One-pot synthesis of heterodimeric agonists that activate the canonical Wnt signaling pathway

Abstract

Fragment antigen-binding domains (Fabs) from anti-Frizzled and anti-LRP6 monoclonal antibodies were conjugated using SpyTag-SpyCatcher chemistry via a one-pot reaction. The resulting synthetic heterodimeric agonist outperformed the natural ligand, Wnt-3a, in activating canonical Wnt signaling in mammalian cells. This approach should be broadly applicable to activate receptor-mediated cellular signaling.

Figure 1.. Schematic of a heterodimer agonist binding to the membrane proteins Frizzled and LRP6.

Fusing Frizzled (Fzd) and LRP6 Fabs via SpyCatcher-SpyTag interaction forms a heterodimer that binds to the canonical Wnt pathway receptors Fzd and LRP6.

Figure 2.. Characterization of expression and binding of anti-Fzd and anti-LRP6 Fabs.

a) Characterization of purified recombinant anti-Fzd Fab (Lane 1) and anti-LRP6 Fab (Lane 2) by SDS-PAGE. b) ELISAs showing specific binding of anti-Fzd and anti-LRP6 Fabs to the recombinant proteins Fzd2 and LRP6 fused to the human Fc region (Fzd2-Fc and LRP6-Fc respectively). BSA served as the negative control. ***p < 0.0001 by ANOVA and Tukey post hoc test; ns: not significant. c) Luciferase assays showing level of canonical Wnt signaling activation in HEK 293T cells when treated with 15 nM of exogenous Wnt-3a in the absence or presence of 1 μM anti-Fzd and anti-LRP6 Fabs respectively. ***p < 0.0001 by Student’s t-test. Plots show mean ± 1 standard deviation (s.d.).

Figure 3.. Synthesis and characterization of the purified Fab Heterodimer and its components.

a) Schematic showing SpyTag and SpyCatcher partner proteins isolated from S. pyogenes FbaB protein and reacting covalently via the formation of an isopeptide bond between them. b) Characterization of the binding of Fzd-Fab-SpyTag and LRP6-Fab-SpyCatcher fusion proteins to Fzd2-Fc, LRP6-Fc, and BSA by ELISA. ***p < 0.0001, **p < 0.01 by ANOVA and Tukey post hoc test; ns: not significant. c) Schematic showing the one-pot synthesis of Fzd-LRP6-Fab heterodimer from individual fusion protein components. d) SDS-PAGE gel confirming expression and purification of Fzd-Fab-SpyTag (Lane 1), LRP6-Fab-SpyCatcher (Lane 2) and the Fab heterodimer product (Lane 3) formed from the one-pot reaction. e) Characterization of the binding of the Fab heterodimer to Fzd2-Fc, LRP6-Fc, and BSA by ELISA. ***p < 0.0001 by ANOVA and Tukey post hoc test. Plots show mean ± 1 s.d.

Figure 4.. Characterizing Fab Heterodimer-Induced canonical Wnt signaling activation.

a) Luciferase assays showing level of canonical Wnt signaling activation in HEK 293T cells when treated with 15 nM of Fab heterodimer, the component Fab fusions, Wnt-3a, and an orthogonal Fab heterodimer. ***p < 0.0001 by ANOVA. b) Bar graph comparing Wnt-responsive luminescence signals at concentrations of Fab heterodimer (dark bar) and Wnt-3a (light bar) ranging from 0 to 15 nM. **p < 0.01 by multiple Bonferroni corrected student’s t-tests; ns: not significant. c) Western blot showing levels of cytosolic (active) β-catenin in HEK 293T cell lysates in the absence (−) and presence (+) of 15 nM Fab heterodimer overnight. d) Luciferase assays showing level of canonical Wnt signaling activation in HEK 293T cells when treated with 15 nM of Fab heterodimer in the absence or presence of 1 μM Fzd and LRP6 Fabs respectively. *p < 0.05 by Student’s t-test. Plots show mean ± 1 s.d.