Role of Extracellular Molecular Chaperones in the Folding of
Oxidized Proteins
REFOLDING OF COLLOIDAL THYROGLOBULIN BY PROTEIN DISULFIDE
ISOMERASE AND IMMUNOGLOBULIN HEAVY CHAIN-BINDING PROTEIN *
Frédéric
Delom,
Bernard
Mallet
,
Pierre
Carayon, and
Pierre-Jean
Lejeune
From the Unité 555 INSERM and Laboratoire de Biochimie
Endocrinienne et Métabolique, Faculté de Médecine,
Université de la Méditerranée,
13385 Marseille Cedex 5, France
Received for publication, February 5, 2000, and in revised form, April 3, 2001
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ABSTRACT |
The process of thyroid hormone
synthesis, which occurs in the lumen of the thyroid follicles, results
from an oxidative reaction leading, as side effects, to the
multimerization of thyroglobulin (TG), the prothyroid
hormone. Although hormone synthesis is a continuous process, the
amount of Tg multimers is relatively constant. Here, we investigated
the role of two molecular chaperones, protein disulfide isomerase (PDI)
and immunoglobulin heavy chain-binding protein (BiP), present in the
follicular lumen, on the multimerization process due to oxidation using
both native Tg and its N-terminal domain (NTD). In vitro,
PDI decreased multimerization of Tg and even suppressed the formation
of NTD multimers. Under the same conditions, BiP was able to bind to Tg
and NTD multimers but did not affect the process of multimerization.
Associating BiP with PDI did not enhance the ability of PDI to limit
the formation of multimers produced by oxidation. However, when BiP and
PDI were reacted together with the multimeric forms and for a longer time (48 h), BiP greatly increased the efficiency of PDI. Accordingly, these two molecular chaperones probably act sequentially on the reduction of the intermolecular disulfide bridges. In the thyroid, a
similar process may also be effective and participate in limiting the
amount of Tg multimers present in the colloid. These results suggest
that extracellular molecular chaperones play a similar role to that
occurring in the endoplasmic reticulum and, furthermore, take part in
the control of multimerization and aggregation of proteins formed by oxidation.
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INTRODUCTION |
In the thyroid gland, the functional unit is the follicle, which
is composed of a monolayer of epithelial cells (the thyrocytes) delimiting a closed space, the follicular lumen, which is filled with
colloid. Thyrocytes are polarized cells that synthesize the prothyroid
hormone: the thyroglobulin
(Tg).1 This protein, which is
the basic component involved in thyroid hormone synthesis, is secreted
into the follicular lumen. Like most newly synthesized proteins, Tg
follows the usual pathway of chaperone-assisted folding in the
endoplasmic reticulum (ER) involving calnexin, calreticulin, Grp78
(BiP), Grp94, PDI, and Grp170 (1-5). Among these molecular chaperones,
PDI certainly plays a fundamental role due to the presence of ~110
cysteine residues in the monomer Tg (330 kDa, 12 S) (6). In normal
conditions, properly folded Tg is dimerized (2 × 330 kDa, 19 S)
without any interchain disulfide bonds, then mature Tg is secreted into
the follicular lumen where Tg19S undergoes the major post-translational modification accompanying the production of thyroid hormones. Hormone
synthesis takes place at the apical surface of the thyrocyte via the
iodination of some tyrosine residues (among 132 tyrosine residues for
Tg19S) and the subsequent coupling of a very limited number of
iodotyrosine residues to form triiodothyronine (T3) and
thyroxine (T4). The iodination and coupling process depend on a H2O2-generating system (NADPH-oxidase) and
thyroperoxidase. During this oxidative reaction, besides participating
in hormone synthesis, Tg19S is also modified by the formation of
interchain covalent cross-links, generating high molecular mass forms
of Tg: the soluble forms, known as Tg27S and Tg37S (4 × 330 kDa
and 8 × 330 kDa, respectively), and the Tg aggregates, which
constitute about 30% of the total Tg in the follicular lumen (7). Tg
aggregates differ from soluble Tg multimers in that they contains about
40% more iodine but almost no hormone (8). In addition, Tg aggregates are formed by several types of covalent cross-links: disulfide, dityrosine, and
-glutamyl-lysine bridges (7-10). The process of
di-, tri-, and multimerization of Tg19S is also observed in vitro as the result of iodination and coupling. The major
difficulty with Tg is its large size, which precludes detailed analysis
in terms of the structure-activity relationships. Therefore, to
circumvent this problem, we have often used a Tg peptide to study the
various process in which the entire molecule is involved. This peptide is obtained from the entire Tg molecule by CNBr treatment. It corresponds to the N-terminal domain (NTD) of the molecule, which contains the main site of hormone synthesis at the Tyr5
(11). The NTD is a dipeptide Asn1-Met127 that
is linked via disulfide bridges to
Glu128-Met171. It contains two sites of
N-glycosylation at Asn57 and Asn91.
