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1.
Toxins (Basel) ; 13(4)2021 04 01.
Article in English | MEDLINE | ID: mdl-33916208

ABSTRACT

The spider family Sicariidae includes three genera, Hexophthalma, Sicarius and Loxosceles. The three genera share a common characteristic in their venoms: the presence of Sphingomyelinases D (SMase D). SMases D are considered the toxins that cause the main pathological effects of the Loxosceles venom, that is, those responsible for the development of loxoscelism. Some studies have shown that Sicarius spiders have less or undetectable SMase D activity in their venoms, when compared to Hexophthalma. In contrast, our group has shown that Sicarius ornatus, a Brazilian species, has active SMase D and toxic potential to envenomation. However, few species of Sicarius have been characterized for their toxic potential. In order to contribute to a better understanding about the toxicity of Sicarius venoms, the aim of this study was to characterize the toxic properties of male and female venoms from Sicarius tropicus and compare them with that from Loxosceles laeta, one of the most toxic Loxosceles venoms. We show here that S. tropicus venom presents active SMases D. However, regarding hemolysis development, it seems that these toxins in this species present different molecular mechanisms of action than that described for Loxosceles venoms, whereas it is similar to those present in bacteria containing SMase D. Besides, our results also suggest that, in addition to the interspecific differences, intraspecific variations in the venoms' composition may play a role in the toxic potential of venoms from Sicarius species.


Subject(s)
Evolution, Molecular , Hemolysis/drug effects , Phosphoric Diester Hydrolases/toxicity , Spider Venoms/toxicity , Spiders/enzymology , Animals , Cell Survival/drug effects , Female , HaCaT Cells , Humans , Keratinocytes/drug effects , Keratinocytes/pathology , Male , Phosphoric Diester Hydrolases/genetics , Phosphoric Diester Hydrolases/metabolism , Sex Factors , Species Specificity , Spider Venoms/enzymology , Spider Venoms/genetics , Spiders/classification , Spiders/genetics
2.
Toxins (Basel) ; 12(11)2020 11 06.
Article in English | MEDLINE | ID: mdl-33171968

ABSTRACT

Envenomation by Loxosceles spiders (Sicariidae family) has been thoroughly documented. However, little is known about the potential toxicity of members from the Sicarius genus. Only the venom of the Brazilian Sicarius ornatus spider has been toxicologically characterized. In Chile, the Sicarius thomisoides species is widely distributed in desert and semidesert environments, and it is not considered a dangerous spider for humans. This study aimed to characterize the potential toxicity of the Chilean S. thomisoides spider. To do so, specimens of S. thomisoides were captured in the Atacama Desert, the venom was extracted, and the protein concentration was determined. Additionally, the venoms were analyzed by electrophoresis and Western blotting using anti-recombinant L. laeta PLD1 serum. Phospholipase D enzymatic activity was assessed, and the hemolytic and cytotoxic effects were evaluated and compared with those of the L. laeta venom. The S. thomisoides venom was able to hydrolyze sphingomyelin as well as induce complement-dependent hemolysis and the loss of viability of skin fibroblasts with a dermonecrotic effect of the venom in rabbits. The venom of S. thomisoides showed intraspecific variations, with a similar protein pattern as that of L. laeta venom at 32-35 kDa, recognized by serum anti-LlPLD1. In this context, we can conclude that the venom of Sicarius thomisoides is similar to Loxosceles laeta in many aspects, and the dermonecrotic toxin present in their venom could cause severe harm to humans; thus, precautions are necessary to avoid exposure to their bite.


Subject(s)
Arthropod Proteins/toxicity , Fibroblasts/drug effects , Hemolysis/drug effects , Phospholipase D/toxicity , Phosphoric Diester Hydrolases/toxicity , Skin/drug effects , Spider Bites/enzymology , Spider Venoms/toxicity , Spiders , Animals , Arthropod Proteins/metabolism , Cell Line , Cell Survival/drug effects , Female , Fibroblasts/pathology , Humans , Hydrolysis , Male , Necrosis , Phospholipase D/metabolism , Rabbits , Skin/pathology , Sphingomyelins/metabolism , Spider Venoms/enzymology
3.
Toxins (Basel) ; 12(10)2020 10 01.
Article in English | MEDLINE | ID: mdl-33019554

