Journal: Nature Communications

Article Title: Engineered botulinum neurotoxin B with improved efficacy for targeting human receptors

doi: 10.1038/s41467-017-00064-y

Figure Lengend Snippet: BACTH screen identifies mutations that enhance BoNT/B binding to h-Syt II. a Sequence alignment of the BoNT/B-binding site in mouse Syt I (m-Syt I), h-Syt I, h-Syt II, and mouse Syt II (m-Syt II), and a close-up view of the interface between H C B and Syt II. Targeted residues in BoNT/B for mutagenesis studies are labeled in green . Syt II residues are marked in purple , with F54, F55, and I58 being highlighted. b A schematic illustration for construction of H C B mutant libraries and the BACTH assay. c H C B mutants identified in the BACTH assay were further analyzed by β-galactosidase activity assay, which reflects the levels of reconstituted adenylate cyclase (Student’s t -test, n = 6, * P < 0.05). Error bars represent SEM. d Point mutations at E1191, W1178, and S1199 were examined in pull-down assays, using immobilized m-Syt II (residues 1–87) or a mouse Syt II (1–87) containing F54L mutation that mimics h-Syt II sequence (designated as h-Syt II). Bound H C B variants were detected by immunoblot analysis detecting the HA-tag fused to H C B. Additional H C B mutations at other sites are shown in Supplementary Fig. . One of two independent experiments is shown

Article Snippet: The following antibodies were purchased from the indicated vendors: Synapsin I (Clone 46.1, Synaptic Systems), VAMP2 (Clone 69.1, Synaptic Systems), HA (16B12, Covance), Syt I (Clone mAB48, The Developmental Studies Hybridoma Bank), Syt II (Clone 26, BD Bioscience), polyclonal rabbit anti-BoNT/B (Metabiologics), and β-tubulin III (ab18207, Abcam).

Techniques: Binding Assay, Sequencing, Mutagenesis, Labeling, Activity Assay, Western Blot

Journal: Nature Communications

Article Title: Engineered botulinum neurotoxin B with improved efficacy for targeting human receptors

doi: 10.1038/s41467-017-00064-y

Figure Lengend Snippet: Combinational mutations in H C B enhance its binding to h-Syt II and Syt I. a H C B double mutations were examined in pull-down assays for their abilities to bind m-Syt II vs. h-Syt II. Three double mutations that showed robust binding to h-Syt II are marked in red . One of two independent experiments is shown. b Binding of H C B to h-Syt II was quantified by the BLI assay. Representative association and dissociation curves are shown for WT H C B and three H C B mutants: E1191M/S1199Y, E1191V/S1199W, and E1191C/S1199W. Binding parameters for all 12 double mutations are listed in Table , and their representative binding traces are shown in Supplementary Fig. . c Binding of WT H C B, H C B (E1191M), and H C B (E1191M/S1199Y) to immobilized GST-tagged h-Syt I (residues 1–80) was examined in pull-down assays, with (+) or without (−) gangliosides (Gangl.). Binding of WT H C B to h-Syt I requires the presence of gangliosides, while H C B (E1191M) and H C B (E1191M/S1199Y) bind to h-Syt I in the absence of gangliosides. One of two independent experiments is shown. Further analysis of H C B binding to h-Syt I by the BLI assay is shown in Supplementary Fig. and Table

Article Snippet: The following antibodies were purchased from the indicated vendors: Synapsin I (Clone 46.1, Synaptic Systems), VAMP2 (Clone 69.1, Synaptic Systems), HA (16B12, Covance), Syt I (Clone mAB48, The Developmental Studies Hybridoma Bank), Syt II (Clone 26, BD Bioscience), polyclonal rabbit anti-BoNT/B (Metabiologics), and β-tubulin III (ab18207, Abcam).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Engineered botulinum neurotoxin B with improved efficacy for targeting human receptors

doi: 10.1038/s41467-017-00064-y

Figure Lengend Snippet: H C B MY shows robust binding to neurons that express h-Syt II. a Humanized neurons were created by KD of endogenous Syt I and by expressing full-length h-Syt II in cultured rat cortical neurons via lentiviral transduction. KD efficiency was validated by immunoblot (Supplementary Fig. ). Neurons that express full-length m-Syt II or m-Syt II (F54L) served as controls. These neurons were exposed to WT H C B (100 nM) followed by immunostaining analysis. Bound H C B was detected via an HA-tag fused to H C B. Synapsin was labeled as a marker for presynaptic terminals. WT H C B bound to WT neurons but not to Syt I KD neurons. Expression of full-length m-Syt II restored binding of H C B. Expression of full-length m-Syt II (F54L) or full-length h-Syt II resulted in only low levels of binding of WT H C B compared to m-Syt II. b Syt I KD abolished binding of H C B MY (100 nM) to neurons. The binding was rescued by expression of full-length m-Syt II, m-Syt II (F54L), or h-Syt II. Scale bars represent 20 µm. One of two independent experiments is shown