Using this model, we have established by performing in vitro iodination and coupling that N-glycosylation was essential
to both hormone formation (12) and multimerization in which covalent cross-links are involved (13).
In a recent study, we reported that in the follicular lumen BiP, Grp94,
and PDI are associated with Tg aggregates. Although these molecular
chaperones carry a retention signal KDEL in the ER, several studies
establish that in secretory cells, some of the molecular chaperones
escape to the ER (14-19).
In the present study, we investigated the role of PDI and BiP in the
process of Tg19S and NTD multimerization during in vitro iodination and coupling. PDI was found to limit the multimerization process more efficiently than BiP. The conjugate action of these two
molecular chaperones did not modify the rate of Tg and NTD multimers
formed by the oxidative iodination and coupling reaction. On the other
hand, when the multimeric forms of Tg were incubated with PDI and BiP
for 48 h, the refolding of the multimeric forms of Tg occurred.
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EXPERIMENTAL PROCEDURES |
Preparation of the Various Forms of Human Tg--
A thyroid
gland was obtained from a patient with a colloid goiter who underwent
thyroidectomy. After the pathological examination, the gland was frozen
in liquid nitrogen and taken to the laboratory where it was immediately
lyophilized. An aliquot of the lyophilized gland was fragmented and
suspended in 0.1 M phosphate buffer, pH 7.2, for 15 min at
+4 °C. After filtration on gauze, the supernatant of the thyroid
homogenate was salted out (1.8 M phosphate buffer). After
being centrifuged, the precipitate was dissolved with distilled water,
dialyzed against 50 mM phosphate buffer, pH 7.2, and
chromatographed on a Bio-Gel A-5m (Bio-Rad) column (100 × 5 cm) equilibrated with the same buffer. Briefly, the typical elution
profile showed in the void volume of the column, a milky fraction
constituted of Tg aggregates followed by two minor peaks corresponding
to Tg37S and Tg27S in that order (fraction "Tg37S+Tg27S"), and a
major peak identified as Tg19S (20). Elution fractions corresponding to
Tg37S+Tg27S and Tg19S were pooled separately, dialyzed against distilled water, and characterized in terms of their amino acid composition and electrophoretic profile.
In Vitro Iodination and Coupling of Tg19S--
In
vitro iodination and coupling (oxidative system) of Tg19S was
carried out as previously described (12) to obtain iodinated and
coupled Tg19S. Briefly, 1 mg of glucose, 20 nmol of KI, and 5 µg of
lactoperoxidase (Sigma) were added to a final volume of 1 ml of 50 mM Tris-HCl buffer, pH 7.2, containing 1 nmol of Tg19S. The
reaction was initiated by 2.5 µg of glucose oxidase (Sigma), continued for 30 min at 37 °C, and stopped by adding 0.1 M hyposulfite. The same reaction was performed with 4 mM reduced glutathione (Roche Molecular Biochemicals) and
0.5 mM oxidized glutathione in the presence or absence of
PDI (Stress-Gen, Victoria, Canada) and/or 100 µM
Ca2+ and 1 mM ATP (Aldrich) in the presence or
absence of BiP (Stress-Gen). When PDI and/or BiP were added to the
sample, the iodide concentration was increased to 20%, since PDI
contains 12 tyrosine residues (21) and BiP contains 13 tyrosine
residues (22).
Preparation of the NTD of Tg19S--
Tg19S was treated with
cyanogen bromide. The CNBr peptides were separated by chromatography on
a Sephadex G-200 column (Amersham Pharmacia Biotech) in 1 M
propionic acid, and the NTD was then purified on a Bio-Gel P-100 column
in 0.05 M ammonium bicarbonate as previously described
(11). Last, it was dialyzed against distilled water and lyophilized.
