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Institut National de la Santé et de la Recherche Médicale (Unité 38), Faculté de Médecine, Université de la Méditerranée, 13385 Marseille, Cedex 5, France
Address all correspondence and requests for reprints to: Jean-Louis Franc, INSERM U38, Faculté de Médecine, 27 Bd Jean 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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15%) of the latter were able to reach the plasma
membrane. The hTPO, unable to fold correctly, was more rapidly degraded
than that recognized by mAb 15 (half-time, 2 h vs.
7 h). Study of the carbohydrate content of hTPO showed that
N-glycans with complex-type structure were found only on hTPO at the
cell surface, whereas intracellular hTPO bore high-mannose-type
structures. Taken together, these data demonstrate that the
intracellular pool of enzyme is formed of newly synthesized molecules
and is not caused by recycling of mature hTPO from the cell surface.
Complete inhibition of hTPO N-glycosylation with tunicamycin led to a
95% decrease in hTPO at the plasma membrane and, thus, to a decrease
in enzymatic activity at the cell surface, emphasizing the role of
N-glycans in the intracellular trafficking of hTPO. However, inhibition
of formation of complex-type structures with deoxymannojirimycin and of
O-glycans with phenyl-
-GalNAc did not influence the intracellular
trafficking and enzymatic activity of hTPO. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CHO cell culture and transfection
CHO cell line (ECCAC number 85050302) was cultured in Ham’s F12
medium supplemented with 10% FBS (Boehringer-Mannheim,
Mannheim, Germany), penicillin (100 IU/ml), streptomycin (100 µg/ml),
and 10 mM sodium butyrate. Cells were transfected with
lipofectamine (Gibco BRL, Cergy-Pontoise, France). Forty-eight hours
after transfection, cells were cultured in a selection medium
containing 400 µg/ml geneticin (Gibco BRL). Cells highly resistant to
geneticin were subcloned by limited dilutions. Resitant colonies were
screened for positive hTPO expression by Western blot analysis. One
selected clone was expended for further study and maintained in the
same selective medium.
Metabolic labeling and extraction of hTPO
After preincubation during 16 h with 10 mM
sodium butyrate, cells were incubated in a methionine- and
cysteine-free DMEM supplemented with 10% FBS, 10 mM sodium
butyrate, and 10 µCi/ml 35S-(Met + Cys)
(Expre35S35S label, NEN, Le Blanc-Menil,
France). Times of incubation with radioactivity varied from 30 min to
5 h. For pulse-chase studies, cells were preincubated for 30 min
in Met- and Cys-free DMEM supplemented with 10% dialyzed FBS and 10
mM sodium butyrate and were subsequently pulsed for 30 min
in the same culture medium supplemented with 10 µCi/ml
35S-(Met + Cys). After the pulse, the radiolabeling medium
was removed, the cell surface was washed twice with 1 ml Ham’s F12
medium, and then cells were chased for times between 30 min and 72
h in culture medium supplemented with 5 mM Met and 5
mM Cys and, when indicated, with 10 mM
cycloheximide. When the chase was completed, cells were kept on ice,
washed twice with 2 ml ice-cold PBS, and scraped in 600 µl extraction
buffer containing Tris-HCl 50 mM (pH 7.4), NaCl 0.15
M, Triton X-100 1%, deoxycholic acid 0.3%, and protease
inhibitor cocktail (Complete, Boehringer-Mannheim). The
cells were then tumbled for 20 min at 4 C (vortexing every 2 min) and
centrifuged for 3 min at 10,000 x g.
