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.

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Teleosts have highly diverged genomes that resulted from whole genome duplication, which leads to an extensive diversity of paralogous genes. Transthyretin (TTR), an extracellular thyroid hormone (TH) binding protein, is thought to have evolved from an ancestral 5-hydroxyisourate hydrolase (HIUHase) by gene duplication at some stage of chordate evolution. To characterize the functions of proteins that arose from duplicated genes in teleosts, we investigated the phylogenetic relationship of teleost HIUHase and TTR aa sequences, the expression levels of Oncorhynchus mykiss HIUHase and TTR mRNA in various tissues and the biological activities of the O. mykiss re-HIUHase and re-TTR. Phylogenetic analysis of the teleost aa sequences revealed the presence of two HIUHase subfamilies, HIUHase 1 (which has an N-terminal peroxisomal targeting signal-2 [PTS2]) and HIUHase 2 (which does not have an N-terminal PTS2), and one TTR family. The tissue distributions of HIUHase 1 and TTR mRNA were similar in juvenile O. mykiss and the mRNA levels were highest in the liver. The O. mykiss re-HIUHase and re-TTR proteins were both 40-50 kDa homotetramers consisting of 14-15 kDa subunits, with 30% identity. HIUHase had 5-hydroxyisourate (5-HIU) hydrolysis activity with Zn(2+) sensitivity, whereas TTR had ligand binding activity with a preference for THs and several environmental chemicals, such as halogenated phenols. Our results suggest that O. mykiss HIUHase and TTR have diverged from a common ancestral HIHUase with no functional complementation.

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