ABSTRACT

The Hemiscorpius lepturus scorpion and brown spider Loxosceles intermedia represent a public health problem in Asia and America, respectively. Although distinct, these organisms contain similar toxins responsible for the principal clinical signs of envenomation. To better understand the properties of these toxins, we designed a study to compare recombinant Heminecrolysin (rHNC) and rLiD1, the major phospholipase D toxins of scorpion and spider venom, respectively. Using a competitive ELISA and a hemolytic inhibition test, we come to spot a cross reaction between scorpion and spider venoms along with an epitopic similarity between rHNC and rLiD1 associated with neutralizing antibodies. Results show that the ability of the rHNC to hydrolyze lysophosphatidylcholine (LPC) is equivalent to that of rLiD1 to hydrolyze sphingomyelin and vice-versa. rHNC exclusively catalyze transphosphatidylation of LPC producing cyclic phosphatidic acid (cPA). The in-silico analysis of hydrogen bonds between LPC and toxins provides a possible explanation for the higher transphosphatidylase activity of rHNC. Interestingly, for the first time, we reveal that lysophosphatidic acid (LPA) can be a substrate for both enzymes using cellular and enzymatic assays. The finding of the usage of LPA as a substrate as well as the formation of cPA as an end product could shed more light on the molecular basis of Hemiscorpius lepturus envenomation as well as on loxoscelism.


Subject(s)
Antivenins/pharmacology , Brown Recluse Spider , Phospholipase D/toxicity , Phosphoric Diester Hydrolases/toxicity , Scorpion Venoms/toxicity , Scorpions , Skin/drug effects , Spider Venoms/toxicity , Animals , Antivenins/immunology , Brown Recluse Spider/enzymology , Brown Recluse Spider/immunology , Cross Reactions , Epitopes , Hemolysis/drug effects , Insect Bites and Stings/enzymology , Lysophosphatidylcholines/metabolism , Necrosis , Phospholipase D/immunology , Phospholipase D/metabolism , Phosphoric Diester Hydrolases/immunology , Scorpion Venoms/enzymology , Scorpion Venoms/immunology , Scorpions/enzymology , Scorpions/immunology , Skin/enzymology , Skin/pathology , Sphingomyelins/metabolism , Spider Venoms/enzymology , Spider Venoms/immunology , Substrate Specificity
4.
J Biol Chem ; 293(6): 2079-2090, 2018 02 09.
Article in English | MEDLINE | ID: mdl-29269415

ABSTRACT

Spider venom neurotoxins and cytolytic peptides are expressed as elongated precursor peptides, which are post-translationally processed by proteases to yield the active mature peptides. The recognition motifs for these processing proteases, first published more than 10 years ago, include the processing quadruplet motif (PQM) and the inverted processing quadruplet motif (iPQM). However, the identification of the relevant proteases was still pending. Here we describe the purification of a neurotoxin precursor processing protease from the venom of the spider Cupiennius salei The chymotrypsin-like serine protease is a 28-kDa heterodimer with optimum activity at venom's pH of 6.0. We designed multiple synthetic peptides mimicking the predicted cleavage sites of neurotoxin precursors. Using these peptides as substrates, we confirm the biochemical activity of the protease in propeptide removal from neurotoxin precursors by cleavage C-terminal of the PQM. Furthermore, the PQM protease also cleaves the iPQM relevant for heterodimerization of a subgroup of neurotoxins. An involvement in the maturing of cytolytic peptides is very likely, due to high similarity of present protease recognition motifs. Finally, bioinformatics analysis, identifying sequences of homolog proteins from 18 spiders of 9 families, demonstrate the wide distribution and importance of the isolated enzyme for spiders. In summary, we establish the first example of a PQM protease, essential for maturing of spider venom neurotoxins. In the future, the here described protease may be established as a powerful tool for production strategies of recombinant toxic peptides, adapted to the maturing of spider venom toxins.


Subject(s)
Neurotoxins/metabolism , Serine Proteases/metabolism , Spider Venoms/enzymology , Spiders/enzymology , Amino Acid Sequence , Animals , Base Sequence , Computational Biology , Enzyme Stability , Hydrogen-Ion Concentration , Molecular Sequence Data , Neurotoxins/chemistry , Neurotoxins/genetics , Protein Processing, Post-Translational , Serine Proteases/chemistry , Serine Proteases/genetics , Serine Proteases/isolation & purification , Spider Venoms/genetics , Spider Venoms/metabolism , Spiders/genetics , Spiders/metabolism
5.
Mol Immunol ; 94: 45-53, 2018 02.
Article in English | MEDLINE | ID: mdl-29257998

