edem3, a soluble edem homolog, enhances glycoprotein erad

1

EDEM3, a Soluble EDEM Homolog, Enhances Glycoprotein

ERAD and Mannose Trimming

Kazuyoshi Hirao*, †, ll, Yuko Natsuka

*, †, ll, Taku Tamura

‡, †, IkuoWada

‡, †, Daisuke

Morito*, †, Shunji Natsuka

§, †, Pedro Romero

¶, Barry Sleno

¶, Linda O. Tremblay

¶,

Annette Herscovics¶, Kazuhiro Nagata

*, †, Nobuko Hosokawa

*, †

From the *Department of Molecular and Cellular Biology, Institute for Frontier Medical

Sciences, Kyoto University, Kyoto 606-8397, Japan, †CREST, JST, Japan, ‡Department ofCell Sciences, Institute of Biomedical Sciences, Fukushima Medical University School ofMedicine, Fukushima 960-1295, Japan, §Department of Chemistry, Osaka UniversityGraduate School of Science, Toyonaka 560-0043, Japan and ¶McGill Cancer Centre,

Montréal, Québec, Canada H3G 1Y6llThese two authors contributed equally to this work

Running title: EDEM homolog enhances glycoprotein ERAD

Address correspondence to: Nobuko Hosokawa, Department of Molecular and Cellular Biology,

Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8397, Japan. Tel:

+81-75-751-3849; Fax: +81-75-751-4646; E-mail: [email protected]

Quality control in the endoplasmic

reticulum ensures that only properly folded

proteins are retained in the cell through

mechanisms that recognize and discard

misfolded or unassembled proteins in a

process called ERAD for endoplasmic

reticulum-associated degradation. We

previously cloned EDEM, ER degradation

enhancing aa-mannosidase-like protein, andshowed that it accelerates ERAD of

misfolded glycoproteins. We now cloned

mouse EDEM3, a soluble homolog of

EDEM. EDEM3 consists of 931 amino

acids and has all the signature motifs of

Class I aa-mannosidases (glycosyl hydrolasefamily 47) in its N-terminal domain and a

protease-associated motif in its C-terminal

region. EDEM3 accelerates glycoprotein

ERAD in transfected HEK293 cells, as

shown by increased degradation of

misfolded aa1-antitrypsin variant (null

Hong Kong, NHK) and of TCRaa.Overexpression of EDEM3 also greatly

stimulates mannose trimming not only

from misfolded NHK but also from total

glycoproteins, in contrast to EDEM which

has no apparent aa1,2-mannosidase activity.Furthermore, overexpression of the E147Q

EDEM3 mutant, which has the mutation in

one of the conserved acidic residues

essential for enzyme activity of

aa1,2-mannosidases, abolishes the

stimulation of mannose trimming and

greatly decreases the stimulation of ERAD

by EDEM3. These results show that

EDEM3 has aa1,2-mannosidase activity invivo suggesting that the mechanism

whereby EDEM3 accelerates glycoprotein

ERAD is different from that of EDEM.

http://www.jbc.org/cgi/doi/10.1074/jbc.M512191200The latest version is at JBC Papers in Press. Published on January 23, 2006 as Manuscript M512191200

Copyright 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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ER quality control is an elaborate

mechanism conserved from yeast to mammals

ensuring that newly synthesized proteins in the

ER fold and assemble correctly, and that only

proteins that acquire their correct

conformations are sorted further into the

secretory pathway (1-4). During this process,

proteins that fail to attain their native

conformation due to mutations of the

polypeptides or to ER stress conditions

adverse for protein folding, as well as orphan

subunits are degraded in a process known as

ERAD for ER-associated degradation (3, 5-7).

The recognition of misfolded proteins for

ERAD is still poorly understood, but there is

increasing evidence for a role of mannose

trimming in the targeting of glycoproteins for

ERAD (8, 9). In mammalian cells,

overexpression of ER a-mannosidase I

stimulates ERAD of misfolded glycoproteins

(10, 11) while the a1,2-mannosidaseinhibitors kifunensine and

1-deoxymannojirimycin stabilize misfolded

glycoproteins (12-16). These observations

suggested that Man8GlcNAc2 isomer B, the

major product of the ER a1,2-mannosidase, isa recognition marker for ERAD of

glycoproteins, but this view is being

challenged since there is increasing evidence

that trimming to smaller oligosaccharides

occurs on ERAD substrates (10, 17-19). We

previously cloned mouse EDEM (ER

degradation enhancing a-mannosidase-likeprotein) as a cDNA whose expression is

upregulated by ER stress, and showed that

EDEM accelerates glycoprotein ERAD (20).

