Characterization of the Thyroxine-Binding Site of Thyroxine-Binding Globulin by Site-Directed Mutagenesis
Christoph Buettner1,
Helmut Grasberger,
Kristine Hermansdorfer,
Bingkun Chen2,
Bettina Treske and
Onno E. Janssen
Department of Medicine Klinikum Innenstadt
Ludwig-Maximilians-University D-80336 Munich, Germany
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ABSTRACT
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The principal transport protein for
T4 in human blood, thyroxine-binding globulin
(TBG), binds T4 with an exceptionally high
affinity (Ka = 1010
M-1). Its homology
to the superfamily of the serpins has recently been used in the design
of chimeric proteins, providing experimental evidence that an
eight-stranded ß-barrel domain encompasses the ligand-binding site.
We have now characterized the T4 binding site
by site-directed mutagenesis. Sequence alignment of TBG from
several species revealed a phylogenetically highly conserved stretch of
amino acids comprising strands 2B and 3B of the ß-barrel motif.
Mutations within this region (Val228Glu,
Cys234Trp, Thr235Trp, Thr235Gln,
Lys253Ala, and Lys253Asp), designed to impose
steric hindrance or restriction of its mobility, had no significant
influence on T4 binding. However, binding affinity was
20-fold reduced by introduction of an N-linked glycosylation site at
the turn between strands 2B and 3B (Leu246Thr) without
compromising the proper folding of this mutant as assessed by
immunological methods. In most other serpins, this glycosylation site
is highly conserved and has been shown to be crucial for cortisol
binding of corticosteroid-binding globulin, the only other member of
the serpins with a transport function. The ligand-binding site could
thus be located to a highly aromatic environment deep within the
ß-barrel. The importance of the binding sites aromatic character
was investigated by exchanging phenylalanines with alanines. Indeed,
these experiments revealed that substitution of Phe249 in
the middle of strand 3B completely abolished T4 binding,
while the substitution of several other phenylalanines had no effect.
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INTRODUCTION
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In human serum, 70% of thyroid hormones is bound to
thyroxine-binding globulin (TBG), a 54-kDa glycoprotein of
hepatic origin (1, 2). Biochemical interest in TBG stems mainly from
its exceptionally high binding affinity for T4
[association constant (Ka) = 1010
M-1] and T3 (Ka
= 109 M-1) (3). In contrast to the
other T4-binding proteins, transthyretin and albumin, its
ligand interaction is undefined at a molecular level as attempts to
crystallize TBG have failed (4), probably due to its microheterogenic
glycosylation (5).
TBG belongs to the superfamily of serine-proteinase inhibitors
(serpins) (6), a functionally heterogeneous group of more than 100
proteins, including
1-proteinase inhibitor
(
1PI),
1-antichymotrypsin, and
corticosteroid-binding globulin (CBG). TBG and CBG are the only serpins
with a transport function for small hydrophobic ligands. The
crystallographic structures of several serpins have been determined and
were found to be highly conserved (reviewed in Refs. 7, 8). The
structure of the archetypal
1PI consists of three large
ß-sheets (AC) and eight well defined helices (9). This structure
model has been successfully used as template for structure-function
correlations of other serpins (7), including the heat-resistant
TBG-Chicago variant (10). Since natural variants with binding defects
invariably also have low expression levels and increased serum
concentrations of the denatured molecule, the correlation of their
mutations with the structure model has been of limited value (reviewed
in Refs. 11, 12).
Binding studies of T4 analogs support the notion that all
parts of the molecule participate in its avid binding to TBG (13).
Considering the high binding affinity, T4 would seem to
bind deep in a binding pocket, similar to T4 binding to
transthyretin (14) rather than binding to the flat pocket in
albumin (15). In analogy to the ligand-binding site of transthyretin, a
barrel of ß-strands, formed by sheets B and C, has been proposed as
the binding domain of TBG (16). This is supported by affinity
cross-linking of Lys253 (17), which maps to the binding
cavity. The ß-barrel of TBG has also recently been transferred to the
1PI scaffold, generating a T4-binding
chimera, confirming that this structural domain encompasses the
ligand-binding site (18). A region corresponding to strands 2B and 3B
of the eight-stranded ß-barrel is highly conserved when compared with
TBG from sheep (19) and rat (20) (Fig. 1
), all of which have the same high
T4-binding affinity (21). In this paper, we present a
characterization of this phylogenetically conserved region by
site-directed mutagenesis.