ABSTRACT

Envenomation by Loxosceles spiders can result in severe systemic and local reactions, which are mainly triggered by Sphingomyelinase D (SMase D), a toxic component of Loxosceles venom. SMase D induces a systemic inflammatory condition similar to the reaction observed during an endotoxic shock. Considering the potent pro-inflammatory potential of Loxosceles venom and the SMase D, in this study we have used the whole human blood model to study the endotoxic-like shock triggered by SMase D. Recombinant purified SMase D from L. intermedia venom, similarly to LPS, induced activation of blood leukocytes, as observed by the increase in the expression of CD11b and TLR4, production of reactive oxygen and nitrogen species (superoxide anion and peroxynitrite) and release of TNF-α. Complement consumption in the plasma was also detected, and complement inhibition by compstatin decreased the SMase D and LPS-induced leukocyte activation, as demonstrated by a reduction in the expression of CD11b and TLR4 and superoxide anion production. Similar results were found for the L. intermedia venom, except for the production of TNF-α. These findings indicate that SMase D present in Loxosceles venom is able to activate leukocytes in a partially complement-dependent manner, which can contribute to the systemic inflammation that follows envenomation by this spider. Thus, future therapeutic management of systemic Loxosceles envenomation could include the use of complement inhibitors as adjunct therapy.


Subject(s)
Complement System Proteins/physiology , Leukocytes/drug effects , Phosphoric Diester Hydrolases/pharmacology , Spider Venoms/enzymology , Animals , Granulocytes/drug effects , Granulocytes/physiology , Humans , Leukocytes/physiology , Macrophage Activation/drug effects , Monocytes/drug effects , Monocytes/physiology , Oxidative Stress/drug effects , Phosphoric Diester Hydrolases/physiology , Reactive Oxygen Species/metabolism , Spider Venoms/pharmacology , Spiders
6.
Toxicon ; 136: 34-43, 2017 Sep 15.
Article in English | MEDLINE | ID: mdl-28688803

ABSTRACT

Theraphosid spider venoms are extremely complex mixtures, composed mainly by low molecular compounds, peptides, and enzymes. The large size of these spiders and their ability to breed in captivity permits access to rather large amounts of venom and an easier venom extraction. In the present study, we conducted a comparative investigation about the content of hyaluronidase-like enzymes in the venoms from several theraphosid spiders, with a special focus on the Poecilotheria species, which are considered as underestimated theraphosids of medical importance. The following species were analyzed: Poecilotheria regalis, Poecilotheria ornata, Poecilotheria rufilata, Poecilotheria vittata, Bonnetina papalutlensis, Aphonopelma sp., Brachypelma smithi, Brachypelma epicureanum, Brachypelma boehmei, Grammostola porteri, Lasiodora klugi, Ceratogyrus darlingi, and Nhandu chromatus. The presence of hyaluronidase-like enzymes was evidenced in all venoms by a turbidimetric method and zymography. Several isoforms of acid-active hyaluronidase-like enzymes were detected in the venoms from Poecilotheria species. These results provide some biochemical characteristics of the high molecular mass proteins of the theraphosid venoms.


Subject(s)
Arthropod Proteins/isolation & purification , Hyaluronoglucosaminidase/isolation & purification , Spider Venoms/enzymology , Spiders/chemistry , Animals , Species Specificity
7.
Toxins (Basel) ; 9(4)2017 04 05.
Article in English | MEDLINE | ID: mdl-28379166

ABSTRACT

Cutaneous loxoscelism envenomation by Loxosceles spiders is characterized by the development of a dermonecrotic lesion, strong inflammatory response, the production of pro-inflammatory mediators, and leukocyte migration to the bite site. The role of phospholipase D (PLD) from Loxosceles in the recruitment and migration of monocytes to the envenomation site has not yet been described. This study reports on the expression and production profiles of cytokines and chemokines in human skin fibroblasts treated with catalytically active and inactive recombinant PLDs from Loxosceles laeta (rLlPLD) and lipid inflammatory mediators ceramide 1-phosphate (C1P) and lysophosphatidic acid (LPA), and the evaluation of their roles in monocyte migration. Recombinant rLlPLD1 (active) and rLlPLD2 (inactive) isoforms induce interleukin (IL)-6, IL-8, CXCL1/GRO-α, and CCL2/monocyte chemoattractant protein-1 (MCP-1) expression and secretion in fibroblasts. Meanwhile, C1P and LPA only exhibited a minor effect on the expression and secretion of these cytokines and chemokines. Moreover, neutralization of both enzymes with anti-rLlPLD1 antibodies completely inhibited the secretion of these cytokines and chemokines. Importantly, conditioned media from fibroblasts, treated with rLlPLDs, stimulated the transmigration of THP-1 monocytes. Our data demonstrate the direct role of PLDs in chemotactic mediator synthesis for monocytes in human skin fibroblasts and indicate that inflammatory processes play an important role during loxoscelism.


