Almost all animal venoms contain secretory phospholipases A2 (PLA2s), 14 kDa disulfide-rich enzymes that hydrolyze membrane phospholipids at the sn-2 position, releasing lysophospholipids and fatty acids. These proteins, depending on their sequence, show a wide variety of biochemical, toxic and pharmacological effects and deserve to be studied for their numerous possible applications, and to improve antivenom drugs. The cellular localization and activity of a protein can be studied by conjugating it with a tag. In this work, we applied an enzymatic labelling method, using Streptomyces mobaraense transglutaminase, on three snake venom PLA2s: a recombinant neuro- and myotoxic group I PLA2 from Notechis scutatus scutatus, and two myotoxic group II PLA2s from Bothrops asper - one of them a natural catalytically inactive variant. We demonstrate that TGase can be used to produce active mono- or bi-derivatives of these three PLA2s modified at specific Lys residues, and that all three of these proteins, conjugated with fluorescent peptides, are internalized in primary myotubes.

Enzymatic labelling of snake venom phospholipase A2 toxins

Spolaore B.
;
Massimino M. L.;Tonello F.
2019

Abstract

Almost all animal venoms contain secretory phospholipases A2 (PLA2s), 14 kDa disulfide-rich enzymes that hydrolyze membrane phospholipids at the sn-2 position, releasing lysophospholipids and fatty acids. These proteins, depending on their sequence, show a wide variety of biochemical, toxic and pharmacological effects and deserve to be studied for their numerous possible applications, and to improve antivenom drugs. The cellular localization and activity of a protein can be studied by conjugating it with a tag. In this work, we applied an enzymatic labelling method, using Streptomyces mobaraense transglutaminase, on three snake venom PLA2s: a recombinant neuro- and myotoxic group I PLA2 from Notechis scutatus scutatus, and two myotoxic group II PLA2s from Bothrops asper - one of them a natural catalytically inactive variant. We demonstrate that TGase can be used to produce active mono- or bi-derivatives of these three PLA2s modified at specific Lys residues, and that all three of these proteins, conjugated with fluorescent peptides, are internalized in primary myotubes.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11577/3316519
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