3URA CNRS 1960, Département dImmunologie Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France, 4Institute of Medical Biochemistry, Department of Molecular Genetics, University and Biocenter Vienna, Dr. Bohr-Gasse 9/2, A-1030 Vienna, Austria, and 5Laboratoire de Chimie Biologique, UMR CNRS 111, Université des Sciences et Technologies de Lille, 59655 Villeneuve dAscq Cedex, France
Received on November 30, 2000; revised on February 1, 2001; accepted on February 6, 2001.
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Abstract |
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Key words: 1,2-mannosidase/CHO glycosylation-defective cell line/ERAD/oligomannoside structure/proteasome
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Introduction |
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Newly synthesized polypeptides enter the ER of eukaryotic cells by crossing the translocon pore (Johnson and van Waes, 1999; Matlack et al., 1999
). Acquisition and processing of N-glycans, particularly the oxidizing environment and folding factors characteristic of this compartment, allow further transport of proteins with a native conformation only (for review see Leitzgen and Haas, 1998
). A quality control machinery assists protein folding (Hammond and Helenius, 1995
; Ellgaard et al., 1999
; Parodi, 2000
) and facilitates elimination of improperly folded or assembled proteins by an ER-associated degradation (ERAD) process defining events that occur in (a) pre-Golgi compartment(s) (Klausner and Sitia, 1990
). Many soluble or membrane proteins of the secretory pathway have now been shown to be substrates for ERAD (Ward et al., 1995
; Wiertz et al., 1996
; Qu et al., 1996
; Hughes et al., 1997
; Yu et al., 1997
; de Virgilio et al., 1998
). In many cases, this pathway results in the cytosolic degradation by the ubiquitin-proteasome system after retrotranslocation of the protein through the Sec61 pore (Sommer and Wolf, 1997
; Bonifacino and Weissman, 1998
; Ivessa et al., 1999
; Plemper and Wolf, 1999
).
The core glycan Glc3Man9GlcNAc2 transferred onto asparagine consensus sites of nascent polypeptides is thereafter subjected to trimming (Hirschberg and Snider, 1987). In the ER, the three glucose residues and a single mannose are removed from carbohydrate side chains by ER glucosidases I and II and ER mannosidase I before glycoproteins become competent to leave this compartment. Interestingly, although monoglucosylated N-glycans, Glc1Man79GlcNAc2 generated by UDP-Glc glycoprotein glucosyltransferase (UGTR), which only reglucosylates malfolded proteins (Sousa et al., 1992
), are important during glycoprotein folding via interaction with lectin-like chaperones, such as calnexin and calreticulin (Ware et al., 1995
; Spiro et al., 1996
), a mannose-trimmed structure of the N-glycan, Man8GlcNAc2, has recently been proposed to play a role in glycoprotein degradation in yeast (Jakob et al., 1998
). In mammals, (a) trimmed structure(s) of N-glycans, thought to be generated by ER mannosidase I, also relate to the degradation process as suggested from indirect studies revealing a stabilization of some ERAD substrates in the presence of mannosidase inhibitors (Yang et al., 1998
; de Virgilio et al., 1999
; Liu et al., 1999
).
It has been demonstrated that monoglucosylated N-glycans interact with calnexin or calreticulin until the glycoproteins acquire their correct conformation, therefore escaping the calnexin or calreticulin/UGTR retention cycle (Ellgaard et al., 1999; Trombetta and Helenius, 1998
). When proteins are unable to fold, the signal to release them from the lectin-like chaperones could then be a trimmed structure of the oligomannoside produced by the action of a mannosidase (de Virgilio et al., 1999
; Liu et al., 1999
; Parodi, 2000
; Wilson et al., 2000
). However, the molecular mechanisms that initiate this process are unknown. Moreover, proteasomal degradation is not always dependent on calnexin binding (Bennett et al., 1998
; Ayalon-Soffer et al., 1999
) or on mannosidase I activity (Yang et al., 1998
; Cabral et al., 2000
), and different interpretations on the role of mannose trimming related to degradation have been proposed.
