From the Laboratoire de Chimie Biologique, CNRS-UMR
8576, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq Cedex, France and the ¶ Department of
Biochemistry, School of Hygiene and Public Health, The Johns Hopkins
University, Baltimore, Maryland 21205
Received for publication, February 5, 2001
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ABSTRACT |
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The Chinese hamster ovary mutant MI8-5 is
known to synthesize
Man9GlcNAc2-P-P-dolichol rather than the
fully glucosylated lipid intermediate
Glc3Man9GlcNAc2-P-P-dolichol. This
nonglucosylated oligosaccharide lipid precursor is used as donor for
N-glycosylation. In this paper we demonstrate that a
significant part of the glycans bound to the newly synthesized
glycoproteins in MI8-5 cells are monoglucosylated. The presence of
monoglucosylated glycans on glycoproteins determines their binding to
calnexin as part of the quality control machinery. Furthermore, we
point out the presence of
Glc1Man5GlcNAc1 in the cytosol of
MI8-5 cells. This indicates that part of the monoglucosylated
glycoproteins can be directed toward a deglycosylation process that
occurs in the cytosol. Besides studies on glycoprotein degradation
based on the disappearance of protein moieties, MI8-5 cells can be
used as a tool to elucidate the various step leading to glycoprotein
degradation by studying the fate of the glycan moieties.
A key reaction of N-glycosylation is the transfer
en bloc of a Glc3Man9GlcNAc2
oligosaccharide from a lipid intermediate to an Asn residue in the
Asn-Xaa-Ser(Thr) consensus sequence of a nascent protein. This
N-glycosylation process is immediately followed by
sequential deglucosylation leading to
Man9GlcNAc2 protein. It is now clearly
established that oligosaccharide trimming in the
ER1 is intimately linked to
the quality control process, leading to the degradation of misfolded
glycoproteins or unassembled multimeric proteins. Indeed the presence
of monoglucosylated oligomannosides on glycoproteins retained in the ER
is essential for their binding to the molecular chaperones calnexin and
calreticulin (1, 2). The presence of a glucosyl residue is controlled
by reglucosylation-deglucosylation cycles involving UDP-glucose :
glycoprotein glucosyltransferase acting as a folding sensor (3) and
glucosidase II, respectively. It has been demonstrated that glucose
trimming and reglucosylation cycles determine the association of a
glycoprotein with calnexin, as well as its folding (4, 5).
During this quality control, part of newly synthesized glycoproteins
are degraded after translocation of the glycosylated polypeptide chains
from the ER to the cytosol. As first demonstrated by Wiertz et
al. (6), using labeled protein with an amino acid precursor, this
retrotranslocation is accompanied by the removal of the glycans by a
cytosolic peptide N-glycanase (PNGase), such as the one
isolated by Suzuki et al. (7). The relationship between the
location of the enzyme and the degradation process has been discussed
by Karaivanova and Spiro (8). However, it is worth mentioning that few
labs follow the fate of newly synthesized proteins by labeling with
[2-3H]mannose. In this case, the
N-glycosylation process is accompanied by the release of
glycans from either lipid intermediates or from newly synthesized
glycoproteins (9-11). The trafficking of the released oligomannosides
from both origins leads to the formation of oligosaccharides possessing
one GlcNAc residue at the reducing end (OSGn1) in the
cytosol (for reviews see Refs. 12-14).
A Chinese hamster ovary mutant, MI8-5, was found to synthesize
Man9GlcNAc2-P-P-Dol rather than fully
glucosylated lipid intermediate (15). This mutant cell line was found
to be deficient in
dolichol-P-Glc:Man9GlcNAc2-P-P-dolichol glucosyl transferase activity. This defect is similar to the one observed for CDGIc (congenital disorders of
glycosylation) (16).
