PKC-α is involved in the signaling of phagocytosis induced by two snake venom secretory PLA2S in macrophages.

Phagocytosis, an essential process for host defense, requires the coordination of a variety of signaling reactions. MT-II, an enzymatically inactive Lys49 phospholipase A2 (PLA2) homolog, and MT-III, a catalytically-active Asp49 PLA2, are known to activate phagocytosis in macrophages. In this study, the signaling pathways mediating phagocytosis, focusing on protein kinases, were investigated. Macrophages from male Swiss mice peritoneum were obtained 96 h after intraperitoneal thioglycolate injection. Phagocytosis was evaluated using non-opsonized zymosan particles in the presence or absence of specific inhibitors, as well as PKC and PKC-α localization by confocal microscopy. Moreover, protein kinase C (PKC) activity was assessed by γP32 ATP in macrophages stimulated by both PLA2s. Data showed that both sPLA2s increased phagocytosis. Cytochalasin D, staurosporine/H7, wortmannin, and herbimycin, inhibitors of actin polymerization, PKC, phosphoinositide 3-kinase (PI3K), and protein tyrosine kinase (PTK), respectively, significantly reduced phagocytosis induced by both PLA2s. PKC activity was increased in macrophages stimulated by both PLA2s. Actin polymerization and talin were evidenced by immunofluorescence and talin was recruited 5 min after both PLA2s stimulation. PKC and PKC-α localization within the cell were increased after 60 min of MT-II and MT-III stimulation. These data suggest that the effect of both PLA2s depends on actin cytoskeleton rearrangements and the activation of PKC, PI3K, and PTK signaling events required for phagocytosis.

Radicicol enhances the regeneration of skeletal muscle injured by crotoxin via decrease of NF-kB activation

This study evaluated cellular and molecular effects of radicicol, a heat shock protein (HSP) inducer, on the regeneration of skeletal muscle injured by crotoxin, the main toxin isolated from Crotalus durissus terrificus venom. Regenerating muscles treated with radicicol had decreased NF-kB activation. Differentiating myoblasts treated with radicicol showed reduced number of NF-kB positive nuclei and increased fusion index. The results suggest that radicicol enhances regeneration of muscle by attenuating NF-kB activation and increasing myogenic differentiation.

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.

The Primary Duct of Bothrops jararaca Glandular Apparatus Secretes Toxins.

Despite numerous studies concerning morphology and venom production and secretion in the main venom gland (and some data on the accessory gland) of the venom glandular apparatus of Viperidae snakes, the primary duct has been overlooked. We characterized the primary duct of the Bothrops jararaca snake by morphological analysis, immunohistochemistry and proteomics. The duct has a pseudostratified epithelium with secretory columnar cells with vesicles of various electrondensities, as well as mitochondria-rich, dark, basal, and horizontal cells. Morphological analysis, at different periods after venom extraction, showed that the primary duct has a long cycle of synthesis and secretion, as do the main venom and accessory glands; however, the duct has a mixed mode venom storage, both in the lumen and in secretory vesicles. Mouse anti-B. jararaca venom serum strongly stained the primary duct's epithelium. Subsequent proteomic analysis revealed the synthesis of venom toxins-mainly C-type lectin/C-type lectin-like proteins. We propose that the primary duct's toxin synthesis products complement the final venom bolus. Finally, we hypothesize that the primary duct and the accessory gland (components of the venom glandular apparatus) are part of the evolutionary path from a salivary gland towards the main venom gland.

Bothrops jararaca accessory venom gland is an ancillary source of toxins to the snake.

In Viperidae snakes, it has been attributed to the main venom gland, a component of the venom gland apparatus, the function of synthesizing all venom toxins and storing them inside a basal-central lumen. However, the role of the accessory gland is still unknown. Here, we analyzed the proteome and the transcriptome of the accessory gland during venom production and secretion cycle. We showed that the accessory gland expresses and synthesizes toxins that are similar to those produced by the main venom gland such as C-type lectin/C-type lectin-like proteins, metalloproteinase, phospholipase A2, cysteine rich secretory protein, nerve growth factor, vascular endothelial growth factor, serine proteinase, and l-amino acid oxidase. Our data have shown that toxin synthesis in the accessory gland is asynchronous when compared to the same process in the venom gland. Moreover, this gland also expresses inhibitors of venom phospholipases A2 and metalloproteinases. Transcriptome analysis showed that the transcripts that correspond to toxins in the accessory gland have a good correlation to the main venom gland transcripts. Therefore, it is proposed that the accessory gland is an ancillary source of toxins to the snake, and provides inhibitors that could control venom toxicity (and integrity) during storage. SIGNIFICANCE: In this study, we propose that the accessory venom gland acts as an important ancillary source of toxins to the snake, in lieu of a depleted main venom gland, and provides inhibiting agents that control venom toxicity (and integrity) during its storage.