In Vitro Iodination and Coupling of the NTD--
NTD (1 nmol),
dissolved in 50 µl of 50 mM Tris-HCl buffer, pH 7.2, was
incubated with 7 nmol of KI, 1 µg of lactoperoxidase, and a
H2O2-generating system consisting of glucose
(0.2 mg) and glucose oxidase (0.5 µg). After 30 min of incubation at
37 °C, the reaction was stopped by adding 0.1 M sodium
hyposulfite. The same reaction was performed with 4 mM
reduced glutathione and 0.5 mM oxidized glutathione in the
presence or absence of PDI and/or 100 µM Ca2+
and 1 mM ATP in the presence or absence of BiP. In the
presence of PDI and/or BiP, iodination was performed with various
amounts of KI taking into account in part the molar ratio molecular
chaperone/NTD and in part the number of tyrosine residues present in
these two molecular chaperones.
In Vitro Iodination of PDI and BiP--
Iodination of PDI and
BiP (1.5 µg each) was performed separately using for each molecular
chaperone 300 pmol of KI, 0.1 µg of lactoperoxidase, and a
H2O2-generating system (20 µg of glucose and
0.1 µg of glucose oxidase). After 30 min of incubation at 37 °C,
the reaction was stopped by adding 0.1 M sodium hyposulfite.
Immunoaffinity Chromatography--
The antibodies anti-PDI or
anti-BiP (Stress-Gen) were coupled to
N-hydroxysuccinimide-activated Sepharose (HiTrap
N-hydroxysuccinimide-activated, Amersham Pharmacia Biotech)
according to the manufacturer's instructions. The ligand was incubated
overnight on the anti-PDI and the anti-BiP affinity columns in 50 mM phosphate buffer, pH 7.2, and 50 mM Tris-HCl
buffer, pH 7.3, 100 mM CaCl2, respectively.
Columns were washed with 6 bed volumes of respective buffers and eluted
with acetic acid solution (pH 3.0). The immunopurified solutions were neutralized and lyophilized. Samples were monitored by performing immunoblotting analysis.
Refolding of Tg19S--
The fraction identified by
chromatography on a Bio-Gel A-5 m column (see above) as Tg37S+Tg27S (10 µg) was dissolved in 200 µl of 50 mM Tris-HCl buffer,
pH 7.2, containing 4 mM reduced glutathione, 0.5 mM oxidized glutathione, 100 µM
Ca2+, 1 mM ATP, and 1 µl of protease
inhibitor mixture (Sigma). Renaturation was initiated by adding
iodinated PDI (0.8 µg) and iodinated BiP (0.6 µg), and the sample
was incubated at 37 °C. At times corresponding to 1, 12, 24, and
48 h, an aliquot (2 µg) was taken and stored at
20 °C for
immunoblotting analysis. The experiment was repeated with native PDI
and BiP (Stress-Gen).
Other Procedures--
SDS-PAGE was performed under nonreductive
conditions using a 5 or 12% acrylamide and a 0.1% SDS gel system.
Protein bands were stained with Coomassie Brilliant Blue.
Immunoblotting was performed with a mouse monoclonal antibody directed
against a specific region of human Tg (monoclonal antibody 2) (23). The second reagent was either gold-conjugate goat anti-mouse IgG (diluted 1/250), detected with a silver enhancing kit (British BioCell International), or horseradish peroxidase-conjugated goat anti-mouse IgG (diluted 1/250,000), detected with Super Signal West Femto maximum
sensitivity substrate (Pierce). Scanning quantitative analysis was
subsequently performed using the NIH Image V1.56 software program.
 |
RESULTS |
Effects of Iodination and Coupling of the Tg19S and
NTD--
After performing in vitro iodination and coupling
of Tg19S or NTD samples for 30 min, we noted upon carrying out SDS-PAGE under nonreducing conditions that Tg (Fig.
1A) and NTD (Fig.
