Release of polymannose oligosaccharides from vesicular stomatitis virus G protein during endoplasmic reticulum–associated degradation

Mary Jane Spiro and Robert G. Spiro1

Departments of Medicine and Biological Chemistry, Harvard Medical School and the Joslin Diabetes Center, 1 Joslin Place, Boston, Massachusetts 02215, USA

Received on April 10, 2001; revised on May 14, 2001; accepted on May 16, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
To further explore the localization of the N-deglycosylation involved in the endoplasmic reticulum (ER)–associated quality control system we studied HepG2 cells infected with vesicular stomatitis virus (VSV) and its ts045 mutant, as in this system oligosaccharide release can be attributed solely to the VSV glycoprotein (G protein). We utilized the restricted intracellular migration of the mutant protein as well as dithiothreitol (DTT), low temperature, and a castanospermine (CST)-imposed glucosidase blockade to determine in which intracellular compartment deglycosylation takes place. Degradation of the VSV ts045 G protein was considerably greater at the nonpermissive than at the permissive temperature; this was reflected by a substantial increase in polymannose oligosaccharide release. Under both conditions these oligosaccharides were predominantly in the characteristic cytosolic form, which terminates in a single N-acetylglucosamine (OS-GlcNAc1); this was also the case in the presence of DTT, which retains the G protein completely in the ER. However when cells infected with the VSV mutant were examined at 15°C or exposed to CST, both of which represent conditions that impair ER-to-cytosol transport, the released oligosaccharides were almost exclusively (> 95%) in the vesicular OS-GlcNAc2 form; glucosidase blockade had a similar effect on the wild-type virus. Addition of puromycin to glucosidase-inhibited cells resulted in a pronounced reduction (> 90%) in oligosaccharide release, which reflected a comparable impairment in glycoprotein biosynthesis and indicated that the OS-GlcNAc2 components originated from protein degradation rather than hydrolysis of oligosaccharide lipids. Our findings are consistent with N-deglycosylation of the VSV G protein in the ER and the subsequent transport of the released oligosaccharides to the cytosol where OS-GlcNAc2 to OS-GlcNAc1 conversion by an endo-ß-N-acetylglucosaminidase takes place. Studies with the ts045 G protein at the nonpermissive temperature permitted us to determine that it can be processed by Golgi endomannosidase although remaining endo H sensitive, supporting the concept that it recycles between the ER and cis-Golgi compartments.

Key words: Vesicular stomatitis virus G protein/endoplasmic reticulum–associated degradation/polymannose oligosaccharide release/endomannosidase/castanospermine


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
It has been appreciated for some time that N-glycosylation of nascent proteins is accompanied by a substantial release of free polymannose oligosaccharides (Anumula and Spiro, 1983Go; Moore and Spiro, 1994Go; Duvet et al., 1998Go; Cacan and Verbert, 1999Go). This phenomenon was postulated to be a function of an endoplasmic reticulum (ER)–associated degradative process, which is involved in the quality control of glycoproteins (Moore and Spiro, 1994Go; Cacan and Verbert, 1999Go; Spiro, 2000Go). Indeed, it has become apparent that glycoproteins that fail to undergo folding and/or oligomerization are retained in the ER by chaperones (Hammond and Helenius (1995)Go and are believed in a number of instances to undergo proteasomal degradation after Sec61-mediated translocation (Wiertz et al., 1996bGo; Kopito, 1997Go; Pilon et al., 1997Go), although alternative routes of proteolysis within the ER have been identified and attributed to quality control (Ivessa et al., 1999Go). Because it is believed that removal of N-linked oligosaccharides precedes proteolytic degradation, deglycosylation through an N-glycanase must occur at an early stage in ER-associated degradation (ERAD) (Wiertz et al., 1996aGo; Kopito, 1997Go). In fact, N-glycanases, which could be involved in oligosaccharide removal from targeted proteins, have been found in the cytosol (Suzuki et al., 1998Go) and in the ER (Suzuki et al., 1997Go; Weng and Spiro, 1997Go) and although the physiological subcellular sites for oligosaccharide release have not yet been established, credible models for both locations have been proposed (Wiertz et al., 1996bGo; Karaivanova and Spiro, 2000Go).

