Glycobiology Advance Access originally published online on June 2, 2004
Glycobiology 2004 14(9):841-849; doi:10.1093/glycob/cwh103
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Glycobiology vol. 14 no. 9 © Oxford University Press 2004; all rights reserved.
Discrimination between lumenal and cytosolic sites of deglycosylation in endoplasmic reticulum-associated degradation of glycoproteins by using benzyl mannose in CHO cell lines
Unité de Glycobiologie Structurale et Fonctionnelle, UMR CNRS 8576, IFR 118, GDR CNRS 2590, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France
Received on April 14, 2004; revised on May 25, 2004; accepted on May 27, 2004
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Abstract |
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Recent studies demonstrated that deglycosylation step is a prerequisite for endoplasmic reticulum (ER)-associated degradation of misfolded glycoproteins. Here, we report the advantages of using benzyl mannose during pulse-chase experiments to study the subcellular location of the deglycosylation step in Chinese hamster ovary (CHO) cell lines. Benzyl mannose inhibited both the ER-to-cytosol transport of oligomannosides and the trimming of cytosolic-labeled oligomannosides by the cytosolic mannosidase in vivo. We pointed out the occurrence of two subcellular sites of deglycosylation. The first one is located in the ER lumen, and led to the formation of Man8GlcNAc2 (isomer B) in wild-type CHO cell line and Man4GlcNAc2 in Man-P-Dol-deficient cell line. The second one was revealed in CHO mutant cell lines for which a high rate of glycoprotein degradation was required. It occurred in the cytosol and led to the liberation of oligosaccharides species with one GlcNAc residue and with a pattern similar to the one bound onto glycoproteins. The cytosolic deglycosylation site was not specific for CHO mutant cell lines, since we demonstrated the occurrence of cytosolic pathway when the formation of truncated glycans was induced in wild-type cells.
Key words: benzyl mannose / Chinese hamster ovary cells / endoplasmic reticulum-associated degradation / free oligomannosides / N-glycosylation / peptide N-glycanase
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Introduction |
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The first event of the N-glycosylation process is the transfer en bloc of Glc3Man9GlcNAc2 from the lipid-linked oligosaccharide (LLO) Glc3Man9GlcNAc2-P-P-dolichol to asparagine residues of nascent proteins. This mechanism occurs at the lumenal face of the endoplasmic reticulum (ER). After folding, glycoproteins were transported to the Golgi apparatus in which their N-linked oligosaccharides are remodeled (for review see Kornfeld and Kornfeld, 1985
). Several lines of evidence indicated that glycoprotein biosynthesis was accompanied by the generation of free oligosaccharides. Accordingly, free oligosaccharides may be generated either by the oligosaccharyl transferase-mediated hydrolysis activity on Glc3Man9GlcNAc2-P-P-dolichol in the lumen of the ER (Spiro and Spiro, 1991) or in the cytosol by peptide N-glycanase (PNGase) activity onto misfolded glycoproteins (for review see Suzuki et al., 2002). In these two cases, the resulting free oligosaccharides possess two GlcNAc residues (OSGn2) at their reducing end and they have been reported in a wide variety of cells, and their processing and trafficking have also been well characterized. Indeed, ER-released OSGn2 were transported into the cytosol (Moore et al., 1995) where they are sequentially trimmed by a chitobiase (Cacan et al., 1996) producing oligosaccharides possessing one GlcNAc residue (OSGn1) species and by a cytosolic mannosidase to yield a specific Man5GlcNAc1 isomer (Kmiecik et al., 1995), which is imported into lysosomes (Saint-Pol et al., 1997) to be degraded further into smaller species and monosaccharides.
