Effect of proteasome inhibitors on the release into the cytosol of free polymannose oligosaccharides from glycoproteins

Velislava K. Karaivanova1 and Robert G. Spiro2

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

Received on December 10, 1999; revised on January 27, 2000; accepted on January 28, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Prompted by previous observations which suggested that the release of polymannose oligosaccharides shortly after the cotranslational N-glycosylation of proteins is a function of the ER-associated quality control system (Moore and Spiro (1994)Go J. Biol. Chem., 269, 12715–12721), we evaluated the effect which proteasome inhibitors have on the appearance of these free saccharide components. Employing as a model system castanospermine-treated BW5147 mouse T-lymphoma cells in which accelerated degradation of the T-cell receptor (TCR) {alpha} subunit takes place (Kearse et al. (1994)Go EMBO J., 13, 3678–3686), we noted that both lactacystin and N-acetyl-L-leucyl-L-leucyl-L-norleucinal, but not leupeptin, brought about a rapid and substantial reduction in the release of free polymannose oligosaccharides into the cytosol during pulse-chase studies, while the oligosaccharides in the intravesicular compartment remained unchanged, as measured by streptolysin O permeabilization. This inhibition was furthermore selective in that it affected solely the components terminating in a single N-acetylglucosamine residue (OS-GlcNAc1) and not the oligosaccharides terminating in a di-N-acetylchitobiose sequence (OS-GlcNAc2), which reside primarily in the intravesicular compartment. Despite the quantitative effect of the proteasome inhibitors on the cytosolic oligosaccharides, the molar distribution of the triglucosyl OS-GlcNAc1 species was unaffected. The decrease in cytosolic oligosaccharides brought about by proteasome inhibition was reflected in a pronounced increase in the stability of the TCR{alpha} subunit. Our findings suggest that the N-deglycosylation and proteasome mediated degradation are coupled events. On the basis of our data and those of others we propose that the quality control mechanism involves proteasomes associated with the cytosolic side of the endoplasmic reticulum acting in concert with a membrane situated N-glycanase. Such a complex by removing the carbohydrate units could facilitate the retrograde ER to cytosol translocation of glycoproteins.

Key words: proteasome inhibitors/polymannose oligosaccharide release/quality control of glycoproteins/ T-lymphocytes/endoplasmic reticulum/cytosol


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
It has become apparent in recent years that a highly sophisticated ER-situated quality control mechanism for glycoproteins exists in eukaryotic cells in which the N-linked oligosaccharides of these molecules play a major role. Indeed, it has been shown that the monoglucosylated polymannose carbohydrate units which are formed shortly after cotranslational N-glycosylation and processing by glucosidases I and II interact in a lectin-like manner with the molecular chaperones calnexin or calreticulin until proper folding and/or oligomerization of the proteins takes place (Helenius et al., 1997Go). This quality control system appears to be monitored by a deglucosylation-reglucosylation cycle involving glucosidase II and UDP-Glc:glycoprotein glucosyltransferase (Sousa et al., 1992Go) in which the former enzyme only prevails when the proper folding and/or assembly has occurred (Helenius et al., 1997Go). If genetic or environmental conditions interfere with proper folding or cause a deficiency in the formation of all of the subunits required for a multipeptide complex, an ER-associated degradation of the peptide appears to take place (Klausner and Sitia, 1990Go; Hammond and Helenius, 1995Go). Furthermore, it has been reported for a number of proteins, including major histocompatibility complex class I heavy chain (Moore and Spiro, 1993Go), TCR {alpha}-chain (Kearse et al., 1994Go), nicotinic acetylcholine receptor {alpha}-subunit (Keller et al., 1998Go), and apolipoprotein B (Chen et al., 1998Go), that inhibition of ER glucosidase action by CST greatly accelerated their degradation presumably due to the failure of the untrimmed triglucosyl sequence to interact with the lectin-like molecular chaperones (Ware et al., 1995Go; Spiro et al., 1996Go).