In this paper, we demonstrate that although in MI8-5 the
oligosaccharide lipids are nonglucosylated, part of the glycans bound to glycoproteins are monoglucosylated. Furthermore, we observe the
formation of Glc1Man5GlcNAc1
species that could only originate from a deglycosylation mechanism
occurring during glycoprotein degradation. Our work demonstrates that
the quality control system for newly synthesized glycoproteins is
intact in MI8-5 cells. This cell line gives us a biological model to
determine the nature of oligosaccharides released from protein
independently of what is happening at the reducing end.
Reagents--
Mutant cell line MI8-5 was isolated as described
previously (15). [2-3H]Mannose (429 Gbq/mmol) and
[U-14C]glucose (10 Gbq/mmol) were from Amersham
Pharmacia Biotech. PNGase F and castanospermine were purchased
from Glyko, (Novato, CA). Metabolic Labeling of Oligosaccharides and Chase
Experiments--
MI8-5 cells were routinely cultured in monolayers in
Analysis of Oligosaccharide Material--
The protein pellet was
digested overnight at room temperature with 0.2 mg of trypsin in 0.1 M ammonium bicarbonate, pH 7.9. The glycan moiety linked to
protein was cleaved by PNGase F as follows: the trypsin-treated protein
was boiled for 10 min to destroy the trypsin activity, and the peptides
were dried and dissolved in 20 mM sodium phosphate buffer,
pH 7.5, containing 50 mM EDTA, 50% (v/v) glycerol, and
0.02% NaN3. PNGase F was then added (0.5 mIU) for
incubation overnight at 37 °C.
Size analysis of the glycan moieties was achieved by HPLC on an
amino-derivatized Asahipak NH2P-50 (250 mm × 4.6 mm)
column (Asahi, Kawasaki-ku, Japan) with a solvent system of
acetonitrile/water from 70:30 (v/v) to 50:50 (v/v) at a flow rate of 1 ml/min over 80 min. Under these conditions the oligomannosides can be
resolved by their numbers of mannose, glucose, and GlcNAc residues from Man to Glc3Man9GlcNAc2.
Oligomannosides were identified by their retention times as described
previously (9); separation of the labeled oligosaccharides was
monitored by continuous-flow detection of the radioactivity with a
Flo-one Jack Bean [U-14C]Glucose Labeling for Glucosyl Residue
Detection--
MI8-5 cells were labeled with 25 µCi/ml of
[U-14C]glucose per dish in
The protein pellet was digested overnight at room temperature with
trypsin in 0.1 M ammonium bicarbonate, pH 7.9, and glycans were then removed by PNGase F treatment. Both oligosaccharides bound to
glycoproteins, and free oligosaccharides were purified by concanavalin
A chromatography. The lectin column (concanavalin A-Sepharose; 5 × 0.5 cm) was equilibrated in 5 mM sodium acetate buffer,
pH 5.2, containing 0.1 M NaCl, 1 mM
MnCl2, 1 mM CaCl2, and 1 mM MgCl2 at room temperature. The fraction
resulting from PNGase F action on glycoprotein fraction and free
oligosaccharides were applied to the column. The strongly retained
[14C]-labeled oligomannosides were eluted with 100 mM Cell Lysis and Immunoprecipitation by Anti-calnexin
Antibody--
[2-3H]Mannose-labeled cells were washed
twice with cold PBS and lysed in buffer (50 mM Hepes, pH
7.4, 200 mM NaCl, 2% CHAPS) containing a mixture of
protease inhibitors (10 µg/ml pepstatin and leupeptin, 1 mg/ml 4-(2
aminoethyl) benzene-sulfonyl fluoride, and 0.1 mM
diisopropylfluorophosphate). The cell lysate was centrifuged at
14000 × g for 3 min at 4 °C to remove insoluble material.
The cell lysate was precleared with protein A-Sepharose beads and
immunoprecipitated for 16 h at 4 °C with anti-calnexin
antibodies prebound to protein A-Sepharose beads. Immunoprecipitates
were washed thrice in high salt buffer (50 mM Hepes, pH
7.4, 200 mM NaCl, 0.5% CHAPS) followed by two washes in 20 mM Hepes, pH 7.4. Immunoprecipitated
[2-3H]mannose-labeled glycoproteins were digested
overnight at room temperature with 0.2 mg of trypsin in 0.1 M ammonium bicarbonate, pH 7.9. After PNGase treatment,
glycan moieties linked to protein were analyzed by HPLC as described above.
