β-micrustoxin (Mlx-9), a PLA2 from Micrurus lemniscatus snake venom: biochemical characterization and anti-proliferative effect mediated by p53

Background: Endogenous phospholipases A2 (PLA2) play a fundamental role in inflammation, neurodegenerative diseases, apoptosis and cellular senescence. Neurotoxins with PLA2 activity are found in snake venoms from the Elapidae and Viperidae families. The mechanism of action of these neurotoxins have been studied using hippocampal and cerebellar neuronal cultures showing [Ca2+]i increase, mitochondrial depolarization and cell death. Astrocytes are rarely used as a model, despite being modulators at the synapses and responsible for homeostasis and defense in the central nervous system. Preserving the cell division ability, they can be utilized to study the cell proliferation process. In the present work cultured astrocytes and glioblastoma cells were employed to characterize the action of β-micrustoxin (previously named Mlx-9), a PLA2 isolated from Micrurus lemniscatus snake venom. The β-micrustoxin structure was determined and the cell proliferation, cell cycle phases and the regulatory proteins p53, p21 and p27 were investigated. Methods: β-micrustoxin was characterized biochemically by a proteomic approach. Astrocytes were obtained by dissociation of pineal glands from Wistar rats; glioblastoma tumor cells were purchased from ATCC and Sigma and cultured in DMEM medium. Cell viability was evaluated by MTT assay; cell proliferation and cell cycle phases were analyzed by flow cytometry; p53, p21 and p27 proteins were studied by western blotting and immunocytochemistry. Results: Proteomic analysis revealed fragments on β-micrustoxin that aligned with a PLA2 from Micrurus lemniscatus lemniscatus previously identified as transcript ID DN112835_C3_g9_i1/m.9019. β-micrustoxin impaired the viability of astrocytes and glioblastoma tumor cells. There was a reduction in cell proliferation, an increase in G2/M phase and activation of p53, p21 and p27 proteins in astrocytes. Conclusion: These findings indicate that β-micrustoxin from Micrurus lemniscatus venom could inhibit cell proliferation through p53, p21 and p27 activation thus imposing cell cycle arrest at the checkpoint G2/M.

Proteomic endorsed transcriptomic profiles of venom glands from Tityus obscurus and T. serrulatus scorpions.

BACKGROUND: Except for the northern region, where the Amazonian black scorpion, T. obscurus, represents the predominant and most medically relevant scorpion species, Tityus serrulatus, the Brazilian yellow scorpion, is widely distributed throughout Brazil, causing most envenoming and fatalities due to scorpion sting. In order to evaluate and compare the diversity of venom components of Tityus obscurus and T. serrulatus, we performed a transcriptomic investigation of the telsons (venom glands) corroborated by a shotgun proteomic analysis of the venom from the two species. RESULTS: The putative venom components represented 11.4% and 16.7% of the total gene expression for T. obscurus and T. serrulatus, respectively. Transcriptome and proteome data revealed high abundance of metalloproteinases sequences followed by sodium and potassium channel toxins, making the toxin core of the venom. The phylogenetic analysis of metalloproteinases from T. obscurus and T. serrulatus suggested an intraspecific gene expansion, as we previously observed for T. bahiensis, indicating that this enzyme may be under evolutionary pressure for diversification. We also identified several putative venom components such as anionic peptides, antimicrobial peptides, bradykinin-potentiating peptide, cysteine rich protein, serine proteinases, cathepsins, angiotensin-converting enzyme, endothelin-converting enzyme and chymotrypsin like protein, proteinases inhibitors, phospholipases and hyaluronidases. CONCLUSION: The present work shows that the venom composition of these two allopatric species of Tityus are considerably similar in terms of the major classes of proteins produced and secreted, although their individual toxin sequences are considerably divergent. These differences at amino acid level may reflect in different epitopes for the same protein classes in each species, explaining the basis for the poor recognition of T. obscurus venom by the antiserum raised against other species.

Proteomic endorsed transcriptomic profiles of venom glands from Tityus obscurus and T. serrulatus scorpions

