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INSERM U-38, Université de la Méditerranée, Faculté de Médecine, 13385 Marseille Cedex 5, France
Address all correspondence and requests for reprint to: J. L. Franc, INSERM U-38, Faculté de Médecine, 27 boulevard J. Moulin, 13385 Marseille Cedex 5, France. E-mail: jean-louis.franc{at}medecine.univ-mrs.fr
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CNX and CRT form transient monovalent associations with a variety of membrane and soluble glycoproteins (9, 10). They specifically recognize monoglucosylated oligosaccharide structures in glycoproteins (11, 12). The oligosaccharide structures are maintained by rapid cycles of glucose removal and reglucosylation catalyzed by the UDP-Glc, glycoprotein glucosyltransferase, which detects any unfolded or misfolded conformations (13, 14). In addition, it has been established that CNX can be associated with protein in a glycan-independent manner (15). CNX and CRT are thought to play an important role in the folding and oligomeric assembly of various major glycoproteins: myeloperoxidase (16), major histocompatibility complex type I (17), influenza hemagglutinin (18), gonadotropin receptor (19), and insulin receptor (20). Some glycoproteins have been reported to associate exclusively with CNX (21). Far less attention has been paid, however, to the possibility that CRT may be part of an ER chaperone apparatus (22).
The 2% of all hTPO molecules that are able to exit from the ER and reach the cell surface are likely to involve interaction with molecular chaperones. No proof has been obtained to date, however, that hTPO associates with any molecular chaperones, either during or after its biosynthesis. In the present study we first demonstrated that hTPO associates with CNX and CRT, and then determined the extent to which these molecular chaperones participate in the folding and intracellular trafficking of hTPO molecules.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cloning of hTPO, CNX, and CRT complementary DNAs (cDNAs)
Full-length 3060-kb cDNA coding for hTPO (obtained from
Dr. B. Rapoport, San Francisco, CA) was cloned into the eukaryotic
transfer vector pcDNA3 as described previously (6). A full-length
1.8-kb cDNA coding for rabbit CRT (provided by Dr. Michalak, Alberta,
Canada) was cloned into the KpnI and XbaI sites
of the pcDNA3.1-Hygro expression vector. A full-length 2.5-kb cDNA
coding for dog CNX (a gift from Dr D. Thomas, Montreal, Canada) was
cloned into the KpnI and NotI sites of the
expression vector pcDNA3.1-Hygro. All cDNAs were expressed by the
cytomegalovirus promoter.
CHO cell culture and transfection procedure
As described previously (6), CHO cells were transfected with
cDNA coding for hTPO and after being selected with geneticin were
subcloned to obtain a clone expressing a high level of hTPO (referred
to as hTPO-CHO cells). hTPO-CHO cells were then transfected with
CNX-cDNA or CRT-cDNA in pcDNA3.1-Hygro or with pcDNA3.1-Hygro alone
using Lipofectamine. Forty-eight hours after the transfection, hTPO-CHO
cell cultures were cultured for 3 weeks in a selection medium
containing 100 µg/ml hygromycin B. Experiments were carried out using
CNX-hTPO-CHO, CRT-hTPO-CHO, and Hygro-hTPO-CHO cell pools.
