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Prof. Silvio Veiga
Universidade Federal do Paraná-UFPR

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0 Toxinology
0 Venom
0 recombinant toxin
0 phospholipases
0 brown spider

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Review article
Published: 17 June 2021 in Frontiers in Molecular Biosciences
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Brown spider (genus Loxosceles) venoms are mainly composed of protein toxins used for predation and defense. Bites of these spiders most commonly produce a local dermonecrotic lesion with gravitational spread, edema and hemorrhage, which together are defined as cutaneous loxoscelism. Systemic loxoscelism, such as hematological abnormalities and renal injury, are less frequent but more lethal. Some Loxosceles venom toxins have already been isolated and extensively studied, such as phospholipases D (PLDs), which have been recombinantly expressed and were proven to reproduce toxic activities associated to the whole venom. PLDs have a notable potential to be engineered and converted in non-toxic antigens to produce a new generation of antivenoms or vaccines. PLDs also can serve as tools to discover inhibitors to be used as therapeutic agents. Other Loxosceles toxins have been identified and functionally characterized, such as hyaluronidases, allergen factor, serpin, TCTP and knottins (ICK peptides). All these toxins were produced as recombinant molecules and are biologically active molecules that can be used as tools for the potential development of chemical candidates to tackle many medical and biological threats, acting, for instance, as antitumoral, insecticides, analgesic, antigens for allergy tests and biochemical reagents for cell studies. In addition, these recombinant toxins may be useful to develop a rational therapy for loxoscelism. This review summarizes the main candidates for the development of drugs and biotechnological inputs that have been described in Brown spider venoms.

ACS Style

Luiza Helena Gremski; Fernando Hitomi Matsubara; Nayanne Louise Costacurta Polli; Bruno Cesar Antunes; Pedro Henrique De Caires Schluga; Hanna Câmara da Justa; João Carlos Minozzo; Ana Carolina Martins Wille; Andrea Senff-Ribeiro; Silvio Sanches Veiga. Prospective Use of Brown Spider Venom Toxins as Therapeutic and Biotechnological Inputs. Frontiers in Molecular Biosciences 2021, 8, 1 .

AMA Style

Luiza Helena Gremski, Fernando Hitomi Matsubara, Nayanne Louise Costacurta Polli, Bruno Cesar Antunes, Pedro Henrique De Caires Schluga, Hanna Câmara da Justa, João Carlos Minozzo, Ana Carolina Martins Wille, Andrea Senff-Ribeiro, Silvio Sanches Veiga. Prospective Use of Brown Spider Venom Toxins as Therapeutic and Biotechnological Inputs. Frontiers in Molecular Biosciences. 2021; 8 ():1.

Chicago/Turabian Style

Luiza Helena Gremski; Fernando Hitomi Matsubara; Nayanne Louise Costacurta Polli; Bruno Cesar Antunes; Pedro Henrique De Caires Schluga; Hanna Câmara da Justa; João Carlos Minozzo; Ana Carolina Martins Wille; Andrea Senff-Ribeiro; Silvio Sanches Veiga. 2021. "Prospective Use of Brown Spider Venom Toxins as Therapeutic and Biotechnological Inputs." Frontiers in Molecular Biosciences 8, no. : 1.

Journal article
Published: 21 May 2021 in International Journal of Biological Macromolecules
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Several classes of toxins are present in the venom of Brown spiders (Loxosceles genus), some of them are highly expressed and others are less expressed. In this work, we aimed to clone the sequence of a little expressed novel toxin from Loxosceles venom identified as a serine protease inhibitor (serpin), as well as to express and characterize its biochemical and biological properties. It was named LSPILT, derived from Loxosceles serine protease inhibitor-like toxin. Multiple alignment analysis revealed high identity between LSPILT and other serpin molecules from spiders and crab. LSPILT was produced in baculovirus-infected insect cells, resulting in a 46-kDa protein fused to a His-tag. Immunological assays showed epitopes in LSPILT that resemble native venom toxins of Loxosceles spiders. The inhibitory activity of LSPILT on trypsin was found both by reverse zymography and fluorescent gelatin-degradation assay. Additionally, LSPILT inhibited the complement-dependent lysis of Trypanosoma cruzi epimastigotes, reduced thrombin-dependent clotting and suppressed B16-F10 melanoma cells migration. Results described herein prove the existence of conserved serpin-like toxins in Loxosceles venoms. The availability of a recombinant serpin enabled the determination of its biological and biochemical properties and indicates potential applications in future studies regarding the pathophysiology of the envenoming or for biotechnological purposes.

ACS Style

Zelinda Schemczssen-Graeff; Hanna Câmara da Justa; Jenifer Nowatzki; Antonielle Beatriz Baldissera; Nayanne Louise Costacurta Polli; Elidiana De-Bona; Izadora Volpato Rossi; Marcel Ivan Ramirez; João Carlos Minozzo; Fernando Hitomi Matsubara; Andrea Senff-Ribeiro; Luiza Helena Gremski; Silvio Sanches Veiga. Description of a serpin toxin in Loxosceles (Brown spider) venoms: Cloning, expression in baculovirus-infected insect cells and functional characterization. International Journal of Biological Macromolecules 2021, 183, 1607 -1620.

AMA Style

Zelinda Schemczssen-Graeff, Hanna Câmara da Justa, Jenifer Nowatzki, Antonielle Beatriz Baldissera, Nayanne Louise Costacurta Polli, Elidiana De-Bona, Izadora Volpato Rossi, Marcel Ivan Ramirez, João Carlos Minozzo, Fernando Hitomi Matsubara, Andrea Senff-Ribeiro, Luiza Helena Gremski, Silvio Sanches Veiga. Description of a serpin toxin in Loxosceles (Brown spider) venoms: Cloning, expression in baculovirus-infected insect cells and functional characterization. International Journal of Biological Macromolecules. 2021; 183 ():1607-1620.

Chicago/Turabian Style

Zelinda Schemczssen-Graeff; Hanna Câmara da Justa; Jenifer Nowatzki; Antonielle Beatriz Baldissera; Nayanne Louise Costacurta Polli; Elidiana De-Bona; Izadora Volpato Rossi; Marcel Ivan Ramirez; João Carlos Minozzo; Fernando Hitomi Matsubara; Andrea Senff-Ribeiro; Luiza Helena Gremski; Silvio Sanches Veiga. 2021. "Description of a serpin toxin in Loxosceles (Brown spider) venoms: Cloning, expression in baculovirus-infected insect cells and functional characterization." International Journal of Biological Macromolecules 183, no. : 1607-1620.

