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INTRODUCTION |
The functional unit of the thyroid gland is the follicle, which is
composed of a monolayer of epithelial cells, the thyrocytes, surrounding a closed space, the follicular lumen. The thyroid prohormone, thyroglobulin
(TG),1 is synthesized by
thyrocytes and then secreted into the follicular lumen in which the
thyroid hormones themselves are synthesized. Hormonal secretion
requires the endocytosis of mature TG (i.e. TG bearing
hormone residues), its targeting to lysosomes, and the subsequent
release of hormones. Each step of this bidirectional trafficking of TG
is subject to regulation, in some cases mediated by molecular
chaperones (1-3).
Thyrocytes synthesize large amounts of TG (up to 50% of protein
synthesis in the gland; Ref. 4). Glycosylation and chaperone-assisted folding of TG monomers (Mr 330,000) take place
in the endoplasmic reticulum (ER), and involve several chaperones
including calnexin, BiP, ERp72, grp94, and grp170 (5-9). The
dimerization of monomers, a process thought to abolish the interactions
between secreted molecules and chaperones, probably determines the
sorting and targeting post-ER of glycoproteins (10). The transit
through the Golgi apparatus of newly synthesized TG leads to the
modification of some high mannose type N-glycans into
N-acetyllactosamine chains. Secreted glycoproteins have an
almost unique property, mediated by an unknown regulatory mechanism, in
that some of these lactosamine chains of TG are incompletely processed
and have accessible GlcNAc moieties (11, 12). TG molecules are then
targeted to small secretory vesicles and released via the regulated
secretory pathway of thyrocytes (13, 14).
In the follicular lumen, the prohormone undergoes several
posttranslational modifications to produce thyroid hormones. These modifications include the iodination of tyrosyl residues, and the
oxidative coupling of some of these residues to form triiodothyronine (T3) and tetraiodothyronine (thyroxine or T4)
(1, 2). They also include the de novo formation of disulfide
bridges, the multimerization of some TG molecules (15), and the
progressive completion of their incompletely processed lactosamine
N-glycans by the addition of galactose and sialic acid sugar
moieties (12).
As TG molecules in the follicular lumen are very heterogeneous in terms
of iodine and hormone content, it has been assumed that there is a
mechanism that either preferentially targets iodine-rich molecules to
lysosomes or prevents the catabolism of iodine-poor molecules. It has
recently been reported that megalin, a 330-kDa glycoprotein associated
with the cell surface, participates, at least partly, in TG
internalization in some some thyroid cell preparations (16, 17).
However, further studies are required to determine whether this process
is selective for a given TG subpopulation, and no receptor specific for
mature thyroglobulin has yet been shown to be responsible for the
selective internalization of TG. The notion of an intrafollicular
retention process for immature molecules arose from three main
findings: 1) there exists, in vivo in the rat, a major
recycling pathway from internalized TG back to the lumen (18); 2)
during TG maturation, galactose and sialic acid are incorporated into
some glycans (12), by a process known to occur in the trans
compartments of the Golgi complex and the trans-Golgi
network (19), and 3) immature TG preferentially binds a membrane
receptor in acidic conditions, suggesting that there may be a means of
recycling TG after endocytosis, in acidic compartments such as the
endosomes and prelysosomes (20, 21). The receptor involved in binding
has not yet identified, but the receptor-binding domain of TG has been
located within the hormonogenic domain
(Ser789-Met1178) of TG, which includes
tyrosine residues involved in hormone formation and a cysteine-rich
type 1 module (22).
Recent reports have established that in secretory cells some chaperones
are secreted or cell surface-associated. For example, calnexin as well
as many other "ER resident proteins" are present at the surface of
thymocytes (23), and grp94 is secreted by pancreatic cells (24, 25).
The best documented example is the protein-disulfide isomerase (PDI).
PDI (Mr 57,000) is an abundant soluble cellular
protein of the secretory pathway (0.4% of total liver protein, Refs.
26-28) found on the cell surface of numerous cell types such as B
cells (29, 30), platelets (31, 32), pancreatic cells (33), and
hepatocytes (34). PDI may be secreted despite possessing the KDEL
retention signal (34, 35). It remains fully active and may be involved
in structural modification of (glyco)proteins in the extracellular
medium. Indeed, PDI catalyzes thiol-disulfide interchanges that may
result in the rearrangement of protein-disulfide bonds. This is why
cell-surface PDI is involved in the reduction of the disulfide-linked
diphtheria toxin heterodimer (36), cell surface events triggering the
entry of the human immunodeficiency virus into lymphoid cells (37),
shedding of the human thyrotropin receptor ectodomain (38), cell
surface recognition during neuronal differentiation of the retina (39), and, more generally, controls the redox state of existing exofacial protein thiols or reactive disulfide bonds (40). PDI, and some other
chaperones such as BiP and grp94, are secreted with TG in in
vivo conditions into the follicular lumen (41, 42), suggesting that these chaperones may be involved in the structural modification of
the prohormone in hormone biogenesis.
We investigated the secretion and cell surface expression of PDI and
other chaperones in the FRTL5 (thyroid) cell line, and then studied the
characteristics of the interaction between TG or derived fusion
proteins and PDI in FRTL5 cells. We report two key observations: 1) PDI
is secreted and associated with membranes, which implies that it may be
present in vivo in post-ER compartments; and 2) PDI
interacts with the membrane-binding domain of TG in acidic
compartments. The potential implications of these observations are discussed.
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MATERIALS AND METHODS |
Chemicals
Amplify, [35S]methionine/cysteine,
Na125I, protein A-Sepharose, and PD10 columns were obtained
from Amersham Pharmacia Biotech; IODOGEN, bovine protein-disulfide
isomerase, fluorescein isothiocyanate (FITC), Sulfo-SHPP, and
chemiluminescence kits and reagents (SuperSignal and SuperSignal Ultra)
from Pierce. Porcine TG and TG-agarose were purchased from
Sigma-Aldrich. Human TG (containing 1% iodine) was obtained from
Scipac (Sittingbourne, United Kingdom). Human TG with a low iodine
content from a patient with a single colloid goiter (2.8 iodine
atoms/mol; traces of T3, 10 mmol T4/mol),
previously characterized in binding studies (22), was kindly donated by B. Mallet (Faculty of Medicine, Marseille, France).
The following antibodies were used: anti-PDI (polyclonal SPA-890
(StressGen) and monoclonals clone 1D3 (StressGen) or clone RL90 (ABR,
Inc.)), anti-KDEL (monoclonal clone 10C3, SPA-827 (StressGen)), anti-BiP (polyclonal SPA-826, and monoclonal SPP-765 (StressGen)), anti-calnexin (polyclonal SPA-865 and monoclonal SPA-866 (StressGen)), anti-grp94 (monoclonal SPA-850 (StressGen)), and anti-
-galactosidase (monoclonal clone GAL-13 (Sigma)).