The occurrence during radiolabeling experiments of substantial quantities of intravesicular free polymannose oligosaccharides in the OS-GlcNAc2 form, which appear to be chased into the cytosol where they can be rapidly converted to the OS-GlcNAc1 species by the endo-ß-N-acetylglucosaminidase (endo H) located in that compartment (Pierce et al., 1979Go; Weng and Spiro, 1997Go), supports the possibility that at least some N-deglycosylation takes place in the ER (Moore and Spiro, 1994Go). Irrespective of whether oligosaccharide release takes place in the ER or the cytosol, it would be important that free polymannose units or those still attached to proteins destined for degradation be segregated from the secretory pathway, where they could interfere with the processing of properly folded glycoproteins. Indeed, the Sec61 channel provides such an escape into the cytosol for intact glycoproteins (Kopito, 1997Go) and apparently also glycopeptides (Gillece et al., 2000Go), and a separate transport mechanism has been identified for free oligosaccharides (Moore et al., 1995Go). Ultimately, after conversion to the OS-GlcNAc1 form in the cytosol, further degradation by a mannosidase residing in this compartment takes place before entrance into the lysosomes for final disposition (Moore and Spiro, 1994Go; Saint-Pol et al., 1997Go).

In the present investigation we have further explored the localization of the oligosaccharide release associated with the protein quality control process by studying cells infected with vesicular stomatitis virus (VSV) and its ts045 mutant. This virus provides the distinct advantage of directing the cells to the formation of a single N-glycosylated protein, the VSV glycoprotein (G protein) (Hunt et al., 1978Go), and consequently in contrast to previous studies makes it possible to attribute oligosaccharide release to a distinct molecular entity. Moreover, the ts045 mutant, which because of improper folding is retained intracellularly at the nonpermissive temperature of 40°C (Balch et al., 1986Go) provides a tool for evaluating the subcellular site of degradation-related oligosaccharide release because it is believed to recycle between the ER and cis-Golgi (Hammond and Helenius, 1994Go) and can be retained exclusively in the ER compartment in the presence of DTT (De Silva et al., 1993Go; Verde et al., 1995Go).

In our studies we availed ourselves of the cytologically different distributions of the two groups of free polymannose oligosaccharides, namely, polymannose oligosaccharides terminating at their reducing end with N-acetylglycosamine or the di-N-acetylchitobiose moiety (OS-GlcNAc1 and OS-GlcNAc2, respectively), with the latter having been shown to occur within the ER vesicles and the former being found in the cytosol (Moore and Spiro, 1994Go; Duvet et al., 1998Go.; Ohashi et al., 1999Go; Karaivanova and Spiro, 2000Go). We also utilized a 6-O-butanoyl derivative of castansopermine (CST)–imposed glucosidase blockade, as well as dithiotreitol (DTT) and low temperature to localize the N-deglycosylation of the G protein. Employing these approaches we conclude that the release of polymannose oligosaccharides from this VSV glycoprotein occurs in the ER compartment. Furthermore, by observing that the G protein from the ts045 VSV mutant can be processed by the Golgi-situated endomannosidase, we have obtained evidence confirming that recycling takes place between the ER and cis-Golgi compartments.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Stability of the VSV G protein in HepG2 cells infected with the ts045 mutant at permissive and nonpermissive temperatures
To determine whether the release of free polymannose oligosaccharides is related to the extent of ER-associated glycoprotein quality control degradation, we chose to examine the G protein of the VSV ts045 mutant, which is known to be incompletely folded at nonpermissive temperature (De Silva et al., 1993Go; Hammond and Helenius, 1994Go). Indeed, during a chase of [35S]methionine-labeled cells at 40°C the degradation of the G protein as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of the cell lysates (Figure 1) was considerably greater (80%) than that noted at the permissive temperature (20%). Under none of these conditions was any radiolabeled virus detected in the incubation medium during the period of the chase.



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Fig. 1. Effect of temperature and DTT on the stability of the G protein produced by VSV ts045-infected HepG2 cells. After infection with the mutant VSV strain the cells on 6-well plates were radiolabeled with [35S]methionine (25 µCi) with or without DTT (5 mM) for 10 min at 40°C after preincubations as described in Materials and methods. This pulse (P) was followed by a 150-min chase (CH) at either 40°C or 32°C in the presence of unlabeled methionine. Equal aliquots of the cell lysates were examined by SDS–PAGE on 10% acrylamide gels. After visualization by fluorography the G protein (Mr = 70 kDa) was quantitated by densitometry and the average of duplicate experiments is shown by the bar graphs. The minor faster moving component is believed to represent the soluble (GS) protein (Schmidt et al., 1992Go).

 
Evaluation of the endo H sensitivity of the G protein under permissive and nonpermissive conditions
After radiolabeling of the ts045-infected cells with [2-3H]mannose, only a single component was observed that migrated to the position of the G protein (Mr = 70 kDa) by SDS–PAGE (Figure 2), which indicated that in this system, polymannose oligosaccharide release can indeed be attributed to a single well-defined glycoprotein. Although the endo H resistance of the [3H]mannose-labeled G protein found at 32°C indicated that it had migrated to medial vesicles of the Golgi apparatus, the protein produced at 40°C, as anticipated (Hammond and Helenius, 1994Go), did not reach this compartment as it remained sensitive to the action of this endoglycosidase (Figure 2). The [35S]methionine-labeled protein produced at 32°C was also endo H resistant (data not shown), whereas in the presence of DTT (Figure 2) it remained endo H sensitive, consistent with previous reports that protein unfolding brought about by the reducing agent causes its retention in the ER (De Silva et al., 1993Go; Verde et al., 1995Go).



