Identification and functional characterization of CXCL17 in cartilaginous fishes reveals an ancient origin of the CXCL17-GPR25 signaling pathway

The newly identified signaling system comprising C-X-C motif chemokine ligand 17 (CXCL17) and G protein-coupled receptor 25 (GPR25) is involved in immune regulation and tumor development. However, the evolutionary origin of this pair has remained unclear because CXCL17 orthologs in lower vertebrates exhibit extreme sequence variation and cannot be identified through conventional homology-based searches. In this study, we identified seven possible CXCL17 orthologs in primitive cartilaginous fishes, including sharks and rays, using an integrated approach based on key amino acid sequence features as well as gene synteny, architecture, and RNA sequencing data in the NCBI gene database. To validate these candidates, a representative ortholog from the cloudy catshark (Scyliorhinus torazame), termed St-CXCL17, was prepared via bacterial overexpression and in vitro refolding. In cell-based functional assays, St-CXCL17 demonstrated high binding affinity and activation potency toward its corresponding receptor, St-GPR25. Further analysis revealed that removing three conserved C-terminal residues almost completely abolished this activity. While these cartilaginous fish CXCL17s share considerable homology with one another, they lack significant overall similarity to orthologs in mammals, amphibians, or bony fishes. These findings identify functional CXCL17 orthologs in cartilaginous fishes for the first time, implying that the CXCL17-GPR25 signaling pair likely originated in ancient cartilaginous fish ancestors or earlier and has been conserved throughout the evolution of jawed vertebrates.