Endoplasmic reticulum-associated degradation of glycoproteinsbearing Man5GlcNAc2 and Man9GlcNAc2 species in the MI8-5 CHO cell line Franc¸ois Foulquier, Sandrine Duvet, Andre´ Klein, Ann
Trang 1Endoplasmic reticulum-associated degradation of glycoproteins
bearing Man5GlcNAc2 and Man9GlcNAc2 species in the MI8-5
CHO cell line
Franc¸ois Foulquier, Sandrine Duvet, Andre´ Klein, Anne-Marie Mir, Fre´de´ric Chirat and Rene´ Cacan Unite´ de Glycobiologie Structurale et Fonctionnelle, CNRS-UMR 8576, IFR 118, GDR CNRS 2590, Universite´ des Sciences
et Technologies de Lille, Villeneuve d’Ascq, France
Endoplasmic reticulum-associated degradation of newly
synthesized glycoproteins has been demonstrated previously
using various mammalian cell lines Depending on the cell
type, glycoproteins bearing Man9 glycans and glycoproteins
bearing Man5 glycans can be efficiently degraded Awide
variety of variables can lead to defective synthesis of
lipid-linked oligosaccharides and, therefore, in mammalian cells,
species derived from Man9GlcNAc2 or Man5GlcNAc2 are
often recovered on newly synthesized glycoproteins The
degradation of glycoproteins bearing these two species has
not been studied We used a Chinese hamster ovary cell
line lacking Glc-P-Dol-dependent glucosyltransferase I to
generate various proportions of Man5GlcNAc2 and Man9GlcNAc2 on newly synthesized glycoproteins By studying the structure of the soluble oligomannosides pro-duced by degradation of these glycoproteins, we demon-strated the presence of a higher proportion of soluble oligomannosides originating from truncated glycans, showing that glycoproteins bearing Man5GlcNAc2 glycans are degraded preferentially
Keywords: degradation signal; lipid intermediates; manno-sidase activity; N-glycosylation; soluble oligomannosides
The N-glycosylation process is characterized by the
transfer en bloc of a preassembled oligosaccharide on
to a nascent protein According to the specificity of the
oligosaccharyltransferase, it has been demonstrated
in vitro that a wide variety of assembly intermediates
Glc(0–3)Man(0–9)GlcNAc2-PP-Dol can serve as the
donor substrate for N-linked glycosylation [1] In vivo,
Gilmore and coworkers have determined that
oligosacch-aryltransferase shows a preference for the full length
oligosaccharide substrate [2]
Once transferred to proteins, the N-linked
oligosaccha-rides play the role of information carrier in the quality
control mechanism of N-glycoproteins in the endoplasmic
reticulum (ER) Indeed, monoglucosylated oligosaccharides
generated either after sequential action of
glucosi-dase I and II or by the soluble UDP-Glc–glycoprotein
glucosyltransferase (UGGT) can be ligands for the mole-cular lectin-like chaperones calnexin and calreticulin until proper folding of the N-glycoproteins [3,4]
With N-glycoproteins that fail to fold correctly, a Man8GlcNAc2 species is generated by an ER a1,2-manno-sidase, probably ER mannosidase I, and this constitutes a putative degradation signal [5] Recently, Frenkel et al [6] demonstrated that the degradation of unstable N-glycopro-teins accumulated in a rough ER subcompartment (QC compartment) involves trimming of the sugar chain to Man(6–5)GlcNAc2 species Furthermore, by using a mutant cell line that synthesizes truncated Man5GlcNAc2, Ermon-val et al [7] showed that the degradation of a soluble form
of ribophorin I required the formation of Man4GlcNAc2 After the trimming of Man residues, these misfolded N-glycoproteins are then degraded by a specific pathway named endoplasmic reticulum-associated degradation (ERAD) This pathway involves a deglycosylation step located in the cytosol, and the soluble oligomannosides released by this process were then submitted to the action of a 1,4-dideoxy-1,4-imino-D-mannitol (DIM)-sensitive cytosolic mannosidase [8], leading to the formation of a specific Man5GlcNAc1 isomer [9] before entering the lysosomal compartment [10]
Thus, it appears that at least three oligomannoside structures may be involved as degradation signal for ERAD (Man8GlcNAc2 and Man6GlcNAc2 for glycoproteins bearing Man9 species and Man4GlcNAc2 for glycoproteins bearing Man5 species) However, this has been observed using different cell lines So we decided to study the degradation of N-glycoproteins when these oligomannoside structures are both transferred to newly synthesized glyco-proteins in the same cell line
Correspondence to R Cacan, Unite´ de Glycobiologie Structurale et
Fonctionnelle, CNRS-UMR 8576, Universite´ des Sciences et
Tech-nologies de Lille, F-59655 Villeneuve d’Ascq Cedex, France.
