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

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.本研究报道了从海栖蚓鳗科鳗鱼中鉴定出的两个新型胆红素诱导型荧光蛋白（fluorescent proteins, FPs）家族成员，并揭示了序列中一段关键区域，该区域可作为非荧光向荧光型脂肪酸结合蛋白（fatty acid-binding proteins, FABPs）演化的分子开关。通过对两种高荧光性裸蚓鳗属鳗鱼（Kaupichthys hyoproroides 及未定种Kaupichthys n. sp.）的转录组学分析，本研究成功鉴定、克隆并表征了两种新型荧光蛋白，分别命名为蚓鳗荧光蛋白I（Chlopsid FP I）与蚓鳗荧光蛋白II（Chlopsid FP II）。随后，本研究对覆盖16个脊椎动物目、共163个脊椎动物类群的210个FABPs序列开展了系统发育分析。结果表明，荧光型FABPs作为一个蛋白家族发生了分化，且与脑型脂肪酸结合蛋白构成姊妹群。本研究结果显示，该蛋白家族的演化过程至少经历了三次基因复制事件。研究发现，荧光型FABPs拥有一段独特且保守的三肽基序Gly-Pro-Pro（甘氨酸-脯氨酸-脯氨酸），该基序在非荧光型脂肪酸结合蛋白中从未被观测到。该基序起源于脑型FABP的一次复制事件，并经历了强烈的纯化选择，最终促成了这一新型荧光蛋白家族的划分。该基序邻近的氨基酸残基则受到强烈的正向选择作用，这表明该鳗鱼蛋白的荧光特性得到了进一步优化。本研究构建了这一新兴荧光蛋白家族的系统发育重建结果，并报道了来自远缘鳗鱼类群的其他荧光型FABP成员。这类特性各异的鱼类荧光蛋白的解析，为基于蛋白的荧光工具开发提供了全新的模板。从脂肪酸结合蛋白向荧光型脂肪酸结合蛋白的演化适应性转变，也引发了学界的诸多疑问：这类栖息于近乎单色的蓝色海洋环境中的隐秘隐居裸蚓鳗属物种，其明亮的绿色荧光究竟承担着何种生物学功能？