Functional inhibition of β-catenin-mediated Wnt signaling by intracellular VHH antibodies

Area of Science:

• Molecular Biology
• Cell Signaling
• Immunology

Background:

• The Wnt signaling pathway regulates crucial biological processes like development and tissue homeostasis.
• Dysregulation of Wnt signaling is implicated in various diseases, including cancer.
• Understanding the precise control mechanisms of beta-catenin, the pathway's master regulator, is essential but remains incomplete.

Purpose of the Study:

• To develop novel tools for investigating the Wnt signaling pathway.
• To explore the intracellular function and regulation of beta-catenin.
• To generate and characterize VHH antibodies targeting beta-catenin for functional studies.

Main Methods:

• Construction and panning of a llama VHH phagemid library against beta-catenin.
• Intracellular expression of VHH antibody fragments.
• Functional assessment using a luciferase-based Wnt signaling reporter bioassay in HEK293 cells.
• Characterization of VHH binding to endogenous beta-catenin and pathway inhibition.
• Mutational analysis of the VHH complementarity-determining region (CDR3) to confirm specificity.

Main Results:

• A VHH phagemid library was successfully screened against beta-catenin.
• Several intracellularly expressed VHH antibodies demonstrated function-modifying activity in the Wnt signaling assay.
• The VHH antibody LL3 specifically bound endogenous beta-catenin.
• LL3 inhibited Wnt signaling downstream of the destruction complex.
• Antibody-mediated inhibition was dependent on the specific CDR3 sequence, confirming targeted activity.

Conclusions:

• Intracellularly expressed llama VHH antibodies can effectively target and modulate beta-catenin activity.
• The VHH antibody LL3 serves as a valuable tool for dissecting Wnt pathway regulation.
• This approach provides a novel method for studying intracellular protein function and signaling pathways.