Expression, purification and application of a recombinant, membrane permeating version of the light chain of botulinum toxin B

Abstract

Botulinum neurotoxins (BoNTs) are valuable tools to unveil molecular mechanisms of exocytosis in neuronal and non-neuronal cells due to their peptidase activity on exocytic isoforms of SNARE proteins. They are produced by Clostridia as single-chain polypeptides that are proteolytically cleaved into light, catalytic domains covalently linked via disulfide bonds to heavy, targeting domains. This format of two subunits linked by disulfide bonds is required for the full neurotoxicity of BoNTs. We have generated a recombinant version of BoNT/B that consists of the light chain of the toxin fused to the protein transduction domain of the human immunodeficiency virus-1 (TAT peptide) and a hexahistidine tag. His6-TAT-BoNT/B-LC, expressed in Escherichia coli and purified by affinity chromatography, penetrated membranes and exhibited strong enzymatic activity, as evidenced by cleavage of the SNARE synaptobrevin from rat brain synaptosomes and human sperm cells. Proteolytic attack of synaptobrevin hindered exocytosis triggered by a calcium ionophore in the latter. The novel tool reported herein disrupts the function of a SNARE protein within minutes in cells that may or may not express the receptors for the BoNT/B heavy chain, and without the need for transient transfection or permeabilization.

Keywords: botulinum toxin B; cell penetrating; exocytosis; sperm cells; synaptobrevin.

Figure 1. Designed His₆-TAT-BoNT/B-LC

(A) Schematic view of designed His₆-TAT-BoNT/B-LC construct on pET28 expression vector. (B) SDS-PAGE stained with Coomassie Brilliant Blue R250. Lanes 1–4 show enriched eluates (buffer containing 250 mM imidazole) from a batch purification. Mr standards (×10³ Da) are indicated on the right. (C) Two orthogonal views, rotated 180°, revealing the solvent-accessible surface of the BoNT/B-LC domain in grey, with the His₆-TAT peptide depicted as a blue cartoon. This representation emphasizes that the N-terminal tag and the BoNT/B-LC active site (coloured in pink) are positioned on opposite sides of the structure. The representation corresponds to that predicted by RoseTTAfold. (D) Secondary structure cartoon representation of His₆-TAT-BoNT/B-LC predicted by RoseTTAfold, showcasing the folding of the His₆-TAT tag as a blue alpha helix over the toxin. The molecular cartoon is color-coded from blue to red, representing the N to C-terminus progression.

Figure 2. His₆-TAT-BoNT/B-LC crosses synaptosomal membranes and is enzymatically active

(A) A rat brain preparation enriched in synaptosomes was incubated for 30 min at 37°C with increasing concentrations of His₆-BoNT/B-LC (left) of His₆-TAT-BoNT/B-LC (right) as described in the Methods section. Reactions were terminated by addition of SDS sample buffer and the amount of intact synaptobrevin-2 was analyzed by Western blot using the 69.1 antibody as probe. The arrow indicates the electrophoretic mobility of synaptobrevin-2 (Syb). Anti-syntaxin 1 (Stx1) blot shows equal loading of synaptosomal proteins. Shown are blots representative of three repetitions. (B) Quantification was expressed as the ratio of the signal intensities of synaptobrevin-2 relative to syntaxin1 when synaptosomes were incubated with His₆-TAT-BoNT/B-LC (squares) or His₆-BoNT/B-LC (circles). (C) 100 nM His₆-BoNT/B-LC and His₆-TAT-BoNT/B-LC (black bar) were introduced into SLO-permeabilized sperm cells by incubating at 37°C for 15 min. The AR was induced with 0.5 mM CaCl₂ and incubating as before. Sperm cells were fixed and the AR was measured by FITC-PSA binding as described in [14]. Gray bars represent controls: background (control), AR stimulated by 0.5 mM CaCl₂ (Ca), lack of effect of the toxins on the basal AR (BoNT/B, TAT-BoNT/B) and inhibitory effect of the not-membrane penetrating version on the AR elicited by CaCl₂. The data represent the mean ± SEM of at least three independent experiments. Data were evaluated before normalization with the program GraphPad Prism 8 using the one way Anova, Dunnett's multiple comparisons test. Different letters indicate statistical significance (P<0.05).