EDEM is an integral ER membrane protein

that has all the signature motifs of Class I

a1,2-mannosidases (glycosyl hydrolase family47), but no detectable enzyme activity as a

processing a-mannosidase in vivo or in vitro.

Recently, it was found that EDEM extracts

terminally misfolded glycoproteins from the

calnexin cycle (21, 22). In S. cerevisiae, theER a1,2-mannosidase as well as

Htm1p/Mnl1p belonging to the same protein

family are also involved in ERAD since

disruption of the genes delays the ERAD of

glycoproteins (23, 24). Although EDEM and

Htm1p/Mnl1p were postulated to be lectins

involved in targeting misfolded glycoproteins

for ERAD, the precise mechanisms whereby

EDEM and Htm1p/Mnl1p recognize and sort

misfolded glycoproteins for degradation is still

unclear, and their role as lectins has not been

established directly. While this manuscript

was in preparation EDEM2 was reported to

stimulate ERAD of misfolded glycoproteins

without affecting mannose trimming (25, 26).

Here, we show that EDEM3 is a soluble

EDEM homolog located in the ER of

transfected mammalian cells that accelerates

ERAD of misfolded glycoproteins through a

mechanism likely to be different from that of

EDEM or EDEM2 since EDEM3 greatly

stimulates mannose trimming in vivo.

EXPERIMENTAL PROCEDURES

Cloning of mouse EDEM homolog—Five ESTclones were sequenced, and one clone

(G431003D06) which was kindly provided by

Dr. Y. Hayashizaki (RIKEN, Japan) (27)

contained the entire EDEM3 cDNA.

Sequencing was performed by PCR-based

dideoxy-termination method using BigDye v.

3 (ABI) and PCR 9700 (Perkin-Elmer), and

then analyzed with the ABI 3100 capillary

sequencer (ABI).

Plasmid construction—The coding region ofEDEM3 cDNA was subcloned into

pcDNA3.1+ by PCR, and the HA-tag was


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introduced prior to the -KDEL ER retention

signal (EDEM3-HA). The NHK-QQQ mutant

was created by replacing Asn residues of

NHK glycosylation sites with Gln using the

Quick ChangeTMsite-directed mutagenesis kit

(STRATAGENE). The EDEM3 E147Q

mutant was constructed by the same method.

FLAG-tagged TCRa was kindly provided byDr. F. Tokunaga (Osaka City Univ. Grad. Sch.

Med.)

In vitro translation andtranslocation—EDEM3-HA cDNA was

linearized with EcoRI, and transcribed in vitroby T7 RNA polymerase (Promega). The

transcript was then translated in vitro in thereticulocyte lysate (Promega, Flexi-lysate)

supplemented with [35S]-methionine with and

without canine pancreas microsomes. The

localization of the EDEM3 product was

determined by the alkali-floatation method

(28), monitored by 10%SDS-PAGE. Mouse

EDEM tagged with HA was used as a control

ER membrane protein (20), and HSP47 as a

control ER lumenal protein (29).

Cell culture and transfection—HEK293,BALB/c3T3, COS7, and HeLa S3 cells

(provided by Japan Health Science Research

Resources Bank) were cultured in DMEM

supplemented with 10% fetal bovine serum

and antibiotics (100 U/ml penicillin G and 100

ng/ml streptomycin), in humidified air

containing 5% CO2 at 37oC. PC12h cells were

cultured in DMEM supplemented with 5%

fetal bovine serum and 5% horse serum.

HepG2 cells stably overexpressing the

EDEM3 were established using the pCX4bsr

plasmid which has a retrovirus LTR (30), and

selected in the presence of 30 mg/ml ofblasticidin (Funakoshi, Japan). Plasmids were

purified with a Plasmid Maxi Kit (Qiagen) and

transfected using the FuGene6 transfection

reagent (Roche) as described previously (10).

