Extended Data Figure 4: CROPs are essential for TcdB binding to CSPG4, but not required for TcdB binding to FZDs. | Nature
a, Schematic drawings of rat CSPG4. Two pools of recombinant extracellular domain (EC) fragments were used: one that does not contain chondroitin sulfate (CS) chains (EC P1), and the other that contains CS (EC P2). TMD-cyto, transmembrane and cytoplasmic domain. b, TcdB, but not TcdB1–1830, binds directly to both EC P1 and EC P2 of CSPG4 in a microtitre plate-based binding assay (error bars indicate mean ± s.d., two independent experiments). c, CSPG4−/− cells transfected with the indicated constructs were exposed to TcdB (10 nM), TcdB1–1830 (10 nM), or the receptor-binding domain of botulinum neurotoxin B (BoNT/BHC; 100 nM) for 10 min. Cell lysates were collected and subjected to immunoblot analysis. IL1RAPL2 and synaptotagmin II (Syt II, a receptor for BoNT/B) served as controls. Transfection of CSPG4 increased binding of TcdB, but not TcdB1–1830, whereas transfection of FZD2 increased binding of both TcdB and TcdB1–1830. One of three independent experiments is shown. d, The CROP domain binds to CSPG4 on cell surfaces in a dose-dependent manner. High concentrations of recombinant CROPs reduced CSPG4-dependent binding of TcdB to cell surfaces, indicating that the CROPs can compete with TcdB for binding to CSPG4 on cell surfaces. One of three independent experiments is shown. e, The CROP domain reduced cytopathic toxicity of TcdB (5 pM) on wild-type (WT) HeLa cells (error bars indicate mean ± s.d., two independent experiments). f, CSPG4−/− cells were transfected with FZD2 and then exposed to TcdB or indicated TcdB fragments. FZD2-mediated binding of TcdB, TcdB1–1830 and TcdB1501–2366, but not the CROPs (TcdB1831–2366). One of three independent experiments is shown.