MINIREVIEW Evolutionary changes to transthyretin: structure–function relationships P Prapunpoj and L Leelawatwattana Department of Biochemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Thailand Keywords binding affinity; evolution; function; plasma protein; protease; retinol-binding protein; splicing; structure; thyroid hormone; transthyretin Correspondence Porntip Prapunpoj, Department of Biochemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Fax: +66 74 446656 Tel: +66 74 288275 E-mail: porntip.p@psu.ac.th (Received February 2009, revised July 2009, accepted 27 July 2009) 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 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 doi:10.1111/j.1742-4658.2009.07243.x Introduction Transthyretin is a major protein in extracellular fluids and it binds thyroid hormones (THs) in both l-3,5,3¢triiodothyronine (T3) and l-thyroxine (T4) forms It was first identified in human cerebrospinal fluid (CSF) and later in human serum [1,2] It is the only TH-binding protein that is synthesized in the cells of the blood–CSF barrier, but its major site of synthesis is the liver Transthyretin is widely distributed among vertebrates and is the only protein in plasma that migrates faster than albumin during electrophoresis at pH 8.6, except for transthyretins from cattle, swine, dog, cat, rabbit, frog and salmon [3–5] Transthyretin exists in vivo mainly as a tetramer of four identical subunits and only a small amount of the monomer [6–8] Each subunit consists of 125 to 136 amino acid residues (depending on the species of animal from which the protein is obtained; Fig 1), which are largely arranged into b-sheet structure (41% b-strand and 5% a-helix) This high b-sheet content is believed to contribute to the extraordinary stability of the molecule [9] Transthyretin in nature is not glycosylated, despite containing potential glycosylation sites Heterogeneity of transthyretin from several species has been described, resulting from phosphorylation, cysteine–glycine conjugation, glutathionylation and the interaction with ligands, such as retinol-binding protein (RBP), in serum and CSF [3,10–14] The primary structure of transthyretins is highly conserved during evolution The predominant changes in amino acid residues are not in the core structure or Abbreviations Ab, amyloid beta; CSF, cerebrospinal fluid; RBP, retinol-binding protein; T3, L-3,5,3¢-triiodothyronine; T4, L-3,5,3¢,5¢-tetraiodothyronine or L-thyroxine; TH, thyroid hormone 5330 FEBS Journal 276 (2009) 5330–5341 ª 2009 The Authors Journal compilation ª 2009 FEBS P Prapunpoj and L Leelawatwattana Structure–function relationships of transthyretin Fig Comparison of the amino acid sequences of transthyretins from 25 vertebrates The complete amino acid sequences and derived amino acid sequences from 25 vertebrate species are aligned The amino acid residues in other species that are identical to those in human transthyretin are indicated by asterisks The numbering of residues is based on human transthyretin: negative numbers, residues in the presegment; positive numbers, residues in the mature protein; a, b, c, d, e, f, g, h and i, positions of residues in noneutherians The first residue in the mature polypeptide is in bold Features of secondary structure of human transthyretin are indicated above the sequences Residues in the core and the central channel of the human transthyretin subunit, according to previous publications [6,21], are single and double underlined, respectively Arrows show the positions of exon borders Sources of transthyretin sequences: human [83,84]; hedgehog and shrew [38]; chimpanzee (accession number Q5U7I5); long-tailed macaque (accession number Q8HXW1); pig [5]; sheep [85]; bovine [86]; rabbit [87]; rat [87–90]; mouse [91,92]; Tammar wallaby [93]; grey kangaroo [15]; sugar glider [43]; stripe-faced dunnart and grey opossum [94]; chicken [95]; crocodile [16]; lizard [34]; bullfrog [96]; Xenopus [39]; sea bream [45]; carp (accession number CAD66520); and sea lamprey and American brook lamprey [40] (Modified from Prapunpoj et al., 2002 [16].) in the binding sites, but in the N-terminal region [15] This structural change influences the ability of transthyretins to bind to THs [16,17] Transthyretin has been recognized as one of the most interesting proteins identified to date, because of its multifunctionality Besides distributes THs in blood, it indirectly transports vitamin A via bound to RBP In addition, proteolytic activity of transthyretin has recently been discovered [18], rising to its more importance in the brain This review summarized the structure of transthyretin and FEBS Journal 276 (2009) 5330–5341 ª 2009 The Authors Journal compilation ª 2009 FEBS 5331 Structure–function relationships of transthyretin P Prapunpoj and L Leelawatwattana the evolutionary changes of the structure particular to the N-terminal region, the shortening mechanism of the N-terminus and the influences of this change on binding to TH and on the functions of transthyretin, including that of