Pattern of Glycans Bound to Glycoproteins Synthesized by
MI8-5 Cell Line--
MI8-5 cells transfer onto proteins only
nonglucosylated oligosaccharides (15). To compare the fate of glycans
bound to glycoproteins synthesized in parental (K1-2 cells) and
MI8-5, these cells were pulse-labeled during 60 min with
[2-3H]mannose in the conditions described under
"Materials and Methods." Fig. 1,
a and b represent the pattern of glycans released
from glycoproteins of K1-2 and MI8-5 after PNGase treatment. Although the oligosaccharide bound to lipid intermediates in K1-2 and MI8-5 cells was different
(Glc3Man9GlcNAc2-PP-Dol for
parental cells and Man9GlcNAc2-PP-Dol for
mutant cells), the oligomannoside species bound to proteins seems to be
similar. In particular, a species having a higher retention time than
Man9GlcNAc2 can be detected in MI8-5 (peak A).
This raised the question of the nature and of the molecular composition
of this oligomannosides species.
Peak A has been isolated, and its sensitivity toward jack bean
Effect of Castanospermine on the Pattern of Glycans Bound onto
Glycoproteins in MI8-5 Cells--
MI8-5 cells were metabolically
labeled with tritiated mannose in the presence of 50 µg/ml of Cst (an
inhibitor of rough ER glucosidases). Comparing panels a and
b of Fig. 3, there was clearly an increase of the radioactivity bound to peak A in the presence of
Cst. As Cst has been demonstrated to prevent deglucosylation of newly
synthesized glycoproteins, this observation strongly suggests that peak
A represents a glucosylated species.
Occurrence of a Glucose Residue on Glycoproteins Synthesized by
MI8-5 Cells--
MI8-5 cells were pulse-labeled with
[U-14C]glucose during 1 h in the presence of
50 µg/ml of Cst as described under "Materials and Methods." After
a PNGase treatment of the protein pellet, oligomannoside-type glycans
were purified by concanavalin A chromatography. [14C]-Labeled peak A (see Fig. 1b) was then
isolated by HPLC and submitted to acid hydrolysis to release
monosaccharides. Fig. 4b shows
chromatography of the [14C]-labeled hydrolysate of peak A
with [2-3H]mannose as an internal standard. The HPLC
profile showed the presence of both [14C]mannose and
glucose residues. All the data from HPLC, Glucosylation of Newly Synthesized Glycoproteins in MI8-5 Allows
Binding to Calnexin--
Because oligomannosides synthesized in MI8-5
are transferred onto nascent proteins as nonglucosylated species, it
can be assumed that the presence of
Glc1Man9GlcNAc2 bound to
glycoproteins originated from a subsequent glucosylation process of the
Man9GlcNAc2 species. At the present time it has
been well demonstrated that the presence of the glucosyl residue is
controlled by reglucosylation-deglucosylation cycles involving the
UDP-glucose : glycoprotein glucosyltransferase and glucosidase II,
respectively (3). Recently it has been demonstrated that the trimming
and readdition of glucose to N-linked oligosaccharides
determine the association of a glycoprotein with calnexin, as well as
its folding (4, 5). We next determined whether the reglucosylation of
oligomannosides onto glycoproteins seen in MI8-5 cells resulted in an
association with calnexin. MI8-5 cells were pulse-labeled for 1 h
with [2-3H]mannose, and the radioactivity was chased for
1 h. To preserve glucosylated oligomannosides, Cst was maintained
in the media during the entire experiment. After immunoprecipitation of
the cell lysate with anti-calnexin antibodies, immunoprecipitated glycoproteins were trypsin-treated and subjected to a PNGase treatment before being analyzed by HPLC. Fig. 5
reveals the presence of two peaks, the first one migrating as
Glc1Man9GlcNAc2 and the second one
as Glc1Man8GlcNAc2. The hydrolysis
of each peak by jack bean Monoglucosylated Free Oligomannosides Are Produced by MI-5
Cells--
It has been shown that the N-glycosylation
process is accompanied by the release of free oligomannoside material
originating from both oligosaccharide-lipid hydrolysis and
degradation of glycoproteins (12-14). It has also been demonstrated in
mutant cell lines lacking mannosylphosphoryldolichol that the
free oligosaccharides released from oligosaccharide-lipid or
glycoprotein are degraded into an oligomannoside isomer,
Man5GlcNAc1 (9). Because MI8-5 cells do not
glucosylate the glycan bound to oligosaccharide-PP-Dol but do
glucosylate the glycans on proteins, this offers a biological model to
discriminate oligosaccharides originating from glycoproteins. Indeed,
after plasma membrane permeabilization with streptolysine O of MI8-5
cells (not shown), free oligomannosides are released from semi-intact
cells indicating their cytosolic location. As shown in Fig.
