Fig. 5.

Binding of BoNT/DC-H_C to ganglioside-containing liposomes. a A schematic drawing of the liposome flotation assay. b Experiments were carried out as depicted in a. Liposomes containing PC alone or PC plus a brain ganglioside mixture (gangl. mix, 1%) were incubated with HA-tagged BoNT/DC-H_C, BoNT/D-H_C, or BoNT/C1-H_C. Samples were centrifuged for 1 h at 240,000 × g in a sucrose gradient. Liposome fractions that floated to the top of the gradient were collected and subjected to immunoblot analysis. Samples without liposomes served as a negative control. BoNT/DC-H_C showed a basal level of binding to PC liposomes. Adding gangliosides increased binding of BoNT/DC-H_C to liposomes. BoNT/D-H_C and BoNT/C1-H_C did not show detectable binding to liposomes under the same assay conditions. c Experiments were carried out as described in b, except with indicated individual ganglioside species instead of a mixture of brain gangliosides. Both simple gangliosides (GM3 and GD3) and complex gangliosides (GM1 and GD1a) enhanced binding of BoNT/DC-H_C to liposomes. GD1b and GT1b also increased binding of BoNT/DC-H_C, but to a much lesser degree than GM1 and GD1a. d Experiments were carried out as described in b, with the indicated mutants of BoNT/DC-H_C and GM3-loaded liposomes. Mutations at the GBS (Y1115F, S1275A, and G1241A) abolished ganglioside-mediated binding to liposomes, as they showed similar levels of binding in PC alone and GM3-containing liposomes. In contrast, Y1165F/N1167A did not affect ganglioside-mediated binding to liposomes. None of these mutations reduced binding of BoNT/DC-H_C to PC liposomes compared to WT. e Model of BoNT/DC-H_C on plasma membranes. BoNT/DC is anchored onto plasma membranes by both Syt-binding and GBS–sialic acid interactions, putting the protein at an ideal angle to allow the extended loop (blue) to penetrate into the lipid membrane. f Experiments were carried out as described in b. Mutating any one of the three residues at the tip of the extended loop (F1253A, Y1251A, or W1252A) abolished binding of BoNT/DC-H_C to PC liposomes. Adding gangliosides did not rescue binding. g Experiments were carried out as described in b. Binding of BoNT/DC-H_C to liposomes containing LacCer or asialo-GM1 was at similar levels as to PC liposomes, while GM1 increased binding of BoNT/DC-H_C. One of three (b–d, f, g) independent experiments is shown. Immunoblot was quantified using ImageJ (n = 3) and normalized to the levels of BoNT/DC-H_C binding to PC liposomes in all panels. Error bars represent standard deviation