[PubMed] [Google Scholar] 26

[PubMed] [Google Scholar] 26

[PubMed] [Google Scholar] 26. of downstream target genes6, 7. Efficient purification of Wnt ligands represents a major impediment in this field of research. Wnt ligands undergo myriad post-translational modifications which are critical for retention of their activity8. For example, the canonical Wnt-3a ligand contains two N-linked glycosylation sites, which are important for its secretion and folding9, 10. Furthermore, the addition of a palmitoleic acid moiety which is essential for binding to Fzd makes the Wnt ligand hydrophobic and water insoluble, which necessitates the use of detergents in workflows designed to purify these ligands. These added difficulties and expenses have traditionally hindered the use of Wnt proteins as therapeutic agents11, 12. To circumvent these issues, several alternate approaches of activating the Wnt signaling pathway have been developed in the past which do not require the natural Wnt ligands. Satisfactorily mimicking the endogenous dynamics of canonical Wnt signalling, however, remains challenging13. One such alternate approach involved treating the cells with Li+. The Li+ inhibits the degradation of -catenin by disrupting the activity of glycogen synthase kinase 3 (GSK3)14. However, Li+ targets several other cellular components such as G-protein coupled receptors, the transcription factor AP-1 and a variety of phosphatases, and is thus not a specific activator of canonical Wnt signaling15. An alternative is the small molecule CHIR99021, which similarly inhibits Exemestane GSK3 but with much greater specificity14. However, it activates Wnt signaling through an alternate mechanism of action and not by the fractional inactivation of the destruction complexes5. Furthermore, the GSK3 enzyme plays a role in transducing other cellular signaling pathways beyond the destruction complex, e.g., insulin signaling, and so inhibition of GSK3 by CHIR99021 may lead to undesired interference with other pathways16. Cong et al. previously showed that heterodimerization of Frizzled and LRP6 receptors may be sufficient to trigger the downstream canonical Wnt signaling cascade17. In a recent elegant study, Janda et al. reported single-chain protein-based canonical Wnt agonists18. These agonists consisted of either an engineered bacterial protein or a single-chain variable fragment specific to Frizzled (Fzd) fused to the C-terminal domain of the natural Wnt antagonist, Dickkopf-1 (DKK1), which binds LRP618. These surrogate agonists simultaneously bound to Fzd and LRP6 and activated canonical Wnt signaling. While this report represents a key milestone in developing surrogate agonists, the approach was based on re-engineering a Exemestane natural ligand of the Wnt co-receptor. We reasoned that it would be advantageous to design Exemestane a strategy that is broadly applicable to designing synthetic agonists for a variety of signaling pathways. Natural ligands for a target receptor may Exemestane not be readily available or easy to express, purify, or re-engineer. Moreover it would be advantageous to develop a modular strategy that allowed experimentalists to easily engineer the agonist by varying properties including but not limited to: specificity (e.g., to change the target receptor or to target a subset of receptors in a family); affinity for one or more target Exemestane receptors; the binding epitope on the target receptor; and the avidity of the agonist. In contrast, the binding epitope for a natural ligand, its affinity for a target receptor, and the avidity of the resulting agonist, are not readily tuneable. To address these limitations, we have developed an alternate approach for designing synthetic heterodivalent agonists that activate canonical Wnt signaling. Specifically, we used SpyTag-SpyCatcher chemistry to covalently link two fragment antigen-binding domains (Fabs) that bound to Frizzled and LRP6 respectively (Fig. 1). Open in a separate window 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. First, to express wild-type Fabs, we obtained sequences of full-length anti-Frizzled and anti-LRP6 antibodies from Gurney et al. and Jenkins et al. respectively19, 20. The sequences of the variable regions within the heavy and light chains of the antibodies were inserted into two separate vectors, TGEX-FH and TGEX-LC respectively (Fig. S1). The anti-Fzd and anti-LRP6 Fabs were expressed by co-transfecting HEK (human embryonic kidney) 293F cells with the plasmids encoding Rabbit Polyclonal to SYTL4 the corresponding heavy and light chains. The Fabs were subsequently purified using immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography in succession. We characterized.