proteolysis Structure of transthyretin 3D structure of human transthyretin The first transthyretin to have its 3D structure revealed was from human plasma [19] Approximately 60 of 127 amino acid residues in the transthyretin monomer are arranged into eight b-strands, named A through H, that are connected by loops to form a sandwich of A 3D structures of other transthyretins B Fig The 3D structures of human transthyretin Ribbon diagrams of (a) transthyretin tetramer and (b) transthyretin dimer The four identical monomers (A, B, C and D) form a tetramer (shown in color ramping from blue to red) with a central channel (along the z axis) where two binding sites for THs exist Two monomers, A and B, join side-by-side to form the dimer AB The eight strands in each monomer are labelled a-h (From Ghosh et al., 2000 [20], copyright of IUCr, http://journals.iucr.org/; reproduced with permission by Professor Louise N Johnson, University of Oxford, UK.) 5332 two b-sheets (Fig 2; [20]) [6] Only 5% of the residues in the monomer, which corresponds to nine amino acid residues, are in a short a-helix [21] Dimers of transthyretin are composed of a pair of twisted eightstranded b-sheets, one inner (strands DAGHH¢G¢A¢D¢) and one outer (strands CBEFF¢E¢B¢C) (Fig 2) The interactions predominantly involved are hydrogen bonding between two F strands (F, F¢) and two H strands (H, H¢); two complex hydrophobic interactions; and two water bridges [6] The association of two dimers results in a tetrameric structure with two pairs of eight-stranded b-sheets The dimer–dimer contacts predominantly involve hydrophobic interactions of residues in two loops (i.e A–B and G–H loops) at the edge of the sheets A large central channel that is ˚ ˚ about A in diameter and 50 A long [22], with two TH-binding sites that differ in their relative binding affinity, is formed as a consequence of the tetrahedral arrangement of the subunits [6,23,24] One of these two TH-binding sites is slightly larger than the other and only one binding site is occupied by TH under physiological conditions [25–27] because of the negative co-operativity [24,27] The movement of Ser117, water displacement in the binding channel and asymmetry of the two binding sites were demonstrated to be responsible for the negative co-operativity The 3D structure of human transthyretin has previously been discussed in great detail by Hamilton and Benson in 2001 [28] To date, transthyretin from only four species other than human have been crystallized and their 3D structures have been reported These included transthyretins from rat [29], mouse [30], chicken [31] and sea bream [32] Analysis of rat and mouse transthyretins showed secondary, tertiary and quaternary structures similar to those of human transthyretin Only a few differences were identified in the flexible loop regions on the surface of rat transthyretin (i.e near residues 30–41, 60–65 and 102–104), leading to more compact monomers of rat transthyretin than those of human transthyretin [29] However, this had no effect on the interaction with THs By contrast, the 3D structure of chicken transthyretin showed several differences in comparison to that of human transthyretin [31] The region showing the greatest number of differences (residues 83–84) is involved in the interaction with RBP The interaction between Tyr116 of one monomer and Glu92 of the nearby monomer, which maintains the monomer–monomer interface of human transthyretin, is absent in chicken transthyretin In addition, chicken FEBS Journal 276 (2009) 5330–5341 ª 2009 The Authors Journal compilation ª 2009 FEBS P Prapunpoj and L Leelawatwattana transthyretin has less of an a-helical structure The overall structure of sea bream transthyretin, in comparison with chicken transthyretin, is much more similar to that of human transthyretin [32,33] However, the entrance to the TH-binding site of sea bream transthyretin is significantly wider, while the channel is narrower, which may result in higher binding affinity to T3 than to T4 [32] Models for the 3D structures of lizard [34] and bullfrog (Rana catesbeiana) [35] transthyretins were produced based on the known crystal structure co-ordinates of human and chicken transthyretins, respectively The secondary and tertiary structures of lizard transthyretin were very similar to those of ˚ human transthyretin, with an rmsd of 0.10A [34] The TH-binding sites and the overall subunit structure of bullfrog transthyretin were similar to those of chicken transthyretin Evolution of the structure of the transthyretin subunit The N-terminal region The primary structures of transthyretins (either partial or full length) from more than 30 animal species have been analyzed These include transthyretins from eutherians (‘placental mammals’), marsupials, birds, reptiles, amphibians and fish (Fig 1) The subunit of transthyretin comprises two parts, namely the presegment that is required for extracellular secretion and the mature polypeptide segment that forms the functioning transthyretin The mature segment of transthyretin