Individual Variability in the Venom Proteome of Juvenile <i>Bothrops jararaca</i> Specimens

Snake venom proteomes/peptidomes are highly complex and subject to ontogenetic changes. Individual variation in the venom proteome of juvenile snakes is poorly known. We report the proteomic analysis of venoms from 21 juvenile specimens of <i>Bothrops jararaca</i> of different geographical origins and correlate it with the evaluation of important venom features. Individual venoms showed similar caseinolytic activities; however, their amidolytic activities were significantly different. Rather intriguingly, plasma coagulant activity showed remarkable variability among the venoms but not the prothrombin-activating activity. LC–MS analysis showed significant differences between venoms; however, an interesting finding was the ubiquitous presence of the tripeptide ZKW, an endogenous inhibitor of metalloproteinases. Electrophoretic profiles of proteins submitted to reduction showed significant variability in total proteins, glycoproteins, and in the subproteomes of proteinases. Moreover, identification of differential bands revealed variation in most <i>B. jararaca</i> toxin classes. Profiles of venoms analyzed under nonreducing conditions showed less individual variability and identification of proteins in a conserved band revealed the presence of metalloproteinases and l-amino acid oxidase as common components of these venoms. Taken together, our findings suggest that individual venom proteome variability in <i>B. jararaca</i> exists from a very early animal age and is not a result of ontogenetic and diet changes

Proteomic and Glycoproteomic Profilings Reveal That Post-translational Modifications of Toxins Contribute to Venom Phenotype in Snakes

Snake venoms are biological weapon systems composed of secreted proteins and peptides that are used for immobilizing or killing prey. Although post-translational modifications are widely investigated because of their importance in many biological phenomena, we currently still have little understanding of how protein glycosylation impacts the variation and stability of venom proteomes. To address these issues, here we characterized the venom proteomes of seven <i>Bothrops</i> snakes using a shotgun proteomics strategy. Moreover, we compared the electrophoretic profiles of native and deglycosylated venoms and, in order to assess their subproteomes of glycoproteins, we identified the proteins with affinity for three lectins with different saccharide specificities and their putative glycosylation sites. As proteinases are abundant glycosylated toxins, we examined the effect of <i>N</i>-deglycosylation on their catalytic activities and show that the proteinases of the seven venoms were similarly affected by removal of <i>N</i>-glycans. Moreover, we prospected putative glycosylation sites of transcripts of a <i>B. jararaca</i> venom gland data set and detected toxin family related patterns of glycosylation. Based on our global analysis, we report that <i>Bothrops</i> venom proteomes and glycoproteomes contain a core of components that markedly define their composition, which is conserved upon evolution in parallel to other molecular markers that determine their phylogenetic classification

Proteomic and Glycoproteomic Profilings Reveal That Post-translational Modifications of Toxins Contribute to Venom Phenotype in Snakes

Snake venoms are biological weapon systems composed of secreted proteins and peptides that are used for immobilizing or killing prey. Although post-translational modifications are widely investigated because of their importance in many biological phenomena, we currently still have little understanding of how protein glycosylation impacts the variation and stability of venom proteomes. To address these issues, here we characterized the venom proteomes of seven <i>Bothrops</i> snakes using a shotgun proteomics strategy. Moreover, we compared the electrophoretic profiles of native and deglycosylated venoms and, in order to assess their subproteomes of glycoproteins, we identified the proteins with affinity for three lectins with different saccharide specificities and their putative glycosylation sites. As proteinases are abundant glycosylated toxins, we examined the effect of <i>N</i>-deglycosylation on their catalytic activities and show that the proteinases of the seven venoms were similarly affected by removal of <i>N</i>-glycans. Moreover, we prospected putative glycosylation sites of transcripts of a <i>B. jararaca</i> venom gland data set and detected toxin family related patterns of glycosylation. Based on our global analysis, we report that <i>Bothrops</i> venom proteomes and glycoproteomes contain a core of components that markedly define their composition, which is conserved upon evolution in parallel to other molecular markers that determine their phylogenetic classification

