Glycoprotein Quality Control in the Endoplasmic Reticulum

MANNOSE TRIMMING BY ENDOPLASMIC RETICULUM MANNOSIDASE I TIMES THE PROTEASOMAL DEGRADATION OF UNASSEMBLED IMMUNOGLOBULIN SUBUNITS*

Claudio FagioliDagger and Roberto SitiaDagger §

From the Dagger  Department of Molecular Pathology and Medicine, DIBIT-San Raffaele Scientific Institute, 20132 Milan, Italy and § Università Vita-Salute San Raffaele, 20132 Milan, Italy

Received for publication, October 20, 2000, and in revised form, January 24, 2001



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Quality control in the endoplasmic reticulum must discriminate nascent proteins in their folding process from terminally unfolded molecules, selectively degrading the latter. Unassembled Ig-µ and J chains, two glycoproteins with five N-linked glycans and one N-linked glycan, respectively, are degraded by cytosolic proteasomes after a lag from synthesis, during which glycan trimming occurs. Inhibitors of mannosidase I (kifunensine), but not of mannosidase II (swainsonine), prevent the degradation of µ chains. Kifunensine also inhibits J chain dislocation and degradation, without inhibiting secretion of IgM polymers. In contrast, glucosidase inhibitors do not significantly affect the kinetics of µ and J degradation. These results suggest that removal of the terminal mannose from the central branch acts as a timer in dictating the degradation of transport-incompetent, glycosylated Ig subunits in a calnexin-independent way. Kifunensine does not inhibit the degradation of an unglycosylated substrate (lambda  Ig light chains) or of chimeric µ chains extended with the transmembrane region of the alpha  T cell receptor chain, implying the existence of additional pathways for extracting proteins from the endoplasmic reticulum lumen for proteasomal degradation.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The endoplasmic reticulum (ER)1 is the port of entry and the main folding compartment for proteins destined to the exocytic pathway. A stringent quality control system is coupled to the folding machinery, ensuring that only structurally mature proteins reach the Golgi (1). Proteins that fail to attain their correct three-dimensional structure are retained in the ER and eventually degraded after a lag that varies among individual substrates (2, 3).

Cytosolic proteasomes are responsible for the degradation of many membrane and soluble ER-synthesized proteins (4-14), implying that proteins targeted for degradation must be delivered to the cytosol (15). Sec61, a component of the translocon complex that mediates the entry of proteins into the ER (16), seems to be involved also in the retrograde translocation, or dislocation, to the cytosol (17, 18). This observation raises intriguing questions on the mechanisms that gate Sec61 for dislocation and determine the directionality of transport across the ER membrane (19-22).

ER quality control prevents the deployment of potentially harmful molecules to their final destinations and maintains homeostasis within the ER. Somehow, the system must be capable of discriminating terminally unfolded and/or unassembled molecules from newly translocated polypeptides, which have not had the time to complete folding. Therefore, the processes of retention and degradation must not only be specific but also be precisely timed.

The oligosaccharide moieties present on many ER-synthesized proteins have been shown to play a crucial role in the quality control (see Ref. 1 for a comprehensive review). A branched oligosaccharide consisting of three glucoses, nine mannoses, and two N-acetylglucosamines (Glc3Man9GlcNAc2) is added to the nascent polypeptide when an appropriate sequence is recognized by oligosaccharyl-transferase (Fig. 1). Immediately after synthesis, the terminal glucoses are removed by two glucosidases (I and II) localized in the ER. Another ER-resident enzyme, UDP-glucose: glycoprotein glucosyltransferase, is capable of adding a glucose residue to the A branch of the glycans present on unfolded molecules, favoring their binding to calnexin or calreticulin. These two ER chaperones bind monoglucosylated proteins and retain them in the folding-promoting ER environment. ER mannosidases I and II remove the terminal mannoses from the B and C branches, respectively. It is thought that glycans lacking the terminal mannoses are poorer substrates of glucosidase II and UDP-glucose: glycoprotein glucosyltransferase, thereby bringing the calnexin cycle to an end (1, 23). Hence, it is possible that the different kinetics of the ER sugar-processing enzymes provide a molecular mechanism to time retention and dislocation/degradation. Evidence for a role of mannose trimming in diverting misfolded glycoproteins to proteasomal degradation has been produced in yeast (24) and in mammalian cells (25-29).


