Identification, Expression And Functional Characterization Of TLP Gene In Amphioxus Branchiostoma Japonicum

Transthyretin is an important thyroid hormone (TH) transport protein in the blood of humans, and can binding to TH and transport it to target tissues. TTR has been only identified in vertebrates. Searches for a TTR progenitor led to the identification of a TTR homolog, TTR-like protein (TLP). TLP have been shown to share remarkable structural similarities to TTR and are able to form a tetramer, it has been identified in a variety of taxa such as bacteria, fungi, plants, invertebrates and vertebrates. TLP has been demonstrated to be an enzyme functioning in the purine catabolism pathway, where it hydrolyzes5-hydroxyisourate (5-HIU). 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. The aims of this study were thus to identify the TLP gene of the amphioxus Branchiostoma japonicum, designated as Bjtlp, to examine its expression and bioactivity and to investigate its phylogenetic relationship to vertebrate TTR.A cDNA of TLP was obtained from B. japonicum, named Bjtlp, which was757bp long and with an ORF of480bp. The ORF encoded a deduced protein of159amino acids with a calculated molecular mass of about17.9kDa. The protein possessed a TLP-HIUase (an enzyme hydrolyzing5-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. qRT-PCR assay was used to examine the expression pattern of Bjtlp in the different tissues of B. japonicum. Bjtlp transcripts were most abundant in the hepatic caecum and hind gut and at a lower level present in the gill, testis and ovary. In contrast, only slight Bjtlp transcripts were detected in the notochord and muscle. These showed that Bjtlp was expressed in a tissue-specific fashion in B. japonicum.For investigating the function of rBjTLP, the recombinant plasmid has been constructed, transferred to E.coli and induced to express. After refold and purification of the rBjTLP, we measured the effect of rBjTLP on the decomposition of5-HIU. The result demonstrated that rBjTLP had the capacity to hydrolyze5-HIU. To test if rBjTLP could bind to T4, fluorescence spectroscopy was performed. It suggesting that rBjTLP can not interacted with T4.We investigated the active site of rBjTLP and the evolution mechanism of TTR and TLP with the method of Site-directed mutagenesis. We converted the amino acid in BjTLP to the residue at the corresponding position in human TTR: R93E, Y156T, and Y156T-R93E (residue numbering is for BjTLP). We have examed the enzymatic and TH-binding activity of the mutants. The5-HIU hydrolytic activity in the mutant BjTLP/Y156T was reduced by97%, and the5-HIU hydrolysis activity in the mutants BjTLP/R93E and BjTLP/Y156T-R93E was nearly completely lost. These indicated that both Y156and R93residues were indispensible for5-HIU hydrolase activity of rBjTLP.The Binding assay indicating that both rBjTLP/Y156T and rBjTLP/Y156T-R93E bound to T4and slightly stronger for rBjTLP/Y156T-R93E. This suggests that Y156T is critical for converting BjTLP HIUase to a transport protein capable of distributing T4. As more effect was observed in Y156T-R93E compared to Y156T, it seems that after the initial event a number of additional mutations were needed to optimize and stabilize ligand binding to the mutated protein.