Bothrops jararaca accessory venom gland is an ancillary source of toxins to the snake

In Viperidae snakes, it has been attributed to the main venom gland, a component of the venom gland apparatus, the function of synthesizing all venom toxins and storing them inside a basal-central lumen. However, the role of the accessory gland is still unknown. Here, we analyzed the proteome and the transcriptome of the accessory gland during venom production and secretion cycle. We showed that the accessory gland expresses and synthesizes toxins that are similar to those produced by the main venom gland such as C-type lectin/C-type lectin-like proteins, metalloproteinase, phospholipase A(2), cysteine rich secretory protein, nerve growth factor, vascular endothelial growth factor, serine proteinase, and L-amino acid oxidase. Our data have shown that toxin synthesis in the accessory gland is asynchronous when compared to the same process in the venom gland. Moreover, this gland also expresses inhibitors of venom phospholipases A(2) and metalloproteinases. Transcriptome analysis showed that the transcripts that correspond to toxins in the accessory gland have a good correlation to the main venom gland transcripts. Therefore, it is proposed that the accessory gland is an ancillary source of toxins to the snake, and provides inhibitors that could control venom toxicity (and integrity) during storage. Significance: In this study, we propose that the accessory venom gland acts as an important ancillary source of toxins to the snake, in lieu of a depleted main venom gland, and provides inhibiting agents that control venom toxicity (and integrity) during its storage.

The primary duct of Bothrops jararaca glandular apparatus secretes toxins

Despite numerous studies concerning morphology and venom production and secretion in the main venom gland (and some data on the accessory gland) of the venom glandular apparatus of Viperidae snakes, the primary duct has been overlooked. We characterized the primary duct of the Bothrops jararaca snake by morphological analysis, immunohistochemistry and proteomics. The duct has a pseudostratified epithelium with secretory columnar cells with vesicles of various electrondensities, as well as mitochondria-rich, dark, basal, and horizontal cells. Morphological analysis, at different periods after venom extraction, showed that the primary duct has a long cycle of synthesis and secretion, as do the main venom and accessory glands; however, the duct has a mixed mode venom storage, both in the lumen and in secretory vesicles. Mouse anti-B. jararaca venom serum strongly stained the primary duct’s epithelium. Subsequent proteomic analysis revealed the synthesis of venom toxins—mainly C-type lectin/C-type lectin-like proteins. We propose that the primary duct’s toxin synthesis products complement the final venom bolus. Finally, we hypothesize that the primary duct and the accessory gland (components of the venom glandular apparatus) are part of the evolutionary path from a salivary gland towards the main venom gland.

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.

Bothrops jararaca accessory venom gland is an ancillary source of toxins to the snake

In Viperidae snakes, it has been attributed to the main venom gland, a component of the venom gland apparatus, the function of synthesizing all venom toxins and storing them inside a basal-central lumen. However, the role of the accessory gland is still unknown. Here, we analyzed the proteome and the transcriptome of the accessory gland during venom production and secretion cycle. We showed that the accessory gland expresses and synthesizes toxins that are similar to those produced by the main venom gland such as C-type lectin/C-type lectin-like proteins, metalloproteinase, phospholipase A(2), cysteine rich secretory protein, nerve growth factor, vascular endothelial growth factor, serine proteinase, and L-amino acid oxidase. Our data have shown that toxin synthesis in the accessory gland is asynchronous when compared to the same process in the venom gland. Moreover, this gland also expresses inhibitors of venom phospholipases A(2) and metalloproteinases. Transcriptome analysis showed that the transcripts that correspond to toxins in the accessory gland have a good correlation to the main venom gland transcripts. Therefore, it is proposed that the accessory gland is an ancillary source of toxins to the snake, and provides inhibitors that could control venom toxicity (and integrity) during storage. Significance: In this study, we propose that the accessory venom gland acts as an important ancillary source of toxins to the snake, in lieu of a depleted main venom gland, and provides inhibiting agents that control venom toxicity (and integrity) during its storage.

