Evolution of Shorter and More Hydrophilic Transthyretin N‐Termini by Stepwise Conversion of Exon 2 into Intron 1 Sequences (Shifting the 3′ Splice Site of Intron 1)

Abstract

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.

Transthyretin gene (TTR) intron 1 elucidates crocodylian phylogenetic relationships

“…Thyroid hormone binding studies have shown that TTR preferentially binds with T 3 (triiodothyronine) in birds and T 4 (thyroxine) in mammals (Power et al, 2000). This is thought to be due to the changes in the 3′ splice site of intron 1, which has led to the shorter N-terminal amino acid sequence (Aldred et al, 1997). Nevertheless, overall sequence identity among diverse species is relatively high due to the conservative structures of hormone binding sites across almost all taxa.…”

Proteolytic activity of Crocodylus porosus transthyretin protease and role of the terminal polypeptide sequences

“…It is a tetramer consisting of four identical subunits in which the amino acid sequence has been highly conserved. The predominant changes in the TTRs from birds, reptiles, amphibians, and fishes are in the N-terminal region which is longer and more hydrophobic in the sequences compared to that in TTRs from mammals 1,2 . In addition, variations in the length of the C-terminal region of TTRs from pig 3 and microbats 4,5 compared with that from human have also been detected.…”

“…In addition, the concentrations of the total and free T4, and the tissue specific and the timing of the expression of the TTR gene in mammals differ from that of other vertebrates [19][20][21] . The stepwise conversion of exon 2 into intron 1 sequences in the TTR gene constitutes the underlying mechanism of the structural change and thus favours TH binding 1,2 . We hypothesized that the activity of TTR also exists and has important roles in other classes of vertebrates, and the selection pressures that led to the structural changes of TTR during evolution would have influenced on this function.…”

Characterization of little skate (Leucoraja erinacea) recombinant transthyretin: Zinc-dependent 3,3′,5-triiodo-l-thyronine binding

“…It has been proposed that the N-terminal region has evolved from a longer and more hydrophobic region in lower vertebrates to a shorter and more hydrophilic region in eutherians, by unidirectional changes (Aldred et al, 1997;Prapunpoj et al, 2000), with functional changes in TH-binding specificity from for 3,3 0 ,5-triiodo-L-thyronine (T3) to for L-thyroxine (T4) (Yamauchi et al, 1993;Chang et al, 1999). Through the survey of several expressed sequence tag and genome databases of elasmobranch fishes, we found TTR cDNA in the little skate Leucoraja erinacea database (http://skatebase.org), and noticed that such unidirectional changes may not be true for the skate TTR.…”