Preparation and Characterization of LGR5 LOOP Region-Specific Nanobodies.

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Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), also known as G-protein-coupled receptor 49 (GPR49), is a class A G-protein-coupled receptor (GPCR) that plays a pivotal role in embryonic development and functions as a marker for adult stem cells in various tissues and organs. LGR5 possesses a large extracellular domain (ecto-domain) enriched with leucine-rich repeats (LRR), primarily responsible for binding to ligands such as R-spondins. The C-terminal LRR extracellular LOOP region of LGR5 refers to the loop structure connecting the C-terminus of LGR5 to the first transmembrane helix. As the LOOP region is located extracellularly, it is readily accessible to exogenous molecules such as antibodies, nanobodies, or small-molecule drugs. The LOOP region of LGR5 modulates receptor conformational changes, thereby influencing signal transduction and contributing to the regulation of cellular functions. In this study, we successfully expressed and purified the LGR5 LOOP region protein in a prokaryotic expression system. The purified protein was subsequently used as an antigen to immunize camels, leading to the generation of specific nanobodies. These nanobodies are composed solely of the variable domain of the heavy-chain antibody (VHH), with a molecular weight of approximately 15 kDa, and exhibit excellent stability. Using the purified LGR5 LOOP region protein as an antigen, we isolated nanobodies that specifically bind to it. Subsequent assays demonstrated that the selected nanobody, NB 4C4 and NB 3E8, exhibited high affinity for the antigen and specifically targeted the LGR5 LOOP region, effectively inhibited β-catenin-mediated Wnt signaling exhibited an inhibitory effect on β-catenin-mediated Wnt signaling to a certain extent. This study provides substantial evidence insights for the development of LGR5-targeted diagnostic reagents and antibody-based therapeutic strategies.