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

This study identifies functional CXCL17 orthologs in cartilaginous fishes through integrated genomic and experimental approaches, demonstrating their high-affinity interaction with GPR25 and establishing that the CXCL17-GPR25 signaling pathway originated in ancient cartilaginous fish ancestors or earlier.

The Gist

The Big Picture: Finding a Lost Key in the Deep Ocean

Imagine the human immune system as a massive, high-tech security force. To do its job, it needs a specific "radio signal" (a chemical messenger) to tell the security guards (immune cells) where to go. For a long time, scientists knew about a signal called CXCL17 and a receiver on the cell surface called GPR25. They knew these two worked together in humans to fight infections and even influence cancer.

However, there was a mystery: Where did this system come from?

Evolution is like a family tree. Usually, if a tool exists in humans, you can find a simpler, older version of it in our distant ancestors, like fish. But when scientists looked at "lower" vertebrates (like frogs or regular fish), they couldn't find the CXCL17 signal. It seemed like humans had invented this radio out of nowhere, which didn't make sense.

This paper is the story of how scientists finally found the "missing link" in the deepest, oldest part of the family tree: Cartilaginous fish (sharks, rays, and skates).

---

The Detective Work: How They Found the Signal

1. The "Needle in a Haystack" Problem
Scientists tried to find the shark version of CXCL17 by looking for DNA that looked similar to the human version. It was like trying to find a specific red car in a parking lot by only looking for cars that are exactly the same shade of red. They failed because the shark version had changed so much over millions of years that it didn't look like the human version anymore. It was a "needle in a haystack" where the needle had been painted a different color.

2. The New Strategy: Looking at the Neighborhood
Instead of just looking at the "car" (the gene sequence), the scientists looked at the "neighborhood" (the surrounding DNA). They knew that in humans, the CXCL17 gene sits next to specific "landmark" genes. They went to the shark genome and said, "If we find these same landmarks, the CXCL17 gene must be hiding nearby, even if it's wearing a disguise."

Using this detective work, combined with RNA data (which acts like a transcript of what genes are currently active), they found seven hidden CXCL17 genes in sharks and rays. One of them came from the Cloudy Catshark (a small, harmless shark), which they named St-CXCL17.

---

The Lab Experiment: Proving It Works

Finding the gene was step one. Step two was proving it actually does something. You can't just find a key; you have to prove it opens a door.

The "Factory" Setup
Since you can't easily run experiments on live sharks, the scientists took the shark gene and put it into bacteria (like E. coli). Think of the bacteria as tiny, fast factories. They programmed the bacteria to mass-produce the shark protein.

The "Refolding" Challenge
When the bacteria made the protein, it came out as a tangled, useless ball (like a ball of yarn that got stuck in a knot). The scientists had to use a special chemical bath to "unravel" and "refold" the protein into its correct, working shape.

The "Lock and Key" Test
Once they had the clean, folded shark protein, they tested it against the shark's version of the receiver (St-GPR25) inside human cells in a petri dish.

• The Result: The shark protein (the key) fit perfectly into the shark receiver (the lock). It triggered a bright light signal in the cells, proving the system works.
• The "Cut-off" Test: They chopped off the very last three letters of the protein's tail. Suddenly, the key didn't fit anymore. This proved that the "tail" of the protein is the most important part for making the connection.

---

Why This Matters: Rewriting the History Book

1. The "Ancient Ancestor" Discovery
The most exciting part is the timeline. Sharks are ancient; they have been swimming in the oceans for over 400 million years. Finding a working CXCL17–GPR25 system in sharks means this signaling pair didn't just appear in humans. It originated in the common ancestor of all jawed vertebrates.

Think of it like this: If you find a specific type of engine in a modern Ferrari, a 1950s Ford, and a 1920s Model T, you know that engine design is very old and fundamental. This paper proves that the "CXCL17 engine" is a fundamental part of our biological heritage, dating back to the age of sharks.

2. The "Disguise" Lesson
The shark protein looks nothing like the human protein. They are like two cousins who grew up in different countries and speak different languages, but they still share the same family name and DNA structure. This teaches scientists that they can't just look for "look-alikes" when studying evolution; they have to look for "functional cousins" hiding in plain sight.

Summary

• The Mystery: Scientists couldn't find the evolutionary ancestors of a human immune signal (CXCL17).
• The Solution: They used "genomic neighborhood" clues to find hidden versions in sharks and rays.
• The Proof: They made the shark protein in bacteria, refolded it, and showed it successfully activates the shark's immune receiver.
• The Takeaway: This immune system is ancient, originating in sharks hundreds of millions of years ago, and has been passed down to humans, frogs, and bony fish ever since.

This discovery is like finding a fossilized blueprint for a car engine that we thought was a modern invention, proving that the basic design has been driving evolution for eons.

Technical Summary

1. Problem Statement

The signaling system comprising the chemokine ligand CXCL17 and its receptor GPR25 is known to regulate immune responses and tumor development in mammals. However, the evolutionary origin of this pair has been unclear due to a phylogenetic discrepancy:

• GPR25 orthologs are widely conserved across vertebrates (from fish to mammals).
• CXCL17 orthologs were previously thought to be restricted to mammals, with no clear homologs identified in lower vertebrates (amphibians, bony fishes, or cartilaginous fishes) using standard homology-based searches.
• The Challenge: CXCL17 sequences in lower vertebrates exhibit extreme sequence divergence, lacking significant overall amino acid similarity to mammalian counterparts, rendering them invisible to conventional BLAST searches and leading to their annotation as "hypothetical proteins."

