 |
INTRODUCTION |
Transthyretin (TTR)1 is
a 54-kDa tetramer, which is present in human plasma (3.6 µM tetramer) and transports thyroid hormones such as
3,5,3'-triiodo-L-thyronine and L-thyroxine (T4)
as well as retinol-binding protein (1, 2). Its structure and ligand binding capabilities have been characterized by x-ray crystallography (3-5). Each subunit contains extensive
-sheet structure and is
arranged within a dimer of dimers to form a compact molecule with two
funnel-shaped hormone ligand binding sites, each defined by a
dimer-dimer interface. The thyroid hormones bind deeply within the
hydrophobic binding channel, their iodinyl moieties residing in
hydrophobic pockets at two different binding sites within the channel
(6). Upon binding of one T4 ligand, the binding affinity for the second
ligand is reduced significantly.
Although the TTR tetramer is inherently very stable (1, 7), in some
circumstances transthyretin has a propensity to form amyloid fibrils.
In diseases such as senile systemic amyloidosis and familial
amyloidotic polyneuropathies, native and mutant TTR, respectively have
been found to form long beta-sheet based fibrillar structures (8, 9).
These amyloid lesions accumulate in specific organs and are implicated
in their dysfunction and ultimately the death of the patients. More
than 70 separate mutations that appear to increase the propensity of
TTR to form amyloid structures underlying familial amyloidotic
polyneuropathy have so far been identified (10-14). It is thought that
the underlying mechanism for TTR fibril formation involves tetramer
dissociation to a monomeric conformational intermediate which
self-assembles to form amyloid fibrils. Any mutation or cellular
condition (such as low pH) that tends to destabilize the tetramer can
result in an increased propensity of the protein to form amyloid
fibrils (9, 11).
The ligand binding properties of TTR have become of major importance
recently, with the discovery that certain thyroid hormone competitors
(e.g. 2,4,6-triiodophenol) are able to decrease the tendency
of TTR to form amyloid fibrils (15, 16). They are reported to act by
binding deeply within the TTR binding channel at both ligand binding
sites and inhibiting the formation of amyloid by stabilizing the normal
fold against the pathogenic conformational change. A range of
nonsteroidal anti-inflammatory drugs are currently being investigated
for their ability to inhibit and reverse amyloid formation (17). While
crystallographic information has revealed the orientations of several
of these drugs bound to TTR, there are many aspects of the ligand
binding that cannot be probed using crystallographic techniques. These
include dynamic aspects of protein-ligand binding in solution and in
the presence of competitors or in the presence of other serum proteins,
including the other thyroid hormone carriers albumin and
thyroxine-binding globulin. For such studies, it is possible that NMR
spectroscopic methods could be employed.
NMR can be used to observe the interaction of a ligand with particular
sites in the protein. Such studies currently depend upon the assignment
of the specific resonances that are perturbed by the ligand (18), which
limits studies to systems in which the protein signal of interest is
either fortuitously distinct or has been fully assigned using isotopic
labeling and heteronuclear NMR techniques. In the latter case, this
also requires that the protein is less than about 40 kDa due to the
broad line widths of the NMR signals and large number of signals that
have similar chemical shifts. The problem can be somewhat reduced by
using chemical ligation strategies in which just one portion of the protein is isotopically labeled (19). Of even greater value would be
the ability to completely control the position of the isotopic label in
a protein for probing the ligand interaction.
The current study thus presents the first stage in the development of a
spectroscopic method for probing a protein-ligand interaction. The
strategy involves the complete synthesis of a TTR analog using solid
phase synthesis and chemical ligation techniques and refolding of the
protein to a tertiary and quaternary structure that approximates the
native form. Since the structure of TTR is known to atomic resolution
from x-ray crystallographic studies, an NMR active probe
(i.e. 15N- or 13C-labeled amino
acid) may be incorporated at any strategic position to enable
subsequent ligand binding studies to be manifest in NMR spectra.
The chemical synthesis of proteins of the size of the monomeric unit of
TTR (127 residues) represents a significant challenge. In the past,
long peptides were synthesized in a stepwise fashion, as exemplified by
human immunodeficiency virus type 1 protease (20) and interleukin-8
(21), but significant purification problems resulted in low yields of
protein. Currently, the synthesis techniques for proteins of this size
rely on chemoselective ligation techniques, where two or more
nonprotected peptides are joined through a highly selective chemical reaction.
There have been several notable protein syntheses described using
chemoselective ligation techniques that incorporate thioester and
thioether surrogate amide bonds. These include the synthesis of linked
heterodimeric basic helix-loop-helix transcription factors (22)
and Cpn10 (23), respectively. Recently, techniques for forming a
native amide bond have been applied to a range of protein types
including serine protease inhibitors (24-26), human II secretory phospholipase A2 (27), and barnase (28, 29).
