Enhancement of ER degradation of misfolded null Hong Kong a1-antitrypsin. by human ER mannosidase I

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1 Enhancement of ER degradation of misfolded null Hong Kong a1-antitrypsin by human ER mannosidase I Nobuko Hosokawa, Linda 0. Tremblay*, Zhipeng You*, Annette Herscovics*, Ikuo Wada**, and Kazuhiro Nagata From the Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto , Japan, Core Research for Evolutional Science and Technology, JST, Japan,*McGill Cancer Centre, McGill University, Montréal H3G1Y6, Québec, Canada, **Department of Biochemistry, Sapporo Medical University School of Medicine, Sapporo , Japan To whom correspondence should be addressed: Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto , Japan. Tel.: ; Fax: ; nobuko@frontier.kyoto-u.ac.jp running title: ER ManI enhances glycoprotein ERAD 1

2 SUMMARY Misfolded glycoproteins synthesized in the endoplasmic reticulum (ER) are degraded by cytoplasmic proteasomes, a mechanism known as ERAD (ER-associated degradation). In the present study, we demonstrate that ERAD of the misfolded genetic variant null Hong Kong a1-antitrypsin is enhanced by overexpression of the ER processing a1,2-mannosidase (ER ManI) in HEK 293 cells, indicating the importance of ER ManI in glycoprotein quality control. We showed previously that EDEM, an enzymatically inactive mannosidase homolog, interacts with misfolded a1-antitrypsin and accelerates its degradation (Hosokawa et al. (2001) EMBO Reports 2: ). Herein we demonstrate a combined effect of ER ManI and EDEM on ERAD of misfolded a1-antitrypsin. We also show that misfolded a1- antitrypsin NHK contains labeled Glc 1 Man 9 GlcNAc and Man 5-9 GlcNAc released by endo-b- N-acetylglucosaminidase H in pulse-chase experiments with [2-3 H] mannose. Overexpression of ER ManI greatly increases the formation of Man 8 GlcNAc, induces the formation of Glc 1 Man 8 GlcNAc and increases trimming to Man 5-7 GlcNAc. We propose a model whereby the misfolded glycoprotein interacts with ER ManI and with EDEM, before being recognized by downstream ERAD components. This detailed characterization of oligosaccharides associated with a misfolded glycoprotein raises the possibility that the carbohydrate recognition determinant triggering ERAD may not be restricted to Man 8 GlcNAc 2 isomer B as previous studies have suggested. 2

3 INTRODUCTION The synthesis of glycoproteins containing N-glycans begins in the endoplasmic reticulum (ER) 1 with the addition of the Glc 3 Man 9 GlcNAc 2 precursor of N-linked glycans to nascent polypeptide chains. Subsequently, the ER lectin chaperones calnexin or calreticulin specifically bind to monoglucosylated N-glycans, promoting proper glycoprotein folding. This ER quality control process is mediated by glucosidases I and II that remove the glucose residues from Glc 3 Man 9 GlcNAc 2 and by UDP-glucose:glycoprotein glucosyl transferase that reglucosylates incompletely folded glycoproteins. Correctly folded glycoproteins exit the ER to their final destinations, whereas misfolded glycoproteins are readily degraded (for reviews, see 1-4). Many terminally misfolded proteins in the ER are degraded by cytoplasmic proteasomes, a mechanism known as ERAD (for reviews, see 5-7). Experimental evidence in yeast and mammalian cells suggests that ER a1,2-mannosidase I (ER ManI) that primarily removes the middle branch mannose from Man 9 GlcNAc 2 to form Man 8 GlcNAc 2 isomer B (Man8B) (8, 9) acts as a signal triggering ERAD of glycoproteins (10-12). Disruption of the ER a-mannosidase gene in yeast (10, 11) and inhibition of ER ManI in mammalian cells (13, 14) both prevent ERAD. We recently reported the molecular cloning of mouse EDEM (ER degradation enhancing a-mannosidase-like protein) and its involvement in glycoprotein ERAD. It was suggested that EDEM is a putative lectin which most likely binds Man8B on misfolded a1-antitrypsin and accelerates its degradation (15). Two groups also reported the yeast homolog of EDEM and its function in yeast ERAD (16, 17), indicating that the ERAD mechanisms involved in the degradation of misfolded glycoproteins are similar in yeast and mammals. Both ER ManI and EDEM are ER resident transmembrane proteins containing characteristic signature motifs of class I a1,2-mannosidases (glycosylhydrolase family 47). In 3

