Maria de Fátima D. Furtado

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.

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.

Interspecific variation in venom composition and toxicity of Brazilian snakes from Bothrops genus

The genus Bothrops spp. is responsible for 90% of envenomation by snakes in Brazil, and the standard treatment for snakebites is the antivenom therapy. The anti-bothropic serum produced by Butantan Institute is prepared by the hyperimmunization of horses with a pool of venoms from Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni and Bothrops neuwiedi. In this study, the biochemical and biological characteristics of the venoms from nineteen snakes of the genus Bothrops, responsible for human accidents in Brazil, were analysed. Venoms, particularly from Crotalidae and Viperidae snakes, are rich sources of serine proteases and metalloproteases and the ability of the Brazilian anti-bothropic serum to neutralize the proteolytic activity of these venoms were also tested. The results obtained here show the existence of a large range of variation in the composition and activities in Bothrops spp. toxins and demonstrate that the anti-bothropic serum is not able to fully neutralize the toxic activities of all analysed venoms. These suggest that for the preparation of a fully effective therapeutic anti-bothropic serum, other venoms should be included in the immunization mixture.

Diversity of Micrurus snake species related to their venom toxic effects and the prospective of antivenom neutralization.

Background Micrurus snake bites can cause death by muscle paralysis and respiratory arrest, few hours after envenomation. The specific treatment for coral snake envenomation is the intravenous application of heterologous antivenom and, in Brazil, it is produced by horse immunization with a mixture of M. corallinus and M. frontalis venoms, snakes that inhabit the South and Southeastern regions of the country. However, this antivenom might be inefficient, considering the existence of intra- and inter-specific variations in the composition of the venoms. Therefore, the aim of the present study was to investigate the toxic properties of venoms from nine species of Micrurus: eight present in different geographic regions of Brazil (M. frontalis, M. corallinus, M. hemprichii, M. spixii, M. altirostris, M. surinamensis, M. ibiboboca, M. lemniscatus) and one (M. fulvius) with large distribution in Southeastern United States and Mexico. This study also analyzed the antigenic cross-reactivity and the neutralizing potential of the Brazilian coral snake antivenom against these Micrurus venoms. Methodology/Principal Findings Analysis of protein composition and toxicity revealed a large diversity of venoms from the nine Micrurus species. ELISA and Western blot assays showed a varied capability of the therapeutic antivenom to recognize the diverse species venom components. In vivo and in vitro neutralization assays indicated that the antivenom is not able to fully neutralize the toxic activities of all venoms. Conclusion These results indicate the existence of a large range of both qualitative and quantitative variations in Micrurus venoms, probably reflecting the adaptation of the snakes from this genus to vastly dissimilar habitats. The data also show that the antivenom used for human therapy in Brazil is not fully able to neutralize the main toxic activities present in the venoms from all Micrurus species occurring in the country. It suggests that modifications in the immunization scheme, with the inclusion of other venoms in the antigenic mixture, should occur in order to generate effective therapeutic coral snake antivenom.

Bothrops jararaca venom proteome rearrangement upon neonate to adult transition

The pharmacological activities displayed by Bothrops jararaca venom undergo a significant ontogenetic shift. Similarly, the diet of this species changes from ectothermic prey in early life to endothermic prey in adulthood. In this study we used large and representative newborn and adult venom samples consisting of pools from 694 and 110 specimens, respectively, and demonstrate a significant ontogenetic shift in the venom proteome complexity of B. jararaca. 2‐DE coupled to MS protein identification showed a clear rearrangement of the toxin arsenal both in terms of the total proteome, as of the glycoproteome. N‐glycosylation seems to play a key role in venom protein variability between newborn and adult specimens. Upon the snake development, the subproteome of metalloproteinases undergoes a shift from a P‐III‐rich to a P‐I‐rich profile while the serine proteinase profile does not vary significantly. We also used isobaric tag labeling (iTRAQ) of venom tryptic peptides for the first time to examine the quantitative changes in the venom toxins of B. jararaca upon neonate to adult transition. The iTRAQ analysis showed changes in various toxin classes, especially the proteinases. Our study expands the in‐depth understanding of venom complexity variation particularly with regard to toxin families that have been associated with envenomation pathogenesis.