Cell Culture
FRTL5 cells (ATCC RL8305) were cultured in Coon's modified
Ham's F-12 medium, pH 7.5, containing bicarbonate (2.67 g/liter) and
5% decomplemented calf serum. A mixture of nonessential amino acids (1 mM) was added, along with six hormones: TSH (10 milliunits/ml), transferrin (5 µg/ml), insulin (10 µg/ml),
somatostatin (10 ng/ml), glycyl-1-histidyl-1-lysine (10 ng/ml), and
hydrocortisone (3.7 ng/ml).
Western Blot Analysis
Proteins were resolved by SDS-PAGE and transferred to
nitrocellulose membrane using a semidry blotter and 180 mA applied for 30 min. The blot was incubated for 1 h with Tris-buffered saline, 10 mM Tris, 0. 15 M NaCl, pH 7.5, containing
5% milk powder, and 0. 5% Tween 20 and was then washed twice with
Tris-buffered saline containing 0. 5% Tween 20 (TBST). The blot was
incubated with TBST containing 2% skim milk powder and primary
antibodies (1/1000 dilution). The blot was washed four times with TBST,
then incubated in 5 ml of TBST containing 2% skim milk powder and the
secondary antibody (1/20,000 dilution). The blot was washed five times
with TBST and incubated with a chemiluminescence substrate, according to the manufacturer's instructions. It was then placed against x-ray
film for detection of the bound primary antibody. The blot was stripped
by incubation with Immunopure elution buffer (Pierce) for 2 h at
room temperature. The blot was washed, incubated with SuperSignal
working solution (Pierce), and placed against x-ray film, to check that
the probes had been adequately removed. The stripped blot was then
washed four times and incubated with blocking reagent before reprobing.
Metabolic Labeling and Immunoprecipitation
Cells were washed with 20 mM sodium phosphate, 0.15 M NaCl, pH 7.4 (PBS), and incubated in cysteine- and
methionine-free labeling mixture containing 10% dialyzed fetal calf
serum, in the presence of [35S]methionine/cysteine (200 µCi/ml, Expre35S35S labeling mix; NEN Life
Science Products). The labeling medium was removed and replaced with
Coon's modified Ham's F-12 medium, and the cells were incubated for
various lengths of time (the chase period). The media were removed
after the chase period, and, protease inhibitors were added (1 mM EDTA, 1 mM phenylmethanesulfonyl fluoride,
and 10 µg/ml each leupeptin and pepstatin) and the mixture centrifuged (1 h at 100,000 × g). For treatment of the
cells with colchicine or brefeldin A, cells were incubated in Coon's
medium containing either 40 µM colchicine or 20 µM brefeldin A for 30 min before labeling and the chase
period in medium containing the same concentrations of these drugs. The
cells were washed four times with ice-cold PBS and harvested by
scraping. They were then centrifuged at 1,500 revolutions/min for 10 min, suspended in 10 ml of ice-cold PBS and recentrifuged. The
resulting cell pellets were suspended in 1 ml of lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet
P-40). The cell lysates were centrifuged at 12,000 × g
for 10 min to remove cell debris. The resulting supernatants were
cleared by incubation with 20 µl of non-immune serum and 20 µl of a
50% suspension of Protein A-Sepharose for 1 h at 4 °C. The
suspension was centrifuged to remove the Protein A-Sepharose, and the
cleared cell lysates and media were incubated with 5 µl of anti-PDI
monoclonal antibody (RL90) and 12 µl of anti-BiP polyclonal antibody
or 12 µl of anti-TG antiserum. Immune complexes were recovered by
addition of 30 µl of a 50% suspension of Protein A-Sepharose and
incubation for 1 h at 4 °C. The suspension was centrifuged at
12,000 × g for 3 min, the supernatant discarded, and
the pellet washed four times with lysis buffer and once with PBS. The
immunoprecipitate was suspended in SDS-PAGE sample buffer containing
2% SDS with or without 1% -mercaptoethanol and subjected to
SDS-PAGE. Gels were dried by conventional methods, and radiolabeled bands were detected by phosphoimaging (Molecular Dynamics, Inc., Sunnyvale, CA). The leakage of proteins from injured cells was assayed
by determining lactate dehydrogenase activity.
Immunodetection of Cell Surface PDI and Chaperones
FRTL5 cells were grown in Lab Tek I chambers, washed briefly in
PBS, and fixed in 2% formaldehyde at room temperature for 15 min. The
cells were then washed twice in PBS and incubated with blocking buffer
(PBS containing 10% BSA) for 10 min at room temperature in a
humidified chamber. The cells were mixed directly, without washing,
with the primary antibody in 0.1% BSA in PBS, and the mixture was
incubated for 1 h. The cells were then washed five times with
0.1% BSA in PBS, and were incubated for 1 h with the
fluorochrome-conjugated secondary antibody in 0.1% BSA in PBS. The
cells were thoroughly washed in PBS, mounted on slides in Mowiol, and
viewed with a confocal microscope (Leitz DMIR BE, Leica TSC 4D,
Heidelberg, Germany).
Quantitation of PDI Secretion
We determined the level of PDI secretion, by subjecting serially
diluted (standardized based on protein content) samples of culture
medium to slot blot analysis on nitrocellulose membranes. The signal
obtained was compared with that given by known amounts of purified PDI.
Blots were probed with anti-PDI antibody, treated for
chemiluminescence, scanned, and analyzed with a PhosphorImager and the
ImageQuant software package (Molecular Dynamics).
Radiolabeling
Sulfo-SHPP was iodinated according to the manufacturer's
instructions. Briefly, 1 mCi of 125I-Na was added to 10 µl of a freshly prepared solution of sulfo-SHPP in dimethyl sulfoxide
(0.1 mg/ml). The following were then added sequentially: 10 µl of
chloramine T (5 mg/ml), 100 µl of hydoxyphenyl acetic acid (1 mg/ml),
and 10 µl of sodium metabisulfite (12 mg/ml). 125I-Labeled sulfo-SHPP (200 µCi) was incubated in
25-cm2 flasks with confluent FRTL5 cells, previously washed
three times with ice-cold PBS, for 1 h each at 2-4 °C. The
labeling medium was removed, and the cells were washed with PBS at
4 °C and recovered by scraping into ice-cold PBS. The cells were
washed, and cell lysates were obtained for immunoprecipitation of PDI
as described above.
PDI (10 µg) was iodinated by incubation with IODOGEN and
125I-Na in 50 µl of 50 mM Tris-HCl buffer, pH
8.0, for 15 min at 4 °C. The reaction was stopped by adding 1 ml of
10 mM phosphate buffer, pH 7.4, 150 mM NaCl, 20 mM KI. Iodinated molecules were isolated by gel filtration
through PD10 columns and extensive dialysis. The same protocol was used
for TG iodination, except that gel filtration was performed through
Sephadex-200 columns (Amersham Pharmacia Biotech).