Journal article
Published: 21 March 2021 in Biomedicines
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Phospholipases-D (PLDs) found in Loxosceles spiders’ venoms are responsible for the dermonecrosis 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 characterized. 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.

ACS Style

Thaís da Silva; Fernando de Castro; Larissa Vuitika; Nayanne Polli; Bruno Antunes; Marianna Bóia-Ferreira; João Minozzo; Ricardo Mariutti; Fernando Matsubara; Raghuvir Arni; Ana Wille; Andrea Senff-Ribeiro; Luiza Gremski; Silvio Veiga. Brown Spiders’ Phospholipases-D with Potential Therapeutic Applications: Functional Assessment of Mutant Isoforms. Biomedicines 2021, 9, 320 .

AMA Style

Thaís da Silva, Fernando de Castro, Larissa Vuitika, Nayanne Polli, Bruno Antunes, Marianna Bóia-Ferreira, João Minozzo, Ricardo Mariutti, Fernando Matsubara, Raghuvir Arni, Ana Wille, Andrea Senff-Ribeiro, Luiza Gremski, Silvio Veiga. Brown Spiders’ Phospholipases-D with Potential Therapeutic Applications: Functional Assessment of Mutant Isoforms. Biomedicines. 2021; 9 (3):320.

Chicago/Turabian Style

Thaís da Silva; Fernando de Castro; Larissa Vuitika; Nayanne Polli; Bruno Antunes; Marianna Bóia-Ferreira; João Minozzo; Ricardo Mariutti; Fernando Matsubara; Raghuvir Arni; Ana Wille; Andrea Senff-Ribeiro; Luiza Gremski; Silvio Veiga. 2021. "Brown Spiders’ Phospholipases-D with Potential Therapeutic Applications: Functional Assessment of Mutant Isoforms." Biomedicines 9, no. 3: 320.

Journal article
Published: 01 January 2021 in Journal of Venomous Animals and Toxins including Tropical Diseases
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Accidents caused by the bites of brown spiders (Loxosceles) generate a clinical condition that often includes a threatening necrotic skin lesion near the bite site along with a remarkable inflammatory response. Systemic disorders such as hemolysis, thrombocytopenia, and acute renal failure may occur, but are much less frequent than the local damage. It is already known that phospholipases D, highly expressed toxins in Loxosceles venom, can induce most of these injuries. However, this spider venom has a great range of toxins that probably act synergistically to enhance toxicity. The other protein classes remain poorly explored due to the difficulty in obtaining sufficient amounts of them for a thorough investigation. They include astacins (metalloproteases), serine proteases, knottins, translationally controlled tumor proteins (TCTP), hyaluronidases, allergens and serpins. It has already been shown that some of them, according to their characteristics, may participate to some extent in the development of loxoscelism. In addition, all of these toxins present potential application in several areas. The present review article summarizes information regarding some functional aspects of the protein classes listed above, discusses the directions that could be taken to materialize a comprehensive investigation on each of these toxins as well as highlights the importance of exploring the full venom repertoire.

ACS Style

Luiza Helena Gremski; Fernando Hitomi Matsubara; Hanna Câmara da Justa; Zelinda Schemczssen-Graeff; Antonielle Beatriz Baldissera; Pedro Henrique De Caires Schluga; Isabel De Oliveira Leite; Marianna Boia-Ferreira; Ana Carolina Martins Wille; Andrea Senff-Ribeiro; Silvio Sanches Veiga. Brown spider venom toxins: what are the functions of astacins, serine proteases, hyaluronidases, allergens, TCTP, serpins and knottins? Journal of Venomous Animals and Toxins including Tropical Diseases 2021, 27, 1 .

AMA Style

Luiza Helena Gremski, Fernando Hitomi Matsubara, Hanna Câmara da Justa, Zelinda Schemczssen-Graeff, Antonielle Beatriz Baldissera, Pedro Henrique De Caires Schluga, Isabel De Oliveira Leite, Marianna Boia-Ferreira, Ana Carolina Martins Wille, Andrea Senff-Ribeiro, Silvio Sanches Veiga. Brown spider venom toxins: what are the functions of astacins, serine proteases, hyaluronidases, allergens, TCTP, serpins and knottins? Journal of Venomous Animals and Toxins including Tropical Diseases. 2021; 27 ():1.

Chicago/Turabian Style

Luiza Helena Gremski; Fernando Hitomi Matsubara; Hanna Câmara da Justa; Zelinda Schemczssen-Graeff; Antonielle Beatriz Baldissera; Pedro Henrique De Caires Schluga; Isabel De Oliveira Leite; Marianna Boia-Ferreira; Ana Carolina Martins Wille; Andrea Senff-Ribeiro; Silvio Sanches Veiga. 2021. "Brown spider venom toxins: what are the functions of astacins, serine proteases, hyaluronidases, allergens, TCTP, serpins and knottins?" Journal of Venomous Animals and Toxins including Tropical Diseases 27, no. : 1.

Journal article
Published: 30 August 2020 in International Journal of Biological Macromolecules
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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 (Loxosceles Allergen-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.

ACS Style

Hanna Câmara da Justa; Fernando Hitomi Matsubara; Elidiana De-Bona; Zelinda Schemczssen-Graeff; Nayanne Louise Costacurta Polli; Thiago Lopes de Mari; Marianna Boia-Ferreira; João Carlos Minozzo; Ana Carolina Martins Wille; Andrea Senff-Ribeiro; Luiza Helena Gremski; Silvio Sanches Veiga. LALLT (Loxosceles Allergen-Like Toxin) from the venom of Loxosceles intermedia: Recombinant expression in insect cells and characterization as a molecule with allergenic properties. International Journal of Biological Macromolecules 2020, 164, 3984 -3999.

AMA Style

Hanna Câmara da Justa, Fernando Hitomi Matsubara, Elidiana De-Bona, Zelinda Schemczssen-Graeff, Nayanne Louise Costacurta Polli, Thiago Lopes de Mari, Marianna Boia-Ferreira, João Carlos Minozzo, Ana Carolina Martins Wille, Andrea Senff-Ribeiro, Luiza Helena Gremski, Silvio Sanches Veiga. LALLT (Loxosceles Allergen-Like Toxin) from the venom of Loxosceles intermedia: Recombinant expression in insect cells and characterization as a molecule with allergenic properties. International Journal of Biological Macromolecules. 2020; 164 ():3984-3999.