3c these oligomannosides recovered after a pulse-chase experiment with [2-3H]mannose possessed one GlcNAc
residue at the reducing end indicating that they have been processed in
the cytosol. Man5GlcNAc1 appeared as the major
species. However, a peak B having a higher retention time could be
observed. The proportion of radioactivity associated with peak B
increased from 20 to 80% in the presence of 50 µg/ml Cst (Fig.
3d). Peak B was isolated after a pulse labeling of
MI8-5 cells for 1 h in the presence of
[U-14C]glucose and 50 µg/ml Cst. As shown in Fig.
6, the cochromatography of this peak B
with free oligomannosides synthesized by B3F7 cells (a
Man-P-Dol-deficient cell line that synthesizes only truncated Man5 bearing oligomannosides) reveals that peak B coeluted
with Glc1Man5GlcNAc1 species. Acid
hydrolysis of the [14C]-labeled peak B revealed the
presence of both mannose and glucose residues (Fig. 4c).
These data indicate that in MI8-5 cells, glucosylated glycans bound to
glycoproteins are released and processed to give a unique intermediate,
Glc1Man5GlcNAc1.
The lack of
dolichol-P-Glc:Man9GlcNAc2-P-P-dolichol
glucosyl transferase activity in MI8-5 cells leads to the synthesis of nonglucosylated Man9GlcNAc2-P-P-dolichol, which
is transferred to protein (15). In this paper, we show that
reglucosylation-deglucosylation cycles occur on glycoproteins of MI8-5
cells, because the presence of monoglucosylated glycans has been
demonstrated. As in all cell lines studied up to now, the presence of
monoglucosylated glycans determines the association of newly
synthesized glycoproteins with calnexin. Indeed, immunoprecipitation of
glycoproteins synthesized in MI8-5 cells by anti-calnexin antibodies
revealed the presence of two oligomannoside species,
Glc1Man8GlcNAc2 and
Glc1Man9GlcNAc2.
The more interesting aspect of this work is the demonstration
that the Glc1Man5GlcNAc1 species is
a component of the soluble free oligomannoside material. These
glucosylated oligosaccharides originated from glycoproteins, because in
MI8-5 cells glucosyl residues are recovered only on glycoproteins.
Endoplasmic reticulum to cytosol transport of oligomannosides
possessing two GlcNAc residues at the reducing end (OSGn2)
is known to be specific for nonglucosylated species (19, 20). Moreover,
it is now well established that OSGn2 are converted to
OSGn1 species by a cytosolic chitobiase (21). Thus the
finding of glucosylated OSGn1 in MI8-5 cells indicates
that the deglycosylation process of glycoproteins occurred in the
cytosol prior to their degradation.
