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Made available in DSpace on 2022-05-01T09:47:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Universidade Federal do Paraná Fundação Araucária Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Accidents involving Brown spiders are reported throughout the world. In the venom, the major toxins involved in the deleterious effects are phospholipases D (PLDs). In this work, recombinant mutated phospholipases D from three endemic species medically relevant in South America (Loxosceles intermedia, L. laeta and L. gaucho) were tested as antigens in a vaccination protocol. In such isoforms, key amino acid residues involved in catalysis, magnesium-ion coordination, and binding to substrates were replaced by Alanine (H12A-H47A, E32A-D34A and W230A). These mutations eliminated the phospholipase activity and reduced the generation of skin necrosis and edema to residual levels. Molecular modeling of mutated isoforms indicated that the three-dimensional structures, topologies, and surface charges did not undergo significant changes. Mutated isoforms were recognized by sera against the crude venoms. Vaccination protocols in rabbits using mutated isoforms generated a serum that recognized the native PLDs of crude venoms and neutralized dermonecrosis and edema induced by L. intermedia venom. Vaccination of mice prevented the lethal effects of L. intermedia crude venom. Furthermore, vaccination of rabbits prevented the cutaneous lesion triggered by the three venoms. These results indicate a great potential for mutated recombinant PLDs to be employed as antigens in developing protective vaccines for Loxoscelism. Department of Cell Biology Federal University of Paraná (UFPR) Production and Research Center of Immunobiological Products (CPPI) State Department of Health Veterinary Hospital Federal University of Paraná (UFPR) Department of Structural Molecular Biology and Genetics State University of Ponta Grossa (UEPG) Multiuser Center for Biomolecular Innovation Departament of Physics Universidade Estadual Paulista (UNESP) Multiuser Center for Biomolecular Innovation Departament of Physics Universidade Estadual Paulista (UNESP) Universidade Federal do Paraná: 02/2020 Universidade Federal do Paraná: 04/2019 Fundação Araucária: 057/2017 CNPq: 303868/2016-3 CNPq: 408633/2018-2

Made available in DSpace on 2021-06-25T11:14:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-03-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Phospholipases-D (PLDs) found in Loxosceles spiders’ venoms are responsible for the der-monecrosis triggered by envenomation. PLDs can also induce other local and systemic effects, such as massive inflammatory response, edema, and hemolysis. Recombinant PLDs reproduce all of the deleterious effects induced by Loxosceles whole venoms. Herein, wild type and mutant PLDs of two species involved in accidents—L. gaucho and L. laeta—were recombinantly expressed and character-ized. The mutations are related to amino acid residues relevant for catalysis (H12-H47), magnesium ion coordination (E32-D34) and binding to phospholipid substrates (Y228 and Y228-Y229-W230). Circular dichroism and structural data demonstrated that the mutant isoforms did not undergo significant structural changes. Immunoassays showed that mutant PLDs exhibit conserved epitopes and kept their antigenic properties despite the mutations. Both in vitro (sphingomyelinase activity and hemolysis) and in vivo (capillary permeability, dermonecrotic activity, and histopathological analysis) assays showed that the PLDs with mutations H12-H47, E32-D34, and Y228-Y229-W230 displayed only residual activities. Results indicate that these mutant toxins are suitable for use as antigens to obtain neutralizing antisera with enhanced properties since they will be based on the most deleterious toxins in the venom and without causing severe harmful effects to the animals in which these sera are produced. Departamento de Biologia Celular Universidade Federal do Paraná Centro de Produção e Pesquisa de Imunobiológicos (CPPI) Departamento de Física Centro Multiusuário de Inovação Biomolecular Universidade Estadual Paulista (UNESP) Departamento de Biologia Estrutural Molecular e Genética Universidade Estadual de Ponta Grossa Departamento de Física Centro Multiusuário de Inovação Biomolecular Universidade Estadual Paulista (UNESP) FAPESP: 2015/13765-0 FAPESP: 2019/10475-2 FAPESP: 2915/18868-2 CNPq: 303868/2016-3 CNPq: 307338/2014-2 CNPq: 309940/2019-2 CNPq: 408633/2018-2

