Sialic acid-containing glycans play a role in the activity of snake venom proteases.

Structural variability is a feature of snake venom proteins, and glycosylation is a post-translational modification that contributes to the diversification of venom proteomes. Studies by our group have shown that Bothrops venoms are distinctly defined by their glycoprotein content, and that most hybrid/complex N-glycans identified in these venoms contain sialic acid. Considering that metalloproteases and serine proteases are abundant components of Bothrops venoms and essential in the envenomation process, and that these enzymes contain several glycosylation sites, the role of sialic acid in venom proteolytic activity was evaluated. Here we show that removal of sialic acid by treatment of nine Bothrops venoms with neuraminidase (i) altered the pattern of gelatinolysis in zymography of most venoms and reduced the gelatinolytic activity of all venoms, (ii) decreased the proteolytic activity of some venoms on fibrinogen and the clotting activity of human plasma of all venoms, and (iii) altered the proteolysis profile of plasma proteins by B. jararaca venom, suggesting that sialic acid may play a role in the interaction of proteases with their protein substrates. In contrast, the profile of venom amidolytic activity on Bz-Arg-pNA did not change after removal of sialic acid, indicating that this monosaccharide is not essential in N-glycans of serine proteases acting on small substrates. In summary, these results expand the knowledge about the variability of the subproteomes of Bothrops venom proteases, and for the first time point to the importance of carbohydrate chains containing sialic acid in the enzymatic activities of venom proteases relevant in human envenomation.

Diversity of Phospholipases A2 from Bothrops atrox Snake Venom: Adaptive Advantages for Snakes Compromising Treatments for Snakebite Patients.

The evolution of snake venoms resulted in multigene toxin families that code for structurally similar isoforms eventually harboring distinct functions. PLA2s are dominant toxins in viper venoms, and little is known about the impact of their diversity on human envenomings and neutralization by antivenoms. Here, we show the isolation of three distinct PLA2s from B. atrox venom. FA1 is a Lys-49 homologue, and FA3 and FA4 are catalytic Asp-49 PLA2s. FA1 and FA3 are basic myotoxic proteins, while FA4 is an acid non-myotoxic PLA2. FA3 was the most potent toxin, inducing higher levels of edema, inflammatory nociception, indirect hemolysis, and anticoagulant activity on human, rat, and chicken plasmas. FA4 presented lower anticoagulant activity, and FA1 had only a slight effect on human and rat plasmas. PLA2s presented differential reactivities with antivenoms, with an emphasis on FA3, which was not recognized or neutralized by the antivenoms used in this study. Our findings reveal the functional and antigenic diversity among PLA2s from B. atrox venom, highlighting the importance of assessing venom variability for understanding human envenomations and treatment with antivenoms, particularly evident here as the antivenom fails to recognize FA3, the most active multifunctional toxin described.

Individual Variability in Bothropsatrox Snakes Collected from Different Habitats in the Brazilian Amazon: New Findings on Venom Composition and Functionality.

Differences in snake venom composition occur across all taxonomic levels and it has been argued that this variation represents an adaptation that has evolved to facilitate the capture and digestion of prey and evasion of predators. Bothrops atrox is a terrestrial pitviper that is distributed across the Amazon region, where it occupies different habitats. Using statistical analyses and functional assays that incorporate individual variation, we analyzed the individual venom variability in B. atrox snakes from four different habitats (forest, pasture, degraded area, and floodplain) in and around the Amazon River in Brazil. We observed venom differentiation between spatially distinct B. atrox individuals from the different habitats, with venom variation due to both common (high abundance) and rare (low abundance) proteins. Moreover, differences in the composition of the venoms resulted in individual variability in functionality and heterogeneity in the lethality to mammals and birds, particularly among the floodplain snakes. Taken together, the data obtained from individual venoms of B. atrox snakes, captured in different habitats from the Brazilian Amazon, support the hypothesis that the differential distribution of protein isoforms results in functional distinctiveness and the ability of snakes with different venoms to have variable toxic effects on different prey.

Functional variability of Bothrops atrox venoms from three distinct areas across the Brazilian Amazon and consequences for human envenomings.

Variability in the composition of snake venoms occurs in different taxa and is usually correlated to snake fitness. Here, we compared B. atrox venoms from three different geographic regions across the Brazilian Amazon and found remarkable functional differences particularly between venoms from two populations separated by the Amazon River, in specimens born, raised and maintained under the same conditions at Instituto Butantan serpentary. Venom from Presidente Figueiredo snakes induced stronger dermonecrosis, but was less procoagulant and lethal to mice; these activities were correlated to the presence of a PI-class SVMP and absence of a SVSP in the venom, respectively. Venom from São Bento snakes was more hemorrhagic, killed mice more efficiently, but induced lower signs of dermonecrosis, which was correlated to the higher proportion of SVMPs and the absence of a PI-class SVMP isoform. Belterra snakes, a reference of wild snakes, presented venoms with intermediate phenotypes. Commercial Bothrops antivenom was effective in neutralizing all biological activities evaluated in this study, including dermonecrosis and pro-coagulant, which are relevant for human snakebite accidents by B. atrox. Functional differences correlated to snake fitness may also imply in different symptomatology for B. atrox snakebite patients and deserve special attention from clinical toxicologists.

Functional and proteomic comparison of Bothrops jararaca venom from captive specimens and the Brazilian Bothropic Reference Venom.