Construction of Human Thyroglobulin Fusion Peptides
RNA Isolation--
Total RNA was extracted from the thyroid
tissues of patients with Graves' disease, as described by Chomczynski
and Sacchi (43). Briefly, tissue (10 mg) was crushed in 3 ml of RNABlue solution (Eurobio). Chloroform (300 µl) was added, and the mixture was vigorously vortexed and centrifuged at 15,000 × g
for 30 min. The aqueous phase was transferred to a microcentrifuge tube
containing an equal volume of isopropanol. RNA was recovered by
precipitation overnight at 
20 °C and centrifugation at 12,000 × g for 20 min. The pellet was washed with 75% ethanol and
suspended in diethyl pyrocarbonate-treated water.
Amplification by Reverse Transcriptase-Polymerase Chain Reaction
(RT-PCR)--
The first strand cDNA was synthesized by incubation
of 1-3 µg of total RNA with Moloney murine leukemia virus reverse
transcriptase (5 units, Life Technologies, Inc.) for 1 h at
42 °C, in a volume of 30 µl. The resulting first strand cDNA
was then used for PCR amplification.
The membrane receptor binding domain of TG
(Ser789-Met1178) was amplified using the
following primers: the forward primer was
5'-GGGATCCGCTGAGCAGGTCTTCGAGTTG-3' (2374-2394) and the reverse primer,
5'-CTCTAGAGAGCACATTTCAGAGGCTTGG-3' (3494-3474). The
cysteine-rich repeat unit 9 (RU9, according to Ref. 44) that forms part
of the binding domain, was amplified using the primers
5'-GGGATCCCTGCAGATTCCACAGGGCCCG-3' (3241-3261; forward) and
5'-CTCTAGAGAGCACATTTCAGAGGCTTGG-3' (3494-3474; reverse). Each of
these primers contained a unique restriction site, BamHI for
the forward primers and XbaI for the reverse primers. The expected protein sequences of the TG-derived fusion proteins included Pro759-Leu1163 for the membrane-binding domain
of TG and Leu1080-Leu1163 for the RU9
cysteine-rich motif.
The reaction mixture for PCR (50 µl) contained 10 mM
amounts of each dNTP, 100 ng of each primer, the buffer supplied by the manufacturer, and 1 unit of Vent polymerase (New England Biolabs). PCR
amplification involved denaturation at 94 °C for 1 min, primer annealing at 55.5 °C (PCR 2) for 90 s, and extension for
90 s, with optimum results obtained using 34 cycles.
Cloning and Sequencing of PCR Products--
PCR fragments were
purified from agarose gel and cut with BamHI and
XbaI endonucleases. They were then ligated into the
BamHI/XbaI-cleaved PMal-CR1 expression vector
(Biolabs) and used to transform Escherichia coli TB1. The TG
fragment inserts of the plasmids of positive clones were checked by
sequencing. The DNA was sequenced using M13 (47) and reverse M13
(48) primers (New England Biolabs). We used the cycle sequencing
method and the dRhodamine Terminator Cycle Sequencing Kit (Applied
Biosystems/Perkin-Elmer). Sequences were determined with an ABI Prism
310 and were aligned with the Tg cDNA sequence using AutoAssembler
software (Applied Biosystems/Perkin-Elmer).
RT-PCR generated two cDNA fragments, one with the expected
sequence, based on the wild-type mRNA (43, 44), and the other with
exons 13, 14 and 15 deleted. Exons 14 and 15 encode the cysteine-rich RU9 motif (His1025-Cys1160, Ref. 45). This
finding is novel, and we therefore tried to determine the incidence of
this deletion in various physiopathological conditions. The results of
this investigation will be published elsewhere.2 Three clones
derived from the TG cDNAs were selected and prepared. One encoded
the full-length binding domain (BD), the second, the alternatively
spliced binding site (BD
RU9), and the third, RU9 (RU9).
Production and Purification of Fusion Proteins
Bacterial clones containing the various constructs were cultured
overnight in 50 ml of LB medium at 37 °C with shaking. An aliquot
(50 ml) of the overnight culture was used to inoculate 1 liter of LB,
which was then incubated at 37 °C until the optical density at 600 nm of the culture reached 0.4 (i.e. 2 × 108 cells/ml). Synthesis of the fusion proteins was induced
by adding isopropyl-1-thio--D-galactopyranoside to a
final concentration of 0.1 mM and incubating at 32 °C
for 90-120 min. The culture was then centrifuged at 1500 × g for 10 min. The bacterial pellet was suspended in 50 ml of
lysis buffer (10 mM phosphate buffer, pH 7.0, 30 mM NaCl, 0.25% Tween 20, 10 mM EDTA, 10 mM EGTA) and placed in a freezer at 20 °C overnight.
The lysate was thawed at 4 °C, in the presence of 1 mM
PMSF and subjected to sonication with 1-min pulses for 20 min. NaCl
(final concentration 0.5 M) was added and the mixture
centrifuged at 40,000 × g for 30 min at 4 °C.
The fusion proteins were purified on amylose resin, according to the
manufacturer's instructions. The protein-containing fractions were
dialyzed and concentrated (1 mg/ml final concentration) against phosphate-buffered saline (PBS) in a MicroProdicon apparatus
(BioMolecular Dynamics). The level of purification of these fusion
proteins was checked by SDS-PAGE and immunoblotting with
anti-maltose-binding protein (MBP) and anti-TG antibodies.
Membrane Preparation and Binding Assay--
FRTL5 plasma
membranes were prepared as described previously (5, 6). Solid-phase
assays of TG and fusion peptide binding to FRTL5 membranes were
performed according to established methods (21, 22). Membranes were
added (20 µg of total protein in 40 µl of PBS buffer) to the
flat-bottomed wells of a microtiter plate (Falcon 3911, Becton
Dickinson, Oxnard, CA) and were incubated for 1 h at 37 °C and
then overnight at 4 °C. The wells were washed three times with 150 µl of PBS and once with 200 µl of PBS containing 2% bovine serum
albumin. Thyroglobulin (5 × 105 to 106
cpm in a binding buffer consisting of 25 mM acetate buffer,
pH 5.0, 150 mM NaCl, 5 mM CaCl2,
and 0.1% BSA) was added and incubated alone or in the presence of
competitors at 4 °C for 90 min. The wells were washed four times
with the binding buffer, and radioactivity was counted. The results are
means of at least three separate experiments performed in duplicate or triplicate.