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Fig. 2. Effect of temperature and DTT on the endo H susceptibility of the G protein from HepG2 cells infected with the VSV ts045 mutant. The virus-infected cells in 75-cm2 flasks were pulse radiolabeled with [2-3H]mannose (300 µCi) at 40°C for 20 min followed by a150-min chase at 40°C or 32°C in the presence of unlabeled mannose and glucose, as described in Materials and methods. Pulse labeling with [35S]methionine (10 min) followed by a 150-min chase at 32°C was performed in the presence of DTT as described in the legend to Figure 1. Aliquots of the cell lysates were analyzed by SDS–PAGE with (+) or without (–) prior endo H digestion. The migration of the G protein as well as the nonglycoprotein VSV constituents (N/NS and M) are indicated to the right of the gel.

 
Characterization and quantitation of the free polymannose oligosaccharides produced by VSV ts045-infected cells at permissive and nonpermissive temperatures
The free polymannose oligosaccharide content of cells chased at 40°C was found to be about twice as great as that observed at the permissive 32°C temperature (Figure 3D) and this correlated with the enhanced G protein degradation noted under the nonpermissive conditions (Figure 1). Examination of the migration of the oligosaccharides on thin-layer chromatography (TLC) with and without prior endo H treatment (Figure 3A) indicated that at both temperatures the components were predominantly in the OS-GlcNAc1 form (Figure 3D). These findings indicated that most of the free oligosaccharides were located in the cytosol as endo-ß-N-acetylglucosaminidase has been reported to be present in this compartment but absent from the ER (Pierce et al., 1979Go; Weng and Spiro, 1997Go) and indeed the OS-GlcNAc1 species have been found to be exclusively located in the cytosol (Moore and Spiro, 1994Go; Duvet et al., 1998Go; Ohashi et al., 1999Go; Karaivanova and Spiro, 2000Go) where rapid conversion of OS-GlcNAc2 to OS-GlcNAc1 has been demonstrated (Moore and Spiro, 1994Go; Duvet et al., 1998Go). Because the oligosaccharides observed in the presence of DTT at the permissive temperature were similar in nature and amount to those found in the absence of the reducing reagent, their release must occur in the ER rather than a more distal compartment as it is known that DTT prevents the exit of proteins from the ER.



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Fig. 3. Effect of temperature, glucosidase blockade and reducing agent on the nature and quantity of free oligosaccharides released by VSV ts045-infected HepG2 cells. (A) TLC examination of oligosaccharides released at nonpermissive (40°C) and permissive (32°C) temperatures and evaluation of the effect of DTT. After radiolabeling of the virus-infected cells with [2-3H]mannose (300 µCi) for 20 min at 40°C in the absence (–) or presence (+) of DTT (5 mM) they were chased at 40°C or 32°C with unlabeled substrate for 90 min as described in Materials and methods. The oligosaccharide fractions were resolved by TLC on silica gel–coated plates in Solvent System A without (–) or with (+) prior endo H treatment. The individual components were visualized by fluorography and their migration compared to radiolabeled standards. GN1 and GN2 refer to OS-GlcNAc1 and OS-GlcNAc2, respectively; the number of glucose (G) and mannose (M) residues present in each oligosaccharide is indicated by the subscripts. (B) Effect of low temperature on the free oligosaccharides produced by the infected cells. After pulse (P) radiolabeling of the cells at 40°C for 20 min as in (A), a 90-min chase was performed at 15°C with unlabeled substrate. TLC and fluorography were carried out on the oligosaccharides without or with prior endo H treatment as described in (A). (C) Nature of the free oligosaccharides formed by glucosidase-inhibited cells. Subsequent to radiolabeling of the cells as in (A) in the presence (+) of 0.2 mM CST, the cells were chased in medium still containing the inhibitor for 90 min at 40°C or 32°C. The free oligosaccharides were then resolved by TLC without (–) or with (+) prior endo H treatment as in (A) and detected by fluorography. (D) Quantitation of the free oligosaccharides and OS-GlcNAc1 species produced by VSV ts 045-infected cells under the conditions shown in (A, B, and C). The total free oligosaccharides formed at the indicated temperatures in the presence and absence of CST or DTT were expressed in molar terms by dividing the radioactivity of each component eluted after TLC resolution by the number of mannose residues it contains and making a summation of these in each sample. The values plotted represent these total moles expressed relative to the oligosaccharides formed at the permissive temperature (32°C) under control conditions without DTT (8.8 x 105 d.p.m. total oligosaccharides = 1.0). The percent of the total oligosaccharides as OS-GlcNAc1 was calculated by quantitating the difference of the oligosaccharide species before and after endo H treatment.