Chicago/Turabian Style

Hanna Câmara da Justa; Fernando Hitomi Matsubara; Elidiana De-Bona; Zelinda Schemczssen-Graeff; Nayanne Louise Costacurta Polli; Thiago Lopes de Mari; Marianna Boia-Ferreira; João Carlos Minozzo; Ana Carolina Martins Wille; Andrea Senff-Ribeiro; Luiza Helena Gremski; Silvio Sanches Veiga. 2020. "LALLT (Loxosceles Allergen-Like Toxin) from the venom of Loxosceles intermedia: Recombinant expression in insect cells and characterization as a molecule with allergenic properties." International Journal of Biological Macromolecules 164, no. : 3984-3999.

Review
Published: 06 March 2020 in Toxins
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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.

ACS Style

Luiza Helena Gremski; Hanna Justa; Thaís Pereira Da Silva; Nayanne Louise Costacurta Polli; Bruno César Antunes; João Carlos Minozzo; Ana Carolina Martins Wille; Andrea Senff-Ribeiro; Raghuvir Krishnaswamy Arni; Silvio Sanches Veiga. Forty Years of the Description of Brown Spider Venom Phospholipases-D. Toxins 2020, 12, 164 .

AMA Style

Luiza Helena Gremski, Hanna Justa, Thaís Pereira Da Silva, Nayanne Louise Costacurta Polli, Bruno César Antunes, João Carlos Minozzo, Ana Carolina Martins Wille, Andrea Senff-Ribeiro, Raghuvir Krishnaswamy Arni, Silvio Sanches Veiga. Forty Years of the Description of Brown Spider Venom Phospholipases-D. Toxins. 2020; 12 (3):164.

Chicago/Turabian Style

Luiza Helena Gremski; Hanna Justa; Thaís Pereira Da Silva; Nayanne Louise Costacurta Polli; Bruno César Antunes; João Carlos Minozzo; Ana Carolina Martins Wille; Andrea Senff-Ribeiro; Raghuvir Krishnaswamy Arni; Silvio Sanches Veiga. 2020. "Forty Years of the Description of Brown Spider Venom Phospholipases-D." Toxins 12, no. 3: 164.

Journal article
Published: 22 November 2019 in Cells
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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.

ACS Style

Marianna Boia-Ferreira; Kamila G. Moreno; Alana B. C. Basílio; Lucas P. Da Silva; Larissa Vuitika; Bruna Soley; Ana Carolina M. Wille; Lucélia Donatti; Katia C. Barbaro; Olga M. Chaim; Luiza Helena Gremski; Silvio S. Veiga; Andrea Senff-Ribeiro. TCTP from Loxosceles Intermedia (Brown Spider) Venom Contributes to the Allergic and Inflammatory Response of Cutaneous Loxoscelism. Cells 2019, 8, 1489 .

AMA Style

Marianna Boia-Ferreira, Kamila G. Moreno, Alana B. C. Basílio, Lucas P. Da Silva, Larissa Vuitika, Bruna Soley, Ana Carolina M. Wille, Lucélia Donatti, Katia C. Barbaro, Olga M. Chaim, Luiza Helena Gremski, Silvio S. Veiga, Andrea Senff-Ribeiro. TCTP from Loxosceles Intermedia (Brown Spider) Venom Contributes to the Allergic and Inflammatory Response of Cutaneous Loxoscelism. Cells. 2019; 8 (12):1489.

Chicago/Turabian Style

Marianna Boia-Ferreira; Kamila G. Moreno; Alana B. C. Basílio; Lucas P. Da Silva; Larissa Vuitika; Bruna Soley; Ana Carolina M. Wille; Lucélia Donatti; Katia C. Barbaro; Olga M. Chaim; Luiza Helena Gremski; Silvio S. Veiga; Andrea Senff-Ribeiro. 2019. "TCTP from Loxosceles Intermedia (Brown Spider) Venom Contributes to the Allergic and Inflammatory Response of Cutaneous Loxoscelism." Cells 8, no. 12: 1489.

Review
Published: 19 June 2019 in Toxins
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Brown spider envenomation results in dermonecrosis with gravitational spreading characterized by a marked inflammatory reaction and with lower prevalence of systemic manifestations such as renal failure and hematological disturbances. Several toxins make up the venom of these species, and they are mainly peptides and proteins ranging from 5-40 kDa. The venoms have three major families of toxins: phospholipases-D, astacin-like metalloproteases, and the inhibitor cystine knot (ICK) peptides. Serine proteases, serpins, hyaluronidases, venom allergens, and a translationally controlled tumor protein (TCTP) are also present. Toxins hold essential biological properties that enable interactions with a range of distinct molecular targets. Therefore, the application of toxins as research tools and clinical products motivates repurposing their uses of interest. This review aims to discuss possibilities for brown spider venom toxins as putative models for designing molecules likely for therapeutics based on the status quo of brown spider venoms. Herein, we explore new possibilities for the venom components in the context of their biochemical and biological features, likewise their cellular targets, three-dimensional structures, and mechanisms of action.

ACS Style

Daniele Moreira; Fernando Hitomi Matsubara; Zelinda Schemczssen-Graeff; Elidiana De Bona; Vanessa Ribeiro Heidemann; Clara Guerra-Duarte; Luiza Helena Gremski; Carlos Chávez-Olórtegui; Andrea Senff-Ribeiro; Olga Meiri Chaim; Raghuvir Krishnaswamy Arni; Silvio Sanches Veiga. Brown Spider (Loxosceles) Venom Toxins as Potential Biotools for the Development of Novel Therapeutics. Toxins 2019, 11, 355 .

AMA Style

Daniele Moreira, Fernando Hitomi Matsubara, Zelinda Schemczssen-Graeff, Elidiana De Bona, Vanessa Ribeiro Heidemann, Clara Guerra-Duarte, Luiza Helena Gremski, Carlos Chávez-Olórtegui, Andrea Senff-Ribeiro, Olga Meiri Chaim, Raghuvir Krishnaswamy Arni, Silvio Sanches Veiga. Brown Spider (Loxosceles) Venom Toxins as Potential Biotools for the Development of Novel Therapeutics. Toxins. 2019; 11 (6):355.

Chicago/Turabian Style

Daniele Moreira; Fernando Hitomi Matsubara; Zelinda Schemczssen-Graeff; Elidiana De Bona; Vanessa Ribeiro Heidemann; Clara Guerra-Duarte; Luiza Helena Gremski; Carlos Chávez-Olórtegui; Andrea Senff-Ribeiro; Olga Meiri Chaim; Raghuvir Krishnaswamy Arni; Silvio Sanches Veiga. 2019. "Brown Spider (Loxosceles) Venom Toxins as Potential Biotools for the Development of Novel Therapeutics." Toxins 11, no. 6: 355.