Chaperones detected in membrane preparations were determined by
immunoassay using microtiter plates coated with membranes (10 µg/well). The plates were coated by incubation for 2 h at 37 °C. The plates were washed with PBS and incubated for 2 h
with PBS containing 3% skim milk powder. They were then incubated with monoclonal antibodies directed against chaperones (1:500 dilution). Bound antibodies were detected by probing with horseradish
peroxidase-linked anti-mouse IgG (1/1000 dilution), extensive wahshing
and enzymatic reaction with OPD (Dako). Absorbance was read at 490 nm
on an IEMS Reader MF (Labsystem).
Cell Surface Labeling of PDI and TG-FITC
Human or porcine TG was conjugated with fluorescein
isothiocyanate (TG-FITC) as follows. FITC (50 µl of a 1 mg/ml
solution) was added to TG in 0.1 M carbonate buffer, pH 9.0 (20 mg in a 1.95-ml final volume), and the mixture was incubated in
darkness for 1 h at room temperature. TG-FITC was recovered by gel
filtration through a PD10 column (Amersham Pharmacia Biotech). It was
dialyzed extensively, and aliquots (200 µl, 10
5
M final concentration) were stored at 20 °C.
FRTL5 cells were grown in Lab Tek I chambers, washed briefly in PBS,
and fixed in 2% formaldehyde at room temperature for 15 min. Cells
were washed twice in PBS and incubated in blocking buffer (PBS
containing 10% BSA) for 10 min at room temperature in a humidified
chamber. For PDI or TG labeling, cells were mixed immediately, without
washing, with primary antibodies in PBS containing 0.1% BSA, and
incubated for 1 h. The cells were washed five times with PBS,
0.1% BSA, and incubated for 1 h with fluorochrome-conjugated secondary antibodies in PBS, 0.1% BSA. They were then washed
extensively in PBS. For TG-FITC binding studies, cells were incubated
with the fluorescent probe (0.5 × 107
M) in 10 mM acetate buffer, pH 5.0, 0.15 M NaCl, for 1 h at 4 °C. They were washed with
acetate buffer and fixed in 2% formaldehyde. They were then mounted on
slides in Mowiol and viewed with a confocal microscope as follows.
Cell samples were examined using a Leica TCS 4D invert confocal
microscope with argon/krypton multilasers giving excitation lines at
568 and 488 nm, respectively. The data from the channels were collected
simultaneously, using the narrow bandpass filter setting built into the
instrument. Double labeling experiments usually employed a combination
of FITC and Texas Red dyes (secondary antibodies) to eliminate channel
overlap effects. Specimen were tested for overlap by turning off
individual laser lines while continuously scanning the two channels.
Data were collected with 8-fold averaging at a resolution of 512 × 512 pixels using optical slices of between 0.5 µm and 1 µm. The
microscope was a Leitz DMIR BE utilizing a 100× oil immersion
objective lens (numeric aperture 1.4). Data sets were processed using
the Scanware software, then exported for preparation for printing using
Photoshop. Data were projected onto a single plane, and the contrast
was adjusted prior to export as TIFF files to Adobe Photoshop.
Surface Plasmon Resonance Analysis of the TG/PDI Interaction
A BIAcore biosensor system (BIAcore AB, Uppsala, Sweden) was
used to study the kinetics of TG binding to immobilized PDI. All
experiments were performed at 25 °C. The sensor chip was washed with
10 mM Hepes, pH 7.5, 150 mM NaCl, 0.005%
BIAcore surfactant P20 (buffer A) (BIAcore AB) between injections.
Purified PDI (100 µg/ml) was directly bound, via its amino groups, to
the sensor surface (CM5 sensor chip), activated by
N-hydroxysuccinimide (50 mM final concentration)
and N-ethyl-N'-(dimethylaminopropyl)carbodiimide (200 mM final concentration) according to the
manufacturer's instructions. The remaining
N-hydroxysuccinimide groups were inactivated with 1 M (pH 8.5) ethanolamine. Acidic acetate buffers (50 mM, pH from 5.0-6.0) were used for equilibration and
binding analysis, and the surfaces were regenerated by washing with 10 mM NaOH, pH 11.8, and equilibration with acetate buffer or
buffer A before reuse. The kinetics of binding to PDI of various TG
samples or TG fragments were determined by injecting various
concentrations of analyte (5-100 µg) in either 10 mM
acetate buffer, pH 5.0, or 10 mM Hepes, pH 7.4, at a flow
rate of 20 µl/min, and then at various flow rates to assess mass
transport limitations. Dissociation was observed in running buffer
without dissociating agents. Regeneration was obtained with 10 µl of
50 mM NaOH. The kinetic parameters were determined using
BIAevaluation 3.0 software (BIAcore AB).
| |
RESULTS |
Detection and Determination of the PDI Content of FRTL5
Cells--
In Western blots of proteins isolated from CEM, BHK21, and
FRTL5 cells, probed with the anti-PDI IgG raised against the whole molecule, a band of Mr 57,000, and sometimes a
faint band of Mr 63,000, were detected. In some
experiments, an additional band of Mr 48,000 to
51,000 was also detected (Fig.
1A). The cellulose membrane
was stripped and reprobed with a monoclonal anti-PDI antibody (clone
RL90). This resulted in the detection of the Mr 57,000 and Mr 51,000 proteins bands only (Fig.
1B), demonstrating that: (i) the Mr
63,000 band was not related to PDI and (ii) the additional
Mr 51,000 band was probably a product of PDI
degradation. This was confirmed by the subsequent finding that the
51-kDa protein was produced after incubation of a freshly prepared
FRTL5 homogenate (Fig. 2, lane
1) for 30 min at 37 °C or at room temperature (Fig. 2,
lanes 2 and 3, respectively).
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Fig. 1.
PDI content of FRTL5, BHK21, and CEM cells,
determined by Western blot analysis. Blots of equivalent amounts
of total proteins (20 µg) isolated from CEM (lane
1), BHK21 (lane 2), and FRTL5 cells
(lane 3), subjected to SDS-PAGE in a 10%
acrylamide gel, transferred to nitrocellulose membranes, probed with
anti-PDI antiserum (A) detected by chemiluminescence or,
after stripping of the nitrocellulose membrane, probed with a
monoclonal anti-PDI antibody (B) detected by
chemiluminescence, as described under "Materials and Methods."
Molecular masses are in kDa.
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Fig. 2.
Identification of the 51-kDa protein
immunologically PDI identified as a degradation product of PDI.
Total protein (20 µg) isolated from FRTL5 cells was treated with an
SDS reducing buffer before (lane 1) or after
incubation for 30 min at 37 °C (lane 2) or
20 °C (lane 3) in 50 mM Tris/HCl,
pH 7.0. Protein were separated by SDS-PAGE in a 10% acrylamide gel,
transferred to nitrocellulose membranes, probed with a monoclonal
anti-PDI antibody, and detected by chemiluminescence as in Fig. 1.