1B) were multimerized. With NTD, quantification of the
various multimerized forms showed that NTD dimer amounted to about 21% ± 8% (n = 8), and another band around 75 kDa
corresponding to NTD trimer was also identified. The percentages of
this latter fraction was variable and low (5 ± 3%). Tg multimer
quantification was more difficult to perform, because the degree of
multimerization was so variable that the fractions were distributed
along the stacking gel, and only some of them reached the running gel.
To evaluate the multimeric forms of Tg, it was therefore necessary to
perform separation on a Bio-Gel A-5m column, which has a better
resolution than SDS-PAGE (data not shown). Under these conditions,
Tg37S+Tg27S amounted to around 25 ± 8% (n = 5).
These values are comparable with those observed in vivo
(24). When the incubation time was extended and when the iodide
concentration was increased, similar results were obtained (data not
shown). Since it was difficult to assess these values when performing
SDS-PAGE, in the forthcoming experiments we designated as Tg multimers
and NTD multimers all the multimerized forms generated during
iodination and coupling.

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Fig. 1.
In vitro multimerization of Tg19S
and NTD. SDS-PAGE was performed with 5% acrylamide in the case of
Tg (A) and 12% acrylamide in that of NTD (B).
Tg19S and NTD (5 µg each) were iodinated and coupled using
lactoperoxidase, the glucose-glucose oxidase system, to produce
H2O2, and iodide (20 mol of I /mol
of Tg19S and 7 mol of I /mol of NTD). After incubation for
30 min at 37 °C, each sample was vacuum-dried before undergoing
electrophoresis. A, lane 1 is Tg19S, and
lane 2 is the result of iodination and coupling of Tg19S.
B, lane 1 is NTD, and lane 2 is the
result of iodination and coupling of NTD. Proteins were stained with
Coomassie Brilliant Blue. In A the arrows
indicate Tg19S and Tg multimers. In B the arrows
indicate NTD and NTD multimers.
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Role of PDI in the Multimerization Process--
It is widely
recognized that PDI can influence the refolding of denatured and/or
reduced proteins in vitro. Because we previously established
that PDI is associated with aggregated Tg (8), we studied the role of
PDI in the oxidation reaction induced by in vitro iodination
and coupling. The presence of PDI in various PDI/ligand ratios
(mol/mol) limited the amount of Tg multimers (Fig.
2A) and NTD multimers (Fig.
2B). These results specifically involved PDI and were not
due to the redox system GSH/GSSG, since in the absence of PDI, this
system did not affect the proportions of the multimers (Fig. 2,
A and B, lanes 1).

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Fig. 2.
Effects of PDI on the multimerization of
Tg19S and NTD. A, Tg19S (0.3 µg) was iodinated and
coupled as in Fig. 1 in presence of the redox system GSH/GSSG (200 µM/50 µM). Lane 1 corresponds
to Tg19S iodinated and coupled without PDI; lanes 2-5
correspond to Tg19S iodinated and coupled (see "Experimental
Procedures") in the presence of PDI at PDI/Tg19S molar ratios of 0.2, 0.5, 1.0, and 2.0, respectively. Tg19S and Tg multimers are indicated.
B, NTD (0.3 µg) was iodinated and coupled as in Fig. 1 in
the presence of the redox system GSH/GSSG (200 µM/50
µM). Lane 1, NTD iodinated and coupled without
PDI; lanes 2-5, NTD iodinated and coupled (see
"Experimental Procedures") in the presence of PDI at PDI/NTD molar
ratios of 0.2, 0.5, 1.0, and 2.0, respectively. NTD and NTD multimer
are indicated. In A and B, immunoblots were
probed with anti-Tg monoclonal antibody and detected by
chemiluminescence.
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With NTD, no multimeric form was obtained with PDI at a molar ratio
1
(Fig. 2B, lanes 4 and 5), whereas PDI
had a slightly less effect on Tg, since at a PDI/Tg molar ratio = 2, around 20% of the Tg multimers were always present (Fig.
2A, lane 5). Although most of the cysteine
residues are already involved in intradisulfide bridges, this
difference was probably due to the number of cysteine residues present
in the Tg19S molecule, which may explain why the efficiency of PDI was
lower with Tg than with NTD (110 versus 6 Cys). Accordingly,
we concluded that PDI was able to limit (Tg) or suppress (NTD) the
multimerization process. However, it was not established whether PDI
reacted with the multimeric forms, causing them to refold into
monomeric forms, or whether it acted on the monomers, thus limiting or
preventing the multimerization process.