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Figure 1. Sequence Alignment of TBG from Different Species
with Other Serpins
The sequence between V228 and E254 of TBG is
phylogenetically highly conserved. The only mismatches are at position
230 and 243. In the 1PI structure model this region
forms strands 2B and 3B, which contribute to the ß-barrel motif. Note
that TBG has an Asn-linked glycosylation consensus site at the
beginning of s2B (boxed), whereas in other serpins a
glycosylation site is found near the turn between s2B and s3B and thus
at the opposite side of the ß-barrel. hTBG, Human TBG; rTBG, rat TBG;
sTBG, sheep TBG; ACh, 1-antichymotrypsin.
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RESULTS
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Design of TBG Mutations
TBG from sheep (19) and rat (20) share 82% and 76% of their
amino acid sequence with human TBG, respectively. However, within the
putative ligand-binding domain, a stretch of 26 residues
(Val228-Lys253) is almost completely conserved
in all known TBG sequences. This region corresponds to two antiparallel
ß-strands, 2B and 3B, of the ß-barrel motif of
1PI.
Comparison of TBG with other serpins reveals several distinct
differences (Fig. 1
). At the entrance to the ß-barrel, the
Thr235 of TBG corresponds to the bulkier Trp238
in
1PI. This substitution has been speculated to block
the access to the ß-barrel in
1PI (7, 22, 23), which
has no known ligand. To test this hypothesis, Thr235 of TBG
was substituted by a tryptophan (TBG-mB). This mutation destroys the
glycosylation site 233235, which is unique to and conserved in all
known TBG sequences. The corresponding glycosylation site of other
serpins is located at residues 247249 (
1PI numbering)
and lies at the opposite end of the ß-barrel. Mutation mA
(Cys234
Trp) introduces a tryptophan at the entrance to
the ß-barrel but keeps the glycosylation site 233235 intact.
Mutation mC (Thr235
Gln) destroys the glycosylation site,
but the small glutamine should not block access to the ß-barrel.
Mutation mD (Leu246
Thr) generates an intact
glycosylation site at 244246, corresponding to the highly conserved
glycosylation site of other serpins. Compared with normal TBG
(TBG-N), mutant mD thus has an additional glycosylation site. Access to
the ß-barrel could also be dependent on the mobility of the involved
ß-strands. In
1PI,
1-antichymotrypsin,
and several other serpins, but not in TBG, the ß-strands 2B and 3B
are held together by a salt bridge. Substitution of Val228
by glutamic acid (mG) allows the formation of a salt bridge between
residues 228 and 253 of TBG and thus might impede T4
binding. Binding studies with T4 analogs (13) suggest that
interactions of the negatively charged oxygen and carboxy groups and
the positively charged amino group of the T4 molecule are
important for the high-affinity binding. An attractive candidate for
electrostatic interactions is the positively charged
Lys253, which has been shown to be near the ligand-binding
domain by affinity labeling (17) and lies at the entrance of the
ß-barrel. Lys253 was substituted with the negatively
charged aspartic acid (mE) and the neutral alanine (mH) to test whether
electrostatic interactions at this position play a role in
T4-binding. However, interactions within the binding cavity
would appear to be more important than those with the entrance of the
ß-barrel. It has been speculated (22) that the iodines of
T4 could interact with aromatic residues, comparable to
mercury-iodide binding to sperm whale myoglobin (24). To test this
hypothesis, phenylalanines thought to contribute to the aromatic
interior of the binding cavity were individually substituted by alanine
(mutations mF249, mF284, mF288, mF371, and mF315). All mutants were
constructed by site-directed mutagenesis and verified by dideoxy
sequencing as described in Materials and Methods.
Expression of Normal and Mutant TBG in Reticulocyte Lysate
The linearized vectors for TBG-N and the mutants were transcribed
in vitro and translated in reticulocyte lysate. All mutants
were synthesized with comparable efficiency and had identical patterns
of nonglycosylated forms on SDS-PAGE (data not shown). After addition
of canine microsomal membranes to the translation reactions, all
variants had a major band at 44 kDa, which corresponded to
unglycosylated TBG after signal peptide processing, and several new
bands of higher mol wt, which reflected the number of available
N-glycosylation sites (Fig. 2
). As
expected, mutants mB and mC had one less glycosylation site and mutant
mD had an additional glycosylation site when compared with TBG-N. The
phenylalanine mutations were not expressed in the reticulocyte lysate
system, since no effect on glycosylation was expected.