To learn more about the role of N-glycans during proteasomal degradation we isolated stable transfectants of an HA-tagged model glycoprotein, the soluble ribophorin I variant (RI332) defined as a substrate for ERAD (de Virgilio et al., 1998). We investigated the oligomannoside content of RI332-3HA expressed in a glycosylation-defective cell line, MadIA214, which synthesizes truncated N-glycans, Man5GlcNAc2-P-P-Dol thereafter transferred onto asparagine consensus sequence of nascent polypeptide chains (Ermonval et al., 1997
). Interestingly, this truncated structure is deprived of the terminal mannose
1,2-linkage that is specifically cleaved by ER mannosidase I, the more abundant mannosidase in the ER, responsible for the formation of Man8GlcNAc2 species from complete core glycans. The MadIA214 mutant has been used to demonstrate the involvement of carbohydrates in secretory processes by interfering with protein folding (Ermonval et al., 2000
). Concomitantly, an alteration of the ER morphology and the detection of high levels of soluble oligomannoside species caused by glycoprotein degradation (Verbert and Cacan, 1999
) were observed in this cell line (Ermonval et al., 1997
). In the present study we show that some mannose trimming also occurs on the truncated N-glycan and plays a role in the degradation of the tagged soluble ribophorin I variant, RI332-3HA. An
1,2-mannosidase activity that is different from the one exhibited by ER mannosidase I generates Man4 (and eventually Man3) derivatives associated with RI332-3HA during its degradation in the glycosylation-defective cell line, and a model accounting for these results is proposed.
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Results |
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We first studied the kinetics of degradation of RI332-3HA in our cellular model system using as control, Hela cells also transfected with the gene encoding RI332-3HA (HelaCl8). The three transfected lines Cl42Cl6, MadCl8, and HelaCl8, were metabolically labeled for 20 min with 35S-Met/Cys amino acid and the decrease of the amount of RI332-3HA was analyzed over a 240-min chase period after electrophoresis of the material immunoprecipitated using antibodies directed against the HA-epitope tag. As shown in Figure 1A, the decay of the signal associated with newly synthesized RI332-3HA was rapid in the control parental CHO transfectant, Cl42Cl6, and in the control HelaCl8 cells, both of which synthesize a complete Glc3Man9GlcNAc2 N-glycan. This conforms to the short half-life of the untagged soluble ribophorin I (RI332) in CHO and Hela cells (de Virgilio et al., 1998, 1999). In MadCl8 cells, where glycoproteins carry a truncated N-glycan with the structure Man5GlcNAc2 (Ermonval et al., 2000
), the rate of degradation of RI332-3HA was quite similar. Phosphorimager analysis (Figure 1B) indicated that RI332-3HA was degraded with a t1/2 of about 1 h in all three cell lines. Because MadIA214 glycosylation defective cell line exhibits some thermosensitivity characteristics (Ermonval et al., 1997
), the experiments were performed at the permissive temperature of 34°C. However, degradation of RI332-3HA was unaffected at the nonpermissive temperature (40°C), being only slightly accelerated (t1/2 about 45 min) as is also the case for the tagged RI332 variant in Cl42 and Hela control cells at this temperature (data not shown).
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Inhibition of mannose trimming results in a stabilization of RI332-3HA in the MadCl8 glycosylation mutant cells
Recent data have pointed to the role of ER mannosidase I in the process of 1-antitrypsin degradation (Liu et al., 1997
, 1999), and a similar observation was made with the truncated variant of ribophorin I, RI332 (de Virgilio et al., 1999
). In a normal situation, the more abundant of two ER mannosidases, the ER mannosidase I (Moremen et al., 1994
), removes a terminal
1,2-mannose on the middle branched arm of the high mannose core (see Figure 6). This mannosidase is sensitive to kifunensine (Kif) and deoxymannojirimycin (DMN), while the ER mannosidase II specific for the outer branched terminal
1,2-mannose is resistant to Kif (Herscovics, 1999
). The truncated moiety synthesized in MadIA214 cells is a "linear Man5." This structure does not contain the middle and outer branched arms present on a complete core and therefore, the terminal
1,2-mannose residues cleaved by ER mannosidase I and II.
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In conclusion, 1,2-mannosidase inhibitors prolong the half-life of RI332-3HA in MadCl8 glycosylation mutant cells, suggesting that some mannose residues on the "linear Man5" structure are important for targeting glycoproteins to degradation.
Specific N-glycan structures are associated with RI332-3HA destined to be degraded in MadCl8 cells
The observation of oligomannoside trimming together with the possibility to accumulate trimmed intermediates of RI332-3HA in MadCl8 cells treated with proteasome inhibitors, prompted us to determine the composition of the N-glycans bound to RI332-3HA during its degradation as compared to the N-glycans found on total proteins.
