From the
Department of Molecular and Cellular
Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto
606-8397, Japan,
Core Research for Evolutional
Science and Technology, Japan Science and Technology Corporation, Saitama
332-0012, Japan, ||McGill Cancer Centre, McGill
University, Montréal, Québec H3G 1Y6, Canada, and
**Department of Biochemistry, Sapporo Medical
University School of Medicine, Sapporo 060-8556, Japan
Received for publication, April 2, 2003 , and in revised form, May 3, 2003.
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ABSTRACT |
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INTRODUCTION |
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In this report, we show that overexpression of human ER ManI accelerates
the degradation of the terminally misfolded 1-antitrypsin
genetic variant-null Hong Kong (A1AT NHK)
(18). We also show the
combined effects of ER ManI and EDEM on the degradation of misfolded
1-antitrypsin. Glycan analysis on misfolded NHK shows that
overexpression of ER ManI greatly increases the formation of
Man8GlcNAc2 and
Glc1Man8GlcNAc2 and also stimulates trimming
of N-linked oligosaccharides to Man5GlcNAc2
in vivo. These experiments suggest that the misfolded glycoprotein
interacts both with ER ManI and EDEM before being recognized by downstream
ERAD components.
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EXPERIMENTAL PROCEDURES |
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ReagentsKifunensine, an inhibitor of ER ManI, was kindly provided by Fujisawa Pharmaceutical Co. (Osaka, Japan) and added to the culture medium at a concentration of 5 µg/ml for 4 h prior to pulse-labeling. The proteasome inhibitor lactacystin was purchased from Kyowa Medics Co. (Tokyo, Japan) and added to the culture medium at a concentration of 1 mM for 4 h prior to pulse-labeling. The inhibitors were also present in the medium during the pulse and the chase periods.
Plasmid ConstructionHuman ER ManI cDNA was subcloned into the pMH vector (Roche Molecular Biochemicals) by PCR. The entire open reading frame (9) was amplified from cDNA using a sense primer containing a HindIII site and a Kozak sequence (5'-AAAAAAGCTTCCACCATGGCTGCCTGCGAGGGCAGGAG-3') and an antisense primer containing a NotI site and omitting the stop codon (5'-AAAAAAAAGCGGCCGCGCTGCAGGGGTCCAGATAGGCAGAG-3'). The open reading frame amplicon was digested with HindIII and NotI and cloned into the HindIII/NotI sites of pMH in-frame with the C-terminal HA-tag. Mouse EDEM cDNA was tagged with HA at its C terminus in pCMV-SPORT2 vector (Invitrogen) as described previously (15). The NHK variant was constructed as described elsewhere (15).
Metabolic Labeling and ImmunoprecipitationCells were preincubated in DMEM lacking methionine (Invitrogen) for 30 min and labeled with Expre35S35S protein labeling mixture (PerkinElmer Life Sciences) for 15 min at a concentration of 8.2 MBq/ml. The cells were then incubated in DMEM during the chase period. After washing twice with phosphate-buffered saline lacking Ca2+ and Mg2+, cells were incubated on ice for 20 min in lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0, supplemented with protease inhibitors) (15). For immunoprecipitation, Nonidet P-40 soluble cell lysates were mixed with various antibodies (1/50 dilution), and the immune complexes were collected using either Protein A- or Protein G-Sepharose beads (Amersham-Pharmacia, Amersham, UK) (19). One-half of the cell lysate was used for immunoprecipitation using antibody against A1AT, and the other half was incubated with anti-HA antibody. In cells co-transfected with NHK and ER ManI or EDEM, the radioactivity of immunoprecipitated NHK decreased compared with cells transfected with NHK and pMH. To obtain approximately equal signals for the immunoprecipitated NHK at chase 0 h, the gels were exposed 2 or 3 times longer for samples co-transfected with ER ManI or EDEM than for those co-transfected with pMH. Alternatively in some experiments, one-half or one-third of the cell lysates was used for immunoprecipitation in pMH transfected cells in comparison with ER ManI or EDEM transfected cells. The conditions are indicated in the figure legends. The immune complex was boiled in Laemmli's sample buffer containing 0.1 M dithiothreitol and separated by 10% SDS-PAGE, unless otherwise indicated in the legends. Radioactivity was quantified by exposing the gels to a PhosphorImager (Storm, Amersham Biosciences), and the gels were exposed to x-ray films (HR-HA, Fuji Photo Film Co. Ltd., Japan) afterward.
