An eel protein illuminates human health

Visualizing individual molecules within the crowded cellular interior is a daunting task. Thanks to the discovery of naturally occurring fluorescent proteins in the 1990s, however, such experiments are now routine. Since the first successful cloning of the gene encoding green fluorescent protein (GFP) from a species of jellyfish, hundreds of other fluorescent proteins have been identified, giving biologists a remarkable array of tools with which to label and image specific molecules, cells or even entire tissues within living organisms.

Atsushi Miyawaki, head of the Laboratory for Cell Function Dynamics at the RIKEN Brain Science Institute in Wako, has spent much of his career searching for and studying fluorescent molecules. His team has now cloned an unusual fluorescent protein with particularly promising clinical utility¹.

Most natural fluorescent proteins isolated to date have been purified from marine invertebrates such as jellyfish and coral. Until quite recently, no such proteins had been identified in vertebrate species. Miyawaki was therefore surprised to learn that Seiichi Hayashi, a food chemist at Kagoshima University, had isolated a GFP from the muscle of the Japanese freshwater eel, commonly known as unagi (Fig. 1).

After learning of Hayashi’s discovery, Miyawaki immediately purchased two eels at a local fish market. “I tried just shining light on the muscle, and I was astonished by the bright green fluorescence,” he says. “After that, I was very curious about the fluorescence in the eel muscle and set about cloning this gene.”

GFP and related proteins exhibit chemical properties that make them inherently fluorescent. This novel ‘UnaG’ protein, however, needs additional assistance from a chemical known as bilirubin in order to fluoresce. Bilirubin is an intermediate molecule in the degradation of heme—the iron-binding component of hemoglobin that gives red blood cells their distinctive color and enables them to transport oxygen throughout the body. UnaG recognizes and binds to bilirubin astonishingly fast, and the two molecules generate bright green fluorescence almost immediately after they combine.

Upon analyzing its sequence and structure, the researchers determined that UnaG belongs to the family of fatty-acid-binding proteins (FABPs), which typically store or transport lipid molecules. Compared to its relatives, however, UnaG is extremely choosy. “Members of the FABP family generally have low affinity for their ligand, and some are ‘broad-spectrum’ and bind to more than one fatty acid ligand,” says Miyawaki. “But UnaG only binds to bilirubin, and with very high specificity and high affinity.”

UnaG is produced in a subset of thin muscle fibers (Fig. 2), where it specifically recognizes what is known as ‘unconjugated’ bilirubin. This heme metabolite is subsequently transported by the blood protein albumin to the liver, where it is converted enzymatically to ‘conjugated’ bilirubin prior to its eventual excretion from the body. However, mounting evidence suggests that the body deliberately maintains a pool of unconjugated bilirubin, which acts as a potent antioxidant that protects tissues against damage from oxidative stress.

Unagi eels migrate for several months at a time, requiring extended muscle exertion that can generate the sort of chemicals that promote oxidative stress. Miyawaki hypothesizes that UnaG may offer a countermeasure to protect muscle tissue in such circumstances. “If you look at locusts or birds, which travel over long distances, their muscles show greatly increased expression of a type of FABP that probably works as a store for some kind of fatty acid for fuel,” he says. “In analogy, we think that the UnaG protein works as storage for this powerful antioxidant.”