Review
Published: 08 February 2017 in Journal of Venomous Animals and Toxins including Tropical Diseases
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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.

ACS Style

Daniele Chaves-Moreira; Andrea Senff-Ribeiro; Ana Carolina Martins Wille; Luiza Helena Gremski; Olga Meiri Chaim; Silvio Sanches Veiga. Highlights in the knowledge of brown spider toxins. Journal of Venomous Animals and Toxins including Tropical Diseases 2017, 23, 6 .

AMA Style

Daniele Chaves-Moreira, Andrea Senff-Ribeiro, Ana Carolina Martins Wille, Luiza Helena Gremski, Olga Meiri Chaim, Silvio Sanches Veiga. Highlights in the knowledge of brown spider toxins. Journal of Venomous Animals and Toxins including Tropical Diseases. 2017; 23 (1):6.

Chicago/Turabian Style

Daniele Chaves-Moreira; Andrea Senff-Ribeiro; Ana Carolina Martins Wille; Luiza Helena Gremski; Olga Meiri Chaim; Silvio Sanches Veiga. 2017. "Highlights in the knowledge of brown spider toxins." Journal of Venomous Animals and Toxins including Tropical Diseases 23, no. 1: 6.

Article
Published: 26 October 2016 in Journal of Cellular Biochemistry
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Loxoscelism refers to the clinical symptoms that develop after brown spider bites. Brown spider venoms contain several phospholipase-D isoforms, which are the main toxins responsible for both the cutaneous and systemic effects of loxoscelism. Understanding of the phospholipase-D catalytic mechanism is crucial for the development of specific treatment that could reverse the toxic effects caused by the spider bite. Based on enzymatic, biological, structural, and thermodynamic tests, we show some features suitable for designing drugs against loxoscelism. Firstly, through molecular docking and molecular dynamics predictions, we found three different molecules (Suramin, Vu0155056, and Vu0359595) that were able to bind the enzyme's catalytic site and interact with catalytically important residues (His12 or His47) and with the Mg2+ co-factor. The binding promoted a decrease in the recombinant brown spider venom phospholipase-D (LiRecDT1) enzymatic activity. Furthermore, the presence of the inhibitors reduced the hemolytic, dermonecrotic, and inflammatory activities of the venom toxin in biological assays. Altogether, these results indicate the mode of action of three different LiRecDT1 inhibitors, which were able to prevent the venom toxic effects. This strengthen the idea of the importance of designing a specific drug to treat the serious clinical symptoms caused by the brown spider bite, a public health problem in several parts of the world, and until now without specific treatment. J. Cell. Biochem. 9999: 1–13, 2016.

ACS Style

Daniele Moreira; Fabio Rogerio de Moraes; Icaro Caruso; Olga Chaim; Andrea Senff-Ribeiro; Anwar Ullah; Luciane Sussuchi da Silva; Jorge Chahine; Raghuvir Arni; Silvio Sanches Veiga. Potential Implications for Designing Drugs Against the Brown Spider Venom Phospholipase-D. Journal of Cellular Biochemistry 2016, 118, 726 -738.

AMA Style

Daniele Moreira, Fabio Rogerio de Moraes, Icaro Caruso, Olga Chaim, Andrea Senff-Ribeiro, Anwar Ullah, Luciane Sussuchi da Silva, Jorge Chahine, Raghuvir Arni, Silvio Sanches Veiga. Potential Implications for Designing Drugs Against the Brown Spider Venom Phospholipase-D. Journal of Cellular Biochemistry. 2016; 118 (4):726-738.

Chicago/Turabian Style

Daniele Moreira; Fabio Rogerio de Moraes; Icaro Caruso; Olga Chaim; Andrea Senff-Ribeiro; Anwar Ullah; Luciane Sussuchi da Silva; Jorge Chahine; Raghuvir Arni; Silvio Sanches Veiga. 2016. "Potential Implications for Designing Drugs Against the Brown Spider Venom Phospholipase-D." Journal of Cellular Biochemistry 118, no. 4: 726-738.

Evaluation study
Published: 15 October 2016 in Insect Molecular Biology
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Loxosceles intermedia venom comprises a complex mixture of proteins, glycoproteins and low molecular mass peptides that act synergistically to immobilize envenomed prey. Analysis of a venom-gland transcriptome from L. intermedia revealed that knottins, also known as inhibitor cystine knot peptides, are the most abundant class of toxins expressed in this species. Knottin peptides contain a particular arrangement of intramolecular disulphide bonds, and these peptides typically act upon ion channels or receptors in the insect nervous system, triggering paralysis or other lethal effects. Herein, we focused on a knottin peptide with 53 amino acid residues from L. intermedia venom. The recombinant peptide, named U2-sicaritoxin-Li1b (Li1b), was obtained by expression in the periplasm of Escherichia coli. The recombinant peptide induced irreversible flaccid paralysis in sheep blowflies. We screened for knottin-encoding sequences in total RNA extracts from two other Loxosceles species, Loxosceles gaucho and Loxosceles laeta, which revealed that knottin peptides constitute a conserved family of toxins in the Loxosceles genus. The insecticidal activity of U2-SCTX-Li1b, together with the large number of knottin peptides encoded in Loxosceles venom glands, suggests that studies of these venoms might facilitate future biotechnological applications of these toxins.

ACS Style

F. H. Matsubara; Gabriel Meissner; Volker Herzig; Hanna Justa; B. C. L. Dias; L. H. Gremski; O. M. Chaim; G. F. King; Dilza Trevisan-Silva; W. Gremski; A. Senff-Ribeiro; S. S. Veiga. Insecticidal activity of a recombinant knottin peptide from Loxosceles intermedia venom and recognition of these peptides as a conserved family in the genus. Insect Molecular Biology 2016, 26, 25 -34.

AMA Style

F. H. Matsubara, Gabriel Meissner, Volker Herzig, Hanna Justa, B. C. L. Dias, L. H. Gremski, O. M. Chaim, G. F. King, Dilza Trevisan-Silva, W. Gremski, A. Senff-Ribeiro, S. S. Veiga. Insecticidal activity of a recombinant knottin peptide from Loxosceles intermedia venom and recognition of these peptides as a conserved family in the genus. Insect Molecular Biology. 2016; 26 (1):25-34.