Subject(s)
Arthropod Proteins/pharmacology , Fibroblasts/drug effects , Monocytes/drug effects , Phospholipase D/pharmacology , Spider Venoms/enzymology , Animals , Cell Line , Cell Movement/drug effects , Ceramides/pharmacology , Cytokines/genetics , Cytokines/metabolism , Fibroblasts/metabolism , Humans , Lysophospholipids/pharmacology , Monocytes/physiology , RNA, Messenger/metabolism , Recombinant Proteins/pharmacology , Skin/cytology , Spiders
8.
Toxicon ; 120: 97-106, 2016 Sep 15.
Article in English | MEDLINE | ID: mdl-27496061

ABSTRACT

Envenoming resulting from Loxosceles spider bites (loxoscelism) is a recognized public health problem in Brazil. However, the pathophysiology of loxoscelism caused by L. similis bites, which is widespread in Brazil, remains poorly understood. In the present work, the RNA sequencing (RNA-Seq - Next Generation sequencing - NGS) of the L. similis venom gland was performed to identify and analyze the sequences of the key component phospholipase D. The sequences were aligned based on their classical domains, and a phylogenetic tree was constructed. In the bioinformatics analysis, 23 complete sequences of phospholipase D proteins were found and classified as Loxtox proteins, as they contained the characteristic domains of phospholipase D: the active site, the Mg(2+)-binding domain, and the catalytic loop. Three phospholipase D sequences with non-canonical domains were also found in this work. They were analyzed separately and named PLDs from L. similis (PLD-Ls). This study is the first to characterize phospholipase D sequences from Loxosceles spiders by RNA-Seq. These results contribute new knowledge about the composition of L. similis venom, revealing novel tools that could be used for pharmacological, immunological, and biotechnological applications.


Subject(s)
Brown Recluse Spider , Insect Proteins/metabolism , Phospholipase D/metabolism , Spider Venoms/enzymology , Amino Acid Sequence , Animals , High-Throughput Nucleotide Sequencing , Insect Proteins/genetics , Phospholipase D/genetics , Phosphoric Diester Hydrolases/genetics , Phylogeny , Sequence Homology, Amino Acid , Spider Venoms/genetics
9.
Vaccine ; 34(33): 3828-34, 2016 07 19.
Article in English | MEDLINE | ID: mdl-27265457

ABSTRACT

In the present investigation we used a recombinant LiD1 toxin, named rLiD1his, from Loxosceles intermedia brown spider to elicit specific antibodies in mice carrying different Human Leukocyte Antigens class II (HLAII) {DRB1.0401 (DR4), DQB1.0601 (DQ6) and DQB1.0302 (DQ8)} as well as in BALB/C and C57BL/6 control mice. All mice strains produced high antibody titers against rLiD1his but DR4 mice antibodies (the lower responder mice) were not able to recognize L. intermedia crude venom. The anti-rLiD1his sera, except from DR4 mice, were able to neutralize dermonecrotic, hemorrhagic and edematogenic effects of rLiD1his in naïve rabbits. Overlapping peptides from the amino acid sequence of LiD1 toxin were prepared by SPOT method and differences in LiD1 epitope recognition were observed using different mice anti-rLiD1his sera. The region (160)DKVGHDFSGNDDISDVGK(177) was recognized by transgenic DQ8 and DQ6 mice sera. Other epitopes were recognized by at least two different animals' sera including (10)MGHMVNAIGQIDEFVNLG(27), (37)FDDNANPEYTYHGIP(51), (70)GLRSATTPGNSKYQEKLV(87) and (259)AAYKKKFRVATYDDN(273). Among these epitopes, the epitopes 37-51 and 160-177 have already been shown in previously studies as good candidates to be used alone or combined with other peptides to induce protective immune response against Loxosceles venoms. The results presented here highlight the importance of HLAII in antibody response and recognition of specific B-cell epitopes of rLiD1his spider toxin according to HLAII type and impact in the epitopic vaccine development against this spider.