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Fig. 1.   Processing of N-glycans in vivo and in vitro. A branched glycan consisting of two N-acetylglucosamines (squares), nine mannoses (open circles), and three glucoses (striped or dotted circles) can be added to the nascent polypeptide when an asparagine (Asn) is followed by the sequence Aaa-(Ser/Thr), where Aaa is any amino acid but proline (59). Soon after translation, glucosidases (Glu) I and II remove the glucoses, whereas UDP-glucose: glycoprotein glucosyltransferase (UGT) can add a single glucose (striped circle) to the terminal mannose of the A branch, yielding a monoglucosylated glycan. ER mannosidases (ER Man) I and II remove the terminal mannoses from the B and C branches, respectively. Castanospermine and dN can be used to inhibit glucosidases, whereas kifunensine and swainsonine block the activity of ER mannosidase I and ER mannosidase II, respectively. dM inhibits both ER mannosidases (1, 43 and references therein). Jack bean alpha -mannosidase (alpha -Man) can be used in vitro to discriminate between glycans carrying or not carrying a glucose on the A branch (46). Only in the absence of the glucose (G0), alpha Man cleaves the mannoses of the A branch.

To investigate how ER-synthesized proteins are diverted from retention into the degradative pathway, we compared the fate of Ig µ, J, and lambda  chains in assembly-deficient myeloma transfectants. These Ig subunits fold primarily under the assistance of BiP (30-34). Whereas µ and J carry five N-linked glycans and one N-linked glycan, respectively, lambda  are not glycosylated. Evidence is presented indicating that the trimming of the B branch terminal mannose by ER mannosidase I times the dislocation of µ and J chains from the ER lumen to the cytosol for proteasomal degradation, without the involvement of calnexin and/or calreticulin.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Lines-- NSO, a myeloma cell line that produces endogenous J chains but no heavy or light chains, and the transfectants expressing wild type secretory (µs) or membrane (µm) µ chains (N[µ1] and N[µm], respectively) or a mutant lambda  chain (N[lambda Gly-214]), were described previously (34-36). N[µ-TCR alpha TM], N[gamma 2b-TCR alpha TM] were generated by stably transfecting NSO with chimeric µ or gamma 2b heavy chains extended at their C termini with the transmembrane region of the T cell receptor alpha  chain (37).

Antibodies and Reagents-- A rabbit anti-mouse J chain antiserum (38) was kindly provided by Dr. R. M. E. Parkhouse (Pirbright Laboratory, Surrey, UK). Horseradish peroxidase-conjugated goat anti-mouse µ, goat anti-mouse Ig, and goat anti-rabbit Ig were from Southern Biotechnology Associates Inc. (Birmingham, AL). Purified rabbit anti-µ and anti-lambda antibodies were obtained from Zymed Laboratories Inc. (San Francisco, CA). Dithiothreitol, leupeptin, N-ethylmaleimide, thapsigargin, and Triton X-100 were purchased from Sigma; the protease inhibitors Complete and endoglycosidase H were from Roche Molecular Biochemicals. The proteasome inhibitor carboxybenzyl-leucyl-leucyl-leucinal (ZL3H) (39, 40), was a kind gift of Drs. M. Bogyo and H. Ploegh (Harvard University, Cambridge, MA). Phenylarsine oxide (Sigma) was used at 20-50 µM (26).

Castanospermine and deoxynojirimycin (dN) were purchased from Sigma and used at the final concentration of 1 mM to inhibit glucosidases I and II. Kifunensine and swainsonine were purchased from Toronto Research Chemicals (Toronto, Canada), dissolved in water, and used at a final concentration of 2 µg/ml and 100 µM, respectively, to selectively block mannosidase I and mannosidase II, respectively (41-44). 1-deoxymannojirimicin (dM, Sigma) was used at 1 mM as an inhibitor of ER mannosidases I and II. The effects of kifunensine and swainsonine on the degradation and electrophoretic mobility of µ and J chains were similar when the drugs were added either before or immediately after the pulse.2 Therefore, to simplify the experimental procedures and to reduce variability in incorporation, cells were pulse-labeled in bulk and then aliquoted and chased with the different drugs.

Pulse-Chase Assays, Immunoprecipitation, and SDS-PAGE-- Pulse-chase assays were performed as previously described (21). In the experiment shown in Fig. 7, cells were chased in Krebs-Ringer Hepes medium (125 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 5.5 mM glucose, 25 mM Hepes, 0.5 mM MgCl2, 2% fetal calf serum, pH 7) supplemented with different concentrations of CaCl2 (3 µM-1 mM). Ionomycin was added at the final concentration of 1 µM. Films were scanned, and relevant bands were quantitated by ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Glucosylation Assay-- Following extensive washings, immunoprecipitates on protein A beads were incubated overnight at 37 °C with jack bean alpha -mannosidase (Sigma) in 20 µl of 50 µM sodium citrate, pH 4.4 before boiling in sample buffer and electrophoresis.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Glycan Trimming Determines Diversion of µ and J Chains into the Proteasomal Degradation Pathway-- In the absence of lambda  chains, µs and J chains cannot assemble into transport-competent IgM polymers and are degraded by cytosolic proteasomes (21). When cells were pulse-labeled for a short period (5 min), a lag was evident before degradation ensued (Fig. 2, A and C). During this period, both subunits undergo some folding, as determined by the formation of intrachain disulfide bonds3 and, in the case of µ, by acquisition of partial protease resistance (21). Changes in the electrophoretic mobility of µ chains were evident during the lag period (Fig. 2A). These mobility shifts probably corresponded to glycan processing, because they were no longer detectable after treatment with endoglycosidase H (data not shown). In contrast, if N[µ1] cells were pulse-labeled for a long period (20 h) so as to label the whole intracellular pool, µ and J chain degradation could be observed immediately (Fig. 2, B and C), consistent with the fact that a fraction of the molecules had reached a degradation-competent state.