The Action of PDI Is Restricted to the Multimeric Forms--
To
determine the forms of Tg and NTD recognized by PDI, we used a
PDI/ligand molar ratio equal to 0.5, which did not significantly reduce
the formation of multimers with either Tg or NTD (see above). After 30 min of incubation for iodination and coupling, the mixture was
chromatographed on an anti-PDI-Sepharose column. After extensively washing the column, the specific fractions were eluted by acidic solution (see "Experimental Procedures"). The acid fractions were analyzed by immunoblotting using a Tg monoclonal antibody. Only the
multimeric forms of Tg and NTD were identified (Fig.
3). Regarding NTD, no band corresponding
to the complex formed by PDI and NTD dimer (150 kDa) was detected; only
two slight bands corresponding to the molecular mass of the multimeric
forms were observed (Fig. 3B, lane 3). This may
have been due to the elution conditions at pH 3.0. In fact, at pH
levels lower than 6.0, it has been reported that PDI, acting as a
molecular chaperone, is dissociated from its ligand (25). On the other
hand, the immunopurification was PDI-specific, since the Tg and NTD
multimers chromatographed on this column at the same concentrations as
those used above were not retained on the anti-PDI-Sepharose column
(data not shown).

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Fig. 3.
Selectivity of PDI for the multimeric
forms. After iodination and coupling of 60 µg of Tg19S
(A) and 5 µg of NTD (B) in the presence of PDI
(PDI/ligand molar ratio = 0.5) and the redox system GSH/GSSG (200 µM/50 µM), the incubation medium was
maintained at 37 °C for 30 min and then immunopurified on an
anti-PDI-Sepharose column (volume = 1 ml). A:
lane 1, Tg19S; lane 2, incubation medium before
immunopurification; lane 3, immunopurified fraction.
B: lane 1, NTD; lane 2, incubation
medium before immunopurification; lane 3, immunopurified
fraction. Proteins were immunoblotted with anti-Tg monoclonal antibody
and detected with immunogold conjugate.
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Interaction of BiP on the Multimerization Process--
BiP is
known to participate in vivo to the correct folding of Tg in
the ER. The action of BiP involves multiple cycles of association and
dissociation, requires Ca2+ and ATP, and interacts
optimally with hydrophobic peptides, which are normally buried in the
hydrophobic core of properly folded proteins (26-28). Moreover, BiP is
not directly involved in disulfide bridge formation and, therefore,
presumably does not participate in the multimerization process. To
confirm this assumption, we performed in vitro iodination
and coupling on Tg19S and NTD in the presence of BiP. With both Tg and
NTD, BiP showed little, if any, ability to limit the multimerization
process whatever the BiP/ligand (mol/mol) ratios tested (Fig.
4). However, this limited ability did not
necessarily mean that BiP did not bind to multimeric forms. We reasoned
that multimeric forms generated by oxidation reaction might express
specific conformational hydrophobic sites recognized by BiP.

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Fig. 4.
Effects of BiP on the multimerization of
Tg19S and NTD. A: lane 1, Tg19S (0.3 µg)
was iodinated and coupled without BiP but in the presence of
CaCl2 (100 µM) and ATP (1 mM);
lanes 2-5, Tg19S iodinated and coupled (see "Experimental
Procedures") in the presence of BiP, corresponding to BiP/Tg19S molar
ratios of 0.2, 0.5, 1.0, and 2.0, respectively. Tg19S and Tg multimers
are indicated by arrows. B: lane 1,
NTD (0.3 µg) was iodinated and coupled without BiP but in the
presence of CaCl2 (100 µM) and ATP (1 mM); lanes 2-5, NTD iodinated and coupled (see
"Experimental Procedures") in the presence of BiP, corresponding to
BiP/NTD molar ratios of 0.2, 0.5, 1.0, and 2.0, respectively. NTD and
NTD multimer are indicated by arrows. Proteins were
immunoblotted with anti-Tg monoclonal antibody and detected by
chemiluminescence.