Northern blotting—Total RNA from culturedcells was extracted using TRIzol

TMreagent

(Invitrogen). After separation on

formaldehyde-denatured gel, RNAs were

blotted onto a nylon membrane (GeneScreen

plus, DuPont-NEN), and then were hybridized

with [32P]-labeled probes for 2 to 16 h in

PerfectHybTM

(TOYOBO, Japan)

hybridization solution. An MTNTM

Blot

membrane (Clontech) was used to examine

mouse tissue distribution. Probes for EDEM,BiP, b-actin, and HSP70 were labeled by themulti-primed labeling method

(Boehlinger-Mannheim). To detect EDEM3mRNA, probes were labeled by unidirectional

PCR using the 571 bp fragment of the EDEM3cDNA near its translational termination site, to

increase the specific activity of the probe, and

to avoid cross reactivity with EDEM.

Metabolic labeling, immunoprecipitation, andSDS-PAGE—Metabolic labeling, cell lysis,immunoprecipitation, and SDS-PAGE were

carried out as previously described (10),

except that DMEM lacking both methionine

and cysteine was used instead of DMEM

lacking methionine.

Antibodies—Antibodies against a1-AT werepurchased from DAKO (rabbit polyclonal).

Polyclonal antibodies against the HA-tag were

obtained from Santa Cruz Biotechnology,

anti-FLAG M2 antibody (mouse monoclonal)

was from Stratagene, and mouse monoclonal

antibody against PDI was purchased from

StressGen.

Reagents—Lactacystin was purchased fromKyowa Medics (Japan), and kifunensine was a

generous gift from Fujisawa Pharmaceutical

Co. (Osaka, Japan). EndoH and PNGaseF

(N-glycanaseF) were purchased from Roche

Applied Science or NEB.


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Immunocytochemistry—COS7 cells were

plated on a cover glass placed in a

3.5-cm-diameter dish approximately 24 h prior

to transfection. Twenty-four hours after

transfection, the cells were fixed with 4%

paraformaldehyde for 15 min at room

temperature, and incubated with anti-a1antibodies (rabbit polyclonal) and anti-PDI

antibody (mouse monoclonal) for 1 h, and

then with an Alexa546-labeled anti-rabbit and

an Alexa488-labeled anti-mouse antibody for

1h. Samples were examined by confocal

microscopy (LSM 510 META, Carl-Zeiss,

Germany).

Oligosaccharide analysis of NHK and totalcellular glycoproteins—Oligosaccharideanalysis of [

3H]-mannose-labeled NHK by

HPLC was performed as described previously

(10, 31). For oligosaccharide analysis of total

glycoproteins in HepG2 and cell line stably

overexpressing EDEM3, N-glycans were

released by hydrazinolysis, fluorescently

labeled using 2-aminopyridine, and were

fractionated by size on an NH2-P HPLC

column (Shodex Asahipak NH2P50, 0.46 x 15

cm, Showa Denko, Japan) as described

previously (32). Briefly, samples were loaded

onto the column and the oligosaccharides were

eluted at 0.5 ml/min by a linear gradient of

solvent A (acetonitrile) and solvent B (50 mM

ammonium acetate, pH 7.0) from 4:1 (v/v) to

3:7 (v/v) for 50 min. Oligosaccharides eluted

in each peak were identified by reverse phase

HPLC.

RESULTS

Cloning of EDEM3, a soluble homolog ofEDEM--A transcript named C1orf22 was

recently mapped in a region susceptible to

human hereditary prostate cancer (33). The

predicted ORF of C1orf22 has a sequence

similar to Class I a1,2-mannosidases (glycosylhydrolase family 47) and shows highest

homology to the KIAA0212 gene product (34).

Since KIAA0212 is the human ortholog of the

mouse EDEM gene (20), we searched the

mouse EST data base for the C1orf22

homolog. Sequencing of EST clones revealed

a mouse cDNA of 6,349 base

(GenBank/EMBL/DDBJ Accession number

AB188342), encoding a protein of 931 amino

acids (Fig. 1A). This ORF consists of a region

(amino acids 60-499) similar to Class I

a1,2-mannosidases (glycosyl hydrolase family47) followed by a C-terminal region

containing a protease-associated motif (amino

acids 686-780) (35) that is lacking in EDEM

and in all Class I a1,2-mannosidases studiedso far. It has a putative signal sequence at the

N-terminus as well as an ER-retrieval signal

(-KDEL) at its C-terminus.