When looking at the reducing end of the released oligomannosides, a
discrepancy appears between the previous demonstration of the PNGase
action (6) releasing OSGn2 and the monoglucosylated OSGn1 observed in this study. Two alternatives can be
proposed, either there was successive action of PNGase and chitobiase,
or there was cleavage by a cytosolic The study of oligomannosides produced by MI8-5 cells suggests the
following pathway for the formation of
Glc1Man5GlcNAc1: after
retrotranslocation of polypeptides bearing monoglucosylated glycans
(Glc1Man8GlcNAc2 and
Glc1Man9GlcNAc2),
deglycosylation occurs in cytosol liberating
Glc1Man8GlcNAc1 and
Glc1Man9GlcNAc1. These
OSGn1 are substrates for the cytosolic
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Mannosidase from jack bean, protease
inhibitors, and trypsin were obtained from Sigma. Polyclonal antibody
anti-calnexin was from StressGen. Protein A-Sepharose CL-4B and
concanavalin A-Sepharose were from Amersham Pharmacia Biotech.
-minimal essential medium with 10% (v/v) fetal calf serum at
34 °C in 10-cm Petri dishes under air/CO2 (19:1).
Cells were labeled with 50 µCi/ml of
[2-3H]mannose in
-minimal essential medium containing
0.5 mM glucose and 10% dialyzed fetal bovine serum. After
incubation, the medium was removed, and the cell layer was rinsed
rapidly twice with ice-cold PBS. When a chase was performed,
pulse-labeled cells were washed twice with PBS and incubated for
different times in
-minimal essential medium containing 5 mM glucose. When used, castanospermine (Cst) was maintained
throughout the incubation and the chase periods at a final
concentration of 50 µg/ml. Sequential extraction and purification of
free oligosaccharide material were then achieved as described
previously (9).
detector (Packard).
-Mannosidase Treatment--
For
enzymatic treatment with jack bean
-mannosidase, oligosaccharides
released after PNGase digestion were dissolved in 20 µl of 50 mM sodium acetate buffer, pH 5. The incubation was achieved overnight at 37 °C with 0.5 units of jack bean
-mannosidase. The
incubation mixture was then analyzed by HPLC.
-minimal essential medium
without glucose containing 5 mM mannose, 50 µg/ml Cst,
and 10% dialyzed fetal bovine serum. Pulse-labeled cells were washed
twice with PBS and chased during 2 h in
-minimal essential
medium containing 5 mM glucose and 50 µg/ml Cst.
Sequential extraction was then achieved as described previously
(9).
-D-mannoside. Peak A (originating from
glycoproteins) and peak B (originating from free oligomannoside
material) were then isolated by preparative HPLC. The isolated peaks A
and B were then submitted to acid hydrolysis (4 M
trifluoroacetic acid at 100 °C during 4 h). After drying under
nitrogen, free monosaccharides were cochromatographed with [2-3H]mannose as an internal standard using HPLC under
isocratic conditions with a solvent system of acetonitrile/water (80:20
(v/v)) at a flow rate of 1 ml/min over 20 min.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Analysis of oligosaccharide species bound to
glycoproteins of parental (K1-2) and MI8-5 cell lines. Cells
were pulse-labeled with [2-3H]mannose for 1 h. After
sequential extraction, glycoproteins were treated with PNGase, and the
released oligosaccharides were analyzed by HPLC as described under
"Materials and Methods." a, glycans from K1-2 cells;
b, glycans from MI8-5 cells. G1M9 indicates
oligomannosides containing one glucose, nine mannose, and two
N-acetyl glucosamine residues; M9 and
M8 indicate oligomannosides containing nine or eight mannose
and two N-acetyl glucosamine residues, respectively.
-mannosidase has been compared with the
Man9GlcNAc2 species used as control. The action
of jack bean
-mannosidase on isolated Man9GlcNAc2 species (Fig.
