom component sphingomyelinase D (SMD) is a contributor to lesion formation and is unknown elsewhere in the animal kingdom. This study reports comparative analyses of SMD activity and venom composition of select Loxosceles species and representatives of closely related Haplogyne genera. The goal was to identify the phylogenetic group of spiders with SMD and infer the timing of evolutionary origin of this toxin. We also preliminarily characterized variation in molecular masses of venom components in the size range of SMD. SMD activity was detected in all (10) Loxosceles species sampled and two species representing their sister taxon, Sicarius, but not in any other venoms or tissues surveyed. Mass spectrometry analyses indicated that all Loxosceles and Sicarius species surveyed had multiple (at least four to six) molecules in the size range corresponding to known SMD proteins (31-35 kDa), whereas other Haplogynes analyzed had no molecules in this mass range in their venom. This suggests SMD originated in the ancestors of the Loxosceles/Sicarius lineage. These groups of proteins varied in molecular mass across species with North American Loxosceles having 31-32 kDa, African Loxosceles having 32-33.5 kDa and Sicarius having 32-33 kDa molecules.     

Astacin-like metalloproteases are a gene family of toxins present in the venom of different species of the brown spider (genus Loxosceles)

Brown spiders have a worldwide distribution, and their venom has a complex composition containing many different molecules. Herein, we report the existence of a family of astacin-like metalloprotease toxins in Loxosceles intermedia venom, as well as in the venom of different species of Loxosceles. Using a cDNA library from the L. intermedia venom gland, we cloned two novel cDNAs encoding astacin-like metalloprotease toxins, LALP2 and LALP3. Using an anti-serum against the previously described astacin-like toxin in L. intermedia venom (LALP1), we detected the presence of immunologically-related toxins in the venoms of L. intermedia, Loxosceles laeta, and Loxosceles gaucho. Zymographic experiments showed gelatinolytic activity of crude venoms of L. intermedia, L. laeta, and L. gaucho (which could be inhibited by the divalent metal chelator 1,10-phenanthroline) at electrophoretic mobilities identical to those reported for immunological cross-reactivity. Moreover, mRNAs extracted from L. laeta and L. gaucho venom glands were screened for astacin-like metalloproteases, and cDNAs obtained using LALP1-specific primers were sequenced, and their deduced amino acid sequences confirmed they were members of the astacin family with the family signatures (HEXXHXXGXXHE and MXY), LALP4 and LALP5, respectively. Sequence comparison of deduced amino acid sequences revealed that LALP2, LALP3, LALP4, and LALP5 are related to the astacin family. This study identified the existence of gene family of astacin-like toxins in the venoms of brown spiders and raises the possibility that these molecules are involved in the deleterious effects triggered by the venom.     

Sphingomyelinase D activity in model membranes: structural effects of in situ generation of ceramide-1-phosphate

The toxicity of Loxosceles spider venom has been attributed to a rare enzyme, sphingomyelinase D, which transforms sphingomyelin to ceramide-1-phosphate. The bases of its inflammatory and dermonecrotic activity, however, remain unclear. In this work the effects of ceramide-1-phosphate on model membranes were studied both by in situ generation of this lipid using a recombinant sphingomyelinase D from the spider Loxosceles laeta and by pre-mixing it with sphingomyelin and cholesterol. The systems of choice were large unilamellar vesicles for bulk studies (enzyme kinetics, fluorescence spectroscopy and dynamic light scattering) and giant unilamellar vesicles for fluorescence microscopy examination using a variety of fluorescent probes. The influence of membrane lateral structure on the kinetics of enzyme activity and the consequences of enzyme activity on the structure of target membranes containing sphingomyelin were examined. The findings indicate that: 1) ceramide-1-phosphate (particularly lauroyl ceramide-1-phosphate) can be incorporated into sphingomyelin bilayers in a concentration-dependent manner and generates coexistence of liquid disordered/solid ordered domains, 2) the activity of sphingomyelinase D is clearly influenced by the supramolecular organization of its substrate in membranes and, 3) in situ ceramide-1-phosphate generation by enzymatic activity profoundly alters the lateral structure and morphology of the target membranes.     

