Identification of key amino acid residues of prolactin closely associated with freshwater adaptation in fish.

Fish living in different saline environments constitute optimal models for studying the osmoregulatory role of prolactin. Analysis of grouper fish prolactins, including those from giant grouper Epinephelus lanceolatus and red-spotted grouper Epinephelus akaara and their hybrid, which are genetically close but exhibit different salinity adaptability, indicated only two amino acid residue differences in prolactin (lysine, K₄₀, and glutamate, E₉₁). An integrated approach, including a comparative analysis of prolactin sequences in fish and habitat salinity, behavior-based monitoring of low-salinity adaptability, and molecular modeling, was employed to explore the functional implications of prolactin sequences. Bioinformatics analysis of the fish prolactin sequence database and habitat information revealed a distinct preference for the K/E pair in prolactins of fish inhabiting or migrating to freshwater environments. This preference contrasts with the wide spectrum of amino acid changes and the rather conserved shift to D at positions corresponding to K and E, respectively, in marine fish prolactin. The results supporting a hypothesis concerning the importance of the K residue in freshwater adaptability were further reinforced by analyses of additional prolactin molecules from marine and freshwater origins and their low-salinity adaptabilities, which revealed prolactin sequences containing K/D > M/D > T/D. Structural modeling of grouper prolactins revealed that the K residue was close to the receptor interface, indicating a possible interaction with the prolactin receptor. These results provide new insights into the role of key amino acid residues in prolactin that are important for osmoregulatory function in fish and their potential applications as indicators of freshwater adaptability.