Enzymatic labelling of snake venom phospholipase A2 toxins. (December 2019)

• Record Type: Journal Article
• Authors: Spolaore, Barbara; Fernández, Julián; Lomonte, Bruno; Massimino, Maria Lina; Tonello, Fiorella
• Is Part Of: Toxicon. Volume 170(2019)
• Page Start: 99
• Page End: 107
• Publication Date: 2019-12
• Subjects: Secretory phospholipase A2 -- Snake venom toxin -- Protein labelling -- Enzymatic conjugation -- Transglutaminase; ACN acetonitrile -- DC dansylcadaverine -- DMSO dimethylsulphoxide -- DNS 5-N-(5′-N′, N′- dimethylamino-1′-naphtalenesulfonly)diaminopentane -- E:S enzyme to substrate ratio -- RP-HPLC reverse-phase high-performance liquid chromatography -- TFA trifluoroacetic acid -- TGase transglutaminase -- ZQG carbobenzoxy-L-glutaminyl-glycine; Toxins -- Periodicals; Venom -- Periodicals; 615.9
• DOI: 10.1016/j.toxicon.2019.09.019
• Languages: English
• ISSNs: 0041-0101

Abstract

Abstract: Almost all animal venoms contain secretory phospholipases A2 (PLA2 s), 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 PLA2 s: a recombinant neuro- and myotoxic group I PLA2 from Notechis scutatus scutatus, and two myotoxic group II PLA2 s 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 PLA2 s modified at specific Lys residues, and that all three of these proteins, conjugated with fluorescent peptides, are internalized in primary myotubes. Graphical abstract: Image 1 Highlights: An efficient and reproducible labelling method is necessary to study the cellular activity of snake venom PLA2 s. Chemical methods are not very reproducible and can cause loss of activity to this type of proteins. The N- and C- terminal parts are relevant for their activity so fusion with fluorescent proteins is inapplicable. We applied enzymatic labelling on three snake venom PLA2 s obtaining the proteins modified to only one or two Lys residues. Modified PLA2 s keep their enzymatic and toxic activity and fluorescence derivatives penetrates inside the cells.