Immunoprecipitation and electrophoresis
The radiolabeled supernatant obtained after extraction and
centrifugation was saved and incubated 2 h at room temperature
with various monoclonal antibodies (mAbs) directed against hTPO. These
mAbs had been previously complexed with protein A-Sepharose (Zymed
Laboratories, San Francisco, CA) by incubation overnight at 4 C. Immune
complexes were then retrieved by a brief centrifugation (10,000 x
g, 10 sec) and washed four times with 1 ml of TPO extraction
buffer and once with 1 ml PBS. The precipitated proteins were separated
from antibody-protein A Sepharose complexes by boiling for 3 min in the
Laemli sample buffer containing 62 mM Tris-HCl (pH6.8), 2%
SDS, 5% glycerol, and 5% 2-mercaptoethanol (2-ME). The samples were
then heated at 100 C for 3 min and subjected to SDS-PAGE (7.5%). The
radioactivity was visualized and quantified by a phosphorimager (Fudjix
BAS 1000, Japan).
Cell surface biotynilation
Confluent cells were radiolabeled for 48 h with
35S-(Met + 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, Pierce, Rockford, IL) was added at the
concentration of 0.5 mg/ml in PBS-CM for 20 min on ice. Cross-linker
was removed, and the same operation was repeated once. Then the medium
was removed, and the remaining reactive NHS-SS-biotin was blocked by
addition of 50 mM NH4Cl in PBS-CM for 10 min on ice and
under gentle agitation. The cells were then washed twice with PBS-CM
before being harvested. After extraction, the proteins were incubated
2 h with previously prepared mAb 47- and mAb 15-protein A
Sepharose complexes. Then immunoprecipitated TPO was separated from the
protein A Sepharose pellet by heating 5 min at 100 C after addition of
10 µl of 10% SDS and 500 µl hTPO extraction buffer. The suspension
was diluted with 500 µl hTPO extraction buffer and then centrifuged 3
min at 10,000 x g. The supernatant was incubated
2 h with avidin-agarose (Pierce). 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 totality of
supernatant corresponding to the cell surface fraction, and only one
tenth of the supernatant corresponding to the intracellular fraction,
were analyzed by SDS-PAGE.
Deglycosylation of hTPO
We adopted the following methods after doing assays to optimize
hTPO deglycosylation. Intracellular and cell surface hTPO were
separated by biotinylation method. After denaturation and reduction
with SDS and 2-ME, the samples were diluted with the buffer used for
incubation with glycosidase [50 mM Tris-HCl (pH 8.5), 1%
Triton X-100 for peptide-N-glycanase F (PNGase F) and 50 mM
acetoacetic buffer (pH 5.0), 1% Triton X-100 for
ß-endo-N-acetylglucosaminidase H (Endo H)] to obtain a final
concentration of 0.1% SDS and 0.05% 2-ME. The samples were incubated
for 16 h at 37 C with 1 U PNGase F or 5 mU Endo H (Boehringer
Mannheim). Controls were done under the same conditions as for the
corresponding assays, except that the deglycosylation enzymes were
omitted. The samples were then prepared for analysis by SDS-PAGE in
denaturing conditions.
O-glycosylation determination
Cells were incubated 48 h with 10 mM sodium
butyrate. In one dish, 2 mM phenyl--GalNAc was added to
the culture medium 1 h before addition of sodium butyrate.
Intracellular and cell-surface TPO were separated by biotinylation
technique, as described above, and were subjected to SDS-PAGE. Proteins
were then electrotransferred overnight to a polyvinylidene difluoride
sheet (Immobilon P. Millipore, St. Quentin Yvelines, France). The
polyvinylidene difluoride blot was blocked with 5% BSA in 20
mM Tris-HCl (pH 7.4), 50 mM NaCl (TBS), rinsed
twice for 10 min with TBS supplemented with 1 mM MgCl2, 1
mM CaCl2, and 1 mM MnCl2 (TBS-C). The blot was
incubated 2 h at room temperature with Jacalin-biotin (Sigma, St.
Louis, MO) diluted at 1/1000 in TBS-C, 1% BSA. The membrane was washed
five times with TBS-C and then incubated 2 h at room temperature
with avidine-horseradish peroxidase (Amersham, Les Ullis,
France) diluted at 1/5000 in TBS-C, 1% BSA. The revelation was done by
chemiluminescence (Pierce).