Oligomannosides associated with total glycoproteins of MadCl8 cells metabolically labeled with 3H-Man were analyzed by high-performance liquid chromatography (HPLC) after a 10-min chase (Figure 4A). As expected from our previous results, a truncated core was found in the glycoprotein fraction and consisted mainly of three different species: Glc1Man5GlcNAc2 (29%), Man5GlcNAc2 (34%), and Man4GlcNAc2 (29%), the Man5GlcNAc2 moiety being present in a larger amount. Indeed, MadIA214 mutant cells synthesize a truncated Man5GlcNAc2-P-P-Dol lipid intermediate, which is elongated neither by ER Dol-P-Man-mannosyl transferases nor by ER Dol-P-Glc-glucosyl transferases normally generating the complete Glc3Man9GlcNAc2 core, due to the mislocalization of some intermediates of the reaction. Therefore, the nonglucosylated Man5GlcNAc2 precursor is transferred onto asparagine consensus sites of newly synthesized proteins in MadIA214 cells. However, glycoproteins carry, in addition, a monoglucosylated derivative, Glc1Man5GlcNAc2, expected then to occur only from reglucosylation (Ermonval et al., 2000) by UGTR, an enzyme and chaperone that is specific for malfolded proteins (Sousa et al., 1992
). In addition, proteins loaded with Man4 oligomannosides accumulate during the chase time in MadIA214 and in another glycosylation-defective cell line, B3F7 (Ermonval et al., 2000
). This is consistent with the three major oligomannoside species found on the pool of glycoproteins of MadCl8 cells.
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Moreover, in agreement with the accumulation of an untrimmed form of RI332-3HA in the presence of 1,2-mannosidase inhibitor (Figure 3B) was the finding of Glc1Man5GlcNAc2 as the predominant moiety associated to RI332-3HA after treatment of MadCl8 cells with Kif (Figure 4C).
These data reveal that under conditions in which Man5GlcNAc2 is the major N-glycan found in the glycoprotein fraction of MadCl8 glycosylation defective cell line, this structure is not detected on the RI332-3HA glycoprotein destined to degradation. In contrast, the truncated N-glycan on RI332-3HA only appears in a reglucosylated form (Glc1Man5 GlcNAc2) or a trimmed form (Man4 GlcNAc2) generated by a mannosidase, the latter form disappearing from cells treated with mannosidase inhibitors.
A mannosidase specific for two terminal linear 1,2-mannose residues is involved in the degradation of RI332-3HA in MadCl8 cells
Because RI332-3HA was rapidly degraded in MadCl8 cells, analysis of its N-glycan structure was only possible shortly after synthesis. However, to follow the evolution of the oligomannoside composition in the course of RI332-3HA degradation, we took advantage of the stabilization of RI332-3HA intermediates in the presence of proteasome inhibitors (Figures 2A and 3B).
HPLC analysis of the N-glycans released from purified RI332-3HA was carried out in MadCl8 cells treated with ZLLL 90 min before, then during the pulse labeling with 3H-Man, as well as during a 4-h chase. The percentage of radioactivity associated to each oligomannoside species was calculated according to the number of mannose residues in each HPLC peak (Figure 5). Compared with the equal amount of Glc1Man5 and Man4 moieties removed from RI332-3HA shortly after synthesis (Figure 5A), Man4 and Man3 N-glycans accumulated at the expense of the Glc1Man5 species no longer detected in MadCl8 cells treated with ZLLL (Figure 5B). This is in agreement with the increase in electrophoretic mobility of RI332-3HA observed under this condition, and with the stabilization of an untrimmed form in the presence of DMN and Kif, indicative of the requirement for a mannosidase to generate the trimmed species (see Figures 3 and 4). The appearance of a Man3 derivative when proteasomes were inhibited demonstrates that the mannosidase activity was able to release a second mannose residue from the truncated Man5 structure. This enzyme is expected, from its specificity to mannosidase inhibitors, to recognize mannose 1,2-linkage. To prove that Man4 and later Man3 structures were produced by cleavage of the terminal mannose on the linear arm rather than by cleavage of the last
1,6-branched mannose residue, half of the oligomannoside sample extracted from RI332-3HA after 4 h chase in presence of ZLLL was also digested by a specific mannosidase from Aspergillus saitoi that removes, on N-glycans, all mannose residues with an
1,2-linkage. As expected, after the enzymatic digestion Man4 moieties were completely converted into Man3GlcNAc2 (Figure 5C).