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BiP Is Bound to the Multimeric Forms--
To investigate the
possibility that BiP might specifically recognize the multimeric forms,
we incubated Tg19S or NTD in the presence of BiP (molar ratio
BiP/ligand = 2) and in the presence of 100 µM
Ca2+. After iodination and coupling for 30 min at 37 °C,
the mixture was chromatographed on an anti-BiP-Sepharose column. After
extensive washing of the column, the specific fractions were eluted
with an acidic solution (see "Experimental Procedures"). The
fractions were neutralized, concentrated, and analyzed by
immunoblotting using a Tg monoclonal antibody (Fig.
5). With Tg and NTD, most of the
specifically retained material was identified as consisting of
multimeric forms, although a small proportion of monomeric forms was
also retained.

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Fig. 5.
Relative selectivity of BiP for the
multimeric forms. After iodination and coupling of Tg19S (30 µg)
or NTD (5 µg) in the presence of BiP (BiP/ligand molar ratio = 1) and Ca2+ (100 µM), the mixture was
incubated for 30 min at 37 °C and then immunopurified on an
anti-BiP-Sepharose column (volume = 1 ml). A,
Tg19S. Lane 1, incubation medium before immunopurification;
lane 2, fraction immunopurified. B, NTD.
Lane 1, incubation medium before immunopurification;
lane 2, fraction immunopurified. In A and
B, immunoblots were probed with anti-Tg monoclonal antibody
and detected with immunogold conjugate.
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Role of the Association PDI/BiP on the Oxidative
Process of Multimerization and on the Refolding of Tg
Multimers--
Depending on the position of the N-linked
glycans in the nascent protein in the ER, the folding of the
glycoprotein is assisted either by BiP or by the calnexin/calreticulin
pathway (29). With Tg, based on the presence of the first
N-linked glycan at position Asn57, an
interaction with BiP can be expected to occur. Moreover, when this
study was in progress, Mayer et al. (30) reported that PDI
and BiP acted synergistically in the in vitro folding of the
denatured and reduced Fab fragment. It therefore seemed to be worth
associating these two molecular chaperones to determine how they
participated in the multimerization process of Tg19S during iodination
and coupling. Used together, BiP and PDI (molar ratios: BiP/Tg19S = 2, PDI/Tg19S = 1) did not affect the yield of Tg multimers. The
pattern of immunoblotting obtained was the same as that obtained with
PDI alone (see Fig. 2A, lane 4) or BiP alone (see
Fig. 4A, lane 5). At this stage in our study, it seemed likely that BiP did not enhance the effects of PDI. However, since BiP very efficiently recognized the multimeric forms of Tg,
we hypothesized that the association PDI+BiP might affect the
renaturation process of the oxidized Tg (Tg multimers). On the other hand, it was possible that the very low ability of BiP to
limit the multimerization of Tg might also be due to the iodination of
certain tyrosine residues of this molecular chaperone. To test these
hypotheses, both native PDI and BiP and iodinated PDI and BiP were
incubated with the Tg37S+Tg27S fraction isolated from a human Tg
preparation. The molecular chaperone/ligand molar ratio worked out at
0.5 (in the case of Tg37S) and 1 (in the case of Tg27S). In any event,
these molar ratios were not large enough to inhibit the Tg19S
multimerization process (see above). We therefore determined the
time course of the refolding of the multimeric forms of Tg. As shown in
Fig. 6, PDI or BiP alone did not
significantly affect the folding of the multimeric forms of Tg after
48 h (lanes 1 and 2, respectively), whereas
the simultaneous presence of PDI and BiP led to the refolding of the
majority of the multimeric forms of Tg (lane 3). It is
noteworthy that iodination of the molecular chaperones did not
significantly affect their activity since after 48-h of incubation
(lane 7), the percentage corresponding to the multimeric
forms decreased in parallel with the decrease observed in the presence
of native molecular chaperones.

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Fig. 6.