Native chemical ligation chemistry is only useful provided there are
suitably positioned cysteines. In order to synthesize a protein like
transthyretin without suitably located cysteines, it becomes necessary
to use chemically modified amino acid substitutes or amino acid
mutations. The thioether ligation strategy we use here introduces
-NH-CH2-CH2-S-CH2-CO-, which mimics
a two-amino acid subunit closely resembling a glycyl-glycine
(-NH-CH2-CO-NH-CH2-CO-). While the thioether
moiety closely resembles the spatial requirements for glycyl-glycine,
it may lack potential hydrogen bond donation/acceptor behavior of the
diamino acid unit, thereby potentially introducing some nonnative
structural characteristics.
Here we demonstrate the total chemical synthesis of an analog of human
TTR through the use of the thioether strategy for the sequential
ligation of three peptides. We also show that this synthetic TTR
(henceforth referred to as sTTR) may be successfully refolded and
reconstituted to form a 54-kDa tetrameric structure able to bind the
thyroid hormone T4. This represents one of the largest active proteins
made synthetically and provides methodology for future protein-ligand
investigations using NMR spectroscopic techniques.
 |
EXPERIMENTAL PROCEDURES |
Chemicals and Reagents--
Trifluoroacetic acid,
dichloromethane, N,N-dimethyl formamide, and
diisopropylethylamine were from Auspep (Melbourne, Australia). O-Benzotriazol-1-yl-N,N,N',N'-tetramethyluronium
hexafluorophosphate was from Richelieu Biotechnologies (St.
Hyacinth, Quebec Canada). Acetonitrile was from BDH Laboratory Supplies
(Poole, United Kingdom). Acetic acid and chloroacetic acid were from
Ajax chemicals (Auburn, Australia), diethyl ether was from Fluka
Biochemicals (Melbourne, Australia), and mercaptoethanol was from
Sigma. Ethanolamine, N,N-diisopropylcarbodiimide,
and bromoacetic acid were from Aldrich. Hydrogen fluoride (HF) was
purchased from Boc Gases (Brisbane, Australia). The
N-Boc-protected L-amino acids Ala, Gly, Ile,
Leu, Phe, Pro, Val, Arg(p-toluenesulphonyl),
Asp(O-cyclohexyl), Asn(xanthyl), Glu
(O-cyclohexyl), His(dinitrophenyl),
Lys(2-chlorobenzyloxycarbonyl), Ser(benzyl), Thr(benzyl), Trp(formyl),
Tyr(2-bromobenzyloxycarbonyl) were purchased either from NovaBiochem
(La Jolla, CA) or Bachem (Switzerland). Human serum was supplied by the
Red Cross Blood Bank (Melbourne, Australia).
Equipment--
Analytical and preparative HPLC was carried out
using a Waters HPLC system composed of a model 600 solvent delivery
system 600E controller and model 484 detector. Vydac C18 columns,
analytical (4.6 × 250 mm) at a flow rate of 1 ml/min,
semipreparative (10 × 250 mm) at a flow rate of 3 ml/min, and
preparative (22 × 250 mm) at a flow rate of 8 ml/min, were used.
All peptides were purified using linear gradients of 0.1% aqueous
trifluoroacetic acid (solvent A) and 90% aqueous acetonitrile 0.09%
trifluoroacetic acid (solvent B).
Mass spectral data were collected using a PerkinElmer Sciex (Toronto,
Canada) API III biomolecular mass analyzer ion-spray mass spectrometer
equipped with an ABI 140B solvent delivery system. Raw data were
analyzed using the program MassSpec (PerkinElmer Sciex). Calculated
masses were obtained using the program MACROMASS (Sunil Vemuri and
Terry Lee, City of Hope, Durate, CA).
Enhanced Chemiluminescence kit was from Amersham Pharmacia Biotech,
x-ray film was from Eastman Kodak Co., methylcellulose and activated
charcoal (Norit PN.5) were from BDH,
L-[125I]thyroxine (1.2 Ci/mg) was from
PerkinElmer Life Sciences (NEN Dupont), SepPak C-18 cartridges
were from Millipore Corp., and thin layer chromatography plates were
from Merck. All reagents were of analytical grade.
Native Human TTR--
Native human TTR was isolated from serum
using an adapted version of the method described by Dwulet and Benson
(30).
Peptide Synthesis--
Peptides were synthesized using the rapid
manual
O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium
hexafluorophosphate in situ neutralization synthesis
technique (31) or using the same technique on a modified ABI 430A
peptide synthesizer (32). The thioether resins were prepared according
the methods of Englebretsen et al. (33), initially on amino
methyl resins and then subsequently Boc-amino acid-Pam resins
(ABI). The bromoacetyl and chloroacetyl groups at the amino termini of
peptides were coupled using the symmetrical anhydride formed from
reaction with N,N-diisopropylcarbodiimide. The
dinitrophenyl group was removed using 20% mercaptoethanol in 10%
diisopropylethylamine/N,N-dimethyl formamide
solution for two or three 30-min treatments. The Trp formyl
deprotection was carried out using ethanolamine prior to HF cleavage.