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Fig. 2.   Newly made µ and J chains are degraded after a lag. N[µ1] cells were pulse-labeled for 5 min (A) or 20 h (B) and chased for the indicated times before lysis and sequential immunoprecipitation with anti-µ and anti-J antibodies. Immunoprecipitates were resolved by SDS-PAGE (12% acrylamide for anti-J and 10% or 9% for the anti-µ immunoprecipitates shown in A and B, respectively). Note the mobility shifts in µ chains during chase after a short pulse (A). Autoradiograms were scanned, and the percentage of µ and J chains present at each time point was determined relative to the intensity at time 0 of chase (C). After a 20-h pulse, the degradation of µ (closed squares) and J (closed circles) chains clearly begins, without the lag that is evident after a short pulse.

Inhibiting Binding to Calnexin and Calreticulin Does Not Alter Significantly the Kinetics of µ or J Chain Degradation-- Because some newly synthesized µ chains can be coprecipitated with calnexin and calreticulin (21), the effects described above could reflect a role of the two chaperones in µ chain quality control, as described for certain alpha 1-antitrypsin mutants (23). To verify this, the glucosidase inhibitor castanospermine was added before the pulse, so as to inhibit the rapid removal of glucose residues from newly made proteins. Consistent with the presence of additional glucoses, µ and J chains synthesized in the presence of castanospermine migrated more slowly (Fig. 3A). Under these conditions, µ chains were not coimmunoprecipitated by anti-calnexin antibodies (data not shown). Preventing the binding to calnexin and calreticulin, however, did not affect the kinetics of µ chain degradation; after the characteristic lag, clearance was efficient (Fig. 3B, lanes 1-4). Castanospermine also had little, if any, effect on the degradation of J chains (Fig. 3C, lanes 2-5). Pre-treatment with castanospermine did not affect the dislocation of J chains, which can be monitored by the appearance of the two faster migrating bands (Fig. 3C, >right-arrow and right-arrow) when cells are chased in the presence of proteasome inhibitors (21). Similar amounts of cytosolic J chains accumulated in the presence or absence of castanospermine (Fig. 3C, compare lanes 1 and 9 and the histograms below).


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Fig. 3.   Preventing the binding to calnexin and calreticulin by continuous exposure to castanospermine does not alter the kinetics of µ and J chain degradation. N[µ1] cells were pre-incubated for 30 min in the presence of castanospermine, then pulsed for 5 min, and chased for the indicated times in the presence or absence of the glucosidase inhibitor alone or in combination with other drugs (C, castanospermine; K, kifunensine; Z, ZL3H). As indicated by the mobility shifts in both µ and J chains (A), pre-treatment with castanospermine was effective in preventing glucose removal from newly synthesized glycoproteins (see also Fig. 6). After chase in the presence of the indicated drugs, cells were lysed and sequentially immunoprecipitated with anti-µ (B) and anti-J (C) antibodies and resolved by SDS-PAGE. The two arrows in the left-hand margin of C (>right-arrow and right-arrow) point to deglycosylated, cytosolic J chains (21). The densitometric quantitation of the relevant bands is shown below the corresponding autoradiograms. Bars represent the percentage of µ and J chains present at each time point relative to the intensity at time 0 of chase. In C, the shaded area of the bars indicates the percentage of deglycosylated J isoforms relative to total J chains accumulating at individual time points.

These results indicated that calnexin and calreticulin do not play a major role in timing the degradation of the two Ig subunits. We therefore investigated other glycan processing steps known to occur in the ER lumen, in particular mannose trimming, which has been implicated in the quality control of mutated alpha 1-antitrypsin (23, 25-27) and thyroglobulin (29).