Fax: + 33 3 20 43 65 55, Tel.: + 33 3 20 43 44 30,
E-mail: rene.cacan@univ-lille1.fr
Abbreviations: CHO, Chinese hamster ovary; DIM,
1,4-dideoxy-1,4-imino- D -mannitol; Dol, dolichol; ERAD, endoplasmic
reticulum-associated degradation; ER, endoplasmic reticulum; LLO, lipid-linked
oligosaccharide; PNGase, peptide N-glycanase F; UGGT,
UDP-glucose–glycoprotein glucosyltransferase; UPR,
unfolded protein response.
Enzymes: peptide N-glycanase F (PNGase; EC 3.2.2.18);
b-galactosi-dase (EC 3.2.1.23); b-hexosaminib-galactosi-dase (EC 3.2.1.30).
(Received 15 September 2003, revised 21 October 2003,
accepted 21 November 2003)
Trang 2Experimental procedures
Materials
[2-3H]Man (429 GBqÆmmol)1) was from Amersham (Little
Chalfont, Bucks, UK) Trypsin and castanospermine were
from Sigma Kifunensine and DIM were from ICN, Orsay,
France Peptide N-glycanase F (PNGase) was from Biolabs
b-Galactosidase and b-hexosaminidase (both isolated from
Jack bean) were from Oxford GlycoSystems (Abingdon,
Oxon, UK) Glucosidase II purified from rat liver was a gift
from T Butters (Oxford Glycobiology Institute, University
of Oxford, UK)
Cell culture
The Glc-P-Dol-dependent glucosyltransferase I-deficient
mutant Chinese hamster ovary (CHO) cell line (MI8-5)
was a gift from S S Krag (Johns Hopkins University,
Baltimore, MD, USA) This cell line was grown in
a-minimal essential medium (Gibco-BRL) supplemented
with 10% (v/v) fetal bovine serum, at 34C under 5%
CO2
Metabolic labeling of oligosaccharides and pulse-chase
experiments
For this purpose, MI8-5 cells were routinely grown in
10-cm Petri dishes Cells were preincubated at low Glc
concentration (0.175 mM) for different times and then
metabolically labeled for 1 h with 3.6 GBqÆmL)1 (4 lM)
[2-3H]Man at the same Glc concentration For pulse-chase
experiments, the radioactive culture medium was replaced
by a-minimal essential medium containing the
physiolo-gical Glc concentration (5 mM) supplemented with 5 mM
Man When used, kifunensine (20 lM) and DIM (1 mM)
were present throughout the experiment (preincubation,
pulse and chase) Sequential extraction and purification of
oligosaccharide material were achieved as previously
described [9]
Analysis of oligosaccharide material and glycosidase
treatments
Soluble oligomannoside fractions obtained after the
sequential extraction were desalted on Bio-Gel P2 eluted
with 5% (v/v) acetic acid Glycoprotein fractions obtained
at the end of the sequential extraction were digested with
trypsin (1 mgÆmL)1) in 0.1M ammonium bicarbonate
buffer, pH 7.9, overnight at room temperature
Elution of the radiolabeled oligosaccharides was moni-tored by continuous-flow detection of the radioactivity with a flo-one b-detector (Packard, Les Ullis, France) For the sequential glycosidase treatments, oligosaccha-rides released after PNGase digestion were first dissolved
in 2Macetic acid, and hydrolysis was carried out at 80C for 2 h to release sialyl residues Incubation with 0.1 U b-galactosidase was performed in 20 lL 100 mM sodium acetate buffer, pH 3.5, overnight at 37C Then 0.1 U b-hexosaminidase was added for an additional overnight incubation The incubation mixture was then analyzed by HPLC
Results
Synthesis and transfer of Man5GlcNAc2 species
in the MI8-5 cell line
We have demonstrated previously that MI8-5 CHO cells are deficient in Glc-P-Dol-dependent glucosyltransferase I and that the glycans transferred on to glycoproteins are thus non-glucosylated [12] By using incubation with labeled Man at low Glc concentration (0.175 mM), we now observed that Man5GlcNAc2-PP-Dol, an inter-mediate in the formation of Man9GlcNAc2-PP-Dol, was transferred to glycoprotein acceptors (Fig 1A,E) Recently, Shang et al [13] reported that, when cells were preincubated at low Glc concentration, the truncated LLOs were extended as a consequence of activation of the unfolded protein response (UPR) To obtain various proportions of Man5 and Man9 species on newly synthesized glycoproteins, MI8-5 cells were preincubated for up to 120 min at low Glc concentration (0.175 mM) and incubated with labeled Man for 60 min Figure 1 shows that, when the preincubation period was increased, the UPR was activated, because, after 20, 40 and 120 min