The primary duct of Bothrops jararaca glandular apparatus secretes toxins

Despite numerous studies concerning morphology and venom production and secretion in the main venom gland (and some data on the accessory gland) of the venom glandular apparatus of Viperidae snakes, the primary duct has been overlooked. We characterized the primary duct of the Bothrops jararaca snake by morphological analysis, immunohistochemistry and proteomics. The duct has a pseudostratified epithelium with secretory columnar cells with vesicles of various electrondensities, as well as mitochondria-rich, dark, basal, and horizontal cells. Morphological analysis, at different periods after venom extraction, showed that the primary duct has a long cycle of synthesis and secretion, as do the main venom and accessory glands; however, the duct has a mixed mode venom storage, both in the lumen and in secretory vesicles. Mouse anti-B. jararaca venom serum strongly stained the primary duct's epithelium. Subsequent proteomic analysis revealed the synthesis of venom toxins-mainly C-type lectin/C-type lectin-like proteins. We propose that the primary duct's toxin synthesis products complement the final venom bolus. Finally, we hypothesize that the primary duct and the accessory gland (components of the venom glandular apparatus) are part of the evolutionary path from a salivary gland towards the main venom gland.

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.

Insulin-regulated aminopeptidase in adipocyte is Cys-specific and affected by obesity.

Insulin-regulated aminopeptidase (IRAP, EC 3.4.11.3) in adipocytes is well known to traffic between high (HDM) and low (LDM) density microsomal fractions toward the plasma membrane (MF) under stimulation by insulin. However, its catalytic preference for aminoacyl substrates with N-terminal Leu or Cys is controversial. Furthermore, possible changes in its traffic under metabolic challenges are unknown. The present study investigated the catalytic activity attributable to EC 3.4.11.3 in HDM, LDM and MF from isolated adipocytes of healthy (C), food deprived (FD) and monosodium glutamate (MSG) obese rats on aminoacyl substrates with N-terminal Cys or Leu, in absence or presence of insulin. Efficacy and reproducibility of subcellular adipocyte fractionation procedure were demonstrated. Comparison among HDM vs LDM vs MF intragroup revealed that hydrolytic activity trafficking from LDM to MF under influence of insulin in C, MSG and FD is only on N-terminal Cys. In MSG the same pattern of anterograde traffic and aminoacyl preference occurred independently of insulin stimulation. The pathophysiological significance of IRAP in adipocytes seems to be linked to comprehensive energy metabolism related roles of endogenous substrates with N-terminal cysteine pair such as vasopressin and oxytocin.

Venom-related transcripts from Bothrops jararaca tissues provide novel molecular insights into the production and evolution of snake venom.

Attempts to reconstruct the evolutionary history of snake toxins in the context of their co-option to the venom gland rarely account for nonvenom snake genes that are paralogous to toxins, and which therefore represent important connectors to ancestral genes. In order to reevaluate this process, we conducted a comparative transcriptomic survey on body tissues from a venomous snake. A nonredundant set of 33,000 unigenes (assembled transcripts of reference genes) was independently assembled from six organs of the medically important viperid snake Bothrops jararaca, providing a reference list of 82 full-length toxins from the venom gland and specific products from other tissues, such as pancreatic digestive enzymes. Unigenes were then screened for nontoxin transcripts paralogous to toxins revealing 1) low level coexpression of approximately 20% of toxin genes (e.g., bradykinin-potentiating peptide, C-type lectin, snake venom metalloproteinase, snake venom nerve growth factor) in body tissues, 2) the identity of the closest paralogs to toxin genes in eight classes of toxins, 3) the location and level of paralog expression, indicating that, in general, co-expression occurs in a higher number of tissues and at lower levels than observed for toxin genes, and 4) strong evidence of a toxin gene reverting back to selective expression in a body tissue. In addition, our differential gene expression analyses identify specific cellular processes that make the venom gland a highly specialized secretory tissue. Our results demonstrate that the evolution and production of venom in snakes is a complex process that can only be understood in the context of comparative data from other snake tissues, including the identification of genes paralogous to venom toxins.

