RESUMO
The present work reports the effects of a C-type lectin (BpLec) isolated from Bothrops pauloensis snake venom upon in vitro and in vivo angiogenesis models. Initially, we noted that BpLec was not cytotoxic to endothelial cells (tEnd) in doses up to 40µg/mL, but lower doses (2.5µg/mL, 5µg/mL, 10µg/mL and 20µg/mL) reduced tEnd cells adhesion to some extracellular matrix proteins and inhibited the in vitro vessel formation in Matrigel assay stimulated by bFGF. ß-galactosides (d-lactose, N-acetyl-d-galactosamine and d-galactose) at 400mM reversed the effect of BpLec on tEnd cells adhesion, whereas d-galactose (400mM) partially reversed BpLec property of inhibiting vessel formation by tEnd cells in Matrigel. In vivo assays showed that BpLec increased hemoglobin content and capillary vessels number in polyether-polyurethane sponge discs subcutaneously implanted into dorsal skin mice. Additionally, BpLec also reduced collagen deposition and did not induce a pro-inflammatory response, as demonstrated by the decreased the secretion of some inflammatory cytokines, whereas myeloperoxidase (MPO) and N-acetylglucosaminidase (NAG) activities were not altered by BpLec. Taken together, our results indicate that BpLec might represent an interesting angiogenesis and inflammatory modulator that could also be used for searching possible therapeutic targets involved in these processes.
Assuntos
Inibidores da Angiogênese/farmacologia , Bothrops , Venenos de Crotalídeos/química , Lectinas Tipo C/metabolismo , Acetilglucosaminidase/metabolismo , Inibidores da Angiogênese/isolamento & purificação , Inibidores da Angiogênese/toxicidade , Animais , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Lectinas Tipo C/isolamento & purificação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Fisiológica/efeitos dos fármacos , Peroxidase/metabolismoRESUMO
Toxoplasmosis affects a third of the global population and presents high incidence in tropical areas. Its great relevance in public health has led to a search for new therapeutic approaches. Herein, we report the antiparasitic effects of BnSP-7 toxin, a Lys49 phospholipase A2 (PLA2) homologue from Bothrops pauloensis snake venom, on Toxoplasma gondii. In an MTT assay, BnSP-7 presented significant cytotoxicity against host HeLa cells at higher doses (200 µg/mL to 50 µg/mL), whereas lower doses (25 µg/mL to 1.56 µg/mL) produced low cytotoxicity. Furthermore, the toxin showed no effect on T. gondii tachyzoite viability when evaluated by trypan blue exclusion, but decreased both adhesion and parasite proliferation when tachyzoites were treated before infection. We also measured cytokines in supernatants collected from HeLa cells infected with T. gondii tachyzoites previously treated with RPMI or BnSP-7, which revealed enhancement of only MIF and IL-6 cytokines levels in supernatants of HeLa cells after BnSP-7 treatment. Our results showed that the BnSP-7 PLA2 exerts an anti-Toxoplasma effect at a lower dose than that required to induce cytotoxicity in HeLa cells, and also modulates the immune response of host cells. In this sense, the anti-parasitic effect of BnSP-7 PLA2 demonstrated in the present study opens perspectives for use of this toxin as a tool for future studies on toxoplasmosis.
Assuntos
Venenos de Crotalídeos/toxicidade , Lisina/química , Fosfolipases A2/toxicidade , Toxoplasma/efeitos dos fármacos , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Venenos de Crotalídeos/enzimologia , Eletroforese em Gel de Poliacrilamida , Células HeLa , Humanos , Fosfolipases A2/química , Homologia de Sequência de AminoácidosRESUMO
Snake venoms constitute a mixture of bioactive components that are involved not only in envenomation pathophysiology but also in the development of new drugs to treat many diseases. Different enzymatic and non-enzymatic proteins, such as phospholipases A2, hyaluronidases, L-amino acid oxidases, metalloproteinases, serine proteinases, lectins and disintegrins have been isolated and their functional and structural properties described in the literature. Many of these studies have also explored their medicinal potential focusing mainly on anticancer, antithrombotic and microbicide therapies. Bothrops pauloensis is a species found in Brazil, whose venom has been the focus of our studies in order to explore the biochemical and functional characteristics of their components. In this review, we have presented the main results of years of research on different toxins from B. pauloensis emphasizing their therapeutic potential. Studies concerning snake venom toxins to search for new therapeutic models open perspectives for new drug discovery.
