Fig 1.
Ganglioside-binding specificity of HC/C.
Sensorgrams were plotted as the mass of HC/C binding (in RU) to immobilized ganglioside (GM1, GD2, GD1a, GD1b, GT1b, and GQ1b)-containing liposomes. A concentration series (15.6, 31.3, 62.5, 125, 250, or 500 nM) of HC/C was injected onto immobilized POPC liposomes containing 1% ganglioside. Triplicate curves were fitted globally using BIAevaluation ver. 4.1 software with a 1:1 (Langmuir) binding model to determine the KD values.
Fig 2.
Effects of knockdown of ganglioside synthase genes on binding/entry of HC/C into P19 neurons.
(A) Synthetic pathway of gangliosides and depletion of ganglioside synthase by siRNA. The red or blue arrow indicates the ganglioside synthesis pathway targeted by GalNAc-T or Siat8, respectively. (B) GalNAc-T (left) and Siat8 (right) mRNA levels of knockdown cells. The mRNA levels obtained from mock-transfected cells were set to 100%. Significance for differences (compared with control) was evaluated by one-way ANOVA (***, p < 0.001). (C) Binding and entry of Alexa Fluor 647-CTB into knockdown cells. Mean fluorescence intensities were measured by flow cytometric analysis. Significance for differences (compared with control) was evaluated by one-way ANOVA (***, p < 0.001). (D) Flow cytometric analysis for the binding and entry of Alexa Fluor 488-HC/C into knockdown cells. Histograms represented the number of cells versus relative fluorescence intensity in the left panel (black: without Alexa Fluor 488-HC/C, green: si-Control, red: si-GalNAc-T, blue: si-Siat8). The right panel indicates the mean fluorescence intensities. Significance for differences (compared with control) was evaluated by one-way ANOVA (***, p < 0.001). (E) Cleavage of syntaxin-1 and SNAP-25 by BoNT/C in si-RNA-transfected P19 neurons. P19 neurons transfected with control- or GalNAc-T-siRNA were exposed to 0, 0.04, 0.16, 0.64, 2.5, or 10 nM BoNT/C for 30 min at 37°C, then the toxin was removed and cells were incubated for 18 h at 37°C in fresh medium. Cells were then harvested and solubilized, and extracts were subjected to SDS-PAGE and western blotting. The membranes were probed with anti-syntaxin-1, anti-SNAP-25, and anti-GAPDH mAbs followed by an HRP-conjugated goat anti-mouse antibody. GAPDH is shown as an internal control. The reactive bands were visualized with SuperSignal West Dura Extended Duration Substrate.
Fig 3.
Generation of GalNAc-T-knockout P19 cells.
(A) Target sequence derived from the genomic sequence of B4galnt1 exon 1 was inserted into the GeneArt CRISPR nuclease vector. P19 cells were transfected with the vector using the Lipofectamine 3000 reagent. (B) Sequencing of the neighboring target region in exon 1 of the B4galnt1 gene of CRIPR/Cas9 vector-transfected P19 cells. Parts of the B4galnt1 gene from 3 different subcloned cells were amplified by PCR and subcloned into a TOPO cloning vector. Eight TOPO vectors derived from 3 subcloned cells (subclones 1 to 3) were sequenced. All target sites in the vectors contained the 14-nucleotide deletion, and neither other deletions nor original sequence were detected. Gray squares indicate the target sequences. (C) P19 neurons were fixed and stained with an Alexa Fluor 647-labeled CTB (magenta) or anti-GT1b mAb and Alexa Fluor 488-labeled anti-mouse IgG antibody (green). DAPI was used to stain the nuclei (blue). Differential interference contrast (DIC) and fluorescence images were collected using confocal microscopy. Neither CTB, which binds GM1, nor GT1b (using an anti-GT1b mAb) were detected on GalNAc-T-knockout P19 cells. WT and KO indicate wild-type and GalNAc-T-knockout cells, respectively. Scale bars indicate 10 μm.
Table 1.
Potential off-target sites in mouse genome.
Fig 4.
Sensitivity of GalNAc-T-knockout P19 cells to BoNT/C.
(A) Uptake of HC/C into P19 neurons. P19 neurons were treated with Alexa Fluor 488-labeled HC/C (green) and NucBlue Live Cell Stain ReadyProbes reagent for nuclear staining (blue), then plasma membranes were stained with CellMask Deep Red Plasma Membrane Stain (red). Differential interference contrast (DIC) and fluorescence images were collected using confocal microscopy. HC/C was detected as punctate signals in P19 neurons derived from wild-type cells, but not in knockout cells. High degrees of HC/C and CellMask overlap in merged images are represented in yellow. Scale bars indicate 10 μm. (B) Cleavage of syntaxin-1 and SNAP-25 in P19 neurons by BoNT/C. P19 neurons were exposed to 0.04, 0.16, 0.64, 2.5, or 10 nM BoNT/C for 30 min at 37°C, then the toxin was removed and cells were incubated for 18 h at 37°C in fresh medium. Cells were harvested, solubilized, and extracts were subjected to SDS-PAGE and western blotting. The membranes were probed with anti-GAPDH, anti-syntaxin-1, and anti-SNAP-25 mAbs followed by an HRP-conjugated goat anti-mouse antibody. GAPDH is shown as an internal control. The reactive bands were visualized with SuperSignal West Dura Extended Duration Substrate (upper). The relative amount of intact protein was calculated as a ratio of the relative intensity of each band (intact band/intact + cleaved band). Each point represents the average of three experiments; and because the standard errors for each point were extremely low (<0.01), the error bars are not shown (lower). WT and KO indicate wild-type and GalNAc-T-knockout cells, respectively. Both syntaxin-1 and SNAP-25 were cleaved by BoNT/C in a dose-dependent manner in wild-type cells, but were barely cleaved in knockout cells. (C) Recovery of BoNT/C-induced SNAP-25 cleavage in P19 neurons derived from GalNAc-T-knockout cells by addition of exogenous gangliosides. P19 neurons were treated with the gangliosides GD1a, GD1b, GT1b (each 9 μM), no gangliosides (control), or a mixture of these 3 gangliosides (total 9 μM, each 3 μM) for 24 h. Cells were then exposed to BoNT/C (4 nM) for 30 min. Western blotting analysis was performed at 18 h after removing the toxin for the detection of SNAP-25 cleavage. The relative amount of intact SNAP-25 cleavage was calculated by measuring the intensity of the bands and calculating the ratio of intact to intact + uncleaved bands. Each column represents the average of three independent experiments; error bar indicate the standard error. Significance for differences was evaluated by one-way ANOVA (*, p < 0.05; **, p < 0.01; ***, p < 0.001).