Snake venom is a variable phenotypic trait, whose plasticity and evolution are critical for effective antivenom production. A significant reduction of the number of snake donations to Butantan Institute (São Paulo, Brazil) occurred in recent years, and this fact may impair the production of the Brazilian Bothropic Reference Venom (BBRV). Nevertheless, in the last decades a high number of Bothrops jararaca specimens have been raised in captivity in the Laboratory of Herpetology of Butantan Institute. Considering these facts, we compared the biochemical and biological profiles of B. jararaca venom from captive specimens and BBRV in order to understand the potential effects of snake captivity upon the venom composition. Electrophoretic analysis and proteomic profiling revealed few differences in venom protein bands and some differentially abundant toxins. Comparison of enzymatic activities showed minor differences between the two venoms. Similar cross-reactivity recognition pattern of both venoms by the antibothropic antivenom produced by Butantan Institute was observed. Lethality and neutralization of lethality for B. jararaca venom from captive specimens and BBRV showed similar values. Considering these results we suggest that the inclusion of B. jararaca venom from captive specimens in the composition of BBRV would not interfere with the quality of this reference venom. BIOLOGICAL SIGNIFICANCE: Snakebite envenomation is a neglected tropical pathology whose treatment is based on the use of specific antivenoms. Bothrops jararaca is responsible for the majority of snakebites in South and Southeastern Brazil. Its venom shows individual, sexual, and ontogenetic variability, however, the effect of animal captivity upon venom composition is unknown. Considering the reduced number of wild-caught snakes donated to Butantan Institute in the last decades, and the increased life expectancy of the snakes raised in captivity in the Laboratory of Herpetology, this work focused on the comparative profiling of B. jararaca venom from captive snakes and the Brazilian Bothropic Reference Venom (BBRV). BBRV is composed of venom obtained upon the first milking of wild-caught B. jararaca specimens, and used to assess the potency of all bothropic antivenoms produced by Brazilian suppliers. The use of proteomic strategies, added to biochemical and neutralization tests, allowed to conclude that, despite some subtle differences detected between these two venoms, venom from captive specimens could be used in the BBRV composition without affecting its quality in antivenom potency assays.

Edema and Nociception Induced by Philodryas patagoniensis Venom in Mice: A Pharmacological Evaluation with Implications for the Accident Treatment.

We have investigated the mechanisms involved in the genesis of edema and nociception induced by Philodryas patagoniensis venom (PpV) injected into the footpad of mice. PpV induced dose-related edema and nociceptive effects. Pretreatment of mice with cyclooxygenase inhibitor (indomethacin), but not with cyclooxygenase 2 inhibitor (celecoxib) markedly inhibited both effects. Pretreatments with H1 receptor antagonist (promethazine) or with dual histamine-serotonin inhibitor (cyproheptadine) failed in inhibiting both effects. In groups pretreated with captopril (angiotensin-converting enzyme inhibitor) the edema was unaltered, but nociception was clearly increased, suggesting the participation of kinins in the pathophysiology of the nociception but not of the edema-forming effect of PpV. When PpV was treated with EDTA, the nociception was similar to the one induced by untreated venom, but edema was markedly reduced. We concluded that PpV-induced edema and nociception have cyclooxygenase eicosanoids as the main mediators and no participation of vasoactive amines. Kinins seem to participate in nociception but not in edema induced by PpV. The results also suggest that metalloproteinases are the main compounds responsible for the edema, but not for the nociception induced by this venom.

Individual variability in the venom proteome of juvenile Bothrops jararaca 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 Bothrops jararaca 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 B. jararaca 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 B. jararaca exists from a very early animal age and is not a result of ontogenetic and diet changes.

Venomics profiling of Thamnodynastes strigatus 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 Thamnodynastes strigatus , 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.

Analysis of the ontogenetic variation in the venom proteome/peptidome of Bothrops jararaca reveals different strategies to deal with prey.

Previous studies have demonstrated that the pharmacological activities displayed by Bothrops jararaca venom undergo a significant ontogenetic shift. Variation in the venom proteome is a well-documented phenomenon; however, variation in the venom peptidome is poorly understood. We report a comparative proteomic and peptidomic analysis of venoms from newborn and adult specimens of B. jararaca and correlate it with the evaluation of important venom features. We demonstrate that newborn and adult venoms have similar hemorrhagic activities, while the adult venom has a slightly higher lethal activity in mice; however, the newborn venom is extremely more potent to kill chicks. The coagulant activity of newborn venom upon human plasma is 10 times higher than that of adult venom. These differences were clearly reflected in their different profiles of SDS-PAGE, gelatin zimography, immunostaining using specific antibodies, glycosylation pattern, and concanavalin A-binding proteins. Furthermore, we report for the first time the analysis of the peptide fraction of newborn and adult venoms by MALDI-TOF mass spectrometry and LC-MS/MS, which revealed different contents of peptides, while the bradykinin potentiating peptides (BPPs) showed rather similar profiles and were detected in the venoms showing their canonical sequences and also novel sequences corresponding to BPPs processed from their precursor protein at sites so far not described. As a result of these studies, we demonstrated that the ontogenetic shift in diet, from ectothermic prey in early life to endothermic prey in adulthood, and in animal size are associated with changes in the venom proteome in B. jararaca species.

Some aspects of the venom proteome of the Colubridae snake Philodryas olfersii revealed from a Duvernoy's (venom) gland transcriptome.

We investigated the putative toxins of Philodryas olfersii (Colubridae), a representative of a family of snakes neglected in venom studies despite their growing medical importance. Transcriptomic data of the venom gland complemented by proteomic analysis of the gland secretion revealed the presence of major toxin classes from the Viperidae family, including serine proteases, metalloproteases, C-type lectins, Crisps, and a C-type natriuretic peptide (CNP). Interestingly, the phylogenetic analysis of the CNP precursor showed it as a linker between two related precursors found in Viperidae and Elapidae snakes. We suggest that these precursors constitute a monophyletic group derived from the vertebrate CNPs.