Article Snippet: The following antibodies were purchased from the indicated vendors: Synapsin I (Clone 46.1, Synaptic Systems), VAMP2 (Clone 69.1, Synaptic Systems), HA (16B12, Covance), Syt I (Clone mAB48, The Developmental Studies Hybridoma Bank), Syt II (Clone 26, BD Bioscience), polyclonal rabbit anti-BoNT/B (Metabiologics), and β-tubulin III (ab18207, Abcam).

Techniques: Binding Assay, Expressing, Cell Culture, Transduction, Western Blot, Immunostaining, Labeling, Marker

Journal: Nature Communications

Article Title: Engineered botulinum neurotoxin B with improved efficacy for targeting human receptors

doi: 10.1038/s41467-017-00064-y

Figure Lengend Snippet: BoNT/B MY shows enhanced functional efficacy in neurons that express h-Syt II. a Humanized neurons were created as described in Fig. . Neurons were exposed to a gradient of full-length WT BoNT/B or BoNT/B MY for 24 h. Cell lysates were harvested and subjected to immunoblot analysis. β-tubulin served as an internal loading control. BoNT/B MY cleaved more VAMP2 than WT BoNT/B at all concentrations tested, indicating that BoNT/B MY entered neurons more efficiently than WT BoNT/B. One of three independent experiments is shown. b , c Humanized neurons were exposed to a gradient of WT BoNT/B or BoNT/B MY for 24 h. The mIPSC was recorded by a whole-cell patch-clamp approach. b Representative mIPSC recordings at 30 pM toxins. c The mIPSC activities vs. toxin concentrations, normalized to neurons that were not exposed to toxins. The numbers of recorded neurons for each data point are noted within parentheses . The same number of neurons was recorded for WT BoNT/B and BoNT/B MY . The half maximum inhibitory concentration (IC 50 ) was determined to be 89 pM for WT BoNT/B and 7.8 pM for BoNT/B MY , demonstrating that enhancing binding to receptors increased functional efficacy of toxins in neurons. Statistical analysis was performed with Student’s t -test (* P < 0.01). All data shown are means ± SEM

Article Snippet: The following antibodies were purchased from the indicated vendors: Synapsin I (Clone 46.1, Synaptic Systems), VAMP2 (Clone 69.1, Synaptic Systems), HA (16B12, Covance), Syt I (Clone mAB48, The Developmental Studies Hybridoma Bank), Syt II (Clone 26, BD Bioscience), polyclonal rabbit anti-BoNT/B (Metabiologics), and β-tubulin III (ab18207, Abcam).

Techniques: Functional Assay, Western Blot, Patch Clamp, Concentration Assay, Binding Assay

Journal: Nature Communications

Article Title: Engineered botulinum neurotoxin B with improved efficacy for targeting human receptors

doi: 10.1038/s41467-017-00064-y

Figure Lengend Snippet: Variations in BoNT/B subtypes influence their binding to h-Syt II and h-Syt I. a Sequence alignment of BoNT/B subtypes in the region around residues E1191/S1199. b H C B4 showed robust binding to m-Syt II, but no detectable binding to h-Syt II without gangliosides, which is similar to H C B. Both showed low levels of binding to h-Syt II in the presence of gangliosides. c There are four residues that differ between H C B4 and H C B within the 19 key residues in the Syt-binding pocket besides E1191Q/S1199Y. Replacing all four residues in H C B4 with the corresponding residues in H C B created a mutant H C B4 (V1113K/S1117P/S1196A/I1197P) that can bind to h-Syt II. The sequence alignment between H C B and H C B4 is shown in Supplementary Fig. . d Binding of H C B and H C B4 to immobilized GST-tagged h-Syt I was examined in pull-down assays. Binding of H C B to h-Syt I requires the presence of gangliosides, while H C B4 is capable of binding to h-Syt I without gangliosides in pull-down assays

Article Snippet: The following antibodies were purchased from the indicated vendors: Synapsin I (Clone 46.1, Synaptic Systems), VAMP2 (Clone 69.1, Synaptic Systems), HA (16B12, Covance), Syt I (Clone mAB48, The Developmental Studies Hybridoma Bank), Syt II (Clone 26, BD Bioscience), polyclonal rabbit anti-BoNT/B (Metabiologics), and β-tubulin III (ab18207, Abcam).

Techniques: Binding Assay, Sequencing, Mutagenesis