Background Except for the northern region, where the Amazonian black scorpion, T. obscurus, represents the predominant and most medically relevant scorpion species, Tityus serrulatus, the Brazilian yellow scorpion, is widely distributed throughout Brazil, causing most envenoming and fatalities due to scorpion sting. In order to evaluate and compare the diversity of venom components of Tityus obscurus and T. serrulatus, we performed a transcriptomic investigation of the telsons (venom glands) corroborated by a shotgun proteomic analysis of the venom from the two species. Results The putative venom components represented 11.4% and 16.7% of the total gene expression for T. obscurus and T. serrulatus, respectively. Transcriptome and proteome data revealed high abundance of metalloproteinases sequences followed by sodium and potassium channel toxins, making the toxin core of the venom. The phylogenetic analysis of metalloproteinases from T. obscurus and T. serrulatus suggested an intraspecific gene expansion, as we previously observed for T. bahiensis, indicating that this enzyme may be under evolutionary pressure for diversification. We also identified several putative venom components such as anionic peptides, antimicrobial peptides, bradykinin-potentiating peptide, cysteine rich protein, serine proteinases, cathepsins, angiotensin-converting enzyme, endothelin-converting enzyme and chymotrypsin like protein, proteinases inhibitors, phospholipases and hyaluronidases. Conclusion The present work shows that the venom composition of these two allopatric species of Tityus are considerably similar in terms of the major classes of proteins produced and secreted, although their individual toxin sequences are considerably divergent. These differences at amino acid level may reflect in different epitopes for the same protein classes in each species, explaining the basis for the poor recognition of T. obscurus venom by the antiserum raised against other species.

Proteomic endorsed transcriptomic profiles of venom glands from Tityus obscurus and T. serrulatus scorpions

Background Except for the northern region, where the Amazonian black scorpion, T. obscurus, represents the predominant and most medically relevant scorpion species, Tityus serrulatus, the Brazilian yellow scorpion, is widely distributed throughout Brazil, causing most envenoming and fatalities due to scorpion sting. In order to evaluate and compare the diversity of venom components of Tityus obscurus and T. serrulatus, we performed a transcriptomic investigation of the telsons (venom glands) corroborated by a shotgun proteomic analysis of the venom from the two species. Results The putative venom components represented 11.4% and 16.7% of the total gene expression for T. obscurus and T. serrulatus, respectively. Transcriptome and proteome data revealed high abundance of metalloproteinases sequences followed by sodium and potassium channel toxins, making the toxin core of the venom. The phylogenetic analysis of metalloproteinases from T. obscurus and T. serrulatus suggested an intraspecific gene expansion, as we previously observed for T. bahiensis, indicating that this enzyme may be under evolutionary pressure for diversification. We also identified several putative venom components such as anionic peptides, antimicrobial peptides, bradykinin-potentiating peptide, cysteine rich protein, serine proteinases, cathepsins, angiotensin-converting enzyme, endothelin-converting enzyme and chymotrypsin like protein, proteinases inhibitors, phospholipases and hyaluronidases. Conclusion The present work shows that the venom composition of these two allopatric species of Tityus are considerably similar in terms of the major classes of proteins produced and secreted, although their individual toxin sequences are considerably divergent. These differences at amino acid level may reflect in different epitopes for the same protein classes in each species, explaining the basis for the poor recognition of T. obscurus venom by the antiserum raised against other species.

Origin and characterization of small membranous vesicles present in the venom of Crotalus durissus terrificus.

Small membranous vesicles are small closed fragments of membrane. They are released from multivesicular bodies (exosomes) or shed from the surface membrane (microvesicles). They contains various bioactive molecules and their molecular composition varies depending on their cellular origin. Small membranous vesicles have been identified in snake venoms, but the origin of these small membranous vesicles in the venom is controversial. The aim of this study was to verify the origin of the small membranous vesicles in venom of Crotalus durissus terrificus by morphological analyses using electron microscopy. In addition, the protein composition of the vesicles was analyzed by using a proteome approach. The small membranous vesicles present in the venom were microvesicles, since they originated from microvilli on the apical membrane of secretory cells. They contained cytoplasmic proteins, and proteins from the plasma membrane, endoplasmic reticulum (ER), and Golgi membrane. The release of microvesicles may be a mechanism to control the size of the cell membrane of the secretory cells after intense exocytosis. Microvesicle components that may have a role in envenoming include ecto-5'-nucleotidase, a cell membrane protein that releases adenosine, and aminopeptidase N, a cell membrane protein that may modulate the action of many peptides.

Bothrops jararaca venom gland secretory cells in culture: Effects of noradrenaline on toxin production and secretion.

Primary culture of snake venom gland secretory cells could be a good model to study the mechanism(s) of toxin(s) production. These cells can produce and secrete venom to the medium with a hemorrhagic activity comparable to that induced by venom collected from snakes. Production of new venom is triggered by the sympathetic outflow, through the release of noradrenaline, but the importance of this neurotransmitter on toxin synthesis has not been addressed. This work led to the identification and comparison of the toxin panel produced by cultured secretory cells, during a 12-day time-course analysis, as well as to the effects of noradrenaline on the process. The results showed that in our culture model the synthesis of new toxins is asynchronous, mimicking data previously published from proteomic analyses of venom glands harvested from animal experimentation. Furthermore, noradrenaline did regulate the synthesis and/or secretion of venom toxins over the analyzed period. Finally, we demonstrated that snake venom metalloproteinases present in these cultured cells secretome were mostly in their zymogen forms; consequently, processing occurs after secretion to the gland lumen. Overall, the data support the use of venom gland secretory cells as a reliable model to investigate the mechanism(s) of toxin(s) synthesis and secretion.