Molecular masses are in kDa.
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Densitometry showed that there was more PDI in FRTL5 cells than in the
other cell types studied. The relative amount of PDI present was
determined by subjecting serially diluted cell lysate samples to slot
blot analysis, probing with monoclonal antibodies, and comparing the
signal obtained with that obtained using known amounts of purified PDI.
The PDI concentrations in CEM cells has been estimated to be 0.42%
(0.33-0.56%, n = 6), that of BHK21 cells, 0.78%
(0.68-0.89%, n = 6) and that of FRTL5 cells to be 1.05% (0.85-1.25%, n = 6) of total protein in the homogenates.
PDI Is Secreted by FRTL5 Cells--
We metabolically radiolabeled
FRTL5 cells and recovered PDI by immunoprecipitation of cell lysates
and media. Cellular PDI was recovered as two major protein bands
corresponding to the monomer (57-kDa monomer) and its principal
degradation product (51 kDa) (Fig. 3,
left). PDI was detected in the medium after a 1-h chase
period, and the amount present in the medium increased between 1 and
2 h (Fig. 3, right).
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Fig. 3.
PDI synthesis and secretion by FRTL5
cells. Cells were radiolabeled by incubation with
[35S]cysteine and [35S]methionine for
1 h and subjected to chase periods of various lengths, as
indicated. PDI (57-kDa monomer, presumed degradation products at 51 kDa) recovered by immunoprecipitation from cell lysates
(panel A) and media (panel
B), was resolved by SDS-PAGE (10% acrylamide gel) and
detected by fluorography as described under "Materials and
Methods." Chase periods were: 0 min (lanes 1 and 6), 15 min (lanes 2 and
7), 30 min (lanes 3 and 8),
60 min (lanes 4 and 9) and 120 min
(lanes 5 and 10).
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It is also possible to isolate PDI from a culture medium in which the
cells have been radiolabeled at equilibrium for 24 h. Apart from
the major 57-kDa protein, a 330-kDa protein was also precipitated (Fig.
4, lane 1). This
protein was identified as the thyroglobulin monomer (Fig. 4,
lanes 5 and 6) and is probably present
as a contaminant (Fig. 4, lane 7). Indeed, the
same pattern was observed, with similar amounts of PDI recovered, in
non-reducing conditions (Fig. 4, lane 2), showing
that most of the PDI and TG molecules were not covalently bound. This
is further supported by the observation that PDI was not detected in
immunoprecipitates of TG, with or without -mercaptoethanol (Fig. 4,
lanes 5 and 6). We also isolated BiP
from the culture medium. Similar fractions were isolated in the
presence and absence of ATP, throughout the immunoprecipitation
experiments. This excludes the possibility of ATP-dependent
binding between the secreted forms of BiP and TG (Fig. 4,
lanes 3 and 4). It is interesting to
note that BiP was recovered using a polyclonal antibody directed
against the eight amino acid residues at the extreme C terminus of BiP,
including the KDEL sequence.
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Fig. 4.
PDI, BiP, and TG secretion by FRTL5
cells. Cells were radiolabeled by incubation with
[35S]cysteine and [35S]methionine for
24 h. The media were concentrated as described under "Materials
and Methods." PDI (lanes 1 and 2),
BiP (lanes 2 and 3), and TG
(lanes 5 and 6) were recovered by
immunoprecipitation, resolved by SDS-PAGE (7% acrylamide gel) and
detected by fluorography. A control sample with Protein A-Sepharose but
without antibodies was shown for comparison (lane
7).
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PDI was probably secreted by FRTL5 cells rather than leaked from
injured cells, because only 0.8% after 24 h, and 1.8% after 48 h, of total lactate dehydrogenase was released from FRTL5
cells, whereas densitometry showed that 7.5% of total PDI was present in the medium (i.e. the medium + cell extracts) after
24 h. To confirm that PDI recovered from the media was secreted
and did not result from cell rupture, we treated cells with either
brefeldin A or colchicine prior to and during the labeling and chase
periods. Densitometric analysis of fluorographs of PDI
immunoprecipitates showed that the amount of newly synthesized PDI in
cells was slightly increased after drug treatment (control: 0.048 ± 0.017; brefeldin A: 0.055 ± 0.018; colchicine: 0.054 ± 0.016; n = 3), whereas there was much less PDI in the
media, with a reduction of 75-95% (control: 0.128 ± 0.016;
brefeldin A: 0.022 ± 0.008; colchicine: 0.032 ± 0.006;
n = 3).
PDI Is Present at the Surface of FRTL5 Cells--
PDI was
recovered by immunoprecipitation following the radioiodination of cell
surface proteins. Similar amounts of PDI were recovered in the absence
(Fig. 5, lane 2)
and presence (Fig. 5, lane 3) of
-mercaptoethanol. This rules out disulfide bridge formation as the
mechanism of PDI cell association at the cell surface. If the anti-KDEL
antibody was used after the anti-PDI antibody, six proteins were
immunoprecipitated from the labeled plasma membrane fraction (Fig. 5,
lane 4) as reported previously in a study of
exocrine pancreatic cells (33). These proteins, thought to include
grp94 and BiP (33), are probably chaperones containing the KDEL
sequence.
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Fig. 5.
Biochemical identification of cell surface
PDI and several proteins that presumably have a KDEL sequence.
FRTL5 cells were surface labeled with iodinated sulfo-SHPP. Cell
lysates were analyzed directly by SDS-PAGE (lane
1) or subjected to immunoprecipitation with anti-PDI
(lanes 2 and 3) or anti-KDEL
antibodies (lane 4) as described under
"Materials and Methods." Molecular mass markers are in kDa.
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PDI was readily detected on the cell surface of FRTL5 cells by confocal
microscopy (Fig. 6a). All
cells were labeled. The labeling was punctate and covered the entire
cell surface, although it was more dense in some regions than in
others, suggesting the formation of aggregates or clusters. Calnexin
was also detected but the labeling pattern differed between cells. The
most strongly labeled cells also showed punctate labeling (Fig.
6b). In contrast, despite numerous studies on different cell
preparations, we failed to detect specific labeling of BiP at the cell
surface (Fig. 6c). A possible explanation is that, whereas
the subcellular distribution of BiP did not markedly differ from that
of PDI (Fig. 6d) and calnexin (Fig. 6e) (all
three chaperones presented the same punctate labeling pattern in
vesicular structures located throughout the cytoplasm) the level of
fluorescence staining was less intense for BiP (Fig. 6f). We
concluded that, if present at the cell surface, BiP was not readily
detectable using this approach, and we did not further investigate the
colocalization of various chaperones by confocal microscopy.
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Fig. 6.