Platelet aggregation and sphingomyelinase D activity of a purified toxin from the venom of loxosceles reclusa

A facile and quantitative assay for measuring the activity of sphingomyelinase D in recluse spider venom has been developed using L-α-[palmitoyl-1-¹⁴C]lysophosphatidylcholine as substrate. This assay avoids the problem of substrate insolubility that occurs when sphingomyelin and other lipids are used as subtrates. This assay has been employed in gel filtration and isoelectric focusing isolation techniques to purify sphingomyelinase D from spider venom. The purified sphingomyelinase exhibits four active enzyme forms in isoelectric focusing with pI values of 8.7, 8.4., 8.2, and 7.8. Each active form when examined in SDS-polyacrylamide gel electrophoresis gave an estimated molecular weight of 32 000. The four active enzyme forms were immunologically cross-reactive with each other as demonstrated with radioimmune assays using an antiserum developed to one of the active forms. Each active form hydrolysed sphingomyelin to release choline and produce N-acylsphingosine phosphate. One of the active enzyme forms was characterized further in dermonecrosis and platelet aggregation measurements. This purified sphingomyelinase D was identified as a poisonous toxin that can develop the typical dermonecrotic spider lesion when injected into experimental animals at levels expected to be delivered in a normal bite. Furthermore, the purified toxin acts to aggregate human blood platelets. The toxin-induced platelet aggregation has been related to serotonin release as aggregation occurs, and it has been shown to be inhibited by EDTA over the range of 0.6 to 3.0 mM EDTA. It is suggested that spider-induced dermonecrosis could result in part from platelet aggregation at and near the site of envenomation.     

Platelet aggregation and sphingomyelinase D activity of a purified toxin from the venom of Loxosceles reclusa

A facile and quantitative assay for measuring the activity of sphingomyelinase D in recluse spider venom has been developed using L-alpha-[palmitoyl-1-14C]lysophosphatidylcholine as substrate. This assay avoids the problem of substrate insolubility that occurs when sphingomyelin and other insoluble lipids are used as substrates. This assay has been employed in gel filtration and isoelectric focusing isolation techniques to purify sphingomyelinase D from spider venom. The purified sphingomyelinase exhibits four active enzyme forms in isoelectric focusing with pI values of 8.7, 8.4, 8.2, and 7.8. Each active form when examined in SDS-polyacrylamide gel electrophoresis gave an estimated molecular weight of 32 000. The four active enzyme forms were immunologically cross-reactive with each other as demonstrated with radioimmune assays using an antiserum developed to one of the active forms. Each active form hydrolysed sphingomyelin to release choline and produce N-acylsphingosine phosphate. One of the active enzyme forms was characterized further in dermonecrosis and platelet aggregation measurements. This purified sphingomyelinase D was identified as a poisonous toxin that can developed typical dermonecrotic spider lesions when injected into experimental animals at levels expected to be delivered in a normal bite. Furthermore, the purified toxin acts to aggregate human blood platelets. The toxin-induced platelet aggregation has been related to serotonin release as aggregation occurs, and it has been shown to be inhibited by EDTA over the range of 0.6 yo 3.0 mM EDTA. It is suggested that spider-induced dermonecrosis could result in part from platelet aggregation at and near the site of envenomation.     

Two new phospholipase D isoforms of Loxosceles laeta: cloning, heterologous expression, functional characterization, and potential biotechnological application

Toxin phospholipases-D present in the venom of Loxosceles spiders is the principal responsible for local and systemic effects observed in the loxoscelism. In this study, we describe the cloning, expression, functional evaluation, and potential biotechnological application of cDNAs, which code for two new phospholipase D isoforms, LIPLD1 and LIPLD2, of the spider Loxosceles laeta. The recombinant protein rLIPLD1 had hydrolytic activity on sphingomyelin and in vitro hemolytic activity on human red blood cells, whereas rLIPLD2 was inactive. The purified recombinant proteins and the venom are recognized by polyclonal anti-rLIPLD1 and rLIPLD2 sera produced in animals and conferred immunoprotection against the venom. These new isoforms reinforce the importance of the multigene family of phospholipases-D present in Loxosceles spiders. A highly immunogenic inactive isoform such as rLIPLD2 raises important expectation for its use as a potential immunogenic inducer of the immunoprotective response to the toxic action of the venom of Loxosceles laeta.