For labeling with [3H]mannose, cells were preincubated in DMEM containing 1 mM glucose (Invitrogen) for 30 min and labeled with 7.4 MBq/ml D-[2-3H]mannose for 1 h. Cells were then chased for the times indicated in DMEM containing 25 mM glucose (Invitrogen) supplemented with 5 mM mannose (20). Cell lysis, immunoprecipitation, and SDS-PAGE were performed as described for the labeling with 35S-protein-labeling mixture, and the samples were blotted onto a PVDF membrane (Bio-Rad) in 5 mM sodium tetraborate buffer. The position of the [3H]mannose-labeled NHK bands on the PVDF membrane were identified by exposing the membrane to a PhosphorImager.
AntibodiesAntiserum against 1-antitrypsin
was purchased from Cappel (ICN Pharmaceuticals, Inc.), and purified IgG
against the HA-tag was obtained from Santa Cruz Biotechnology, Inc. Human ER
ManI antibody was prepared by immunizing rabbits with a keyhole limpet
hemocyanin-conjugated synthetic peptide (GRRDVEVKPADRHNLLRPET). The antiserum
was affinity-purified using the peptide. For the antibody against mouse EDEM,
multiple antigenic peptide was synthesized using the following peptide
sequence, DERRYSLPLKSIYMRQID. Whole rabbit serum was used for the
experiments.
Western BlottingNonidet P-40-soluble cell lysates or immunoprecipitates prepared as described above (see "Metabolic Labeling and Immunoprecipitation") were adjusted to 1 x Laemmli's buffer containing 0.1 M dithiothreitol and separated by 10% SDS-PAGE. For Western blotting, 40 µg of protein was loaded per lane. For Western blotting following immunoprecipitation, 400 µg of protein of Nonidet P-40-soluble cell lysate was used as starting material. After blotting to a nitrocellulose membrane, antigen-antibody complexes were detected by ECL (Amersham Biosciences) and exposed to x-ray films.
Characterization of OligosaccharidesThe
[3H]mannose-labeled NHK bands on the PVDF membranes were cut into
small pieces. The pieces were first rinsed with methanol and then with 0.1
M sodium citrate, pH 5.5, three times for 15 min at 37 °C, and
the washes were discarded. The membrane pieces were then incubated with Endo-H
(New England Biolabs) in the same buffer at 37 °C for 48 h. Endo-H (1000
units) was added at 0, 12 and 36 h of incubation, and the membrane pieces were
washed three times with water. The extracts and washes were combined, boiled
for 3 min, and lyophilized. The labeled oligosaccharides completely released
by this procedure were dissolved in water, mixed with standard
14C-labeled Glc3Man9GlcNAc and fractionated
by HPLC as described previously
(21). In some cases, an
internal standard of 14C-labeled
Glc1Man8GlcNAc was also used. It was prepared by
treatment of labeled Glc1Man9GlcNAc for 45 min at 37
°C with 0.2 mg of purified recombinant 1,2-mannosidase of
Saccharomyces cerevisiae
(22).
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RESULTS |
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NHK immunoprecipitated from cell lysates transfected with ER ManI migrated
slightly faster on SDS-PAGE than that from mock transfected cells
(Fig. 1A, closed
arrow). Kifunensine inhibited the degradation of NHK in cells
co-transfected with ER ManI (Fig. 2,
A and C) and also resulted in a small decrease
in NHK electrophoretic mobility, consistent with inhibition of ER
1,2-mannosidase activity (Fig.
2A, black arrow, compare lanes
13 with lanes 46). Therefore overexpression of ER
ManI enhances ERAD of misfolded NHK by accelerating mannose trimming from its
N-linked glycans. This conclusion was further supported by NHK
oligosaccharide analyses described below.
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Co-immunoprecipitation of ER ManI with NHK was detected in cells
transfected with ER ManI (Fig.
1A, open arrow). Notably, the transfected ER
ManI was degraded very rapidly (Fig.
1B, lanes 46, and see below). A broad
uncharacterized protein band of 170 kDa was co-immunoprecipitated with ER
ManI using anti-HA antibody (Fig.
1B, lanes 46). This 170-kDa band
decreased in parallel with the HA-tagged ER ManI during chase.
The degradation of co-transfected ER ManI was not inhibited by the addition of kifunensine (Fig. 2B, compare lanes 13 with lanes 46), in accordance with the fact that human ER ManI is not a glycoprotein (8, 9). Kifunensine also decreased the mobility of the 170-kDa co-immunoprecipitated protein band (Fig. 2B, compare lanes 1 and 4), suggesting that the 170-kDa band is glycosylated.