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Figure 2. Synthesis of Normal and Mutant TBG in Reticulocyte
Lysate
In vitro transcribed RNA of the TBG mutants was
translated in rabbit reticulocyte lysate. In the presence of canine
microsomal membranes (CMM), all variants showed a major band (44 kDa)
corresponding to unglycosylated TBG with a processed signal peptide.
The number of the slower migrating bands reflects the number of
N-glycosylation sites available for core glycosylation within the
microsomes. Note the introduction of an additional glycosylation site
in mutant mD, the deletion of a glycosylation site in mutants mB and
mC, and the detrimental effect of mutant mA on the amount of the
completely glycosylated product. MWM, Mol wt marker (69K, 46K).
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Expression of Normal and Mutant TBG in Xenopus
Oocytes
Oocytes were removed from Xenopus laevis, culled,
injected with TBG synthetic messenger RNA (sRNAs), and incubated in
medium with [35S]methionine. The TBG mutants synthesized
and secreted into the medium were then submitted to SDS-PAGE (Fig. 3
). The broad appearance of the bands is
due to the microheterogenic glycosylation, which is faithfully
reproduced in the Xenopus system. In addition, variants
mBmH differed by size depending on the number of glycosylation sites.
However, no significant differences of the amount of secreted protein
could be observed when compared with TBG-N except for mutant mA, which
was not produced by the oocytes in several experiments (Fig. 3A
). Of
the phenylalanine mutants, mF284mF371 were synthesized by the oocytes
in the same amount as TBG-N (data not shown). TBG mF249 was also
expressed with a mol wt equal to TBG-N, but in slightly lower amounts
(Fig. 3B
).

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Figure 3. SDS-PAGE Analysis of TBG Variants Expressed in
Xenopus Oocytes
Oocytes injected with sRNAs were incubated in the presence of
[35S]methionine. A, The mutants mBmH differed by size
depending on the number of glycosylation sites. While mutant mA was not
secreted into the medium, no significant differences in the efficiency
of synthesis and secretion of the other TBG mutants compared with TBG-N
were found in four independent experiments. B, Of the Phe Ala
mutants, mF249 was secreted in slightly reduced amounts compared with
TBG-N, while mF284, mF288, mF315, and mF371 differed neither in
expression level nor in apparent mol wt from TBG-N (not shown). MWM:
14C-labeled mol wt markers.
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Analysis of T4 Binding to TBG
The amount of functionally active TBG synthesized in
Xenopus oocytes was examined by measurement of the
T4 binding characteristics. The TBG mutants were expressed
as described above, but without the addition of
[35S]methionine. Scatchard analysis of the secreted TBG
mutants revealed no significant differences in T4 binding
affinity and capacity/oocyte of the variants mB, mC, mG, and mH (Fig. 4A
and Table 1
). However, the T4 binding
affinity of mD was 20 times lower than that of TBG-N. Of the
phenylalanine mutations, only mF249 showed a significant reduction of
binding affinity, by at least 2 orders of magnitude (Fig. 4B
and Table 1
).

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Figure 4. Scatchard Analysis of T4-Binding to TBG
Variants Expressed in Xenopus Oocytes
TBG-N and the TBG mutants were expressed in oocytes. The secreted TBGs
were incubated with [125I]T4 and increasing
amounts of unlabeled T4. Of all mutants, no significant
differences in T4-binding affinity (slope) and binding
capacity/oocyte (intercept) were found, except for TBG mD and mF249. A,
The binding affinity (Ka) of mutant mD was almost 20-fold
lower than that of TBG-N (108 M-1
vs. 1010 M-1). (), TBG-N; ,
mB; , mC; , mD; , mE; , mG; , mH. B, No Scatchard
analysis could be obtained from mutant mF249 due to its substantially
reduced specific T4 binding with an estimated upper limit
of its Ka of at least 2 orders of magnitude lower than
TBG-N or the other F A variants, respectively. , TBG-N; ,
mF284; , mF288; , mF315; , mF371.
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Heat Resistance of TBG Variants
To determine the functional stability of the TBG variants, heat
denaturation was performed. At 58 C, no significant differences in the
rate of heat denaturation of TBG-N and the variants mB, mC, mE, mG, and
mH were found (Fig. 5
).