A Transcriptomic View of the Proteome Variability of Newborn and Adult Bothrops jararaca Snake Venoms

Background Snake bite is a neglected public health problem in communities in rural areas of several countries. Bothrops jararaca causes many snake bites in Brazil and previous studies have demonstrated that the pharmacological activities displayed by its venom undergo a significant ontogenetic shift. Similarly, the venom proteome of B. jararaca exhibits a considerable variation upon neonate to adult transition, which is associated with changes in diet from ectothermic prey in early life to endothermic prey in adulthood. Moreover, it has been shown that the Brazilian commercial antibothropic antivenom, which is produced by immunization with adult venom, is less effective in neutralizing newborn venom effects. On the other hand, venom gland transcripts of newborn snakes are poorly known since all transcriptomic studies have been carried out using mRNA from adult specimens. Methods/Principal Findings Here we analyzed venom gland cDNA libraries of newborn and adult B. jararaca in order to evaluate whether the variability demonstrated for its venom proteome and pharmacological activities was correlated with differences in the structure of toxin transcripts. The analysis revealed that the variability in B. jararaca venom gland transcriptomes is quantitative, as illustrated by the very high content of metalloproteinases in the newborn venom glands. Moreover, the variability is also characterized by the structural diversity of SVMP precursors found in newborn and adult transcriptomes. In the adult transcriptome, however, the content of metalloproteinase precursors considerably diminishes and the number of transcripts of serine proteinases, C-type lectins and bradykinin-potentiating peptides increase. Moreover, the comparison of the content of ESTs encoding toxins in adult male and female venom glands showed some gender-related differences. Conclusions/Significance We demonstrate a substantial shift in toxin transcripts upon snake development and a marked decrease in the metalloproteinase P-III/P-I class ratio which are correlated with changes in the venom proteome complexity and pharmacological activities.

Pro-inflammatory activities in elapid snake venoms.

1 Snake venoms from the genera Micrurus (M. ibiboboca and M. spixii) and Naja (N. naja, N. melanoleuca and N. nigricollis) were analysed, using biological and immunochemical methods, to detect pro‐inflammatory activities, cobra venom factor (COF), proteolytic enzymes, thrombin‐like substances, haemorrhagic and oedema‐producing substances. 2 The venoms of the five snake species activate the complement system (C) in normal human serum (NHS) in a dose‐related fashion, at concentrations ranging from 5 μg to 200 μg ml−1 serum. Electrophoretic conversion of C3 was observed with all venoms in NHS containing normal concentrations of Ca2+ and Mg2+, but only by venoms from N. naja and N. melanoleuca when Ca2+ was chelated by adding Mg2+‐EGTA. 3 Purified human C3 was electrophoretically converted, in the absence of other C components, by the venoms from N. naja, N. nigricollis and M. ibiboboca. However, only the venoms from N. naja and N. melanoleuca contained a 144 kDa protein revealed in Western blot with sera against COF or human C3. 4 All venoms, at minimum concentrations of 30 ng ml−1, were capable of lysing sheep red blood cells, also in a dose‐related fashion, when incubated with these cells in presence of egg yolk as a source of lecithin. Although the venoms from M. spixii and N. nigricollis showed detectable thrombin‐like activity, these and the other venoms were free of proteolytic activity when fibrin, gelatin and casein, were used as substrates. 5 When tested on mice skin, all five venoms were capable of inducing an increase in vascular permeability and oedema, but were devoid of haemorrhagic producing substances (haemorrhagins). 6 These data provide evidence indicating that Elapidae venoms contain various pro‐inflammatory factors which may be important in the spreading of neurotoxins throughout the tissues of the prey or human victim.