The nonlysosomal proteolytic system which functions in quality control has now been shown to be primarily represented by the action of proteasomes with or without prior ubiquitination (Orlowski, 1990Go; Rivett, 1993Go; Hilt and Wolf, 1996Go) and it has been proposed that degradation takes place subsequent to the retrotranslocation of the protein from the luminal to the cytosolic side of the ER (Wiertz et al., 1996bGo). Since removal of N-linked oligosaccharides would have to precede or accompany proteasomal degradation of the proteins, it has been postulated that the free polymannose oligosaccharides which are released into the cytosol shortly after cotranslational carbohydrate attachment are the products of this ER-associated quality control process (Moore and Spiro, 1994Go). Indeed, N-glycanases which are probably involved in the deglycosylation of proteins destined for degradation have been found in both ER (Suzuki et al., 1997Go; Weng and Spiro, 1997Go) and cytosolic (Kitajima et al., 1995Go; Suzuki et al., 1998Go) locations and furthermore endo-ß-N-acetylglucosaminidase (Pierce et al., 1979Go) and chitobiase (Cacan et al., 1996Go) are known to be present in the latter site.

It was the purpose of the present study to explore the relationship between the release of the free polymannose oligosaccharides and the ER-associated protein degradation by determining the effect of proteasome inhibitors on the former event. For this purpose we employed as a model system CST-treated BW5147 mouse T-lymphoma cells for which the studies of Kearse et al. (1994)Go demonstrated that a rapid degradation of TCR{alpha} subunit takes place. Our findings clearly indicate that in the presence of these agents there was a substantial reduction in the appearance of oligosaccharides in the cytosol and this decrease was limited to the OS-GlcNAc1 species in contrast to the unchanged level of the primarily intravesicularly situated OS-GlcNAc2 components. These observations suggest that N-deglycosylation and proteasome-mediated degradation of proteins are coupled events.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Effect of proteasome inhibitors on CST-treated BW5147 mouse T-lymphoma cells
In order to determine if the release of free polymannose oligosaccharides during the initial stages of glycoprotein biosynthesis can be influenced by inhibitors of ER-associated proteasomal degradation, we used as an experimental model BW5147 cells incubated in the presence of CST. The choice of this system was guided by the finding of Kearse et al. (1994)Go that the {alpha}-subunits of the TCR complex which are defectively assembled in this cell line (Lippincott-Schwartz et al., 1988Go) are rapidly degraded in the presence of a glucosidase inhibitor because of the absence of monoglucosylated N-linked polymannose oligosaccharides to interact with calnexin or calreticulin (Van Leeuwen and Kearse, 1996Go).

When the cells were incubated with the proteasomal inhibitor ALLN ( Rock et al., 1994Go; Ward et al., 1995Go; Hughes et al., 1996Go) or lactacystin (Ward et al., 1995Go; Dick et al., 1996Go), a substantial decrease in the total free neutral polymannose oligosaccharides was observed after [3H]mannose labeling followed by a 30-min chase although the oligosaccharide-lipid level was essentially unchanged (Table I). In contrast, the inclusion in the incubation of the lysosomal protease inhibitor leupeptin (Hyman and Froehner, 1983Go; Renfrew and Hubbard, 1991Go) did not result in a reduction of oligosaccharide release (Table I).


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Table I. Effect of several protease inhibitors on free polymannose and lipid-linked oligosaccharides in CST-treated BW5147 mouse T-lymphoma cellsa
 
Effect of proteasome inhibitors on the distribution of OS-GlcNAc1 and OS-GlcNAc2
When the released oligosaccharides were separated into their OS-GlcNAc1 and OS-GlcNAc2 families, it became apparent that the decrease in the total oligosaccharide level brought about by the proteasomal inhibitors was exclusively due to a substantial reduction in the former group (Table II). Since it has been previously reported that the OS-GlcNAc1 species formed in the presence or absence of CST are present predominantly in the cytosol while the oligosaccharides terminating in the di-N-acetylchitobiose moiety remain primarily inside the vesicles (Moore and Spiro, 1994Go), we evaluated the effect of proteasomal inhibitors on the intracellular location of the free oligosaccharides.