Detection by immunofluorescence of PDI and
calnexin on the surface of FRTL5 cells. Non-permeabilized
(a-c) and Triton X-100-permeabilized (d-f)
cells were incubated at 4 °C with anti-PDI (a and
d), anti-calnexin (b and e), and
anti-BiP (c and f) antibodies. The cells were
fixed and incubated with polyclonal antibodies, and then with goat
anti-rabbit IgG conjugated with FITC. Extracellular (a-c)
and intracellular (d-f) labeling were compared.
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As stated in the Introduction, TG is thought to interact with various
molecules, either constituents of the plasma membrane (megalin,
interaction at neutral pH) or molecules thought to pass via the surface
of the cell during their intracellular transport (the putative TG
receptor, interaction in acidic conditions). The cell membrane
chaperones associate with misfolded (3, 5-9) and denatured TG (47).
This raises the question as to whether these chaperones also associate
with particular subpopulations of TG molecules in preparations that we
use. To clarify this issue, we fist tried to identify the locations of
the putative receptors for TG and PDI at the cell surface (Fig.
7). In our conditions, endogenously
expressed rat TG was not detected on the surface of FRTL5 cells,
regardless of the type of anti-TG antibody used (monoclonal or
polyclonal, data not shown). As deduced from biochemical studies of the
TG binding characteristics on plasma membrane preparations (21, 22),
cell surface-associated TG could only be detected after addition and
incubation of exogenous TG in acidic conditions at 4 °C (data not
shown). In the present study, we have used fluorescein isothiocyanate-conjugated TG to visualize directly the binding sites of
TG on the cell surface. This binding was as specific as that observed
on membrane preparations, since in control experiments we noted that it
could be specifically prevented by preincubation with non-derivatized
TG (10-5 M concentration, data not shown). Fig. 7 showed
that PDI was readily detected on the surface of these FRTL5 control
cells (Fig. 7a). TG-FITC did not bind to cells at neutral pH
(Fig. 7b), but binding was abundant and clearly detectable
in acidic conditions (Fig. 7c). The detection in the same
cellular preparation of TG bound to cells in acidic conditions (Fig.
7d) and of PDI (Fig. 7e) demonstrated that this
receptor and PDI were located in the same plasma membrane domains (Fig. 7f). Thus, PDI is either located in the vicinity of the
receptor or is itself the receptor that binds TG in acidic
condition.
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Fig. 7.
Presence at the cell surface and
determination of the distribution of PDI and TG binding sites on FRTL5
cells by confocal microscopy. Polyclonal anti-PDI antibodies
detected PDI (a) on the surface of fixed cells. As described
under "Materials and Methods," the incubation of TG-FITC with
living cells at 4 °C in either PBS or acetate buffer at pH 5.0 indicated that TG did not bind to membranes at neutral pH
(b) but did bind to membranes in acidic conditions
(c). On the same cell preparation, PDI was detected using
the anti-PDI monoclonal antibody (d), the TG binding sites
were detected using TG-FITC in acidic binding conditions
(e), and both were co-localized at the surface of the cells
(f) and analyzed as described under "Materials and
Methods."
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PDI Is Involved in the Binding of TG to FRTL5 Membranes--
We
have reported that the membrane receptor that binds TG in acidic
conditions interacts with the various species of TG and preferentially
binds molecules with a low iodine content (21). The domain of TG
responsible for the binding of the molecule to membranes is located
within the Ser789-Met1172 sequence (22). This
domain contains two N-linked glycan moieties and a
cysteine-rich motif encoded by exons 14 and 15. The cysteine-rich motif
is not involved in the interaction (22). The glycan moieties are
involved in the interaction because their enzymatic cleavage reduces
binding affinity by an order of magnitude (21). Bearing these previous
findings in mind, we assessed whether TG fusion proteins encoding all
or part of the binding domain were fully active, and investigated
whether PDI was involved in the recognition of this binding domain.
The binding activities of TG fusion proteins at pH 5.0 were analyzed
using 125I-TG, TG and TG fusion proteins as competitors,
and microwell plates coated with FRTL5 plasma membranes (Fig.
8). Unlike the control proteins, MBP and
MBP-paramyosin, the TG-fusion protein containing the binding domain
(Pro759-Leu1163 fragment, BD in Fig. 8)
inhibited the interaction between 125I-TG and membranes.
However, as expected for a non-glycosylated binding region (21), the
binding affinity of this peptide was lower than that of TG itself
(K0.5 = 0.2 × 108
M and 0.3 × 107 M). The
peptide fragment lacking RU9, due to the alternative splicing of exons
14 and 15 (BDRU9), had the same degree of inhibitory activity as BD.
The lack of involvement of RU9 in binding was also demonstrated by its
lack of inhibitory activity, similar to that observed for the
nonspecific binding of MBP to membranes (Fig. 8). We thus concluded
that the binding domain encoded by TG fusion proteins had the same
binding properties as those encoded by native TG molecules.
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Fig. 8.
Inhibition of the binding of
125I-TG to thyroid membranes coated on multiwell microtiter
plates by TG-derived fusion proteins. The binding of labeled
porcine TG to membranes was challenged with human TG, porcine TG, MBP,
the TG-derived MBP fusion proteins BD (BD is the membrane-binding
domain of human TG (Pro759-Leu1163)), BDRU9
(BDRU9 is that of the human TG variant with the cysteine-rich module
RU9 (His1025-Cys1160) deleted), and RU9
(RU9 is the RU9 cysteine-rich module
(Leu1080-Leu1163)). MBP and MBP-paramyosin
were used as controls.
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We investigated whether PDI, alone or with other chaperones, was
directly involved in this binding. We first identified the chaperones
that were present in our preparations of plasma membranes. We found, as
observed previously (Fig. 5, lane 4; Fig.
6b) that in addition to PDI, other chaperones such as
calnexin, grp94, and BiP were present in these preparations (Fig.
9, left). Monoclonal antibodies directed against PDI inhibited TG binding to these membrane
preparations, confirming that PDI is either in the vicinity of the
receptor or is directly involved in TG binding to membranes (Fig. 9,
right). In contrast, monoclonal antibodies directed against grp94, BiP, and calnexin had no effect on the binding of TG (Fig. 9,
right), demonstrating that none of these chaperones were
involved in binding. TG binding was strongly inhibited (80%) at a
concentration of 1 µg/ml, by a monoclonal antibody with an epitope
close to one of the thioredoxin sites (monoclonal antibody RL90) and
was inhibited by about 30% by a monoclonal antibody directed against the C-terminal domain of PDI (Fig. 9, right). The same
pattern of selective inhibition by the anti-PDI antibody was observed for two TG-derived fusion proteins, MBP BD and MBP BDRU9 (Fig. 9,
right), demonstrating that they interact with the same
binding site.
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Fig. 9.