Peptide resins were cleaved using HF with p-cresol and
p-thiocresol as scavengers at
5 to 0 °C for 1-2 h. The
HF was removed in vacuo, the peptide product was triturated
with cold diethyl ether (3 × 50 ml), and the precipitated peptide
was collected and dissolved in 50% acetonitrile with 0.1%
trifluoroacetic acid.
The crude peptides were purified by reverse phase (RP)-HPLC, and
fractions were collected and analyzed by analytical RP-HPLC and
electrospray mass spectrometry (ESMS). Fractions containing the
purified peptide were combined and lyophilized.
Solid Phase Synthesis of Br-Ac-PRRYTIAALLSPYSYSTTAVVTNPKE-OH
(Bromoacetyl-102-127 TTR) (I)--
Peptide
I (see Fig. 2) was synthesized on a
Boc-Glu(O-benzyl)-Pam-polystyrene resin (ABI) on a 0.5-mmol
scale. Amino acid couplings averaged 99.5% efficiency.
N-Boc deprotection and coupling of bromoacetic acid followed
by HF cleavage gave the crude peptide, which was purified by
preparative HPLC using a linear gradient of 0-70% B. The peptide was
then analyzed by HPLC and ESMS. The purified peptide was characterized
as the desired product (I) by ESMS (observed mass = 3021 ± 0.3; calculated for C133 H210
N34 O41 Br1 = 3021.24 (average
isotope composition)).
Solid Phase Synthesis of
Cl-Ac-ELHGLTTEEEFVEGIYKVEIDTKSYWKALGISPFHEHAEVVFTAND-NH-CH2-CH2-SH
(Chloroacetyl-54-99-NH-CH2-CH2-SH)
(II)--
The C-terminal thiol peptide II
was manually synthesized using the thiol linker attached to Boc-Ala-Pam
resin in the first synthesis and then Boc-Gly-Pam resin for the second.
The average amino acid coupling for the syntheses was 99.5 and 99.6%, respectively. The dinitrophenyl protecting group was removed followed by N-Boc and formyl group removal. The chloroacetyl group
was coupled, and then the peptide was cleaved by HF. The crude
peptide was purified by preparative HPLC using a linear gradient of
0-70% B and then analyzed by HPLC and ESMS. The purified peptide was characterized as the desired product (II) by ESMS (observed mass = 5416.46 ± 1.1; calculated for C246
H364 N58 O76 S1
Cl1 = 5417.46 (average isotope composition)).
Solid Phase Synthesis of
H-GPTGTGESKAPLMVKVLDAVRGSPAINVAVHVFRKAADDTWEPFASGKTSE-NH-CH2-CH2-SH
(1-51-NH-CH2-CH2-SH)
(III)--
The C-terminal thiol peptide III was
synthesized using machine-assisted synthesis. The peptide was
synthesized using the thiol linker attached to Boc-Gly-Pam resin. The
average amino acid coupling was 99.6% (1st coupling) for the
synthesis, which was routinely double coupled. The dinitrophenyl
protecting group was removed, followed by N-Boc and then the
formyl group. The peptide was cleaved from the resin, and the crude
peptide was purified by preparative HPLC using a linear gradient of
0-70% B. The peptide was then analyzed by HPLC and ESMS. The purified
peptide was characterized as the desired product (III) by
ESMS (observed mass = 5355 ± 1.0; calculated for
C236 H379 N66 O72
S2 = 5357.14 (average isotope composition)).
Formation of
Cl-Ac-ELHGLTTEEEFVEGIYKVEIDTKSYWK- ALGISPFHEHAEVVFTAND-NH-CH2-CH2-S-CH2-CO-PRRYTIAALLSPYSYSTTAVVTNPKE-OH
(Chloroacetyl-54-99-
-102-127) (IV)--
The
ligation reaction was initiated by mixing the two peptides,
bromoacetyl-102-127 TTR (I) (5.44 mg, 1.8 mmol) and chloroacetyl-54-99-NH-CH2-CH2-SH
(II) (6.84 mg, 1.26 mmol), in 1 ml of 6 M urea
0.1 M NaHCO3, pH 8.3, under nitrogen. After mixing, the reaction mixture was left to stand at room temperature for
24 h. Samples were withdrawn at 0, 1, 2, and 20 h during this period for HPLC and ESMS analysis. The ligated peptide IV was isolated
from the reaction mixture by semipreparative HPLC using a linear
gradient of 0-70% B. The fractions were analyzed by HPLC and ESMS;
the fractions containing the ligated peptide were then lyophilized. The
purified peptide IV (6.01 mg) was characterized as the
desired product IV by ESMS (observed mass = 8355 ± 1.2; calculated for
C379H572N92O117S1Cl1 = 8356.8 (average isotope composition)).