Chicago/Turabian Style

F. H. Matsubara; Gabriel Meissner; Volker Herzig; Hanna Justa; B. C. L. Dias; L. H. Gremski; O. M. Chaim; G. F. King; Dilza Trevisan-Silva; W. Gremski; A. Senff-Ribeiro; S. S. Veiga. 2016. "Insecticidal activity of a recombinant knottin peptide from Loxosceles intermedia venom and recognition of these peptides as a conserved family in the genus." Insect Molecular Biology 26, no. 1: 25-34.

Journal article
Published: 01 September 2016 in Biochimie
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Loxosceles spiders’ venom comprises a complex mixture of biologically active toxins, mostly consisting of low molecular mass components (2-40 kDa). Amongst, isoforms of astacin-like metalloproteases were identified through transcriptome and proteome analyses. Only LALP1 (Loxosceles Astacin-Like protease 1) has been characterized. Herein, we characterized LALP3 as a novel recombinant astacin-like metalloprotease isoform from L. intermedia venom. LALP3 cDNA was cloned in pET-SUMO vector, and its soluble heterologous expression was performed using a SUMO tag added to LALP3 to achieve solubility in E. coli SHuffle T7 Express LysY cells, which express the disulfide bond isomerase DsbC. Protein purification was conducted by Ni-NTA Agarose resin and assayed for purity by SDS-PAGE under reducing conditions. Immunoblotting analyses were performed with specific antibodies recognizing LALP1 and whole venom. Western blotting showed linear epitopes from recombinant LALP3 that cross-reacted with LALP1, and dot blotting revealed conformational epitopes with native venom astacins. Mass spectrometry analysis revealed that the recombinant expressed protein is an astacin-like metalloprotease from L. intermedia venom. Furthermore, molecular modeling of LALP3 revealed that this isoform contains the zinc binding and Met-turn motifs, forming the active site, as has been observed in astacins. These data confirmed that LALP3, which was successfully obtained by heterologous expression using a prokaryote system, is a new astacin-like metalloprotease isoform present in L. intermedia venom.

ACS Style

Adriano M. Morgon; Matheus R. Belisario-Ferrari; Dilza Trevisan-Silva; Gabriel Meissner; Larissa Vuitika; Brenda Marin; Alexandre Keiji Tashima; Luiza H. Gremski; Waldemiro Gremski; Andrea Senff-Ribeiro; Silvio S. Veiga; Olga M. Chaim. Expression and immunological cross-reactivity of LALP3, a novel astacin-like metalloprotease from brown spider (Loxosceles intermedia) venom. Biochimie 2016, 128-129, 8 -19.

AMA Style

Adriano M. Morgon, Matheus R. Belisario-Ferrari, Dilza Trevisan-Silva, Gabriel Meissner, Larissa Vuitika, Brenda Marin, Alexandre Keiji Tashima, Luiza H. Gremski, Waldemiro Gremski, Andrea Senff-Ribeiro, Silvio S. Veiga, Olga M. Chaim. Expression and immunological cross-reactivity of LALP3, a novel astacin-like metalloprotease from brown spider (Loxosceles intermedia) venom. Biochimie. 2016; 128-129 ():8-19.

Chicago/Turabian Style

Adriano M. Morgon; Matheus R. Belisario-Ferrari; Dilza Trevisan-Silva; Gabriel Meissner; Larissa Vuitika; Brenda Marin; Alexandre Keiji Tashima; Luiza H. Gremski; Waldemiro Gremski; Andrea Senff-Ribeiro; Silvio S. Veiga; Olga M. Chaim. 2016. "Expression and immunological cross-reactivity of LALP3, a novel astacin-like metalloprotease from brown spider (Loxosceles intermedia) venom." Biochimie 128-129, no. : 8-19.

Journal article
Published: 01 September 2016 in Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids
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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.

ACS Style

L. Vuitika; Daniele Moreira; Icaro Caruso; M.A. Lima; F.H. Matsubara; M.T. Murakami; H.K. Takahashi; Marcos Toledo; M.A. Coronado; Helena Nader; Andrea Senff-Ribeiro; Olga Chaim; Raghuvir Arni; S.S. Veiga. Active site mapping of Loxosceles phospholipases D: Biochemical and biological features. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 2016, 1861, 970 -979.

AMA Style

L. Vuitika, Daniele Moreira, Icaro Caruso, M.A. Lima, F.H. Matsubara, M.T. Murakami, H.K. Takahashi, Marcos Toledo, M.A. Coronado, Helena Nader, Andrea Senff-Ribeiro, Olga Chaim, Raghuvir Arni, S.S. Veiga. Active site mapping of Loxosceles phospholipases D: Biochemical and biological features. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. 2016; 1861 (9):970-979.

Chicago/Turabian Style

L. Vuitika; Daniele Moreira; Icaro Caruso; M.A. Lima; F.H. Matsubara; M.T. Murakami; H.K. Takahashi; Marcos Toledo; M.A. Coronado; Helena Nader; Andrea Senff-Ribeiro; Olga Chaim; Raghuvir Arni; S.S. Veiga. 2016. "Active site mapping of Loxosceles phospholipases D: Biochemical and biological features." Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 1861, no. 9: 970-979.

Book chapter
Published: 06 January 2016 in Venom Genomics and Proteomics
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Brown spiders (Loxosceles genus) are venomous arthropods that use venom for predation and defense. These spiders have also been associated with human accidents, and the primary clinical manifestations are dermonecrosis with gravitational lesion spreading, hematological disturbances, and acute renal failure. Loxosceles venom comprises a complex mixture of toxins enriched in low molecular mass proteins (5–40 kDa). Characterization of this venom revealed three highly expressed protein classes: phospholipase-D family proteins, astacin-like proteases, and inhibitor cystine knot (ICK) peptides. A recent study also showed the presence of several other venom proteins, such as serine proteases, protease inhibitors, hyaluronidases, allergen-like toxins, and translationally controlled tumor protein (TCTP), expressed at low levels in Loxosceles venom. The Brown spider phospholipase-D family proteins have been well studied, and these toxins alone induce inflammatory responses, dermonecrosis, hemolysis, thrombocytopenia, and renal failure. In addition, the functional role of hyaluronidases as spreading factors in loxoscelism has been demonstrated. However, the biological characterization of other toxins has not been reported. Nevertheless, the mechanism by which Loxosceles toxins exert noxious effects is not fully elucidated. The aim of this chapter is to provide insights into Brown spider toxins, including the identification of novel toxins using molecular and proteomics approaches, and the biological characterization and structural description of toxins using X-ray crystallography. Putative biotechnological uses of Loxosceles toxins and future perspectives in this field will also be discussed.

