SNARE tagging allows stepwise assembly of a multimodular medicinal toxin

Abstract

Generation of supramolecular architectures through controlled linking of suitable building blocks can offer new perspectives to medicine and applied technologies. Current linking strategies often rely on chemical methods that have limitations and cannot take full advantage of the recombinant technologies. Here we used SNARE proteins, namely, syntaxin, SNAP25, and synaptobrevin, which form stable tetrahelical complexes that drive fusion of intracellular membranes, as versatile tags for irreversible linking of recombinant and synthetic functional units. We show that SNARE tagging allows stepwise production of a functional modular medicinal toxin, namely, botulinum neurotoxin type A, commonly known as BOTOX. This toxin consists of three structurally independent units: Receptor-binding domain (Rbd), Translocation domain (Td), and the Light chain (Lc), the last being a proteolytic enzyme. Fusing the receptor-binding domain with synaptobrevin SNARE motif allowed delivery of the active part of botulinum neurotoxin (Lc-Td), tagged with SNAP25, into neurons. Our data show that SNARE-tagged toxin was able to cleave its intraneuronal molecular target and to inhibit release of neurotransmitters. The reassembled toxin provides a safer alternative to existing botulinum neurotoxin and may offer wider use of this popular research and medical tool. Finally, SNARE tagging allowed the Rbd portion of the toxin to be used to deliver quantum dots and other fluorescent markers into neurons, showing versatility of this unique tagging and self-assembly technique. Together, these results demonstrate that the SNARE tetrahelical coiled-coil allows controlled linking of various building blocks into multifunctional assemblies.

Fig. 1.

SNARE tagging allows Rbd-mediated delivery of quantum dots to synaptic endings. (A) Diagram showing structure of native botulinum neurotoxin, composed of the Light chain (Lc), Translocation domain (Td), and Receptor-binding domain (Rbd). (B) Schematic diagram showing SNARE tagging scheme for linking streptavidin-coated quantum dot (Qdot) with Receptor-binding domain of botulinum neurotoxin (Rbd). Biotin(star)-syntaxin peptide (syx, red) allows SNARE tagging of Qdot, whereas synaptobrevin peptide (syb, blue) is fused to Rbd. Addition of SNAP25 (green) allows attachment of Rbd to Qdot. (C) Coomassie-stained SDS gel showing irreversible assembly of biotinylated syntaxin3 peptide, SNAP25, and syb-Rbd into an SDS-resistant complex, biotin-SNARE-Rbd. (D) Qdots carrying Rbd exhibit synaptic binding, as evidenced by coincidence of Qdot fluorescence (green) and immunostaining of synaptic vesicle marker synaptophysin (red) at axonal extensions of cultured hippocampal neurons. Omission of SNAP25 during assembly prevents targeting of Qdots to synaptic terminals.

Fig. 2.

SNARE tagging allows stepwise assembly of individual parts of BoNT/A into a single molecular entity. (A) Structure of SNARE-tagged botulinum neurotoxin. Positions of disulphide bond and engineered SNARE tags between LcTd and the Rbd part of BoNT/A are indicated. (B) LcTd, tagged with SNAP25, can be purified and broken into Lc and Td-SNAP25 following treatment with 50 mM DTT. Coomassie-stained SDS gel. (C) LcTd, tagged with SNAP25, can be united with Rbd, tagged with synaptobrevin, upon addition of the syntaxin3 peptide as evidenced by the Coomassie-stained and fluorescently imaged SDS gels.

Fig. 3.

SNARE-linked botulinum neurotoxin exhibits synaptic localization and cleaves its intrasynaptic target. (A) Fluorescein-labeled LcTd-SNARE-Rbd binds to axonal extensions of hippocampal neurons. Immunostaining with antisynaptophysin antibody highlights presynaptic terminals of cultured hippocampal neurons. (B) Immunoblot showing cleavage of intrasynaptic SNAP25 by assembled neurotoxin in a fashion similar to that of native BoNT/A. (C) Immunoblot showing that cleavage of SNAP25 by LcTd-SNARE-Rbd (1 nM) is blocked by bafilomycin A1, which prevents acidification of recycled synaptic vesicles.

Fig. 4.

SNARE-linked botulinum neurotoxin inhibits neurotransmitter release from brain nerve endings and in neuromuscular junctions. (A and B) Fluorometric measurements of glutamate release from isolated rat brain synaptic endings (synaptosomes) indicate similar degree of inhibition between LcTd-SNARE-Rbd and BoNT/A. Real-time glutamate release graph (A) and dose-dependence graph (B, assessed after 15-min stimulation with 35 mM KCl and 2 mM CaCl₂) were obtained following 1-h incubation of synaptosomes with toxins. (C) Individual SNARE-tagged neurotoxin parts do not block glutamate release following 1-h incubation with synaptosomes, as assessed after 15-min stimulation. (D) LcTd-SNARE-Rbd blocks spontaneous synaptic activity (MEPPs) in mouse flexor digitorum brevis preparations. Degree of inhibition is further enhanced when exocytosis is stimulated with 30 mM KCl during toxin application (activity dependent). Data were recorded for five myofibers for each condition in eight muscle preparations (shown with SEM). (E) Example of endplate potentials following application of 15 mM KCl in control and LcTd-SNARE-Rbd–treated synapses.

Fig. 5.

SNARE-linked botulinum neurotoxin inhibits neuromuscular and CNS functions. (A) Graph showing dose-dependent inhibition of isometric contractions of mouse diaphragm by LcTd-SNARE-Rbd. Error bars represent SD; n = 3. (B) Representative trace of circadian bioluminescence rhythms of suprachiasmatic nucleus (SCN) slice treated with 6 nM LcTd-SNARE-Rbd. Note rapid and sustained suppression of amplitude of circadian oscillation following application of the assembled toxin (arrow).