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Table II. Effect of proteasome inhibitors on the nature of the free polymannose oligosaccharides released by CST-treated BW5147 mouse T-lymphoma cellsa
 
Intracellular distribution of free oligosaccharides in the presence of proteasome inhibitors
Examination of the oligosaccharides present in the cytosolic and vesicular compartments of radiolabeled BW5147 cells indicated that in the presence of ALLN or lactacystin there was a pronounced decrease in the cytosolically situated components while the vesicular constituents showed only a minimal reduction (Table III). The inclusion of leupeptin in the incubations did not effect either the cytosolic or vesicular oligosaccharide content (Table III) as anticipated from the lack of effect which this lysosomal protease inhibitor had on the total free oligosaccharides (Table I). The specificity of the SLO permeabilizing agent for the plasma membrane was confirmed by the finding that the Glc3Man tetrasaccharide product of endomannosidase action was exclusively confined to the vesicular fraction of the BW5147 cells (data not shown) as was previously observed in HepG2 cells (Moore and Spiro, 1994Go).


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Table III. Effect of proteasome inhibitors and leupeptin on the distribution of free polymannose oligosaccharides between cytosolic and vesicular components of CST-treated mouse T-lymphoma cellsa
 
The time course for the appearance of the radiolabeled oligosaccharide in the cytosol and the effect of ALLN on this process indicated that a maximum was reached in a 30-min chase (Figure 1). At each of the time periods examined the proteasomal inhibitor brought about a substantial reduction in the cytosolic components (Figure 1). The radiolabel associated with the vesicular oligosaccharides remained unaffected by the proteasomal inhibitor at 15 min and 45 min chase time in a manner already shown for the 30 min chase (Table III).



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Fig. 1. Effect of ALLN on the release of free glucosylated polymannose oligosaccharides into the cytosol by CST-treated mouse T-lymphoma cells as a function of time. After a 30-min preincubation with 6-O-butanoyl-CST (0.2 mM) followed by a 15-min incubation with or without ALLN (120 µM), the cells were radiolabeled with [2-3H]mannose (100 µCi) followed by a chase of various lengths of time. After permeabilization with SLO the radioactivity in the cytosolic oligosaccharides was determined as described in Materials and methods. The hatched bars represent incubation conducted in the presence of ALLN, while the stippled bars indicate control incubations.

 
Nature of the cytosolic oligosaccharides
Thin layer chromatographic examination of the oligosaccharides present in the cytosol after a 30-min chase indicated that they were present predominantly in the triglucosylated OS-GlcNAc1 form (Figure 2), as previously noted in CST-treated HepG2 cells (Moore and Spiro, 1994Go). The chromatographic patterns indicated that the presence of lactacystin or ALLN in the incubations resulted in a decrease in cytosolic oligosaccharides while leupeptin had no effect (Figure 2) and this was consonant with the data presented in Table III. The observation that the oligosaccharide species were almost exclusively larger than Glc3Man6 can be attributed to the fact that the chase, during which processing of the unsubstituted branches of the polymannose unit can take place (Moore and Spiro, 1994Go) was relatively short.



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Fig. 2. Effect of proteasome inhibitors and leupeptin on the free triglucosyloligosaccharides appearing in the cytosol of CST-treated mouse BW5147 T-lymphoma cells. After a 30-min preincubation with 6-O-butanoyl-CST (0.2 mM) followed by a 15-min incubation without (-) or with lactacystin (48 µM), ALLN (120 µM) or leupeptin (120 µM), respectively, the cells were radiolabeled with [2-3H]mannose (75 µCi) for 15 min followed by a 30-min chase as described in Materials and Methods. Subsequent to permeabilization with SLO, equal aliquots of the deproteinized and desalted cytosolic oligosaccharide families were resolved by thin layer chromatography on a silica gel–coated plate in 1-propanol/acetic acid/water, 3:3:2 for 26 h; the components were visualized by fluorography. The migration of standard oligosaccharides are indicated on the left of the chromatograms. The abbreviations are G3M9, Glc3Man9; G3M8, Glc3Man8; G3M7, Glc3Man7; oligosaccharides terminating in GlcNAc1 and di-N-acetylchitobiose are indicated by GN1 and GN2, respectively.

