Snake Venom Extracellular vesicles (SVEVs) reveal wide molecular and functional proteome diversity

Sergio Paulo Bydlowski,Giuseppe Palmisano,Martin R Larsen,Paulo Baldasso,Livia Rosa-Fernandes,Victor Corassolla Carregari,Sergio Marangoni

doi:10.1038/s41598-018-30578-4

Sergio Paulo Bydlowski, Giuseppe Palmisano + Show 5 more

Open Access

https://doi.org/10.1038/s41598-018-30578-4

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Abstract

Proteins constitute almost 95% of snake venom’s dry weight and are produced and released by venom glands in a solubilized form during a snake bite. These proteins are responsible for inducing several pharmacological effects aiming to immobilize and initiate the pre-digestion of the prey. This study shows that proteins can be secreted and confined in snake venom extracellular vesicles (SVEVs) presenting a size distribution between 50 nm and 500 nm. SVEVs isolated from lyophilized venoms collected from four different species of snakes (Agkistrodon contortrix contortrix, Crotalus atrox, Crotalus viridis and Crotalus cerberus oreganus) were analyzed by mass spectrometry-based proteomic, which allowed the identification of proteins belonging to eight main functional protein classes such as SVMPs, serine proteinases, PLA2, LAAO, 5′nucleotidase, C-type lectin, CRISP and Disintegrin. Biochemical assays indicated that SVEVs are functionally active, showing high metalloproteinase and fibrinogenolytic activity besides being cytotoxic against HUVEC cells. Overall, this study comprehensively depicts the protein composition of SVEVs for the first time. In addition, the molecular function of some of the described proteins suggests a central role for SVEVs in the cytotoxicity of the snake venom and sheds new light in the envenomation process.

Highlights

Proteins constitute almost 95% of snake venom’s dry weight and are produced and released by venom glands in a solubilized form during a snake bite
The SDS-PAGE protein pattern was different between the unretained fraction and the whole venom, suggesting a concentration/enrichment of particular proteins in the non-retained fraction. These first evidences suggested the presence of larger proteins complexes or extracellular vesicles (EVs) in the lyophilized snake venom
Two manuscripts have shown EVs isolation from freshly extracted venom from Crotalus durissus terrificus and Gloydius blomhoffii blomhoffii using differential ultracentrifugation and SEC, respectively[5,33], suggesting that, in this study, the unretained SEC fraction could contain proteins encapsulated in EVs

Summary

Introduction

Proteins constitute almost 95% of snake venom’s dry weight and are produced and released by venom glands in a solubilized form during a snake bite. This study shows that proteins can be secreted and confined in snake venom extracellular vesicles (SVEVs) presenting a size distribution between 50 nm and 500 nm. Toxins with hemorrhagic and myonecrotic activity are generally found in the venoms of the Viperidae family due to synergic action of proteolytic enzymes, such as metalloproteinases and serine proteinases[4] Such venom components are formed in the venom gland as pre-pro-proteins containing a signal peptide that, once cleaved, produces a mature protein released in its soluble form[5]. N-terminal amino acid sequence analysis of the purified DPP IV revealed that the N-terminus is not processed This molecular feature raised questions regarding the mechanism of protein secretion into venom[5,6,7], which opened up the possibility of alternative routes for snake venom proteins secretion. Despite a morphological characterization of EVs in snake venom freshly collected, comprehensive studies on the biological content of these vesicles have never been conducted

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