Inhibitors of ER Mannosidase I Prevent µ Chain Degradation-- Inhibiting the activity of ER mannosidase I with kifunensine prevented the degradation of µ chains synthesized in the presence of castanospermine as efficiently as did proteasome inhibitors (Fig. 3B, compare lanes 6 and 9). The simultaneous presence of kifunensine and castanospermine did not have synergistic effects (Fig. 3B, lane 8). As shown in Fig. 3C, kifunensine partially inhibited J chain degradation as well (see below). These results suggested that the trimming of the B branch mannose is important in timing the degradation of unassembled Ig subunits.

To further confirm this, specific inhibitors of the main ER glycosidases were added after a 5-min pulse without castanospermine preincubation (Fig. 4). As observed above in castanospermine pre-treated cells, kifunensine was as effective as ZL3H in preventing the degradation of µ chains (Fig. 4A, compare lanes 2, 3, 6, and 9). dM, an inhibitor of both ER mannosidase I and ER mannosidase II, also inhibited µ chain degradation (Fig. 4A, lane 17). In contrast, the ER mannosidase II inhibitor swainsonine was not effective (Fig. 4A, lane 12). Consistent with the presence of additional mannoses, µ chains accumulating in the presence of dM, kifunensine, or swainsonine migrated more slowly than those present in untreated cells (Fig. 4A, lane 2) or in cells incubated with proteasome inhibitors (lane 3). When both swainsonine and kifunensine were present during the chase, the effects of the ER mannosidase I inhibitor prevailed, and abundant µ chains accumulated intracellularly (Fig. 4A, lane 22). The mobility shift was more pronounced than that induced by kifunensine or swainsonine alone. Densitometric quantitations (Fig. 4A, see bars below each lane) confirmed that the removal of the terminal mannose from the central branch is crucial for the degradation of µ chains.


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Fig. 4.   Mannosidase I is involved in timing the degradation of glycosylated Ig subunits. N[µ1] cells were pulse-labeled for 5 min and chased for 1, 2, or 4 h in the presence of the indicated combinations of inhibitors (Z, ZL3H; K, kifunensine; C, castanospermine; S, swainsonine). Lanes 1-13 and 14-22 derive from two independent experiments. Lysates were sequentially immunoprecipitated with anti-µ (A) and anti-J (B) antibodies and resolved by SDS-PAGE. The densitometric quantitation of the relevant bands is shown below the corresponding autoradiograms. Arrows and bars are as in the legend to Fig. 3.

Because the removal of mannoses may alter the substrate recognition by glucosidases and UDP-glucose: glycoprotein glucosyltransferase, it was possible that the drugs influenced the binding of µ chains to calnexin. To exclude this possibility, castanospermine or dN was added after the pulse so as to block glycans in a monoglucosylated state. The effects of castanospermine and dN were confirmed by the mobility shift of µ chains accumulating in treated cells (Fig. 4A, compare lanes 18-20) and by mannosidase sensitivity assays (see Fig. 6 below). Under these experimental conditions, which should favor the binding of unfolded glycoproteins to calnexin and/or calreticulin (1), castanospermine and dN had minor effects on µ chain degradation.

Analysis of the anti-J immunoprecipitates confirmed the role of mannose trimming in targeting unassembled Ig subunits to degradation. Castanospermine and dN had minor effects (Fig. 4A, lanes 10, 18, 19). Kifunensine and dM inhibited the degradation of J chains, although to a lesser extent than µ chains. A single J chain band was precipitated from treated cells, with mobility slightly slower than the upper band accumulating in the presence of proteasome inhibitors (Fig. 4B, compare lanes 3 and 4). Inhibitors of ER mannosidase I prevented only in part the dislocation of J chains, as indicated by the appearance of the two bands with faster mobility, corresponding to deglycosylated, cytosolic J chains (21), when proteasome inhibitors were added during the chase (Fig. 4B, compare lanes 4-9). Therefore, glycan trimming is not essential for the dislocation of J chains. The differences between µ and J chains may reflect the number of N-glycans present on the two substrates and may correlate with our previous observation that some J but no µ chains can dislocate to the cytosol when proteasomal degradation is inhibited (21).

Neither Kifunensine nor Castanospermine Inhibits the Degradation of Unglycosylated lambda  Chains-- To exclude the possibility that the different drugs acted on general cellular functions, we investigated their effects on lambda , a nonglycosylated Ig subunit. Neither kifunensine nor castanospermine had any effect on the degradation of lambda G213, a mutant that is also degraded by proteasomes (Fig. 5), albeit with slower kinetics than µ or J chains (36, 45).4


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Fig. 5.   Kifunensine does not affect the proteasomal degradation of lambda  chains. N[lambda G213] cells were pulse-labeled for 5 min and chased for 4 h in the presence of the indicated inhibitors (Z, ZL3H; C, castanospermine; K, kifunensine). Lysates were immunoprecipitated with anti-lambda antibodies and resolved by SDS-PAGE. Because anti-lambda antibodies react poorly with unfolded lambda  chains, they fail to bring down all radioactive lambda  present at the end of the pulse.