The mouse EDEM3 cDNA sequence

exhibits 90% identity with its human ortholog

C1orf22 in the coding region, and 76% overall

identity. The mouse and human orthologs are

91% identical in amino acid sequence. The

mouse EDEM3 has 44 additional amino acids

at the N-terminus compared to the C1orf22

translation product. The a-mannosidasedomain shows 44% and 30% amino acid

identity between EDEM3 and EDEM, and

between EDEM3 and ER a-mannosidase I(ER ManI), respectively. All nine acidic

amino acids which are essential for a-1,2mannosidase activity (36) are conserved

between these three proteins (Fig. 1A),

although the two Cys important for activity of

the yeast ER a1,2-mannosidase (37) are notconserved in either EDEM3 or EDEM. The

PA motif is a consensus sequence found in

several proteases (35), the significance of


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which in EDEM3 is currently unknown.

Expression of EDEM3 and effects of ERstress--Northern blotting shows a major

EDEM3 transcript of approximately 6.5 kb inall mouse tissues, as expected from the cloned

cDNA, with relatively high levels in liver,

heart and kidney (Fig. 2A), as reported for the

human homolog C1orf22 (33). EDEM mRNAis highly expressed in liver, moderately in

kidney, whereas the expression is low in heart,

brain and skeletal muscle (Fig. 2A). We

examined whether EDEM3 mRNA expressionis regulated by ER stress, as previously shown

for EDEM (20, 38). Addition of tunicamycin,

which induces ER stress by inhibiting

N-glycosylation of proteins, causes a mild

induction of EDEM3mRNA depending on thecell lines (Fig. 2B). The EDEM3 transcript isincreased about 1.5- 2-fold in BALB/c 3T3,

293 and PC12h cells, whereas no induction is

observed in COS7 and in HeLa S3 cells.

EDEM3mRNA is also upregulated by treatingcells with the glucose analogue

(2-deoxyglucose) or the calcium ionophore

A23187 (ER stress), but the level of EDEM3mRNA does not change in cells exposed to

cytosolic stress that greatly stimulates HSP70

expression (Fig. 2C).

EDEM3 is localized in the ER lumen--Toestablish whether the hydrophobic region near

the N-terminus acts as a cleavable signal

sequence upon co-translational translocation

into the ER, or whether it serves as a

transmembrane region, we separated integral

membrane proteins from soluble proteins by

alkali-floatation. When EDEM3 RNA is

translated in vitro, most of the radioactiveEDEM3 is recovered in the soluble fraction

(Fig. 3A). We observed a shift in the size by

SDS-PAGE of 110 kDa to 120 kDa when

EDEM3 was translocated into microsomes

(data not shown). Treatment with EndoH or

PNGaseF shows the removal of high-mannose

type N-glycans from EDEM3 (Fig. 3B), as

predicted from the sequence (Fig. 1B).

Next, we examined the intracellular

localization of EDEM3 by transfecting

HA-tagged EDEM3 transiently into COS7

cells. Indirect immunofluorescence shows a

fine reticular network pattern around the

nucleus that colocalizes with the ER resident

protein PDI (Fig. 3C). EDEM3 is not secreted

into the medium in pulse-chase experiments of

transiently transfected HEK293 cells (data not

shown). Thus, we conclude that EDEM3 is an

ER lumenal protein.

EDEM3 accelerates glycoprotein ERAD--Wethen investigated whether EDEM3 affects

glycoprotein ERAD. We used the

a1-antitrypsin genetic variant null (Hong

Kong) (NHK) as a soluble ERAD substrate

(13, 39). Co-transfection of EDEM3 enhances

the ERAD of NHK (Fig. 4A, B).

Co-immunoprecipitation of EDEM3 with

NHK was observed using antibodies to either

a1-AT or HA-tag (Fig. 4A), indicating thatEDEM3 interacts with NHK in the cells.

Co-immunoprecipitation was most prominent

after 1h and 2h chase. At these times the shift

in mobility of NHK indicates additional

trimming of the oligosaccharides. This

observation suggests that the interaction

between EDEM3 and NHK is stronger with

increased mannose trimming from NHK (Fig.

4A, compare lanes 5 and 6 with lane 4, and

lanes 8 and 9 with lane 7).

NHK degradation is inhibited by lactacystin

in the presence of co-transfected EDEM3,

showing that EDEM3 accelerates glycoprotein


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ERAD by proteasomes (Fig. 4C). The

mobility shift of NHK after chase periods was

consistently larger in cells co-transfected with

EDEM3 than in cells co-transfected with

mock vector (Fig. 4A, compare lanes 4-6 with

1-3). The electrophoretic mobility shift of

NHK in EDEM3 overexpressing cells is

compared with that of deglycosylated NHK

prepared by PNGaseF digestion (Fig. 4D).