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Discussion |
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To evaluate the role of mannosidases during ERAD, we used the MadIA214 glycosylation-defective cell line, which is impaired in oligomannoside elongation (Ermonval et al., 1997). We showed in the present study that RI332-3HA, an HA-epitope tagged version of a soluble variant of ribophorin I, is rapidly degraded in MadIA214 cells dependent on proteasomal activity. Interestingly, mannose trimming of the truncated Man5GlcNAc2 N-glycan bound to RI332-3HA in the glycosylation defective cell line is a prerequisite for its degradation. When mannosidase
1,2-activity was blocked with DMN or Kif, an untrimmed form of RI332-3HA bearing a Glc1Man5GlcNAc2 accumulated in MadCl8 transfected cells instead of being degraded. In addition, our determination of the structure of the N-glycans associated with RI332-3HA demonstrates that a Man4GlcNAc2 moiety is an intermediate of degradation that accumulates in the glycosylation-defective cell line when proteasomal activity is inhibited by ZLLL. This led us to speculate that this particular N-glycan structure could serve as a signal targeting RI332-3HA to degradation in glycosylation mutant cells. Moreover, the observation (1) that degradation was inhibited both by DMN and Kif, which stabilize the monoglucosylated Man5 derivative of RI332-3HA, (2) that Man3GlcNAc2 appeared together with Man4GlcNAc2 in the presence of ZLLL, and (3) that all the Man4GlcNAc2 can be converted to Man3GlcNAc2 by an exogenous
1,2-mannosidase, strongly support the role of a mannosidase specific for mannose
1,2-linkages but different from ER mannosidase I, in the process of glycoprotein degradation in MadIA214 mutant cells. In fact, mannose trimming was obtained although the Man5GlcNAc2 structure does not contain the branched mannose
1,2-linkage recognized by the highly specific ER mannosidase I (Tremblay and Herscovics, 1999
). We can also exclude the involvement of the endomannosidase that generates Man8GlcNac2 isomers A in wild-type cells (Weng and Spiro, 1996b
) and therefore could directly transform a Glc1Man5GlcNAc2 N-glycan into a Man4GlcNAc2 structure, because this enzyme is insensitive to Kif and was not detectable in CHO cells (Karaivanova et al., 1998
; Weng and Spiro, 1996a
).
In mammalian cells, two ER mannosidases (I and II) are able to generate Man8GlcNAc2 isomers (B and C respectively) by digesting one single terminal mannose residue present, respectively, on the inner and the outer branched arm of the high mannose N-glycan. Both are sensitive to DMN, but only mannosidase I is inhibited by Kif (Weng and Spiro, 1996a). In a normal situation, only the isomer B produced by ER mannosidase I is detected and is an intermediate in the processing of complex oligomannosides associated to secretory glycoproteins (Atkinson and Lee, 1984
; Daniel et al., 1994
). Mannosidases not only operate as processing enzymes but, as already pointed out, also operate in ERAD processes. Because glycoproteins to be degraded are initially retained in the ER by molecular chaperones and because the ER mannosidase I is the most abundant among this family of enzymes in this compartment, many studies concluded that the Man8GlcNAc2 structure generated by ER mannosidase I constitutes a signal for degradation (Liu et al., 1997
; Jakob et al., 1998
; Yang et al., 1998
; de Virgilio et al., 1999
; Parodi, 2000
; Wilson et al., 2000
). This was supported by results from experiments utilizing the mannosidase inhibitors DMN and Kif, but the structure of the N-glycans was not determined in these studies. One direct argument in favor of the role of a particular oligomannoside structure in ERAD comes from analysis of the degradation of misfolded mutant carboxypeptidase (CPY*) in yeast mutants blocked at different steps in their N-glycan processing. Optimal degradation of CPY* occurred with the Man8GlcNAc2 structure, but Man6, Man7, Man9 derivatives did not efficiently support degradation of this glycoprotein. Therefore, the authors (Jakob et al., 1998
) speculated that trimming of N-glycans by the yeast ER mannosidase I could be a timer for the disposal of misfolded proteins.
In contrast to yeast, more mannosidases have been found in mammals and one or more of these could account for the particular trimmed structure of N-glycan that we detected on a glycoprotein only destined to be degraded in MadIA214 mutant cells. Of particular interest in this context is the Man9-mannosidase (Bause et al., 1992, 1993) which has been shown to remove three
1,2-mannosidic linkages on a Man9GlcNAc2 structure, leading to a Man6GlcNAc2 isomer. Although this enzyme has a low affinity for the terminal mannose of the inner branched arm cleaved by ER mannosidase I, it removes terminal linear mannose residues on a truncated Man5GlcNAc2 (Bause et al., 1992
) and thus is able to generate Man4GlcNAc2 and, less efficiently, Man3GlcNAc2. This accommodates well our results revealing these two N-glycan structures on RI332-3HA in MadCl8 cells when proteasome-mediated degradation was inhibited. Man3GlcNAc2 moieties should indeed be better detectable under conditions in which glycoproteins are retained artificially in the ER (as with ZLLL), allowing for the cleavage of the
1,2-mannosidic linkage exhibiting a lower affinity for the mannosidase.