ACS Style

Daniele Chaves-Moreira; Dilza Trevisan-Silva; Luiza Helena Gremski; Silvio Sanches Veiga. Brown Spider Venom: The Identification and Biotechnological Potential of Venom Toxins. Venom Genomics and Proteomics 2016, 125 -147.

AMA Style

Daniele Chaves-Moreira, Dilza Trevisan-Silva, Luiza Helena Gremski, Silvio Sanches Veiga. Brown Spider Venom: The Identification and Biotechnological Potential of Venom Toxins. Venom Genomics and Proteomics. 2016; ():125-147.

Chicago/Turabian Style

Daniele Chaves-Moreira; Dilza Trevisan-Silva; Luiza Helena Gremski; Silvio Sanches Veiga. 2016. "Brown Spider Venom: The Identification and Biotechnological Potential of Venom Toxins." Venom Genomics and Proteomics , no. : 125-147.

Comparative study
Published: 01 December 2015 in Toxicon
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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.

ACS Style

Daniela Regina Buch; Fernanda Nunes Souza; Gabriel Meissner; Adriano Marcelo Morgon; Luiza Gremski; Valéria Pereira Ferrer; Dilza Trevisan-Silva; Fernando Hitomi Matsubara; Marianna Boia Ferreira; Youssef Sade; Daniele Moreira; Waldemiro Gremski; Silvio Sanches Veiga; Olga Chaim; Andrea Senff-Ribeiro. Brown spider (Loxosceles genus) venom toxins: Evaluation of biological conservation by immune cross-reactivity. Toxicon 2015, 108, 154 -166.

AMA Style

Daniela Regina Buch, Fernanda Nunes Souza, Gabriel Meissner, Adriano Marcelo Morgon, Luiza Gremski, Valéria Pereira Ferrer, Dilza Trevisan-Silva, Fernando Hitomi Matsubara, Marianna Boia Ferreira, Youssef Sade, Daniele Moreira, Waldemiro Gremski, Silvio Sanches Veiga, Olga Chaim, Andrea Senff-Ribeiro. Brown spider (Loxosceles genus) venom toxins: Evaluation of biological conservation by immune cross-reactivity. Toxicon. 2015; 108 ():154-166.

Chicago/Turabian Style

Daniela Regina Buch; Fernanda Nunes Souza; Gabriel Meissner; Adriano Marcelo Morgon; Luiza Gremski; Valéria Pereira Ferrer; Dilza Trevisan-Silva; Fernando Hitomi Matsubara; Marianna Boia Ferreira; Youssef Sade; Daniele Moreira; Waldemiro Gremski; Silvio Sanches Veiga; Olga Chaim; Andrea Senff-Ribeiro. 2015. "Brown spider (Loxosceles genus) venom toxins: Evaluation of biological conservation by immune cross-reactivity." Toxicon 108, no. : 154-166.

Review
Published: 01 June 2014 in Toxicon
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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.

ACS Style

Luiza Gremski; Dilza Trevisan-Silva; Valéria Pereira Ferrer; Fernando Hitomi Matsubara; Gabriel Meissner; Ana Carolina Martins Wille; Larissa Vuitika; Camila Dias-Lopes; Anwar Ullah; Fabio Rogerio de Moraes; Carlos Delfin Chávez Olórtegui; Katia Cristina Barbaro; Mario Tyago Murakami; Raghuvir Arni; Andrea Senff-Ribeiro; Olga Chaim; Silvio Sanches Veiga. Recent advances in the understanding of brown spider venoms: From the biology of spiders to the molecular mechanisms of toxins. Toxicon 2014, 83, 91 -120.

AMA Style

Luiza Gremski, Dilza Trevisan-Silva, Valéria Pereira Ferrer, Fernando Hitomi Matsubara, Gabriel Meissner, Ana Carolina Martins Wille, Larissa Vuitika, Camila Dias-Lopes, Anwar Ullah, Fabio Rogerio de Moraes, Carlos Delfin Chávez Olórtegui, Katia Cristina Barbaro, Mario Tyago Murakami, Raghuvir Arni, Andrea Senff-Ribeiro, Olga Chaim, Silvio Sanches Veiga. Recent advances in the understanding of brown spider venoms: From the biology of spiders to the molecular mechanisms of toxins. Toxicon. 2014; 83 ():91-120.

Chicago/Turabian Style

Luiza Gremski; Dilza Trevisan-Silva; Valéria Pereira Ferrer; Fernando Hitomi Matsubara; Gabriel Meissner; Ana Carolina Martins Wille; Larissa Vuitika; Camila Dias-Lopes; Anwar Ullah; Fabio Rogerio de Moraes; Carlos Delfin Chávez Olórtegui; Katia Cristina Barbaro; Mario Tyago Murakami; Raghuvir Arni; Andrea Senff-Ribeiro; Olga Chaim; Silvio Sanches Veiga. 2014. "Recent advances in the understanding of brown spider venoms: From the biology of spiders to the molecular mechanisms of toxins." Toxicon 83, no. : 91-120.

Comparative study
Published: 01 December 2013 in Toxicon
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Loxosceles bites have been associated with characteristic dermonecrotic lesions with gravitational spreading and systemic manifestations. Venom primarily comprises peptides and protein molecules (5-40 kDa) with multiple biological activities. Although poorly studied, metalloproteases have been identified in venoms of several Loxosceles species, presenting proteolytic effects on extracellular matrix components. The characterization of an Astacin-like protease (LALP) in Loxosceles intermedia venom was the first report of an Astacin family member as a component of animal venom. Recently, these proteases were described as a gene family in L. intermedia, Loxosceles laeta and Loxosceles gaucho. Herein, the whole venom complexity of these three Loxosceles species was analyzed using two-dimensional electrophoresis (2DE). Subproteomes of LALPs were explored through 2DE immunostaining using anti-LALP1 antibodies and 2DE gelatin zymogram. Proteins presented molecular masses ranging from 24 to 29 kDa and the majority of these molecules had basic or neutral isoelectric points (6.89-9.93). Likewise, the measurement of gelatinolytic effects of Loxosceles venom using fluorescein-gelatin showed that the three venoms have distinct proteolytic activities. The metalloprotease fibrinogenolytic activities were also evaluated. All venoms showed fibrinogenolytic activity with different proteolytic effects on Aα and Bβ chains of fibrinogen. The results reported herein suggest that the LALP family is larger than indicated in previously published data and that the complex profile of the gelatinolytic activity reflects their relevance in loxoscelism. Furthermore, our investigation implicates the brown spider venom as a source of Astacin-like proteases for use in loxoscelism studies, cell biology research and biotechnological applications.