Addition of the a1,2-mannosidase inhibitorkifunensine greatly inhibits NHK degradation

in cells overexpressing EDEM3, and reduces

the mobility of NHK on SDS-PAGE (Fig. 4E).

This suggests that the mobility shift of NHK in

cells co-transfected with EDEM3 is caused by

the mannose trimming from the N-linked

oligosaccharides.

Since different mechanisms for ERAD of

soluble and transmembrane proteins have been

proposed (40-42), we examined the effect of

EDEM3 on a FLAG-tagged TCRa, a

glycosylated transmembrane ERAD substrate

(43). Co-expression of EDEM3 enhances the

degradation of TCRa-FLAG (Fig. 5A, B),

which is partly inhibited by kifunensine

treatment (Fig. 5A). However, EDEM3 does

not affect the degradation of NHK lacking all

three N-glycosylation sites (NHK-QQQ),

demonstrating its specificity for glycoproteins

(Fig. 5C, D). NHK-QQQ is degraded faster

than NHK which bears the three N-glycans,

and we have confirmed that NHK-QQQ was

also degraded by ERAD (data not shown).

These data indicate that the acceleration of

glycoprotein ERAD by EDEM3 depends on

mannose trimming from the N-glycans.

Effect of EDEM3 on mannose trimming fromN glycans of NHK and of total glycoproteins--Since the results in Fig. 4 suggested that

overexpression of EDEM3 stimulates

mannose trimming from N-glycans on NHK,

the oligosaccharides were examined after

labeling 293 cells with [3H]-mannose. The

N-glycans released from NHK by EndoH

were analyzed by HPLC. Overexpression of

EDEM3 greatly stimulates trimming of

N-glycans from NHK to Man7GlcNAc2 and

Man6GlcNAc2 (Fig. 6A, B), but there is

relatively little Man8GlcNAc2 found on NHK

in cells overexpressing EDEM3 compared to

mock transfected cells. At 0h chase, there is

only a trace amount of Man6GlcNAc2 and

Man7GlcNAc2 on NHK from mock

transfected cells whereas significant labeled

Man6GlcNAc2 and Man7GlcNAc2 are

observed in cells transfected with EDEM3.

The relative amount of Man6GlcNAc2 and

Man7GlcNAc2 increases with time of chase in

EDEM3 transfected cells and is greater at 1h

chase than the proportion of Man6GlcNAc2

and Man7GlcNAc2 in mock transfected cells.

Furthermore, at all time points the percent

labeled Man6GlcNAc2 and Man7GlcNAc2 is

much greater in EDEM3 transfected cells than

in the control. In contrast, the percent

radioactivity in Man8GlcNAc2 is always

much lower in the presence of EDEM3. This

pattern of oligosaccharides indicates that

overexpression of EDEM3 stimulates

mannose trimming from NHK. To determine

whether the increased mannose trimming is

due to intrinsic mannosidase activity of

EDEM3, the effects of the E147Q mutant on

NHK oligosaccharides was studied. Glu147is a

conserved residue corresponds to Glu132and to

Glu330in the active site of yeast and human ER

a1,2-mannosidases, respectively (44, 45). It isessential for enzyme activity since mutation of

this residue abolishes a1,2-mannosidaseactivity (36).

The pattern of oligosaccharides on NHK


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obtained from cells overexpressing E147Q is

identical to that of mock transfected cells. And

the relative amounts of Man6GlcNAc2 and

Man7GlcNAc2 are the same as in control cells

(Fig. 6A, B).

Stimulation of mannose trimming by

EDEM3 was also demonstrated by analyzing

N-glycans of total glycoproteins from HepG2

cells stably overexpressing EDEM3 (Fig. 6C).

There is a large increase of Man6GlcNAc2

concomitant with a relative decrease of

Man7-8GlcNAc2, compared to the parental

HepG2 cells. All these results demonstrate that

EDEM3 has a1,2-mannosidase activity invivo.

Following labeling with

[35S]-methionine/cysteine, NHK degradation

was greatly reduced in 293 cells

overexpressing the E147Q mutant compared

to wild-type EDEM3 (Fig. 6D), indicating that

the mannosidase activity of EDEM3 is

important for its effect on ERAD of NHK.