has been found to vary in size among species, ranging from 125 amino acid residues in hedgehog to 136 amino acid residues in lamprey The amino acid sequence alignment of the vertebrate transthyretin subunits (Fig 1) shows that the residues in all 17 positions in the central channel, including those involved in the binding interaction with THs [22,24,36], are conserved and have not been altered for more than 400 million years By contrast, the predominant changes during evolution occurred in the N-terminal region of the transthyretin subunit These N-terminal segments of transthyretins in birds, reptiles, amphibians and fish are longer and relatively more hydrophobic than those in mammalian transthyretins The N-terminal segments are not defined by X-ray crystallography, so are thought to move freely in solution [6] A structure determined by Hamilton et al [23] revealed that the N-termini had a 0.25 occupancy of curved rods at the entrance to the central channel This suggested that the structure of the N-termini determined the affinity of T3 and T4 binding to transthyretins [15] A detailed study by Chang et al., Structure–function relationships of transthyretin 1999 [37] supported a strong correlation between the character of the N-terminus and the preference of ligand binding: transthyretins with shorter and more hydrophilic N-termini had higher affinity for T4 [37] The interference of the N-termini with the accessibility of TH to the binding site is discussed in the ‘Functions of transthyretin’ section below Mechanism of N-terminus shortening Comparison of transthyretin cDNA and genomic DNA sequences revealed that the region coding for the N-terminus of the transthyretin subunit was at the 3¢ end of exon Compared with human transthyretin, two (for marsupials) or three to nine (for birds, reptiles, amphibians and fish) additional amino acids are present in the N-termini of transthyretins (see Fig 1) The systematic analysis and comparison of the nucleotide sequences flanking the exon ⁄ intron and intron ⁄ exon borders of transthyretin mRNAs from eight species (eutherians, marsupials and a bird) revealed shifting in successive steps of the intron ⁄ exon splice site in the 3¢ direction during evolution [15] The nucleotide sequences at the exon ⁄ intron border were unchanged (Fig 3A) However, changes occurred at the intron ⁄ exon border (Fig 3B) Shifting of the intron ⁄ exon splice site in the 3¢ direction was postulated to occur in successive steps, which led to a successive shortening of the transthyretin N-terminal region (Fig 4) [15] The same successive changes resulting in shortening and an increase in hydrophilicity of the N-terminal region of the transthyretin subunit was also demonstrated in a reptile, an amphibian and fish [16,38–40] The mechanism underlying the splice site movement is a series of single base mutations that converted specific amino acid codons into new splice-recognition sites For example, a single base mutation of A, C or U to G in the codons CAA (for glutamine), CAC (for histidine) or CAU (for histidine) can lead to changing of these amino acid codons to the 3¢ splice-site recognition sequence, CAG During evolution, the histidine codon, CAU, at the 5¢ end of exon of marsupial transthyretin genes may have been converted into CAG by a single base change from U to G In addition, other single base substitutions (i.e G to U or C), have occurred to inactivate the former 3¢ splice site recognition sequence that operates in marsupial transthyretin genes (Fig 3B) These changes led to a progressive movement of the intron ⁄ exon splice site in successive steps in transthyretin genes from fish to amphibian, to reptilian and avian, to marsupial and, finally, to eutherian species [15,16,38–40] FEBS Journal 276 (2009) 5330–5341 ª 2009 The Authors Journal compilation ª 2009 FEBS 5333 Structure–function relationships of transthyretin P Prapunpoj and L Leelawatwattana A B Fig Comparison of nucleotide and amino acid sequences of transthyretins at the exon ⁄ intron border (A) and intron ⁄ exon border (B) The 5¢ and 3¢ splice sites of intron of transthyretin precursor mRNAs from 13 vertebrate species are aligned with those of human transthyretin precursor mRNA The splice sites are indicated by arrows The consensus recognition sequences for splicing [97] are indicated above the position of the splice sites in human transthyretin precursor mRNA Nucleotides identical to those in the consensus sequence for the 3¢ splice site branch point are underlined Nucleotides in exons are in upper case; those in introns are in lower case The amino acid residues at the N-terminus, determined by Edman degradation of the mature native or recombinant transthyretin, and their corresponding codons are shown in bold (Modified from Prapunpoj et al., 2002 [16].) 