Enzymatic activity
Enzymatic activity was investigated as in Ref. 15 , with slight
modifications. Forty-eight hours before assaying hTPO enzymatic
activity, we supplemented the cell culture medium with 10
mM sodium butyrate. Then the medium was removed, and cells
were washed twice with ice-cold PBS buffer. The incubation mixture
contained BSA (5 mg/ml in PBS) and Na125I (106
cpm/ml). The reaction was initiated by addition of
H2O2 to obtain a final concentration of 0.5
mM. Cells were incubated for 20 min at room temperature. At
the end of this incubation time, the medium was transferred to cold
reaction tubes, and the cell surface was washed once with 0.5 ml PBS.
Then 1 ml ice-cold 20% (wt/vol) trichloroacetic acid, supplemented
with 10-4 M KI, was added to each tube. After
20 min at 4 C, the suspension was centrifuged (2000 x
g, 6 min). The supernatant was discarded, and the
acid-insoluble iodinated material obtained was washed 3 times with 2 ml
of 10% trichloroacetic acid (TCA). The radioactivity remaining
in the pellet was counted.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The use of the couple mAb 15 + mAb 47
in the immunoprecipitation experiments allowed us to recover more than
90% of hTPO expressed in CHO cells. The same results were obtained
using polyclonal antibodies directed against hTPO or using mAb 47 alone
if the cell extract was obtained with 1% SDS (data not shown). After
the pulse, newly synthesized hTPO molecules were strongly recognized by
mAb 47 but were not recognized by mAb 15. After 0.5 h of chase, an
increased amount of hTPO was immunoprecipitated with the mAb 47. This
is certainly caused by a synthesis or a completion of
35S-(Met + Cys) hTPO that continues during some minutes
after the addition of the chase medium. More interestingly, at this
time, 18% of the hTPO recognized by mAb 15 + mAb 47 was recognized by
mAb 15. At longer chase periods, there was no increase in these
molecules, and they were relatively stable: we noted a decrease of only
50% between 1 and 24 h of chase. In contrast, hTPO molecules
recognized only by mAb 47 were rapidly degraded. Depending on the
experiments, the half-life of hTPO molecules varied from 2–4 h for the
hTPO molecules recognized by mAb 47 and from 7–11 h for the hTPO
molecules recognized by mAb 15. The quantity of hTPO immunoprecipitated
by mAb 15 + mAb 47 was similar to or slightly higher than the sum of
the hTPO immunoprecipitated by mAb 15 and by mAb 47. These results
showed that mAb 47 is unable to immunoprecipitate all hTPO molecules;
thus, the linear epitope for the mAb 47 is accessible only when hTPO is
in an unfolded state. This was confirmed by an immunofluorescence
study, in which, in contrast to mAb 15, mAb 47 was not able to
recognize the hTPO at the level of the plasma membrane (data not
shown).
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).
Of the hTPO molecules, 85% were recovered in the intracellular
compartments and only 15% at the cell surface (Fig. 2, lanes 2 and 4).
Thus, as in thyroid cells, the majority (85–90%) of the hTPO
molecules are localized in the intracellular compartments. Another
interesting aspect was to determine the time needed by hTPO molecules
to reach the cell surface. We thus monitored pulse-chase experiments
(Fig. 3), using mAb 15 (because mAb 47 is
unable to react with TPO present at the cell surface). The maximum of
hTPO at the cell surface was found after 5 h of chase, and the
majority of the hTPO molecules never reached the plasma membrane. Note
that the recognition for intracellular hTPO was maximal after 5 h
of chase, because we used mAb 15 and, as we previously showed (Fig. 1
),
it took at least 30 min for hTPO to acquire the three-dimensional
structure suitable for the recognition by mAb 15.
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).
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Very little is known about the O-linked carbohydrate structure of hTPO.