Bites evoked by Brown spiders (Loxosceles genus) are associated with skin injuries (cutaneous rash, itching, swelling, erythema and dermonecrosis) and systemic manifestations. Transcriptome analyses of Loxosceles venom glands showed that the venom has a complex composition containing toxins such as phospholipases-D, metalloproteases and hyaluronidases. Here, by screening the RNA from L. intermedia venom glands, we cloned a novel allergen toxin, and named LALLT (LoxoscelesAllergen-Like Toxin). Sequence analysis showed that LALLT is closely related to allergens from other spiders and RNA screening indicated the presence of LALLT orthologues in the venom of other Loxosceles spiders. Recombinant LALLT was expressed (~45 kDa) in baculovirus-infected insect cells and purified by affinity chromatography. Antibodies against different Loxosceles venoms cross-reacted with LALLT and antibodies against LALLT recognized three Loxosceles venoms, revealing epitopes identity. LALLT triggered paw edema in mice and erythema, edema and leukocyte infiltration into the dermis of rabbit skin. Also, LALLT induced vascular permeability in mice, degranulation of rat mesentery mast cells, as well as prompted degranulation and increased calcium influx in RBL-2H3 cells. Data reported suggest for the first time the existence of allergens in Loxosceles venoms and make LALLT available for clinical studies about allergenic events arisen by Loxosceles envenoming.

Spiders of the genus Loxosceles, popularly known as Brown spiders, are considered a serious public health issue, especially in regions of hot or temperate climates, such as parts of North and South America. Although the venoms of these arachnids are complex in molecular composition, often containing proteins with distinct biochemical characteristics, the literature has primarily described a family of toxins, the Phospholipases-D (PLDs), which are highly conserved in all Loxosceles species. PLDs trigger most of the major clinical symptoms of loxoscelism i.e., dermonecrosis, thrombocytopenia, hemolysis, and acute renal failure. The key role played by PLDs in the symptomatology of loxoscelism was first described 40 years ago, when researches purified a hemolytic toxin that cleaved sphingomyelin and generated choline, and was referred to as a Sphingomyelinase-D, which was subsequently changed to Phospholipase-D when it was demonstrated that the enzyme also cleaved other cellular phospholipids. In this review, we present the information gleaned over the last 40 years about PLDs from Loxosceles venoms especially with regard to the production and characterization of recombinant isoforms. The history of obtaining these toxins is discussed, as well as their molecular organization and mechanisms of interaction with their substrates. We will address cellular biology aspects of these toxins and how they can be used in the development of drugs to address inflammatory processes and loxoscelism. Present and future aspects of loxoscelism diagnosis will be discussed, as well as their biotechnological applications and actions expected for the future in this field.

LiTCTP is a toxin from the Translationally Controlled Tumor Protein (TCTP) family identified in Loxosceles brown spider venoms. These proteins are known as histamine-releasing factors (HRF). TCTPs participate in allergic and anaphylactic reactions, which suggest their potential role as therapeutic targets. The histaminergic effect of TCTP is related to its pro-inflammatory functions. An initial characterization of LiTCTP in animal models showed that this toxin can increase the microvascular permeability of skin vessels and induce paw edema in a dose-dependent manner. We evaluated the role of LiTCTP in vitro and in vivo in the inflammatory and allergic aspects that undergo the biological responses observed in Loxoscelism, the clinical condition after an accident with Loxosceles spiders. Our results showed LiTCTP recombinant toxin (LiRecTCTP) as an essential synergistic factor for the dermonecrotic toxin actions (LiRecDT1, known as the main toxin in the pathophysiology of Loxoscelism), revealing its contribution to the exacerbated inflammatory response clinically observed in envenomated patients.