Inhibition of the binding to FRTL5 membranes
of 125I-TG or TG-derived fusion proteins by anti-PDI
antibodies. Left, membranes (10 µg) were used to coat
multiwell microtiter plates, and chaperones were detected by probing
with antibodies and revealed, the primary antibodies then being
detected by enzymatic reaction using horseradish peroxidase-linked
anti-mouse IgGs as described under "Materials and Methods." A
monoclonal anti--galactosidase was used as control.
Right, membranes (20 µg) were coated on multiwell
microtiter plates and the binding of 125I-labeled human TG
or fusion proteins was challenged using anti-grp94, BiP, calnexin, and
PDI monoclonal antibodies (1 µg/ml). The results shown are those of
three experiments performed in duplicate.
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We then used a biochemical approach to demonstrate that PDI is directly
involved in the binding of TG to the membrane (Fig. 10). The surface proteins of FRTL5
cells were radiolabeled with 125I. The cells were lysed,
and the cell lysates were incubated, in various conditions, with TG
adsorbed onto agarose beads. If the agarose beads were incubated and
washed in neutral pH conditions, the material associated with the beads
gave four major bands in SDS-PAGE. Three of these bands had molecular
masses below 26.6 kDa. The fourth had a molecular mass of about 57 kDa
(Fig. 10, lane 1), identical to that of the
125I-labeled PDI used as a standard (Fig. 10,
lane 5). If the agarose beads were incubated with
cell lysate in acidic conditions (pH 5.0), even with an aliquot
one-fifth the volume of that used in neutral conditions, a larger
amount of the 57-kDa protein was recovered and the low molecular weight
contaminating bands were not detected (Fig. 10, lane
2). The 57-kDa band was not recovered if the lysate was
cleared with an anti-PDI monoclonal antibody prior to the experiment
(Fig. 10, lane 3). Therefore, PDI was the only
radioiodinated cell membrane protein that bound TG in acidic conditions.
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Fig. 10.
Isolation and SDS-PAGE analysis of the FRTL5
cell surface TG-binding protein. FRTL5 cells were cell
surface-labeled and lysed as described under "Materials and
Methods." The lysate was aliquoted. Aliquots were incubated and
washed in the presence of 250 µl of TG-agarose beads in: 1.50 ml of
lysate in PBS, pH 7.2 (lane 1), 0.3 ml of lysate
in acetate buffer pH 5.0 (lane 2); 0.3 ml of
lysate cleared by a monoclonal anti-PDI antibody (clone RL90) prior to
use (lane 3). Lanes 4 and
5 correspond to aliquots of total cell surface iodinated
proteins and 125I-PDI, respectively. Proteins were resolved
by electrophoresis in a 10% acrylamide gel in the presence of
-mercaptoethanol. Molecular masses in kDa are shown.
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Surface Plasmon Resonance Analysis of the TG:PDI
Interaction--
Finally, we investigated whether PDI was itself
sufficient for both TG binding and the selection of immature molecules,
via binding to the binding domain. Real time studies of TG binding to
immobilized PDI were recorded on a biosensor using PDI as a ligand and
various sources of TG as analytes. No binding occurred at neutral pH,
but binding did occur in acidic conditions (data not shown). We decided
to analyze the interactions at pH 5.0 to enable us to compare the
results to those obtained previously. We found that TG from pigs and
humans bound PDI (Fig. 11). Similar amounts of mature human and porcine prohormone were bound by PDI (Fig.
11), but the binding affinity was higher for human TG
(KD = 1.6 × 109
M and 2.1 × 108 M,
respectively, see Table I). About three
times as much iodine-poor TG (iTG, iodine content = 0.1%) as
normal TG (TG, iodine content = 1%) bound to PDI (Fig. 11). The
binding affinity for iodine-poor TG was also higher than that for
normal TG (KD = 1.0 × 109
M and 1.6 × 109 M,
respectively, Table I). Thus, these results are consistent with
previous finding for membrane preparations indicating: 1) that TG
molecules are heterogeneous and only a subset is capable of binding,
and 2) that modifications clearly correlated with maturation affect the
binding activity. We found that fusion proteins encoding the binding
domain also bound to PDI. BD bound to PDI with an affinity one-tenth
that of native TGs (KD = 3.8 × 108), as expected for the non glycosylated binding domain
(Fig. 8; see also Fig. 4 in Ref. 21). Deletion of the type I
cysteine-rich module did not reduce this binding (it even seemed, for
still unknown reasons, to increase binding by a factor of 3:
KD = 1.1 × 108). No surface
plasmon resonance signal was detected with either RU9 or MBP (data not
shown).
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Fig. 11.
Typical sensorgrams (response in arbitrary
units (RU) versus time in s) of TG
binding to PDI (50 µg/ml in acetate buffer, pH
5.0). Curve 1, normal human TG
(hTG); curve 5, iodine-poor human TG
(ihTG); curve 3, porcine TG
(pTG); curve 4, TG fragment BD;
curve 2, TG fragment BDRU9.
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Table I
Kinetics and constants for the binding of various TG forms and
TG-derived protein fusions to PDI
KD is calculated from the ratio of
kd to ka. ihTG, hTG, and pTG
stand for iodine-poor human TG, normal human TG, and porcine TG,
respectively. BD is the PDI-binding domain of human TG
(Pro759-Leu1163), BDRU9 is that of the human TG
variant with the cysteine-rich module RU9
(His1025-Cys1160) deleted, and RU9 is the RU9
cysteine-rich module (Leu1080-Leu1163).
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DISCUSSION |
We report the following novel findings. 1) PDI is secreted by and
associated with the surface of FRTL5 thyroid cells. 2) The binding of
TG to FRTL5 cell membranes in acidic conditions is selectively
inhibited by anti-PDI antibodies. These antibodies also inhibit the
binding of fusion proteins encoding the
Ser789-Met1178 membrane receptor binding
domain. 3) Surface plasmon resonance analysis of TG/PDI interaction
characteristics indicated: (i) that PDI binds TG, but only in acidic
conditions, and that it preferentially recognizes immature molecules,
and (ii) that the Ser789-Met1178 domain is
involved in binding, even if the cysteine-rich module is deleted. These
results, and the potential function of PDI as a binder of secreted TG
molecules in acidic post-ER compartments, are of particular importance.
FRTL5 thyroid cells are an additional secretory cell type in which some
molecular chaperones can escape the ER compartment, even if they
possess the C-terminal KDEL retention signal (33, 34). The mechanism of
secretion by the various cell types is unknown. Secretion may be due to
the high level of chaperones in the ER of these cells, or may be an
overflow of the KDEL or other ER retention mechanisms (48, 49).
However, the specific overproduction of PDI in CHO cells (50) or HT
1080 fibrosarcoma cells (40) increases the secretion of PDI but not
other resident endoplasmic reticulum peroteins containing the
KDEL-sequence, such as grp78 and grp94, suggesting that "PDI
secretion and cell-localization is a specific event" (40), probably
related to a specific function.
