Adaptive Evolution of Eel Fluorescent Proteins from Fatty Acid Binding Proteins Produces Bright Fluorescence in the Marine Environment

Abstract

We report the identification and characterization of two new members of a family of bilirubin-inducible fluorescent proteins (FPs) from marine chlopsid eels and demonstrate a key region of the sequence that serves as an evolutionary switch from non-fluorescent to fluorescent fatty acid-binding proteins (FABPs). Using transcriptomic analysis of two species of brightly fluorescent Kaupichthys eels (Kaupichthys hyoproroides and Kaupichthys n. sp.), two new FPs were identified, cloned and characterized (Chlopsid FP I and Chlopsid FP II). We then performed phylogenetic analysis on 210 FABPs, spanning 16 vertebrate orders, and including 163 vertebrate taxa. We show that the fluorescent FPs diverged as a protein family and are the sister group to brain FABPs. Our results indicate that the evolution of this family involved at least three gene duplication events. We show that fluorescent FABPs possess a unique, conserved tripeptide Gly-Pro-Pro sequence motif, which is not found in non-fluorescent fatty acid binding proteins. This motif arose from a duplication event of the FABP brain isoforms and was under strong purifying selection, leading to the classification of this new FP family. Residues adjacent to the motif are under strong positive selection, suggesting a further refinement of the eel protein's fluorescent properties. We present a phylogenetic reconstruction of this emerging FP family and describe additional fluorescent FABP members from groups of distantly related eels. The elucidation of this class of fish FPs with diverse properties provides new templates for the development of protein-based fluorescent tools. The evolutionary adaptation from fatty acid-binding proteins to fluorescent fatty acid-binding proteins raises intrigue as to the functional role of bright green fluorescence in this cryptic genus of reclusive eels that inhabit a blue, nearly monochromatic, marine environment.

Fig 1. Biofluorescent Chlopsidae (lower right) photographed in Little Cayman Island.

Fig 2. Kaupichthys hyoproroides collected in Bahamas and used in this study.

A) White light; B) Green fluorescence.

Fig 3. Details of green fluorescence of Kaupichthys sp.

A) Head fluorescence, B) Cross-section through demonstrating fluorescence in the musculature. C) Close up of skin revealing dark pigmented regions interspersed with fluorescence arising from both the skin and internal musculature.

Fig 4. Native gel of tissue homogenate from Kaupichthys hyoproroides.

A) Coomassie stained gel; B) Fluorescent bands imaged under illumination with blue light.

Fig 5. Sequence alignment of fluorescent FABPs from eels with a non-fluorescent FABP from Kaupichthys hyoproroides (Chlopsid NFP) and human brain FABP-7.

Residues highlighted in blue show areas of homology. The GPP sequence motif is highlighted in red.

Fig 6. Excitation/emission spectra of Chlopsid FP I and UnaG.

Fig 7. Phylogenetic tree generated by Maximum likelihood analysis in RaxML Blackbox.

See text for details of analysis. The arrows in the tree indicate potential branches where duplications occur to explain the paralog patterns in the gene family. The purple circles indicate the two branches where significant dN/dS skew occurs, and the number inside of the circle refers to the magnitude of the dN/dS skew.

Fig 8. Diagram showing the alignment of FABP’s and structure of UnaG.

A) Alignment of human brain FABP-7, UnaG and Chlopsid FP I. Red rectangles show residues that are inserted in the eel FP. Green rectangles show residues under dN/dS skew. Grey rectangles indicate polarity index. Blue highlights secondary structure factors. Pink highlights volume. Light green highlights refractivity/heat capacity and purple highlights charge/iso-electric point. When multiple colors appear at a site, this means that more than a single method detected changes or skew at those positions. B) Crystal structure of UnaG (pdb 4I3B) showing areas that are inserted in the eel FP as well as green showing residues under dN/dS skew.

Fig 9. Two photon image of eel fluorescence.

UnaG distribution in HEK293 cells (top panel), two photon localization of Chlopsid FP I in HEK293 cells (bottom panel) and confocal images of CiVSP-UnaG fusions in HEK293 cells.