Discovery and Characterization of a Bilirubin Inducible Green Fluorescent Protein From the Moray Eel Gymnothorax zonipectis

Abstract

Since the initial discovery of Aqueoria victoria’s green fluorescence off the coast of Washington’s Puget Sound, biofluorescent marine organisms have been found across the globe. The variety of colors of biofluorescence as well as the variability in the organisms that exhibit this fluorescence is astounding. The mechanisms of biofluorescence in marine organisms are also variable. To fluoresce, some organisms use fluorescent proteins, while others use small molecules. In eels, green biofluorescence was first identified in Anguilla japonica. The green fluorescence in A. japonica was discovered to be caused by a fatty acid binding protein (UnaG) whose fluorescence is induced by the addition of bilirubin. Members of this class of proteins were later discovered in Kaupichthys eels (Chlopsid FP I and Chlopsid FP II). Here, we report the discovery and characterization of the first member of this class of green fluorescent fatty acid binding proteins from the moray eel Gymnothorax zonipectis. This protein, GymFP, is 15.6 kDa with a fluorescence excitation at 496 nm and an emission maximum at 532 nm upon addition of bilirubin. GymFP is 61% homologous to UnaG and 47% homologous to Chlopsid FP I. Here, we report de novo transcriptome assembly, protein expression, and fluorescence spectroscopic characterization of GymFP. These findings extend the fluorescent fatty acid binding proteins into a third family of true eels (Anguilliformes).

Genetically encodable fluorescent protein markers in advanced optical imaging

“…Its 139 amino acids fold into a ten-stranded β-barrel that is capped by two α-helices (figure 4). Meanwhile, representatives of this protein class have been identified in other eel species as well [111][112][113]. UnaG binds BR non-covalently with high specificity and affinity (K D =98 pM) in an interior pocket.…”

Biofluorescent response in lumpfish Cyclopterus lumpus to a therapeutic stressor as assessed by hyperspectral imaging

“…sp. ), Anguilla japonica and Gymnothorax zonipectis 31 – 33 . As biliverdin is transported within the serum, it circulates broadly within fish.…”

Diversity and function of fluorescent molecules in marine organisms

“…(5) Bilirubin binding to reduce cellular, oxidative stress ((a)) Chordata -Vertebrata -Osteichthyes (fish) Green and red fluorescent patterns have been observed in moray eels (e.g. Muraenidae) (Guarnaccia et al, 2021), false morays (Chlopsidae) (Sparks et al, 2014) and freshwater eels (Anguillidae) (Hayashi & Toda, 2009;. The FPs are believed to have regulatory functions in stress mitigation (Funahashi et al, 2016).…”

“…Other members of the bilirubin binding GFPs have also been identified in two chlopsid eels Kaupichthys hyoproroides and Kaupichthys n. sp. (Chlopsid FP I and II) and in the moray eel Gymnothorax zonipectis (GymFP) (Gruber et al, 2015;Guarnaccia et al, 2021). Since most moray eels do not show fluorescence in a comparable habitat and do not migrate for long-distances the ecological and physiological function of GymFP should be investigated further.…”

“…The nocturnal moray eel Gymnothorax zonipectis reveals bilirubin inducible fluorescence (GymFp). It has been suggested that the visual detection of this fluorescence might be involved in synchronized spawning during full moon (Munz & McFarland, 1973;Lee et al, 2008;Wang, Tang & Yan, 2011;Guarnaccia et al, 2021).…”