Origin and characterization of small membranous vesicles present in the venom of Crotalus durissus terrificus

Small membranous vesicles are small closed fragments of membrane. They are released from multivesicular bodies (exosomes) or shed from the surface membrane (microvesicles). They contains various bioactive molecules and their molecular composition varies depending on their cellular origin. Small membranous vesicles have been identified in snake venoms, but the origin of these small membranous vesicles in the venom is controversial. The aim of this study was to verify the origin of the small membranous vesicles in venom of Crotalus durissus terrificus by morphological analyses using electron microscopy. In addition, the protein composition of the vesicles was analyzed by using a proteome approach. The small membranous vesicles present in the venom were microvesicles, since they originated from microvilli on the apical membrane of secretory cells. They contained cytoplasmic proteins, and proteins from the plasma membrane, endoplasmic reticulum (ER), and Golgi membrane. The release of microvesicles may be a mechanism to control the size of the cell membrane of the secretory cells after intense exocytosis. Microvesicle components that may have a role in envenoming include ecto-5'-nucleotidase, a cell membrane protein that releases adenosine, and aminopeptidase N, a cell membrane protein that may modulate the action of many peptides.

Bothrops jararaca venom gland secretory cells in culture: effects of noradrenaline on toxin production and secretion

Primary culture of snake venom gland secretory cells could be a good model to study the mechanism(s) of toxin(s) production. These cells can produce and secrete venom to the medium with a hemorrhagic activity comparable to that induced by venom collected from snakes. Production of new venom is triggered by the sympathetic outflow, through the release of noradrenaline, but the importance of this neurotransmitter on toxin synthesis has not been addressed. This work led to the identification and comparison of the toxin panel produced by cultured secretory cells, during a 12-day time-course analysis, as well as to the effects of noradrenaline on the process. The results showed that in our culture model the synthesis of new toxins is asynchronous, mimicking data previously published from proteomic analyses of venom glands harvested from animal experimentation. Furthermore, noradrenaline did regulate the synthesis and/or secretion of venom toxins over the analyzed period. Finally, we demonstrated that snake venom metalloproteinases present in these cultured cells secretome were mostly in their zymogen forms; consequently, processing occurs after secretion to the gland lumen. Overall, the data support the use of venom gland secretory cells as a reliable model to investigate the mechanism(s) of toxin(s) synthesis and secretion.

Caracterização das microvesículas presentes no veneno de Crotalus durissus terrificus / Caracterization of the microvescles present in Crotalus durissus terrificus venom

The Crotalus durissus terrificus (Cdt) venom is constituted by toxins that are responsible for the clinical complications caused by envenoming. These toxins are not the only components presents in the venom; numerous electron-dense microvesicles (40 – 80 nm in diameter) are observed on the luminal face of secretory cells of venom gland and in the venom of Cdt. This microvesicles originate from microvilli by fragmentation or membrane budding and have intramembranous particles on the cytoplasmic leaftet, suggesting the presence of transmembrane proteins. The aim of this study was to identify and characterize specific proteins in the microvesicles from Cdt venom. The venom used was manually extracted from Cdt snakes maintained in the Laboratory of Herpetology at Instituto Butantan. The microvesicles were isolated by ultracentrifugation. Supernatant (S2) from the first ultracentrifugation, venom without microvesicles, and crude venom were used as controls. Morphological analysis showed that after two ultracentrifugations the microvesicles kept their morphology. One-dimensional electrophoresis assay showed band of approximately 72, 148, 176, 272 and 323 kDa that were present only in the microvesicle extract. Analysis of two-dimensional electrophoresis confirms the presence these specific microvesicle proteins. When S2 was used as a control, 18 spots were detected only in microvesicle extract. These spots are: 1) approximately 180 kDa with pI around 4; 2) ranging from 60 to 70 kDa with pI ranging from 7 to 9; 3) 11 kDa with PI 4; 4) approximately 10 kDa with pI ranging from 5 to 7 or 9 to 10. However, when fresh crude venom was used as a control, besides the 18 spots, 10 more spots were detected in S2 and microvesicle extract that is not present in the crude venom (spots of 36 kDa and pI ranging from 5 to 7 and spots of 32 kDa and pI ranging from 9 to 10). Western Blotting analysis showed that the Crotalus antivenom recognize only some microvesicle proteins. The identification of proteins by mass spectrometry showed the presence of cellular membrane and intracellular proteins. Identified membrane cellular proteins are ecto-5´nucleotidase, aminopeptidase N and angiotensin-converting enzyme-like. Identified intracellular proteins are Ankyrin repeat domain-containing protein 62, N-acylglucosamine 2-epimerase, leucine-rich repeat, immunoglobulin-like domain and transmembrane,domain-containing protein 3 DNA fragmentation factor subunit alpha isoform 1, protein phosphatase 1 regulatory subunit 26. In conclusion, we showed that after ultracentrifugations the microvesicles kept their morphology, but some of them seem to be lysed since 10 spots were found only in S2 and microvesicles extract, but not in the crude venom. Besides, the protein identified could have a role in metabolic pathways that regulates the integrity of the secretory cells. In addition, these proteins could have a relevant role in envenoming according to their functions described in the literature. The participation of the microvesicles in pathophysiology process shows its importance and open new avenues towards an understanding of their role in the envenoming.