Assuntos
Bothrops , Descoberta de Drogas/métodos , Venenos de Serpentes/química , Toxinas Biológicas/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Antiprotozoários/química , Antiprotozoários/isolamento & purificação , Antiprotozoários/farmacologia , Brasil , Fibrinolíticos/química , Fibrinolíticos/isolamento & purificação , Fibrinolíticos/farmacologia , Humanos , Leishmaniose/tratamento farmacológico , Toxinas Biológicas/química , Toxinas Biológicas/isolamento & purificaçãoRESUMO
Here we evaluate the effects of BpLec, a C-type lectin isolated from Bothrops pauloensis snake venom, on Toxoplasma gondii parasitism. BpLec (0.195-12.5 µg/mL) did not interfere with HeLa (host cell) viability by MTT assay, whereas higher doses decreased viability and changed HeLa morphology. In addition, the host cell treatment before infection did not influence adhesion and proliferation indexes. BpLec did not alter T. gondii tachyzoite viability, as carried out by trypan blue exclusion, but decreased both adhesion and parasite replication, when tachyzoites were treated before infection. Galactose (0.4 M) inhibited the BpLec effect on adhesion assays, suggesting that BpLec probably recognize some glycoconjugate from T. gondii membrane. Additionally, we performed cytokine measurements from supernatants collected from HeLa cells infected with T. gondii tachyzoites previously treated with RPMI or BpLec. MIF and IL-6 productions by HeLa cells were increased by BpLec treatment. Also, TGF-ß1 secretion was diminished post-infection, although this effect was not dependent on BpLec treatment. Taken together, our results show that BpLec is capable of reducing T. gondii parasitism after tachyzoite treatment and may represent an interesting tool in the search for parasite antigens involved in these processes.
Assuntos
Bothrops/metabolismo , Coccidiostáticos/química , Coccidiostáticos/farmacologia , Lectinas Tipo C/química , Toxoplasma/efeitos dos fármacos , Peçonhas/química , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Coccidiostáticos/isolamento & purificação , Citocinas/metabolismo , Células HeLa , Humanos , Lectinas Tipo C/isolamento & purificaçãoRESUMO
BACKGROUND: Hyaluronate is one of the major components of extracellular matrix from vertebrates whose breakdown is catalyzed by the enzyme hyaluronidase. These enzymes are widely described in snake venoms, in which they facilitate the spreading of the main toxins in the victim's body during the envenoming. Snake venoms also present some variants (hyaluronidases-like substances) that are probably originated by alternative splicing, even though their relevance in envenomation is still under investigation. Hyaluronidases-like proteins have not yet been purified from any snake venom, but the cDNA that encodes these toxins was already identified in snake venom glands by transcriptomic analysis. Herein, we report the cloning and in silico analysis of the first hyaluronidase-like proteins from a Brazilian snake venom. METHODS: The cDNA sequence of hyaluronidase was cloned from the transcriptome of Bothrops pauloensis venom glands. This sequence was submitted to multiple alignment with other related sequences by ClustalW. A phylogenetic analysis was performed using MEGA 4 software by the neighbor joining (NJ) method. RESULTS: The cDNA from Bothrops pauloensis venom gland that corresponds to hyaluronidase comprises 1175 bp and codifies a protein containing 194 amino acid residues. The sequence, denominated BpHyase, was identified as hyaluronidase-like since it shows high sequence identities (above 83%) with other described snake venom hyaluronidase-like sequences. Hyaluronidases-like proteins are thought to be products of alternative splicing implicated in deletions of central amino acids, including the catalytic residues. Structure-based sequence alignment of BpHyase to human hyaluronidase hHyal-1 demonstrates a loss of some key secondary structures. The phylogenetic analysis indicates an independent evolution of BpHyal when compared to other hyaluronidases. However, these toxins might share a common ancestor, thus suggesting a broad hyaluronidase-like distribution among venomous snakes. CONCLUSIONS: This work is the first report of a cDNA sequence of hyaluronidase from Brazilian snake venoms. Moreover, the in silico analysis of its deduced amino acid sequence opens new perspectives about the biological function of hyaluronidases-like proteins and may direct further studies comprising their isolation and/or recombinant production, as well as their structural and functional characterization.
RESUMO
Hyaluronate is one of the major components of extracellular matrix from vertebrates whose breakdown is catalyzed by the enzyme hyaluronidase. These enzymes are widely described in snake venoms, in which they facilitate the spreading of the main toxins in the victims body during the envenoming. Snake venoms also present some variants (hyaluronidases-like substances) that are probably originated by alternative splicing, even though their relevance in envenomation is still under investigation. Hyaluronidases-like proteins have not yet been purified from any snake venom, but the cDNA that encodes these toxins was already identified in snake venom glands by transcriptomic analysis. Herein, we report the cloning and in silico analysis of the first hyaluronidase-like proteins from a Brazilian snake venom.
Assuntos
Animais , Clonagem Molecular/métodos , Hialuronoglucosaminidase/análise , Venenos de VíborasRESUMO
Hyaluronate is one of the major components of extracellular matrix from vertebrates whose breakdown is catalyzed by the enzyme hyaluronidase. These enzymes are widely described in snake venoms, in which they facilitate the spreading of the main toxins in the victims body during the envenoming. Snake venoms also present some variants (hyaluronidases-like substances) that are probably originated by alternative splicing, even though their relevance in envenomation is still under investigation. Hyaluronidases-like proteins have not yet been purified from any snake venom, but the cDNA that encodes these toxins was already identified in snake venom glands by transcriptomic analysis. Herein, we report the cloning and in silico analysis of the first hyaluronidase-like proteins from a Brazilian snake venom.
