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Exploring the Enzymatic Potential of Olindias sambaquiensis Extracts 
 

Knittel PS1, Long PF2, Marques AC3, Almeida MT1, Padilla G4, Moura-da-Silva AM1

1 Laboratório de Imunopatologia, Instituto Butantan, Brasil; 2 Institute of Pharmaceutical Science, King's College London, UK; 3 Instituto de Biociências Universidade de São Paulo, Brasil, Instituto de Biociências Universidade de São Paulo, Brasil ; 4 Instituto de Ciências Biomédicas Universidade de São Paulo, Brasil, Instituto de Ciências Biomédicas Universidade de São Paulo, Brasil

Introduction: Jellyfish are amongst the most familiar of venomous marine animals. Jellyfish encounters with humans are common, although subsequent envenomation has varying toxicity ranging from usually mild symptoms to sometimes lethal consequences. Jellyfish venoms are of biotechnological importance, with toxins displaying antimicrobial, analgesic and anti-tumor activities. Although proteolytic enzymes have also been described, detailed characterisation of these proteins is scant. Recently, the proteomic profile of putative toxins isolated from nematocysts of the hydromedusae Olindias sambaquiensis was reported, which included protease and phospholipase enzymes. Here we present the results of a study to experimentally confirm proteolytic activity in extracts of O. sambaquiensis tentacles.  Objectives: This work aims to use extracts of O. sambaquiensis, in order to examine the functional activity of the proteins identified by proteome. Methods: Specimens of O. sambaquiensis were collected at the coast of São Sebastião. A procedure to generate tentacle extracts was standardised according to Weston et al. (2013). The extracts were assayed for the presence of metalloproteases, serine proteases and phospholipase A2 activities using (FRET) Fluorescence Resonance Energy Transfer substrates designed for SVMPs (Snake Venom Metalloproteases), MMPs (Matrix Metalloproteases) and ADAMs (A Disintegrin And Metalloprotease) and chromogenic substrates designed for serine proteases and phospholipase A2. Furthermore, the inhibitory potential of O. sambaquiensis extracts on commercial enzymes (ADAM-17, MMP-2/MMP-9 and Trypsin), Jararhagin (PIII SVMP from Bothrops jararaca venom) and Crotoxin B (phospholipase A2 subunit of crotoxin from Crotalus durissus terrificus venom) was also tested.  Results and Discussion: The procedure to generate tentacle extracts yielded high quantities of proteins of different molecular mass.Tentacle extracts of O. sambaquiensis displayed serineproteinase, metalloproteinase and phospholipase activities, confirming previous proteomic evidence for the presence of these enzymes.  In addition, proteolytic activity was also detected by zymography assays on casein. The inhibition tests showed no decrease in activity of ADAM-17, MMP-2/MMP-9, Trypsin and Crotoxin B after incubation with the extract, however, a slight reduction about 30 % in the proteolytic activity of Jararhagin was detected. 
These data provide a better understanding of the toxin arsenal of this underexplored venomous animal.
 

Supported by FAPESP



12 - PAP PROGRAM 21th Annual Scientific Meeting of Instituto Butantan.