µ Chains Accumulating in the Presence of Kifunensine Are Not Glucosylated-- The presence of a terminal glucose on the A branch of an N-glycan can be monitored by a mannosidase sensitivity assay (46). When the glucose is present (Fig. 1, G1), the mannosidase cannot remove the underlying mannoses, yielding a small mobility shift. In contrast, glycans lacking the glucose (see Fig. 1, G0) are digested to a greater extent. As expected, µ chains present in castanospermine-treated cells were only marginally affected by mannosidase (Fig. 6A, compare lanes 9 and 10). In contrast, ZL3H (Fig. 6A, lanes 5 and 6) and kifunensine (lanes 7 and 8) caused the accumulation of µ chains that were sensitive to the enzyme. Similarly, J chains appeared to be sensitive to mannosidase except after chase in the presence of castanospermine (Fig. 6B). µ and J chains accumulating in the presence of swainsonine (Fig. 6, A, lanes 11 and 12 and B, lane 6) were largely resistant to mannosidase, suggesting that the presence of a terminal mannose on the C branch inhibits glucosidase activity on the two Ig subunits.


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Fig. 6.   µ and J chains accumulating in kifunensine-treated cells are not glucosylated. N[µ1] cells were pulse-labeled for 5 min and chased for 4 h in the presence of the indicated inhibitors (Z, ZL3H; K, kifunensine; C, castanospermine; S, swainsonine). Aliquots of the anti-µ (A) and anti-J (B) immunoprecipitates were digested in vitro with jack bean alpha -mannosidase (alpha -Man) (before electrophoresis. The arrow at the right-hand margin indicates the mobility of untreated J chains.

Calcium Is Required for the Degradation of Unassembled µ Chains-- The recent solution of the structure of yeast class I alpha 1,2-mannosidase I revealed a crucial role for calcium ions in enzymatic activity (28). Therefore, we investigated whether perturbing calcium homeostasis in the ER lumen affected µ chain degradation. As shown in Fig. 7, the ER Ca2+-ATPase inhibitor thapsigargin caused the intracellular accumulation of µ chains, which migrated with a mobility slower than that of those present in untreated cells (Fig. 7, compare lanes 2 and 3). Similar to that reported above for kifunensine, thapsigargin also partially inhibited J chain degradation (data not shown).


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Fig. 7.   Calcium is required for µ chain degradation. N[µ1] cells were pulse-labeled for 5 min and chased for 4 h in the presence of thapsigargin or ionomycin in calcium-free Krebs-Ringer Hepes medium before lysis and immunoprecipitation with anti-µ antibodies. Ionomycin-treated cells were chased in the presence of the indicated concentrations of CaCl2.

The requirements for calcium were further confirmed by chasing pulse-labeled N[µ1] cells with ionomycin and different Ca2+ concentrations (Fig. 7, lanes 4-8). In the presence of the ionophore, the Ca2+ levels in the ER are expected to be similar to those in the extracellular medium. Based on this assumption, it would seem that 100 µM Ca2+ is required to sustain the processes underlying µ chain degradation. The reduced degradation of µs in this experiment (Fig. 7, lane 2) could be due to the low extracellular Ca2+ concentration.

Mannose Trimming Is Important for the Degradation of µm Chains as Well-- Both substrates analyzed so far, µs and J chains, are soluble proteins retained in the ER lumen. It was of interest to analyze the role of glycan processing on an integral membrane protein. Alternate RNA processing generates two forms of µ chains, µs and µm, which differ only in their C-terminal ends (47) and are degraded with similar kinetics (35). Clearly, kifunensine prevented the degradation of membrane bound µ chains (Fig. 8). Cytosolic proteasomes are responsible for the degradation of µm, because ZL3H caused their accumulation (Fig. 8, lane 11). Castanospermine had little, if any, effect on the clearance of membrane-bound µm chains (Fig. 8, lane 10).


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Fig. 8.   Kifunensine prevents the proteasomal degradation of membrane µ chains. N[µm] cells were pulse-labeled for 5 min and chased for the indicated times with kifunensine (K), castanospermine (C), or ZL3H (Z) before lysis and immunoprecipitation with anti-µ antibodies.

Kifunensine Does Not Inhibit IgM Secretion-- In the presence of lambda  chains, most µ chains synthesized by plasma cells are assembled into IgM polymers and secreted (34). Only part of them, corresponding to molecules that fail to assemble with lambda  or to polymerize, are degraded (37). We therefore tested the effects of kifunensine and ZL3H on J[µ1] cells, which express µ, lambda , and J chains. As shown in Fig. 9, kifunensine did not inhibit IgM secretion (compare lanes 5 and 6). The proteasome inhibitor ZL3H had only a marginal effect (Fig. 9, lane 7). Both kifunensine and ZL3H caused a small accumulation of intracellular µ chains (Fig. 9, lanes 2 and 3), suggesting that secretory µ chains that failed to polymerize were degraded by proteasomes. These results imply that the molecular mechanisms that recognize µ chains lacking the central mannose(s) and that divert them to proteasomal degradation are specific for structurally immature molecules (either unassembled or unpolymerized).