ACS Style

Dilza Trevisan-Silva; Aline Viana Bednaski; Luiza Helena Gremski; Olga Meiri Chaim; Silvio Sanches Veiga; Andrea Senff-Ribeiro. Differential metalloprotease content and activity of three Loxosceles spider venoms revealed using two-dimensional electrophoresis approaches. Toxicon 2013, 76, 11 -22.

AMA Style

Dilza Trevisan-Silva, Aline Viana Bednaski, Luiza Helena Gremski, Olga Meiri Chaim, Silvio Sanches Veiga, Andrea Senff-Ribeiro. Differential metalloprotease content and activity of three Loxosceles spider venoms revealed using two-dimensional electrophoresis approaches. Toxicon. 2013; 76 ():11-22.

Chicago/Turabian Style

Dilza Trevisan-Silva; Aline Viana Bednaski; Luiza Helena Gremski; Olga Meiri Chaim; Silvio Sanches Veiga; Andrea Senff-Ribeiro. 2013. "Differential metalloprotease content and activity of three Loxosceles spider venoms revealed using two-dimensional electrophoresis approaches." Toxicon 76, no. : 11-22.

Journal article
Published: 13 September 2013 in Journal of Cellular Biochemistry
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Brown spider (Loxosceles genus) bites have been reported worldwide. The venom contains a complex composition of several toxins, including phospholipases-D. Native or recombinant phospholipase-D toxins induce cutaneous and systemic loxoscelism, particularly necrotic lesions, inflammatory response, renal failure, and hematological disturbances. Herein, we describe the cloning, heterologous expression and purification of a novel phospholipase-D toxin, LiRecDT7 in reference to six other previously described in phospholipase-D toxin family. The complete cDNA sequence of this novel brown spider phospholipase-D isoform was obtained and the calculated molecular mass of the predicted mature protein is 34.4 kDa. Similarity analyses revealed that LiRecDT7 is homologous to the other dermonecrotic toxin family members particularly to LiRecDT6, sharing 71% sequence identity. LiRecDT7 possesses the conserved amino acid residues involved in catalysis except for a conservative mutation (D233E) in the catalytic site. Purified LiRecDT7 was detected as a soluble 36 kDa protein using anti-whole venom and anti-LiRecDT1 sera, indicating immunological cross-reactivity and evidencing sequence-epitopes identities similar to those of other phospholipase-D family members. Also, LiRecDT7 exhibits sphingomyelinase activity in a concentration dependent-manner and induces experimental skin lesions with swelling, erythema and dermonecrosis. In addition, LiRecDT7 induced a massive inflammatory response in rabbit skin dermis, which is a hallmark of brown spider venom phospholipase-D toxins. Moreover, LiRecDT7 induced in vitro hemolysis in human erythrocytes and increased blood vessel permeability. These features suggest that this novel member of the brown spider venom phospholipase-D family, which naturally contains a mutation (D233E) in the catalytic site, could be useful for future structural and functional studies concerning loxoscelism and lipid biochemistry. 1- Novel brown spider phospholipase-D recombinant toxin contains a conservative mutation (D233E) on the catalytic site. 2-LiRecDT7 shares high identity level with isoforms of Loxosceles genus. 3-LiRecDT7 is a recombinant protein immunodetected by specific antibodies to native and recombinant phospholipase-D toxins. 4-LiRecDT7 shows sphingomyelinase-D activity in a concentration-dependent manner, but less intense than other isoforms. 5-LiRecDT7 induces dermonecrosis and inflammatory response in rabbit skin. 6-LiRecDT7 increases vascular permeability in mice. 7-LiRecDT7 triggers direct complement-independent hemolysis in erythrocytes.

ACS Style

Larissa Vuitika; Luiza Helena Gremski; Valéria Pereira Ferrer; Matheus Regis Belisário‐Ferrari; Daniele Chaves‐Moreira; Andrea Senff‐Ribeiro; Olga Meiri Chaim; Silvio Sanches Veiga. Brown spider phospholipase-D containing a conservative mutation (D233E) in the catalytic site: Identification and functional characterization. Journal of Cellular Biochemistry 2013, 114, 2479 -2492.

AMA Style

Larissa Vuitika, Luiza Helena Gremski, Valéria Pereira Ferrer, Matheus Regis Belisário‐Ferrari, Daniele Chaves‐Moreira, Andrea Senff‐Ribeiro, Olga Meiri Chaim, Silvio Sanches Veiga. Brown spider phospholipase-D containing a conservative mutation (D233E) in the catalytic site: Identification and functional characterization. Journal of Cellular Biochemistry. 2013; 114 (11):2479-2492.

Chicago/Turabian Style

Larissa Vuitika; Luiza Helena Gremski; Valéria Pereira Ferrer; Matheus Regis Belisário‐Ferrari; Daniele Chaves‐Moreira; Andrea Senff‐Ribeiro; Olga Meiri Chaim; Silvio Sanches Veiga. 2013. "Brown spider phospholipase-D containing a conservative mutation (D233E) in the catalytic site: Identification and functional characterization." Journal of Cellular Biochemistry 114, no. 11: 2479-2492.

Journal article
Published: 01 September 2013 in Toxicon
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The venom of a Loxosceles spider is composed of a complex mixture of biologically active components, consisting predominantly of low molecular mass molecules (3-45 kDa). Transcriptome analysis of the Loxosceles intermedia venom gland revealed ESTs with similarity to the previously described LiTx peptides. Sequences similar to the LiTx3 isoform were the most abundant, representing approximately 13.9% of all ESTs and 32% of the toxin-encoding messengers. These peptides are grouped in the ICK (Inhibitor Cystine Knot) family, which contains single chain molecules with low molecular mass (3-10 kDa). Due to their high number of cysteine residues, ICK peptides form intramolecular disulfide bridges. The aims of this study were to clone and express a novel ICK peptide isoform, as well as produce specific hyperimmune serum for immunoassays. The corresponding cDNA was amplified by PCR using specific primers containing restriction sites for the XhoI and BamHI enzymes; this PCR product was then ligated in the pET-14b vector and transformed into E. coli AD494 (DE3) cells. The peptide was expressed by IPTG induction for 4 h at 30 °C and purified by affinity chromatography with Ni-NTA resin. Hyperimmune serum to the recombinant peptide was produced in rabbits and was able to specifically recognize both the purified recombinant peptide and the native form present in the venom. Furthermore, the recombinant peptide was recognized by antisera raised against L. intermedia, L. gaucho and L. laeta whole venoms. The recombinant peptide obtained will enable future studies to characterize its biological activity, as well as investigations regarding possible biotechnological applications.

