Evolution of the thyroid hormone-binding protein, transthyretin.

Transthyretin (TTR) belongs to a group of proteins, which includes thyroxine-binding globulin and albumin, that bind to and transport thyroid hormones in the blood. TTR is also indirectly implicated in the carriage of vitamin A through the mediation of retinol-binding protein (RBP). It was first identified in 1942 in human serum and cerebrospinal fluid and was formerly called prealbumin for its ability to migrate faster than serum albumin on electrophoresis of whole plasma. It is a single polypeptide chain of 127 amino acids (14,000 Da) and is present in the plasma as a tetramer of noncovalently bound monomers. The major sites of synthesis of TTR in eutherian mammals, marsupials, and birds are the liver and choroid plexus but in reptiles it is synthesised only in the choroid plexus. The observation that TTR is strongly expressed in the choroid plexus but not in the liver of the stumpy-tailed lizard and the strong conservation of expression in the choroid plexus from reptiles to mammals have been taken as evidence to suggest that extrahepatic synthesis of TTR evolved first. The identification and cloning of TTR from the liver of an amphibian, Rana catesbeiana, and a teleost fish, Sparus aurata, and its absence from the choroid plexus of both species suggest an alternative model for its evolution. Protein modelling studies are presented that demonstrate differences in the electrostatic characteristics of the molecule in human, rat, chicken, and fish, which may explain why, in contrast to TTR from human and rat, TTR from fish and birds preferentially binds triiodo-l-thyronine.

Binding of thyroxine to pig transthyretin, its cDNA structure, and other properties.

Thyroxine binding to proteins in pig plasma during electrophoresis was observed in the albumin, but not in the prealbumin and post-albumin regions. Transthyretin could be identified in medium from in vitro pig choroid plexus incubations by size and number of subunits and a very high rate of synthesis and secretion. Its electrophoretic mobility was intermediate between that of thyroxine-binding globulin and albumin. It bound thyroxine, retinol-binding protein, anti-(rat transthyretin) antibodies and behaved similarly to transthyretins from other vertebrate species when plasma was extracted with phenol. Inhibition experiments with the synthetic flavonoid F 21388, analysing the binding of thyroxine, suggested that transthyretin is not a major thyroxine carrier in the bloodstream of pigs. Cloning and sequencing of transthyretin cDNA from both choroid plexus and liver showed that the same transthyretin mRNA is expressed in pig choroid plexus and liver. The amino acid sequence derived from the nucleotide sequence revealed that pig transthyretin differs from the transthyretins of all other studied vertebrate species by an unusual C-terminal extension consisting of the amino acids glycine, alanine and leucine. This extension results from the mutation of a stop codon into a codon for glycine. The unusual C-terminal extensions do not seem to interfere with the access of thyroxine to its binding site in the central channel of transthyretin.

Transthyretin gene expression in choroid plexus first evolved in reptiles.

The presence of transthyretin in mammals and birds, but not amphibia, suggested that transthyretin expression first appeared in stem reptiles. Therefore, transthyretin synthesis was studied in a lizard. Transthyretin synthesis in choroid plexus pieces from Tiliqua rugosa was demonstrated by incorporation of radiactive amino acids. Oligonucleotides corresponding to conserved regions of transthyretin were used as primers in polymerase chain reaction with lizard choroid plexus cDNA. Amplified DNA was used to screen a lizard choroid plexus cDNA library. A full-length transthyretin cDNA clone was isolated and sequenced. A three-dimensional model of lizard transthyretin was obtained by homology modeling. The central channel of transthyretin, containing the thyroxine-binding site, was found to be completely conserved between reptiles and mammals. Transthyretin expression was not detected in lizard liver. These data suggest that transthyretin first evolved in the choroid plexus of the brain. Due to a change in tissue distribution of gene expression, occurring much later during evolution, transthyretin also became a plasma protein, synthesized in the liver.