 
Effect of low temperature and CST on the nature of the released oligosaccharides
Because it has been shown that the deglycosylation step of glycoprotein degradation during quality control takes place through an N-glycanase action (Wiertz et al., 1996aGo), we sought to determine whether in the VSV-infected cells the formation of the OS-GlcNAc2 species resulting from this enzymatic action takes place in the ER or in the cytosol. For this purpose we chased cells at 15°C or alternatively incubated them in the presence of CST, both of which represent conditions that would be expected to reduce or prevent oligosaccharide transport from ER to cytosol and result in their being trapped in the former compartment. When the cells subsequent to radiolabeling at 40°C were chased at 15°C, TLC of the free oligosaccharides with and without prior endo H treatment (Figure 3B) indicated that they started as and remained predominantly (> 95%) in the OS-GlcNAc2 form (Figure 3D). This would be consistent with their release in the ER during the 40°C pulse (Figure 3B) and their subsequent decreased transport at this low temperature due to reduced availability of adenosine triphosphate (ATP) (Klingenberg et al., 1982Go), which is required for oligosaccharide transport (Moore et al., 1995Go) and/or a reduced rate of ER transmembrane movement (Römisch and Schekman, 1992Go). Their retention in the OS-GlcNAc2 form indicates their failure to reach the cytosol where the endo-ß-N-acetylglucosaminidase is situated.

Incubation of the cells in the presence of a CST-imposed glucosidase blockade resulted in the appearance of triglucosylated oligosaccharides that were exclusively in the OS-GlcNAc2 form irrespective of temperature or the presence of DTT (Figure 3C and D). This finding is consistent with the report that triglucosylated oligosaccharides cannot be transported through the ER membrane (Moore et al., 1995Go), which would result in their retention in this compartment and again is indicative of an ER N-glycanase action. The total amount of oligosaccharides formed in the presence of CST was very much enhanced over controls at 32°C (Figure 3D), as anticipated from several studies showing that the glucosidase inhibition accelerates glycoprotein degradation (Moore and Spiro, 1993Go; Kearse et al., 1994Go; Keller et al., 1998Go). Examination by SDS–PAGE of the [35S]methionine-labeled G protein in the presence of CST also showed a substantial increase in degradation at the permissive temperature (data not shown). At 40°C the stimulated oligosaccharide formation was not evident, nor was there an increase in the already pronounced degradation of the misfolded G protein.

Effect of glycosidase inhibitors on oligosaccharide release in HepG2 cells infected with wild type VSV
To determine whether the effect of CST in bringing about a retention of oligosaccharides exclusively in the OS-GlcNAc2 form was a function solely of the G protein produced by the ts045 mutant, we examined the effect of glycosidase inhibitors on HepG2 cells infected with the wild-type virus. Although the released oligosaccharides in control incubations and in the presence of kifunensine (KIF), a mannosidase inhibitor (Elbein, 1991Go), were predominantly in the OS-GlcNAc1 form (Figure 4, lanes 5 and 6), the further addition of CST resulted in the appearance of exclusively OS-GlcNAc2 species (Figure 4, lane 7), similar to the observation made in cells infected with the mutant virus (Figure 3D). Indeed the failure of KIF to arrest processing of the OS-GlcNAc1 components (Figure 4, lane 6) is consistent with their presence in the cytosol where a mannosidase resistant to this inhibitor resides (Weng and Spiro, 1996Go).



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Fig. 4. Evaluation of the effect of glycosidase inhibitors and puromycin on the formation of free oligosaccharides and G protein N-linked polymannose oligosaccharides by HepG2 cells infected with the wild-type VSV. The infected cells were radiolabeled with [2-3H]mannose (300 µCi) at 37°C for 25 min in the absence (–) or presence (+) of 0.2 mM CST, 0.1 mM KIF, and 0.2 mM puromycin (PM) for 25 min at 37°C followed by a 70-min chase as described in Materials and methods. The inhibitors were present throughout the incubations. The oligosaccharides released from the Pronase digested glycoprotein (GP) by endo H as well as those present in the free oligosaccharide fraction (OS) without endo H treatment were analyzed by TLC on a silica gel–coated plate in solvent system A. The components were detected by fluorography and their migration compared to radiolabeled standards; the abbreviations are the same as in Figure 3.

 
Effect of puromycin on the release of oligosaccharides in virus-infected cells
Since the release of oligosaccharides in the OS-GlcNAc2 form could be a function of both N-glycanase action on the proteins undergoing quality control as well as cleavage of oligosaccharide-lipids through the hydrolase action of the oligosaccharyltransferase (Spiro and Spiro, 1991Go), we employed puromycin to assess the contribution of each of these two possibilities. Addition of this agent to the glycosidase-inhibited cell resulted in a pronounced reduction (91%) of the oligosaccharide release (Figure 4, lane 8), which reflected the decrease in glycoprotein formation (Figure 4, lane 4) although no change in the oligosaccharide-lipid level was noted (data not shown).