Proteomic and Glycoproteomic Profilings Reveal That Post-translational Modifications of Toxins Contribute to Venom Phenotype in Snakes

Snake venoms are biological weapon systems composed of secreted proteins and peptides that are used for immobilizing or killing prey. Although post-translational modifications are widely investigated because of their importance in many biological phenomena, we currently still have little understanding of how protein glycosylation impacts the variation and stability of venom proteomes. To address these issues, here we characterized the venom proteomes of seven <i>Bothrops</i> snakes using a shotgun proteomics strategy. Moreover, we compared the electrophoretic profiles of native and deglycosylated venoms and, in order to assess their subproteomes of glycoproteins, we identified the proteins with affinity for three lectins with different saccharide specificities and their putative glycosylation sites. As proteinases are abundant glycosylated toxins, we examined the effect of <i>N</i>-deglycosylation on their catalytic activities and show that the proteinases of the seven venoms were similarly affected by removal of <i>N</i>-glycans. Moreover, we prospected putative glycosylation sites of transcripts of a <i>B. jararaca</i> venom gland data set and detected toxin family related patterns of glycosylation. Based on our global analysis, we report that <i>Bothrops</i> venom proteomes and glycoproteomes contain a core of components that markedly define their composition, which is conserved upon evolution in parallel to other molecular markers that determine their phylogenetic classification

Dynamic Rearrangement in Snake Venom Gland Proteome: Insights into <i>Bothrops jararaca</i> Intraspecific Venom Variation

We carried out an analysis of the venom gland proteome of <i>Bothrops jararaca</i> taking into account two distinct phases of its ontogenetic development (i.e., newborn and adult) and the marked sexual dimorphism recently reported on its venom proteome. Proteomic data analysis showed a dynamic rearrangement in the proteome landscape of <i>B. jararaca</i> venom gland upon development and gender-related changes. Differentially expressed proteins covered a number of biological pathways related to protein synthesis, including proteins associated with transcription and translation, which were found to be significantly higher expressed in the newborn venom gland. Our results suggest that the variation in the expression levels of cellular proteins might give rise to an even higher variation in the levels of the expressed toxins. Upon aging, the venom gland proteome repertoire related to the protein synthesis together with ecological traits would have an impact on the toxin repertoire, which, in the case of <i>B. jararaca</i> species, would enable the species to deal with different prey types during its lifespan. Proteomic data are available via ProteomeXchange with identifier PXD004186

Exploring Potential Virulence Regulators in <i>Paracoccidioides brasiliensis</i> Isolates of Varying Virulence through Quantitative Proteomics

Few virulence factors have been identified for <i>Paracoccidioides brasiliensis</i>, the agent of paracoccidioidomycosis. In this study, we quantitatively evaluated the protein composition of <i>P. brasiliensis</i> in the yeast phase using minimal and rich media to obtain a better understanding of its virulence and to gain new insights into pathogen adaptation strategies. This analysis was performed on two isolates of the Pb18 strain showing distinct infection profiles in B10.A mice. Using liquid chromatography/tandem mass spectrometry (LC–MS/MS) analysis, we identified and quantified 316 proteins in minimal medium, 29 of which were overexpressed in virulent Pb18. In rich medium, 29 out of 295 proteins were overexpressed in the virulent fungus. Three proteins were found to be up-regulated in both media, suggesting the potential roles of these proteins in virulence regulation in <i>P. brasiliensis</i>. Moreover, genes up-regulated in virulent Pb18 showed an increase in its expression after the recovery of virulence of attenuated Pb18. Proteins up-regulated in both isolates were grouped according to their functional categories. Virulent Pb18 undergoes metabolic reorganization and increased expression of proteins involved in fermentative respiration. This approach allowed us to identify potential virulence regulators and provided a foundation for achieving a molecular understanding of how <i>Paracoccidioides</i> modulates the host–pathogen interaction to its advantage

