title: 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.
authors.affiliation: Laboratory for Evolution & Development, Institute of Evolution & Marine Biodiversity and Department of Marine Biology, Ocean University of China, Qingdao 266003, China.
authors.forename: Zhaohe
authors.initials: Z
authors.lastname: Li
authors.forename: Feng
authors.initials: F
authors.lastname: Yao
authors.forename: Mengyang
authors.initials: M
authors.lastname: Li
authors.forename: Shicui
authors.initials: S
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002816:Chordata, Nonvertebrate / Q000187:drug effects / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D019143:Evolution, Molecular; D005470:Fluorometry; D020869:Gene Expression Profiling; D016678:Genome / Q000235:genetics; D006728:Hormones / Q000378:metabolism*; D006867:Hydrolases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D011228:Prealbumin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012015:Reference Standards; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014527:Uric Acid / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: C510200:5-hydroxyisourate; D006728:Hormones; D011228:Prealbumin; D011994:Recombinant Proteins; D014527:Uric Acid; D006867:Hydrolases