Subject(s)
Antibodies/immunology , Epitopes, B-Lymphocyte/immunology , Phosphoric Diester Hydrolases/immunology , Spider Venoms/enzymology , Amino Acid Sequence , Animals , Genetic Background , Immune Sera/immunology , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Neutralization Tests , Rabbits
10.
Biochim Biophys Acta ; 1861(9 Pt A): 970-979, 2016 09.
Article in English | MEDLINE | ID: mdl-27233517

ABSTRACT

Brown spider phospholipases D from Loxosceles venoms are among the most widely studied toxins since they induce dermonecrosis, triggering inflammatory responses, increase vascular permeability, cause hemolysis, and renal failure. The catalytic (H12 and H47) and metal-ion binding (E32 and D34) residues in Loxosceles intermedia phospholipase D (LiRecDT1) were mutated to understand their roles in the observed activities. All mutants were identified using whole venom serum antibodies and a specific antibody to wild-type LiRecDT1, they were also analyzed by circular dichroism (CD) and differential scanning calorimetry (DSC). The phospholipase D activities of H12A, H47A, H12A-H47A, E32, D34 and E32A-D34A, such as vascular permeability, dermonecrosis, and hemolytic effects were inhibited. The mutant Y228A was equally detrimental to biochemical and biological effects of phospholipase D, suggesting an essential role of this residue in substrate recognition and binding. On the other hand, the mutant C53A-C201A reduced the enzyme's ability to hydrolyze phospholipids and promote dermonecrosis, hemolytic, and vascular effects. These results provide the basis understanding the importance of specific residues in the observed activities and contribute to the design of synthetic and specific inhibitors for Brown spider venom phospholipases D.


Subject(s)
Catalytic Domain/genetics , Phospholipase D/chemistry , Phospholipids/chemistry , Spider Venoms/enzymology , Animals , Brown Recluse Spider/chemistry , Brown Recluse Spider/enzymology , Capillary Permeability , Circular Dichroism , Hemolysis , Mutation , Phospholipase D/metabolism , Phospholipids/metabolism , Phosphoric Diester Hydrolases/chemistry , Spider Venoms/chemistry
11.
PLoS One ; 11(4): e0153090, 2016.
Article in English | MEDLINE | ID: mdl-27078876

ABSTRACT

Envenomation by Loxosceles spider is characterized by the development of dermonecrosis. In previous studies, we have demonstrated that increased expression/secretion of matrix metalloproteinases 2 and 9, induced by Loxosceles intermedia venom Class 2 SMases D (the main toxin in the spider venom), contribute to the development of cutaneous loxoscelism. In the present study we show that the more potent venom containing the Class 1 SMase D from Loxosceles laeta, in addition to increasing the expression/secretion of MMP2 and MMP9, also stimulates the expression of MMP7 (Matrilysin-1), which was associated with keratinocyte cell death. Tetracycline, a matrix metalloproteinase inhibitor, prevented cell death and reduced MMPs expression. Considering that L. laeta venom is more potent at inducing dermonecrosis than L. intermedia venom, our results suggest that MMP7 may play an important role in the severity of dermonecrosis induced by L. laeta spider venom SMase D. In addition, the inhibition of MMPs by e.g. tetracyclines may be considered for the treatment of the cutaneous loxoscelism.


Subject(s)
Arthropod Proteins/pharmacology , Keratinocytes/drug effects , Phosphoric Diester Hydrolases/pharmacology , Spider Venoms/pharmacology , Spiders/enzymology , Animals , Anti-Bacterial Agents/pharmacology , Apoptosis/drug effects , Blotting, Western , Cell Line , Cell Survival/drug effects , Dose-Response Relationship, Drug , Humans , Keratinocytes/metabolism , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 7/metabolism , Matrix Metalloproteinase 9/metabolism , Necrosis/prevention & control , Rabbits , Skin/drug effects , Skin/pathology , Spider Venoms/enzymology , Tetracycline/pharmacology , Time Factors
12.
PLoS One ; 10(12): e0143963, 2015.
Article in English | MEDLINE | ID: mdl-26630650