2a) leads to the formation of
88% of free mannose and 12% of
Man1GlcNAc2 species (see Fig. 2b and
Table I). These results indicate that the
totality of
-linked mannose residues has been removed. In contrast,
the same incubation of isolated peak A (Fig. 2c) with jack
bean
-mannosidase leads to the formation of 30% of free mannose and
70% of higher species migrating as
hexose(6-8)GlcNAc2, peaks 1, 2, and 3, respectively (see Fig. 2d and Table I). The absence of a
resulting Man1GlcNAc2 species suggests that
only partial hydrolysis of peak A was performed by the enzyme. It has
to be noted that when incubation was achieved with higher enzyme
concentration, the proportion of peak 1 increases, but peaks 2 and 3 remained detectable. This suggests that peak 1 is the limit
product of the hydrolysis of peak A by jack bean
-mannosidase.
Moreover, peak 1 comigrated with the
Glc1Man5GlcNAc2 species that
corresponds to the major oligomannoside species synthesized by
mannosylphosphoryldolichol-deficient cell line B3F7 (not shown). Partial digestion of
Glc3Man9GlcNAc2 species isolated
from the glucosidase I-deficient cell line Lec23 has also been obtained leading to a mixture of three species (17). As in our case, complete
digestion to a single product was not observed even after longer
incubations. Furthermore, it has been clearly demonstrated on bovine
rhodopsin that jack bean
-mannosidase is not able to cleave an
unsubstituted
1,6-linked mannose from the core
-mannose (18). Our
results are consistent with these observations, because the smallest
species is Glc1Man5GlcNAc2.
According to our results, we can postulate that peaks 1, 2, and 3 correspond to Glc1Man5GlcNAc2, Glc1Man6GlcNAc2, and
Glc1Man7GlcNAc2, respectively.
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Fig. 2.
Jack bean
-mannosidase digestion of oligosaccharide species
bound to glycoproteins isolated from MI8-5 cells. MI8-5 cells
were pulse-labeled with [2-3H]mannose for 1 h. After
sequential extraction, glycoproteins were treated with PNGase, and
Man9GlcNAc2 (panel a) and peak
A (panel c) were isolated by HPLC. 50,000 dpm of each
peak was treated overnight by jack bean
-mannosidase as described
under "Materials and Methods." Hydrolysates were then analyzed by
HPLC. Panel b, analysis of
Man9GlcNAc2 hydrolysis product; panel
d, analysis of peak A hydrolysis product. M9 and
M1 indicate oligomannosides containing nine or one mannose
and two N-acetyl glucosamine residues, respectively.
Man indicates free mannose.
Proportion of various species obtained after jack bean -mannosidase
treatment of [2-3H]mannose-labeled oligomannoside species
isolated from MI8-5 glycoproteins
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Fig. 3.
Effect of castanospermine on the pattern of
glycans bound to glycoproteins and free oligomannosides synthesized by
MI8-5 cells. MI8-5 cells were pulse labeled with
[2-3H]mannose for 1 h in the absence (panels
a and c) or in the presence (panels b and
d) of 50 µg/ml Cst. After sequential extraction, the free
oligosaccharide material (panels c and d) and the
glycans released from glycoproteins with PNGase (panels a
and b) were analyzed by HPLC as described under "Materials
and Methods." M9, M8, M5,
M4, and M3 indicate oligomannosides containing
nine, eight, five, four, and three mannose residues, respectively.
Open peaks represent oligomannosides possessing two
N-acetyl glucosamine residues at the reducing end, and
hatched peaks represent oligomannosides possessing one
N-acetyl glucosamine residue at the reducing end.
-mannosidase treatment,
and composition analysis demonstrate that the species migrating as peak
A is glucosylated and possesses the following molar composition:
Glc1Man9GlcNAc2.
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Fig. 4.
Glycoproteins and free oligomannosides
synthesized by MI8-5 cells are glucosylated. MI8-5 cells were
pulse-labeled with [U-14C]glucose for 1 h and then
the radioactivity was chased for 2 h in the presence of 50 µg/ml
Cst. After sequential extraction, the glycans released from
glycoproteins with PNGase and the free oligomannosides were purified by
concanavalin A chromatography. Peaks A and B (see Figs. 1 and 3) were
then isolated from glycoproteins and free oligomannosides,
respectively, and submitted to acid hydrolysis as described under
"Materials and Methods." The [14C]-labeled
monosaccharides are then cochromatographed with
[2-3H]mannose using HPLC under isocratic conditions.