of Glc deprivation, the radioactivity shifted from the truncated species (Man3GlcNAc2-PP-Dol and Man5Glc-NAc2-PP-Dol) to Man9GlcNAc2-PP-Dol (Fig 1A–C)
To demonstrate that glycans bound to proteins are not the result of modification by processing enzymes, the same experiment was performed in the presence of kifunensine, an inhibitor of the demannosylation process Figure 1D,H shows that incubation in the presence of
20 lMkifunensine did not affect the proportion of Man5 and Man9 species bound to LLOs and glycoproteins This demonstrates that both species were transferred with different proportions according to the relative abundance
of each species present on the LLOs (Fig 1E–G)
Trang 3Dual fate of Man5GlcNAc2 bound to glycoproteins
in the MI8-5 cell line
As described previously for MI8-5 cells, the transfer of
Man9GlcNAc2 to glycoproteins led to the formation of
Man8GlcNAc2 and Glc1Man9GlcNAc2, as the formation
of monoglucosylated glycans by UGGT was not affected
in this cell line In the same manner, the transfer of
Man5GlcNAc2 species to glycoproteins was confirmed by
the presence of three peaks: Man4GlcNAc2,
Man5Glc-NAc2 and peak X migrating as Glc1Man5GlcMan5Glc-NAc2
(Fig 1E–H)
To demonstrate that peak X may be the result of the
reglucosylation of the Man5GlcNAc2 species, MI8-5 cells
were metabolically labeled with [2-3H]Man with or without
100 lgÆmL)1castanospermine, an inhibitor of glucosidases I
and II In the presence of castanospermine, the levels of
both Glc1Man9GlcNAc2 and peak X were increased
(Fig 2A,B) Under these conditions, the decreasing
amounts of Man9GlcNAc2 and Man5GlcNAc2 correlated
with the increasing amounts of Glc1Man9GlcNAc2 and
peak X, respectively Furthermore, as expected, peak X and
Glc1Man9GlcNAc2 were completely sensitive to treatment with purified glucosidase II, which resulted in Man5Glc-NAc2 and Man9GlcMan5Glc-NAc2, respectively (Fig 2C) As Glc-P-Dol-dependent glucosyltransferase was not active
in MI8-5 cells, the monoglucosylated species detected on glycoproteins could only originate from the action of UGGT Furthermore, when the incubation was performed
in the presence of 20 lM kifunensine, the formation of Man4GlcNAc2 and Man8GlcNAc2 species was strongly inhibited (Fig 1H) This suggests the involvement of class I mannosidase in these demannosylation processes [14]
Fate of glycoproteins bearing Man5GlcNAc2 and Man9GlcNAc2
Depending on the preincubation time at low Glc concen-tration, various proportions of Man5GlcNAc2 and Man9GlcNAc2 species could be transferred to proteins
To obtain an equal distribution of the radioactivity between the Man5 and Man9 populations bound to the glycopro-teins, cells were preincubated for 40 min in 0.175 mMGlc, labeled for 1 h, and chased in the culture medium (5 m
Fig 1 HPLC analysis of LLO and oligo-mannoside species bound to newly synthesized glycoproteins during Glc deprivation of MI8-5 cells MI8-5 cells were preincubated in 0.175 m M Glc for 20, 40 and 120 min, and then pulsed for 1 h with [2- 3 H]Man in the same medium Cells were then submitted to the sequential extraction procedure Oligo-mannosides bound to LLO (A, B, C and D) and glycoproteins (E, F, G and H) were ana-lyzed by HPLC as described in Experimental procedures (D) and (H) correspond to the pattern of oligomannosides bound to LLO and glycoproteins, respectively, when the incubation was performed in the presence of
20 l M kifunensine after a 20 min preincuba-tion G1M9 indicates oligomannosides con-taining one Glc, nine Man, and two GlcNAc residues M3, M4, M5, M8 and M9 indicate oligomannosides containing three, four, five, eight and nine Man residues and two GlcNAc residues, respectively X indicates an uniden-tified peak.