ER-associated protein degradation (ERAD) has been well documented and different pathways have been described. According to the model, the degradation process involved either ubiquitin proteasome machinery after the retrotranslocation of the protein from the ER into the cytosol (Wiertz et al., 1996a,b; Yu et al., 1997; De Virgilio et al., 1998; Plemper and Wolf, 1999) or ER-located proteases (Loo and Clarke, 1998; Fayadat et al., 2000). For N-glycoproteins, a prerequisite for these degradation processes is the deglycosylation step (Hirsch et al., 2003). Even though a PNGase activity has been well characterized in the cytosol (Suzuki et al., 1998, 2002), interestingly, part of free oligosaccharides are believed to arise from a ER lumen PNGase activity (Weng and Spiro, 1997; Spiro and Spiro, 2001). Indeed, Karaivanova and Spiro (2000) proposed that N-deglycosylation could take place in both the ER and cytosolic compartments, and presented schematic models for this dual location. Recently, a yeast strain deficient in the PNGase encoded by the PNG1 gene was found to be unable to produce free oligomannosides (Chantret et al., 2003).
Benzyl 
-D-mannopyranoside (Bz-man), a mannose derivative has been described to inhibit the ER-to-cytosol transport of free oligomannosides (Moore, 1998). In this present study, we use Bz-man in vivo as a tool to discriminate cytosolic and lumenal oligomannosides released during the N-glycosylation process. In wild-type Chinese hamster ovary (CHO) cells, metabolic labeling with [2-3H]mannose in the presence of Bz-man led to the release of a unique lumenal oligomannoside isomer: Man8GlcNAc2 (isomer B). Experiments in the presence of cycloheximide (CH) indicated that a large majority of Man8Gn2 is derived from deglycosylation of newly synthesized glycoproteins. In contrast, in glycosylation mutant cell lines, the lumenal process was accompanied by the release of cytosolic oligomannosides which possess the same pattern as the one linked to proteins. In addition, we clearly demonstrated that these two deglycosylation processes could simultaneously occur in the same cell line depending on the nature of the glycans which were transferred onto proteins. Thus the use of Bz-man allowed us to point out lumenal and cytosolic deglycosylation processes in a variety of CHO cell lines.
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Results |
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Effect of Bz-man on soluble oligomannoside trafficking in wild-type CHO cell line
Bz-man has been described to inhibit in vitro the transport of soluble oligomannosides from the ER lumen to the cytosol (Moore, 1998
). We used this mannose derivative in vivo during metabolic labeling of Pro–5 CHO cell line. According to the N-glycosylation process, Pro–5 CHO cell line was taken as a reference since the nature of LLO precursor was Glc3Man9GlcNAc2-P-P-dolichol, the profile recovered onto the newly synthesized glycoproteins revealed three species, Man8-, Man9-, and Glc1Man9GlcNAc2, and the only free oligomannoside recovered after 1 h incubation with radiolabeled mannose was Man5GlcNAc1 (Kmiecik et al., 1995). Figure 1A shows the effect of increasing concentrations of Bz-man (from 0 to 5 mM) on the nature of soluble oligomannosides released during 1 h incubation. As widely demonstrated (see Kmiecik et al., 1995), the control incubation without Bz-man led to the formation of Man5GlcNAc1, which resulted from the sequential action of the cytosolic chitobiase and mannosidase onto soluble oligomannosides. Increasing concentration of Bz-man induced the formation of Man8GlcNAc2 which was the unique species recovered at 5 mM of Bz-man. Indeed, incubation of this species with specific 2-mannosidase from Aspergillus saitoi led to the formation of Man5GlcNAc2 (data not shown). Furthermore, high-performance liquid chromatography (HPLC) analysis indicated that this species co-migrates with Man8GlcNAc2 released from glycoproteins after PNGase treatment which corresponds to the isomer B (Figure 1B). It is important to note that in CHO cell line, the lack of endomannosidase implies that isomer A of Man8GlcNAc2 could not be formed (Hiraizumi et al., 1993). The fact that this oligomannoside possess two GlcNAc residues demonstrated that it was localized in the ER lumen (Kmiecik et al., 1995) and shows that the oligomannoside transporter has been inhibited in vivo. In these conditions, inhibition was spontaneously reversible because during a chase experiment in which Bz-man was added only during the 1 h pulse, the formation of Man5GlcNAc1 was completely restored after 2 h chase (Figure 2). This experiment clearly demonstrates the substrate product relationship between lumenal Man8GlcNAc2 and cytosolic Man5GlcNAc1. Moreover, it has to be noted that the nature of LLO precursor Glc3Man9GlcNAc2-P-P-dolichol and the processing of the glycan bound onto nascent glycoproteins were unaffected by the presence of 5 mM of Bz-man (data not shown).