Efeito do 17-beta-estradiol ICI 182.780 e G1 sobre as células MCF-7: Uma abordagem proteômica

O GPER, identificado como um possível receptor das ações rápidas do estrógeno e caracterizado como mecanismo da via não-genômica do estrógeno, é o mais recente receptor de estrógeno descoberto acoplado à proteína G e seu estudo tem sido bastante amplo desde sua identificação diante da sua grande importância na implicação do tratamento realizado no câncer de mama, uma vez que os moduladores seletivos utilizados no tratamento do câncer de mama possuem também uma ação agonista nesse receptor e sua possível ativação pode resultar no desenvolvimento da resistência desses moduladores utilizados no tratamento do câncer mamário. Neste trabalho, realizamos um estudo proteômico das células MCF-7 de carcinoma mamário tratadas com Estradiol, ICI 182, 780 (antagonista dos receptores clássicos do estrógeno, mas agonista do GPER) e G1 (agonista seletivo do GPER), a fim de comparar a ação genômica dos diferentes receptores de estrógeno. Neste estudo, pudemos verificar um panorama geral da estimulação tanto dos receptores clássicos de estrógeno, como do GPER sobre a regulação da síntese de proteínas. Verificamos ainda, que poucas proteínas identificadas são exclusivamentes reguladas pela ativação dos receptores clássicos de estrógeno; detectamos somente quatro. Portanto, parece haver um sinergismo na ativação desses receptores com o intuito de promover a síntese de proteínas e a manutenção da homeostase celular. Foram encontradas também proteínas já descritas em outros trabalhos que tem envolvimento e grande importância no câncer de mama e que tiveram sua síntese aumentada nos três tratamentos, como no caso da Calreticulina, a proteína Heat Shock 70kDa e a Fosfoproteína 1 induzida por estresse (STP1). Dessa forma, este trabalho mostra que, como os receptores clássicos de estrógeno, o GPER também tem uma ação genômia, além da não genômica.

Unraveling the processing and activation of snake venom metalloproteinases.

Snake venom metalloproteinases (SVMPs) are zinc-dependent enzymes responsible for most symptoms of human envenoming. Like matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase (ADAM) proteins, SVMPs are synthesized as zymogens, and enzyme activation is regulated by hydrolysis of their prodomain, but the processing of SVMPs is still unclear. In this study, we attempted to identify the presence of prodomain in different compartments of snake venom glands as zymogens or in the free form to elucidate some mechanism involved in SVMP activation. Using antibodies obtained by immunization with a recombinant prodomain, bands of zymogen molecular mass and prodomain peptides were detected mostly in gland extracts all along the venom production cycle and in the venom collected from the lumen at the peak of venom production. Prodomain was detected in secretory cells mostly in the secretory vesicles near the Golgi. We hypothesize that the processing of SVMPs starts within secretory vesicles and continues in the lumen of the venom gland just after enzyme secretion and involves different steps compared to ADAMs and MMPs but can be used as a model for studying the relevance of peptides resulting from prodomain processing and degradation for controlling the activity of metalloproteinases.

Activation of Bothrops jararaca snake venom gland and venom production: a proteomic approach.

Viperidae venom glands have a basal-central lumen where the venom produced by secretory cells is stored. We have shown that the protein composition of venom gland changes during the venom production cycle. Here, we analyzed the venom gland proteins during the venom production cycle by proteomic approach. We identified specific proteins in each stage of the cycle. Protein species from endoplasmic reticulum (PDI and GPR78) and cytoplasm (actin, vimentin, tropomyosin, proteasome subunit alpha type-1, thioredoxin, and 40S ribosomal protein) are more abundant in the activated stage, probably increasing the synthesis and secretion of toxins. We also showed for the first time that many toxins are present in the secretory cells during the quiescent stage. C-type lectin-like and serine proteinases were more abundant in the quiescent stage, and GPIb-BP and coagulation factor IX/X were present only in this stage. Metalloproteinases, L-amino acid oxidases, PLA2 and snake venom metalloproteinase and PLA2 inhibitors, and disintegrins were more abundant in the activated stage. Regarding metalloproteinases, the presence of peptides corresponding to the pro-domain was observed. These results allow us to better understand the mechanism of venom gland activation and venom production, contributing to studies about snake toxins and their diversity. BIOLOGICAL SIGNIFICANCE: In this study we identified, for the first time, the presence of different toxins in the snake venom gland in its quiescent stage. Furthermore, we showed that not all toxins are synthesized during the activated stage of the gland, suggesting an asynchronous synthesis for different toxins. Besides, the synthesis of some protein species from endoplasmic reticulum and cytoplasm, which are related to the synthesis and secretion processes, are more abundant in the activated stage of this gland. The knowledge of the proteomic composition of the venom gland in different stages of the venom production cycle will give us new insights into the mechanism of venom gland activation and venom production, contributing to studies about snake toxins and their diversity.