Assuntos
Animais , Clonagem Molecular/métodos , Hialuronoglucosaminidase/análise , Venenos de VíborasRESUMO
Background Hyaluronate is one of the major components of extracellular matrix from vertebrates whose breakdown is catalyzed by the enzyme hyaluronidase. These enzymes are widely described in snake venoms, in which they facilitate the spreading of the main toxins in the victim's body during the envenoming. Snake venoms also present some variants (hyaluronidases-like substances) that are probably originated by alternative splicing, even though their relevance in envenomation is still under investigation. Hyaluronidases-like proteins have not yet been purified from any snake venom, but the cDNA that encodes these toxins was already identified in snake venom glands by transcriptomic analysis. Herein, we report the cloning and in silicoanalysis of the first hyaluronidase-like proteins from a Brazilian snake venom.Methods The cDNA sequence of hyaluronidase was cloned from the transcriptome of Bothrops pauloensisvenom glands. This sequence was submitted to multiple alignment with other related sequences by ClustalW. A phylogenetic analysis was performed using MEGA 4 software by the neighbor joining (NJ) method.Results The cDNA from Bothrops pauloensis venom gland that corresponds to hyaluronidase comprises 1175 bp and codifies a protein containing 194 amino acid residues. The sequence, denominated BpHyase, was identified as hyaluronidase-like since it shows high sequence identities (above 83%) with other described snake venom hyaluronidase-like sequences. Hyaluronidases-like proteins are thought to be products of alternative splicing implicated in deletions of central amino acids, including the catalytic residues. Structure-based sequence alignment of BpHyase to human hyaluronidase hHyal-1 demonstrates a loss of some key secondary structures. The phylogenetic analysis indicates an independent evolution of BpHyal when compared to other hyaluronidases. However, these toxins might share a common ancestor, thus suggesting a broad hyaluronidase-like distribution among venomous snakes.Conclusions This work is the first report of a cDNA sequence of hyaluronidase from Brazilian snake venoms. Moreover, the in silico analysis of its deduced amino acid sequence opens new perspectives about the biological function of hyaluronidases-like proteins and may direct further studies comprising their isolation and/or recombinant production, as well as their structural and functional characterization.(AU)
Assuntos
Animais , Filogenia , Venenos de Serpentes , Clonagem Molecular , Bothrops , HialuronoglucosaminidaseRESUMO
Hyaluronate is one of the major components of extracellular matrix from vertebrates whose breakdown is catalyzed by the enzyme hyaluronidase. These enzymes are widely described in snake venoms, in which they facilitate the spreading of the main toxins in the victims body during the envenoming. Snake venoms also present some variants (hyaluronidases-like substances) that are probably originated by alternative splicing, even though their relevance in envenomation is still under investigation. Hyaluronidases-like proteins have not yet been purified from any snake venom, but the cDNA that encodes these toxins was already identified in snake venom glands by transcriptomic analysis. Herein, we report the cloning and in silico analysis of the first hyaluronidase-like proteins from a Brazilian snake venom.(AU)
Assuntos
Animais , Clonagem Molecular/métodos , Venenos de Víboras , Hialuronoglucosaminidase/análiseRESUMO
In this work, we describe the molecular cloning and pharmacological properties of an acidic phospholipase A(2) (PLA(2)) isolated from Bothrops pauloensis snake venom. This enzyme, denominated BpPLA(2)-TXI, was purified by four chromatographic steps and represents 2.4% of the total snake venom protein content. BpPLA(2)-TXI is a monomeric protein with a molecular mass of 13.6 kDa, as demonstrated by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis and its theoretical isoelectric point was 4.98. BpPLA(2)-TXI was catalytically active and showed some pharmacological effects such as inhibition of platelet aggregation induced by collagen or ADP and also induced edema and myotoxicity. BpPLA(2)-TXI displayed low cytotoxicity on TG-180 (CCRF S 180 II) and Ovarian Carcinoma (OVCAR-3), whereas no cytotoxicity was found in regard to MEF (Mouse Embryonic Fibroblast) and Sarcoma 180 (TIB-66). The N-terminal sequence of forty-eight amino acid residues was determined by Edman degradation. In addition, the complete primary structure of 122 amino acids was deduced by cDNA from the total RNA of the venom gland using specific primers, and it was significantly similar to other acidic D49 PLA(2)s. The phylogenetic analyses showed that BpPLA(2)-TXI forms a group with other acidic D49 PLA(2)s from the gender Bothrops, which are characterized by a catalytic activity associated with anti-platelet effects.