 
While the proteasome inhibitors reduced the total amount of cytosolic oligosaccharides, the molar distribution of the oligosaccharide species remained unaffected (Table IV).


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Table IV. Effect of proteasome inhibition on the molar distribution of free oligosaccharides released into the cytosol by CST-treated mouse T-lymphoma cellsa
 
Effect of proteasome inhibition on the stability of TCR{alpha} chains
In order to assess whether the decrease in oligosaccharide release brought about by proteasome inhibition reflected a decrease in glycoprotein degradation we examined by PAGE the TCR{alpha} subunit immunoprecipitates subsequent to pulse-chase [3H]mannose labeling of CST-treated BW5147 cells. As previously noted by Kearse et al. (1994)Go with [35S]methionine, there was a substantial degradation of this protein during a 30-min chase (Figure 3). This instability was obviated in the presence of lactacystin (Figure 3); a similar stabilizing effect was noted with ALLN (data not shown).



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Fig. 3. Effect of lactacystin on the stability of TCR{alpha} chain from CST-treated BW5147 mouse T-lymphoma cells. After a 30 min preincubation with 6-O-butanoyl-CST (0.2 mM) followed by a 15 min incubation without (-) or with (+) lactacystin (48 µM) the cells were pulsed for 15 min with [2-3H]mannose (200 µCi) followed by a 30-min chase as described in Materials and methods. Subsequent to treatment with the lysis buffer, equal aliquots (30%) were immunoprecipitated with monoclonal anti-TCR{alpha} antibodies and submitted to 13% SDS-PAGE. Visualization of the immunoprecipitated proteins was achieved by fluorography.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The finding in previous studies from our laboratory (Anumula and Spiro, 1983Go; Spiro and Spiro, 1991Go, Moore and Spiro, 1994Go) and those of others (Hanover and Lennarz, 1982Go; Cacan et al., 1987Go; Cacan et al., 1992Go; Duvet et al., 1998Go) that polymannose oligosaccharides are released shortly after N-glycosylation prompted us to postulate (Moore and Spiro, 1994Go) that their appearance is a function of the ER-associated quality control system which degrades misfolded and/or unassembled proteins. The more recent understanding that this molecular editing is mediated by proteasomes (Sommer and Wolf, 1997Go) provided us with an approach for exploring the relationship between oligosaccharide release to proteolysis through the use of proteasome inhibitors.

Indeed the use of these agents in the present investigation did provide new insights into the connection between the deglycosylation and the polypeptide degradation steps of the protein quality control process and suggested that these two events are coupled. It was observed that proteasome inhibitors bring about a rapid reduction in the appearance of free polymannose oligosaccharides in the cytosol. This inhibition was quite selective, as it affected solely the OS-GlcNAc1 species which as previously reported (Moore and Spiro, 1994Go) are predominantly present in this cellular locale while the level of the OS-GlcNAc2 components which reside primarily in the intravesicular compartment remained unchanged. This discriminating effect of the proteasome inhibitors on the two families of free polymannose oligosaccharides is consistent with previous observations which provided evidence that OS-GlcNAc1 components originate from a protein source as puromycin specifically prevented their appearance in contrast to the OS-GlcNAc2 species (Spiro and Spiro, 1991Go). On the basis of in vitro studies it is believed that the latter group of oligosaccharides originate in the ER primarily by the hydrolytic action of oligosaccharyltransferase on dolichol-linked OS-GlcNAc2 (Anumula and Spiro, 1983Go; Spiro and Spiro, 1991Go). The presence of N-glycanases both in the ER and soluble form suggests that the protein-derived oligosaccharides are released as OS-GlcNAc2 and subsequently converted to OS-GlcNAc1 by the action of endo-ß-N-acetylglucosaminidase.