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Hydrolysis of Bz-man by the cytosolic mannosidase
The spontaneous reversibility of the action of Bz-man onto the oligomannoside trafficking could be explained through their hydrolysis by cellular mannosidases. Table I indicates that addition of Bz-man during a pulse experiment in Pro–5 cells led to a significant decrease (35%) in the incorporation of the radioactivity onto LLO, glycoproteins, and soluble oligomannosides. This result suggested that Bz-man was hydrolyzed and the resulting mannose was used as an unlabeled precursor. Because the class II cytosolic
-mannosidase has been described clearly to act onto aryl-mannose, such as p-nitrophenyl--D-mannopyranoside (Grard et al., 1994; Weng and Spiro, 1996), we postulated that this enzyme could be a potential candidate for Bz-man hydrolysis. An additional experiment was performed with Bz-man in the presence of 1,4-dideoxy-1,4-imino-D-mannitol (DIM), an inhibitor of class II -mannosidases (Daniel et al., 1994; Weng and Spiro, 1996). In these conditions, the action of DIM abolished the isotopic dilution (Table I) indicating that the cytosolic mannosidase was directly involved in the hydrolysis of Bz-man.
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Soluble Man8GlcNAc2 synthesized in the presence of Bz-man originate from glycoprotein degradation
OSGn2 can be generated from LLO by the hydrolytic activity of the oligosaccharyl transferase as well as by PNGase action onto newly synthesized glycoproteins. Figure 3A shows that in the presence of CH and 5 mM of Bz-man, although the radioactivity bound to LLO was not affected, a decrease in 98% of the radioactivity bound to glycoproteins and soluble oligomannosides was observed indicating a clear relationship between newly synthesized glycoproteins and the release of Man8GlcNAc2. As an additional approach, Pro–5 CHO cells (auxotrophic to proline) were preincubated in a proline-free medium supplemented with 20 mM of L-azetidine-2-carboxylic acid (AZC), a proline analog that interferes with the folding of protein. AZC induced a 4-fold decrease in the incorporation of radiolabeled mannose onto glycoproteins (Figure 3A) and an increase in the formation of Glc1Man9GlcNAc2 bound to glycoproteins (Figure 3B: compare glycoproteins Bz-man and Bz-man + AZC), a species which is involved in the quality control of glycoproteins indicating that this agent interferes with the folding of glycoproteins. As this experiment was performed in the presence of 5 mM of Bz-man, the only oligomannoside species which was detected correspond to Man8GlcNAc2 (Figure 3B) but it is interesting to note that taking into account the incorporation onto glycoproteins AZC induced a production of Man8GlcNAc2, which was 3.4-fold higher than in control cells without AZC. This indicated a clear relationship between the folding state of glycoproteins and the production of free lumenal Man8GlcNAc2. The effects of CH and AZC clearly indicated that a high proportion of the Man8GlcNAc2 observed in the presence of Bz-man originated from the deglycosylation step occurring during the degradation process of newly synthesized glycoproteins.