DISCUSSION

The present work shows that EDEM3 is a

soluble homolog of EDEM that accelerates

ERAD of both soluble NHK and

membrane-bound TCRa in an

N-glycan-dependent manner. The extent of

ERAD stimulation on NHK degradation is

similar to that previously reported following

overexpression of EDEM and of ER ManI (10,

11, 20). However, the role of EDEM3 in

ERAD of NHK is likely to be different from

that of EDEM, since EDEM3 overexpression

greatly stimulates mannose trimming of

N-glycans from NHK whereas overexpression

of EDEM does not (10).

Furthermore, when EDEM3 is

overexpressed, the pattern of oligosaccharides

released from NHK is very different from that

observed on NHK isolated from ER

a1,2-mannosidase I transfected cells. In cellsoverexpressing EDEM3, there is extensive

trimming of N-glycans to Man6-7GlcNAc2

with relatively little Man8GlcNAc2 (Fig. 6A,

B) whereas in cells overexpressing ER ManI,

there is increased accumulation of

Man8GlcNAc2 and Glc1Man8GlcNAc2

concomitant with increased trimming to

smaller oligosaccharides (10). Importantly, the

effect of EDEM3 on the trimming of

N-glycans is abolished by mutating the

essential acidic residue Glu147to Gln (Fig. 6A,

B, E147Q), and the increased ERAD of NHK

due to EDEM3 overexpression is greatly

reduced by this mutation (Fig. 6D). This

residue is found in the active site of processing

Class I a-mannosidases by X-ray

crystallography (44-48) and is essential for

enzyme activity (36). Although these results

strongly indicate that EDEM3 stimulation of

NHK ERAD is caused by its

a1,2-mannosidase activity, an alternative lesslikely interpretation is that the mutation of

Glu147to Gln affects the conformation of

EDEM3 and thus abolishes the effect on NHK

degradation independently of enzyme activity.

Since overexpression of EDEM3 stimulates

mannose trimming from total glycoproteins as

well as from the misfolded glycoprotein NHK

(Fig. 6), EDEM3 is most likely acting as a

processing a1,2-mannosidase in vivo,accelerating trimming of Man8GlcNAc2

oligosaccharides to Man6-7GlcNAc2. Its

specificity appears to be different from that of

ER ManI that greatly stimulates trimming to

Man8GlcNAc2 and Glc1Man8GlcNAc2 (10).

The present results indicate that

Man8GlcNAc2 is not an exclusive targeting

signal for ERAD of glycoproteins and that


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smaller oligosaccharides (Man5-7GlcNAc2)

attached to misfolded glycoproteins participate

in this recognition, in agreement with other

studies (10, 17-19, 49).

While this manuscript was in preparation,

two groups reported studies on another EDEM

homolog, which they named EDEM2 (25, 26).

Although EDEM2 stimulates glycoprotein

ERAD, it has no effect on mannose trimming

from misfolded glycoproteins, indicating that

its mechanism of action is different from that

of EDEM3 described in the present work. In

both manuscripts, the existence of EDEM3 is

mentioned, but its function is not further

analyzed.

The mechanisms involved in ERAD is an

area of active investigation at the present time,

not only for fundamental cell biology, but also

for clinical applications, because ERAD is

important in the pathogenesis of a large

number of genetic diseases caused by protein

misfolding. Yet the mechanisms whereby the

cell recognizes misfolded proteins and targets

them to ERAD are not fully understood. Since

earlier studies showed ER ManI and EDEM

both stimulate ERAD of glycoproteins, a

relatively simple mechanism has been

proposed whereby targeting of misfolded

glycoproteins depends on Man8B formed by

ERManI which is then recognized by EDEM.

However, it is clear from more recent studies

and from the work presented in this

manuscript that the targeting for ERAD is far

more complicated, since there are two

additional EDEM proteins implicated and

trimming of oligosaccharides on misfolded

glycoproteins to species smaller than Man8

occurs. Thus, the cloning and characterization

of EDEM3 makes a novel contribution to the

understanding of the quality control of

misfolded proteins.


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Abbreviations

ER: endoplasmic reticulum, ERAD: ER-associated degradation, EDEM: ER degradation enhancing

a-mannosidase-like protein, ER ManI: ER a1,2-mannosidase I, PA domain: protease-associateddomain, HA: influenza hemagglutinin epitope, a1AT: a1-antitrypsin, NHK: a1AT null (Hong Kong),TCRa: T cell receptor a subunit, Ab: antibody, BiP: Immunoglobulin heavy chain binding protein,PDI: protein disulfide isomerase.

