Wednesday, October 16, 2013

Variability in the venom proteome of juvenile Bothrops jararaca

Bothrops jacaraca juvenile. Photo credit: Fernando Tatagiba
Snake venoms are complex mixtures but it is important that we understand them so that we can develop effective antivenoms. For snakes of the viper family, a number of studies have illustrated that the picture is complicated by the fact that the venom composition can vary between members of the same species. These changes are inherited, so have a genetic background.

However, the snake venom of individual viperids also changes with the age and diet of the creatures, so there are other factors at play. These so-called ontogenetic effects are particularly striking in the jararaca, one of the most abundant venomous snakes found in Brazil, which causes many deaths in the local population. This snake, Bothrops jararaca, has caught the attention of Brazilian researchers who have been studying variations in the venom.

Solange Serrano and colleagues from the Butantan Institute, Sao Paulo, and the University of Sao Paulo have found that the proteome of young jararacas differs from that of adults, and that there are gender differences in the venoms too. However, there have been very few studies on juvenile jacaracas, so they undertook a comprehensive proteomic study of the venoms of young snakes in order to define any compositional differences that occur as the juveniles grow.

The research team selected 21 young specimens of Bothrops jacaraca that were born to 10 different mothers originating from different geographical regions of Brazil, all of which were within the states of Sao Paulo, Minas Gerais and Santa Catarina. The mothers were housed in the same lab so that the young could be brought up on exactly the same diet and environmental conditions, to rule out the ontogenetic influences.

Venom was milked from the snakes over 9 months and the proteins were subjected to Western blotting using polyclonal antibodies raised against venom metalloproteinases (SVMPs) and serine proteinases (SVSPs). They were also separated by SDS-PAGE and the proteins in each band were identified by tandem mass spectrometry by searching against the NCBI Serpentes database.

The various properties associated with different venom components, such as caseinolytic, amidolytic and coagulant activity and prothrombin activation, were also investigated and compared between the individual snakes as they matured.

The protein profiles of the snakes showed a wide variation between individuals regardless of gender or the geographical origin of their mothers, even for the eight that were born to the same mother. In addition, the degree of glycosylation of the venom proteins varied between individuals and this was supported by the variations between SVMPs and the SVSPs.

Some of the activities of the venom were similar between snakes while others varied markedly. The abilities to break down casein and activate prothrombin were comparable whereas the amidolytic properties and the plasma coagulant activity differed strongly. So, the variation in the ability to clot the blood of victims, which is a characteristic feature of jararaca venom, cannot be attributed to age, gender or diet and is likely to be a genetic trait.

Although there were large differences in the actual protein compositions, especially within most of the principal toxin classes, there were some areas on the SDS-PAGE bands which were conserved between individuals. For instance, the region corresponding to proteins of molecular mass 45-50 kDa contained SVMPs and L-amino acid oxidase. For 10 venoms, aminopeptidase A was also identified for the first time in the venom of this snake.

A small tripeptide of composition ZKW was also detected in all venoms. This component inhibits SVMPs and acts to prevent the uncontrolled breakdown of proteins within the venom gland.

So, there appears to be a central core of components that are conserved across all jararaca venoms but there are also large variations in the protein compositions. The lack of influence of gender and geographic origin was confirmed by a multivariate analysis. Overall, the findings point strongly to genetic inheritance as the major factor in determining venom composition in juvenile Bothrops jararaca snakes.

Gabriela S. Dias, Eduardo S. Kitano, Ana H. Pagotto, Sávio S. Sant’anna, Marisa M. T. Rocha, André Zelanis, and Solange M. T. Serrano 2013. Individual Variability in the Venom Proteome of Juvenile Bothrops jararaca Specimens Journal of Proteome Research 2013 12 (10), 4585-4598

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