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Fig. 9.   Neither kifunensine nor proteasome inhibitors significantly perturb IgM secretion. J[µ1] cells, which produce µ, lambda , and J chains and secrete IgM polymers (34), were pulse-labeled for 5 min and chased for 4 h in the presence or absence (-) of the indicated inhibitors (K, kifunensine; Z, ZL3H). The anti-µ immunoprecipitates from the cell lysates and supernatants were resolved by SDS-PAGE under reducing conditions. The diagonal arrow on lane 3 highlights the altered migration of intracellular µ chains present in kifunensine-treated cells.

The Degradation of µ-TCR alpha TM Chains Does Not Require Mannose Trimming-- We have previously shown that appending the transmembrane region of the TCR alpha  chain to the C terminus of Ig µ and gamma 2b chains induces brefeldin A-insensitive degradation of chimeric immunoglobulins (37). When expressed in the absence of light chains in NSO, degradation of µ-TCR alpha TM was rapid. Unlike in the case of µs and µm chains, however, kifunensine did not alter the kinetics of µ-TCR alpha TM degradation (Fig. 10A). Thapsigargin failed to inhibit the proteasomal disposal of µ-TCR alpha TM as well (Fig. 10B), in agreement with the notion that Ca2+ is not required for the degradation of T cell receptor subunits (48). In addition, the proteasomal degradation of gamma 2b-TCR alpha TM chains, which occurs with kinetics slower than µ-TCR alpha TM (37), was insensitive to glycosidase inhibitors and thapsigargin (Fig. 10C), suggesting that the extraction of these chimeric molecules follows a different pathway, independent from the activity of ER mannosidase I. 


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Fig. 10.   Proteasomal degradation of µ-TCR alpha TM is not inhibited by kifunensine or thapsigargin. A, kifunensine does not inhibit the degradation of µ-TCR alpha TM. N[µ-TCR alpha TM] cells were pulse-labeled for 5 min and chased for the indicated times in the presence or absence of kifunensine. Anti-µ immunoprecipitates were resolved by SDS-PAGE, and the intensity of the µ-TCR alpha TM chain bands were quantitated by densitometry. The graph shows the percentage of µ-TCR alpha TM present at individual chase points relative to time 0. B, unlike µs, µ-TCR alpha TM chains are degraded in the presence of thapsigargin. N[µ-TCR alpha TM] and N[µ1] cells were pulse-labeled for 5 min and chased for the indicated times in the presence or absence (-) of thapsigargin (Tg) or ZL3H (Z). Bars represent the densitometric quantitation, performed as described in the legend to Fig. 3. C, the more slowly degraded chimeric gamma 2b-TCR alpha TM chains are also insensitive to kifunensine. N[gamma 2b-TCR alpha TM] cells were pulse-labeled for 5 min and chased for the indicated times in the presence or absence (-) of kifunensine (K), thapsigargin (Tg), or ZL3H (Z). D, inhibitors of tyrosine phosphatases block the degradation of both µs and µ-TCR alpha TM chains. N[µ-TCR alpha TM] and N[µ1] cells were pulse-labeled for 5 min and chased for 4 h with phenylarsine oxide (P), ZL3H (Z), or both drugs (PZ). Bars represent the densitometric quantitation, performed as described in the legend to Fig. 3. The absence of deglycosylated J chains (>right-arrow and right-arrow) in cells treated with both phenylarsine oxide and ZL3H suggests that phenylarsine oxide inhibits J chain dislocation. Chase in the presence of pervanadate (data not shown) or diamide (21) gave similar results.

