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Article Dans Une Revue Toxins Année : 2022

Chemical Synthesis of a Functional Fluorescent-Tagged α-Bungarotoxin

Oliver Brun
  • Fonction : Auteur
Claude Zoukimian
  • Fonction : Auteur
Barbara Oliveira-Mendes
Jérôme Montnach
Benjamin Lauzier
Michel Ronjat
  • Fonction : Auteur
Rémy Béroud
  • Fonction : Auteur
Frédéric Lesage
Michel de Waard

Résumé

α-bungarotoxin is a large, 74 amino acid toxin containing five disulphide bridges, initially identified in the venom of Bungarus multicinctus snake. Like most large toxins, chemical synthesis of α-bungarotoxin is challenging, explaining why all previous reports use purified or recombinant α-bungarotoxin. However, only chemical synthesis allows easy insertion of non-natural amino acids or new chemical functionalities. Herein, we describe a procedure for the chemical synthesis of a fluorescent-tagged α-bungarotoxin. The full-length peptide was designed to include an alkyne function at the amino-terminus through the addition of a pentynoic acid linker. Chemical synthesis of α-bungarotoxin requires hydrazide-based coupling of three peptide fragments in successive steps. After completion of the oxidative folding, an azide-modified Cy5 fluorophore was coupled by click chemistry onto the toxin. Next, we determined the efficacy of the fluorescent-tagged α-bungarotoxin to block acetylcholine (ACh)-mediated currents in response to muscle nicotinic receptor activation in TE671 cells. Using automated patch-clamp recordings, we demonstrate that fluorescent synthetic α-bungarotoxin has the expected nanomolar affinity for the nicotinic receptor. The blocking effect of fluorescent α-bungarotoxin could be displaced by incubation with a 20-mer peptide mimicking the α-bungarotoxin binding site. In addition, TE671 cells could be labelled with fluorescent toxin, as witnessed by confocal microscopy, and this labelling was partially displaced by the 20-mer competitive peptide. We thus demonstrate that synthetic fluorescent-tagged α-bungarotoxin preserves excellent properties for binding onto muscle nicotinic receptors.
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Origine : Publication financée par une institution

Dates et versions

hal-03543053 , version 1 (25-01-2022)

Identifiants

Citer

Oliver Brun, Claude Zoukimian, Barbara Oliveira-Mendes, Jérôme Montnach, Benjamin Lauzier, et al.. Chemical Synthesis of a Functional Fluorescent-Tagged α-Bungarotoxin. Toxins, 2022, 14 (2), pp.79. ⟨10.3390/toxins14020079⟩. ⟨hal-03543053⟩
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