ABSTRACT

STRUCTURE OF CUPIENNIUS SALEI VENOM HYALURONIDASE: Hyaluronidases are important venom components acting as spreading factor of toxic compounds. In several studies this spreading effect was tested on vertebrate tissue. However, data about the spreading activity on invertebrates, the main prey organisms of spiders, are lacking. Here, a hyaluronidase-like enzyme was isolated from the venom of the spider Cupiennius salei. The amino acid sequence of the enzyme was determined by cDNA analysis of the venom gland transcriptome and confirmed by protein analysis. Two complex N-linked glycans akin to honey bee hyaluronidase glycosylations, were identified by tandem mass spectrometry. A C-terminal EGF-like domain was identified in spider hyaluronidase using InterPro. The spider hyaluronidase-like enzyme showed maximal activity at acidic pH, between 40-60°C, and 0.2 M KCl. Divalent ions did not enhance HA degradation activity, indicating that they are not recruited for catalysis. FUNCTION OF VENOM HYALURONIDASES: Besides hyaluronan, the enzyme degrades chondroitin sulfate A, whereas heparan sulfate and dermatan sulfate are not affected. The end products of hyaluronan degradation are tetramers, whereas chondroitin sulfate A is mainly degraded to hexamers. Identification of terminal N-acetylglucosamine or N-acetylgalactosamine at the reducing end of the oligomers identified the enzyme as an endo-ß-N-acetyl-D-hexosaminidase hydrolase. The spreading effect of the hyaluronidase-like enzyme on invertebrate tissue was studied by coinjection of the enzyme with the Cupiennius salei main neurotoxin CsTx-1 into Drosophila flies. The enzyme significantly enhances the neurotoxic activity of CsTx-1. Comparative substrate degradation tests with hyaluronan, chondroitin sulfate A, dermatan sulfate, and heparan sulfate with venoms from 39 spider species from 21 families identified some spider families (Atypidae, Eresidae, Araneidae and Nephilidae) without activity of hyaluronidase-like enzymes. This is interpreted as a loss of this enzyme and fits quite well the current phylogenetic idea on a more isolated position of these families and can perhaps be explained by specialized prey catching techniques.


Subject(s)
Hyaluronoglucosaminidase/metabolism , Spider Venoms/enzymology , Amino Acid Sequence , Animals , Base Sequence , Glycosylation , Hyaluronoglucosaminidase/chemistry , Molecular Sequence Data , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spiders , Tandem Mass Spectrometry
13.
BMC Evol Biol ; 15: 290, 2015 Dec 21.
Article in English | MEDLINE | ID: mdl-26690570

ABSTRACT

BACKGROUND: Sphingomyelinase D is the main toxin present in the venom of Loxosceles spiders. Several isoforms present in these venoms can be structurally classified in two groups. Class I Sphingomyelinase D contains a single disulphide bridge and variable loop. Class II Sphingomyelinase D presents an additional intrachain disulphide bridge that links a flexible loop with a catalytic loop. These classes exhibit differences in their toxic potential. In this paper we address the distribution of the structural classes of SMase D within and among species of spiders and also their evolutionary origin by means of phylogenetic analyses. We also conducted tests to assess the action of natural selection in their evolution combined to structural modelling of the affected sites. RESULTS: The majority of the Class I enzymes belong to the same clade, which indicates a recent evolution from a single common ancestor. Positively selected sites are located on the catalytic interface, which contributes to a distinct surface charge distribution between the classes. Sites that may prevent the formation of an additional bridge were found in Class I enzymes. CONCLUSIONS: The evolution of Sphingomyelinase D has been driven by natural selection toward an increase in noxiousness, and this might help explain the toxic variation between classes.


Subject(s)
Evolution, Molecular , Phosphoric Diester Hydrolases/genetics , Spider Venoms/enzymology , Spiders/classification , Spiders/genetics , Animals , Models, Molecular , Phosphoric Diester Hydrolases/chemistry , Phylogeny , Selection, Genetic , Spider Venoms/genetics , Spiders/enzymology
14.
Toxicon ; 108: 176-80, 2015 Dec 15.
Article in English | MEDLINE | ID: mdl-26482933

ABSTRACT

Phospholipase D (PLD) toxins from sicariid spiders, which cause disease in mammals, were recently found to convert their primary substrates, sphingomyelin and lysophosphatidylcholine, to cyclic phospholipids. Here we show that two PLD toxins from pathogenic actinobacteria and ascomycete fungi, which share distant homology with the spider toxins, also generate cyclic phospholipids. This shared function supports divergent evolution of the PLD toxins from a common ancestor and suggests the importance of cyclic phospholipids in pathogenicity.


Subject(s)
Bacterial Toxins/chemistry , Mycotoxins/chemistry , Phospholipase D/physiology , Spider Venoms/enzymology , Actinobacteria/chemistry , Actinobacteria/enzymology , Amino Acid Sequence , Animals , Ascomycota/chemistry , Ascomycota/enzymology , Evolution, Molecular , Lysophosphatidylcholines/metabolism , Molecular Sequence Data , Phospholipase D/chemistry , Sequence Alignment , Sphingomyelins/metabolism , Spiders
15.
Toxicon ; 108: 154-66, 2015 Dec 15.
Article in English | MEDLINE | ID: mdl-26474948