Trang 4Glc with 5 mMMan) for 0, 3 and 6 h At each time point,
glycans bound to glycoproteins were released by PNGase
and sequentially treated to release sialic acid, galactose and
GlcNAc residues As previously described [15], the
appear-ance of Man3GlcNAc2 species after these treatments
reveals the level of Golgi processing Figure 3 shows that
the percentage of radioactivity bound to Man3GlcNAc2
species increased from 18% after the pulse to 38% after the
6 h chase Formation of complex-type glycans correlated
with the decrease in the percentage of radioactivity
associ-ated with oligomannoside-type glycans, mainly the Man9
population If we look at the end products of processing,
most of the Man5GlcNAc2 was converted into
Man4Glc-NAc2, in contrast with the small amount of Man6GlcNAc2
originating from the Man9GlcNAc2 species
ERAD of glycoproteins bearing Man5 and Man9
populations in MI8-5 cells
The N-glycosylation process is accompanied by the release
of soluble oligomannosides At least some of this soluble
material has been shown to originate from glycoprotein
degradation [16,17] after the quality control mechanism
Figure 4Ashows the pattern of oligomannosides bound to
glycoproteins after 20 min of preincubation at 0.175 mM
Glc followed by 1 h of labeling and a 1 h chase in culture
medium containing 5 mMGlc and 5 mMMan Under these
conditions as described previously [17], the profile of soluble
oligomannosides contained three major species:
Man4Glc-NAc1, Man5GlcNAc1 and Glc1Man5GlcNAc1 (Fig 4B)
The smaller species (from Man3GlcNAc1 to
Man1Glc-NAc1) correspond to lysosomal degradation of the soluble
oligomannosides, as previously demonstrated [18] As these
species may result from the action of cytosolic mannosidase
[8], the same experiment was performed in the presence of
DIM to avoid cytosolic demannosylation of glycans
released during the degradation process Figure 4C shows
the profile of soluble oligomannosides when the same
experiment was performed in the presence of 1 mMDIM
Although the pattern of oligosaccharides bound to
glyco-proteins was not affected by the inhibitor (not shown),
Man5 and Man9 species were recovered in the soluble
oligomannoside fraction However, if we compare the
pattern of soluble oligomannosides with that obtained with
glycans bound to proteins (Fig 4C,A, respectively), it is
clear that a higher proportion of soluble oligomannosides
originate from the Man5 population than from the Man9
population
However, it has been shown that some of these soluble oligomannosides may originate from hydrolytic activity of the oligosaccharyltransferase complex using water as accep-tor for the oligosaccharide-PP-Dol donor [19] As glucosy-lated oligomannosides are present only on glycoproteins in MI8-5 cells, Glc1Man5GlcNAc1 and Glc1Man9GlcNAc1 can be used as markers for glycoprotein degradation Table 1 shows that the ratio Glc1Man5GlcNAc1/
Trang 5Glc1Man9GlcNAc1 obtained with soluble
oligomanno-sides was twofold higher than the ratio
Glc1Man5Glc-NAc2/Glc1Man9GlcNAc2 obtained with the glycoprotein
pattern This indicates that, when glycoproteins bearing
Man9 and Man5 are synthesized in the same cell line, those
bearing Man5 are more efficiently degraded
Furthermore, when various proportions of Man5 were
transferred to glycoproteins as a result of different
preincu-bation times at low Glc concentration, a direct relationship
was observed between the level of soluble oligomannosides
released and the proportion of Man5 species bound to the
glycoprotein fraction (Fig 4D)
Glycoproteins bearing only Man9 species can be obtained
in two ways: (a) induction of UPR by extensive
preincuba-tion at low Glc concentrapreincuba-tion (this treatment produces
extension of oligomannosides bound to lipid intermediates)
as in Fig 4D (0% of Man5 population); (b) incubation of
cells in the culture medium (5 mMGlc) which considerably
