Transthyretin is one of the three major thyroid hormone‐binding proteins in plasma and/or cerebrospinal fluid of vertebrates. It transports retinol via binding to retinol‐binding protein, and exists mainly as a homotetrameric protein of ∼ 55 kDa in plasma. The first 3D structure of transthyretin was an X‐ray crystal structure from human transthyretin. Elucidation of the structure–function relationship of transthyretin has been of significant interest since its highly conserved structure was shown to be associated with several aspects of metabolism and with human diseases such as amyloidosis. Transthyretin null mice do not have an overt phenotype, probably because transthyretin is part of a network with other thyroid hormone distributor proteins. Systematic study of the evolutionary changes of transthyretin structure is an effective way to elucidate its function. This review summarizes current knowledge about the evolution of structural and functional characteristics of vertebrate transthyretins. The molecular mechanism of evolutionary change and the resultant effects on the function of transthyretin are discussed.

During vertebrate evolution, the N-terminal region of transthyretin (TTR) subunit has undergone a change in both length and hydropathy. This was previously shown to change the binding affinity for thyroid hormones (THs). However, it was not known whether this change affects other functions of TTR. In the present study, the effect of these changes on the binding of TTR to retinol-binding protein (RBP) was determined. Two wild-type TTRs from human and Crocodylus porosus, and three chimeric TTRs, including a human chimeric TTR in which its N-terminal sequence was changed to that of C. porosus TTR (croc ⁄ huTTR) and two C. porosus chimeric TTRs (hu ⁄ crocTTR in which its N-terminal sequence was changed to that of human TTR and xeno ⁄ crocTTR in which its N-terminal sequence was changed to that of Xenopus laevis TTR), were analyzed for their binding to human RBP by native-PAGE followed by immunoblotting and a chemilluminescence assay. The K d of human TTR was 30.41 ± 2.03 lM, and was similar to that reported for the second binding site, whereas that of crocodile TTR was 2.19 ± 0.24 lM. The binding affinities increased in croc ⁄ huTTR (K d = 23.57 ± 3.54 lM) and xeno ⁄ crocTTR (K d = 0.61 ± 0.16 lM) in which their N-termini were longer and more hydrophobic, but decreased in hu ⁄ crocTTR (K d = 5.03 ± 0.68 lM) in which its N-terminal region was shorter and less hydrophobic. These results suggest an influence of the N-terminal primary structure of TTR on its function as a co-carrier for retinol with RBP.

The thyroid hormone (TH)-disrupting activity of effluents and environmental water samples in Thailand was surveyed by three in vitro bioassays with different endpoints. These assays test the potency of competitive binding with the active form of TH, 3,3',5-[(125)I]triiodo-l-thyronine (T(3)), to the plasma transport protein transthyretin (TTR) and TH receptor (TR; the TTR assay and TR assay, respectively) and the interference with the cellular T(3)-signaling pathway through TR-mediated luciferase gene activation (the luc assay). The TH-disrupting activity in water samples collected from paper manufacturing plants (PMPs), the canal Khlong U-Taphao, and a sewage-treatment plant (STP) was detected predominantly in the dichloromethane/methanol or methanol fractions of solid-phase extraction, suggesting a similar hydrophobic nature of the causative contaminants. The TR assay was relatively more sensitive than the TTR assay to the competitively potent contaminants. The luc assay indicated that the dichloromethane/methanol fractions of most water samples contained anti-T(3)-like activity. Our assays demonstrated that wastewater treatment effectively removed the TH-disrupting contaminants from wastewater in the PMP and the STP. The potencies for TH disruption at the three sampling points of the STP exhibited positive correlations among the three bioassays, whereas those from the canal and PMP water were not correlated among the three bioassays. Furthermore, the influent contaminants that were competitively potent in the TTR assay partially affected the luc assay. These bioassays are useful monitoring tools that give results relevant for evaluating the health of amphibian populations.

Transthyretin is responsible for a series of highly progressive, degenerative, debilitating, and incurable protein misfolding disorders known as transthyretin (TTR) amyloidosis. Since dissociation of the homotetrameric protein to its monomers is crucial in its amyloidogenesis, stabilizing the native tetramer from dissociating using small-molecule ligands has proven a viable therapeutic strategy. The objective of this study was to determine the potential role of the medicinal herb Centella asiatica on human transthyretin (huTTR) amyloidogenesis. Thus, we investigated the stability of huTTR with or without a hydrophilic fraction of C. asiatica (CAB) against acid/urea-mediated denaturation. We also determined the influence of CAB on huTTR fibrillation using transmission electron microscopy. The potential binding interactions between CAB and huTTR was ascertained by nitroblue tetrazolium redox-cycling and 8-anilino-1-naphthalene sulfonic acid displacement assays. Additionally, the chemical profile of CAB was determined by liquid chromatography quadruple time-of-flight mass spectrometry (HPLC-QTOF-MS). Our results strongly suggest that CAB bound to and preserved the quaternary structure of huTTR in vitro. CAB also prevented transthyretin fibrillation, although aggregate formation was unmitigated. These effects could be attributable to the presence of phenolics and terpenoids in CAB. Our findings suggest that C. asiatica contains pharmaceutically relevant bioactive compounds which could be exploited for therapeutic development against TTR amyloidosis.

Human transthyretin (TTR), a recently identified protease, participates in the biology of high density lipoprotein and in the nervous system. In the present study, we determined whether TTR from a non-mammal Crocodylus porosus (crocTTR) has proteolytic activity and whether the N-and C-termini of the TTR polypeptide affect the proteolytic activity. The proteolytic activity of crocTTR and three chimeric crocTTRs: xenoN/crocTTR (crocTTR in which the N-terminal sequence was replaced with that of Xenopus laevis TTR), pigC/crocTTR (crocTTR in which the C-terminal sequence was replaced with that of Sus scrofa TTR), and xenoN/pigC/crocTTR (crocTTR in which the N-and C-terminal sequences were replaced with that of X. laevis TTR and S. scrofa TTR, respectively) were studied and compared. Using either casein or apoAI as a substrate, crocTTR had a lower proteolytic activity than human TTR. Replacing the C-terminal sequence of crocTTR increased the activity (casein: 1008 ± 36 pmol/min; apoAI: 231 ± 43 pmol/min), whereas replacing the N-terminal sequence decreased the activity (casein: 299 ± 26 pmol/min; apoAI: 31.5 ± 2.0 pmol/min). The activity of xenoN/pigC/crocTTR (casein: 502 ± 11 pmol/min; apoAI: 371 ± 23 pmol/min) was higher than those of crocTTR or xenoN/crocTTR, but similar to that of pigC/crocTTR. These results are the first to show the proteolytic activity of reptile TTR, and that the activity is changed when the N-and/or C-terminal amino acid sequences of the TTR subunit are changed.