Identification and functional characterization of two CXCL17 paralogs from zebrafish

C-X-C motif chemokine ligand 17 (CXCL17) and its receptor G protein-coupled receptor 25 (GPR25) have been identified as a significant pair in regulating immunity, but CXCL17 orthologs have not yet been identified from non-mammalian vertebrates. This study aimed to identify and characterize non-mammalian CXCL17 orthologs based on key features of mammalian CXCL17s, such as a C-terminal Xaa-Pro-Yaa motif, a signal peptide, and six cysteine residues. Two possible CXCL17 paralogs were identified from zebrafish (Danio rerio): Dr-CXCL17 (encoded by zgc:158701) and Dr-CXCL17-like (encoded by si:dkey-112a7.5). Both are previously uncharacterized proteins with unknown functions because they lack overall sequence similarity to known mammalian CXCL17s. For functional characterization, recombinant Dr-CXCL17 and Dr-CXCL17-like were prepared via overexpression in Escherichia coli and subsequent in vitro refolding, and their activity was tested using NanoLuc Binary Technology (NanoBiT)-based β-arrestin recruitment assays, NanoBiT-based ligand‒ receptor binding assays, and chemotaxis assays. The results showed that both Dr-CXCL17 and Dr-CXCL17-like tightly bound to and efficiently activated zebrafish GPR25 (Dr-GPR25) and induced chemotactic movement in transfected human embryonic kidney (HEK) 293T cells expressing the receptor. Deletion of three C-terminal residues in both paralogs almost eliminated their binding, activation, and chemotactic effects, which suggests that this fragment is crucial for their function. Homologs of Dr-CXCL17 or Dr-CXCL17-like were retrieved from several other ray-finned fish species, indicating that two CXCL17 paralogs are present in certain fish species and function as endogenous agonists for the fish GPR25 receptor. The identification of fish CXCL17 orthologs suggests that the CXCL17‒GPR25 pair likely originated in ancient fishes and was conserved across vertebrate lineages. This work represents the first identification of CXCL17 orthologs in non-mammalian vertebrates, paving the way for future functional studies of this ligand‒receptor pair.