Chloro-Iodo Exchange of (IV) to give
I-Ac-ELHGLTTEEEFVEGIYKVEIDTKSYWKALGISPFHEHAEVVFTAND-NH-CH2-CH2-S-CH2-CO-PRRYTIAALLSPYSYSTTAVVTNPKE-OH
(Iodoacetyl-54-99-
-102-127) (V)--
Peptide IV
(5.46 mg, 0.654 mmol) was dissolved in 8 M urea 0.01 M NaOAc pH 7.5 (1 ml), and KI was added to saturation (~8 M). After placing under nitrogen, a sample was removed at
30 min, purified by HPLC, and analyzed by ESMS to check completion of the iodo exchange. The iodoacetyl peptide V was then
purified by semipreparative HPLC and lyophilized to give 3.64 mg. The
peptide was characterized as the desired product V by ESMS (observed
mass = 8447 ± 1.0; calculated for
C379H572N92O117S1Cl1 = 8448.2 (average isotope composition)).
Formation of
H-GPTGTGESKAPLMVKVLDAVRGSPAINVAVHVFRKAADDTWEPFASGKTSE-NH-CH2-CH2-S-CH2-COELHGLTTEEEFVEGIYKVEIDTKSYWKALGISPFHEHAEVVFTAND-NH-CH2-CH2-S-CH2-CO-PRRYTIAALLSPYSYSTTAVVTNPKE-OH
(1-51-
-54-99-
-102-127 TR)
(VI)--
Ligation of the two peptides
iodoacetyl-54-99-NH-CH2-CH2-SH V (3.46 mg, 0.43 mmol) and
1-51-NH-CH2-CH2-SH III (3.58 mg, 6.68 mmol) was initiated
by mixing the two peptides in 6 M urea, 0.2 M
NaHCO3, pH 8.3 (500 ml) and placing under nitrogen. The
reaction was mixed and then left to stand at room temperature for
5 h. Samples were withdrawn at 0, 1.5, and 4 h for HPLC and ESMS analysis. The ligated peptide VI was isolated from the
reaction mixture by semipreparative HPLC using a linear gradient of
0-70% B. The fractions were analyzed by HPLC and ESMS; the fractions
containing the ligated peptide were then lyophilized to give
VI (2.76 mg). The purified peptide was characterized as the
desired product VI by ESMS (observed mass = 13,676 ± 1.3; calculated for
C615H949N158O189S3 = 13,676.4 (average isotope composition)).
Formation of the Tetrameric Complex--
The ligated peptide
(1-51-
-54-99-
-102-127) (VI) (0.25 mg) was dissolved
in 0.075 M NH4HCO3, pH 8.3 (100 µl) and then diluted with 100 µl of H2O. To this
solution was added 5 µl of thyroxine T4 (5 mg/ml in 0.1 M
NaOH). After equilibrating at room temperature for 18 h, the
tetrameric protein was isolated by gel filtration on a Superdex 75 column (HR10/30; Amersham Pharmacia Biotech; calibrated with
phosphorylase, 97 kDa; bovine albumin, 66 kDa; native TTR, 54 kDa;
ovalbumin, 45 kDa; carbonic anhydrase, 30 kDa; soya bean trypsin
inhibitor, 20.1 kDa, and
-lactalbumin, 14.4 kDa) with 0.075 M NH4HCO3 10% CH3CN as
the eluent at 0.3 ml/min. The tetrameric protein was isolated at a
retention time of 22 min as determined by its equivalent retention time
to that of native TTR. It was kept in solution in the presence of
excess T4 prior to further analysis.
Western Analysis of Synthetic TTR--
2 µl of human serum, 25 µl (0.5 µg) of sTTR solution, and 50 µl (1.0 µg) of sTTR were
separated in a 0.1% SDS-polyacrylamide gel, using a stacking gel of
4.5% acrylamide, pH 6.8, and a resolving gel of 15% acrylamide, pH
8.6 (34). Proteins were transferred onto a nitrocellulose membrane,
following which the membrane was blocked. The primary antibody was
1:5000 antiserum raised in a rabbit against a mixture of TTRs purified
from serum from human (Homo sapiens) wallaby (Macropus
eugenii), and chicken (Gallus gallus), and the
secondary antibody was 1:10,000 anti-rabbit Ig raised in sheep
(Silenus), as described previously (35). Detection achieved was using
enhanced chemiluminescence against x-ray film.
Preparation of
L-[125I]Thyroxine--
Commercially
available L-[125I]thyroxine was found to
contain up to 5% 125I
on the reference date.
Therefore, [125I]thyroxine was separated from
125I
and other degradation products by
reversed phase chromatography using a SepPak C-18 cartridge column
(36). Purification was checked by thin layer chromatography followed by
autoradiography (37).