Immunoprecipitation of TBG-N and Variant mD
To test for the immunological integrity of variant mD, samples
were immunoprecipitated with either a polyclonal antibody against both
nTBG and dnTBG or an antibody specific for dnTBG. Both TBG-N and mD
were almost exclusively recognized by anti-nTBG (Fig. 6A
). In addition, immunoprecipitation in
the presence of excess nTBG or dnTBG, respectively, revealed that both
TBG-N and TBG mD were competed only by nTBG, indicating that both
molecules were correctly folded (Fig. 6B
).

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Figure 6. Immunoprecipitation of TBG-N and TBG mD synthesized
in Xenopus Oocytes
A, [35S]methionine-labeled samples were
immunoprecipitated with either a polyclonal antibody that recognizes
both nTBG (n) and dnTBG (dn) or an antibody that recognizes only dnTBG
and then submitted to SDS-PAGE and autoradiographed. Both TBG-N and TBG
mD were recognized by anti-n/dnTBG (lanes 2 and 4) but only weakly by
the anti-dnTBG antibody (lanes 3 and 5). B, Samples were
immunoprecipitated with the anti-n/dnTBG antibody without (-) and with
competition; n denotes residual nTBG from samples competed with dnTBG;
dn denotes residual dnTBG from samples competed with nTBG. Both TBG-N
and TBG mD were competed only by nTBG, indicating that the molecules
were correctly folded. MWM, 14C-labeled mol wt markers.
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DISCUSSION
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In this paper we describe a rational approach to define the impact
of individual residues of TBG on T4 binding by
site-directed mutagenesis. Previous attempts to characterize the
binding site of TBG have relied on the identification of naturally
occurring mutations. So far, all of the six binding-deficient variants
characterized on a molecular level exhibit lower expression levels than
TBG-N and more or less explicit heat lability (11, 12, 25). Since these
natural variants are also characterized by increased dnTBG serum
concentrations, they most likely decrease T4 binding by
gross structural changes. Thus, the analysis of natural occurring TBG
variants has failed to contribute to our understanding of the
structural requirements of TBG for T4 binding, consistent
with the location of the affected residues throughout the molecule but
not within the ß-barrel.
Apart from mutant mA, all TBG mutants generated in this study were
synthesized and secreted by Xenopus oocytes in similar
amounts as TBG-N. The respective amino acid substitutions can thus be
assumed not to interfere with the folding and processing of the
molecule, because this has been found to lead to lower expression
levels in all known natural mutations (26, 27). Mutants mB, mC, mG, and
mH even had a normal heat stability. The secretion defect of mA most
likely results from inefficient glycosylation of the neighboring
Asn233, as demonstrated by the reduced utilization of this
glycosylation site in the cell-free translation system (Fig. 2
). This
finding is compatible with a previous report on the glycosylation
efficiency of N-linked core glycosylation, where glycosylation sites
with Trp in the X-position of an Asn-X-Ser sequon were found to be
inefficiently processed (28).
Neither the introduction of a putative salt bridge between the
ß-strands, nor the sterical hindrance by introduction of a bulky
tryptophan, nor modification of the glycosylation at the entrance to
the ligand-binding site had an impact on the T4 binding
affinity. A major effect, however, was seen after introduction of a new
glycosylation site at position 244 (mutant mD), a site highly conserved
in other serpins (Fig. 1
). The integrity of mutant mD could not be
ascertained by heat denaturation due to its low binding capacity.
However, immunological studies showed that it was detected only with
antibodies against nTBG and not dnTBG and could only be displaced with
nTBG and not dnTBG from polyclonal antibodies (Fig. 6
). Therefore,
introduction of the additional glycosylation site in mutant mD appears
to cause a specific binding defect. As shown in a model of the TBG
ß-barrel (Fig. 7
), Leu246
faces into the solvent rather than into the binding pocket. Its
substitution by threonine in TBG-mD should thus not interfere with
binding per se, but rather by causing glycosylation of
Asn244.