Exploring Potential Virulence Regulators in <i>Paracoccidioides brasiliensis</i> Isolates of Varying Virulence through Quantitative Proteomics

Few virulence factors have been identified for <i>Paracoccidioides brasiliensis</i>, the agent of paracoccidioidomycosis. In this study, we quantitatively evaluated the protein composition of <i>P. brasiliensis</i> in the yeast phase using minimal and rich media to obtain a better understanding of its virulence and to gain new insights into pathogen adaptation strategies. This analysis was performed on two isolates of the Pb18 strain showing distinct infection profiles in B10.A mice. Using liquid chromatography/tandem mass spectrometry (LC–MS/MS) analysis, we identified and quantified 316 proteins in minimal medium, 29 of which were overexpressed in virulent Pb18. In rich medium, 29 out of 295 proteins were overexpressed in the virulent fungus. Three proteins were found to be up-regulated in both media, suggesting the potential roles of these proteins in virulence regulation in <i>P. brasiliensis</i>. Moreover, genes up-regulated in virulent Pb18 showed an increase in its expression after the recovery of virulence of attenuated Pb18. Proteins up-regulated in both isolates were grouped according to their functional categories. Virulent Pb18 undergoes metabolic reorganization and increased expression of proteins involved in fermentative respiration. This approach allowed us to identify potential virulence regulators and provided a foundation for achieving a molecular understanding of how <i>Paracoccidioides</i> modulates the host–pathogen interaction to its advantage

Venomics Profiling of <i>Thamnodynastes strigatus</i> Unveils Matrix Metalloproteinases and Other Novel Proteins Recruited to the Toxin Arsenal of Rear-Fanged Snakes

Rear-fanged and aglyphous snakes are usually considered not dangerous to humans because of their limited capacity of injecting venom. Therefore, only a few studies have been dedicated to characterizing the venom of the largest parcel of snake fauna. Here, we investigated the venom proteome of the rear-fanged snake <i>Thamnodynastes strigatus</i>, in combination with a transcriptomic evaluation of the venom gland. About 60% of all transcripts code for putative venom components. A striking finding is that the most abundant type of transcript (∼47%) and also the major protein type in the venom correspond to a new kind of matrix metalloproteinase (MMP) that is unrelated to the classical snake venom metalloproteinases found in all snake families. These enzymes were recently suggested as possible venom components, and we show here that they are proteolytically active and probably recruited to venom from a MMP-9 ancestor. Other unusual proteins were suggested to be venom components: a protein related to lactadherin and an EGF repeat-containing transcript. Despite these unusual molecules, seven toxin classes commonly found in typical venomous snakes are also present in the venom. These results support the evidence that the arsenals of these snakes are very diverse and harbor new types of biologically important molecules

Venomics Profiling of <i>Thamnodynastes strigatus</i> Unveils Matrix Metalloproteinases and Other Novel Proteins Recruited to the Toxin Arsenal of Rear-Fanged Snakes

Rear-fanged and aglyphous snakes are usually considered not dangerous to humans because of their limited capacity of injecting venom. Therefore, only a few studies have been dedicated to characterizing the venom of the largest parcel of snake fauna. Here, we investigated the venom proteome of the rear-fanged snake <i>Thamnodynastes strigatus</i>, in combination with a transcriptomic evaluation of the venom gland. About 60% of all transcripts code for putative venom components. A striking finding is that the most abundant type of transcript (∼47%) and also the major protein type in the venom correspond to a new kind of matrix metalloproteinase (MMP) that is unrelated to the classical snake venom metalloproteinases found in all snake families. These enzymes were recently suggested as possible venom components, and we show here that they are proteolytically active and probably recruited to venom from a MMP-9 ancestor. Other unusual proteins were suggested to be venom components: a protein related to lactadherin and an EGF repeat-containing transcript. Despite these unusual molecules, seven toxin classes commonly found in typical venomous snakes are also present in the venom. These results support the evidence that the arsenals of these snakes are very diverse and harbor new types of biologically important molecules