Analysis of Thyroxine Binding to Synthetic Human
TTR--
Commercially purchased [125I]thyroxine was
purified from degradation products and 125I
as described above. Methylcellulose charcoal was prepared as described
by Chang et al. (38). In order to remove thyroxine from the
solution containing sTTR, 40 µl of methylcellulose-charcoal (1%) in
Tris-HCl, pH 8.9, was centrifuged, and the supernatant was removed. The
methylcellulose-charcoal was resuspended in a 160-µl solution
containing 3.2 µg of sTTR. The mixture was kept at 4 °C for half
an hour, with mixing each 10 min. The solution was centrifuged, and the
supernatant was removed for analysis of [125I]thyroxine
binding to sTTR.
10 µl of human serum and 80 µl of solution containing sTTR (1.6 µg) were incubated with 1.1 fmol (2.4 nCi) of purified
[125I]thyroxine (room temperature for 1 h), and a
second aliquot of 80 µl of solution containing sTTR (1.6 µg) was
incubated with 4.4 fmol (9.6 nCi) of purified
[125I]thyroxine (room temperature for 1 h). 5 µl
of human serum and the total amounts of sTTR solutions were analyzed by
nondenaturing polyacrylamide gel electrophoresis, 10% acrylamide, 0.05 M Tris-HCl, pH 8.9, 4 °C (39) followed by autoradiography.
 |
RESULTS |
Synthetic Strategy--
The choice of ligation sites for the
preparation of sTTR was based on both the amino acid sequence and the
known tertiary structure of the TTR molecule (Fig.
1, A and B) and
involved the ligation of three peptides (Fig. 1C). While the
ligation of two peptides, each of 60-65 residues in length, is a
possible alternative strategy, the degree of difficulty of preparing
and purifying peptides of this length is comparable with the difficulty
of a second ligation. In addition, since all of the residues considered for future labeling studies occur within the last 30 residues, it was
desirable to prepare a relatively small C-terminal fragment. It was
anticipated that this approach would increase the ease of preparing
several sTTR molecules with selective labels, since only this fragment
would need to be resynthesized.

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 1.
A, sequence of human transthyretin
1-127 and depiction of ligation points within the monomeric sequence.
The chemoselective thioether ligation sites are underlined.
The cysteine at position 10 was changed to Ala in the current
synthesis. B, ribbon diagram of the
TTR monomer and tetramer showing the sites of chemical ligation in the
folded molecule. C, names and sequences of the three
peptides synthesized for the current study of sTTR.
|
|
The thioether linker spans a distance equivalent to two amino acids, is
highly flexible, and is nonfunctionalized. While Gly-Gly sequences are
thus ideally suited as ligation points, the TTR sequence contains no
Gly-Gly site, although several Ser-Gly sites are present at convenient
positions. Two of these Ser-Gly sites exist in loop positions within
the TTR structure, which were considered to be potentially more
tolerant of surrogate amide bonds than
-sheet or helical regions.
Ser100-Gly101 occurs in the loop between
-strands F and G, and Ser52-Gly53 occurs in
the loop between
-strands C and D. Neither are close to the hormone
binding site or at points of intersubunit contact. A third Ser-Gly site
(Ser46-Gly47) occurs in the center of
-strand C and was ruled out as a potential ligation point due to the
likely disruption of the
-sheet by a surrogate amide.
The selection of Ser52-Gly53 and
Ser100-Gly101 ligation sites required the
synthesis of three peptides of 51, 46, and 26 residues. The ligation
strategy for the three peptides is outlined in Fig.
2. The N-bromoacetylated
102-127 (peptide I) is ligated to the
N-chloroacetylated, C-thiolated 54-99 (peptide
II). The ligation proceeds preferentially between the thiol
and the bromoacetyl groups, so that polymerization of the peptide
II should not occur. The purified chloroacetylated 54-127
(peptide IV) may then undergo simultaneous or sequential
exchange to the iodoacetylated form (peptide V) and ligation
with C-thiolated 1-51 (peptide III) to produce fully
ligated 1-127 (peptide VI).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 2.
Ligation scheme for the synthesis of
transthyretin showing the three-step process to give synthetic
transthyretin.
|
|
In addition to nonnative bonds, one other modification was made to
sTTR. Cysteine 10 was replaced with alanine in order to avoid any
problems of competition for the iodoacetyl peptide fragment during the
ligation reaction and to exclude the possibility of oxidation when the
protein was folded. Alanine was chosen due to its similarity in bulk
and hydrophobicity to cysteine. This was considered unlikely to have
any detrimental effect on the folding or activity of TTR.