We therefore consider the possibility likely that the Man9-mannosidase could trigger the delivery of glycoproteins to degradation in our mutant cell line by generating a Man4GlcNAc2 trimmed structure (see model, Figure 6A). Shortly after biosynthesis, Man5GlcNAc2 represents the major species associated to total proteins. As expected from previous work (Ermonval et al., 2000) the Man5GlcNAc2 oligomannoside precursor, which is transferred on newly synthesized proteins, can be reglucosylated on improperly folded proteins in MadIA214 cells, and Glc1Man5GlcNAc2 is detected on total proteins. A considerable proportion of Man4GlcNAc2 is also present and could correspond to a pool of proteins destined to be degraded (see Figure 4). Earlier studies have shown that in MadIA214 mutant cells and in another mutant (B3F7), both synthesizing truncated N-glycans (Man5GlcNAc2 and Glc3Man5GlcNAc2, respectively) differing only at the level of glucosylation, a trimmed Man4GlcNAc2 oligomannoside is the major structure associated with total protein, at long periods of time after synthesis, while concomitantly, proteins are maintained longer in the ER of these glycosylation-defective cells (Duvet et al., 2000
; Ermonval et al., 2000
). In the present study we describe a remarkable selection of the population of N-glycans attached to RI332-3HA shortly after synthesis, consisting only of Glc1Man5GlcNAc2 and Man4GlcNAc2, while Man5GlcNAc2 is no longer present. We believe this difference in N-glycan structure distribution to be due to the fact that the glycoprotein pool consists of proteins that will either be degraded or follow the secretory pathway. As the truncated ribophorin I variant is unable to assemble with other partners of the oligosaccharyl transferase complex in the ER and is not secreted either (Ivessa et al., 1992
; Tsao et al., 1992
), its only fate is to be degraded. Our results support the idea that the major N-glycan, Man5GlcNAc2, transferred onto proteins in MadIA214 cells is very transient on a glycoprotein destined to degradation. In contrast, this structure is expected to be carried by proteins that have acquired a native conformation and would not be reglucosylated by UGTR anymore, therefore escaping from chaperone retention. The finding of a high amount of Man5GlcNAc2 on total proteins substantiates this assumption. Finally, the truncated N-glycan associated to RI332-3HA appears either as reglucosylated moieties specific for unfolded or malfolded proteins that accumulate in cells treated with Kif, or as a mannose-trimmed form that accumulates at the expense of the Glc1Man5GlcNAc2 in the presence of drugs blocking the proteasome and could signal the protein for targeting to disposal. Importantly, the RI332-3HA degradation rate and pathway in MadIA214 cells were shown to be similar to control cells, arguing that they result from the particularity of the protein rather than from the abnormal glycosylation. Altogether, the use of an ERAD substrate loaded with a truncated N-glycan made the characterization of N-glycans associated to degradation substrates easier and has revealed an alternative mannosidase activity to that of the ER mannosidase I in this system.
It is to be noted that more mannosidases are being characterized even though their role in oligomannoside processing as well as their precise subcellular locations are not always defined and can vary among cell types and species (Daniel et al., 1994; Tremblay et al., 1998
; Igdoura et al., 1999
). Therefore, we cannot eliminate the possibility that a not yet described or a mislocalized mannosidase, or the Golgi mannosidase I that exhibits common specificities with the Man9-mannosidase, could generate the trimmed Man4 derivative revealed in our mutant cells. For instance, unfolded proteins could leave the ER in association with folding factors, such as the ER chaperone BiP, and be retrieved from the Golgi apparatus, where they could have encountered other mannosidases (Figure 6). Interrestingly, RI332-3HA interacts with BiP in MadIA214 cells (data not shown). Accelerated degradation in the presence of brefeldin A (Tsao et al., 1992
; Dusseljee et al., 1998
), a drug blocking transport and redistributing Golgi enzymes to the ER, suggests the role of a post-ER compartment in ERAD.
