Identification and bioactivity analysis of transthyretin-like protein in amphioxus: A case demonstrating divergent evolution from an enzyme to a hormone distributor

Abstract

Transthyretin-like proteins are a family of proteins that share remarkable structural similarities to transthyretin, that have been identified in a variety of taxa such as bacteria, fungi, plants, invertebrates and vertebrates. Despite the enormous progress in the study of transthyretin-like protein, little is known about it in amphioxus, a model organism for insights into the origin and evolution of vertebrates. Here we identified a transthyretin-like protein gene in Branchiostoma japonicum, named Bjtlp, which possessed a TLP-HIUase (an enzyme hydrolyzing 5-hydroxyisourate) domain and a consensus C-terminal tetrapeptide Tyr-Arg-Gly-Ser that are both characteristics of all known transthyretin-like proteins. Phylogenetic and intron-exon structure analyses support that TTR likely arose from a vertebrate specific duplication after vertebrates diverged from invertebrate chordates. Quantitative real-time PCR analysis revealed that Bjtlp was expressed in a tissue-specific fashion, with the transcript levels being most abundant in the hepatic caecum and hind gut. Enzymatic activity assays demonstrated that recombinant BjTLP had the capacity to hydrolyze 5-hydroxyisourate. Site-directed mutagenesis showed that both Y156 and R93 residues were critical for 5-hydroxyisourate hydrolase activity of recombinant BjTLP. Moreover, the single mutation, Y156T, at the active site of BjTLP caused approximately 97% loss of its enzymatic activity, and meanwhile gained the thyroxine binding activity. All these data together suggest that the single mutation Y156T is critical for converting BjTLP to a new transport protein capable of distributing thyroxine.