We chose BW5147 mouse T-lymphoma cells as a model to explore the relation of oligosaccharide release to the ER-associated proteasomal quality control system as it has been shown that in these cells there is a defect in TCR assembly which results in the failure of the subunits of this receptor to exit the ER and makes them susceptible to nonlysosomal proteolysis (Lippincott-Schwartz et al., 1988Go). More specifically, Kearse et al. (1994)Go reported that while the {alpha} subunit of TCR remains stable in these cells during at least a 60-min chase under normal circumstances, it undergoes a rapid degradation in the presence of CST. Indeed such rapid and/or accelerated degradation brought about by this glucosidase I and II inhibitor has been noted for a variety of other glycoproteins and has been attributed to the fact that the untrimmed triglucosyl sequence of newly synthesized glycoproteins precludes association with the monoglucosyl specific lectin-like chaperones; indeed, an association of the {alpha} subunit with calnexin and to a lesser extent with calreticulin has been demonstrated in untreated BW5147 cells (Van Leeuwen and Kearse, 1996Go).

The rapid glycoprotein degradation in glucosidase-inhibited cells proved to be very useful in our studies and we were able to confirm the instability of the TCR{alpha} subunit by employing [3H]mannose pulse-chase labeling; furthermore, our findings indicate that proteasome inhibitors block this degradation and this was reflected in a reduced release of oligosaccharides into the cytosol. However, in agreement with the findings of Kearse et al. (1994)Go, no discernible degradation of the {alpha} subunit was apparent in the cells without CST in the time interval examined (data not shown). It is likely that in the CST-treated cells, other glycoproteins in addition to the TCR{alpha} subunit, which we used as an indicator, may have contributed to the released cytosolic oligosaccharides and responded to the proteasomal inhibition in the presence of CST. Indeed, in a recent report the CST-induced accelerated protein degradation noted in a HepG2 system was likewise prevented by proteasome inhibition (Chen et al., 1998Go).

Although it is not possible at the present time to completely decipher the mechanism for the effect of proteasome inhibitors on cytosolic OS-GlcNAc1 release, sufficient information is currently available to place our findings into a more general context. The observations we report do suggest that the deglycosylation and proteolysis are coupled events. Although it has been suggested that the two processes are distinct and cytosolic subsequent to a Sec61-mediated retrotranslocation of the glycoprotein (Wiertz et al., 1996bGo), other findings support the possibility that they take place in association with the ER membrane. Indeed, subpopulations of proteasomes have been localized to the ER by electron microscopic and immunochemical approaches in liver (Rivett et al., 1992Go; Rivett, 1998Go) and a recent report by Enenkel et al. (1998)Go indicated that in yeast the nuclear envelope-ER network is the major site for proteasomal peptide cleavage. Furthermore, although soluble N-glycanases have been reported to be present in a variety of cells (Kitajima et al., 1995Go; Suzuki et al., 1998Go), other studies have indicated that this deglycosylating enzyme is present in the ER (Weng and Spiro, 1997Go). Moreover, in the studies of Suzuki et al. (1997)Go, an accounting of the subcellular distribution of N-glycanase indicated that a substantial portion of this enzyme at relatively high specific activity was associated with a microsomal fraction. While the identification of deglycosylated forms of glycoproteins destined for proteasomal degradation have been observed in the cytosol in several cell systems (Wiertz et al., 1996aGo,b; Tortorella et al., 1998Go), the presence of these proteins in various stages of deglycosylation was however also evident in the microsomal fractions (Wiertz et al., 1996aGo,b; Tortorella et al., 1998Go). Indeed, in the studies of Wiertz et al. (1996b)Go the deglycosylated glycoprotein under study was observed to associated with the Sec61 in the ER membrane indicating that its luminal domain must be accessible to the action of an N-glycanase. Clearly, while these conflicting observations (i.e., cytosolic vs. ER-membrane associated) are not mutually exclusive, a number of recent studies tend to provide further support for a membrane-associated coupled process. A report by Mayer et al. (1998)Go identified membrane-embedded degradation intermediates in yeast which suggested to these investigators that ER-localized proteasomes are involved in the proteolytic process; indeed from their data it was postulated that the membrane-bound proteasome provides the driving force of the reverse translocation process. Investigations on a quality control degradation of the cystic fibrosis membrane conductance regulator likewise provided evidence that the proteolysis is mediated by proteasomes present on the ER as membrane-bound rather than cytosolic degradation intermediates were identified and it was proposed that degradation of this conductance regulator is tightly coupled to the removal of the polypeptide from the lipid bilayer (Xiong et al., 1999Go). Since experimental observations indicate that N-deglycosylation precedes proteolysis in the quality control system, an ER location of the proteasome would favor a membrane site for the N-glycanase. The studies of de Virgilio et al. (1998)Go on the degradation of ribophorin I identified deglycosylation intermediates in the ER-membranes which suggested to these investigators that the carbohydrate removal process is initiated in this organelle and completed in the cytosol; this would be consistent with the presence of deglycosylated intermediates in both membrane and soluble fractions in other systems. Further pertinent observations were made by Yu et al. (1997)Go in studies with TCR{alpha}- transfected cells which indicated that while proteasome inhibitors stabilized the membrane associated glycosylated form of this protein, deglycosylated TCR{alpha} species were also present in the microsomal fraction.