Panel a indicates the separation of free mannose and
glucose; panel b shows the analysis of monosaccharides
obtained after the hydrolysis of oligosaccharide A isolated from
glycoproteins; panel c shows the analysis of monosaccharides
obtained after the hydrolysis of oligosaccharide B isolated from free
oligomannosides.
-mannosidase leads to the same
pattern as the one obtained after hydrolysis of
Glc1Man9GlcNAc2 (peak A) isolated
from glycoproteins (Table I). These results indicate that the
reglucosylated oligomannoside glycans of MI8-5 could associate with
calnexin. Therefore, the quality control system for newly synthesized
glycoproteins appeared intact in MI8-5 cells.
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Fig. 5.
Oligomannosides species
bound to glycoproteins associated with calnexin in MI8-5 cells.
MI8-5 cells were pulse-labeled with [2-3H]mannose for
1 h and then the radioactivity was chased for 1 h in the
presence of 50 µg/ml Cst. Immunoprecipitation of the cell lysate with
anti-calnexin antibody was performed as described under "Materials
and Methods." The immunoprecipitates were then treated by PNGase, and
the oligosaccharide material was analyzed by HPLC after purification.
G1M9 and G1M8 indicate oligomannosides containing one glucose, nine or
eight mannose, and two N-acetyl glucosamine residues.
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Fig. 6.
Cochromatography of free oligomannosides
synthesized by the Man-P-Dol-deficient cell line B3F7 and peak B
isolated from free oligomannosides synthesized by MI8-5 cells.
MI8-5 cells were pulse-labeled with [U-14C]glucose for
1 h and then the radioactivity was chased for 2 h in the
presence of 50 µg/ml Cst. After sequential extraction, the free
oligomannosides were purified by concanavalin A chromatography.
Peak B was then isolated and cochromatographed together with
free oligomannosides purified from B3F7 cells pulse-labeled with
[2-3H]mannose. G1M5 indicates oligomannoside
possessing one glucose residue and five mannose residues, and
M5, M4, and M3 indicate
oligomannosides containing five, four, and three mannose residues,
respectively. Hatched peaks represent oligomannosides
possessing one N-acetyl glucosamine residue at the reducing
end.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-endoglucosaminidase (22, 23).
-mannosidase (24) generating the observed
Glc1Man5GlcNAc1. The fate of this
oligosaccharide is still in question, because up to now, no
-glucosidase activity has been detected in the cytosol. Although its
transport into lysosomes has not yet been studied (25), our results
suggest a lysosomal degradation into
Man5GlcNAc1 and lower species. The question
remains whether glycoproteins bearing monoglucosylated glycans and
being directed toward degradation must be linked to calnexin, as
demonstrated by Liu et al. (26) for the degradation of
misfolded
1-antitrypsin.
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FOOTNOTES |
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* This work was supported in part by European Economic Communautee Contract ERB FMRX CT96 0025 (Carenet-2) from CNRS and Université des Sciences et Technologies de Lille, Grant 9736 from the Association pour la Recherche sur le Cancer, and National Institutes of Health Grant R01 CA 20421 (to S. S. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We dedicate this article to the memory of our coauthor, André Verbert. We will miss his friendship and enthusiasm for science.
§ To whom correspondence should be addressed. Tel.: 33-3-20-43-44-30; Fax: 33-3-20-43-65-55; E-mail: Rene.Cacan@univ-lille1.fr.
Published, JBC Papers in Press, April 6, 2001, DOI 10.1074/jbc.M101077200
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ABBREVIATIONS |
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The abbreviations used are: ER, endoplasmic reticulum; PNGase, peptide N-glycanase; OSGn1, one GlcNAc residue at the reducing end. Dol, dolichol; PBS, phosphate-buffered saline; Cst, castanospermine; HPLC, high pressure liquid chromatography; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio[-1-propanesulfonic acid.
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1. |
Ware, F. E.,
Vassilakos, A.,
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