Fig 1: The LC of BoNT/X cleaves VAMPs at a unique site.(a) X-LC was incubated with BDE. Immunoblot analysis was carried out to detect syntaxin 1, SNAP-25 and VAMP2. Synaptophysin (Syp) served as a loading control. A-LC and B-LC were analysed in parallel. Cleavage of VAMP2 by B-LC results in loss of immunoblot signals, while cleavage of SNAP-25 by A-LC generates a smaller fragment (marked with an asterisk). EDTA blocked the activity of X-, A- and B-LCs. (b) VAMP2 (1–93) was incubated with X-LC. Samples were analysed by SDS–PAGE and Coomassie Blue staining. X-LC converted VAMP2 (1–93) into two smaller fragments. (c–e) VAMP2 (1–93) was incubated with X-LC. Samples were analysed by mass spectrometry (LC–MS/MS) to determine the molecular weight of cleaved fragments. Eluted peptide peaks from the HPLC column are plotted over running time (RT, X axis). The mass spectrometry data for the two cleavage products are colour-coded, with mass-to-charge ratio (m/z) noted. The molecular weight is deduced by multiplying m with z, followed by subtracting z. The protein sequences for the two cleavage products are colour-coded and listed in c. (f) Sequence alignment between VAMP family members, with the cleavage sites for BoNT/B, D, F, G and X marked in red, and the two SNARE motifs in blue shade. (g) HA-tagged VAMP1, 3, 7 and 8, and Myc-tagged Sec22b and Ykt6 were expressed in 293T cells via transient transfection. Cell lysates were incubated with X-LC and subjected to immunoblot analysis. Actin is a loading control. (h) GST-tagged Ykt6 was incubated with X-LC (100 nM). Samples were analysed by SDS–PAGE and Coomassie Blue staining. (i) GST-tagged VAMP2 (33–86), VAMP4 (1–115) and VAMP5 (1–70) were incubated with X-LC (100 nM). Samples were analysed by SDS–PAGE and Coomassie Blue staining. X-LC cleaved both VAMP4 and VAMP5. We note that VAMP5 protein contains a contaminant band that runs close to the cleavage product. (j) Experiments were carried out as described in a, except that VAMP4 and Sec22b were detected. Synaptotagmin I (Syt I) is a loading control. X-LC cleaved native VAMP4 in BDE. One of two (b,g,j) or three (a,h,i) independent experiments is shown.