While variation in cell types and proteins under study could account for different findings in regard to the subcellular site of the deglycosylation and proteolytic events which are involved in protein quality control, the observations made in the present study are consistent with a hypothetical model shown in Figure 4A. In this scheme the proteasomes are located on the cytosolic face of the ER membrane and could provide, as suggested by Kopito (1997)Go, at least part of the driving force for the retrotranslocation of glycoproteins from the ER lumen to the cytosol. This model depicts the ER N-glycanase as a translocon component which acts to release the polymannose oligosaccharides as the glycoprotein passes through the transmembrane channel. After this co-retrotranslocational cleavage of the oligosaccharides, they enter the cytosol where they become substrate for the endo-ß-N-acetylglucosaminidase located in this compartment to yield the predominant OS-GlcNAc1 species, while the deglycosylated protein is fragmented by the proteasomes. In this scheme proteasome inhibitors would be expected to stabilize the N-glycosylated protein and to bring about a reduction in the amount of cytosolic oligosaccharides, as was indicated by our data. Indeed, such an involvement of the N-glycanase in retrotranslocation could be considered to be analogous to the function of the oligosaccharyltransferase in the import of proteins from the ribosomes through the translocon channel of the ER membrane (Johnson and van Waes, 1999Go). In an alternate model (Figure 4B), such as proposed by Wiertz et al. (1996a)Go, proteasomes not associated with the ER membrane and a soluble N-glycanase, both located in the cytosol, serve to sequentially deglycosylate and proteolyze the retrotranslocated glycoprotein. In this scheme, stabilization of the N-glycosylated protein should not occur nor would there be the reduction in the cytosolic oligosaccharides which we observe. The two models need not be mutually exclusive and only further studies will clarify the complex series of events which are involved in protein quality control and retrotranslocation.



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Fig. 4. Schematic proposals for the retrotranslocation and N-deglycosylation of glycoproteins. Hypothetical model A, which is consistent with the observations made in the present study, indicates that proteasomes (PR) located on the cytosolic side of the ER membrane provide part of the machinery which translocates the glycoprotein from the ER lumen to the cytosol. The N-glycanase (NG) which is shown as a translocon-associated protein cleaves the polymannose oligosaccharides (solid triangles, OS-GlcNAc2) from the peptide chain in a co-retrotranslocational manner as it passes through the translocon. As the released oligosaccharides reach the cytosol they are converted from OS-GlcNAc2 to OS-GlcNAc1 by the action of the endo-ß-N-acetylglucosaminidase (EH) located in that compartment while the proteasome continues to produce peptide fragments (-). In the presence of the proteasome inhibitors, lactacystin (Lact) and ALLN, there would be a stabilization of the N-glycosylated protein and a decrease in the cytosolic oligosaccharides. In an alternate model (B), such as proposed by Wiertz et al. (1996a,b), both the proteasomes and the N-glycanase are located in the cytosol and accordingly deglycosylation and proteolysis occur sequentially on the retrotranslocated glycoprotein. This scheme would not be consistent with the data of the present study since the proteasome inhibitors would not bring about a stabilization of the N-glycosylated protein nor would there be a reduction in the level of cytosolic oligosaccharides such as we noted. Because of variation in quality control among different glycoproteins and cell types, the two models are not mutually exclusive. The ubiquitination machinery and role of chaperones are not depicted in these models.