Evaluation of whether the VSV ts045-infected cells can utilize the endomannosidase deglucosylation pathway in processing the G-protein
An examination by TLC of the endo H released oligosaccharides from the glycoprotein of the wild-type VSV-infected HepG2 cells incubated in the presence of CST and KIF revealed the pattern characteristic of endomannosidase action (Figure 4, lane 3) in which the Man9GlcNAc processing stage is circumvented with the appearance of large amounts of Man8GlcNAc and smaller quantities of Man7GlcNAc from Glc3Man9GlcNAc and Glc3Man8GlcNAc, respectively (Moore and Spiro, 1990Go).

As it has been proposed (Hammond and Helenius, 1994Go) that the G protein of the ts045 mutant recycles between the ER and cis-Golgi compartments at the nonpermissive temperature (40°C), we examined the N-linked processing intermediates formed in the VSV mutant–infected cells in the presence of CST and KIF. It was evident from the TLC pattern that in the presence of these glycosidase inhibitors glucose removal was achieved by endomannosidase action as Man8GlcNAc but not Man9GlcNAc could be seen (Figure 5A) and therefore indicated that even at the nonpermissive temperature some of the G protein did reach the Golgi compartment. When DTT was further added to the incubation to retain the protein in the ER and prevent its access to the endomannosidase, only the triglucosylated N-linked oligosaccharides were seen (Figure 5A).



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Fig. 5. Evaluation of the processing of G protein N-linked oligosaccharides by the endomannosidase route in VSV ts045-infected HepG2 cells at permissive (32°C) and nonpermissive (40°C) temperatures in the absence and presence of DTT. After pulse-chase radiolabeling with [2-3H]mannose of the viral infected cells, as described in Figure 3, in the presence of (A) CST (0.2 mM) plus KIF (0.1 mM) or (B) CST without KIF, with or without DTT (5 mM) at 32°C and 40°C, equal aliquots of the polymannose oligosaccharides released by endo H treatment from the delipidated protein (A) as well as of the free oligosaccharides (B) were resolved by TLC as described in Materials and methods. To separate the small molecular size oligosaccharide products of endomannosidase action (B) from the larger components remaining near the origin, chromatography was carried out on a cellulose-coated plate in Solvent System B, whereas the resolution of the endo H-released polymannose oligosaccharides (A) was achieved on a silica-coated plate in Solvent System A. The components were visualized by fluorography and their migration compared to radiolabeled standards. The abbreviations are the same as in Figure 3 and in addition G3M designates Glc3Man.

 
High-performance liquid chromatography (HPLC) analysis of the aminopyridine derivatives of the Man8GlcNAc formed indicated that although this product was predominantly isomer B in the absence of CST and KIF, in the presence of these glycosidase inhibitors the A-isomer was almost exclusively formed at both the permissive and nonpermissive temperatures (Table I), as would be expected by endomannosidase action on the glucose carrying branch of the N-linked oligosaccharide (Moore and Spiro, 1990Go). Examination of the products of acetolysis fragmentation of the Man8GlcNAc confirmed these findings (data not shown).


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Table I. Nature of the Man8GlcNAc isomers occurring on the G protein of VSV ts045 infected cells during N-linked oligosaccharide processing under various conditionsa
 
To further document the operation of the endomannosidase-mediated deglucosylation route in ts045 VSV-infected cells, incubations were carried out in the presence of CST alone to determine if the characteristic Glc3Man product of this pathway is evident. TLC analyses clearly indicated that at both permissive and nonpermissive temperature Glc3Man was indeed formed (Figure 5B); moreover these findings paralleled those observed in Figure 5A by indicating that in the presence of DTT the endomannosidase catalyzed deglucosylation does not occur.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The present study has shown that at 40°C the radiolabeled G protein formed by HepG2 cells infected with the temperature sensitive ts045 mutant of VSV undergoes a more rapid destruction than at the permissive temperature (32°C) and remains endo H sensitive, indicating its failure to interact with the processing enzymes of the medial Golgi compartment. Some of this misfolded protein must, however, reach the cis-Golgi vesicles, because in the presence of CST glucose removal could be achieved through the action of endomannosidase, a Golgi processing enzyme that is not present in the ER (Lubas and Spiro, 1987Go) and that has been immunohistochemically localized to the cis/medial cisternae (Zuber et al., 2000Go); this observation provides confirmation of the ER-Golgi recycling model proposed by Hammond and Helenius (1994)Go.

