Green fluorescent protein (GFP) fro

Green fluorescent protein (GFP) from eel (Anguilla japonica) muscle (eelGFP) is unique in the vertebrates and
requires bilirubin as a ligand to emit fluorescence. This study was performed to clarify the physiological function
of the unique GFP. Investigation of susceptibility to oxidative stress was carried out using three types of cell lines
including jellyfish (Aequorea coerulescens) GFP (jfGFP)-, or eel GFP (eelGFP)-expressing HEK293 cells, and control
vector-transfected HEK293 cells. Binding of eelGFP to bilirubin was confirmed by the observation of green fluorescence
in HEK293-eelGFP cells. The growth rate was compared with the three types of cells in the presence or
absence of phenol red which possessed antioxidant activity. The growth rates of HEK293-CV and HEK293-jfGFP
under phenol red-free conditions were reduced to 52 and 31% of those under phenol red. Under the phenol redfree
condition, HEK293-eelGFP had a growth rate of approximately 70% of the phenol red-containing condition.
The eelGFP-expressing cells were approximately 2-fold resistant to oxidative stress such as H2O2 exposure. The
fluorescence intensity partially decreased or disappeared after exposure to H2O2, and heterogeneous intensity
of fluorescence was also observed in isolated eel skeletal muscle cells. These results suggested eelGFP, but
not jfGFP, coupled with bilirubin provided the antioxidant activity to the cells as compared to non-bound
free bilirubin.