ACS Style

Fernando Hitomi Matsubara; Luiza Gremski; Gabriel Meissner; Eduardo Soares Constantino Lopes; Waldemiro Gremski; Andrea Senff-Ribeiro; Olga Chaim; Silvio Sanches Veiga. A novel ICK peptide from the Loxosceles intermedia (brown spider) venom gland: Cloning, heterologous expression and immunological cross-reactivity approaches. Toxicon 2013, 71, 147 -158.

AMA Style

Fernando Hitomi Matsubara, Luiza Gremski, Gabriel Meissner, Eduardo Soares Constantino Lopes, Waldemiro Gremski, Andrea Senff-Ribeiro, Olga Chaim, Silvio Sanches Veiga. A novel ICK peptide from the Loxosceles intermedia (brown spider) venom gland: Cloning, heterologous expression and immunological cross-reactivity approaches. Toxicon. 2013; 71 ():147-158.

Chicago/Turabian Style

Fernando Hitomi Matsubara; Luiza Gremski; Gabriel Meissner; Eduardo Soares Constantino Lopes; Waldemiro Gremski; Andrea Senff-Ribeiro; Olga Chaim; Silvio Sanches Veiga. 2013. "A novel ICK peptide from the Loxosceles intermedia (brown spider) venom gland: Cloning, heterologous expression and immunological cross-reactivity approaches." Toxicon 71, no. : 147-158.

Research article
Published: 02 May 2013 in PLoS Neglected Tropical Diseases
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Loxoscelism is the designation given to clinical symptoms evoked by Loxosceles spider's bites. Clinical manifestations include skin necrosis with gravitational spreading and systemic disturbs. The venom contains several enzymatic toxins. Herein, we describe the cloning, expression, refolding and biological evaluation of a novel brown spider protein characterized as a hyaluronidase. Employing a venom gland cDNA library, we cloned a hyaluronidase (1200 bp cDNA) that encodes for a signal peptide and a mature protein. Amino acid alignment revealed a structural relationship with members of hyaluronidase family, such as scorpion and snake species. Recombinant hyaluronidase was expressed as N-terminal His-tag fusion protein (∼45 kDa) in inclusion bodies and activity was achieved using refolding. Immunoblot analysis showed that antibodies that recognize the recombinant protein cross-reacted with hyaluronidase from whole venom as well as an anti-venom serum reacted with recombinant protein. Recombinant hyaluronidase was able to degrade purified hyaluronic acid (HA) and chondroitin sulfate (CS), while dermatan sulfate (DS) and heparan sulfate (HS) were not affected. Zymograph experiments resulted in ∼45 kDa lytic zones in hyaluronic acid (HA) and chondroitin sulfate (CS) substrates. Through in vivo experiments of dermonecrosis using rabbit skin, the recombinant hyaluronidase was shown to increase the dermonecrotic effect produced by recombinant dermonecrotic toxin from L. intermedia venom (LiRecDT1). These data support the hypothesis that hyaluronidase is a “spreading factor”. Recombinant hyaluronidase provides a useful tool for biotechnological ends. We propose the name Dietrich's Hyaluronidase for this enzyme, in honor of Professor Carl Peter von Dietrich, who dedicated his life to studying proteoglycans and glycosaminoglycans. Accidents involving brown spiders (Loxosceles genus) are reported throughout the world. South and Southeast of Brazil are endemic areas for this spider. Loxosceles bites commonly trigger local signs as swelling, erythema, hemorrhage and the hallmark symptom: a dermonecrotic lesion with gravitational spreading. Systemic effects are less common; however, are implicated in more severe cases. Hyaluronidases are referred in several venoms as “spreading factors” due to their enzymatic activity upon extracellular components. This activity facilitates the permeation of other toxins through the victim's body. In fact, a previous study identified the activity of L. intermedia venom upon glycosaminoglycans which are abundant components in the extracellular matrix of many tissues. Disclosing a little more about the role of hyaluronidases within this venom, we investigated the activities of a recombinant hyaluronidase from L. intermedia venom. Dietrich's hyaluronidase, as it was designated, was produced as a recombinant protein. By performing a rabbit skin dermonecrosis assay using Dietrich's Hyaluronidase and a dermonecrotic toxin, we showed that Dietrich's Hyaluronidase increased the dermonecrotic area induced by the dermonecrotic toxin. Our results confirm that hyaluronidases are a “spreading factor” of L. intermedia venom.

ACS Style

Valéria Pereira Ferrer; Thiago Lopes De Mari; Luiza Gremski; Dilza Trevisan-Silva; Rafael Bertoni Da Silveira; Waldemiro Gremski; Olga Chaim; Andrea Senff-Ribeiro; Helena Nader; Silvio Sanches Veiga. A Novel Hyaluronidase from Brown Spider (Loxosceles intermedia) Venom (Dietrich's Hyaluronidase): From Cloning to Functional Characterization. PLoS Neglected Tropical Diseases 2013, 7, e2206 .

AMA Style

Valéria Pereira Ferrer, Thiago Lopes De Mari, Luiza Gremski, Dilza Trevisan-Silva, Rafael Bertoni Da Silveira, Waldemiro Gremski, Olga Chaim, Andrea Senff-Ribeiro, Helena Nader, Silvio Sanches Veiga. A Novel Hyaluronidase from Brown Spider (Loxosceles intermedia) Venom (Dietrich's Hyaluronidase): From Cloning to Functional Characterization. PLoS Neglected Tropical Diseases. 2013; 7 (5):e2206.

Chicago/Turabian Style

Valéria Pereira Ferrer; Thiago Lopes De Mari; Luiza Gremski; Dilza Trevisan-Silva; Rafael Bertoni Da Silveira; Waldemiro Gremski; Olga Chaim; Andrea Senff-Ribeiro; Helena Nader; Silvio Sanches Veiga. 2013. "A Novel Hyaluronidase from Brown Spider (Loxosceles intermedia) Venom (Dietrich's Hyaluronidase): From Cloning to Functional Characterization." PLoS Neglected Tropical Diseases 7, no. 5: e2206.