Because only the G protein becomes radiolabeled with [2-3H]mannose on incubation of the VSV ts045-infected HepG2 cells, oligosaccharide release in the present investigation can be attributed exclusively to this one glycoprotein. The increased quantity of oligosaccharides released at 40°C reflected the enhanced degradation of this molecule at the nonpermissive temperature. Moreover, in the presence of CST at the permissive temperature a pronounced increase in oligosaccharide formation accompanied the accelerated G protein degradation. Enhanced glycoprotein destruction resulting from glucosidase blockade has been previously noted (Moore and Spiro, 1993Go; Kearse et al., 1994Go; Keller et al., 1998Go) and has been attributed to the failure of the untrimmed triglucosyl sequence to interact with calnexin and calreticulin (Hammond and Helenius, 1995Go; Ware et al., 1995Go; Spiro et al., 1996Go),

The most striking finding noted in the infected cells incubated in the presence of CST was the occurrence at both 32°C and 40°C of essentially all of the oligosaccharides in the OS-GlcNAc2 form, which stands in marked contrast to the situation without glucosidase inhibition in which the predominant components belonged to the OS-GlcNAc1 family. Because it has been reported that triglucosylated polymannose oligosaccharides cannot be transported through the ER membrane (Moore et al., 1995Go) while glycopeptides containing such glucosylated carbohydrate units still linked to peptide can be exported (Ali and Field, 2000Go; Suzuki and Lennarz, 2000Go) we believe that our observations indicate that N-deglycosylation of the G protein takes place in the ER compartment. Failure of the glucosylated oligosaccharides to reach the cytosol where the endo-ß-N-acetylglucosaminidase resides (Pierce et al., 1979Go; Weng and Spiro, 1997Go) would prevent conversion of the OS-GlcNAc2 species to the OS-GlcNAc1 form.

Our observation that the oligosaccharides released during radiolabeling at 40°C remain almost exclusively in the OS-GlcNAc2 form after a chase at 15°C, presumably due to a deficiency in ATP or a reduction in the rate of ER to cytosolic transport, again is consistent with the contention that N-deglycosylation products of the G protein are formed within the ER. Indeed, both glucosidase blockade and low-temperature incubation have provided us with an approach by which the released oligosaccharides can be trapped in the ER.

Although OS-GlcNAc2 components can be generated from oligosaccharide lipids by the hydrolytic action of the oligosaccharyltransferase as well as by N-glycanase action on glycoproteins, our studies with puromycin on glucosidase-inhibited cells indicated that the latter route is predominant because inhibition of glycoprotein synthesis reduced oligosaccharide release to minimal amounts, as was previously noted in thyroid cells without glucosidase inhibition (Spiro and Spiro, 1991Go). Indeed hydrolase activity toward oligosaccharide-lipids has only been clearly demonstrated in vesicular systems in which glycoprotein synthesis is negligible (Spiro and Spiro, 1991Go).

In the present investigation we are in a position to attribute the oligosaccharide release associated with quality control to a single glycoprotein in contrast to previous studies (Moore and Spiro, 1994Go; Villers et al., 1994Go; Duvet et al., 1998Go; Karaivanova and Spiro, 2000Go) in which a large number of N-glycosylated proteins were being produced concurrently. Although N-glycanases, which could potentially be involved in oligosaccharide removal during ERAD, have been found in soluble (Suzuki et al., 1998Go), ER-enriched (Suzuki et al., 1997Go), and purified ER (Weng and Spiro, 1997Go) cell fractions, the physiological sites of the deglycosylation step have remained unclear. Models in which the N-glycanase is relegated to a cytosolic (Wiertz et al., 1996bGo) or alternatively to an ER location (Karaivanova and Spiro, 2000Go) have been proposed. Because neither fully accounts for all the experimental observations, the location of the deglycosylation step may differ depending on the glycoprotein undergoing degradation. In the present investigation the glucosylated polymannose carbohydrate units released from the G protein during glucosidase blockade were present only as OS-GlcNAc2, presumably due to the specificity of the ER-cytosolic transport mechanism (Moore et al., 1995Go), whereas in previous studies in which a mixture of glycoproteins were studied in this cell line (Moore and Spiro, 1994Go) triglucosylated OS-GlcNAc1 components were observed, suggesting that export from the ER occurred while the carbohydrate was still attached to the peptide moiety by a transporter that is not sensitive to the fine structure of the oligosaccharide (Ali and Field, 2000Go; Suzuki and Lennarz, 2000Go). Though triglucosylated OS-GlcNAc1 components were also observed in a CST-treated mouse lymphoma cells line, it was evident that substantial amounts of such glucosylated polymannose oligosaccharides remained inside the ER in the OS-GlcNAc2 form (Karaivanova and Spiro, 2000Go) consistent with the concept that some oligosaccharides were cleaved from glycoproteins in that compartment while others were transported into the cytosol in their peptide-linked form with their subsequent release and conversion to OS-GlcNAc1.

The study with lymphoma cells furthermore demonstrated that when protein degradation was decreased by proteasomal inhibitors there was a substantial reduction in the cytosolic OS-GlcNAc1 components while the vesicular OS-GlcNAc2 species remained unaffected (Karaivanova and Spiro, 2000Go), indicating that only the formation of the former was coupled to proteasomal activity. In the present investigation when the infected HepG2 cells were examined under similar conditions of proteasomal inhibition, no effect on oligosaccharide release or G protein degradation was observed (data not shown), suggesting that at least the initial steps in the quality control machinery responsible for degradation of this particular glycoprotein are located in the ER itself. Although protein retrotranslocation into the cytosol followed by proteasomal degradation has been well documented (Wiertz et al., 1996bGo; Kopito, 1997Go; Pilon et al., 1997Go), alternate routes for quality control involving proteolysis in the ER have been suggested for a variety of glycoproteins (Wu et al., 1997Go; Moriyama et al., 1998Go; Loo and Clarke, 1998Go; Ivessa et al., 1999Go; Fayadat et al., 2000Go).

The G protein produced by the ts045 VSV mutant has in the past proved to be a valuable model for the investigation of the folding and migration of newly synthesized glycoproteins (Balch et al., 1986Go; De Silva et al., 1993Go; Presley et al., 1997Go) and the present report suggests that it is also useful in studying protein N-deglycosylation and proteolysis involved in ERAD.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Cell culture and virus infection
HepG2 cells obtained from ATCC (Rockville, MD) were grown in RPMI-1640 (Gibco) supplemented with 10% fetal bovine serum (FBS) and containing streptomycin (100 µg/ml) and penicillin (100 U/ml) at 37°C in an atmosphere of 95% air and 5% CO2.

Infection with VSV (Indiana strain, ATCC) or its ts045 mutant (a gift from Dr. William E. Balch, Scripps Research Institute, La Jolla, CA) was carried out on cell monolayers in 75-cm2 flasks or 6-well plates. The virus was added to the cells in growth medium containing 20 mM HEPES, pH 7.4, buffer, followed by 90-min incubations at 32°C or room temperature for the mutant and wild-type respectively. After removal of the virus, growth medium was added for further incubation for 16 h at 32°C for the mutant-infected cells or 5 h at 37°C for the cells containing the wild-type virus.

Radiolabeling of cells
Prior to radiolabeling with [2-3H]mannose (20 Ci/mmol, DuPont-NEN), the virus-infected cells in 75-cm2 flasks were washed twice with glucose-free medium (Sigma) containing 1 mM pyruvate, 20 mM HEPES, pH 7.4, and 2% (v/v) dialyzed FBS, followed by preincubation in 2 ml of this medium for 30 min at 37°C with or without 0.2 mM CST (a gift from Dr. M. Kang, Merrell Dow Research Institute, Cincinnati, OH) or 0.2 mM CST plus 0.1 mM KIF (Toronto Research Chemicals) with or without puromycin (0.2 mM).

In the case of the VSV mutant-infected cells, the flasks were then equilibrated with or without DTT (5 mM) at 40°C in the water bath for 5 min prior to addition of 300 µCi of the [2-3H]mannose for a 20-min pulse at this temperature to retain the G protein in the ER during radiolabeling. At the end of this time unlabeled glucose (5 mM) and mannose (2 mM) were added and the cells were then chased at 40°C, 32°C, or 15°C. Radiolabeling of cells infected with the wild-type VSV were carried out in the same manner except that all incubations were performed at 37°C.

For radiolabeling with [35S]methionine (25 µCi, 1198 Ci/mmol, DuPont-NEN) VSV ts045-infected cells on 6-well plates were washed with methionine-free medium (Sigma) containing 20 mM HEPES, pH 7.4, and 2% (v/v) dialyzed FBS followed by a 30-min preincubation at 37°C in 0.5 ml of this medium. After equilibration for 5 min at 40°C with or without 5 mM DTT the [35S]methionine was added for a 10-min pulse. Subsequently unlabeled methionine (2 mM) was added for chases at 40°C or 32°C.

The radiolabeled cells were washed with phosphate buffered saline (PBS) containing unlabeled substrate, phenylmethylsulfonyl fluoride (1 mM) and aprotinin (10 U/ml). The cells were dislodged by scraping in the PBS solution and recovered by centrifugation (10 min at 600 x g). The [35S]methionine-radiolabeled pellets were lysed at 4°C in a 100 mM NaMES, pH 6.5, buffer, containing 400 mM NaCl, 2% (v/v) Triton X-100 and a mixture of protease inhibitors (5 mM EDTA, 10 µg/ml leupeptin, 10 U/ml aprotinin, 10 mM iodoacetamide, and 2 mM phenylmethylsulfonyl fluoride). After centrifugation (14,000 x g for 20 min) in an Eppendorf model 5415C microcentrifuge the clear supernatant was submitted to electrophoretic examination.

For the isolation of free oligosaccharides, glycoproteins and oligosaccharide-lipids, the [2-3H]mannose-labeled cells were extracted with a 3:2:1 (v/v/v) mixture of chloroform:methanol:0.15 M Tris–HCl, pH 7.4, buffer containing 4 mM MgCl2 (Spiro et al., 1976Go). To obtain the neutral oligosaccharides, the upper phase of this extract, after evaporation of the organic solvent, was fractionated by Dowex 50 (H+)-Dowex 1 (acetate) and charcoal-Celite chromatography (50% ethanol eluate) in a manner similar to that previously described (Moore and Spiro, 1994Go). After extraction of the oligosaccharide-lipids from the interphase material with chloroform:methanol:water (10:10:3) the delipidated protein pellet was digested with Pronase to obtain glycopeptides (Moore and Spiro, 1990Go) and the latter were then digested with endo H to release polymannose oligosaccharides, which were recovered in the effluent and water wash from coupled Dowex 50 (H+) and Dowex 1 (acetate) columns (Moore and Spiro, 1990Go). An aliquot of the unextracted [2-3H]mannose cells was also taken for electrophoretic examination with and without prior endo H digestion.

TLC
Chromatography of large free oligosaccharides as well as those released from glycopeptides by endo H digestion was carried out on plastic sheets coated with Silica Gel 60 (0.2 mm thickness, Merck) in 1-propanol:acetic acid:water, 3:3:2 (Solvent System A) while resolution of small oligosaccharides and acetolysis fragments was achieved on cellulose-coated plastic sheets (0.1 mm thickness, Merck) in pyridine:ethyl acetate:water:acetic acid, 5:5:3:1 (Solvent System B). A wick of Whatman 3 MM paper was clamped to the thin-layer plates during chromatography. The components were detected by fluorography and quantitated by scintillation counting after elution with water. Radiolabeled oligosaccharide standards were prepared as previously described (Lubas and Spiro, 1988Go).

Characterization of Man8GlcNAc isomers
The radiolabeled Man8GlcNAc component released from the glycopeptides by endo H digestion was eluted subsequent to preparative TLC in solvent System A and after coupling to 2-aminopyridine by reductive amination was resolved into its three isomers by HPLC as described by Moore and Spiro (1994)Go. Alternatively, the eluted oligosaccharides after reduction with NaBH4 were subjected to acetolysis as previously reported (Lubas and Spiro, 1987Go) and the desalted fragments were identified by TLC.

Endo H digestion
Cellular glycoproteins were incubated with endo H (4 mU, Genzyme, Cambridge, MA) as previously described (Spiro and Spiro, 2000Go) prior to SDS–PAGE. Glycopeptides as well as purified free oligosaccharides were treated with the enzyme followed by desalting by passage through Dowex 50 (H+) and Dowex 1 (acetate) (Moore and Spiro, 1990Go). TLC of the free oligosaccharides with and without endo H treatment served to distinguish OS-GlcNAc1 from OS-GlcNAc2 components (Anumula and Spiro, 1983Go).

SDS–PAGE
Electrophoresis of radiolabeled proteins was carried out by the procedure of Laemmli (1970)Go on 10% polyacrylamide gels (1.5 mm thick) that were overlaid by 3.5% stacking gels; the radioactive components were detected by fluorography.

Radioactivity measurements
Liquid scintillation counting was carried out with Ultrafluor (National Diagnostics) with a Beckman LS7500 instrument. Detection of radioactive components on thin-layer plates was accomplished by fluorography at –80°C after treatment with ENHANCE (DuPont-New England Nuclear) using X-Omatic film. The bands were quantified by densitometry using a Model 300A Molecular Dynamics Densitometer (Sunnyvale, CA).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This work was supported by Grant DK17477 from the National Institutes of Health. We thank Vishnu Bhoyroo for help in some aspects of this investigation.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
ATP, adenosine triphosphate; CST, 6-O-butanoyl derivative of castanospermine; DTT, dithiothreitol; endo H, endo-ß-N-acetylglucosaminidase; ER, endoplasmic reticulum; ERAD, ER-associated degradation; FBS, fetal bovine serum; G protein, VSV glycoprotein; HPLC, high-performance liquid chromatography; KIF, kifunensine; MES, 2-(N-morpholino)ethanesulfonic acid; OS-GlcNAc1 and OS GlcNAc2, polymannose oligosaccharides terminating at their reducing end with N-acetylglucosamine or the di-N-acetylchitobiose moiety, respectively; PBS, phosphate buffered saline; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TLC, thin-layer chromatography; VSV, vesicular stomatitis virus.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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