Whichever is the mannosidase implicated in the production of the Man4 structure, considering the high specificity of ER mannosidase I (Tremblay and Herscovics, 1999), its role in the degradation of RI332-3HA in MadCl8 cells remains quite improbable. It has been recently shown that it is necessary to introduce a mutation in the active site of the ER mannosidase I to extend its specificity to other mannose
1,2-linkages (Romero et al., 2000
). In addition, determination of the crystal structure revealed interaction between amino acid residues of the active site and some mannose residues of the Man9 chain absent from the "linear Man5" moiety (Vallee et al., 2000
), and to our knowledge there is no evidence in the literature of ER mannosidase I specificity for such truncated N-glycans. It is thus tempting to extrapolate our data to a wild-type situation. The ER mannosidase I could produce the Man8 isomer B, an intermediate in the processing of complex type carbohydrates, while other mannosidases, namely, the Man9-mannosidase, will generate structures (Man8 isomer A, Man7 and Man6 species) for which trimming of the terminal mannose on the linear arm of the high mannose chain could be of importance for glycoprotein degradation (see model Figure 6B). In cell types other than CHO, the endomannosidase located in the intermediate compartment or the cis-Golgi could also produce a Man8 isomer A (Weng and Spiro, 1996b
). Accounting for the various ER mannosidases that have been described so far, the possibility that they play different roles in processing and degradation has already been proposed (Daniel et al., 1994
; Ayalon-Soffer et al., 1999
). We think that this is consistent with the observation that Man8 isomer B is present on secretory glycoproteins (Atkinson and Lee, 1984
) and on long-lived glycoproteins resident in the ER (Rosenfeld et al., 1984
; Duvet et al., 1998
). Moreover, our findings and model are in accordance with data showing that under particular conditions, such as after long-term residence in the ER (Rosenfeld et al., 1984
) or in particular crystalloid structures detected in cells overexpressing HGM-CoA (Liscum et al., 1983
; Bischoff et al., 1986
), the appearance of trimmed N-glycans consisting of Man6GlcNAc2 was observed.
Once a particular structure is produced by one or more mannosidases, the mechanism of the delivery of the trimmed glycoprotein intermediate to proteasomal degradation still has to be defined and a role for a lectin recognizing such a structure has to be proposed (Jakob et al., 1998; Wilson et al., 2000
). We believe that it might be possible for some mannosidases to exhibit a dual function, such as an enzyme activity and a lectin specificity for improperly folded proteins as described for UGTR (Sousa et al., 1992
), and, by being part of a network of proteins made up of chaperones, folding factors, and the translocon, to target proteins to degradation.
It is also to be noted that for a common pathway of degradation, the intermediate reactions were shown to be variable from one protein to another, depending or not on calnexin interaction (glucose trimming), mannosidase activity, and ubiquitination of the substrate (Yang et al., 1998; Ayalon-Soffer et al., 1999
; Liu et al., 1999
). Also, ER mannosidase II has been recently proposed to act as a signal for nonproteasomal degradation (Cabral et al., 2000
). It is probable that the precise mechanism will vary according to the substrate protein. Our present data add information on the role of a particular trimmed structure, and therefore of
1,2-mannosidase activities, in regulating the ERAD process. We think that such an approach will help determine intermediate reactions taking place in the ER and preceding proteasomal degradation in wild-type conditions.
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Materials and methods |
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RI332 and RI332-Thr were tagged close to the C terminus with three repeats of the HA epitope YPYDVDYA (Field et al., 1988). The tag was amplified by polymerase chain reaction with the forward primer TTTTGGTGACCTTTACCCATACGATGTTCC and the reverse primer TTTTGGTCACCCTGAGCAGCGTAATCTGG using a plasmid DNA that harbored a cDNA containing three HA epitope repeats as template (provided by K. Kuchler). This way BstEII restriction sites were generated at both ends of the DNA fragment, which was cloned into pCR2.1 (Invitrogen). The 3HA-tagged fragment was excised from the resulting plasmid with BstEII and inserted at the same site in RI332 or RI332-Thr (at position 1038 bp of the cDNA, or at Gly322 of the mature protein) within pCI-neo. Clones of pCI-neo containing the tagged soluble variants of ribophorinI, designated RI332-3HA and RI332-Thr-3HA, were isolated and their identities were confirmed by sequencing. These constructs were used for expression in CHO derived cells and in Hela cells.
Cell lines
The CHO-derived hamster cells used in this study were grown at 34°C and 7% CO2 in alpha-Minimum Essential Medium (Biological Industries) supplemented with 7% fetal calf serum (FCS), 1 mM HEPES, and G418 at 1 mg/ml. Hela cells were grown at 37°C in Minimal Essential Medium (Life Technologies) supplemented with 7% FCS, 1 mM HEPES, and 2 mM glutamine. FCS and additives are from Life Technologies.
MadIA214, a glycosylation mutant line, and its Cl42 parental clone are CHO cells expressing two heterologous secretory glycoproteins and the gene encoding neomycin resistance. The mutant cell line synthesized a truncated N-glycan consisting of Man5GlcNAc2. Characteristics of these cell lines have been described (Ermonval et al., 1997).