reduces the labeling In the two cases, glycoprotein
degra-dation, measured by the ratio radioactivity bound to soluble
oligomannosides/radioactivity bound to glycoproteins, is
the same (10–13%) As to the physiological relevance of the
incubation conditions used, this result suggests that Glc
deprivation does not significantly affect the degradation of
glycoproteins
Discussion
The synthesis of oligomannosides linked to lipid
inter-mediates depends on several cellular and extracellular
variables The Glc concentration in the extracellular
medium [20], the capacity of the cell to synthesize the
different metabolic precursors (GDP-Man, UDP-Glc,
Man-P-Dol, Glc-P-Dol), and the variations in the level of
expression of the various glycosytransferases, as observed
in patients with congenital disorders of glycosylation
[21,22], are variables that can modify the quality of the
LLO donors
However, several mechanisms avoid the transfer of truncated lipid donors, especially Man5GlcNAc2, to pro-teins: the activation of UPR, which promotes extension of such truncated LLO intermediates to Glc3Man9GlcNAc2-PP-Dol [13]; the high turnover rate of lipid donors These adaptive events involve only the lipid intermediate donors and not the glycans bound to the protein moiety Thus the fate of glycoproteins bearing Man5GlcNAc2glycans has to
be envisaged
For this, we decided to generate glycoproteins bearing Man5 and Man9 populations in the same cell type To avoid preferential transfer of glucosylated species from LLO, we used MI8-5 CHO cells deficient in Glc-P-Dol-dependent glucosyltransferase I As observed previously for other cell lines [23], the stress induced by preincubation at low Glc concentration leads to a cellular response named UPR, the first level of which is the extension of LLOs Depending on the preincubation time, various proportions
of Man5GlcNAc2 and Man9GlcNAc2 are transferred to proteins The ER processing of these two species has been studied As expected, Man9 species gave Glc1Man9Glc-NAc2, which revealed the action of UGGT For Man5Glc-NAc2 species, although it has been claimed that UGGT is not active on this species, we have clearly demonstrated the formation of Glc1Man5GlcNAc2 The formation of this species has been observed previously in mutant cell lines [24,25] However, this result indicates that this reglucosy-lation step is not specific to the Man-P-Dol-deficient mutant cell line, but can be observed when Man9 species are present on newly synthesized glycoproteins Similarly, the Man9 and Man5 species undergo demannosylation reactions Man5GlcNAc2 is converted into Man4Glc-NAc2, which cannot be reglucosylated, and Man9Glc-NAc2 can be demannosylated first into Man8GlcMan9Glc-NAc2 and, after a longer chase period, into Man6GlcNAc2 This species has been observed on resident glycoproteins, as demonstrated for ribophorin I [26] and unstable glyco-proteins [6]
Fig 3 Evolution of Man9 and Man5 populations bound to proteins during a pulse-chase experiment MI8-5 CHO cells were pulse-labeled with [2- 3 H]Man for 1 h after 40 min of preincubation in 0.175 m M Glc (T0 h) and chased in the culture medium containing 5 m M Glc and 5 m M Man for 3 and 6 h (T3 h and T6 h, respectively) The cells were then submitted to the sequential extraction procedure Glycans were released from the glycoprotein fraction by the action of PNGase They were then analyzed by HPLC after mild acid treatment and the sequential action of b-galactosidase and b-hexosaminidase as described in Experimental procedures M3, M4, M5, M6, M7, M8, M9 indicate oligomannosides with three, four, five, six, seven, eight and nine Man residues and two GlcNAc residues at the reducing end G1M5 indicates oligomannosides with five Man and two GlcNAc residues at the reducing end and one Glc residue, respectively G1M9 indicates oligomannosides with one Glc residue and nine Man and two GlcNAc residues at the reducing end.