Acknowledgements

This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports and

Technology of Japan (to I.W., K.N and N.H.,), and by a grant from the Canadian Institutes of Health

Research (CIHR, to A.H.). We thank Dr. Y. Hayashizaki (RIKEN, Japan) for the cDNA clones, Dr. F.

Tokunaga (Osaka City Univ. Graduate Sch. Med) for the FLAG-tagged TCRa plasmid, and Ms. K.Kanamori for technical assistance.


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FIGURE LEGENDS

Figure 1. Similarity between EDEM3, EDEMand ERManI.

A. Amino acid sequence of mouse EDEM3. Region of similarity with Class I a1,2-mannosidasefamily (glycosyl hydrolase family 47), the protease associated domain, and the signal sequence are

shaded in orange, blue, and green, respectively. KDEL ER retrieval signal is underlined. The nine

conserved acidic amino acids are indicated by closed triangles, and putative N-glycosylation sites

are shown by dots.

B. Domain organization of mouse EDEM3, mouse EDEM and human ERManI.

Figure 2. Tissue distribution of EDEM3 and EDEM mRNAs and effect of stress on EDEM3mRNA in cultured cell lines.

A. Northern blots of mouse tissues showing EDEM3 and EDEM transcripts. Two mg of polyA RNAwere loaded (Clontech). Arrow indicates the position of EDEM3 mRNA of approx. 6.4 kb, andarrowheads show EDEMmRNAcorresponding to 5.8 and 2.4 kb.

B. Effect of tunicamycin treatment. Cells were treated with 5mg/ml tunicamycin (Tu) for 6-7 h.Twenty mg of total RNAwere analyzed by northern blotting with probes for EDEM3, EDEM, BiPand b-actin.

C. BALB/c 3T3 cells were subjected to ER stress (tunicamycin, 2-deoxyglucose, and A23187) or to

cytosolic stress (arsenite and heat shock). The same blot was rehybridized with BiP (induced byER stress), b -actin (loading control), and HSP70 (induced by cytosolic stress).

Figure 3. EDEM3 is expressed in the ER as a lumenal protein.

A. In vitro translation of [35S]-methionine-labeled EDEM3-HA. EDEM3-HA, Hsp47, and

EDEM-HA were translated in rabbit reticulocyte lysates supplemented with canine pancreas

microsomes. Proteins were separated into membrane and soluble fractions by alkali floatation,

and then analyzed by 10% SDS-PAGE and autoradiography. M: membrane fraction, S: soluble

fraction. The positions of the molecular weight markers are shown on the left side of the panels.

B. EndoH and PNGaseF digestion of EDEM3. 293 cells were transfected with EDEM3-HA, and

labeled for 3 h with [35S]-methionine/cysteine. After immunoprecitation by a-HA-tag Ab,

EDEM3-HA was digested with EndoH for 2.5 h, or with PNGaseF for 14 h at 37oC, and

separated by 10% SDS-PAGE. The positions of the molecular weight markers are indicated on

the left side of the panel.

C. Immunolocalization of EDEM3-HA. COS7 cells were transiently transfected with EDEM3-HA,

and stained with anti-HA Ab (2ndAb: Alexa546-labeled anti-rabbit) and anti-PDI Ab (2

ndAb:

Alexa488-labeled anti-mouse). Samples were examined by confocal microscopy. Bar indicates 10

mm.

Figure 4. EDEM3 accelerates glycoprotein ERAD.

A. Effect of EDEM3 on NHK degradation. 293 cells were transiently transfected with NHK and

EDEM3-HA/mock, pulse-labeled for 15 min with [35S]-methionine/cysteine, and then chased for


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the periods indicated. After immunoprecipitation using anti-a1AT or anti-HA Ab, samples wereseparated by 10% SDS-PAGE until the Evans Blue reaches near the bottom of the gel. The

positions of the molecular weight markers are shown on the left side of the panel.

B. Quantification of NHK degradation. Average of three independent experiments were plotted with

standard deviations. Relative radioactivity of NHK at chase 0 h was set to 100%.

C. Effect of proteasome inhibition. 293 cells were transfected with NHK and EDEM3-HA, and

treated with (+) or without (-) lactacystin (20 mM) added 3 h prior to pulse-labeling with[35S]-methionine/cysteine for15 min, followed by chase for the times indicated. Lactacystin was

also present during the chase in lactacystin-treated samples. NHK immunoprecipitated with

a1-AT Ab was subjected to 10% SDS-PAGE. Arrow indicates the position of NHK after a 15min pulse.