O veneno de Crotalus durissus terrificus (Cdt), é constituído por toxinas responsáveis pelas complicações clínicas que ocorrem durante o envenenamento, entretanto, estas toxinas não são os únicos componentes presentes no veneno, numerosas microvesículas de conteúdo elétron-denso (40-80 nm de diâmetro) são observadas na face luminal das células secretoras da glândula de veneno, bem como no veneno coletado. Estas estruturas originam-se de microvilos por fragmentação ou brotamento e possuem partículas intramembranosas na face citoplasmática sugerindo a presença de proteínas transmembrânicas. O objetivo deste trabalho foi identificar e caracterizar as proteínas presentes nas microvesículas do veneno de Cdt, correlacionando suas possíveis funções através de dados já conhecidos na literatura. Em nossos estudos, utilizamos veneno extraído de Cdt, mantidas em cativeiro no Laboratório de Herpetologia do Instituto Butantan. Isolamos as microvesículas do veneno de Cdt através de ultracentrifugações. O Sobrenadante (S2) da primeira ultracentrifugação, veneno sem microvesículas, e o veneno bruto foram utilizados como controles. A análise morfológica por microscopia eletrônica de transmissão mostrou que a morfologia das microvesículas se mantém mesmo após as duas ultracentrifugações. Em eletroforese unidimensional detectamos bandas em torno de 72, 148, 176, 272 e 323 kDa que estão presentes somente no extrato de microvesículas. A análise do perfil protéico em eletroforese bidimensional confirmou a presença de proteínas específicas de microvesículas. Quando S2 foi utilizado como controle, 18 spots foram detectados somente no extrato de microvesículas (spots de180 kDa com pI em torno de 4; spots entre 60 a 70 kDa com pI em torno de 7 a 9; spots de 11 kDa com pI 4; spots de 10 kDa com pI entre 5 a 7 e 9 a 10). Entretanto, quando o veneno bruto foi utilizado como controle, mais 10 spots foram detectados em S2 e extrato de microvesículas (spots de 36 kDa e pI em torno de 5 a 7 e spots de 32 kDa e pI em torno de 9 a 10). A análise por Western blotting, utilizando soro anticrotálico como fonte de anticorpos, mostrou que somente algumas proteínas das microvesículas são reconhecidas. As identificações das proteínas, por espectrometria de massa, mostraram a presença de proteínas de membrana celular e proteínas intracelulares. As proteínas de membrana celular identificadas foram: ecto-5´-nucleotidase, aminopeptidase N e enzima conversora de angiotensina. As proteínas intracelulares identificadas foram: proteína contendo domínios de anquirina, N-acylglucosamine 2-epimerase, proteína com domínios ricos em leucina, domínio imunoglobulina e domínio transmembrânico, isoforma 1 da subunidade alfa do fator de fragmentação de DNA e subunidade regulatória da fosfatase 1. Em conclusão, mostramos que após as ultracentrifugações, as microvesículas mantêm sua morfologia, mas algumas delas são lisadas, visto que mais 10 spots foram identificados em S2 e extrato de microvesículas, mas não no veneno bruto. Além disso, as proteínas identificadas podem fazer parte de vias metabólicas que regulam a integridade da célula secretora bem como ter um papel relevante no envenenamento, de acordo com as funções já descritas em literatura. A participação de microvesículas em processos fisiológicos ou patológicos mostra sua importância e abre novas perspectivas de investigação de seu papel no envenenamento.