Báo cáo khoa học: Evolutionary changes to transthyretin: structure–function relationships ppt

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
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-bind-
ing 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 gly-
cosylated, despite containing potential glycosylation
sites. Heterogeneity of transthyretin from several spe-
cies has been described, resulting from phosphoryla-
tion, cysteine–glycine conjugation, glutathionylation

and the interaction with ligands, such as retinol-bind-
ing 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
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:
(Received 2 February 2009, revised 5 July
2009, accepted 27 July 2009)
doi:10.1111/j.1742-4658.2009.07243.x
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 amy-
loidosis. Transthyretin null mice do not have an overt phenotype, probably

because transthyretin is part of a network with other thyroid hormone dis-
tributor proteins. Systematic study of the evolutionary changes of transthy-
retin structure is an effective way to elucidate its function. This review
summarizes current knowledge about the evolution of structural and func-
tional characteristics of vertebrate transthyretins. The molecular mechanism
of evolutionary change and the resultant effects on the function of trans-
thyretin are discussed.
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
in the binding sites, but in the N-terminal region [15].
This structural change influences the ability of trans-
thyretins to bind to THs [16,17]. Transthyretin has been
recognized as one of the most interesting proteins iden-
tified to date, because of its multifunctionality. Besides
distributes THs in blood, it indirectly transports vita-
min 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
Fig. 1. 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 pre-
segment; positive numbers, residues in the mature protein; a, b, c, d, e, f, g, h and i, positions of residues in noneutherians. The first resi-
due 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].)
P. Prapunpoj and L. Leelawatwattana Structure–function relationships of transthyretin
FEBS Journal 276 (2009) 5330–5341 ª 2009 The Authors Journal compilation ª 2009 FEBS 5331
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
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 trans-
thyretin are composed of a pair of twisted eight-
stranded 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 inter-
actions; 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 con-
tacts 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 8 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].
3D structures of other transthyretins
To date, transthyretin from only four species other
than human have been crystallized and their 3D struc-
tures 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 differ-
ences 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 mono-
mers of rat transthyretin than those of human trans-
thyretin [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 (res-
idues 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
A
B
Fig. 2. 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 mono-
mer are labelled a-h. (From Ghosh et al., 2000 [20], copyright of IUCr,
reproduced with permission by Professor
Louise N. Johnson, University of Oxford, UK.)
Structure–function relationships of transthyretin P. Prapunpoj and L. Leelawatwattana
5332 FEBS Journal 276 (2009) 5330–5341 ª 2009 The Authors Journal compilation ª 2009 FEBS
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]. How-
ever, 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 bull-
frog (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 preseg-
ment 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 align-

ment 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 inter-
action with THs [22,24,36], are conserved and have not
been altered for more than 400 million years. By con-
trast, 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 rela-
tively 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.,
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 1. Compared with human transthyretin,

two (for marsupials) or three to nine (for birds, rep-
tiles, amphibians and fish) additional amino acids are
present in the N-termini of transthyretins (see Fig. 1).
The systematic analysis and comparison of the nucleo-
tide sequences flanking the exon 1 ⁄ intron 1 and intron
1 ⁄ exon 2 borders of transthyretin mRNAs from eight
species (eutherians, marsupials and a bird) revealed
shifting in successive steps of the intron 1 ⁄ exon 2 splice
site in the 3¢ direction during evolution [15]. The nucle-
otide sequences at the exon 1 ⁄ intron 1 border were
unchanged (Fig. 3A). However, changes occurred at
the intron 1 ⁄ exon 2 border (Fig. 3B). Shifting of the
intron 1 ⁄ exon 2 splice site in the 3¢ direction was pos-
tulated to occur in successive steps, which led to a suc-
cessive shortening of the transthyretin N-terminal
region (Fig. 4) [15]. The same successive changes
resulting in shortening and an increase in hydrophilic-
ity of the N-terminal region of the transthyretin sub-
unit 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 spe-
cific 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 recogni-
tion sequence, CAG. During evolution, the histidine
codon, CAU, at the 5¢ end of exon 2 of marsupial
transthyretin genes may have been converted into

CAG by a single base change from U to G. In addi-
tion, 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 trans-
thyretin genes (Fig. 3B). These changes led to a
progressive movement of the intron 1 ⁄ exon 2 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].
P. Prapunpoj and L. Leelawatwattana Structure–function relationships of transthyretin
FEBS Journal 276 (2009) 5330–5341 ª 2009 The Authors Journal compilation ª 2009 FEBS 5333
B
A
Fig. 3. Comparison of nucleotide and amino acid sequences of transthyretins at the exon 1 ⁄ intron 1 border (A) and intron 1 ⁄ exon 2 border
(B). The 5¢ and 3¢ splice sites of intron 1 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].)
Structure–function relationships of transthyretin P. Prapunpoj and L. Leelawatwattana
5334 FEBS Journal 276 (2009) 5330–5341 ª 2009 The Authors Journal compilation ª 2009 FEBS
The C-terminal region
In comparison with the N-terminal region, much less
change occurred in the C-terminal region of the trans-
thyretin subunit during evolution. This region in fish,
amphibians, reptiles and birds is relatively more hydro-
phobic 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 tis-
sues 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 mem-
branes of the endothelial cells of brain capillaries and
the epithelial cells of choroids plexus. The concentra-
tion 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 bar-
rier [48,49]. However, this is not likely to be the situa-
tion 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
Fig. 4. 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 1 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].)
P. Prapunpoj and L. Leelawatwattana Structure–function relationships of transthyretin
FEBS Journal 276 (2009) 5330–5341 ª 2009 The Authors Journal compilation ª 2009 FEBS 5335
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 affini-
ties 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 repro-
ducible and sensitive method [37]. The K
d
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 K
d
values of T3 and T4 for the chi-
meric 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, respec-
tively, providing a K
d
T3 : T4 ratio higher than that of
native crocodile transthyretin [17]. By contrast, the
N-terminal truncated transthyretin had similar affinities
for both T3 (K
d
= 57.78 ± 5.65 nm) and T4 (K
d
=
59.72 ± 3.38 nm). These data led to the postulation
that the N-terminal region has a role in determining
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 secre-
tion of RBP into the blood is initiated by the binding
of retinol. In the bloodstream, RBP is bound to trans-
thyretin 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 trans-
thyretin–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 crystallogra-
phy, up to two binding sites for RBP per transthyretin
tetramer, in the same or opposite dimers, were demon-
strated [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 spec-
trometry revealed a 1 : 1 molar ratio of the complex
formation and the dissociation constants of the trans-
thyretin–RBP complex to be 1.9 ± 1 · 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), apolipopro-
tein 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
Structure–function relationships of transthyretin P. Prapunpoj and L. Leelawatwattana
5336 FEBS Journal 276 (2009) 5330–5341 ª 2009 The Authors Journal compilation ª 2009 FEBS
aggregates is unclear. Several extracellular proteins in
the CSF that bind and sequester Ab have been identi-
fied [69–71]. Sequestration of Ab by these proteins is
believed to prevent amyloidosis, and failure of the pro-
cess can lead to development of Alzheimer’s disease

[72]. Transthyretin is the major Ab sequestering pro-
tein in human CSF [72]. In the presence of this pro-
tein, aggregation of Ab decreased and toxicity of the
Ab was abolished [73]. Transthyretin binds to the solu-
ble nonaggregated Ab with a K
d
of 28 ± 5 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 fibrillo-
genesis [73]. The cleavage of Ab by transthyretin was
recently reported [76]. Several cleavage sites (e.g. after
tyrosine and phenylalanine; after lysine; and after ala-
nine) 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 high-
density lipoproteins via binding to apolipoprotein A-I,
which is a major protein component of the lipopro-
teins. Recently, it has been shown that human trans-
thyretin can specifically cleave the C-terminus of the
apolipoprotein after the phenylalanine residue 225 [18].
The proteolytic activity of transthyretin was demon-
strated both in vitro and in vivo. Activity was optimum
at pH 6.8 (K
m
=29lm) and could be specifically
inhibited by several serine protease inhibitors (e.g.

Pefabloc and phenylmethylsulfonylfluoride) [18]. In
addition, inhibitors of chymotrypsin-like serine prote-
ase, such as chymostatin, could also abolish the activ-
ity. This led to the postulation of a chymotrypsin-like
serine protease activity of transthyretin. The transthy-
retin-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 mam-
mals 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 posi-
tions 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
the minor sites of transthyretin synthesis [79,80]. Trans-
thyretin 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 compo-
nent in normal pancreatic b-cell stimulus-secretory cou-
pling and acts to protect against the apoptosis of b-cells
induced by apolipoprotein CIII [82]. As only a tetra-
meric (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 1 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 ‘improve-
ment ⁄ 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 1 ⁄ exon 2 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 transthy-
retin are involved in the binding to RBP. The proteo-
lytic 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 exter-
nal surface of transthyretin [18]. For multifunction
proteins, such as transthyretin, one could expect the
evolutionary changes of the primary structure, in par-
ticular of N- and C-terminal regions, to effect more
than one function. The evolution of more recently dis-
covered 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 compar-
ative 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–func-
tion relationships of transthyretin not only elucidates
how and why the evolutionary adaptations occurred,
P. Prapunpoj and L. Leelawatwattana Structure–function relationships of transthyretin
FEBS Journal 276 (2009) 5330–5341 ª 2009 The Authors Journal compilation ª 2009 FEBS 5337
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
amyloidoses.
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