It has been recently reported that inhibitors of tyrosine phosphatases, such as phenylarsine oxide prevent the degradation of certain alpha 1-antitrypsin mutants (26). Phenylarsine oxide clearly inhibited the degradation of both µs and µ-TCR alpha TM chains (Fig. 10D). Unlike kifunensine, phenylarsine oxide inhibited the dislocation of J chains, because the simultaneous presence of phenylarsine oxide and proteasome inhibitors during the chase did not yield the characteristic cytosolic J isoforms (Fig. 10D, see arrows) in either N[µ-TCR alpha TM] or N[µ1] cells (lanes 5 and 9). Pervanadate (data not shown) and the oxidant diamide (21) gave similar results.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To mediate folding efficiency and homeostasis, the processes of retention and degradation that underlie quality control within the ER must be precisely coordinated. Sufficient time must be allocated to allow the folding of newly made polypeptides in a milieu that provides optimal assistance by specialized chaperones and enzymes. On the other hand, molecules that fail to attain the correct three-dimensional structure after some time should be degraded to prevent their accumulation or aggregation in the ER lumen (49). The data presented in this paper demonstrate that the trimming of N-linked glycans provides a molecular mechanism for coordinating the quality control of two glycosylated unassembled Ig subunits. This timer function seems to be based on the sequential activity of the ER sugar-processing enzymes. Whereas glucosidases act rapidly as substrates emerge from the translocon, ER mannosidases I and II come into the scene later (1). The absence of the terminal mannose on the B branch of asparagine-linked glycans enables the targeting of terminally unfolded glycoproteins for dislocation to the cytosol and proteasomal destruction.

The distinct patterns of µ and J chain electrophoretic mobility and in vitro sensitivity to jack bean alpha -mannosidase confirmed the specificity of the pharmacological inhibitors. Interestingly, µ chains isolated from cells chased in the presence of swainsonine are largely resistant to mannosidase, whereas kifunensine leads to the accumulation of sensitive molecules. This suggests that, at least in myeloma cells, removal of the B branch mannose does not impair glucosidase II to a greater extent than UDP-glucose: glycoprotein glucosyltransferase. On the other hand, on glycans lacking a C branch terminal mannose, the activity of UDP-glucose: glycoprotein glucosyltransferase prevails, and glucosylated molecules accumulate, which are nonetheless dislocated and degraded by proteasomes. Thus, it appears that calnexin has a minor role in the clearance of µ chains. In contrast, kifunensine and dM were as effective as proteasome inhibitors in preventing µ chain degradation. Under both conditions, µ chain dislocation across the ER membrane was blocked, and glycosylated heavy chains accumulated. Kifunensine (Figs. 3 and 4) and proteasome inhibitors (21) only partially blocked the dislocation of J chains, perhaps reflecting the different number of glycans or folding state of the two subunits.

The involvement of mannose trimming in targeting glycoproteins for proteasomal destruction has been demonstrated in several experimental models. In yeast, biochemical and genetic evidence indicates that the degradation of CPY* requires the activity of a Class I alpha 1,2-mannosidase (50, 51). Trimming of the central mannose is important also for the degradation of yeast pre-pro-alpha -factor ectopically expressed in mammalian cells (52) and of certain alpha 1-antitrypsin mutants associated with familial emphysema (23, 25-27). The degradation of a truncated alpha 1-antitrypsin mutant was shown to depend on a prolonged interaction with calnexin (23). In this respect the degradation of µ and J chains seems to be mechanistically different, because it is only marginally affected by castanospermine. This indicates that more than one pathway can coordinate the dislocation/degradation of glycoproteins, a notion that is not surprising because at least two parallel systems exist in the ER to assist glycoprotein folding. Whether a glycoprotein selects calnexin/calreticulin or BiP seems to depend on the location of the glycan(s). The closer the oligosaccharide moieties are to the N terminus, the higher are the chances that the protein engages with calnexin (53). This may explain why µ chains, in which the first of the five N-linked glycans is located at position 171 (see Ref. 54 and references therein), fold primarily on BiP. However, a fraction of newly made µ chains can be coprecipitated with calnexin (21). It is not known whether these have detached from a first round of interaction with BiP or represent direct binding to the lectin, possibly facilitated by the numerous glycan moieties present on µ chains. Although the single glycan of J chains is bound to asparagine 48, we failed to detect interactions of this small Ig subunit with calnexin or calreticulin (21). In contrast, J chains can be easily coprecipitated with BiP, especially in tunicamycin-treated cells.2 Because unassembled µ and J chains preferentially associate with BiP, it is not surprising that pre-incubation with castanospermine (which prevents binding to calnexin/calreticulin) fails to affect their degradation. The minor effects observed when the glucosidase inhibitor is administered after the pulse could be due to a reduced activity or accessibility of ER mannosidase I on monoglucosylated substrates. Therefore, our results suggest the involvement of glycan trimming in timing glycoprotein degradation independently from interactions with calnexin or calreticulin.

Many ER resident proteins bind Ca2+, and perturbing the homeostasis of this ion is bound to have pleiotropic effects. However, because ER mannosidase I contains Ca2+ (28), thapsigargin could inhibit µ chain degradation by interfering with the function of this enzyme, as also suggested by the similar electrophoretic mobility of µ chains accumulating in thapsigargin- or kifunensine-treated cells.