Metabolic labeling and immunoprecipitation procedure
After a 2-h preincubation in methionine- and cysteine-free DMEM
supplemented with 10% FBS and 10 mM sodium butyrate, cells
were radiolabeled with [35S]Met and
[35S]Cys (10 µCi/ml). The incubation times
varied from 30 min to 5 h. For the pulse-chase studies, cells were
preincubated for 2 h in Met- and Cys-free DMEM supplemented with
10% dialyzed FBS and sodium butyrate (10 mM) and
subsequently pulsed for 30 min in the same culture medium supplemented
with [35S]Met and
[35S]Cys (10 µCi/ml). After the pulse, the
radiolabeling medium was removed, the cell surface was washed twice
with 1 ml Ham’s F-12 medium, and the cells were then chased four times
for 5–48 h in culture medium supplemented with 5 mM Met
and 5 mM Cys. Once the chase was over, cells were kept on
ice, washed twice with 2 ml ice-cold PBS, and scraped in 600 µl hTPO
extraction buffer containing 50 mM Tris-HCl (pH 7.4), 0.15
M NaCl, 1% Triton X-100, 0.3% deoxycholate, protease inhibitor
cocktail, and 100 mM iodoacetamide when required. The cells
were then tumbled for 20 min at 4 C and centrifuged for 3 min at
10,000 x g. The radiolabeled supernatant obtained was
saved and incubated for 2 h at room temperature with association
of two mAbs directed against hTPO (mAb 15 and mAb 47). The use of this
couple in the immunoprecipitation experiments allowed us to recover
more than 90% of the hTPO molecules expressed in CHO cells (6). On the
other hand, when used separately, these two mAbs immunoprecipitated two
different populations of hTPO molecules; mAb 47 immunoprecipitated
unfolded hTPO forms, and mAb 15 immunoprecipitated both partially and
completely folded hTPO forms. The populations of hTPO molecules that
overlapped between mAb 15 and mAb 47 did not amount to more than 10%
of each population (data not shown). These mAbs had been previously
complexed with protein A-Sepharose by incubating them overnight at 4 C.
Immune complexes were then retrieved by performing a brief
centrifugation (10,000 x g, 10 sec) and washed four
times with 1 ml hTPO extraction buffer and once with 1 ml PBS. The
precipitated proteins were separated from antibody-protein A-Sepharose
complexes by boiling for 5 min in the Laemmli buffer, and samples were
subjected to SDS-PAGE analysis.
Sequential immunoprecipitation experiments
Metabolic labeling and immunoprecipitation were performed as
described above, except that 1% CHAPS was used instead of 1% Triton
X-100 and 0.3% deoxycholate in the extraction buffer and for the
binding of anti-CRT and anti-CNX antibodies to protein A-Sepharose.
CHO-hTPO cells were incubated for 2 h with 10 mM
sodium butyrate and radiolabeled for 30 min with
[35S]Met and [35S]Cys.
The immunoprecipitation step with the first antibody (anti-CRT or
anti-CNX) was performed as described above. After the first
immunoprecipitation step, the proteins were separated from the protein
A-Sepharose pellet by heating it for 5 min at 100 C after adding 10
µl 10% SDS diluted with 500 µl CHAPS buffer. After centrifugation
for 3 min at 10,000 x g, the protein A-Sepharose
pellet was discarded, and the supernatant was incubated overnight at 4
C with the two associated anti-hTPO mAbs (mAbs 15 and 47) preabsorbed
on protein A-Sepharose beads. After immunoprecipitation and a washing
step, the second precipitates were resuspended in Laemmli buffer,
boiled for 5 min, and then loaded on SDS-PAGE.
Cell surface biotinylation
Confluent cells were radiolabeled for 48 h with
[35S]Met and [35S]Cys.
Cell monolayers were then washed twice with ice-cold PBS (pH 8.0)
supplemented with 1 mM CaCl2 and 1
mM MgCl2 (PBS-CM) before the
cross-linker
sulfosuccinimidyl-2-(biotinamide)ethyl-1,3'-dithiopropionate
(NHS-SS-biotin) was added at a concentration of 0.5 mg/ml in PBS-CM for
20 min on ice. The cross-linker was removed, and the above operation
was repeated once. The medium was then removed, and the remaining
reactive NHS-SS-biotin was blocked by adding 50 mM
NH4Cl in PBS-CM for 10 min on ice under gentle
agitation. The cells were then washed twice with PBS-CM before being
harvested. After being extracted, the proteins were incubated for
2 h with previously prepared mAb 47- and mAb 15-protein
A-Sepharose complexes. Immunoprecipitated hTPO was then separated from
the protein A-Sepharose pellet by heating it for 5 min at 100 C after
adding 10 µl 10% SDS and 500 µl hTPO extraction buffer. The
suspension was diluted with 500 µl hTPO extraction buffer and then
centrifuged for 3 min at 10,000 x g. The supernatant
was incubated for 2 h with avidin-agarose (Pierce Chemical Co., Rockford, IL). Biotinylated surface hTPO and intracellular
hTPO were separated by centrifugation (10 sec, 10,000 x
g). The beads were washed four times with hTPO extraction
buffer and once with PBS, resuspended in electrophoretic buffer, and
boiled for 5 min. The total supernatant corresponding to the cell
surface fraction and only 1/10th of the supernatant corresponding to
the intracellular fraction were analyzed by SDS-PAGE.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, lanes 1 and 4).
