Grilled freshwater eel is just another item on the menu at many Japanese restaurants, but the fish has now been introduced to microscopists, who are always hungry for new imaging tools to advance their research.
“UnaG can be used as a direct fluorescent sensor for bilirubin,” said RIKEN Brain Science Institute researcher Atsushi Miyawaki, who led the study. A low concentration of bilirubin is an indicator of proper liver function. But liver dysfunction causes unconjugated to bilirubin buildup in the circulation system and extravascular tissue, leading to jaundice, kernicterus, and other related diseases.
The team named the new protein UnaG, in recognition of its source, the Unagi eel, as well as its green fluorescence. UnaG is the first fluorescent protein identified in a vertebrate species, which means it might be more biocompatible for imaging human tissue.
The glass eel’s ability to glow was first reported in 2009. Because Miyawaki’s lab is interested in imaging technologies, the team set out to identify the protein causing the fluorescence. The scientists synthesized complementary DNA, or cDNA, sequences from five glass eels. They then developed degenerative primers representing variations in nine partial peptides whose amino acid sequences were identified in 2009 (2) as possible sources of fluorescence.
The results revealed a cDNA clone encoding a full-length protein composed of 139 amino acids that was likely the protein that caused the eels to glow. The team expressed the protein in bacteria, but it did not fluoresce. However, when expressed in HeLa cells, the protein did glow—with an intensity comparable to the widely-used green fluorescent protein (GFP), which is found in jellyfish.
Since the protein fluoresced in mammalian cells but not in bacteria, the scientists realized that UnaG likely needed another molecule to switch it on. They were surprised to find that the purified protein only fluoresced when bound to unconjugated bilirubin.
To test if UnaG could sense the buildup of bilirubin seen in patients with poor liver function, the researchers added the protein to normal blood serum and to samples spiked with bilirubin. The bilirubin-spiked samples produced stronger fluorescent signals than serum alone. Miyawki and colleagues think UnaG could be used to study how bilirubin is distributed through muscle tissue, what role it plays in muscle endurance, and how buildup of bilirubin could be linked to cardiovascular disease, diabetes mellitus, and other diseases caused by oxidative stress.
Other experiments showed that, unlike GFP, UnaG can fluoresce in low-oxygen conditions, suggesting that the protein could be used in tests of cell and animal systems under harsh, anaerobic conditions.
The scientists think that the UnaG protein relates in some way to the glass eel’s migratory habits. They found that two other long-distance migratory freshwater eels, Anguilla anguilla and Anguilla rostrata, also have green fluorescence in their white muscle tissue. But, the sea eel Conger myriaster, which sticks mostly to the ocean, does not.
“It may also help preserve freshwater eel species,” which are threatened in Japan said Miyawaki. Because of the eels’ newfound importance for biomedicine, Miyawaki said he hopes new legislation will help preserve eel species and possibly reverse their recent drastic decline worldwide.
1. Kumagai, A., R. Ando, H. Miyatake, P. Greimel, T. Kobayashi, Y. Hirabayashi, T. Shimogori, and A. Miyawaki. 2013. A Bilirubin-Inducible fluorescent protein from eel muscle. Cell (June).
2. Hayashi, S., and Y. Toda. 2009. A novel fluorescent protein purified from eel muscle. Fisheries Science 75(6):1461-1469.