Trang 6Most of the evidence for the occurrence of degradation
signals involved in ERAD has been obtained by studying
the effect of inhibitors of demannosylation (kifunensine and
deoxymannojirimycine) on glycoprotein degradation As we
demonstrated that the glucosylated soluble
oligomanno-sides released during the N-glycosylation process of MI8-5
originate only from glycoprotein degradation [17], we
compared the ratio Glc1Man5/Glc1Man9 on glycoproteins
and the soluble oligomannoside fractions in experiments
performed in the presence of DIM, an inhibitor of the
cytosolic mannosidase We observed that glycoproteins
bearing Man5 were more efficiently degraded (twofold)
than those bearing Man9 It is noteworthy that the release
of the glucosylated species was accompanied by the release
of Man8–5GlcNAc1 and Man4GlcNAc1, which are puta-tive degradation signals for glycoproteins bearing Man9 and Man5, respectively As we only took into account mono-glucosylated species, the degradation of the Man5 popula-tion was probably underestimated
We have reported previously [27] that the level of soluble oligomannosides released during N-glycosylation is higher
in Man-P-Dol-deficient cell lines than in wild-type cells, which are able to elongate their lipid intermediates We have also demonstrated that the pattern of these oligomannosides
is related to the pattern of the glycans bound to the newly
Table 1 Radioactivity bound to Glc1Man5 and Glc1Man9 species during chase experiments in MI8-5 cells in the presence of 1 m M DIM MI8-5 cells in the presence of 1 m M DIM were pulsed for 1 h and chased in culture medium containing 5 m M Glc and 5 m M Man for 1 h after 20 min preincubation with 0.175 m M Glc Cells were then sub-mitted to the sequential extraction procedure The radioactivity bound
to Glc1Man5 and Glc1Man9 species was measured after counting of the glycoprotein and soluble oligomannoside fractions, taking into account the percentage of these species as determined by HPLC Values (d.p.m.) from three different experiments are shown.
Glc1Man5 Glc1Man9
Glc1Man5/ Glc1Man9 Glycoprotein fraction 113512 127650 0.88
114070 134000 0.85
114418 134350 0.85 Soluble oligomannoside
released during the chase experiment
14520 7100 2.04
13250 8206 1.61
13510 8214 1.64
0.175 m M Glc before the 1-h pulse (from 120 min preincubation for 0% Man5 species to 20 min preincubation for 50% Man5 species).
OS, Soluble oligomannosides M4, M5, M6, M7, M8, M9 indicate oligomannosides with four, five, six, seven, eight and nine Man resi-dues and two GlcNAc resiresi-dues at the reducing end G1M5 indicates oligomannosides with five Man and two GlcNAc residues at the reducing end and one Glc residue, respectively G1M9 indicates oligo-mannosides with one Glc residue and nine Man and two GlcNAc residues at the reducing end M4Gn1, M5Gn1, M6Gn1, M7Gn1, M8Gn1, and M9Gn1 indicate oligomannosides with four, five, six, seven, eight and nine Man residues and one GlcNAc residue at the reducing end G1M5Gn1 and G1M9Gn1 indicate oligomannosides with one Glc residue, five or nine Man residues and one GlcNAc residue at the reducing end.