BiP and PDI affect the refolding of Tg
multimers. Twenty micrograms of the fraction Tg37S+Tg27S isolated
by gel filtration (see "Experimental Procedures") were incubated in
50 mM Tris-HCl buffer, pH 7.2, the redox system GSH/GSSG
(200 µM/50 µM), CaCl2 (100 µM), ATP (1 mM), and protease inhibitor in
the presence of 0.8 µg of PDI for 48 h (lane 1), 0.6 µg of BiP for 48 h (lane 2), or the mixture PDI+BiP
(0.8 µg and 0.6 µg, respectively) for 48 h (lane
3). In vitro iodinated PDI and BiP (0.8 µg and 0.6 µg, respectively) were incubated with 20 µg of the fraction
Tg37S+Tg27S in 50 mM Tris-HCl buffer, pH 7.2, the redox
system GSH/GSSG (200 µM/50 µM),
CaCl2 (100 µM), ATP (1 mM), and
protease inhibitor; the sample was analyzed after 1 h (lane
4), 12 h (lane 5), 24 h (lane 6),
and 48 h (lane 7). Lane 8, 2 µg of the
Tg37S+Tg27S fraction. Lane 9, Tg19S. Tg and Tg multimers are
indicated by arrows. Immunoblots were probed with anti-Tg
monoclonal antibody and detected with immunogold conjugate.
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DISCUSSION |
Under physiological conditions, the native Tg19S secreted into the
follicular lumen undergoes the oxidative iodination and coupling
reaction leading to hormone formation. This reaction results in a Tg
with a high hormonal content but also in the formation of covalent
cross-links, which take the form of either dityrosine bridges or
disulfide bridges (intermolecular or intramolecular cross-links) (8).
Soluble multimeric forms of Tg constitute the first step toward the
insolubility and aggregation of Tg molecules (7). Several mechanisms
may be involved in limiting these aggregates in the follicular lumen,
such as that based on the presence of the lysosomial enzymes,
cathepsins B, L (31), and K (32), at the surface of the thyrocyte as
well as that based on the presence of oxygen free radicals generated by
the system H2O2 + peroxidase (8). The results
of all the previous studies have suggested that the limitation of the
multimeric forms of Tg present into the follicular lumen may be
attributable to their degradation. In the present study, it was
proposed to test the hypothesis that the molecular chaperones present
in the follicular lumen might contribute to both limiting the formation
of the multimeric forms of Tg19S produced during the process of hormone
synthesis and reducing the proportions of the soluble multimeric forms
of Tg after their formation.
Numerous studies have described the role of PDI and/or BiP in the
folding of proteins in vitro. However, to our knowledge, molecular chaperones were used in these studies to rescue the unfolded
proteins generated by reduction-denaturation (33, 34) or heating (35).
Here we investigated the role of PDI and/or BiP using an original
process to avoid the formation of unfolded protein from a folded
protein (Tg19S) and the secondary misfolding, which occurs due to the
oxidative process of hormone synthesis. For this purpose, we used the
entire Tg molecule (Tg19S) and its NTD. In vitro the
iodination and coupling reaction multimerized ~20% of all the Tg and
NTD, although in the case of Tg, the rate of multimerization was more
difficult to assess exactly. In vitro, PDI either limited
(in the case of Tg) or prevented (in that of NTD) the formation of
multimeric forms. In addition, when PDI was used at various
concentrations relative to Tg or NTD, no anti-chaperone activity was
evidenced. This finding differs from those of previous studies where
the PDI, present at substoichiometric concentrations relative to
denatured lysozyme, exhibited anti-chaperone activity (36). This
difference is probably due to the fact that none of the substrates (Tg
and NTD) used in the present experiments tends to aggregate
spontaneously. It is worth noting that PDI acted specifically on the
multimeric forms, since the immunopurification procedure performed
using a polyclonal antibody directed against PDI yielded no Tg19S
forms. The fact that Tg19S was not bound to PDI during the
immunopurification step was not in disagreement with Mezghrani et
al. (19), since under the conditions used here (subneutral pH
buffer) we ruled out the possibility that Tg19S may have bound to
PDI via a "receptor-binding domain" expressed only
under acidic conditions.