Figure 3. His₆-TAT-BoNT/B-LC penetrates into human sperm cells

(A) 1 μg purified His₆-TAT-BoNT/B-LC was incubated with increasing amounts of trypsin as indicated in the Methods section. Top: Coomassie Brilliant Blue R250 (CB) stained gel; 2/3 of each sample were run per lane. The full length protein (arrow) disappeared and a band with higher mobility (asterisk) appeared with the higher amounts of trypsin tested. Mr standards (×10³ Da) are indicated on the right. Bottom: aliquots of the same samples were subjected to anti-His₆ Western blot; 1/3 of each sample were run per lane. The antibodies detected full length His₆-TAT-BoNT/B-LC (arrow). Shown is an experiment representative of three repetitions. (B) Quantification of the anti-His₆ signal in uncleaved His₆-TAT-BoNT/B-LC depicted as mean ± SD from all replicates. (C) Capacitated human sperm cells were incubated with 1 μM His₆-TAT-BoNT/B-LC, washed and treated with 0.1 μg/ml trypsin as indicated in the Methods section. Samples were centrifuged and proteins from both the sperm cells pellet (incorporated, lane 3) and the extracellular supernatant (unincorporated, lane 2) were processed for anti-His₆ Western blot. Lane 1 shows His₆-TAT-BoNT/B-LC equivalent to that found in the supernatant of mock experiments conducted without trypsin (1 µg). Shown is an experiment representative of four repetitions.

Figure 4. His₆-TAT-BoNT/B-LC translocated into human sperm cells inhibits the AR

(A) Capacitated human sperm cells were exposed to increasing concentrations of His₆-TAT-BoNT/B-LC for 15 min at 37°C. Acrosomal exocytosis was initiated with 10 µM A23187 and incubating as before. (B) 350 nM His₆-BoNT/B-LC and His₆-TAT-BoNT/B-LC (black bar) were introduced into capacitated sperm cells incubating at 37°C for 15 min. The AR was induced with 10 µM A23187 and incubating as before. Sperm cells were fixed and the AR was measured by FITC-PSA binding as described in [14]. Gray bars represent controls: background (control), AR stimulated by 10 µM A23187 (A23187), lack of effect of the elution buffer and of the not-membrane penetrating version on the AR elicited by A23187. The data represent the mean ± SEM of at least three independent experiments. Data were evaluated before normalization with the program GraphPad Prism 8 using the one-way Anova, Dunnett’s multiple comparisons test. Different letters indicate statistical significance (P<0.05).

Figure 5. His₆-TAT-BoNT/B-LC translocated into human sperm cells cleaves synaptobrevin

(A) Sperm cells incubated as indicated in the figure key were fixed and triple stained with an anti-synaptobrevin-2 antibody followed by a fluorescent secondary antibody (anti-mouse-Cy3, red, left panels), FITC-PSA (green, central panels), and Hoechst 33342 (blue, right panels). Samples were examined with an 80i Nikon microscope equipped with a Plan Apo 60x/1.40 oil objective. All images were captured - with the same length of exposure for the acquisitions - with a Nikon DS-Fi1 camera operated with NIS software (Nikon). ImageJ (freeware from N.I.H.) was used to subtract background and adjust brightness/contrast to render all-or nothing labeling patterns. The presence of immunostaining in the acrosomal region was scored in ≈ 20 digital images from at least ≈200 cells. Shown are representative images of sperm cells with intact acrosomes and synaptobrevin-2 staining and without synaptobrevin-2 immunostaining due to toxin cleavage (asterisks). Bars = 5 μm. The silhouette of the head of a sperm cell is delineated with dashed bars (bottom, green). (B) Quantification of the percentage of non-exocytosing sperm cells with synaptobrevin-2 staining. The data represent the mean ± SEM of three independent experiments: BoNT/B: 208-201-206 cells counted; BoNT/B+A23187: 227-222-210 cells counted; TAT-BoNT/B: 205-232-218 cells counted; TAT-BoNT/B+A23187: 230-254-223 cells counted. Data were evaluated with the program GraphPad Prism 8 using the one-way Anova, Dunnett’s test multiple comparisons test. Different letters indicate statistical significance (P<0.05).