Identification of the Wnt signal peptide that directs secretion on extracellular vesicles

Wnt proteins are hydrophobic glycoproteins that are nevertheless capable of long-range signaling. We found that Wnt7a is secreted long distance on the surface of extracellular vesicles (EVs) following muscle injury. We defined a signal peptide region in Wnts required for secretion on EVs, termed exosome-binding peptide (EBP). Addition of EBP to an unrelated protein directed secretion on EVs. Palmitoylation and the signal peptide were not required for Wnt7a-EV secretion. Coatomer was identified as the EV-binding protein for the EBP. Analysis of cocrystal structures, binding thermodynamics, and mutagenesis found that a dilysine motif mediates EBP binding to coatomer with a conserved function across the Wnt family. We showed that EBP is required for Wnt7a bioactivity when expressed in vivo during regeneration. Overall, our study has elucidated the structural basis and singularity of Wnt secretion on EVs, alternatively to canonical secretion, opening avenues for innovative therapeutic targeting strategies and systemic protein delivery.