At this stage, it was possible to assume that PDI in the follicular
lumen contributes in limiting the formation of the multimeric forms of
Tg, which would otherwise be generated by in vitro
iodination and coupling. This reaction probably also resulted in the
iodination of the PDI present in the incubation medium, and this effect
is certainly very similar to what occurs in vivo. In the
colloid, the molecular chaperones may also be iodinated as the result
of the process of hormone synthesis, like several other colloidal proteins (37). On the other hand, to considerably reduce the formation
of the multimeric forms of Tg, we used an amount of PDI corresponding
to ~1/5 of the Tg in weight. This amount of PDI may seem rather low
for an in vitro study, but it is very high given the
physiological concentrations of PDI that are to be found in the
follicular lumen (38). It therefore seemed to be interesting to examine
the role of another molecular chaperone, BiP, used both alone and in
association with PDI. BiP is known to contribute in the ER to the
folding of several proteins (39). This molecular chaperone is
relatively abundant in the colloid of human thyroid glands (8). BiP
possesses a peptide binding groove lined with hydrophobic side chains
and interacts with heptapeptides containing a subset of aromatic and
hydrophobic amino acids in alternating positions (40). Such linear
motifs are present in Tg but not in NTD (11). With these two ligands,
BiP was not effective in changing the amount of multimers, even at a
molar ratio equal to 2. However, BiP was efficiently bound to the
multimeric forms of Tg and NTD, although in the latter case, the
"native" peptide did not possess a specific peptide binding groove
for BiP. Therefore, it is possible that the multimerization of NTD may
have led to the expression of a three-dimensional heptapeptide-like structure recognized by BiP. In the case of Tg19S, the nascent molecule
present in the ER expresses the heptapeptide recognized by BiP, and
this site is buried in mature Tg19S. Since BiP binds to the multimeric
forms of Tg, we concluded that the Tg multimers underwent
conformational changes revealing the heptapeptide. The association of
BiP with PDI at various molar ratios did not affect the ability of PDI
to modify the multimerization process. These results are not in line
with those reported on the oxidative folding of antibodies in
vitro (30). This difference was not due to the iodination of the
molecular chaperone but partly to the fact that the chaperone/ligand
molar ratios were low (never above 2) in our study and partly to the
incubation time being restricted to 30 min. When directly applied to
the multimeric forms for a longer time (48 h), BiP actually greatly
increases the efficiency of PDI, since Tg37S+Tg27S were almost
completely transformed in Tg19S. This result confirmed both that BiP
was bound to multimeric forms of Tg and that BiP acted synergistically
with PDI to restore the initial structure of the Tg19S. The difference
in the efficiency of the association PDI/BiP observed between the
inhibition of the formation and the refolding of the multimeric forms
was probably due to the kinetic competition occurring between the
disulfide bridge formation resulting from the oxidation of Tg19S,
reduction of the disulfide bridges by PDI, and the speed with which BiP bound to the multimeric forms. Once formed, most of the disulfide bridges become rapidly buried deeply in the core of the multimeric Tg
molecules, which explains why PDI alone had little effect, even after
48 h. On the other hand, Tg multimers may expose specific conformational peptides recognized by BiP, as described in the case of
NTD, and accordingly, PDI and BiP may act sequentially on the reduction
of the intermolecular disulfide bridges, so that the Tg molecules would
gradually detach themselves.
In conclusion, in these in vitro experiments performed using
two molecular chaperones present in the colloid, it was possible both
to limit the formation of multimeric forms of Tg and to rescue the
prothyroid hormone from its multimers. Moreover, because the colloid
contains at least one other molecular chaperone (Grp94) (8) known to
interact sequentially with BiP (41), our results support the idea that
a whole network of folding helper proteins exists in the follicular
lumen and takes part in the control of multimerization and aggregation
of proteins formed by oxidation.
 |
ACKNOWLEDGEMENTS |
Special thanks to J. L. Franc for
helpful discussions and for critical reading of the manuscript and to
L. Vinet and R. Galibert for their excellent technical assistance.
 |
FOOTNOTES |
*
This work was supported in part by the Association pour le
Développement des Recherches Biologiques et Médicales.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence and reprint requests should be addressed.
Tel.: 33 4.91.32.43.92; Fax: 33 4.91.79.77.74; E-mail: bernard. mallet@medecine.univ-mrs.fr.
Published, JBC Papers in Press, April 9, 2001, DOI 10.1074/jbc.M101086200
 |
ABBREVIATIONS |
The abbreviations used are:
Tg, thyroglobulin;
NTD, N-terminal domain;
PAGE, polyacrylamide gel electrophoresis;
Grp, glucose-regulated protein;
PDI, protein disulfide isomerase;
BiP, immunoglobulin heavy chain binding protein;
ER, endoplasmic reticulum;
GSH, reduced glutathione;
GSSG, oxidized glutathione.
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