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Oligomannosides released in the presence of Bz-man in various CHO glycosylation mutant cell lines
Previous studies on a Man-P-Dol deficient CHO cell line (B3F7) (Villers et al., 1994
; Duvet et al., 1998) and on a Glc-P-Dol-dependent glucosyltransferase I deficient CHO cell line (MI8-5) (Cacan et al., 2001) clearly indicated the release of cytosolic OSGn1 oligomannosides during the N-glycosylation process. Figure 4 shows the pattern of glycoproteins and soluble oligomannosides obtained after incubation of MI8-5 and B3F7 in the presence of 5 mM of Bz-man. Surprisingly, in MI8-5 cells, although the pattern bound to glycoprotein was similar to the one observed onto wild-type Pro–5 cell line (Figure 3B, glycoprotein Bz-man), the pattern of soluble oligomannosides was largely represented by OSGn1 species (Man8GlcNAc1, Man9GlcNAc1, and Glc1Man9GlcNAc1) which are present onto glycoproteins, only a minor peak of Man8GlcNAc2 appeared. As published previously (Cacan et al., 2001) control incubation of MI8-5 cell line led to the formation of Man5- and Glc1Man5GlcNAc1 species. The fact that the cytosolic-free oligomannosides remained untrimmed was due to a competition between these labeled oligomannosides and Bz-man for hydrolysis by the cytosolic mannosidase. Indeed after a 2 h chase, the complete hydrolysis of Bz-man led all these oligomannoside species to be converted into Man5GlcNAc1 species as already pointed out in Figure 2. Similar results were observed for B3F7 cells in which soluble oligomannosides corresponded predominantly to the pattern recovered onto glycoproteins (Man4GlcNAc1, Man5GlcNAc1, and Glc1Man5GlcNAc1), the only OSGn2 species was now Man4GlcNAc2, a species which has been demonstrated to originate from the action of a kifunensine-sensitive class I mannosidase onto Man5GlcNAc2 species (Duvet et al., 2000; Foulquier et al., 2002). Thus the use of Bz-man allowed us to point out that besides the deglycosylation process which occurred in the ER lumen, the previously described cytosolic deglycosylation mechanism was operating in the CHO mutant cell lines for which a high degradation rate of newly synthesized glycoproteins has been demonstrated.
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Occurrence of lumenal and cytosolic deglycosylation mechanism in wild-type CHO cell line
As recently demonstrated by Foulquier et al. (2004)
, glycoproteins bearing truncated glycans have a higher degradation rate than the one bearing fully extended glycans. To generate glycoproteins bearing both Man5GlcNAc2 and Man9GlcNAc2 species in Pro–5 cells in the presence of Bz-man, we relied upon studies which have demonstrated that glucose (Glc) starvation led to the synthesis of truncated LLO (Rearick et al., 1981). Figure 5A (control) shows that when incubation of Pro–5 cell line was performed in a Glc-free medium, a transfer of truncated species was observed onto glycoproteins because Man4, Man5, and Glc1Man5GlcNAc2 appeared besides the fully extended species (Man8-, Man9-, and Glc1Man9GlcNAc2). When Bz-man was added these truncated species disappeared due to the hydrolysis of Bz-man and recycling of the released unlabeled mannose used as precursor for the extention of LLO (Figure 5A, Bz-man). This extension was abolished when incubation was performed in the presence of both Bz-man and DIM (Figure 5A, Bz-man + DIM) in accordance with the inhibition of the cytosolic mannosidase activity. In these latter conditions, the soluble oligomannosides released during the 1 h pulse were represented by two populations: Man8GlcNAc2 originating from the deglycosylation of fully extended species as in the control, and a pattern similar to the one observed in B3F7 (Figure 4B), i.e. OSGn2 species (Man4- and Man5GlcNAc2) and OSGn1 species (Man4-, Man5-, and Glc1Man5GlcNAc1).
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Thus these findings indicated that, in wild-type CHO cell line, an additional cytosolic deglycosylation site was revealed when truncated LLO were transferred onto glycoproteins.