Trang 7synthesized glycoproteins [18] We have shown here that,
when Man5GlcNAc2 oligosaccharides are transferred to
glycoproteins in cells able to synthesize and transfer
Man9GlcNAc2, the glycoproteins bearing these truncated
glycans are preferentially degraded It means that the
mechanism of degradation found in mutant cells is also used
by normal cells
Acknowledgements
This work was supported by the Centre National de la Recherche
Scientifique and the Ministe`re de l’Education Nationale et de la
Recherche Technologique We are grateful to Dr J.-C Michalski
director of UMR 8576, Glycobiologie Structurale et Fonctionnelle.
We gratefully acknowledge Dr T Butters for generously supplying
glucosidase II, and Dr S S Krag for her gift of MI8-5 cell lines.
References
1 Jackson, B.J., Warren, C.D., Bugge, B & Robbins, P.W (1989)
Synthesis of lipid-linked oligosaccharides in Saccharomyces
cere-visiae: Man 2 GlcNAc 2 and Man 1 GlcNAc 2 are transferred from
dolichol to protein in vivo Arch Biochem Biophys 272, 203–209.
2 Karaoglu, D., Kelleher, D.J & Gilmore, R (2001) Allosteric
regulation provides a molecular mechanism for preferential
utili-zation of the fully assembled dolichol-linked oligosaccharide by
the yeast oligosaccharyltransferase Biochemistry 40, 12193–12206.
3 Lehrman, M.A (2001) Oligosaccharide-based information in
endoplasmic reticulum quality control and other biological
sys-tems J Biol Chem 276, 8623–8626.
4 Helenius, A & Aebi, M (2001) Intracellular functions of N-linked
glycans Science 291, 2364–2369.
5 Jakob, C.A., Burda, P., Roth, J & Aebi, M (1998) Degradation
of misfolded endoplasmic reticulum glycoproteins in
Saccharo-myces cerevisiae is determined by a specific oligosaccharide
structure J Cell Biol 142, 1223–1233.
6 Frenkel, Z., Gregory, W., Kornfeld, S & Lederkremer, G.Z.
(2003) ER-associated degradation of mammalian glycoproteins
involves sugar chain trimming to Man6)5GlcNAc 2 J Biol Chem.
278, 34119–34124.
7 Ermonval, M., Kitzmuller, C., Mir, A.M., Cacan, R & Ivessa,
N.E (2001) N-glycan structure of a short-lived variant of
ribo-phorin I expressed in the MadIA214 glycosylation-defective cell
line reveals the role of a mannosidase that is not ER mannosidase I
in the process of glycoprotein degradation Glycobiology 11,
565–576.
8 Weng, S & Spiro, R.G (1996) Endoplasmic reticulum
kifu-nensine-resistant alpha-mannosidase is enzymatically and
immunologically related to the cytosolic alpha-mannosidase.
Arch Biochem Biophys 325, 113–123.
9 Kmie´cik, D., Herman, V., Stroop, C.J., Michalski, J.C., Mir,
A.M., Verbert, A & Cacan, R (1995) Catabolism of glycan
moieties of lipid intermediates leads to a single Man 5 GlcNAc
oligosaccharide isomer: a study with permeabilized CHO cells.
Glycobiology 5, 483–494.
10 Saint-Pol, A., Bauvy, C., Codogno, P & Moore, S.E (1997)
Transfer of free polymannose-type oligosaccharides from the
cytosol to lysosomes in cultured human hepatocellular carcinoma
HepG2 cells J Cell Biol 136, 45–59.
11 Foulquier, F., Harduin-Lepers, A., Duvet, S., Marchal, I., Mir,
A.M., Delannoy, P., Chirat, F & Cacan, R (2002) The unfolded
protein response in a dolichyl phosphate mannose-deficient
Chinese hamster ovary cell line points out the key role of a
demannosylation step in the quality-control mechanism of N-glycoproteins Biochem J 362, 491–498.