Peptide Synthesis and Purification--
Trial syntheses of the
peptides I, II, and III were carried
out to see how each of the peptides would behave during the synthesis,
cleavage, and ligation reactions. This preliminary work showed that all
three peptides could be readily synthesized, cleaved from the resin,
and purified, excepting the middle fragment, which tended to retain the
methylphenoxyacetic acid (AMPA) linker at the C-terminal thiol after HF
treatment. This was detected as a result of cleavage at the C-terminal
amino acid attached to the resin on which the peptide-AMPA linker was synthesized (see "Experimental Procedures" for details). Despite this, the three target peptides were obtained in good yield and purity.
Ligation of Peptides Iand II--
Ligation
of peptides I and II proceeded cleanly to give
peptide IV (Fig. 2). The purification and monitoring of the
ligation reaction was complicated by the fact that the ligated peptide
IV co-elutes with the starting peptide II; thus,
the use of an excess of peptide I was required to drive the
reaction to completion. After 2 h, the starting peptide
II could not be detected, and the ligated peptide IV was present in high yield as judged by HPLC and ESMS (Fig. 3, A and B).
The ligation was left overnight for completion of the reaction. The
broad HPLC profile of peptide II was attributed to the
chloroacetyl and the thiol functionalities at the N and C termini of
the peptide. The absence of the chloroacetyl group, in particular,
significantly reduces the broadness of the peak shape.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 3.
A, HPLC traces showing the peptide
ligation mixture for peptides I and II at time 0 (top) and 2 h (bottom). The resultant
ligated peptide IV elutes at the same position that the
starting peptide II elutes. B, the mass spectrum
of the peak for peptide IV, showing the formation of the
ligated peptide. *, daughter ions of starting peptide II
still present after 2 h. Inset, mass spectrum
reconstruct showing the correct mass for the ligated peptide
IV.
|
|
Formation of the Iodoacetyl Peptide V--
The initial
chloro-iodo exchange reaction and the subsequent ligation were carried
out in situ (peptide III was added to peptide
IV in saturated KI solution). Most of the thiol peptide
III was found to form disulfide dimers, and the iodoacetyl
peptide V also lost a small percentage of its iodofunctionality. In subsequent ligations, the iodoacetyl peptide V was either purified by RP-HPLC and the ligation reaction was carried out immediately after lyophilization, or it was purified by
rapid desalting using a PD10 column (Amersham Pharmacia Biotech) followed by ligation.
Ligation of Peptides III and V to Give
VI--
After purification of the iodoacetyl peptide
V, its ligation to peptide III (1-51 TTR) was
carried out as rapidly as possible under nitrogen. Samples were removed
from the ligation mixture and analyzed by HPLC and ESMS. The rate of
the ligation reaction, affording the ligated 1-127 TTR VI,
was monitored by a slow HPLC gradient (0.5%/min B) as the ligated
product eluted very close to the two starting peptides (III
and V) and the disulfide-linked peptide formed by the
unreacted excess of peptide III (Fig.
4A). The ligated 1-127
peptide VI was easily purified by RP-HPLC in excellent
yield, and its identity was confirmed by ESMS (Fig. 4B).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 4.
A, HPLC traces showing the peptide
ligation mixture for peptides III and V at time 0 (above)
and 2 h (below). B, the mass spectrum of a
sample of the peak for the ligated peptide VI, 1-127.
Inset, mass spectrum reconstruct showing the correct mass
for the ligated synthetic TTR, peptide VI (13,675 Da), as
well as the presence of a deletion product (13,522 Da).
|
|
Folding of Synthetic TTR to Give the 54-kDa Tetrameric
Complex--
The sTTR spontaneously folded to its tetramer
complex in the presence of the ligand T4, in 0.075 M
NH4HCO3. In addition to the formation of
tetramer (as initially determined by its equivalent retention time to
that of native TTR upon size exclusion chromatography), two other
products were also formed. These corresponded to species with an
intermediate molecular weight, likely to be a dimeric form of sTTR, as
well as a high molecular weight aggregated sTTR (Fig.
5, top trace). The ratio of
these products altered over time in the refolding buffer, with a
gradual accumulation of the high molecular weight aggregate. After
isolating the tetrameric complex, it was equilibrated at 37 °C for
48 h in the presence of excess T4 ligand. Some reequilibration
between the monomeric, tetrameric, and high molecular weight aggregates
occurred in the initial 2 h, but the ratios of tetramer to monomer
and the high molecular weight aggregates remained constant after that
time (Fig. 5, bottom trace).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 5.
Gel filtration profiles of the solution used
for folding the ligated synthetic transthyretin VI. An Amersham
Pharmacia Biotech Superdex 75 10/30 column used 0.075 M NH4HCO3, pH 8.3, with 10%
CH3CN and a flow rate of 0.3 ml/min. The protein was
detected at 214 nm. The top trace shows the
protein VI (3.65 nM) after 18 h at room
temperature in 0.375 M NH4HCO3 with
0.5 equivalents of T4 (excess T4 precipitates from solution). The
bottom trace shows the isolated synthetic
transthyretin VI tetrameric complex after reequilibration at
37 °C for 48 h. Monomeric and dimeric species, as ascertained
by their retention time, are indicated.
|
|
Western Analysis of Synthetic TTR--
Western analysis was
employed to determine the subunit molecular mass and confirm the
recognition of the sTTR by anti-TTR antiserum (Fig.
