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

Composition of toxin transcripts in newborn and adult cDNA libraries from <i>B. jararaca</i> venom glands.

<p>Composition of toxin transcripts in newborn and adult cDNA libraries from <i>B. jararaca</i> venom glands.</p

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

Exclusive SVMP clusters from newborn and adult venom gland cDNA libraries.

<p>Amino acid sequences deduced from the most abundant SVMP clusters (for details on cluster identification see the text). Pro (white), metalloproteinase (gray), disintegrin/disintegrin-like (black), and cysteine-rich (dark gray) domains are shaded.</p

Graphical overview of SVMP clusters from newborn and adult venom gland cDNA libraries.

<p>Newborn (N) exclusive clusters are shown on the left, shared clusters in the middle and adult (A) exclusive clusters on the right. P-III class SVMPs are shown in red; P-II class SVMPs are shown in blue and SVMP clusters whose sequence matched only to UTR regions, signal peptide, pro-domain and/or catalytic domain are shown in green. The area of each circle is proportional to the number of clones comprised in that cluster. Circles are labeled as either cluster (C) or singleton (S). Graphical view generated with <i>Cytoscape</i> v. 2.8.1 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001554#pntd.0001554-Cline1" target="_blank">[39]</a>.</p

SDS-PAGE and Western blotting of the isolated fractions.

<p>Isolated fractions enriched with SVMPs (10 µg) were subjected to SDS-PAGE under non-reducing conditions and stained by Coomassie blue (A), silver stained (B) or electrotransferred to nitrocellulose membranes and revealed with anti-jararhagin antibody followed by peroxidase labeled anti-rabbit IgG and enzyme substrate (C). The bands with asterisks were cut and analyzed by mass spectrometry.</p

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

Relative comparison of functional activities of isolated fractions.

<p>The biological activities shown above were classified as low (+), moderate (++), or high (+++) for graphical representation.</p><p>Relative comparison of functional activities of isolated fractions.</p