To determine whether hTPO was one of these proteins, lysates of
radiolabeled CHO-hTPO cells were precipitated with anti-CNX or anti-CRT
antibodies. The precipitated proteins were then released by boiling for
5 min in the presence of 1% SDS and incubation in the presence of mAbs
directed against hTPO molecules (see Materials and Methods)
or an irrelevant mAb directed against thyroglobulin. As shown in Fig. 1
(lines 3 and 6), the specific 110-kDa band corresponding to hTPO
indicated that in CHO cells, hTPO molecules associated with both CNX
and CRT.
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, lanes 2). In the presence of MG132, a
proteasome inhibitor that prevents the degradation of hTPO (see Fig. 4), a similar result was obtained (Fig. 2, lanes 3).
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), approximately the same amount of hTPO molecules was
recognized by both mAb 47 and mAb 15. The quantity of hTPO molecules
able to acquire the three-dimensional structure making them
recognizable by mAb 15 was much lower when the cells were treated with
CST. When interactions between CNX and/or CRT were inhibited by CST,
the hTPO molecules, therefore, remained mostly in the unfolded form
recognized by the mAb 47 (Fig. 3).
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). The presence of these two mAbs
allowed immunoprecipitation of more then 90% of the hTPO (6). The data
indicated that CST strongly enhanced the degradation rate of hTPO
molecules, as after 4 h of chase, only 39% of the hTPO were
immunoprecipitated compared with that in the control experiments, and
the half-life of hTPO during CST treatment was also dramatically
reduced (2.5 h, vs. 17 h for the control experiments).
Note that under control conditions, a greater amount of hTPO was
immunoprecipitated after 4 h of chase than after the pulse. This
was probably due to a process of [35S]hTPO
synthesis or completion that continued for some minutes after the chase
medium was added. As cycloheximide, which is known to be a potent
protein synthesis inhibitor, has been reported to be a degradation
inhibitor, we did not add this compound to the chase medium.
To obtain further insights into the mechanism regulating hTPO molecule
degradation, further experiments were performed with a view to
ascertaining whether treatment with CST might increase the
proteasome-dependent ER-associated protein degradation process, as the
results of recent studies have indicated that proteasomes were involved
in the degradation of several proteins when glucose trimming was
inhibited with CST (24). In this study, pulse-chase experiments were
carried out; cells were treated with or without 1 mM CST
and with or without the proteasome inhibitor MG132 added to the
preincubation, pulse, and chase media. The addition of MG132
significantly diminished the degradation of hTPO in CST-treated cells
(Fig. 4). Similar results were obtained with lactacystin, an
irreversible proteasomal inhibitor. Interestingly, these data indicate
that proteasome activity is necessary for the degradation of hTPO
molecules with an impaired CNX and CRT association, as MG132 and
lactacystin strongly inhibited the degradation of hTPO during CST
treatment, but also had much less noticeable effects in these
experiments on the degradation of hTPO under the control conditions.
These results indicate that when the association with CNX and CRT is
impaired, hTPO is degraded rapidly via the proteasome pathway.
Effects of CST on the delivery of hTPO at the cell surface
As the degradation of hTPO was strongly increased in the presence
of CST, we investigated the consequence of CST treatment on the
delivery of hTPO at the cell surface. To quantitate the percentage of
hTPO present at the cell surface in the steady state, the cells were
radiolabeled for 48 h with [35S]Met and
[35S]Cys with or without CST, and the cell
surface proteins were then biotinylated. In the presence of CST, only
7.5% of the hTPO molecules were able to reach the cell surface,
vs. 30% under control conditions (Fig. 5). These data are consistent with the
previous results (Figs. 3 and 4), which showed that CST treatment
prevents hTPO folding and increases its degradation.
