Chemical Synthesis of a Functional Fluorescent-Tagged α-Bungarotoxin

Abstract

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.

Keywords: TE671 cells; automated patch-clamp; click chemistry; fluorescent peptide; native chemical ligation; nicotinic acetylcholine receptor; peptide chemistry; toxins; α-bungarotoxin.

Figure 1

Syntheses strategies of PA-α-BgTx by NCL. (A) Synthesis strategy from the N-terminus towards the C-terminus. Only the first step is shown, since it was a dead-end procedure. a = 0.2 M NaH₂PO₄, H₂O, 6 M Gn.HCl, pH 3, −15 °C, 10 eq. NaNO₂, 20 min, then 100 eq. 4-mercatophenylacetic acid (MPAA), pH 6.5, room temperature (RT), overnight. (B) Synthesis strategy from the C-terminus towards the N-terminus. b = Acm deprotection: 0.2 M NaH₂PO₄, H₂O, 6 M Gn.HCl, pH 7.0, 10 eq. PdCl₂, 10 min, then 100 eq. dithiothreitol (DTT). c = oxidative folding: 0.1 M Tris pH 8.2, 5 mM glutathione (GSH), 0.5 mM glutathione disulphide (GSSG), 48 h.

Figure 2

RP-HLPC and MS profiles of synthetic peptides. RP-HPLC A to C were performed on an AdvanceBio Peptide C18 column (10 cm, 214 nm, 5–65% B solvent in 12 min), RP-HPLC D on an AdvanceBio Peptide C18 column (25 cm, 214 nm, 15–65% B solvent in 25 min) and RP-HPLC E on a XSelect Peptide CSH C18 column (15 cm, 214 nm, 5–60% B solvent in 20 min). Retention times were t_R = 6.5 min (A), 7.2 min (B), 9.1 min (C), 21.1 min (D) and 16.9 min (E).

Figure 3

Effect of native α-BgTx and Cy5-α-BgTx on ACh-mediated current in TE671 cells. (A) Representative ACh-mediated currents following two consecutive 3.33 μM ACh doses. First ACh pulse illustrated in black, second dose in grey. Mean ± SEM (n = 82 cells, **** p < 0.0005). The reduction in current amplitude with the second ACh application is due to desensitisation of mAChR. (B) Representative current traces in response to a first application of 3.33 ACh (black trace), or a second identical application in presence of increasing concentrations of native α-BgTx (blue) or Cy5-α-BgTx (red). The dotted line represents the expected current after a second ACh application in the absence of any toxin. (C) Concentration-response curves for the native α-BgTx and Cy5-α-BgTx-mediated block of ACh response (n = 8–41 cells/concentration, total N = 339 cells), **** p < 0.0005.

Figure 4

BgTx as measured by MST. A total of 5 nM Cy5-α-BgTx was incubated for 15 min at room temperature with a range of BSpep concentrations. The change in thermophoretic signal yields a K_D = 1.04 µM in these experimental conditions.

Figure 5

Effect of the BSpep on native α-BgTx and Cy5-α-BgTx inhibition of ACh-mediated current in TE671 cells. (A) Representative current traces in response to a first application of 3.33 μM ACh (black) or a second identical application in the presence of α-BgTx (blue), BSpep (yellow), α-BgTx + BSpep (green), Cy5-α-BgTx (red) or Cy5-α-BgTx + BSpep (pink). The dotted line represents the expected current after a second ACh control dose in the absence of toxins. (B) Mean normalised current after the second 3.33 μM ACh dose in the presence of α-BgTx (blue), BSpep (yellow), α-BgTx + BSpep (green), Cy5-α-BgTx (red) or Cy5-α-BgTx + BSpep (pink). Mean ± SEM. Kruskal-Wallis test with Dunn’s multiple comparison post-test, **** p < 0.0005.

Figure 6

Distribution of nAChR in TE671 cells as labelled with Cy5-α-BgTx. (A) Confocal microscopic imaging of TE671 cells labelled with 5 µg/mL Hoechst and either 200 nM Cy5-α-BgTx (left) or 200 nM Cy5-α-BgTx pre-incubated with 100 µM BSpep (right). Top: X60 magnification, Hoechst (cyan) and Cy5-α-BgTx (magenta) displayed; Middle: X60 magnification, only Cy5-α-BgTx displayed; Bottom: X240 magnification, Hoechst and Cy5-α-BgTx displayed. (B) Quantification of Cy5-α-BgTx fluorescence after labelling with either 200 nM Cy5-α-BgTx (red) or 200 nM Cy5-α-BgTx pre-incubated with 100 µM BSpep (pink). Mean ± SEM. Two-tailed t-test, ** p < 0.05.