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Discussion |
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A general scheme for the degradation of newly synthesized glycoproteins after the quality control mechanism involves sequential events, such as the retrotranslocation of the glycoprotein from the ER lumen to the cytosol, a deglycosylation step catalyzed by a cytosolic PNGase, and the proteolytic cleavage by the ubiquitin proteasome machinery. This pathway has been established following the fate of radiolabeled glycoprotein during chase experiment and by using proteasome inhibitors, such as lactacystin. Previous works using [2-3H]mannose labeling of glycosylation mutant cell lines have confirmed that the deglycosylation step occurred in the cytosol and that the released glycans were rapidly converted into oligomannosides possessing one GlcNAc residue at the reducing end due to the action of a cytosolic chitobiase. By using Bz-man, a membrane-permeable molecule, we demonstrated that the inhibition of the ER-to-cytosol oligomannoside transport observed in vitro (Moore, 1998
) was also recovered in vivo. Furthermore when labeled oligomannosides were released in the cytosol, Bz-man competes for their hydrolysis with the help of DIM-sensitive cytosolic mannosidase. In these conditions, these cytosolic oligomannosides remained untrimmed until Bz-man was entirely hydrolyzed.
The use of Bz-man in wild-type CHO cell line revealed the release of lumenal Man8GlcNAc2 (isomer B) concomitantly with the glycosylation process. This oligomannoside species was known to be the product of ER mannosidase I. This Man8GlcNAc2 appeared during the radiolabeling period thus demonstrating that its formation was consequent upon oligosaccharyl transferase activity. CH, which caused an inhibition in protein synthesis (98% decrease in mannose incorporation into glycoproteins) without affecting LLO production, inhibited the formation of Man8GlcNAc2 by the same factor indicating that the bulk of Man8GlcNAc2 arises from deglycosylation of newly synthesized glycoproteins. The same result was recently obtained by Chantret et al. (2003) in yeast in which a reduced level of Man8GlcNAc2 has been observed in a strain deficient in PNGase. In addition to the fact that in AZC-treated cells, the ratio: radioactivity bound to newly synthesized glycoproteins/radioactivity bound to Man8GlcNAc2 was 3.4-fold higher than in control cells strongly support a direct relationship between the formation of Man8GlcNAc2 and the glycoprotein degradation process.
The lumenal deglycosylation process was also observed in MI8-5 (Man8GlcNAc2) and in B3F7 (Man4GlcNAc2) indicating that the lumenal deglycosylation process also occurred in mutant cell lines. But in addition, the incubation of MI8-5 and B3F7 in the presence of Bz-man revealed the release of cytosolic OSGn1 with a pattern similar to the one bound onto ER glycoproteins indicating that a cytosolic deglycosylation step occurred besides the formation of lumenal OSGn2.
The present paper constitutes the first description of the simultaneous occurrence of lumenal and cytosolic deglycosylation sites of newly synthesized glycoproteins in the same cell line, presumably as prerequisite steps for different degradation pathways. Surprisingly, only the lumenal deglycosylation occurred in wild-type CHO cells but when these cells were induced to synthesize and transfer onto proteins truncated glycans, an efficient deglycosylation mechanism occurred in the cytosol. Thus, we can hypothesize that the cytosolic route for deglycosylation was used when high glycoprotein degradation rate was requested. However, the relationship between the lumenal and the cytosolic deglycosylation sites and the proteasomal and non-proteasomal degradation machineries remains to be elucidated.
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Materials and methods |
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Materials
[2-3H]mannose (429 GBq/mmol) was from Amersham Pharmacia Biotech (Little Chalfont, UK). Bz-man, trypsin, AZC, and CH were obtained from Sigma-Aldrich Chimie SARL (Saint Quentin, Fallavier, France). DIM was purchased from ICN (Orsay, France). PNGase F was obtained from New England Biolabs (Beverly, MA). Endo-ß-N-acetylglucosaminidase H was from Boehringer Mannheim (Germany). Kifunensine was from Calbiochem (Darmstadt, Germany).
2-mannosidase from A. saitoi was from Glyko (Novato, CA).
Cell culture
The Glc-P-Dol-dependent glucosyltransferase I-deficient CHO mutant cell line (MI8-5) and the mannosylphosphoryldolichol-deficient CHO mutant cell line (B3F7) were a gift from S.S. Krag (Johns Hopkins University, Baltimore, MD). Wild-type CHO cell line (Pro–5), MI8-5, and B3F7 cells were routinely grown in monolayers in alpha-minimal essential medium (Gibco) supplemented with 10% (v/v) fetal bovine serum at 34°C in 10-cm Petri dishes under 5% CO2.