6). An aliquot of human serum was
analyzed as a positive control. TTR has a subunit molecular mass of
~15 kDa, as estimated by SDS-polyacrylamide gel electrophoresis (39). The interactions between monomers to form the dimer are very strong, and even after boiling in the presence of 2% SDS for 20 min, some TTR
still exists as a dimer (38). Thus, bands with molecular masses of
about 15 and of about 34 kDa correspond to the TTR monomer and dimer,
respectively. Bands are also apparent at higher molecular masses. These
result from nonspecific binding of the antibodies to other proteins in
serum as is commonly observed (35).

View larger version (58K):
[in this window]
[in a new window]
|
Fig. 6.
Western analysis for the presence of
transthyretin in human serum and in solution containing synthetic
transthyretin. Samples were separated in an SDS-polyacrylamide
gel, and then proteins were transferred onto a nitrocellulose membrane
and probed with antiserum against a mixture of transthyretins purified
from human, wallaby, and chicken sera (raised in a rabbit), and the
secondary antibody was anti-rabbit Ig (raised in sheep). Detection was
performed using enhanced chemiluminescence (see "Experimental
Procedures" for details). Lane 1, 2 µl of
human serum; lane 2, 25 µl of solution
containing 0.5 µg of synthetic transthyretin; lane
3, 50 µl of solution containing 1.0 µg of synthetic
transthyretin. Molecular weight markers (× 10 3) were "Mark 12" from Novex:
-galactoside, 116.3; phosphorylase b, 97.4; serum
albumin, 66.3; glutamic dehydrogenase, 55.4; lactate dehydrogenase,
36.5; carbonic anhydrase, 31; trypsin inhibitor, 21.5; lysozyme, 14.4;
aprotinin, 6; insulin B chain, 3.5; and insulin A chain, 2. The
positions of the origin and migration of transthyretin dimer,
transthyretin monomer, and the electrophoretic front are
indicated.
|
|
Synthetic TTR gave rise to bands corresponding to the molecular masses
of the sTTR monomer and dimer (Fig. 6, lanes 2 and 3). The bands were discrete, and no indication of
partially synthesized or partially degraded sTTR was apparent.
Analysis of Thyroxine Binding to Synthetic TTR--
The correct
folding and formation of the tetramer was assessed by analyzing
nondenaturing polyacrylamide gel migration combined with a
[125I]thyroxine binding assay. The analysis of
[125I]thyroxine binding to proteins in human serum was
used as the reference. This revealed the presence of thyroxine-binding
globulin, albumin, and TTR (Fig. 7,
lanes 1 and 4). The binding of
[125I]thyroxine by sTTR was clearly demonstrated in both
the aliquots of 80 µl of solution containing 1.6 µg of sTTR,
following incubation with both 1 µl (1.1 fmol) and 4 µl (4.4 fmol)
of [125I]thyroxine (Fig. 7, lanes 2 and 3, respectively). The position of migration was almost
identical to that in serum, indicating that the tetrameric size, shape,
and charge distribution of sTTR were almost identical to native TTR.
There was no evidence for the existence of aggregates.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 7.
Analysis of thyroxine binding to proteins in
human serum and to synthetic transthyretin. Aliquots of human
serum (10 µl) and of synthetic TTR solution (80 µl containing 1.6 µg) were incubated with [125I]thyroxine prior to
separation in a nondenaturing polyacrylamide gel, pH 8.6. The gel was
dried and then exposed to autoradiographic film for 7 days (see
"Experimental Procedures"). Lane 1, 5 µl of
human serum; lanes 2 and 3, 80 µl of
solution containing 1.6 µg of synthetic transthyretin with 1.1 and
4.4 fmol of [125I]thyroxine respectively; lane
4, 5 µl of human serum. The positions of migration of
human thyroxine-binding globulin (TBG), albumin, and
transthyretin (TTR) are indicated. The positions of the
origin and front of the gel are also indicated.
|
|
 |
DISCUSSION |
We have shown that it is possible to chemically synthesize and
correctly fold a transthyretin analog (sTTR) from its monomeric unfolded state to produce the 54-kDa tetrameric quaternary structure in
the presence of TTR's strongest binding native ligand, T4. This is
probably due to the stabilizing effect of T4, which has previously been
reported to stabilize native TTR against acid denaturation, leading to
the formation of amyloid fibrils (15). The integrity of the final
product was confirmed by native TTR antibody recognition and ligand
binding studies. Since the binding site for thyroid hormone ligands is
formed only upon the formation of the tetrameric species, ligand
binding also demonstrated tetramer formation.