ABSTRACT

Loxosceles spiders are responsible for serious human envenomations worldwide. The collection of symptoms found in victims after accidents is called loxoscelism and is characterized by two clinical conditions: cutaneous loxoscelism and systemic loxocelism. The only specific treatment is serum therapy, in which an antiserum produced with Loxosceles venom is administered to the victims after spider accidents. Our aim was to improve our knowledge, regarding the immunological relationship among toxins from the most epidemiologic important species in Brazil (Loxosceles intermedia, Loxosceles gaucho and Loxosceles laeta). Immunoassays using spider venoms and L. intermedia recombinant toxins were performed and their cross-reactivity assessed. The biological conservation of the main Loxosceles toxins (Phospholipases-D, Astacin-like metalloproteases, Hyaluronidase, ICK-insecticide peptide and TCTP-histamine releasing factor) were investigated. An in silico analysis of the putative epitopes was performed and is discussed on the basis of the experimental results. Our data is an immunological investigation in light of biological conservation throughout the Loxosceles genus. The results bring out new insights on brown spider venom toxins for study, diagnosis and treatment of loxoscelism and putative biotechnological applications concerning immune conserved features in the toxins.


Subject(s)
Antivenins/immunology , Spider Venoms/immunology , Spiders , Animals , Arthropod Proteins/chemistry , Computational Biology , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Spider Venoms/chemistry , Spider Venoms/enzymology , Tumor Protein, Translationally-Controlled 1
16.
Toxicon ; 93: 11-9, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25449105

ABSTRACT

The medical importance of Loxosceles spiders has promoted extensive research on different aspects of their venoms. Most of the reported cases of loxoscelism have occurred in the Americas, and thus, much work has focused on North and South American Loxosceles species. Interestingly, loxoscelism cases are rare in the Mediterranean Basin although Loxosceles rufescens, endemic to the Mediterranean, is an abundant spider even in human-altered areas. Thus, it has been suggested that the venom of L. rufescens could be of less medical relevance than that of its congeners. In this study, we challenge this hypothesis by using multiple approaches to study venom variation in selected species and lineages from the Mediterranean Basin and the Canary Islands. We found that SMase D activity, the key bioactive component of Loxosceles venom, is comparable to American species that are confirmed to have medically relevant bites. The venom protein composition using SDS-PAGE presents some differences among regional Loxosceles taxa in banding pattern and intensity, mostly between the Canarian and L. rufescens lineages. Differences between these species also exist in the expression of different paralogs of the SicTox gene family, with the Canarian species being less diverse. In conclusion, our results do not support the challenged hypothesis, and suggest that venom of these species may indeed be as potent as other Loxosceles species. Pending confirmation of loxoscelism with direct evidence of Loxosceles bites with species identification by professionals, Loxosceles in the Mediterranean region should conservatively be considered medically relevant taxa.


Subject(s)
Phosphoric Diester Hydrolases/genetics , Phosphoric Diester Hydrolases/toxicity , Phylogeny , Spider Bites/epidemiology , Spider Bites/physiopathology , Spider Venoms/enzymology , Base Sequence , Bayes Theorem , Cloning, Molecular , DNA Primers/genetics , DNA, Complementary/genetics , Electrophoresis, Polyacrylamide Gel , Humans , Mediterranean Region/epidemiology , Models, Genetic , Molecular Sequence Data , Oxazines , Sequence Analysis, DNA , Spain/epidemiology , Species Specificity , Spider Venoms/toxicity
17.
Toxicon ; 93: 37-40, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25448393

ABSTRACT

In this work, an anti-loxoscelic serum was produced by immunizing horses with a recombinant dermonecrotic protein from Loxosceles intermedia (rLiD1). Anti-rLiD1 antibodies were able to recognize different species of Loxosceles venoms by Western Blot and ELISA. The efficacy of anti-rLiD1 serum against the toxic effects of Loxosceles laeta (Peru) venom was tested, showing that anti-rLiD1 serum can neutralize those effects. This study confirms that recombinant proteins can be good candidates to replace crude venoms for antivenom production.


Subject(s)
Antivenins/immunology , Phosphoric Diester Hydrolases/chemistry , Phosphoric Diester Hydrolases/metabolism , Recombinant Proteins/metabolism , Spider Venoms/chemistry , Animals , Antivenins/pharmacology , Blotting, Western , Brazil , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Horses , Neutralization Tests , Peru , Phosphoric Diester Hydrolases/analysis , Species Specificity , Spider Venoms/enzymology
18.
Acta Crystallogr F Struct Biol Commun ; 70(Pt 10): 1418-20, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25286953

ABSTRACT

Brown spider envenomation results in dermonecrosis, intravascular coagulation, haemolysis and renal failure, mainly owing to the action of sphingomyelinases D (SMases D), which catalyze the hydrolysis of sphingomyelin to produce ceramide 1-phosphate and choline or the hydrolysis of lysophosphatidylcholine to produce lysophosphatidic acid. Here, the heterologous expression, purification, crystallization and preliminary X-ray diffraction analysis of LgRec1, a novel SMase D from Loxosceles gaucho venom, are reported. The crystals belonged to space group P21212, with unit-cell parameters a = 52.98, b = 62.27, c = 84.84 Šand diffracted to a maximum resolution of 2.6 Å.