2. Methodology

The authors employed an integrated multi-step strategy to identify and validate CXCL17 orthologs in cartilaginous fishes:

• Bioinformatic Identification:

  • Instead of relying solely on sequence homology, the team used a strategy combining key amino acid sequence features (e.g., signal peptides, specific cysteine patterns, C-terminal motifs), gene synteny (genomic neighborhood), and gene architecture (exon-intron structure).
  • They screened the NCBI gene database and RNA sequencing data from primitive cartilaginous fishes (sharks and rays).
  • Manual assembly of transcript exons was performed to reconstruct full-length cDNAs for unannotated candidates.

• Protein Production and Refolding:

  • A representative ortholog from the cloudy catshark (Scyliorhinus torazame), designated St-CXCL17, was selected.
  • The gene was chemically synthesized and expressed in E. coli as inclusion bodies.
  • The protein was solubilized using an S-sulfonation strategy, purified via Ni²⁺ affinity chromatography, and refolded in vitro.
  • A C-terminally truncated mutant (St-[desC3]CXCL17, lacking the last three residues) and a SmBiT-tagged version for binding assays were also generated.

• Functional Assays (NanoBiT Technology):

  • β-Arrestin Recruitment: HEK293T cells co-expressing St-GPR25-LgBiT and SmBiT-β-arrestin 2 were used to measure receptor activation via bioluminescence complementation.
  • Calcium Mobilization: A NanoBiT-based calcium sensor (CALM1-SmBiT + LgBiT-MYLK2) was used to detect intracellular calcium flux upon receptor activation.
  • Ligand-Receptor Binding: A homogeneous binding assay using SmBiT-tagged St-CXCL17 and sLgBiT-fused St-GPR25 was conducted to determine dissociation constants ($K_d$) and competition kinetics.
  • Sulfation Analysis: Co-expression of human tyrosylprotein sulfotransferases (TPST1/2) was tested to determine if receptor tyrosine sulfation affects binding affinity.

3. Key Contributions

• Discovery of Ancient Orthologs: The study successfully identified seven functional CXCL17 orthologs in cartilaginous fishes (including sharks and rays), expanding the known phylogenetic range of this ligand family.
• Methodological Innovation: Demonstrated that combining synteny, gene architecture, and specific structural motifs is essential for identifying highly divergent chemokines that evade standard homology searches.
• Structural Characterization: Revealed unique structural features of cartilaginous fish CXCL17s, specifically the presence of eight cysteine residues (forming four disulfide bonds) and a unique C-terminal motif containing a hydrophilic threonine, distinct from the six-cysteine mammalian/bony fish variants and four-cysteine amphibian variants.
• Functional Validation: Provided the first experimental proof that cartilaginous fish CXCL17s are biologically active and specifically activate their cognate GPR25 receptors.

4. Key Results

• Sequence and Structure:
  • St-CXCL17 and other cartilaginous fish candidates possess an N-terminal signal peptide and a mature peptide with 8 cysteines.
  • They share high sequence similarity with each other but low similarity to mammalian/amphibian/bony fish CXCL17s.
  • The mature proteins are highly basic (pI 10.2–10.9), facilitating interaction with negatively charged glycosaminoglycans.
• Receptor Activation:
  • Recombinant St-CXCL17 activated St-GPR25 in a dose-dependent manner.
  • EC50 values: ~60 nM for β-arrestin recruitment and ~140 nM for calcium mobilization.
  • Critical C-Terminal Residues: Deletion of the three C-terminal residues (St-[desC3]CXCL17) almost completely abolished activity in both β-arrestin and calcium assays, confirming the C-terminus is essential for function.
• Binding Affinity:
  • Direct binding assays yielded a dissociation constant ($K_d$) of 33.0 ± 4.1 nM.
  • Co-expression of sulfotransferases improved affinity ($K_d$ ~19.5 nM), suggesting tyrosine sulfation on the receptor enhances binding.
  • The truncated mutant failed to compete for binding, reinforcing the importance of the C-terminus.
• Evolutionary Implication: The conservation of the CXCL17–GPR25 pair in cartilaginous fishes (which diverged ~450 million years ago) implies the signaling system originated in early jawed vertebrates or earlier and has been conserved throughout vertebrate evolution.

5. Significance

• Evolutionary Biology: Resolves the long-standing phylogenetic discordance between CXCL17 and GPR25, establishing that the signaling pair is an ancient feature of jawed vertebrates, not a mammalian innovation.
• Chemokine Diversity: Expands the understanding of chemokine structural plasticity, showing that the core CXCL17 function can be maintained despite drastic changes in sequence length, cysteine count, and overall homology.
• Immunology and Oncology: Since CXCL17 is involved in immune cell recruitment and tumor progression, understanding its ancient evolutionary roots may provide new insights into the fundamental mechanisms of immune regulation and cancer biology across vertebrates.
• Future Discovery: The identification strategy used in this paper provides a blueprint for discovering other "hidden" or highly divergent signaling molecules in non-model organisms.