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Figure 7. Structure Model of the TBG ß-Barrel
TBG was modeled based on the structure of the archetypical serpin
1PI. The ß-barrel motif is depicted in
green, strands s2B and s3B are depicted in
brown, and residues relevant for T4 binding
are highlighted in different colors. Phe249
faces into the binding cavity, where it could interact with one of the
T4s iodines. The Leu246, which
is substituted by Thr in TBG-mD, faces away from the binding pocket
into the solvent. Its substitution per se should thus not interfere
with T4 binding, but rather by causing glycosylation of
Asn244. Substitution of
Lys253, which can be affinity labeled (17 ),
had no significant effect on T4 binding, which supports the
concept that T4 binds deep in the ß-barrel. Also shown is
Ala191, which is substituted by Thr in the
binding deficient variant TBG-Aborigine (39 ).
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The new carbohydrate could cause the loss of binding either by a direct
sterical effect or by modulation of the processing of the
ligand-binding site during synthesis of the molecule. The respective
carbohydrate attached to the corresponding Asn247 of
1PI does extend through the interior of the binding
pocket, as shown by its interaction with Trp238 at the
opposite end of the ß-barrel (29). Molecular modeling of
1PI (9) also reveals that the carbohydrate moiety linked
to this site anchors the turn between strands 2B and 3B to helix D.
Accordingly, the binding pocket in
1PI is smaller than
in ovalbumin, the only other serpin lacking glycosylation of the
corresponding site (30). Introduction of the mutation mD in TBG could
thus reduce binding by diminishing the available space for
T4. In contrast, this glycosylation site has been
previously shown to be crucial and sufficient for corticosteroid
binding of CBG (31). CBG normally contains six glycosylation sites and
inactivation of only the Asn238, which corresponds to the
Asn244 of TBG, abolished steroid binding. A mutant
containing only this glycosylation site (the other five were
inactivated) had normal steroid binding, which was not altered by
deglycosylation of the molecule. This suggests that interaction of the
polypeptide with the carbohydrate at the glycosylation site
Asn238 is necessary for the folding and creation of the
steroid-binding site only during CBG biosynthesis (32) and implicates
substantial differences in the structure of the ligand-binding sites of
TBG and CBG.
The reduced binding of mD taken together with the lack of effect of the
mutations at the putative entrance of the ß-barrel (7) would be
compatible with T4 entering the binding cavity from the
opposite side of the molecule, as proposed by Terry and Blake (22) and
Jarvis et al. (23). While the latter entrance would be
conceivably wider, the entrance at the opposite side would still be
wide enough to allow T4 with its dimensions of 6x12 Å to
enter and would directly be restricted by the additional carbohydrate
group in TBG-mD.
Binding of T4 by TBG depends on the presence of its
iodines, especially at the 5'-position (13). It has been speculated
that these might interact with the aromatic, rather than just
hydrophobic, interior of the binding site, namely phenylalanine, and
possibly also tyrosine residues (22). Of the four phenylalanines within
the binding cavity of TBG (Phe249, Phe284,
Phe288, and Phe371) and the Phe315
outside the ß-barrel, only substitution of Phe249 with
alanine (mF249) substantially diminished T4 binding (Table 1
). This residue is located in the middle of strand 3B and thus in
close spatial proximity to mutation mD, as shown in the model of the
interior of the ligand-binding site (Fig. 7
).
In conclusion, mutations mD and mF249 locate the binding site deep
within the ß-barrel of TBG. A more precise structural analysis of
T4 binding awaits crystallization of TBG, attempts at which
have now been intensified.
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MATERIALS AND METHODS
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Site-Directed Mutagenesis
The cDNA of normal TBG (TBG-N) had been subcloned
previously into the pSELECT expression vector (33). Site-directed
mutagenesis (Altered Sites Kit, Promega Corp., Madison,
WI) with oligonucleotides (see Table 2
)
was performed to obtain vectors coding for the TBG mutants mAmH and
the F
A variants (mF249, mF284, mF288, mF315, and mF371). The coding
regions of all TBG mutants were sequenced in its entirety by the
dideoxy-nucleotide termination method (34) using the Sequenase-2 kit
from United States Biochemical Corp. (Cleveland, OH).
In Vitro Transcription
sRNA was prepared with the Gemini-II in vitro
transcription kit and T7 RNA polymerase according to the
recommendations of the supplier (Promega Corp.).
Cell-Free Translation
Translations were carried out in rabbit reticulocyte lysate in a
final volume of 50 µl according to the protocol supplied by the
manufacturer (Promega Corp.). Samples were metabolically
labeled with [35S]methionine (DuPont-NEN, Boston, MA).
For the analysis of early cotranslational events, eight equivalents of
canine microsomal membranes were added.