Cl42 and MadIA214 cells were cotransfected by the lipofection method (according to the manufacturer, Life Technologies) with the expression vectors pCI-neo-RI332-3HA containing the RI332-3HA cDNA, or pCI-neo-RI332-Thr-3HA containing the cDNA corresponding to the nonglycosylated variant RI332-Thr-3HA. The plasmids were mixed with pZeoSV (Invitrogen) used as a selection plasmid at a ratio of 3 µg:0.3 µg in 10 µl of lipofection reagent. Cells cultured in a 6-cm dish were incubated for 24 h with the lipofection/DNA transfection reagent. Stable clones Cl42-RI332-3HA-Cl6, Mad-RI332-3HA-Cl8 and Mad-RI332-Thr-3HA-Cl7 were selected in the presence of zeocin (Invitrogen) at 0.2 mg/ml and tested for expression of the tagged soluble ribophorin I variant by pulse labeling, followed by immunoprecipitation with anti-ribophorin I antibodies and anti-HA antibodies. To facilitate the reading, transfectants names have been shortened as follows: Cl42Cl6, MadCl8, and Mad*Cl7 for Cl42-RI332-3HA-Cl6; Mad-RI332-3HA-Cl8 and Mad-RI332-Thr-3HA-Cl7, respectively.
Hela cells were transfected according to the same protocol with pCI-neo-RI332-3HA conferring resistance to G418 antibiotic. A stable transfectant, Hela-RI332-3HA-Cl8, expressing the tagged soluble ribophorin I variant was selected in medium supplemented with G418. This transfected clone is referred to as HelaCl8 in the present study.
Transfected cells were maintained in their corresponding selective medium containing zeocin (0.2 mg/ml) and/or G418 (1 mg/ml).
Antibodies
The polyclonal rabbit antibody against rat liver ribophorin I (Marcantonio et al., 1984; Yu et al., 1990
; Tsao et al., 1992
) was a kind gift from G. Kreibich. This antibody works on SDS-denatured cell extracts and recognizes both the endogenous ribophorin I (RI) and its soluble variant. The tagged soluble ribophorin was specifically detected using a polyclonal rabbit antibody directed against HA, purchased from BabCo. For N-glycan analysis requiring purification of big quantities of the soluble variant of RI, we employed the rabbit polyclonal RI-LB antibody reacting with the luminal domain of RI. To prepare the anti-RI-LB antiserum, the peptide CPSYEYLYNLGDQYALK corresponding to amino acids 335 to 350 of the rat RI preprotein plus a C-terminal cysteine residue was synthesized and purified by HPLC (Research Genetics). The peptide was coupled to activated KLH, and the resulting product was separated from low molecular weight compounds by chromatography on Excellulose GF-5 (Pierce). The peptide/KLH conjugate was mixed with Freunds complete adjuvant and used to immunize an adult female New Zealand white rabbit. The anti-RI-LB antibody works with Triton X-100 extracts, but less well with SDS extracts, and therefore reacts preferentially with the soluble RI variant, because the endogenous ribophorin I is better solubilized in SDS than in Triton X-100.
Immunoprecipitations were performed by precoupling of the antibodies to Protein A-Sepharose CL-4B beads obtained from Amersham Pharmacia Biotech.
Pulse-chase analysis and immunoprecipitation
Degradation of the tagged soluble ribophorin in the different transfected lines was studied using previously described conditions (Ermonval et al., 1997). Cells (1.52.5 x 105) were cultured overnight in 24-well plates at 34°C, then pulsed for 15 min with 100 µCi of a radioactive [35S]Met-[35S]Cys amino acid mixture (TRANS35S-LABEL, ICN) in FCS-, Met-, and Cys-free medium (ICN). Radioactivity was chased for the indicated period of time.
In cells treated with proteasome inhibitors (ZLLL, 50100 µM or ZLLNva 40 µM purchased from Peptides International) or with mannosidase inhibitors (DMN, 1 mM from Boehringer Mannheim or Kif, 10 µM from ICN); the drugs were added prior to labeling. Cultures were pretreated for 1 h with the drug, which was then kept for all the following incubation: starvation for 30 min in FCS-, Met-, and Cys-free medium, pulse for 20 min, and chased for the indicated periods of time.
