Chemical Synthesis of a Functional Fluorescent-Tagged α-Bungarotoxin
α-bungarotoxin is a large, 74 amino acid toxin containing five disulphide bridges, initially identified in the venom of <i>Bungarus multicinctus</i> snake. Like most large toxins, chemical synthesis of α-bungarotoxin is challenging, explaining why all previous reports use purified or rec...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-01-01
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| Series: | Toxins |
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| Online Access: | https://www.mdpi.com/2072-6651/14/2/79 |
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| author | Oliver Brun Claude Zoukimian Barbara Oliveira-Mendes Jérôme Montnach Benjamin Lauzier Michel Ronjat Rémy Béroud Frédéric Lesage Didier Boturyn Michel De Waard |
| author_facet | Oliver Brun Claude Zoukimian Barbara Oliveira-Mendes Jérôme Montnach Benjamin Lauzier Michel Ronjat Rémy Béroud Frédéric Lesage Didier Boturyn Michel De Waard |
| author_sort | Oliver Brun |
| collection | DOAJ |
| description | α-bungarotoxin is a large, 74 amino acid toxin containing five disulphide bridges, initially identified in the venom of <i>Bungarus multicinctus</i> 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. |
| first_indexed | 2024-03-09T20:56:51Z |
| format | Article |
| id | doaj.art-d2b5f17dd06b4fd9980cd477e3fd9b7b |
| institution | Directory Open Access Journal |
| issn | 2072-6651 |
| language | English |
| last_indexed | 2024-03-09T20:56:51Z |
| publishDate | 2022-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Toxins |
| spelling | doaj.art-d2b5f17dd06b4fd9980cd477e3fd9b7b2023-11-23T22:21:02ZengMDPI AGToxins2072-66512022-01-011427910.3390/toxins14020079Chemical Synthesis of a Functional Fluorescent-Tagged α-BungarotoxinOliver Brun0Claude Zoukimian1Barbara Oliveira-Mendes2Jérôme Montnach3Benjamin Lauzier4Michel Ronjat5Rémy Béroud6Frédéric Lesage7Didier Boturyn8Michel De Waard9L’institut Du Thorax, Nantes Université, INSERM, CNRS, INSERM UMR 1087/CNRS UMR 6291, 8 Quai Moncousu, F-44007 Nantes, FranceSmartox Biotechnology, 6 Rue des Platanes, F-38120 Saint-Egrève, FranceL’institut Du Thorax, Nantes Université, INSERM, CNRS, INSERM UMR 1087/CNRS UMR 6291, 8 Quai Moncousu, F-44007 Nantes, FranceL’institut Du Thorax, Nantes Université, INSERM, CNRS, INSERM UMR 1087/CNRS UMR 6291, 8 Quai Moncousu, F-44007 Nantes, FranceL’institut Du Thorax, Nantes Université, INSERM, CNRS, INSERM UMR 1087/CNRS UMR 6291, 8 Quai Moncousu, F-44007 Nantes, FranceL’institut Du Thorax, Nantes Université, INSERM, CNRS, INSERM UMR 1087/CNRS UMR 6291, 8 Quai Moncousu, F-44007 Nantes, FranceSmartox Biotechnology, 6 Rue des Platanes, F-38120 Saint-Egrève, FranceResearch Center, Montréal Heart Institute, Montréal, QC H1T 1C8, CanadaUMR 5250, Département de Chimie Moléculaire, CNRS, Université Grenoble Alpes, CEDEX 09, F-38058 Grenoble, FranceL’institut Du Thorax, Nantes Université, INSERM, CNRS, INSERM UMR 1087/CNRS UMR 6291, 8 Quai Moncousu, F-44007 Nantes, Franceα-bungarotoxin is a large, 74 amino acid toxin containing five disulphide bridges, initially identified in the venom of <i>Bungarus multicinctus</i> 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.https://www.mdpi.com/2072-6651/14/2/79toxinspeptide chemistrynative chemical ligationα-bungarotoxinclick chemistryautomated patch-clamp |
| spellingShingle | Oliver Brun Claude Zoukimian Barbara Oliveira-Mendes Jérôme Montnach Benjamin Lauzier Michel Ronjat Rémy Béroud Frédéric Lesage Didier Boturyn Michel De Waard Chemical Synthesis of a Functional Fluorescent-Tagged α-Bungarotoxin Toxins toxins peptide chemistry native chemical ligation α-bungarotoxin click chemistry automated patch-clamp |
| title | Chemical Synthesis of a Functional Fluorescent-Tagged α-Bungarotoxin |
| title_full | Chemical Synthesis of a Functional Fluorescent-Tagged α-Bungarotoxin |
| title_fullStr | Chemical Synthesis of a Functional Fluorescent-Tagged α-Bungarotoxin |
| title_full_unstemmed | Chemical Synthesis of a Functional Fluorescent-Tagged α-Bungarotoxin |
| title_short | Chemical Synthesis of a Functional Fluorescent-Tagged α-Bungarotoxin |
| title_sort | chemical synthesis of a functional fluorescent tagged α bungarotoxin |
| topic | toxins peptide chemistry native chemical ligation α-bungarotoxin click chemistry automated patch-clamp |
| url | https://www.mdpi.com/2072-6651/14/2/79 |
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