Preparation of Oocytes and RNA Injection
Ovaries were removed from mature Xenopus laevis (H.
Köhler, Hamburg, Germany) (35) and suspended in OR-IIa medium (83
mM NaCl, 2.5 mM KCl, 1 mM
MgCl2, 1 mM Na2HPO4,
and 5 mM HEPES, pH 7.6) (36). After manual dissection of
the follicles, oocytes were dissociated from the surrounding connective
tissue by incubation in OR-IIa containing 0.2% collagenase type IA
(Sigma Chemical Co., St. Louis, MO) for 2 h with
shaking at room temperature. The liberated oocytes were then rinsed
extensively in OR-IIb medium (OR-IIa with 1 mM
CaCl2 and 100 mg/ml gentamycin), and stage VI oocytes (37)
were separated and kept up to 3 days in OR-IIb with daily medium
changes. After injection with 100 nl of sRNA (0.5 mg/ml), oocytes were
kept on ice for 1 h and then for 26 h at 19 C in OR-IIb. Intact
oocytes (routinely >95%) were transferred to 24-well plates
(Costar) and kept in OR-IIc (OR-IIb with 1 mM
sodium pyruvate), 5 µl/oocyte, at 19 C for up to 4 days, with daily
exchange of medium. Typically, 100 oocytes were injected with each sRNA
preparation. Control oocytes were either injected with water or
noninjected, with identical results. In some experiments, proteins
synthesized by the oocytes were metabolically labeled by addition of
250 mCi [35S]methionine per 500 µl medium.
SDS-Gel Electrophoresis
Products of cell-free translation or proteins synthesized in
Xenopus oocytes were analyzed by the method of Laemmli (38),
using 10% polyacrylamide gels. Gels were dried and autoradiographed at
-90 C on X-AR5 film (Eastman Kodak Co.) with an
intensifying screen.
Measurement of T4 Binding to TBG
Parameters of T4-binding to TBG were measured by a
method previously described in detail (39). Briefly, TBG preparations
were incubated with [125I]T4 (DuPont-NEN) in
the presence of increasing amounts of unlabeled T4.
TBG-bound T4 was separated from free T4 with
anion exchange resin beads (Mallinckrodt, Inc., St. Louis,
MO) and the protein-bound [125I] activity was determined.
The affinity constants (Ka) of TBG preparations were
determined by the method of Scatchard (40).
Immunoprecipitation
Immunoprecipitation was performed according to Kessler (41),
with minor modifications as described (33). The polyclonal antibodies
used in this study (against both nTBG and dnTBG and against dnTBG only)
have been described (42).
Heat Denaturation
The functional stability of the TBG variants was quantified by
thermal denaturation in a water bath at 58 ± 0.1 C for various
periods of time. The samples were then cooled on ice and centrifuged
for 15 min at 13,000 x g to remove precipitated
protein. Residual specific T4 binding capacity was
expressed relative to controls kept at 4 C. The half-lives
(t1/2) of heat denaturation were calculated by least square
analysis of semilogarithmic plots of remaining specific T4
binding vs. time of incubation.
Experimental Animals
All animal studies were conducted in accord with the principles
and procedures outlined in the "Guidelines for Care and Use of
Experimental Animals."
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ACKNOWLEDGMENTS
|
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We thank S. Refetoff (University of Chicago, Chicago, IL) for
kindly providing us with anti-TBG antisera and preparations of nTBG and
dnTBG. We also thank P. Gardner (Howard Hughes Medical Institute,
University of Chicago, Chicago, IL) for the synthesis of
oligonucleotide primers and R. Huber and R. Engh (Max Planck Institute
for Biochemistry, Martinsried, Germany) for help with the structural
data.
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FOOTNOTES
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Address requests for reprints to: Onno E. Janssen, M.D., Molecular Thyroid Study Unit, Department of Medicine, Klinikum Innenstadt, Ludwig-Maximilians-University, Ziemssenstrasse 1, D-80336 Munich, Germany.
1 Current address: Thyroid Division, Department of Medicine, Brigham
and Womens Hospital and Harvard Medical School, Boston, Massachusetts
02115. 
2 Current address: Department of Pathophysiology, Jiamusi Medical
College, Jiamusi, Heilongjiang, 154002 China. 
Received for publication April 30, 1999.
Revision received July 16, 1999.
Accepted for publication July 19, 1999.
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