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.
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Abstract |
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Key words: proteasome inhibitors/polymannose oligosaccharide release/quality control of glycoproteins/ T-lymphocytes/endoplasmic reticulum/cytosol
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Introduction |
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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, 1990; Rivett, 1993
; Hilt and Wolf, 1996
) 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., 1996b
). 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, 1994
). Indeed, N-glycanases which are probably involved in the deglycosylation of proteins destined for degradation have been found in both ER (Suzuki et al., 1997
; Weng and Spiro, 1997
) and cytosolic (Kitajima et al., 1995
; Suzuki et al., 1998
) locations and furthermore endo-ß-N-acetylglucosaminidase (Pierce et al., 1979
) and chitobiase (Cacan et al., 1996
) 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) demonstrated that a rapid degradation of TCR
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.
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Results |
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When the cells were incubated with the proteasomal inhibitor ALLN ( Rock et al., 1994; Ward et al., 1995
; Hughes et al., 1996
) or lactacystin (Ward et al., 1995
; Dick et al., 1996
), 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, 1983
; Renfrew and Hubbard, 1991
) did not result in a reduction of oligosaccharide release (Table I).
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Discussion |
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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, 1994) 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, 1991
). 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, 1983
; Spiro and Spiro, 1991
). 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., 1988). More specifically, Kearse et al. (1994)
reported that while the
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
subunit with calnexin and to a lesser extent with calreticulin has been demonstrated in untreated BW5147 cells (Van Leeuwen and Kearse, 1996
).
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 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)
, no discernible degradation of the
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
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., 1998
).
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., 1996b), 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., 1992
; Rivett, 1998
) and a recent report by Enenkel et al. (1998)
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., 1995
; Suzuki et al., 1998
), other studies have indicated that this deglycosylating enzyme is present in the ER (Weng and Spiro, 1997
). Moreover, in the studies of Suzuki et al. (1997)
, 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., 1996a
,b; Tortorella et al., 1998
), the presence of these proteins in various stages of deglycosylation was however also evident in the microsomal fractions (Wiertz et al., 1996a
,b; Tortorella et al., 1998
). Indeed, in the studies of Wiertz et al. (1996b)
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)
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., 1999
). 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)
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)
in studies with TCR
- transfected cells which indicated that while proteasome inhibitors stabilized the membrane associated glycosylated form of this protein, deglycosylated TCR
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), 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, 1999
). In an alternate model (Figure 4B), such as proposed by Wiertz et al. (1996a)
, 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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Materials and methods |
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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, 1994).
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, 1994). 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., 1976
). 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 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 428710.16 against TCR
(a generous gift of Dr. John Moorehead, University of Colorado Health Science Center, Denver, CO) followed by protein ASepharose as previously described (Rabouille and Spiro, 1992
; Karaivanova and Spiro, 1998
).
SDS-PAGE
Electrophoresis of the immunoprecipitates was carried out by the procedure of Laemmli (1970) 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) 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, 1994
). 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, 1988).
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, 1985). 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.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 To whom correspondence should be addressed at: Joslin Diabetes Center, One Joslin Place, Boston, MA 02215
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