Caracterizacion de las actividades biologicas de los venenos ‘amarillo’ y ‘blanco’ de Crotalus durissus ruruima comparados con el veneno de Crotalus durissus terrificus. Poder neutralizante de los antivenenos frente a los venenos de Crotalus durissus ruruima

Abstract The biological activities of ‘yellow’ and ‘white’ venom of a rattlesnake Crotalus durissus ruruima Hoge, 1965, found in the savanna-like vegetation (cerrado) of northern Brazil (Roraima) and Venezuela have been studied, and compared to the reference Crotalus durissus terrificus venom. The lethal activity of venoms depended on the inoculation route. The most toxic venom was the white one. The venoms of C. d. terrificus and the yellow of C. d. ruruima had similar lethalities. The yellow venom of C. d. ruruima showed a caseinolytic activity three times higher than that obtained with either the venom of C. d. terrificus or the white one of the C. d. ruruima . Hemorrhagic and necrotic activities were found only in the yellow venom. White and yellow venoms from C. d. ruruima showed a similar action on fibrinogen; this thrombin-like action was greater with C. d. terrificus venom. On histopathological sections local and pulmonary hemorrhage was found only with the yellow venom, but myonecrotic activity was observed with both venoms of C. d. ruruima . Among all antivenoms studied, the anti-bothropic-crotalic was the best at neutralizing hemorrhagic and hemolytic activities. These results suggest that antivenom bothropic-crotalic should be used in the treatment of patients with snakebite by C. d. ruruima : besides its neutralization on lethal activity, it also neutralizes the hemorrhagic activity present in some venoms.

A eficácia do antiveneno botrópico-crotálico na neutralização das principais atividades do veneno de Bothrops jararacussu

A mionecrose e um dos efeitos causados pelo veneno de Bothrops jararacussu. Uma miotoxina com homologia estrutural a fosfolipase A2 (PLA2), mas sem atividade enzimatica, foi isolada desse veneno. O veneno de Crotalus durissus terrificus apresenta tambem atividade miotoxica, que vem sendo atribuida a crotoxina e a PLA2 (crotoxina B), o componente basico do complexo crotoxina. O veneno de Bothrops jararacussu apresenta tres proteinas, que tem identidade imunologica com a PLA2 da crotoxina. O presente trabalho comparou a eficiencia dos antivenenos polivalentes comerciais produzidos pelo Instituto Butantan - o antiveneno botropico (AB) e o antiveneno botropico-crotalico (AB/C) - na neutralizacao das atividades letal, hemorragica, coagulante e miotoxica do veneno de B. jararacussu. Os dois antivenenos neutralizaram de maneira semelhante a atividade hemorragica, mas o AB/C foi tres vezes mais potente que o AB em neutralizar a acao miotoxica e duas vezes mais potente na neutralizacao da letalidade e na acao coagulante do veneno de B. jararacussu. Os dados sugerem que a utilizacao do AB/C pode ser vantajosa no tratamento de pacientes picados por serpentes dessa especie

Analysis of the subproteomes of proteinases and heparin-binding toxins of eight Bothrops venoms.

Viperid snakes show the most complex snake‐venom proteomes and offer an intriguing challenge in terms of understanding the nature of their components and the pathological outcomes of envenomation characterized by local and systemic effects. In this work, the venom complexity of eight Bothrops species was analyzed by 2‐DE, and their subproteomes of proteinases were explored by 2‐D immunostaining and 2‐D gelatin zymography, demonstrating the diversity of their profiles. Heparin, a highly sulfated glycosaminoglycan released from mast cells, is involved in anti‐coagulant and anti‐inflammatory processes. Here, we explored the hypothesis that heparin released upon envenomation could interact with toxins and interfere with venom pathogenesis. We first identified the Bothrops venom subproteome of toxins that bind with high‐affinity for heparin as composed of mainly serine proteinases and C‐type lectins. Next, we explored the Bothrops jararaca toxins that bind to heparin under physiological conditions and identified a relationship between the subproteomes of proteinases, and that of heparin‐binding toxins. Only the non‐bound fraction, composed mainly of metalloproteinases, showed lethal and hemorrhagic activities, whereas the heparin‐bound fraction contained mainly serine proteinases associated with coagulant and fibrinogenolytic activities. These data suggest that heparin binding to B. jararaca venom components in vivo has a minor protective effect to venom toxicity.