How can removal of the B branch terminal mannose activate dislocation? Terminally unfolded substrates, in particular soluble molecules like µs and J chains, must be recruited in the vicinities of a functional dislocon, which must eventually be opened and activated. Dislocation across the ER membrane, which in the case of µ chains requires active proteasomes (21), is probably facilitated by unfolding and reduction of the substrate. It is tempting to speculate that lectin molecules endowed with specificity for Man8 glycans play an important role in this chain of events (24). In principle, if such lectins had a binding specificity similar to ER mannosidase I, kifunensine, thapsigargin, and other drugs may compete with or inhibit the lectin itself. The observation that kifunensine does not affect IgM secretion indicates that, if a lectin plays a role in timing dislocation, it must be specific for unfolded molecules. It also suggests that the inhibition of degradation caused by kifunensine is not due to prolonged retention in the ER lumen. Taken together, these results are consistent with an active role of Man8-binding lectin molecules in targeting misfolded or unassembled glycoproteins to dislocation/degradation.

The degradation of the PiZ and null variants of alpha 1-antitrypsin seems to follow different pathways (23, 26, 27). The results obtained with µ-TCR alpha TM indicate that indeed more than one pathway can be utilized to dislocate a protein from the ER to the cytosol. In µ-TCR alpha TM, the last residues of µs, including the C-terminal N-glycan (54) and Cys-575, had been replaced with the transmembrane and cytosolic domains of the alpha -TCR chain, known to contain a dominant ER-associated degradation (ERAD) targeting signal (11, 14, 55, 56). Like µm, µ-TCR alpha TM contains four of the five glycans present in wild type µs and is an integral membrane protein with a short cytosolic tail. Therefore, neither the number of sugars nor the different topology can explain the distinct requirements of the three ERAD substrates for dislocation and degradation. In addition, gamma 2b-TCR alpha TM, whose slower kinetics of degradation allow glycan processing by ER mannosidase I, is efficiently degraded in the presence of kifunensine. These observations suggest that information contained in the transmembrane and cytosolic tail of the alpha -TCR chain can induce mannosidase I-independent degradation of a polypeptide that normally requires removal of the B terminal mannoses. In addition, this pathway seems to utilize cytosolic proteasomes, because it can be inhibited by ZL3H (Fig. 10), and in this respect differs from the pathway described for the PiZ alpha 1-antitrypsin variant (26). In view of the importance of maintaining ER homeostasis and fidelity of quality control, it is not surprising that multiple pathways are operative in mammalian cells to eliminate misfolded proteins from the ER.

Although differing in sensitivity to kifunensine and thapsigargin, µs and µ-TCR alpha TM share the property of being stabilized by inhibitors of tyrosine phosphatases, such as phenylarsine oxide (Fig. 10D) and pervanadate (data not shown). In addition, the degradation of endogenous J chains is blocked by phenylarsine oxide. The absence of deglycosylated J isoforms suggests that this drug blocks dislocation from the ER lumen to the cytosol, a molecular phenotype similar to that induced by the reversible oxidant diamide (21). The involvement of tyrosine phosphatases could be important for integrating ERAD with other cellular signaling pathways, including the responses to unfolded proteins and ER overload (57, 58 and references therein). However, because many steps in the complex process of ERAD are redox-sensitive, the precise targets of phenylarsine oxide, pervanadate, and diamide remain to be identified.

    ACKNOWLEDGEMENTS

We thank Drs. M. Aebi, M. Alessio, A. Cabibbo, A. Fassio, A. M. Fra, A. Helenius, C. Jakob, R. Mancini, A. Mezghrani, and T. Simmen for helpful discussions and suggestions; Drs. M. Bogyo, H. Ploegh, and R. M. E. Parkhouse for generously providing reagents; and S. Trinca for impeccable secretarial assistance.

    FOOTNOTES

* This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro, Consiglio Nazionale delle Ricerche (Target Project on Biotechnology, 97.01296.PF49 and 5% 99.0089.PF31), and Ministero della Sanità (Special AIDS Project and Ricerca Finalizzata RF 98.53).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Via Olgettina 58, 20132 Milan, Italy. Tel.: 39 02 2643 4763; Fax: 39 02 2643 4723; E-mail: r.sitia@hsr.it.

Published, JBC Papers in Press, January 24, 2001, DOI 10.1074/jbc.M009603200

2 C. Fagioli and R. Sitia, unpublished observations.

3 A. Mezghrani, C. Fagioli, and R. Sitia, unpublished results.

4 A. Sparvoli and R. Sitia, unpublished results.

    ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; µs, secretory µ; µm, membrane µ; TM, tunicamycin; ZL3H, carboxybenzyl-leucyl-leucyl-leucinal; dN, deoxynojirimycin; dM, deoxymannojirimicin; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; TCR, T cell receptor; ERAD, ER-associated degradation.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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