4 spite of this significant sequence similarity, EDEM lacks a1,2-mannosidase activity with Man 9 GlcNAc as substrate (15). In addition, these two proteins differ in their response to ER stress. EDEM is induced by various forms of ER stress, but ER ManI is not (15). In this report, we show that overexpression of human ER ManI accelerates the degradation of the terminally misfolded a1-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 a1-antitrypsin. Glycan analysis on misfolded NHK shows that overexpression of ER ManI greatly increases the formation of Man 8 GlcNAc 2 and Glc 1 Man 8 GlcNAc 2 and also stimulates trimming of N-linked oligosaccharides to Man 5 GlcNAc 2 in vivo. These experiments suggest that the misfolded glycoprotein interacts both with ER ManI and EDEM before being recognized by downstream ERAD components. 4

5 EXPERIMENTAL PROCEDURES Cell Culture and Transfection--Human HEK 293 cells were cultured in DMEM supplemented with 10 % fetal bovine serum, and transfection of plasmids encoding human ER ManI, mouse EDEM, and human a1-antitrypsin null Hong Kong were performed using FuGENE6 transfection reagents (Boehringer-Mannheim, Indianapolis, IN) according to the protocol recommended by the manufacturer. Approximately 1.5 x 10 5 cells were plated on poly-l lysine coated 3.5-cm dishes. After 24 h of plating, 0.5 mg of plasmid encoding ER ManI, EDEM, or pmh vector were mixed with 1 mg of plasmid encoding the a1-antitrypsin variant A1AT NHK for transfection, and cells were pulse-labeled and harvested 36 hours post transfection. Reagents--Kifunensine, 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 mg/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 construction--human ER ManI cdna was subcloned into the pmh vector (Roche) by PCR. The entire ORF (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 ORF amplicon was digested with HindIII and NotI, and cloned into the HindIII/NotI sites of pmh 5

6 in frame with the C-terminal HA tag. Mouse EDEM cdna was tagged with HA at its C- terminus in pcmv SPORT2 vector (Gibco-BRL, Rockville, MD) as described previously (15). The NHK variant was constructed as described elsewhere (15). Metabolic Labeling and Immunoprecipitation--Cells were preincubated in DMEM lacking methionine (Gibco-BRL, Rockville, MD) for 30 min, and labeled with Express [ 35 S] protein labeling mixture (NEN, Boston, MA) 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 PBS lacking Ca 2+ and Mg 2+, cells were incubated on ice for 20 min in lysis buffer (1% NP-40, 150 mm NaCl, 50 mm Tris-HCl, ph 8.0, supplemented with protease inhibitors) (15). For immunoprecipitation, NP-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 to cells transfected with NHK and pmh. To obtain approximately equal signals for the immunoprecipitated NHK at chase 0 h, the gels were exposed two or three 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 were 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 DTT, and separated by 10% SDS-PAGE, unless otherwise indicated in the legends. Radioactivity was quantified by exposing the gels to a phosphoimager (Storm, Amersham- 6

7 Pharmacia), and the gels were exposed to X-ray films (HR-HA, Fuji Photo Film Co. Ltd., Japan) afterwards. For labeling with [ 3 H]-mannose, cells were preincubated in DMEM containing 1 mm glucose (GIBCO BRL) for 30 min, and labeled with 7.4 MBq/ml of D-[2-3 H(N)]-mannose for 1 h. Cells were then chased for the times indicated in DMEM containing 25mM glucose (GIBCO BRL) supplemented with 5 mm mannose. Cell lysis, immunoprecipitation and SDS-PAGE were performed as described for the labeling with [ 35 S] protein labeling mixture, and the samples were blotted onto a PVDF membrane (BIO-RAD) in 5 mm sodium tetraborate buffer. The position of the [ 3 H]-mannose-labeled NHK bands on the PVDF membrane were identified by exposing the membrane to a phosphoimager. Antibodies--Antiserum against a1-antitrypsin was purchased from Cappel (ICN Pharmaceuticals, Inc., Ohio), and purified IgG against the HA-tag was obtained from Santa Cruz Biotechnology. Human ER ManI antibody was prepared by immunizing rabbits with a KLH-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 blotting--np40-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 DTT, and separated by 10% SDS-PAGE. For western blotting, 40 mg protein was loaded per lane. For western blotting following immunoprecipitation, 400 mg protein of NP40-soluble cell lysate was used as starting material. After blotting to a nitrocellulose membrane, antigen-antibody complexes were detected by ECL (Amersham- 7