Subject(s)
Arthropod Proteins/chemistry , Phosphoric Diester Hydrolases/chemistry , Spider Venoms/enzymology , Amino Acid Sequence , Crystallization , Crystallography, X-Ray , Molecular Sequence Data
19.
Biol Res ; 47: 2, 2014 Mar 26.
Article in English | MEDLINE | ID: mdl-25027855

ABSTRACT

BACKGROUND: Loxoscelism is the envenomation caused by the bite of Loxosceles spp. spiders. It entails severe necrotizing skin lesions, sometimes accompanied by systemic reactions and even death. There are no diagnostic means and treatment is mostly palliative. The main toxin, found in several isoforms in the venom, is sphingomyelinase D (SMD), a phospholipase that has been used to generate antibodies intended for medical applications. Nucleic acid aptamers are a promising alternative to antibodies. Aptamers may be isolated from a combinatorial mixture of oligonucleotides by iterative selection of those that bind to the target. In this work, two Loxosceles laeta SMD isoforms, Ll1 and Ll2, were produced in bacteria and used as targets with the aim of identifying RNA aptamers that inhibit sphingomyelinase activity. RESULTS: Six RNA aptamers capable of eliciting partial but statistically significant inhibitions of the sphingomyelinase activity of recombinant SMD-Ll1 and SMD-Ll2 were obtained: four aptamers exert ~17% inhibition of SMD-Ll1, while two aptamers result in ~25% inhibition of SMD-Ll2 and ~18% cross inhibition of SMD-Ll1. CONCLUSIONS: This work is the first attempt to obtain aptamers with therapeutic and diagnostic potential for loxoscelism and provides an initial platform to undertake the development of novel anti Loxosceles venom agents.


Subject(s)
Aptamers, Nucleotide/isolation & purification , Aptamers, Nucleotide/metabolism , Phosphodiesterase Inhibitors/isolation & purification , Phosphoric Diester Hydrolases , Spider Venoms/enzymology , Animals , Aptamers, Nucleotide/therapeutic use , Brown Recluse Spider/enzymology , Chromatography, Affinity , Cloning, Molecular , Gene Expression/genetics , Phosphodiesterase Inhibitors/pharmacology , Phosphoric Diester Hydrolases/classification , Sequence Analysis, DNA/methods , Spider Bites/drug therapy , Spider Venoms/classification
20.
Vaccine ; 32(18): 2086-92, 2014 Apr 11.
Article in English | MEDLINE | ID: mdl-24565754

ABSTRACT

We report the production of a neutralizing monoclonal antibody able to recognize the venoms of three major medically important species of Loxosceles spiders in Brazil. The mAb was produced by immunization of mice with a toxic recombinant L. intermedia sphingomyelinase D {SMases D isoform (rLiD1)} [1] and screened by enzyme-linked immunosorbent assay (ELISA) using L. intermedia, L. laeta and L. gaucho venoms as antigens. One clone (LiD1mAb16) out of seventeen anti-rLiD1 hybridomas was cross-reactive with the three whole Loxosceles venoms. 2D Western blot analysis indicated that LiD1mAb16 was capable of interacting with 34 proteins of 29-36kDa in L. intermedia, 33 in L. gaucho and 27 in L. laeta venoms. The results of immunoassays with cellulose-bound peptides revealed that the LiD1mAb16 recognizes a highly conserved linear epitope localized in the catalytic region of SMases D toxins. The selected mAb displayed in vivo protective activity in rabbits after challenge with rLiD1. These results show the potential usefulness of monoclonal antibodies for future therapeutic approaches and also opens up the perspective of utilization of these antibodies for immunodiagnostic assays in loxoscelism.


Subject(s)
Antibodies, Monoclonal/immunology , Epitopes/immunology , Phosphoric Diester Hydrolases/immunology , Spider Venoms/enzymology , Amino Acid Sequence , Animals , Antibodies, Neutralizing/immunology , Cross Reactions , Epitope Mapping , Hybridomas , Mice , Mice, Inbred BALB C , Models, Molecular , Molecular Sequence Data , Neutralization Tests , Rabbits , Recombinant Proteins/immunology , Spider Venoms/immunology , Spiders/enzymology
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