Endoplasmic 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. A wide 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 Ôen blocÕ 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 ´ desSciencesetTech- 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:peptideN-glycanase F (PNGase; EC 3.2.2.18); b-galactosi- dase (EC 3.2.1.23); b-hexosaminidase (EC 3.2.1.30). (Received 15 September 2003, revised 21 October 2003, accepted 21 November 2003) Eur. J. Biochem. 271, 398–404 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03938.x Using the MI8-5 cell line which is unable to synthesize glucosylated lipid-linked oligosaccharides (LLOs), but in which UGGT remains active, we show that non-glucosyl- ated Man5GlcNAc2-PP-Dol could be efficiently transferred to glycoproteins. In contrast with that observed in vitro, Man5GlcNAc2 bound to glycoproteins was efficiently glucosylated. By studying the degradation process, as revealed by the structure of the released soluble oligoman- nosides, we demonstrate that glycoproteins bearing Man5 are preferentially degraded over glycoproteins bearing Man9. Experimental procedures Materials [2- 3 H]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 34 °C under 5% CO 2 . 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 m M ) for different times and then metabolically labeled for 1 h with 3.6 GBqÆmL )1 (4 l M ) [2- 3 H]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 m M ) supplemented with 5 m M Man. When used, kifunensine (20 l M )andDIM(1m M ) 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 )in0.1 M ammonium bicarbonate buffer, pH 7.9, overnight at room temperature. Glycopeptides were then treated with 0.5 U PNGase in 50 m M phosphate buffer, pH 7.2, for 4 h to release oligosaccharides. Oligosaccharide moieties were released from LLO by mild acid treatment (0.1 M HCl in tetrahydrofuran) for 2 h at 50 °C. The oligosaccharide fractions were then desalted on Bio-Gel P2 eluted with 5% (v/v) acetic acid. Analysis was performed by HPLC on an amino-derivatized Asahipak NH2P-50 column (250 mm · 4.6 mm; 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 mLÆmin )1 over 90 min. Oligomannosides were identified on the basis of their retention times compared with well-defined standards [11]. 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 2 M acetic acid, and hydrolysis was carried out at 80 °C for 2 h to release sialyl residues. Incubation with 0.1 U b-galactosidase was performed in 20 lL 100 m M sodium acetate buffer, pH 3.5, overnight at 37 °C. 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 m M ), 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 forupto120minatlowGlcconcentration(0.175m M ) 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 l M kifunensine 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). Ó FEBS 2003 Endoplasmic reticulum-associated degradation (Eur. J. Biochem. 271) 399 Dual 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 Glc1Man5GlcNAc2 (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- 3 H]Man with or without 100 lgÆmL )1 castanospermine, 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 Man9GlcNAc2, 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 l M 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 m M Glc, labeled for 1 h, and chased in the culture medium (5 m 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]Maninthe 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. 400 F. Foulquier et al.(Eur. J. Biochem. 271) Ó FEBS 2003 Glc with 5 m M Man) 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 4A shows the pattern of oligomannosides bound to glycoproteins after 20 min of preincubation at 0.175 m M Glc followed by 1 h of labeling and a 1 h chase in culture medium containing 5 m M Glc and 5 m M Man. 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 m M DIM. 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/ Fig. 2. Reglucosylation of Man5GlcNAc2 and Man9GlcNAc2 species in MI8-5 cells. MI8-5 cells were preincubated for 20 min in the pres- ence of 0.175 m M Glc and pulse-labeled with [2- 3 H]Man for 1 h without (A) or with (B) 100 lgÆmL )1 castanospermine. After incuba- tion and sequential extraction, oligomannosides bound to glycopro- teins were analyzed by HPLC as described in Experimental procedures. (C) HPLC profile of oligomannosides obtained after incubation with purified glucosidase II. G1M9 indicates oligoman- nosides containing one Glc, nine Man, and two GlcNAc residues. M4, M5, M8 and M9 indicate oligomannosides containing four, five, eight and nine Man residues and two GlcNAc residues, respectively. X indicates an unidentified peak. Ó FEBS 2003 Endoplasmic reticulum-associated degradation (Eur. J. Biochem. 271) 401 Glc1Man9GlcNAc1 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 concentration (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 m M Glc) 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 Man 5 GlcNAc 2 glycans 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 Man8GlcNAc2 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. 402 F. Foulquier et al.(Eur. J. Biochem. 271) Ó FEBS 2003 Most 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 Fig. 4. Pattern of oligomannosides released by MI8-5 CHO cells during the N-glycosylation process. MI8-5 cells were pulse-labeled with [2- 3 H]Manfor1hafter20minpreincubationin0.175m M Glc and chased for 1 h in culture medium containing 5 m M Glc and 5 m M Man in the presence (C) or absence (A, B) of 1 m M DIM. Cells were then submitted to the sequential extraction procedure. Glycoproteins were extracted and glycan species were analyzed by HPLC (A). The soluble oligomannoside material was purified and analyzed by HPLC in the absence (B) or presence (C) of DIM. (D) Relationship between the radioactivity bound to soluble oligomannoside material and the pro- portion of Man5 species (Man4GlcNAc2, Man5GlcNAc2 and Glc1Man5GlcNAc2) transferred to glycoproteins. These different proportions were obtained by using different preincubation times in 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 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 oligo- mannosides with one Glc residue and nine Man and two GlcNAc residuesatthereducingend.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. Ó FEBS 2003 Endoplasmic reticulum-associated degradation (Eur. J. Biochem. 271) 403 synthesized glycoproteins [18]. 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(1994) Release of oligomannoside-type glycans as a marker of the degradation of newly synthesized glycoproteins. Biochem. J. 298, 135–142. 404 F. Foulquier et al.(Eur. J. Biochem. 271) Ó FEBS 2003 . Endoplasmic reticulum-associated degradation of glycoproteins bearing Man5GlcNAc2 and Man9GlcNAc2 species in the MI8-5 CHO cell line Franc¸ois. newly synthesized glycoproteins. The degradation of glycoproteins bearing these two species has not been studied. We used a Chinese hamster ovary cell line