To determine whether hTPO expressed in CHO cells bore O-glycans, we
used a Western blot with Jacalin-biotin to detect O-glycan structures
(18). Experiments were done with hTPO immunopurified from cell cultures
treated or not with phenyl--GalNAc, a specific inhibitor of
O-glycosylation (19). Intracellular and cell-surface hTPO were
separated. After Western blot, the Jacalin lectin clearly recognized a
band at 110 kDa corresponding to cell surface hTPO (Fig. 5, lane 2), but the lectin did not bind
to intracellular hTPO (lane 1). When the cells were treated with
phenyl--GalNAc, the intensity of the band corresponding to cell
surface hTPO was reduced by 80% (lane 4).
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-GalNAc, a specific inhibitor of
O-glycosylation.
After labeling the cells, 16 h with 35S-(Met + Cys)
and with or without Tu, dMM, or phenyl--GalNAc, we biotinylated the
cell-surface proteins. In another set of experiments, cells were
treated 16 h with drugs, and then hTPO cell-surface activity was
measured. In the control, 1.1% of 35S-hTPO was present at
the cell surface (Fig. 6A, lanes 1 and
3). When cells were cultured in the presence of Tu, only 0.05% of hTPO
reached the cell surface (Fig. 6A, lanes 2 and 4; and Fig. 6B). In the
same conditions, the cell-surface activity was decreased by 50% in the
presence of Tu, relative to control (Fig. 6C). This decrease is similar
to that obtained with cycloheximide. The decrease in cell-surface
activity obtained with Tu was not caused by an effect on the protein
synthesis, because, in our conditions, Tu did not inhibit the protein
synthesis by more than 5%. All these results show that the N-glycans
are essential for intracellular transport of hTPO to the cell surface.
Given these results, we thus questioned the stability and
three-dimensional structure of unglycosylated hTPO. We first did a
pulse-chase experiment, in the presence or absence of Tu (Fig. 7). The half-time of unglycosylated hTPO
was strongly reduced: 3 h vs. 11 h for
glycosylated hTPO. The three-dimensional structure of unglycosylated
TPO was checked using a panel of 12 mAbs directed against hTPO. One of
these mAbs recognizes a linear sequence, whereas the others recognize
conformational epitopes. After being labeled with 35S-(Met
+ Cys) for 16 h, native hTPO or unglycosylated hTPO was
immunoprecipitated with each of the 12 mAbs (Fig. 8, A and B). As expected, hTPO
recognition by mAbs differed with the conformation state of the protein
(Fig. 8C). A strong recognition was observed for mAbs 15, 53, 59, and
64. In contrast, unglycosylated hTPO was strongly recognized by mAb 47,
whereas the other mAbs exhibited only a slight reactivity. The
almost-complete loss of binding of several mAbs demonstrated the
crucial contribution of N-glycans to protein conformation.
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, lanes 8 and 11). Thus dMM led to
formation of hTPO, bearing only high-mannose type structures.
Quantification of the bands showed that dMM did not affect the
intracellular trafficking of TPO (Fig. 9B). This drug did not affect
hTPO cell surface activity (Fig. 9C).
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-GalNAc. This led, as described above, to a
decrease of 80% in O-glycosylation. This treatment only slightly
altered hTPO traffic and activity (data not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) show that only a small part
(18%) of hTPO acquired the three-dimensional structure suitable for
the recognition by mAb 15, whereas the other part was rapidly degraded.
This degradation process most likely occurs in the ER, the site where
nascent proteins acquire their final tertiary and quaternary structure
(20). Through a pulse chase experiment, we determined that only
10–15% of the molecules recognized by mAb 15 were recovered at the
cell surface. Therefore, only 1.5–2% of total synthesized hTPO
reaches the cell surface. This suggests that mAb 15 recognizes both an
incompletely folded intermediate that cannot reach out of the ER and
the completely folded protein that is able to reach the plasma
membrane. Note that the incompletely folded intermediate has a longer
half life than the unfolded protein recognized only by mAb 47. At
steady state, the rate of TPO expressed at the cell surface was about
the same in thyroid and in CHO cells. This strongly suggests that
intracellular traffic of hTPO does not greatly differ between the two
cell types, but we cannot totally exclude that, in thyroid cells, a
somewhat-greater part of hTPO could arrive at the cell surface.