Inhibition of NHK Degradation by LactacystinThe effect of the proteasome inhibitor lactacystin on NHK degradation was then examined. The accelerated degradation of NHK in cells transfected with ER ManI was repressed by lactacystin (Fig. 3A, arrow, compare lanes 13 with lanes 46), indicating that the enhanced NHK degradation induced by ER ManI overexpression occurs through the ERAD pathway. Again, it is notable that the transfected ER ManI is rapidly degraded (Fig. 3B, lanes 13, open arrow, see also Figs. 1B and 2B). General protein synthesis examined by the incorporation of [35S]methionine into the trichloroacetic acid-insoluble fraction was not inhibited by transfection of ER ManI. The degradation of ER ManI is independent of proteasomes (Fig. 3B, compare lanes 13 with lanes 46). The degradation of ER ManI was not inhibited by other protease inhibitors including serine or cysteine protease inhibitors, calpain inhibitor, nor aspartic protease inhibitor.2
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Combined Effects of ER ManI and EDEM on NHK
DegradationRecently, we reported that mouse EDEM, which has
sequence homology to 1,2-mannosidases but lacks enzyme activity with
Man9GlcNAc as substrate, accelerates ERAD of misfolded NHK when
transfected into 293 cells
(15). EDEM was shown to
interact with NHK in co-immunoprecipitation experiments and was postulated to
recognize misfolded glycoproteins to sort them for retrotranslocation. We
first compared the effects of transfection with ER ManI and with EDEM on ERAD
of NHK. ER ManI and EDEM expressed individually shortened the intracellular
half-life of NHK (Fig.
4A, closed arrow, compare lanes
13 with lanes 46 or 79).
Co-immunoprecipitation of either ER ManI or EDEM was detected with NHK, using
antibody raised against A1AT (Fig.
4A, lanes 46, open arrow showing ER ManI;
lanes 79, thin arrow indicating EDEM). In the
immunoprecipitates using anti-HA antibody, protein bands of
170 and 150
kDa on SDS-PAGE were observed in ER ManI and EDEM transfected cells,
respectively (Fig. 4B,
lanes 46, open arrowhead for ER ManI; lanes 79,
closed arrowhead for EDEM). Although transfected ER ManI is rapidly
degraded, transfected EDEM is stable during the 2-h chase period
(Fig. 4B, compare
lanes 46 with lanes 79).
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Co-transfection of 293 cells with both ER ManI and EDEM further enhanced ERAD of NHK, compared with ER ManI alone, showing that there is a combined effect of these two ER resident proteins on NHK degradation (Fig. 5A, compare lanes 13 with lanes 46). Enhancement of degradation of NHK was also evident in cells co-transfected with both ER ManI and EDEM compared with EDEM alone (Fig. 5A, compare lanes 46 with lanes 79). Because of the rapid degradation of NHK observed following transfection of both ER ManI and EDEM in this experiment, cell lysates were examined at shorter chase periods. The levels of expression of ER ManI, EDEM, and NHK were reduced when both ER membrane proteins and substrate proteins were co-transfected. However, when the proteasome inhibitor lactacystin was added, similar amounts of labeled NHK were recovered from the immunoprecipitates of cell lysates (Fig. 5B, lanes 3, 4, 7, and 8), whereas only a very small amount of NHK was detected in cells overexpressing both ER ManI and EDEM after a 45-min chase in the absence of lactacystin (Fig. 5B, lane 6). These results confirm that the reduced amount of labeled NHK immunoprecipitated from cells co-transfected with ER ManI and EDEM was actually due to degradation by the proteasomes and not due to reduced synthesis of NHK.
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We then investigated whether ER ManI and EDEM form a complex with each other. We prepared antisera against synthetic peptides from both ER ManI and EDEM. Each anti-serum recognized its corresponding overexpressed protein by immunoprecipitation and Western blot analysis but hardly detected endogenous levels of the proteins. Accordingly, we co-transfected HA-tagged ER ManI with untagged EDEM or untagged ER ManI with HA-tagged EDEM. We performed immunoprecipitation of labeled cell lysates, as well as immunoprecipitation of cell lysates followed by Western blotting using either HA-tag or synthetic peptide antibodies. We did not detect any interaction of overexpressed ER ManI with EDEM (data not shown). The co-immunoprecipitated band observed in cells transfected with ER ManI was consistently larger than that observed in EDEM transfected cells (Fig. 4B, lanes 49, open and closed arrowheads), indicating that the proteins differ.
Characterization of Oligosaccharides on NHKTo determine the
structure of the oligosaccharides present on NHK, cells were labeled with
[2-3H]mannose, and the oligosaccharides released by Endo-H from
immunoprecipitated NHK were analyzed by HPLC
(Fig. 6). In mock transfected
cells the major labeled oligosaccharides on NHK were
Glc1Man9GlcNAc and Man9GlcNAc following
pulse-labeling, along with smaller amounts of Man8GlcNAc
(Fig. 6A). The
identity of the oligosaccharides was confirmed by exhaustive treatment with
jack bean 1,2-mannosidase followed by HPLC of the products.
Glc1Man9GlcNAc yielded about 47% labeled
Glc1Man4GlcNAc and mannose whereas Man9GlcNAc
was entirely converted to mannose (data not shown). With increasing times of
chase, trimming of NHK glycans to Man7GlcNAc, Man6GlcNAc
(Fig. 6, B and
C), and even to Man5GlcNAc after 4 h of chase
(data not shown) was observed. Therefore, the oligosaccharides on misfolded
NHK are trimmed beyond Man8GlcNAc2 by endogenous
1,2-mannosidase activity.
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DISCUSSION |
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In mock transfected cells, Glc1Man9GlcNAc,
Man9GlcNAc, and Man8GlcNAc were the major
oligosaccharides found on NHK, but smaller amounts of Man7GlcNAc
and Man6GlcNAc were also present. It is therefore possible that
oligosaccharides other than Man8B, or in addition to Man8B, may act as
recognition markers for ERAD. The relatively small amounts of
Man7GlcNAc and Man6GlcNAc found on NHK might be the
result of selective ERAD of specific NHK glycoforms bearing these
oligosaccharides. The presence of Glc1Man9GlcNAc on NHK
is consistent with previous studies demonstrating NHK association with
calnexin (26). The results
also show that the additional trimming beyond Man8GlcNAc is not due
to ER -mannosidase II because it was sensitive to kifunensine
(Fig. 8).
Overexpression of ER ManI, which increased ERAD of NHK, also greatly stimulated trimming of mannose residues to Man5GlcNAc, Man6GlcNAc, Man7GlcNAc, and Man8GlcNAc, as well as the formation of Glc1Man8GlcNAc. With the exception of the latter, the same oligosaccharides were formed in mock transfected cells, showing that the stimulation of NHK degradation caused by overexpression of ER ManI is not likely caused by the formation of atypical glycans. The increased trimming of N-glycans beyond Man8GlcNAc observed in cells overexpressing ER ManI is consistent with previous experiments with recombinant ER ManI in vitro indicating that ER ManI is less specific than previously believed (25).
The pattern of oligosaccharides found in EDEM and mock transfected cells is very similar, but there is always a shoulder ahead of the Man9GlcNAc peak that corresponds to a small amount of Glc1Man8GlcNAc, suggesting that EDEM somehow facilitates either trimming of Glc1Man9GlcNAc to Glc1 Man8GlcNAc or glucosylation of Man8GlcNAc by UDP-glucose: glycoprotein glucosyltransferase.
We demonstrated combined effects of ER ManI and of EDEM on ERAD. We previously reported that EDEM interacts with and accelerates the degradation of misfolded NHK, suggesting that EDEM may be a putative lectin, which recognizes misfolded glycoproteins for ERAD (15). NHK degradation is faster in cells co-transfected with both ER ManI and EDEM than in cells transfected with ER ManI or EDEM alone (Fig. 5A).
We also obtained evidence indicating that ER ManI and EDEM are part of different complexes because different proteins were co-immunoprecipitated with each of them, and no evidence that ER ManI and EDEM form a complex with each other was obtained. We propose a model whereby misfolded glycoproteins are recognized independently by ER ManI and by EDEM. The identities of the proteins co-immunoprecipitated with ER ManI and EDEM remain to be determined, which may aid in the elucidation of productive glycoprotein folding and in understanding the machinery leading to retrotranslocation and subsequent degradation by proteasomes.
Notably, ER ManI transfected into 293 cells was degraded very rapidly through a mechanism independent of cytoplasmic proteasomes (Fig. 3B, lanes 16). Despite its rapid turnover, we could detect the expression of transfected ER ManI by Western blot analysis using antibody against HA-tag or human ER ManI peptide. The level of expression of ER ManI in transfected cells is higher than that of endogenous ER ManI because we could not detect the latter by Western blot analysis using anti-peptide antibody. Although the molecular mechanism and the reason for the rapid degradation of ER ManI will require further investigation, the results suggest that the level of ER ManI expression is stringently controlled.
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FOOTNOTES |
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Present address: Dept. of Cell Science, Institute of Biomedical Sciences,
Fukushima Medical University School of Medicine, Fukushima 960-1295,
Japan.
¶ To whom correspondence should be addressed. Tel.: 81-75-751-3849; Fax: 81-75-751-4646; E-mail: nobuko{at}frontier.kyoto-u.ac.jp.
1 The abbreviations used are: ER, endoplasmic reticulum; ERAD, ER-associated
degradation; A1AT NHK, 1-antitrypsin-null Hong Kong; ER
ManI, ER
1,2-mannosidase I; EDEM, ER degradation enhancing
-mannosidase-like protein; Man8B, Man8GlcNAc2
isomer B; HEK 293, human embryonic kidney 293; Endo-H,
endo-
-N-acetylglucosaminidase H; DMEM, Dulbecco's modified
Eagle's medium; HA, hemagglutinin; PVDF, polyvinylidene difluoride.
2 N. Hosokawa, I. Wada, and K. Nagata, unpublished observation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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