Anti-TTR Nanobodies Allow the Identification of TTR Neuritogenic Epitope Associated with TTR-Megalin Neurotrophic Activities

Transthyretin (TTR) has intrinsic neurotrophic physiological activities independent from its thyroxine ligands, which involve activation of signaling pathways through interaction with megalin. Still, the megalin binding motif on TTR is unknown. Nanobodies (Nb) have the ability to bind “hard to reach” epitopes being useful tools for protein/structure function. In this work, we characterize two anti-TTR Nanobodies, with similar mouse TTR binding affinities, although only one is able to block its neuritogenic activity (169F7_Nb). Through epitope mapping, we identified amino acids 14–18, at the entrance of the TTR central channel, to be important for interaction with megalin, and a stable TTR K15N mutant in that region was constructed. The TTR K15N mutant lacks neuritogenic activity, indicating that K15 is critical for TTR neuritogenic activity. Thus, we identify the putative binding site for megalin and describe two Nanobodies that will allow research and clarification of TTR physiological properties, regard...