Xenopus laevis transthyretin (xTTR) cDNA was cloned and sequenced. The derived amino acid sequence was very similar to those of other vertebrate transthyretins (TTR). TTR gene expression was observed during metamorphosis in X. laevis tadpole liver but not in tadpole brain nor adult liver. Recombinant xTTR was synthesized in Pichia pastoris and identified by amino acid sequence, subunit molecular mass, tetramer formation, and binding to retinol-binding protein. Contrary to mammalian xTTRs, the affinity of xTTR was higher for L-triiodothyronine than for L-thyroxine. The regions of the TTR genes coding for the NH(2)-terminal sections of the polypeptide chains of TTR seem to have evolved by stepwise shifts of mRNA splicing sites between exons 1 and 2, resulting in shorter and more hydrophilic NH(2) termini. This may be one molecular mechanism of positive Darwinian evolution. Open reading frames with xTTR-like sequences in the genomes of C. elegans and several microorganisms suggested evolution of the TTR gene from ancestor TTR gene-like "DNA modules." Increasing preference for binding of L-thyroxine over L-triiodothyronine may be associated with evolving tissue-specific regulation of thyroid hormone action by deiodination.

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.

Structure and function were studied for Crocodylus porosus transthyretin (crocTTR), an important intermediate in TTR evolution. The cDNA for crocTTR mRNA was cloned and sequenced and the amino acid sequence of crocTTR was deduced. In contrast to mammalian TTRs, but similar to avian and lizard TTRs, the subunit of crocTTR had a long and hydrophobic NH(2)-terminal region. Different from the situation in mammals, triiodothyronine (T(3)) was bound by crocTTR with higher affinity than thyroxine (T(4)). Recombinant crocTTR and a chimeric construct, with the NH(2)-terminal region of crocTTR being replaced by that of Xenopus laevis TTR, were synthesized in the yeast Pichia pastoris. Analysis of the affinity of the chimeric TTRs showed that the NH(2)-terminal region modulates T(4) and T(3) binding characteristics of TTR. The structural differences of the NH(2)-terminal regions of reptilian and amphibian TTRs were caused by a shift in splice sites at the 5' end of exon 2. The comparison of crocodile and other vertebrate TTRs shows that TTR evolution is an example for positive Darwinian evolution and identifies its molecular mechanism.

Transthyretin cDNA was cloned from Eastern Grey Kangaroo liver and its nucleotide sequence determined. Analysis of the derived amino acid sequence of kangaroo transthyretin, together with data obtained previously for transthyretins from other vertebrate species [Duan, W., Richardson, S. J., Babon, J. J., Heyes, R. J., Southwell, B. R., Harms, P. J., Wettenhall, R. E. H., Dziegielewska, K. M., Selwood, L., Bradley, A. J., Brack, C. M. & Schreiber, G. (1995) Eur. J. Biochem. 227, 396–406], showed that the N‐terminus is the region which changes most distinctly during evolution. It has been shown for human, mouse and rat transthyretins, that this region is encoded by DNA at the border of exon 1 and exon 2. Therefore, this section of transthyretin genomic DNA was amplified by PCR and directly sequenced for the Buffalo Rat, Tammar Wallaby, Eastern Grey Kangaroo, Stripe‐faced Dunnart, Short‐tailed Grey Opossum and White Leghorn Chicken. The splice sites at both ends of intron 1 were identified by comparison with the cDNA sequences. The obtained data suggest that the N‐termini of transthyretin evolved by successive shifts of the 3’ splice site of intron 1 in the 3’ direction, resulting in successive shortening of the 5’ end of exon 2. At the protein level, this resulted in a shorter and more hydrophilic N‐terminal region of transthyretin. Successive shifts in splice sites may be an evolutionary mechanism of general importance, since they can lead to stepwise changes in the properties of proteins. This could be a molecular mechanism for positive Darwinian selection.

The relationship between the structure of the N‐terminal sequence of transthyretin (TTR) and the binding of thyroid hormone was studied. A recombinant human TTR and two derivatives of Crocodylus porosus TTRs, one with the N‐terminal sequence replaced by that of human TTR (human/crocTTR), the other with the N‐terminal segment removed (truncated crocTTR), were synthesized in Pichia pastoris. Subunit mass, native molecular weight, tetramer formation, cross‐reactivity to TTR antibodies and binding to retinol‐binding protein of these recombinant TTRs were similar to TTRs found in nature. Analysis of the binding affinity to thyroid hormones of recombinant human TTR showed a dissociation constant (Kd) for triiodothyronine (T3) of 53.26 ± 3.97 nm and for thyroxine (T4) of 19.73 ± 0.13 nm. These values are similar to those found for TTR purified from human serum, and gave a Kd T3/T4 ratio of 2.70. The affinity for T4 of human/crocTTR (Kd = 22.75 ± 1.89 nm) was higher than those of both human TTR and C. porosus TTR, but the affinity for T3 (Kd = 5.40 ± 0.25 nm) was similar to C. porosus TTR, giving a Kd T3/T4 ratio of 0.24. A similar affinity for both T3 (Kd = 57.78 ± 5.65 nm) and T4 (Kd = 59.72 ± 3.38 nm), with a Kd T3/T4 ratio of 0.97, was observed for truncated crocTTR. The obtained results strongly confirm the hypothesis that the unstructured N‐terminal region of TTR critically influences the specificity and affinity of thyroid hormone binding to TTR.