8 Pharmacia), and exposed to X-ray films. Characterization of oligosaccharides The [ 3 H]mannose-labeled NHK bands on the PVDF membranes were cut into small pieces. The pieces were first rinsed with methanol and then with 0.1M sodium citrate, ph 5.5, three times for 15 min at 37 o C and the washes discarded. The membrane pieces were then incubated with Endo-H (New England Biolabs) in the same buffer at 37 o 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 [ 14 C]-labeled Glc 3 Man 9 GlcNAc and fractionated by HPLC as described previously (21). In some cases, an internal standard of [ 14 C]-labeled Glc 1 Man 8 GlcNAc was also used. It was prepared by treatment of labeled Glc 1 Man 9 GlcNAc for 45 min at 37 C with 0.2 mg of purified recombinant a-1,2-mannosidase of Saccharomyces cerevisiae (22). 8

9 RESULTS Enhanced Degradation of A1AT NHK by overexpression of ER ManI--To examine the effect of ER mannosidase I on ERAD of misfolded glycopoteins, human ER ManI cdna was transfected into HEK 293 cells along with the a1-antitrypsin (A1AT) variant null Hong Kong (NHK). A1AT is a serum protein belonging to a serine protease inhibitor superfamily. Mutations in a1-antitrypsin cause emphysema or liver cirrhosis (23). The NHK genetic variant of A1AT misfolds terminally in the ER and is degraded by ERAD (12, 15). Transfection of human ER ManI greatly enhanced the degradation of misfolded A1AT NHK in comparison with mock-transfected cells (Fig. 1A closed arrow, compare lanes 1-3 with 4-6). NHK was not secreted into the medium of cells co-transfected with ER ManI (data not shown), nor of cells co-transfected with vector alone (15, 24). Quantitative analysis revealed that the T 1/2 of transfected NHK in 293 cells was approximately 100 min. Co-transfection of ER ManI shortened the T 1/2 of NHK to about 50 min (Fig. 1C). 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. 2A, C), and also resulted in a small decrease in NHK electrophoretic mobility, consistent with inhibition of ER a1,2-mannosidase activity (Fig. 2A, black arrow, compare lanes 1-3 with 4-6). 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. 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. 9

10 1B, lanes 4-6, and see below). A broad uncharacterized protein band of approximately 170 kda was co-immunoprecipitated with ER ManI using anti-ha antibody (Fig. 1B, lanes 4-6). This 170kDa 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 1-3 with 4-6), in accordance with the fact that human ER ManI is not a glycoprotein (8, 9). Kifunensine also decreased the mobility of the 170kDa coimmunoprecipitated protein band (Fig. 2B, compare lanes 1 and 4), suggesting that the 170kDa band is glycosylated. Inhibition of NHK Degradation by Lactacystin --The 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 1-3 with 4-6), 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 1-3, open arrow, see also Figs. 1B, and 2B). General protein synthesis examined by the incorporation of [ 35 S] methionine into TCA-insoluble fraction was not inhibited by transfection of ER ManI. The degradation of ER ManI is independent of proteasomes (Fig. 3B, compare lanes 1-3 with 4-6). The degradation of ER ManI was not inhibited by other protease inhibitors including serine- or cysteine-protease inhibitors, calpain inhibitor, nor aspartic protease inhibitor (Hosokawa, N. et al., unpublished observation). Combined Effects of ER ManI and EDEM on NHK Degradation-- Recently, we reported that 10