 

    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Culture and radiolabeling of cells
Mouse BW5147.3 T-lymphoma cells were obtained from ATCC (Rockville, MD) and were grown as previously described (Karaivanova and Spiro, 1998Go). Subsequent to a 30 min preincubation with 0.2 mM 6-O-butanoyl derivative of CST (a gift of Dr. M.Kang, Merell Dow Research Institute, Cincinnati, OH) followed by a 15 min incubation with or without a proteolytic inhibitor (lactacystin or ALLN, both purchased from Calbiochem, La Jolla, CA, or leupeptin hemisulfate obtained from Sigma, St. Louis, MO), the cells were radiolabeled with 50 to 100 µCi of [2-3H]mannose (21 Ci/mmol, DuPont-New England Nuclear) for 15 min in glucose-free DMEM containing 5 mM pyruvate and 2 mM glutamine. This pulse radiolabeling was followed by a chase (15–45 min) by the addition of mannose and glucose to final concentrations of 2 mM and 5 mM, respectively. The incubations were carried out in duplicate with the cells suspended in 600 µl of medium on 6-well plates (35 mm diameter) at 37°C in a humidified atmosphere with 5% CO2. The proteasome inhibitors lactacystin or ALLN were added to the incubations in DMSO (6 µl) and an equal volume of the solvent was added to the control incubation. Inhibitors present during the pulse phase of the incubations remained present in the chase.

Permeabilization of cells
At the end of the chase period the cells were collected by low-speed centrifugation (500 x g for 10 min) and washed twice with ice-cold phosphate-buffered saline prior to being incubated with 2 ml of cold permeabilization buffer (20 mM MOPS, containing 250 mM mannitol and 2 mM CaCl2) containing 1.6 units/ml SLO (Murex Diagnostics, Inc., Atlanta, GA) for 20 min at 4°C with gentle shaking. Upon completion of the permeabilization period, the SLO-containing medium was removed and combined with a subsequent wash with 1 ml of cold permeabilization buffer. Permeabilization buffer (2 ml) warmed to 37°C was then added to the cells which were incubated at this temperature for 5 min. The cells were then cooled by the addition of 3 ml of ice-cold permeabilization buffer and incubated further for 10 min in an ice bath to ensure diffusion of cytosolic components. The combined permeabilization media and washes were regarded as the cytosolic fraction while the residual cellular material was considered as the vesicular fraction (Moore and Spiro, 1994Go).

Preparation of free oligosaccharides and oligosaccharide-lipids
After deproteinization of the cytosolic fraction with ice-cold trichloroacetic acid, neutral oligosaccharides were obtained by Dowex 50-Dowex 1 and charcoal-Celite chromatography (50% ethanol eluate) in a manner similar to that previously described (Moore and Spiro, 1994Go). Vesicular oligosaccharides were isolated after extraction of the permeabilized cells 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. The upper phase of this extract after evaporation of the organic solvent was fractionated as an aqueous solution on the ion exchange and charcoal columns as described for the cytosolic fraction to yield neutral oligosaccharides. To obtain the oligosaccharide-lipids the interphase material from the chloroform/methanol/buffer (3:2:1) extraction of the permeabilized cells was further treated with a chloroform/methanol/water (10:10:3) mixture as previously described (Spiro et al., 1976Go). Free oligosaccharides and oligosaccharide-lipids were also isolated directly from the washed cells in a manner similar to that described for the permeabilized cells.

