Preferential Entry of Botulinum Neurotoxin A Hc Domain through Intestinal Crypt Cells and Targeting to Cholinergic Neurons of the Mouse Intestine
Figure 5
Transcytosis of BoNT/A through intestinal and neuroendocrine cell monolayers, and SV2C-dependent entry of HcA into cells.
(A) Transcytosis of BoNT/A trough the intestinal neuroendocrine cell line STC-1, m-ICcl2, and Caco-2 cells. Cells were grown on filters (Transwell) until confluence. Integrity of cell monolayers was confirmed by ZO-1 labeling and non-permeability to FITC-labeled dextran (4300 Da). BoNT/A was added to the upper chamber and 60 min after incubation at 37°C, the toxin was assayed in the lower chamber by mouse bioassay. The results were expressed as the mean percentage (n = 9) of transcytosed BoNT/A corresponding to the ratio of mouse lethal dose 50 between the lower and upper well (inoculation titer). BoNT/A transport was significantly higher (*) in m-ICcl2 and in cells treated with anti-proteases (Student's t test). (B) Immunoblotting of cell lysates separated by SDS-PAGE with anti-chromograninA, anti-SV2A, anti-SV2B, anti-SV2C, and anti SV2C-L4 antibodies. (C) m-ICcl2 and STC-1 grown on glass cover slips were exposed to HcA-Cy3, alone or in combination with a 10-fold more molar concentration of SV2C/L4-GST for 10 min at 37°C. SV2C/L4 impaired HcA uptake in both m-ICcl2 and STC-1 cells. (D) The number of HcA fluorescent patches per µm2 in m-ICcl2 and STC-1 cells were reduced by 97% and 94% respectively (p<0.0001) by incubation with SV2C/L4. Each column represents the mean ± SEM (n = 250).