12 Quellhorst, G.J Jr, O’Rear, J.L., Cacan, R., Verbert, A & Krag, S.S (1999) Nonglucosylated oligosaccharides are transferred to protein in MI8-5 Chinese hamster ovary cells Glycobiology 9, 65–72.
13 Shang, J., Korner, C., Freeze, H & Lehrman, M.A (2002) Extension of lipid-linked oligosaccharides is a high-priority aspect
of the unfolded protein response: endoplasmic reticulum stress in Type I congenital disorder of glycosylation fibroblasts Glyco-biology 12, 307–317.
14 Herscovics, A (2001) Structure and function of Class I alpha 1,2-mannosidases involved in glycoprotein synthesis and endo-plasmic reticulum quality control Biochimie 83, 757–762.
15 Verbert, A & Cacan, R (1999) Trafficking of oligomannosides released during N-glycosylation: a clearing mechanism of the rough endoplasmic reticulum Biochim Biophys Acta 1473, 137–146.
16 Moore, S.E.H & Spiro, R.G (1994) Intracellular compartmen-talization and degradation of free polymannose oligosaccharides released during glycoprotein synthesis J Biol Chem 269, 12715– 12721.
17 Cacan, R., Duvet, S., Labiau, O., Verbert, A & Krag, S.S (2001) Monoglucosylated oligomannosides are released during the degradation process of newly synthesized glycoproteins J Biol Chem 276, 22307–22312.
18 Duvet, S., Labiau, O., Mir, A.M., Kmiecik, D., Krag, S.S., Ver-bert, A & Cacan, R (1998) Cytosolic deglycosylation process of newly synthesized glycoproteins generates oligomannosides pos-sessing one GlcNAc residue at the reducing end Biochem J 335, 389–396.
19 Spiro, M.J & Spiro, R.G (1991) Potential regulation of N-glycosylation precursor through oligosaccharide-lipid hydro-lase action and glucosyltransferase-glucosidase shuttle J Biol Chem 266, 5311–5317.
20 Rearick, J.I., Chapman, A & Kornfeld, S (1981) Glucose star-vation alters lipid-linked oligosaccharide biosynthesis in Chinese hamster ovary cells J Biol Chem 256, 6255–6261.
21 Marquardt, T & Freeze, H (2001) Congenital disorders of gly-cosylation: glycosylation defects in man and biological models for their study Biol Chem 382, 161–177.
22 Freeze, H.H (2002) Human disorders in N-glycosylation and animal models Biochim Biophys Acta 1573, 388–393.
23 Doerrler, W.T & Lehrman, M.A (1999) Regulation of the dolichol pathway in human fibroblasts by the endoplasmic reticulum unfolded protein response Proc Natl Acad Sci USA
96, 13050–13055.
24 Ermonval, M., Duvet, S., Zonneveld, D., Cacan, R., Buttin, G & Braakman, I (2000) Truncated N-glycans affect protein folding in the ER of CHO-derived mutant cell lines without preventing calnexin binding Glycobiology 10, 77–87.
25 Duvet, S., Chirat, F., Mir, A.M., Verbert;, A., Dubuisson, J & Cacan, R (2000) Reciprocal relationship between alpha 1,2 mannosidase processing and reglucosylation in the rough endoplasmic reticulum of Man-P-Dol deficient cells Eur J Bio-chem 267, 1146–1152.
26 Rosenfeld, M.G., Marcantonio, E.E., Hakimi, J., Ort, V.M., Atkinson, P.H., Sabatini, D & Kreibich, G (1984) Biosynthesis and processing of ribophorins in the endoplasmic reticulum.
J Cell Biol 99, 1076–1082.
27 Villers, C., Cacan, R., Mir, A.M., Labiau, O & Verbert, A (1994) Release of oligomannoside-type glycans as a marker of the degradation of newly synthesized glycoproteins Biochem J 298, 135–142.