11 mouse EDEM which has sequence homology to a1,2-mannosidases, but lacks enzyme activity with Man 9 GlcNAc as substrate, accelerates ERAD of misfolded NHK when transfected into 293 cells (15). EDEM was shown to interact with NHK in coimmunoprecipitation 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 1-3 with 4-6 or 7-9). Co-immunoprecipitation of either ER ManI or EDEM was detected with NHK, using antibody raised against A1AT (Fig. 4A, lanes 4-6, open arrow showing ER ManI; lanes 7-9, closed arrow indicating EDEM). In the immunoprecipitates using anti-ha antibody, protein bands of approximately 170 kda and 150 kda on SDS-PAGE were observed in ER ManI and EDEM transfected cells, respectively (Fig. 4B, lanes 4-6, open arrowhead for ER ManI; lanes 7-9, thin closed arrow for EDEM). Although transfected ER ManI is rapidly degraded, transfected EDEM is stable during the 2h chase period (Fig. 4B, compare lanes 4-6 with lanes 7-9). Co-transfection of 293 cells with both ER ManI and EDEM further enhanced ERAD of NHK, compared to ER ManI alone, showing that there is a combined effect of these two ER resident proteins on NHK degradation (Fig. 5A, compare lanes 1-3 with 4-6). Enhancement of degradation of NHK was also evident in cells co-transfected with both ER ManI and EDEM compared to EDEM alone (Fig. 5A, compare lanes 4-6 with 7-9). 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 11

12 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, 8), while 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. 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 antiserum recognized its corresponding overexpressed protein by immunoprecipitation and western blot analysis, but hardly detected endogenous levels of the proteins. Accordingly, we cotransfected HA-tagged ER ManI with untagged EDEM or untagged ER Man I with HAtagged 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 consistantly larger than that observed in EDEM transfected cells (Fig. 4B, lanes 4-9, open and closed arrowheads), indicating that the proteins differ. Characterization of oligosaccharides on NHK -- To determine the structure of the oligosaccharides present on NHK, cells were labeled with [2-3 H]-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 12

13 Glc 1 Man 9 GlcNAc and Man 9 GlcNAc following pulse-labeling, along with smaller amounts of Man 8 GlcNAc (Fig. 6 A). The identity of the oligosaccharides was confirmed by exhaustive treatment with jack bean a1,2-mannosidase followed by HPLC of the products. Glc 1 Man 9 GlcNAc yielded about 47% labeled Glc 1 Man 4 GlcNAc and mannose whereas Man 9 GlcNAc was entirely converted to mannose (data not shown). With increasing times of chase, trimming of NHK glycans to Man 7 GlcNAc, Man 6 GlcNAc (Fig. 6 B, C ) and even to Man 5 GlcNAc after 4h of chase (data not shown) was observed. Therefore, the oligosaccharides on misfolded NHK are trimmed beyond Man 8 GlcNAc 2 by endogenous a1,2-mannosidase activity. Overexpression of ER ManI caused a large increase in Man 8 GlcNAc, and an appearance of Glc 1 Man 8 GlcNAc (Fig. 6 D, E, F and Fig. 7 A). At the same time, there was a large decrease in the proportion of Glc 1 Man 9 GlcNAc and Man 9 GlcNAc 2 as well as an increase in Man 5-7GlcNAc compared to mock-transfected cells. An aliquot of the sample from Fig. 6F subjected to HPLC with an internal standard of Glc 1 Man 8 GlcNAc (Fig. 7A) confirmed the identity of the large peak of Glc 1 Man 8 GlcNAc, and the disappearance of Man 9 GlcNAc in cells transfected with ER ManI compared to mock-transfected cells. The Glc 1 Man 8 GlcNAc may have been formed by the action of ER ManI on Glc 1 Man 9 GlcNAc or by the addition glucose to the increased Man 8 GlcNAc by UDP-glucose:glycoprotein glucosyltransferase. These results show that ER ManI is capable of trimming beyond Man 8 GlcNAc 2 in vivo as was recently reported for recombinant ER ManI in vitro (25). In cells overexpressing EDEM the major labeled oligosaccharides were Glc 1 Man 9 GlcNAc and Man 9 GlcNAc, as in mocktransfected cells with smaller proportions of Man 6-8 GlcNAc (Fig. 6 G, H, I). When a sample similar to that shown in Fig. 6I was fractionated in the presence of the Glc 1 Man 8 GlcNAc 13

14 internal standard (Fig. 7B), Man 9 GlcNAc and a small amount of Glc 1 Man 8 GlcNAc were found. When cells were treated with kifunensine the formation of Man 6-8 GlcNAc was nearly completely inhibited (Fig. 8B) showing that ER a1,2-mannosidase II is not responsible for trimming NHK oligosaccharides in vivo. 14