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) indicated that CNX and/or
CRT played a crucial role in the folding, stability, and delivery of
hTPO to the cell surface. To obtain further insights into the
contribution of CNX and CRT to the maturation and stability of hTPO, we
transfected CNX or CRT into hTPO-CHO cells to determine whether the
inefficient folding of the hTPO molecules reflected a limited
availability of endogenous CNX or CRT in CHO cells. On the other hand,
it seemed likely that these transfections might make it possible to
distinguish between the roles of CNX and CRT in the folding of hTPO
molecules. Western blot analysis was performed on hTPO-CHO cells and on
the pool of cells selected with hygromycin to compare the expression of
CNX and CRT in CHO-hTPO cells transfected with the cDNA coding for the
chaperones (CNX-hTPO-CHO cells or CRT-hTPO-CHO cells) or with the
pcDNA3-Hygro vector alone (Hygro-hTPO-CHO cells). We detected 5 times
more CNX and 6 times more CRT expression in the transfected cells than
in the control cells (Hygro hTPO-CHO cells and hTPO-CHO cells) (data
not shown).
In the first set of experiments we investigated the question of whether
overexpressing CNX or CRT might affect the folding of hTPO molecules.
Here we repeated the same experiment as that shown in Fig. 2, but
without adding CST. Under control conditions, a greater proportion of
the hTPO molecules was recognized by mAb 47 than by mAb 15 (Fig. 6). Overexpression of CNX and CRT led to
an increase in the percentage of hTPO molecules able to acquire the
three-dimensional structure making them recognizable by mAb15. The
folding enhancement resulting from CNX or CRT overexpression was
similar in both cases and ranged from approximately 45–60%.
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. The results showed that
CNX and CRT were associated with the unfolded forms immunoprecipitated
by mAb 47 as well as with the partially folded forms immunoprecipitated
by mAb 15 (Fig. 7).
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), and the extraction supernatant was immunoprecipitated with the mAb
15 and mAb 47 pair. No significant difference was observed in the
half-life of the hTPO among the three cell lines (Fig. 8), but after
the pulse chase, the amount of hTPO immunoprecipitated was always
significantly higher in CNX-hTPO-CHO and CRT-hTPO-CHO cells than in
Hygro-hTPO-CHO-cells (four different experiments). We therefore
hypothesized that these differences might be attributable to a rapid
proteasomal degradation of the hTPO molecules occurring just after
their synthesis. This finding suggests that CNX and CRT may play a
vital protective role and significantly decrease the degradation rate
of newly synthesized hTPO molecules, whereas they may have no
noticeable effect on the stability of hTPO molecules during long chase
periods. In the same way, overexpression of CNX and CRT had no
noticeable effect on the efficiency of the delivery of hTPO to the cell
surface (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results of the first set of experiments showed that 1) hTPO was
coimmunoprecipitated with these chaperones; and 2) the quantity of hTPO
molecules able to be recognized by mAb 15 was strongly decreased when
the binding of hTPO to CNX and CRT was inhibited by CST. These findings
are in agreement with previous descriptions of the roles of CNX and/or
CRT in the early stages of maturation of many glycoproteins: influenza
hemagglutinin (18, 25), insulin receptor (20), major histocompatibility
complex type I (26), and vesicular stomatitis virus G
glycoprotein (27), although folding into the native conformation has
been described as not being CNX or CRT dependent in the case of other
proteins (28). It is worth mentioning, however, that to our knowledge,
there exist no other data indicating that CNX or CRT plays such a
crucial role in the folding of a glycoprotein. Here we established that
after 5 h of [35S]Met and
[35S]Cys labeling in the presence of CST, less
than 10% of all hTPO molecules were able to acquire the
three-dimensional structure necessary for them to be recognized by mAb
15, in contrast with what occurred under the control conditions (Fig. 3).
In the second set of experiments (Fig. 4), the pulse-chase data
indicated that CST treatment dramatically increased the degradation of
hTPO molecules. Contrary to what was observed under the control
conditions, where the association with CNX and/or CRT was prevented, we
detected a 61% loss of protein after 4 h of chase. Our data are
consistent with the results of other studies, which showed that the ER
contains a unique quality control machinery in which the association of
proteins with CNX prevents these proteins from being degraded until
they have acquired the appropriate three-dimensional structure, and
hence, that intracellular disposal requires protein dissociation from
CNX (29). Over the last few years, an increasing amount of studies on
soluble and membrane-bound protein degradation have suggested that the
ubiquitin proteasome pathway is the main site of intracellular
degradation. After undergoing reverse transport from the ER back into
the cytoplasm, proteins may be degraded by the proteasome (30). To
date, very few studies have established any evidence for the existence
of a link between CNX association and proteasome degradation. Recently,
Keller and collaborators (24) reported that inhibiting glucose trimming
with CST led to proteasomal degradation of the 
-subunit of
nicotinamic acetylcholine receptor. It was also proposed by Chen and
collaborators (31) that inhibitory effects on the interaction between
CNX and apolipoprotein B might increase the ubiquitinylation of this
protein.