5334 FEBS Journal 276 (2009) 5330–5341 ª 2009 The Authors Journal compilation ª 2009 FEBS P Prapunpoj and L Leelawatwattana Structure–function relationships of transthyretin Fig Comparison of the transthyretin exon1 ⁄ exon2 border The amino acid residues in the presegment and in the N-terminal region of transthyretins from 14 vertebrate species are aligned with that of human transthyretin Arrows, positions of the intron splice site; bold letter, the first amino acid at the N-terminus of the mature transthyretin subunit Sources of the splice site data: human [84]; rat [90]; tammar wallaby, grey kangaroo, stripe-faced dunnart, grey opossum, chicken and lizard [15]; hedgehog, shrew, mouse, crocodile, Xenopus, sea lamprey (as referenced in Fig 1) (Modified from Prapunpoj et al., 2006 [17].) The C-terminal region In comparison with the N-terminal region, much less change occurred in the C-terminal region of the transthyretin subunit during evolution This region in fish, amphibians, reptiles and birds is relatively more hydrophobic than that in mammals (Fig 1) In addition, the C-terminal region of the transthyretin from pig, amphibians and lampreys contains two to three amino acids more than that of human transthyretin (Fig 1) As the C-terminal segments are near the entrances to the central channel of transthyretin [23], the C-terminal segments may influence the accessibility of THs to the binding sites This is currently under investigation The involvement of the C-terminal regions on the functions of transthyretin that have been revealed to date includes the binding with RBP and pathogenesis of senile systemic amyloidosis These are discussed in the ‘Functions of transthyretin’ section (below) Functions of transthyretin As a thyroid hormone distributor In plasma Transthyretin, albumin and thyroxine-binding globulin are the three major TH distributor proteins that are synthesized in the liver and secreted into the blood of larger mammals In blood, these three proteins ensure the appropriate distribution of the THs throughout tissues in the body and maintain the free hormone pool in the blood and CSF Transthyretin is believed to be the most important distributor for T4 in the blood of humans [41] because of its association and dissociation rates for TH that are between those of albumin and thyroxine-binding globulin In other vertebrates, including diprotodont marsupials [42], birds [43], young reptiles [44], premetamorphic amphibians [35,39] and juvenile fish [45–47], transthyretin is the major TH transport protein in the blood In brain The brain is separated from the bloodstream by the blood–brain barrier, which includes the blood–CSF barrier that is located at the tight junctions and membranes of the endothelial cells of brain capillaries and the epithelial cells of choroids plexus The concentration of most proteins in the CSF is much lower than in blood, and most proteins in the CSF (including albumin and thyroxine-binding globulin) originate from the blood and move across the blood–brain barrier [48,49] However, this is not likely to be the situation for transthyretin Only a small amount of transthyretin in the CSF is derived from the blood [50] The epithelial cells of the choroid plexus are the major synthesis site of transthyretin, which is secreted into the CSF [48,51] However, the transthyretin gene in the choroid plexus is differently regulated from that in the liver [48] For example, the absolute levels of transthyretin mRNA in rat choroid plexus are 11.3 times higher than those in the liver, and the activity of FEBS Journal 276 (2009) 5330–5341 ª 2009 The Authors Journal compilation ª 2009 FEBS 5335 Structure–function relationships of transthyretin P Prapunpoj and L Leelawatwattana transthyretin in CSF is more specific than in serum [52] Transthyretin is the major TH distributor protein in the CSF of reptiles, birds and mammals [12] For more discussion on this topic, see the review in this miniseries by Richardson By using a two-chamber cell-culture system [53], three mechanisms of T4 transport from the blood via the choroid plexus into the CSF were proposed First, free THs in blood can partition into choroid plexus cells Second, T4 may bind to transthyretin synthesized in the choroid plexus epithelial cells or pass through the choroid plexus and bind transthyretin in the CSF Finally, T4 could be drawn across the blood–brain barrier by the presence of transthyretin in the CSF As deiodinases were not detected in the choroid plexus cells, the intact T4 was proposed to enter into the CSF through the choroid plexus cells without deiodination, and is subsequently converted to T3 by deiodinases within the brain [53] Recently, transthyretin-mediated delivery of T4 to stem cells and progenitor cells within the brain has been demonstrated [54] Influence of the N-terminal structure on the TH distributor function During the evolution of vertebrates, the binding affinities of transthyretin to THs varied [8,16,37,39,55] The binding to T4 increased, while the binding to T3 decreased, during the evolution of eutherians from their ancestors The crystallographic studies revealed that amino acid residues in the binding cavity which are directly involved in binding THs are conserved [6,21] Because the change in affinities of T3 and T4 [37] was directly correlated with the change in the structure of the N-termini [15], it was suggested that the N-termini could affect the access of THs to the binding sites To test this hypothesis, recombinant native and chimeric transthyretins were produced from salt-water crocodile (Crocodylus porosus) and analysed for affinities to T3 and T4 [16,17] using a highly reproducible and sensitive method [37] The Kd values of T3 and T4 for the native crocodile transthyretin were 7.56 ± 0.84 nm and 36.73 ± 2.38 nm, respectively [16] However, the Kd values of T3 and T4 for the chimeric transthyretin in which the N-terminal sequence had been replaced with that of human transthyretin were 5.40 ± 0.25 nm and 22.75 ± 1.89 nm, respectively, providing a Kd T3 : T4 ratio higher than that of native crocodile transthyretin [17] By contrast, the N-terminal truncated transthyretin had similar affinities for both T3 (Kd = 57.78 ± 5.65 nm) and T4 (Kd = 59.72 ± 3.38 nm) These data led to the postulation that the N-terminal region has a role in determining 5336 the binding affinities of T3 and T4 for transthyretin This hypothesis was subsequently supported by others using fish-truncated transthyretin [55] As a carrier for retinol via binding to RBP In blood, the transport of retinol is mediated by RBP [56] Liver is the site of RBP synthesis, and the secretion of RBP into the blood is initiated by the binding of retinol In the bloodstream, RBP is bound to transthyretin with affinities in the range of 1.0 · 10)6 to 3.4 · 10)7 m, depending on the animal species and forms of transthyretin and RBP [13,57–59] The transthyretin–RBP complex is formed before the complex is secreted into the blood This complex is believed to prevent the loss of RBP through glomerular filtration by the kidney [59–62] The binding with transthyretin was postulated as a positive regulator in the delivery of RBP-bound retinol from plasma into liver cells, possibly via a receptor-mediated mechanism However, excess transthyretin inhibited the retinol uptake of the transthyretin–RBP complex [63] The nature of the transthyretin-binding site for RBP has been studied extensively Based on crystallography, up to two binding sites for RBP per transthyretin tetramer, in the same or opposite dimers, were demonstrated [59,63,64] In the binding interaction, RBP and transthyretin each contribute 21 amino acids to the protein–protein recognition interface and most of these residues are in the C-terminal regions of the two proteins [65] The affinity of transthyretin for RBP is sensitive to several factors (e.g pH, ionic strength, the binding of retinol to RBP and the hydrophobicity at the interaction interface) Analysis using electrospray ionization combined with time-of-flight mass spectrometry revealed a : molar ratio of the complex formation and the dissociation constants of the transthyretin–RBP complex to be 1.9 ± · 10)7 m for the first binding site and 3.51 · 10)5 m for the second binding site [66], indicating negative co-operativity As a plasma protease Proteolytic activity is a newly discovered function of transthyretin Only a few natural substrates have been identified These include amyloid b (Ab), apolipoprotein A-I and amidated neuropeptide Y Ab is the major component of senile plaques that deposit in the brain and leptomenings of patients with Alzheimer’s disease [67,68] It also exists in a soluble form in the CSF and blood Although the deposition of Ab aggregates has been known to be a critical step of the disease, the mechanism by which Ab forms FEBS Journal 276 (2009) 5330–5341 ª 2009 The Authors Journal compilation ª 2009 FEBS P Prapunpoj and L Leelawatwattana aggregates is unclear Several extracellular proteins in the CSF that bind and sequester Ab have been identified [69–71] Sequestration of Ab by these proteins is believed to prevent amyloidosis, and failure of the process can lead to development of Alzheimer’s disease [72] Transthyretin is the major Ab sequestering protein in human CSF [72] In the presence of this protein, aggregation of Ab decreased and toxicity of the Ab was abolished [73] Transthyretin binds to the soluble nonaggregated Ab with a Kd of 28 ± nm [73], via amino acid residues on the surface of its monomer [74] Different transthyretin variants bound Ab with different strengths [73,75], but there was no correlation with the degree of inhibition ⁄ disruption of Ab fibrillogenesis [73] The cleavage of Ab by transthyretin was recently reported [76] Several cleavage sites (e.g after tyrosine and phenylalanine; after lysine; and after alanine) were identified Transthyretin cleaved both the soluble and the aggregated forms of Ab, and the Ab amyloidogenicity was diminished upon cleavage [76] Under physiological conditions, a fraction (1–2%) of transthyretin in human plasma circulates in highdensity lipoproteins via binding to apolipoprotein A-I, which is a major protein component of the lipoproteins Recently, it has been shown that human transthyretin can specifically cleave the C-terminus of the apolipoprotein after the