PDI has been implicated in the reduction of proteins present at, or
interacting with, the cell surface, including thrombospondin (51),
plasmin (52), diphtheria toxin (36), and the human immunodeficiency
virus type 1 envelope glycoprotein (37). More recently, it has been
reported that secreted PDI manipulates the redox state of exofacial
extracellular protein thiols/disulfides, thereby controlling the
function of extracellular proteins (40). It is therefore possible that
in vivo PDI is involved in the structural modification of
TG, particularly the formation of disulfide bridges between and within
chains, that takes place in the follicular lumen during the synthesis
of thyroid hormones (15).
We quantified PDI secretion by FRTL5 cells and found that it was as
high as 15.01 ± 3.71 µg of PDI/5 ml of culture medium per
48 h (range: 18.7-13.1, n = 4) for semiconfluent
cells. This estimation was made in a cell line known to produce large
amount of secreted molecules, at least in the case of TG. Further
studies are therefore required to compare the secretion of PDI in
thyroid cells, primary cultures, and other cell lines.2
Nonetheless, the quantity outside the ER appears to be far from negligible, and PDI may therefore be involved in TG metabolism in the
follicular lumen in vivo. Studies in vitro have
shown that PDI has two distinctive features independent of its function
as a catalyst; at high concentration, it acts as a chaperone,
inhibiting aggregation, but at low concentration, it facilitates
aggregation (anti-chaperone behavior) (28, 53). One example of possible anti-chaperone behavior by PDI is the small quantities of PDI molecules
that have been found associated with highly cross-linked aggregates in
the follicular lumen (41). These aggregates are 50-500 µm in size,
may reach concentrations of 590 mg/ml, and consist of TG molecules with
high iodine but no hormone (41). The formation of these aggregates,
which are also known as "colloid globules" (41), therefore prevents
the homing of misfolded and hormone-poor prohormones to the endocytotic
secretory pathway of thyrocytes.
However, our results suggested a novel function for PDI, different from
those previously described. First, the interaction between TG and PDI
occurs in acidic conditions. For reasons explained elsewhere (20, 21),
we decided to analyze this interaction at pH 5.0. However, it also
occurs to a significant extent at pH 6.0, and at pH 5.75 fixation was
about 30% the maximum (20, 21), which suggests that it may take place
in vivo, in any of the acidic compartments of the cellular
exocytosis (54-56) and endocytosis (57-59) pathways. Second, in
contrast to the situation for TG misfolded during synthesis (3, 5-9)
and for denatured TG (47), this interaction involves only one domain of
TG and takes place between TG and PDI only, to the exclusion of other chaperones such as grp94 and BiP. Third, the catalytic function of PDI
is not involved because carboxymethylated TG molecules bind to
membranes (Fig. 4 in Ref. 21), and cysteine-rich modules are not
involved in the binding (Ref. 22 and this work). Thus, this TG binding
property of PDI is activated in acidic post-ER compartments such as
those of the exocytotic and endocytotic pathways.
There is morphological and functional evidence for the existence of a
regulated secretion for TG in thyrocytes (see Ref. 3 for a recent
review). It is generally accepted that biogenesis of the vesicles of
the regulated secretion pathway begins in the trans
compartment of the Golgi apparatus and continues via a gradual process
of enrichment and condensation of products destined for secretion (60).
The vesicles become more acidic as they cross the Golgi apparatus, and
this is known to play an essential role in the secretion pathway (60).
It is probable that PDI and TG interact in these compartments. Indirect
evidence for this is provided by the presence in the TG-binding domain
of N-linked glycans with accessible GlcNAC residues, which
may be involved in PDI recognition (Refs. 21 and 22; this report). It
may be that the addition of galactose and sialic acid, which is known to occur in the trans-Golgi network (19), is prevented by
competition between PDI and glycosyl transferases and steric hindrance
on the "receptor-binding domain" of TG. If this were the case: 1) all or some of the secreted PDI would be released into the regulated secretory pathway, and 2) interaction between PDI in this acidic compartment would probably be involved in the efflux of this chaperone from FRTL5 cells.
As previously speculated for the membrane receptor of TG that functions
in acidic conditions, the interaction between PDI and TG may occur
after their internalization, in the endosomes or prelysosomes. The
receptor responsible for the interaction has yet to be identified
because attempts at its molecular cloning have not been successful (61,
62). PDI possesses several of the biochemical characteristics
previously reported for this receptor. It is an acidic non-glycosylated
protein (63), abundant in the intracellular compartments (63). The
molecular mass of the protein initially isolated by large scale
purification from membrane preparations (51 kDa; Ref. 63) corresponds
to that of the major degradation product of PDI in thyroid FRTL5 cells
(this study). In addition, binding experiments with the biosensor have
shown that, like the membrane receptor, PDI recognizes ovomucoid (data
not shown), a GlcNAc-bearing glycoprotein that competes with TG for the
binding site on the membrane (20). As we exhausted our supply of the antibody directed against the receptor (61), we were unable to test the
antigenicity of the receptor (NAGR1, Ref. 63) and PDI and were
therefore unable to draw the conclusions about the identity of the two
molecules. However, we demonstrate here that PDI is a
membrane-associated protein capable of binding preferentially to
immature prohormones in acidic conditions as it recognizes the
Ser789-Met1172 domain of TG. Thus,
membrane-associated PDI is a prime candidate for involvement in the
quality control thought to be responsible for the recycling of immature
TG from the endosomal compartment back to the lumen.
In conclusion, PDI may bind to certain form of immature TG in the
exocytosis and endocytosis pathways of thyrocytes. It is possible that
PDI acts as an "escort," preventing structural modification of the
binding domain of TG during its cellular transit, and that it
coordinates the modifications of TG associated with hormonogenesis. There are several lines of evidence for this. 1) The binding domain of
Tg contains two clusters of tyrosine residues involved in iodination and hormone formation (22); 2) the binding domain is located in the
N-terminal region of TG, which is highly cross-linked via 11 repeated
cysteine-rich motifs and undergoes profound structural changes during
its maturation in the follicular lumen (46); and 3) iodination and
hormonogenesis are thought to occur after excocytosis, at the cell
surface, when TG comes into contact with thyroperoxidase. We are
investigating this possibility further in two ways. First, as PDI is
not an integral membrane protein, we are trying to identify the
partners responsible for its association with the membrane and to
determine whether and how PDI is transported with TG in the secretory
and endocytotic pathway. Second, we are trying to identify the
determinants of the binding domain that acts as sensors, that promote
or prevent the interaction with TG during its intracellular transit in
post-ER compartments.
§,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed. Tel.:
33-4-91-96-20-69; Fax: 33-4-91-65-75-95.
