Characterization of a membrane binding loop leads to engineering botulinum neurotoxin B with improved therapeutic efficacy
Fig 5
Characterizing binding of HC/BWW on cultured neurons and BoNT/BMY-WW in vivo.
(A) Binding of WT HC/B, HC/BW, and HC/BWW to cultured rat cortical neurons was examined by immunoblot analysis. Neurons were exposed to HA-tagged HC/B, HC/BW, and HC/BWW for 5 minutes, washed, and harvested for immunoblot analysis. Actin served as an internal loading control. The total represents 50 ng of HC proteins. Representative blots and the intensity relative to total HC/B quantification results are shown. (B) Binding of HC/B and HC/BWW to cultured mouse cortical WT and GD2 KO neurons was examined by immunostaining analysis. Neurons were exposed to HA-tagged HC/B, HC/BWW, or HC/BWW plus GST-tagged recombinant Syt II (1–61, 10-fold of HC/BWW concentration) for 5 minutes, washed, fixed, and subjected to immunostaining analysis. Synapsin serves as an internal marker for presynaptic terminals. HC/BWW showed enhanced binding to neurons compared with HC/B and colocalized with Synapsin. Binding of HC/BWW is lower on GD2 KO neurons compared with WT neurons, and residual binding of HC/BWW on GD2 KO neurons is eliminated by recombinant Syt II fragment protein as a receptor decoy. Scale bar represents 30 μm. (C) The control BoNT/BMY elicited a dose-dependent response in the DAS mean score. Data are means ± SEM of n = 7 or 8. The response within the first 4 days is shown in the enlarged insert. (D) BoNT/BMY-WW showed a more potent response and longer duration in the DAS assay than the control BoNT/B (panel C). Data are means ± SEM of n = 7 or 8. Numerical values for (A), (C), and (D) are available in S1 Data. BoNT, botulinum neurotoxin; DAS, Digit Abduction Score; GD2 KO, Galgt1 knockout; GST, glutathione S-transferase; HA, human influenza hemagglutinin; HC, C-terminal receptor-binding domain; Syt, synaptotagmin; WT, wild-type.