phenylalanine residue 225 [18] The proteolytic activity of transthyretin was demonstrated both in vitro and in vivo Activity was optimum at pH 6.8 (Km = 29 lm) and could be specifically inhibited by several serine protease inhibitors (e.g Pefabloc and phenylmethylsulfonylfluoride) [18] In addition, inhibitors of chymotrypsin-like serine protease, such as chymostatin, could also abolish the activity This led to the postulation of a chymotrypsin-like serine protease activity of transthyretin The transthyretin-cleaved apolipoprotein A-I showed a decrease in the ability to promote cholesterol efflux and had a high tendency to aggregate to form amyloid fibrils [77] Neuropeptide Y is the most abundant neuropeptide in the brain and autonomic nervous system of mammals and has a role in numerous physiologic processes Its amidated form was identified very recently to be another natural substrate for transthyretin [78] The amidated peptide was cleaved after the arginine positions 33 and 35, and this cleavage was demonstrated to promote the axonal regeneration of neurons As a protector against apoptosis Besides liver and choroid plexus, which are the main sites of transthyretin synthesis, the pancreas is one of Structure–function relationships of transthyretin the minor sites of transthyretin synthesis [79,80] Transthyretin is synthesized by the alpha (glucagons) cells in the islets of Langerhans, stored in the secretory granules and released upon exocytosis [81] It is also a component in normal pancreatic b-cell stimulus-secretory coupling and acts to protect against the apoptosis of b-cells induced by apolipoprotein CIII [82] As only a tetrameric (not a monomeric) form was responsible for this role, the conversion of transthyretin tetramer to the monomer was postulated to be associated with b-cell failure ⁄ destruction in type diabetic patients [82] Conclusion and future directions The amino acids in the central channel of transthyretin that are involved in binding THs have not changed in more than 400 million years However, the amino acids in the N-terminal regions of transthyretins have changed in a stepwise manner These changes have been selected for and have remained in the population, so could be considered as representing an ‘improvement ⁄ adaptation’ of transthyretin function Selection pressure has apparently operated on the length and composition of transthyretin N-termini by a series of single base mutations that resulted in the movement of the intron ⁄ exon border in the 3¢ direction This leads to a stepwise change in primary structure and, as a consequence, in function of the binding affinities to T3 and T4 of transthyretin Specific residues on the external surface of transthyretin are involved in the binding to RBP The proteolytic site has not been clearly identified; however, because binding to RBP (but not to T4) abolishes the enzyme activity, the site may be located on the external surface of transthyretin [18] For multifunction proteins, such as transthyretin, one could expect the evolutionary changes of the primary structure, in particular of N- and C-terminal regions, to effect more than one function The evolution of more recently discovered functions of transthyretin (cleavage of Ab, apolipoprotein A-I, neuropeptide Y; protection against apoptosis) should be investigated in transthyretins from birds, reptiles, amphibians and fish Here, we have shown how evolution of the structure–function relationship of a protein can be studied using comparative biochemistry and how hypotheses regarding the structure–function relationship can be proved by producing chimeric and truncated proteins As structure determines function, and because much current research is associated with human diseases such as amyloidoses, insight into the structure–function relationships of transthyretin not only elucidates how and why the evolutionary adaptations occurred, FEBS Journal 276 (2009) 5330–5341 ª 2009 The Authors Journal compilation ª 2009 FEBS 5337 Structure–function relationships of transthyretin P Prapunpoj and L Leelawatwattana but also points to its clinical significance (although it should be noted that this study was not clinical and any connection with other research findings remains to be established) and the future potential of transthyretin as a therapeutic agent for preventing or treatment of 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The Authors Journal compilation ª 2009 FEBS 5341 ... These changes led to a progressive movement of the intron ⁄ exon splice site in successive steps in transthyretin genes from fish to amphibian, to reptilian and avian, to marsupial and, finally, to. .. Langerhans, stored in the secretory granules and released upon exocytosis [81] It is also a component in normal pancreatic b-cell stimulus-secretory coupling and acts to protect against the apoptosis... compilation ª 2009 FEBS 5331 Structure–function relationships of transthyretin P Prapunpoj and L Leelawatwattana the evolutionary changes of the structure particular to the N-terminal region, the