15 DISCUSSION In the present study, we have shown that overexpression of human ER ManI in HEK 293 cells enhances ERAD of the terminally misfolded glycoprotein NHK. Accelerated degradation of NHK in cells overexpressing ER ManI was inhibited by lactacystin and by kifunensine, indicating that misfolded NHK was degraded through the ERAD pathway and that overexpression of ER ManI enhanced ERAD by accelerating trimming of its N-glycans. It has been postulated that the processing of N-linked oligosaccharides to Man8B by ER ManI acts as a signal for degradation of misfolded glycoproteins (1, 7, 13, 14). However, all studies to-date were based on the assumption that Man8B is the only oligosaccharide found on misfolded glycoproteins due to ER ManI activity and on observations of carbohydrate trimming from misfolded glycoproteins by shifts in their mobility upon SDS-PAGE. In this first study reporting HPLC analysis of the glycans on terminally misfolded NHK, the results clearly show that a mixture of oligosaccharides is found on NHK, and that the accepted assumption that only Man8B is formed by ER ManI needs to be revised. 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 a-mannosidase II since it was sensitive to kifunensine (Fig. 8). 15

16 Overexpression of ER ManI which increased ERAD of NHK also greatly stimulated trimming of mannose residues to Man 5 GlcNAc, Man 6 GlcNAc, Man 7 GlcNAc, Man 8 GlcNAc, as well as the formation of Glc 1 Man 8 GlcNAc. 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 Man 8 GlcNAc 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 are very similar, but there is always a shoulder ahead of the Man 9 GlcNAc peak that corresponds to a small amount of Glc 1 Man 8 GlcNAc, suggesting that EDEM somehow facilitates either trimming of Glc 1 Man 9 GlcNAc to Glc 1 Man 8 GlcNAc or glucosylation of Man 8 GlcNAc by UDPglucose: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 since 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 16

17 remain to be determined, which may aid in the elucidation of productive glycoprotein folding and in understanding the machinery leading to retro-translocation 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 1-6). In spite of 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 since 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. 17

18 References 1. Ellgaard, L., Molinari, M., and Helenius, A. (1999) Science 286, Herscovics, A. (1999) Biochim. Biophys. Acta 1473, Parodi, A. J. (2000) Annu. Rev. Biochem. 69, Helenius, A., and Aebi, M. (2001) Science 291, Bonifacino, J.S., and Weissman, A. M. (1998) Annu. Rev. Cell Dev. Biol. 14, Plemper, R. K., and Wolf, D. H. (1999) Trends Biochem. Sci. 24, Cabral, C. M., Liu, Y., and Sifers, R. (2001) Trends Biochem. Sci. 26, Gonzalez, D. S., Karaveg, K., Vandersall-Nairn, A. S., Lal, A., and Moremen, K. W. (1999) J. Biol. Chem. 274, Tremblay, L. O., and Herscovics, A. (1999) Glycobiology 9, Knop, M., Hauser, N., and Wolf, D. H. (1996) Yeast 12, Jakob, C. A., Burda, P., Roth J., and Aebi, M. (1998) J. Cell Biol. 142, Liu, Y., Choudhury, P., Cabral, C. M., and Sifers, R. N. (1999) J. Biol. Chem. 274, Tokunaga, F., Brostrom C., Koide, T. and Arvan, P. (2000) J. Biol. Chem. 275, Fagioli, C. and Sitia, R. (2001) J. Biol. Chem. 276, Hosokawa, N., Wada, I., Hasegawa, K., Yorihuzi, T., Tremblay, L.O., Herscovics, A., and Nagata, K. (2001) EMBO reports 2, Jakob, C.A., Bodmer, D., Spirig, U., Bättig, P., Marcil, A., Dignard, D., Bergeron, J.J.M., Thomas, D.Y., and Aebi, M. (2001) EMBO reports 2, Nakatsukasa, K., Nishikawa S-I, Hosokawa, N., Nagata, K., and Endo, T. (2001) J. Biol. 18

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20 ACKNOWLEDGEMENTS We thank Björn Stork for assistance in construct preparation and Dr. Pedro Romero for comments on the manuscript. FOOTNOTES This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (N.H., I.W. and K.N.) and by a grant from CIHR (A.H.). 1 The abbreviations used are: ER, endoplasmic reticulum; ERAD, ER-associated degradation; A1AT NHK, a1-antitrypsin null (Hong Kong); ER ManI, ER a1,2-mannosidase I; EDEM, ER degradation enhancing a-mannosidase-like protein; Man8B, Man 8 GlcNAc 2 isomer B; ORF, open reading frame; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; HEK 293, human embryonic kidney 293; TCA, trichroloacetic acid, Endo-H, endo-b-nacetylglucosaminidase H. 20