Hyaluronidases are low expressed toxins of brown spider venoms, but, as highly active molecules, they present an important role as spreading factors. By degrading extracellular matrix components, these enzymes favor the diffusion of toxins in the affected tissue and at systemic level. Here, a novel isoform of hyaluronidase of Loxosceles intermedia Mello-Leitão (1934) venom was cloned, expressed in a baculovirus-insect cell expression system and fully active purified. This recombinant enzyme, named LiHyal2 (Loxosceles intermedia Hyaluronidase isoform 2), shares high identity with hyaluronidases of other spiders and scorpions. The catalytic and sugar binding amino acid residues are conserved in LiHyal2, human, and honeybee venom hyaluronidases and the molecular model of LiHyal2 shares major similarities with their crystal structures, including the active site. LiHyal2 was expressed as a 45 kDa protein and degraded hyaluronic acid (HA) and chondroitin sulphate as demonstrated by HA zymography and agarose gel electrophoresis. Lectin blot analysis revealed that LiHyal2 is post-translationally modified by the addition of high mannose N-linked carbohydrates. In vivo experiments showed that LiHyal2 potentialize dermonecrosis and edema induced by a recombinant phospholipase-D (PLD) of L. intermedia venom, as well as enhance the increase in capillary permeability triggered by this PLD, indicating that these toxins act synergistically during envenomation. Altogether, these results introduce a novel approach to express spider recombinant toxins, contribute to the elucidation of brown spider venom mechanisms and add to the development of a more specific treatment of envenomation victims.

The mechanism through which brown spiders (Loxosceles genus) cause dermonecrosis, dysregulated inflammatory responses, hemolysis and platelet aggregation, which are effects reported following spider bites, is currently attributed to the presence of phospholipase-D in the venom. In the present investigation, through two-dimensional immunoblotting, we observed immunological cross-reactivity for at least 25 spots in crude Loxosceles intermedia venom, indicating high expression levels for different isoforms of phospholipase-D. Using a recombinant phospholipase-D from the venom gland of L. intermedia (LiRecDT1) in phospholipid-degrading kinetic experiments, we determined that this phospholipase-D mainly hydrolyzes synthetic sphingomyelin in a time-dependent manner, generating ceramide 1-phosphate plus choline, as well as lysophosphatidylcholine, generating lysophosphatidic acid plus choline, but exhibits little activity against phosphatidylcholine. Through immunofluorescence assays with antibodies against LiRecDT1 and using a recombinant GFP-LiRecDT1 fusion protein, we observed direct binding of LiRecDT1 to the membrane of B16-F10 cells. We determined that LiRecDT1 hydrolyzes phospholipids in detergent extracts and from ghosts of B16-F10 cells, generating choline, indicating that the enzyme can access and modulate and has activity against membrane phospholipids. Additionally, using Fluo-4, a calcium-sensitive fluorophore, it was shown that treatment of cells with phospholipase-D induced an increase in the calcium concentration in the cytoplasm, but without altering viability or causing damage to cells. Finally, based on the known endogenous activity of phospholipase-D as an inducer of cell proliferation and the fact that LiRecDT1 binds to the cell surface, hydrolyzing phospholipids to generate bioactive lipids, we employed LiRecDT1 as an exogenous source of phospholipase-D in B16-F10 cells. Treatment of the cells was effective in increasing their proliferation in a time- and concentration-dependent manner, especially in the presence of synthetic sphingomyelin in the medium. The results described herein indicate the ability of brown spider phospholipase-D to induce the generation of bioactive phospholipids, calcium influx into the cytoplasm and cell proliferation, suggesting that this molecule can be used as a bioactive tool for experimental protocols in cell biology.