For immunoprecipitation using anti-HA or RI-LB antibodies, cells were lysed with 1% Triton X-100 (Merck) in NET buffer (150 mM NaCl, 50 mM Tris, pH 7.4, 5 mM EDTA). Specific immunoprecipitations were performed overnight at 4°C with 15 µl per sample of protein-A Sepharose beads precoated with either 1 µl of anti-HA antiserum or 2.5 µl of anti-RI-LB. After washing in high salt (0.5 M NaCl) NET buffer the immunoprecipitates were eluted from the beads with reducing sample buffer containing 5% ß-mercaptoethanol. Using the polyclonal anti-RI antibody, cell lysis and immunoprecipitations were performed under stringent conditions using an SDS method, as reported elsewhere (Tsao et al., 1992). Samples were analyzed by SDSPAGE on 8% gels, and the signal was amplified by fluorography and detected on Amershams Hyperfilm. Quantitations of immunoprecipitations were performed either by scanning densitometry or by phosphorimager using the ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The relative intensities at different chase times were expressed as percentages of the 5-min value corresponding to the highest intensity of labeling of the truncated ribophorin I variants.
HPLC analysis of the oligosaccharides bound to RI332-3HA in MadCl8 cells
To determine the structure of the N-glycans associated to total proteins, MadCl8 cells (510 x 106) were metabolically labeled at 34°C with 200 µCi of 2-(3H)Man (42.9 Gbq/mmol) from Amersham Pharmacia Biotech as described previously (Ermonval et al., 1997). The protein fraction was prepared by sequential extraction of cell monolayers with organic solvents as already described (Kmiécik et al., 1995
). Proteins were digested overnight at room temperature with 0.2 mg of TPCK-treated trypsin, and the oligosaccharides were cleaved from the peptides by incubating overnight with 5 U of Peptide N-Glycanase F (Boehringer). After separation and desalting through a biogel-P2 column, oligosaccharides were analyzed by HPLC on an ASAHIPAK-NH2-P-50 column (Asahi, Kawasaki-ku, Japan). The oligomannosides eluted over an 80-min period of time in a 70:30 to 50:50 (v/v) acetonitrile/water gradient (flow rate 1 ml/min) were monitored with a Flo-one ß detector (Flotec).
Analysis of the N-glycans specifically associated to RI332-3HA had to face the difficulty of a protein representing a small amount of total proteins and in addition being degraded. To end up with sufficient material for HPLC analysis, the number of cells normally used was doubled and the labeling conditions were changed as to increase 3H-mannose incorporation. MadCl8 cells were preincubated for 30 min at 34°C in culture medium containing 10% dialyzed FCS at 0.5 mM glucose. They were metabolically labelled for 45 min in the same medium without glucose but containing 300 µCi of 2-(3H)Man per flask (about 107 cells). After washing the cells, the radioactivity was chased in normal culture medium for 10 min only, corresponding to a time where maximum level of truncated RI is detected. RI332-3HA was purified from MadCl8 Triton X-100 extracts by immunoprecipitation with protein A-sepharose beads (500 µl) coupled to anti-RI-LB antiserum (200 µl). Oligomannosides were released from RI332-3HA by directly treating the beads in the same way as indicated above for the protein fraction, then analyzed by HPLC. The same procedure was used to prepare the N-glycans associated to RI332-3HA from MadCl8 cells treated with Kif to inhibit 1, 2-mannosidases, except that the cells were pretreated for 1 h with Kif at 10 µM and the drug was maintained during all the subsequent steps of labeling and for 1 h of chase.
HPLC analysis of the N-glycans associated to intermediate of degradation of RI332-3HA in MadCl8 cells
The same protocol as described for HPLC analysis of oligomannosides bound to RI332-3HA was used, but for this study cell cultures were treated with the proteasome inhibitor ZLLL at 50 µM. The MadCl8 culture was preincubated in normal culture medium for 1 h with the drug, which was then maintained during the starvation in 0.5 mM glycose, the pulse labeling without glucose, and the chase in normal medium, which lasted for 4 h. Extraction and HPLC analysis of N-glycans released from RI332-3HA was proceeded as above.
To determine more precisely the mannose linkages present in the oligomannoside structure associated to RI332-3HA during its degradation, half of the material obtained from MadCl8 cells treated with ZLLL was digested overnight at 37°C with 5 µU of a mannosidase from A. saitoi (Oxford Glycosystem), specific for all the 1,2-mannose linkages, then digested oligomannosides were prepared for HPLC analysis.
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Acknowledgment |
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Abbreviations |
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Footnotes |
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2 Present address: Différenciation Cellulaire et Prions, UPR 1983 CNRS, Institut André Lwoff, 7 rue Guy Moguet, BP 8, 94800 Villejuif, France
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References |
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