Our pulse-chase experiments in which CST was associated with the proteasome inhibitor MG132 clearly demonstrated that CNX and/or CRT association with hTPO prevented these molecules from being rapidly degraded via the proteasome pathway. This shows the crucial importance of CNX and/or CRT in the folding process that enables hTPO molecules to evolve from an unfolded to a partially folded structure recognized by mAb15. To distinguish between the role of CNX and CRT in the folding of newly synthesized molecules, we also investigated whether CNX availability was a limiting factor in CHO cells, and we overexpressed these two chaperones in hTPO-CHO cells. In both cases, this led to a 20% increase in the forms recognized by mAb15, but at the same time, it did not lead to any enhancement of the long term stability and delivery of hTPO to the cell surface. These data showed the stage of hTPO folding and maturation in which CNX and CRT are involved and suggested the existence of two distinct degradation systems: one for the unfolded hTPO molecules (possibly the proteasome) and another for the partially folded hTPO. The latter hypothesis is currently under investigation in our laboratory (Fayadat, L., et al., manuscript in preparation).
We then integrated the present findings into our previously proposed
model (6). CNX and CRT are both of crucial importance in the first step
of hTPO folding (i.e. unfolded form 47+/15-
partially
folded 15+), but once hTPO is in the partially folded form 15+,
overexpression of CNX or CRT has no noticeable effect during the
following folding process, which yields mature hTPO molecules. These
data are among the first (16, 26, 27) to show precisely at which
folding step CNX or CRT is absolutely necessary to efficient protein
folding. On the other hand, CNX and CRT still interacted with the
partially folded form (15+). This binding may 1) not have any effect on
the further folding of the hTPO; 2) protect the partially folded form
15+ or, on the contrary, activate its degradation; or 3) affect the
further folding of hTPO with or without an association with other
molecular chaperones. It is tempting to postulate that other molecular
chaperones (such as BiP or GRP94) might also contribute importantly to
1) this first folding step (unfolded form 47+/15- partially folded
15+), and then to 2) partially folded form 15+totally folded form
15+ and subsequently for delivery to the cell surface. An increasing
number of proteins have been reported to interact sequentially with
several molecular chaperones during the folding process (21, 27, 32).
Furthermore, heme insertion into hTPO has been found to be a
prerequisite for the exit of hTPO from the ER to occur (33). On the
other hand, the possibility cannot be ruled out that the lack of effect
of CNX and CRT on the delivery of hTPO to the cell surface may have
been due to the limited availability of ERp57, a protein homologous to
protein disulfide isomerase that has been reported to
considerably enhance the folding of CNX-bound substrate (34, 35, 36).
In conclusion, the present study on hTPO is among the first, besides
studies on the cystic fibrosis transmembrane conductance regulator, to
describe a wild-type protein in which the majority of the molecules
fail to reach their intended site of biological action. Several mutant
proteins (
F508CFTR, 1-antitrypsin, low
density lipoprotein receptor, etc.) have, in fact, been
reported to be involved in ER storage disease and ER-associated
degradation (37). One of the most noteworthy findings obtained in the
present study was that the binding of CNX and CRT to hTPO is a
prerequisite for the initial molecular folding step to be possible.
Further investigations are now required to identify the cellular
components involved in the final folding steps.
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Acknowledgments |
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Footnotes |
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Received July 23, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, cell
treated with 1 mM CST; 
, cells treated with MG132; 
,
cells treated with CST and MG132. Samples were analyzed by SDS-PAGE,
and the band corresponding to hTPO was quantitated by a phosphorimager.
The maximum intensity of the hTPO bands recorded at each treatment was
taken to be 100%. These experiments were repeated three times with
very similar results.
), CNX-hTPO-CHO cells (