Brown spiders are venomous arthropods that use their venom for predation and defense. In humans, bites of these animals provoke injuries including dermonecrosis with gravitational spread of lesions, hematological abnormalities and impaired renal function. The signs and symptoms observed following a brown spider bite are called loxoscelism. Brown spider venom is a complex mixture of toxins enriched in low molecular mass proteins (4–40 kDa). Characterization of the venom confirmed the presence of three highly expressed protein classes: phospholipases D, metalloproteases (astacins) and insecticidal peptides (knottins). Recently, toxins with low levels of expression have also been found in Loxosceles venom, such as serine proteases, protease inhibitors (serpins), hyaluronidases, allergen-like toxins and histamine-releasing factors. The toxin belonging to the phospholipase-D family (also known as the dermonecrotic toxin) is the most studied class of brown spider toxins. This class of toxins single-handedly can induce inflammatory response, dermonecrosis, hemolysis, thrombocytopenia and renal failure. The functional role of the hyaluronidase toxin as a spreading factor in loxoscelism has also been demonstrated. However, the biological characterization of other toxins remains unclear and the mechanism by which Loxosceles toxins exert their noxious effects is yet to be fully elucidated. The aim of this review is to provide an insight into brown spider venom toxins and toxicology, including a description of historical data already available in the literature. In this review article, the identification processes of novel Loxosceles toxins by molecular biology and proteomic approaches, their biological characterization and structural description based on x-ray crystallography and putative biotechnological uses are described along with the future perspectives in this field.

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.

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 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.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:25:55Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:36:16Z : No. of bitstreams: 1 2-s2.0-79959208567.pdf: 1564068 bytes, checksum: e4ff2769199a62af31ebb67b9ff28280 (MD5) Made available in DSpace on 2014-05-27T11:25:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-06-17 Phospholipases D (PLDs) are principally responsible for the local and systemic effects of Loxosceles envenomation including dermonecrosis and hemolysis. Despite their clinical relevance in loxoscelism, to date, only the SMase I from Loxosceles laeta, a class I member, has been structurally characterized. The crystal structure of a class II member from Loxosceles intermedia venom has been determined at 1.7. Å resolution. Structural comparison to the class I member showed that the presence of an additional disulphide bridge which links the catalytic loop to the flexible loop significantly changes the volume and shape of the catalytic cleft. An examination of the crystal structures of PLD homologues in the presence of low molecular weight compounds at their active sites suggests the existence of a ligand-dependent rotamer conformation of the highly conserved residue Trp230 (equivalent to Trp192 in the glycerophosphodiester phosphodiesterase from Thermus thermophofilus, PDB code: 1VD6) indicating its role in substrate binding in both enzymes. Sequence and structural analyses suggest that the reduced sphingomyelinase activity observed in some class IIb PLDs is probably due to point mutations which lead to a different substrate preference. © 2011 Elsevier Inc. Laboratório Nacional de Biociências (LNBio) Centro Nacional de Pesquisa em Energia e Materiais, Campinas, 13083-970 SP Centro Multiusuário de Inovação Biomolecular Departamento de Física Universidade Estadual Paulista (UNESP), São José do Rio Preto, 15054-000 SP Departamento de Biologia Celular Universidade Federal do Paraná, Curitiba, 80060-000 PR Department of Structural Molecular Biology and Genetics State University of Ponta Grossa, Ponta Grossa, Paraná Centro Multiusuário de Inovação Biomolecular Departamento de Física Universidade Estadual Paulista (UNESP), São José do Rio Preto, 15054-000 SP

Made available in DSpace on 2013-08-28T14:10:43Z (GMT). No. of bitstreams: 1 WOS000287030600013.pdf: 142138 bytes, checksum: dc227d9e9af74a3255e4e8fd5be8eb6b (MD5) Made available in DSpace on 2013-09-30T18:43:35Z (GMT). No. of bitstreams: 2 WOS000287030600013.pdf: 142138 bytes, checksum: dc227d9e9af74a3255e4e8fd5be8eb6b (MD5) WOS000287030600013.pdf.txt: 15752 bytes, checksum: b414a9f7408062b13033ef55c9777133 (MD5) Previous issue date: 2011-02-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T14:02:41Z No. of bitstreams: 2 WOS000287030600013.pdf: 142138 bytes, checksum: dc227d9e9af74a3255e4e8fd5be8eb6b (MD5) WOS000287030600013.pdf.txt: 15752 bytes, checksum: b414a9f7408062b13033ef55c9777133 (MD5) Made available in DSpace on 2014-05-20T14:02:41Z (GMT). No. of bitstreams: 2 WOS000287030600013.pdf: 142138 bytes, checksum: dc227d9e9af74a3255e4e8fd5be8eb6b (MD5) WOS000287030600013.pdf.txt: 15752 bytes, checksum: b414a9f7408062b13033ef55c9777133 (MD5) Previous issue date: 2011-02-01 Secretaria de Estado de Ciencia, Tecnologia e Ensino Superior (SETI) do Parana Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná (FAADCT/PR) TWAS Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Phospholipases D are the major dermonecrotic component of Loxosceles venom and catalyze the hydrolysis of phospholipids, resulting in the formation of lipid mediators such as ceramide-1-phosphate and lysophosphatidic acid which can induce pathological and biological responses. Phospholipases D can be classified into two classes depending on their catalytic efficiency and the presence of an additional disulfide bridge. In this work, both wild-type and H12A-mutant forms of the class II phospholipase D from L. intermedia venom were crystallized. Wild-type and H12A-mutant crystals were grown under very similar conditions using PEG 200 as a precipitant and belonged to space group P12(1)1, with unit-cell parameters a = 50.1, b = 49.5, c = 56.5 angstrom, beta = 105.9 degrees. Wild-type and H12A-mutant crystals diffracted to maximum resolutions of 1.95 and 1.60 angstrom, respectively. Univ Estadual Paulista, UNESP, Dept Fis, Ctr Multiusuario Inovacao Biomol, BR-15054000 Sao Jose do Rio Preto, SP, Brazil Ctr Nacl Pesquisa Energia & Mat, LNBio, BR-13083970 Campinas, SP, Brazil Univ Fed Parana, Dept Biol Celular, BR-81531960 Curitiba, Parana, Brazil Univ Estadual Ponta Grossa, Dept Biol Estrutural Biol Mol & Genet, BR-84030900 Ponta Grossa, PR, Brazil Univ Estadual Paulista, UNESP, Dept Fis, Ctr Multiusuario Inovacao Biomol, BR-15054000 Sao Jose do Rio Preto, SP, Brazil

Bites from brown spiders (Loxosceles genus) have clinical manifestations including skin necrosis with gravitational spreading, and systemic involvement that may include renal failure, hemolysis, and thrombocytopenia. Mice were exposed to recombinant wild-type phospholipase-D, or to an isoform with a mutation in the catalytic domain resulting in no phospholipasic activity. Renal biopsies from mice treated with the wild-type toxin showed glomerular edema, erythrocytes and collapse of Bowman's space, edema and deposition of proteinaceous material within the tubular lumen. Ultrastructural analyses confirmed cytotoxicity by demonstrating disorders of glomerulus at foot processes and at fenestrated endothelium. Tubule alterations include deposits of amorphous material and edema, as well as an increase of epithelial cytoplasmic multivesicular bodies and electron-dense structures. There was an absence of nephrotoxicity in mice treated with the mutated toxin. Analyses of urine and blood showed that wild type toxin induced hematuria and elevation of blood urea, while treatment with mutated toxin caused no changes. Mouse lethality experiments also showed oliguria and mortality after treatment with wild-type toxin, but not following exposure to the mutated toxin. Immunofluorescence using antibodies to phospholipase-D toxin showed deposition of both toxins along the renal tubular structures as detected by confocal microscopy. Immunoblots of urine showed a 30 kDa band in samples from animals treated with wild-type toxin, but no band from mice exposed to mutated toxin. Wild-type toxin treatment caused cytoplasmic vacuolization, impaired spreading, reduction of cellular viability, and cell-cell and cell-substratum detachment in MDCK cells, while treatment with mutated isoform had no effect. Finally, there is a direct correlation between toxin activity on cell membrane phospholipids generating choline and cytotoxicity. We have defined for the first time a molecular mechanism for Loxosceles venom nephrotoxicity that is dependent on the catalytic activity of phospholipase-D toxin.