Immunoprecipitation
In order to examine the TCR{alpha} subunits, CST-treated BW5147 cells were pulse radiolabeled for 15 min with 200 µCi of [2-3H]mannose in the presence and absence of proteasome inhibitor followed by a 30-min chase. The cells were then separated from the medium by centrifugation (500 x g for 10 min) and, after two washes with ice-cold phosphate-buffered saline, were lysed at 4°C for 45 min with a 100 mM Tris/HCl, pH 7.6, 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 µg/ml aprotinin, 10 mM iodoacetamide and 2 mM phenylmethylsulfonyl fluoride). After centrifugation (14,000 x g for 20 min) in an Eppendorf 5415C microcentrifuge, equal aliquots of the clear supernatants were incubated with hamster monoclonal antibodies 428–710.16 against TCR{alpha} (a generous gift of Dr. John Moorehead, University of Colorado Health Science Center, Denver, CO) followed by protein A–Sepharose as previously described (Rabouille and Spiro, 1992Go; Karaivanova and Spiro, 1998Go).

SDS-PAGE
Electrophoresis of the immunoprecipitates was carried out by the procedure of Laemmli (1970)Go on 13% polyacrylamide gels (1.5 mm thick) overlaid with 3.5% stacking gels. The radioactive components were detected by fluorography and quantitated by densitometry (model 300A Molecular Dynamics Densitometer, Sunnyvale, CA).

Separation of OS-GlcNAc1 and OS-GlcNAc2 oligosaccharides
Oligosaccharides were coupled to 2-aminopyridine (Aldrich) by the procedure of Hase et al. (1984)Go and then desalted on columns of Bio-Gel P-2 equilibrated with 0.1 N formic acid. After endo H digestion of the aminopyridine derivatives of the oligosaccharides, separation of OS-GlcNAc1 and OS-GlcNAc2 was accomplished on coupled Dowex 50 (H+ form) and Dowex 1 (acetate) columns as previously described (Moore and Spiro, 1994Go). In this procedure the endo H sensitive OS-GlcNAc2 components are obtained in the effluent and water wash from these columns while the endo H-resistant OS-GlcNAc1 species are recovered by elution of the Dowex 50 resin with NH4OH.

Thin layer chromatography
Chromatography of large oligosaccharides 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, while resolution of small saccharides was achieved on cellulose-coated plastic sheets (0.1 mm thickness, Merck) in pyridine/ethyl acetate/water/acetic acid, 5:5:3:1. 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 into water. Radiolabeled oligosaccharide standards were prepared as previously described (Lubas and Spiro, 1988Go).

Radioactivity measurements
Liquid scintillation counting was carried out with Ultrafluor (National Diagnostics) with a Beckman LS 7500 instrument. Detection of radioactive components on thin layer plates was accomplished with X-Omatic AR film (Eastman Kodak) after spraying with a mixture containing 2-methylnaphthalene (Spiro and Spiro, 1985Go). The components on electrophoretic gels were visualized by fluorography at –80°C after treatment with ENHANCE (DuPont-New England Nuclear) using the X-Omatic film.


    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; V.K.K. was supported by an American Diabetes Association Mentor-based postdoctoral fellowship.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
ER, endoplasmic reticulum; TCR, T cell antigen receptor; CST, castanospermine; ALLN, N-acetyl-L-leucyl-L-leucyl-L-norleucinal; OS-GlcNAc1 and OS-GlcNAc2, polymannose oligosaccharides terminating at their reducing end with N-acetylglucosamine or a di-N-acetylchitobiose moiety, respectively; SLO, streptolysin O; MOPS, 4-morpholinepropanesulfonic acid; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.


    Footnotes
 
1 Present address: Department of Chemistry, East Carolina University, NC 27858 Back

2 To whom correspondence should be addressed at: Joslin Diabetes Center, One Joslin Place, Boston, MA 02215 Back


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 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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