Competing folding pathways involving the formation of high molecular
weight aggregate as well as the appearance of smaller molecular weight
species, possibly corresponding to dimeric and monomeric sTTR, were
also apparent. The former pathway was expected, considering the
predisposition for TTR to form amyloid fibrils and, in particular, the
increased propensity of many mutant forms of TTR to do so. The
appearance of dimeric-like species of sTTR, however, was unexpected,
since this species has never previously been reported. The current
model of the unfolding pathway of TTR involves a transition between
correctly folded TTR to its monomeric form via a perturbed tetramer and
an extremely transient dimeric form (40). The monomeric TTR is thought
to be in equilibrium with a molten globule-like monomeric structure or
to be sequestered to irreversible amyloid formation. Our refolding
studies of sTTR have revealed a species that appears to have the
molecular weight of the dimeric form of sTTR. It was not possible to
isolate this dimeric species for further identification.
Rechromatographing the species using gel diffusion chromatography only
gave rise to the high molecular weight aggregate and some of the
monomeric species. This highly unstable form may be unique to sTTR or
represent an alternative species that could be the basic unit of
TTR-based amyloid fibrils.
It is not surprising that the sTTR construct has a high propensity to
form aggregates. Investigations into mutant forms of TTR have shown
that even in the absence of apparent perturbations to the tertiary and
quaternary structure of TTR, most mutant forms of TTR display a higher
propensity to form amyloid fibril than native TTR (41-43). The
mutations, rather than underlying an alternative TTR conformation, are
thought to cause subtle perturbations to the equilibrium between the
different forms of TTR. Any such movement toward the monomeric form of
TTR thus results in the increased opportunity for TTR monomer to be
irreversibly incorporated into amyloid.
As observed for native TTR, the tetrameric form of sTTR is its most
stable form. Early studies of recombinant TTR and TTR isolated from
serum showed that it is very difficult to dissociate the tetrameric
structure into its monomeric subunits. TTR was reported as stable in
chaotropic solutions of 8 M urea and 6 M guanadinium hydrochloride (7). More recent studies of the denaturation pathway of recombinant TTR showed that the tetrameric unit does not
begin to dissociate into unfolded monomer until the chaotrope concentration exceeds 4 M guanadinium hydrochloride (44).
Fully unfolded recombinant TTR (denatured in 7 M
guanadinium hydrochloride) does not refold until the denaturant
concentration has dropped well below 2 M guanadinium
hydrochloride, showing that the tetrameric TTR structure is kinetically
highly stable.
In the current study, sTTR could not be refolded by slowly decreasing
the denaturant concentration by dialysis. This only led to the
formation of high molecular weight aggregates. sTTR did, however, fold
to the tetrameric structure at a low salt concentration in the presence
of the native T4 ligand. Folding the protein using ligands more soluble
than T4 such as 3,5,3'-triiodothyronine, 3',5',3-triiodothyronine,
diiodothyronine, and thyronine did not give rise to the tetrameric form
of the protein. This may have been due to the lower
Kd of these ligands to TTR relative to the T4
ligand, thereby not stabilizing the sTTR complex to the same degree as
the T4 ligand.
It is not known whether the sTTR tetramer forms and then is stabilized
by T4 or the tetramer spontaneously forms about the T4. The
stabilization of sTTR by a high affinity ligand, however, appears
analogous to the stabilization observed for TTR in the presence of
several nonsteroidal anti-inflammatory drugs currently under
investigation for their ability to inhibit human transthyretin amyloid
disease (17). These molecules, which include flufenamic acid,
diclofenac, and flurbiprofen, bind in the T4 binding site of TTR (45),
forming similar polar and nonpolar contacts as do the natural ligands.
These interactions are thought to stabilize the native quaternary
structure of TTR against pH-mediated dissociation and conformational
changes associated with amyloid formation (16). It may be that some of
these drugs would also promote the folding of sTTR from its unfolded
monomeric state.
In conclusion, the current study reports a methodology for the total
chemical synthesis of a TTR analog (sTTR) through use of a thioether
strategy for the sequential ligation of three peptides and successful
folding and formation of the sTTR tetramer in the presence of the
native ligand T4. It is remarkable that this macromolecule can be
synthesized, however, since other multimeric forms of sTTR also formed
readily, the construct may not be not ideal for biophysical studies. It
is possible that other TTR analogs made using thioether linkages placed
in alternative positions may provide closer mimics of native structure.
Such constructs potentially allow the incorporation of 15N
or 13C isotopic labels at TTR ligand binding sites and may
facilitate future TTR-ligand studies using NMR spectroscopy. In
particular, the kinetics of the interaction between sTTR and
TTR-binding drugs of current interest for their antiamyloidogenic
capacity may be carried out in the presence of other competitive
binding proteins to provide a more complete picture of their mode of
action in serum.