21 FIGURE LEGENDS Fig. 1. Transient transfection of ER ManI enhances the degradation of A1AT NHK variant in 293 cells. 293 cells transfected with plasmids encoding A1AT NHK and either ER ManI (+) or the pmh vector (-) were labeled with EXPRESS 35 S protein labeling mixture for 15 min, and chased for the times indicated. The closed arrow indicates the position of A1AT NHK, and the open arrows show that of ER ManI. Molecular weight marker positions are noted on the left. A, autoradiogram of SDS-PAGE of the immunoprecipitates using anti-a1at antibody. Because the radioactivity of NHK co-transfected with ER ManI at 0 h chase was always weaker than that from cells co-transfected with the vector (pmh), lanes 4-6 were exposed twice as long as lanes 1-3. B, autoradiogram of the immunoprecipitates from the same samples as in A, except using anti-ha antibody. The open arrowhead indicates proteins that were coimmunoprecipitated with transfected ER ManI. C, quantitative analysis of the NHK degradation. The rate of NHK degradation in cell lysates is plotted on a semi-log graph. The data shown are the means with standard deviations of five independent experiments. Radioactivity was quantified using a phosphoimager. The value at 0 h chase was set arbitrarily to 1 in both mock and ER ManI transfected cells. Fig. 2. Kifunensine inhibits the degradation of NHK in ER ManI transfected cells. Kifunensine (5 mg/ml) was added to the culture medium 4 h prior to metabolic labeling, and was present in the medium during the pulse labeling and chase period. Both NHK and ER ManI were transfected in all the cells. Immunoprecipitates were prepared as in Fig. 1. Arrow notations are as described in Fig.1. A, B, autoradiogram of the immunoprecipitates using 21

22 antibody against A1AT (A) and HA tag (B), respectively. C, quantification of NHK within the cell. The radioactivity of NHK at 0 h chase was set arbitrarily to 1.0, and the means with standard deviations of three independent experiments were shown. Fig. 3. Lactacystin inhibits the degradation of NHK in ER ManI transfected cells. Cells were transfected with ER ManI and NHK, and treated with or without lactacystin (1mM) for 4 h prior to metabolic labeling. In lactacystin + cells, drug was present during pulse labeling and during chase. Autoradiogram of SDS-PAGE of immunoprecipitates with anti-a1at antibody (A) or with anti-ha tag antibody (B). Arrows and an arrowhead indicate the same species as in Fig. 1. Fig. 4. Comparison of the effects of ER ManI and EDEM on NHK degradation. Cells were transfected with pmh (vector), ER ManI or EDEM, and immune complexes with anti-a1at antibody (A) and anti-ha antibody (B) were separated by SDS-PAGE. To obtain nearly equal amount of NHK radioactivity at chase 0 h in all the transfectants, 3 and 2 volume of cell lysate was used for immunoprecipitation in ER ManI and EDEM transfected cells to that of mock transfected cells, respectively. Open arrows indicate the position of ER ManI, and thin arrows show that of EDEM. Open and closed arrowheads indicate the bands that coimmunoprecipitated with ER ManI and EDEM, respectively. Closed arrow indicates the position of NHK. C, immunoprecipitates with anti-a1at antibody prepared at each chase period were separated in adjacent lanes on SDS-PAGE. Three and two volumes of cell lysate was used for immunoprecipitation in ER ManI and EDEM transfected cells compared to that of mock transfected cells, respectively. 22