Additional studies are needed to define features important for hTPO
maturation. In particular, it would be necessary to establish: 1) the
hypothetical role of heme in the folding of the hTPO [it has been
suggested that heme is required to achieve the proper conformation of
myeloperoxidase (21)]; and 2) the mechanisms of hTPO retention and
degradation in the ER. The major limiting step in the protein export
from the ER is a quality control in which unfolded or misfolded
proteins are retained and then degraded (12). The lumen of the ER
contains numerous molecular chaperones (BiP, GRP 94, hsp 90,
calnexin... ); those molecules play a central role in the folding
and the quality control process that limits the exportation from the ER
of misfolded, unfolded, or unassembled molecules (22). hTPO interaction
with molecular chaperones and potential roles of this association in
the retention-degradation process remain to be elucidated. Such
retention phenomena have been described for many proteins. In thyroid,
a mutant Tg has been reported to be abnormally folded, retained, and
degraded within the ER (23). That leads to a congenital goiter, with
hypothyroidism. On the other hand, by means of an interaction with the
molecular chaperone BiP, partially folded Ig light chains are retained
and then degraded within the ER (24).
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The inhibition of N-glycosylation by treatment with Tu led to a 50% diminution of enzymatic activity of TPO at the cell surface. When cells were treated with dMM (which leads only to high mannose-type structures), the activity was not (or only slightly) decreased. N-glycans thus play a major role, but complex type N-glycan formation is not essential to the expression of hTPO activity at the cell surface. The work presented here extends that of Giraud et al. (26), who reported that the enzymatic activity of hTPO was inhibited by Endo H deglycosylation through alteration of the active site domain of the enzyme. In our study, because a direct interference between N-glycans and the active site of the enzyme remains uncertain, we examined more closely whether the diminution of the enzymatic activity, when cells were treated with Tu, was caused by a direct role of N-glycans on the catalytic site or by a decrease in the expression of the hTPO molecules at the cell surface. After a 16-h incubation with Tu, the cell-surface expression of newly synthesized hTPO was decreased by 95%.
Our data indicate that unglycosylated TPO has a reduced half-time (3 h vs. 11 h for glycosylated hTPO). Because a reduced half-life can be related to a misfolding of the protein, we thus determined the role of N-glycans on the three-dimensional structure of the protein. We have recently shown that an alternatively spliced form of hTPO (TPO2) has an improper folding, causing an inhibition of intracellular trafficking and a rapid degradation (28). Among the 12 mAbs directed against hTPO that we tested, only mAb 47, directed against a linear epitope (17), strongly recognizes unglycosylated hTPO. The other mAbs exhibit little (if any) reactivity toward this molecule. Thus, unglycosylated hTPO exhibits conformational changes.
Kiso et al. reported that hTPO bore O-glycans (27), whereas Foti et al. (25) reported that hTPO expressed in CHO cells did not contain O-glycans. In our system, we determined, after separation of intracellular and cell-surface TPO that (as expected) only hTPO (present at the cell surface) bears O-glycans, because O-glycosylation processing occurs in the Golgi apparatus. Furthermore it seems that O-glycans have no effect on the activity and the expression of hTPO at the cell surface. However, we cannot exclude a possible role of O-glycans in the protection of hTPO against proteolysis.
Overall, our results demonstrate that only a little part of the synthesized hTPO is able to fold correctly and to reach the cell surface. In contrast to O-glycans, N-glycans play an essential role for the correct folding, the intracellular trafficking, and the activity of hTPO.
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Acknowledgments |
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Footnotes |
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Received February 12, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-galactosaminides
in human colon cancer cells. J Biol Cell 264:19271–19277
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), or with mAb 15 and mAb 47
(
).