Metabolic labeling of oligosaccharides and chase experiments
Cells were preincubated at low-Glc concentration (0.5 mM) during 30 min and then metabolically labeled for 1 h with 3.6 GBq/mL (4 µM) [2-3H]mannose at the same Glc concentration. For pulse-chase experiment, the radioactive culture medium was then replaced by alpha-minimal essential medium containing the physiological Glc concentration (5 mM) supplemented with 5 mM of mannose. Bz-man (at mentioned concentrations) was added only during the pulse. When used, DIM, CH, and AZC were present throughout the experiment (preincubation, pulse, and chase) at a concentration of 2, 1, and 20 mM, respectively. Sequential extraction and purification of oligosaccharide material were achieved as described previously (Kmiecik et al., 1995).
Analysis of oligosaccharide material
Soluble oligomannoside fractions obtained after the sequential extraction were desalted on Bio-Gel P2 eluted with 5% acetic acid. The protein pellet was digested overnight at room temperature with trypsin (1 mg/mL) in 0.1 M of ammonium bicarbonate buffer, pH 7.9. Glycopeptides were then treated with 0.5 U PNGase in 50 mM of phosphate buffer, pH 7.2, for overnight incubation at 37°C 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% acetic acid. Analysis was performed by HPLC on an amino-derivatized Asahipak NH2P-50 column (250 mm x 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 mL/min over 90 min. Oligomannosides were identified on the basis of their retention times compared with well-defined standards (Foulquier et al., 2002). Elution of the radiolabeled oligosaccharides was monitored by continuous-flow detection of the radioactivity with a flo-one ß-detector (Perkin-Elmer, Les Ullis, France).
Preparation of radiolabeled oligomannosides
Radiolabeled Man8Gn2 (isomer B) was prepared from wild-type CHO glycoproteins after [2-3H]mannose incubation for 1 h. The labeled oligosaccharide moieties released after PNGase digestion was isolated by preparative HPLC in conditions described above to isolate Man8Gn2 (isomer B) from Man9Gn2 and Glc1Man9Gn2. The Man8Gn1 isomer B was prepared in the same conditions but the labeled oligomannosides were released by endo-ß-N-acetylglucosaminidase H treatment. Radiolabeled Man8Gn2 (isomer C) was prepared according to Weng and Spiro (1996) by incubating the previously isolated Man9Gn2 with rat liver microsomes prepared according to Lubas and Spiro (1987) in the presence of 40 µM kifunensine. After sequential extraction, the aqueous phase was submitted to a preparative HPLC as described by Yamagishi et al. (2002) to isolate Man8Gn2 isomer C from remaining Man9Gn2. The Man8Gn1 isomer C was prepared in the same conditions but using Man9Gn1 as substrate.
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Acknowledgements |
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We are grateful to Dr J.C. Michalski director of UMR 8576, Glycobiologie Structurale et Fonctionnelle. We gratefully acknowledge Dr S.S. Krag for her gift of MI8-5 and B3F7 cell lines. This work was supported by the Centre National de la Recherche Scientifique.
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Footnotes |
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1 To whom correspondence should be addressed; e-mail: rene.cacan{at}univ-lille1.fr
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Abbreviations |
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AZC, L-azetidine-2-carboxylic acid; Bz-man, benzyl
-D-mannopyranoside; CH, cycloheximide; CHO, Chinese hamster ovary; DIM, 1,4-dideoxy-1,4-imino-D-mannitol; ER, endoplasmic reticulum; ERAD, ER-associated protein degradation; HPLC, high-performance liquid chromatography; LLO, lipid-linked oligosaccharide; OSGn1, oligosaccharides possessing one GlcNAc residue; OSGn2, oligosaccharides possessing two GlcNAc residues; PNGase, peptide N-glycanase |
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