23 Fig. 5. Combined effects of ER ManI and EDEM on NHK degradation. NHK degradation in cells co-expressing EDEM in addition to ER ManI. Equal amount of cell lysates were used for immunoprecipitation of all the samples. A, cells were pulse-labeled for 15 min, and chased for the periods indicated. For lanes 4-6, the film was exposed twice as long. The result of one representative experiment is shown, and the relative radioactivity of the NHK is indicated below each lane (radioactivity at chase 0 h of each transfection was arbitrarily set to 1.0). B, cells were pulse-labeled for 15 min (P) and chased for 45 min (C) with or without lactacystin. Specific immunoprecipitates using anti-a1at antibody are shown, and the relative radioactivity was indicated below each lane (radioactivity of lane 1 was arbitrarily set to 1.0). Fig. 6. Oligosaccharides on misfolded NHK transfected into 293 cells. 293 cells transfected with A1AT NHK and either the pmh vector (A-C), ER ManI (D-F) or EDEM (G-I) were metabolically labeled with [ 3 H]-mannose for 1 h, and chased for 0 h (A, D, G), 1 h (B, E, H) or for 2 h (C, F, I). Following immunoprecipitation using anti-a1at antibody, samples were separated by 10% SDS-PAGE, and were blotted onto a PVDF membrane. The oligosaccharides released from NHK by Endo-H were fractionated by HPLC. Arrows indicate the position of the Glc 3 Man 9 GlcNAc internal standard; M5-M9 show [ 3 H]labeled Man 5-9 GlcNAc and G1M8 corresponds to Glc 1 Man 8 GlcNAc (shown in Fig. 7), G1M9, which co-eluted with [ 14 C]labeled Glc 1 Man 9 GlcNAc (not shown). Fig. 7. Co-chromatography of oligosaccharides with Glc 1 Man 8 GlcNAc standard. 23

24 293 cells transfected with A1AT NHK and either ER Man I (A) and EDEM(B) were metabolically labeled with [3H]-mannose for 1 h, chased for 2 h and then analyzed, as described in Fig. 6. The arrow indicates the position of the Glc 3 Man 9 GlcNAc internal standard and the dotted line the position of [ 14 C]-labeled Glc 1 Man 8 GlcNAc internal standard. Fig. 8. Effect of kifunensine on the oligosaccharides of misfolded NHK. Cells were transfected with NHK, and analyzed as described in Fig. 6. Cells were harvested after a 3 h chase. The cells were either preincubated in the absence (A) or presence (B) of 5 mg/ml kifunensine for 4 h prior to metabolic labeling with [ 3 H]-mannose. The size of the oligosaccharides is indicated as in Fig

25 A B α-a1at Ab α-ha Ab C ERManI - + ERManI - + Chase (h) Chase (h) mock ERManI relative radioactivity chase period 2 h Fig. 1. Hosokawa et al.

26 A B C Kifunensine α-a1at Ab - + Kifunensine α-ha Ab - + Chase (h) Chase (h) mock Kifnensine relative radioactivity chase period 2 h Fig. 2. Hosokawa et al.

27 A α-a1at Ab Lactacystin - + B α-ha Ab Lactacystin - + Chase (h) Chase (h) Fig. 3. Hosokawa et al.

28 A α-a1at Ab B α-ha Ab plasmid pmh perm pedem plasmid pmh perm pedem Chase (h) Chase (h) C Chase α-a1at Ab 0 1 (h) plasmid pmh permani pedem pmh permani pedem Fig. 4. Hosokawa et al.

29 A α-a1at Ab Plasmids ERManI ERManI + EDEM EDEM Chase (h) B α-a1at Ab Plasmids Lact ERManI ERManI + EDEM P C P C P C P C Fig. 5. Hosokawa et al.

30 Chase 0 h Chase 1 h Chase 2 h Mock 500 A 250 M9 B 120 M9 C M G1M9 G1M M8 100 G1M9 M8 40 M M7 M7 M6 M7 M ER Man I EDEM Radioactivity (cpm) M6 M7 M8 G1M8 G1M M7 M8 G1M9 M D G M6 M7 M8 G1M8 G1M9 E M7 M6 G1M8 M8 G1M M9 G1M9 H M M7 M8 M7 M Fraction Number M5 M9 G1M9 F I

31 M8 G1M8 A Radioactivity (dpm) M7 M6 G1M9 M G1M9 B M9 M8 M7 M Fraction Number

32 M9 A 150 G1M9 100 M8 Radioactivity (cpm) 50 M6 M B M G1M9 100 M Fraction Number

33 Enhancement of ER degradation of misfolded null Hong Kong α1-antitrypsin by human ER mannosidase I Nobuko Hosokawa, Linda O. Tremblay, Zhipeng You, Annette Herscovics, Ikuo Wada and Kazuhiro Nagata J. Biol. Chem. published online May 6, 2003 Access the most updated version of this article at doi: /jbc.M Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts