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; splicin
Trang 1Evolutionary 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 conjugaphosphoryla-tion, 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: porntip.p@psu.ac.th
(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.
Trang 2in 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].)
Trang 3the 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,
http://journals.iucr.org/; reproduced with permission by Professor
Louise N Johnson, University of Oxford, UK.)
Trang 4transthyretin 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 evolurecogni-tion, 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]
Trang 5A
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].)
Trang 6The 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].)
Trang 7transthyretin 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 Kdvalues 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 Kdvalues 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 KdT3 : 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
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)7m, 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)7m for the first binding site and 3.51 · 10)5m 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
Trang 8aggregates 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
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 Kd 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 (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
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 monotetra-meric) 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 relastructure–func-tionships of transthyretin not only elucidates how and why the evolutionary adaptations occurred,
Trang 9but 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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