D. Effect of PNGaseF on NHK. NHK expressed in 293 cells was immunoprecipitated as described in

C, and the electrophoretic mobility shift of NHK in EDEM3-transfected cells during chase was

compared with that of deglycosylated NHK prepared by PNGaseF digestion. The positions of the

molecular weight markers are indicated on the left side of the panel.

E. Effect of kifunensine on NHK degradation. Cells were metabolically labeled with

[35S]-methionine/cysteine for 15 min (P: pulse) and chased for 90 min (C: chase). In

kifunensine-treated cells, drug (5 mg/ml) was added 3 h prior to pulse-labeling and during thechase.

Figure 5. EDEM3 accelerates ERAD of membrane-bound TCRaa but not unglycosylated NHK.A. Effect of EDEM3 on TCRa degradation. 293 cells were transiently transfected with TCRa-FLAGand EDEM3-HA/mock, pulse-labeled for 15 min with [

35S]-methionine/cysteine, and then chased

for the periods indicated. After immunoprecipitation using anti-FLAG Ab, samples were separated

by 10% SDS-PAGE. Kifunensine (5 mg/ml) was added 3 h prior to pulse-labeling and during thechase where indicated (+). The positions of the molecular weight markers are shown on the left

side of the panel.

B. Quantification of TCRa degradation. Average of three independent experiments were plotted withstandard deviations. Relative radioactivity of TCRa at chase 0 h was set to 100%.

C. Effect of EDEM3 on the degradation of unglycosylated NHK-QQQ. 293 cells were transfected

with NHK-QQQ and EDEM3-HA/mock, and pulse-chase experiment was performed as described

in A. NHK-QQQ immunoprecipitated with a1-AT Ab was subjected to 10% SDS-PAGE.

Arrowhead indicates the position of NHK-QQQ. The position of the molecular weight marker is

shown on the left side of the panel.

D. Quantification of NHK-QQQ degradation. Average of three independent experiments were plotted

with standard deviations. Relative radioactivity of NHK-QQQ at chase 0 h was set to 100%.

Figure 6. EDEM3 stimulates mannose trimming of N-linked oligosaccharides.

A. Effect of EDEM3 on mannose trimming of N-linked glycans on NHK. 293 cells were transiently

transfected with NHK and either mock or EDEM3. After labeling for 30 min in medium

containing 1mM glucose and [2-3H]-mannose, cells were chased in medium containing 25mM


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glucose with or without lactacystin (20 mM). Immunoprecipitated NHK was separated by

SDS-PAGE, and transferred to PVDF membrane. NHK bands were excised from the blotted

membrne, and the oligosaccharides were released with endo-H and fractionated by HPLC. Arrows

indicate the position of the [14C]-labeled Glc3Man9GlcNAc internal standard.

B. Effect of EDEM3 E147Q mutant on mannose trimming of N-linked glycans on NHK. 293 cells

were transfected with NHK and either mock, EDEM3 or E147Q mutant plasmids. Samples were

analyzed as described in A, and relative amount of each oligosaccharides are shown as bar graph.

C. Effect of EDEM3 overexpression on oligosaccharides from total cellular glycoproteins from

HepG2 cells. The fluorescent-labeled oligosaccharides were fractionated by HPLC as described in

materials and methods.

D. Effect of the EDEM3 E147Q mutant on NHK degradation in 293 cells. Pulse-chase experiment

using [35S]-methionine/cysteine was performed as described in Fig. 4A. The average of four

independent experiments is shown on the graph.


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MSKAGGCRGCGCRVPQRASWSLVAATAALCLVLATSVCTAGAAPMSREEKQKLGNQVLEMFDHAYGNYMEHAYPADELMP 80

LTCRGRVRGQEPSRGDVDDALGKFSLTLIDSLDTLVVLNKTKEFEDAVRKVLRDVNLDNDVVVSVFETNIRVLGGLLGGH 160

SLAIMLKEKGEHMQWYNDELLHMAKQLGYKLLPAFNTTSGLPYPRINLKFGIRKPEARTGTETDTCTACAGTLILEFAAL 240

SRFTGATIFEEYARKALDFLWEKRQRSSNLVGVTINIHTGDWVRKDSGVGAGIDSYYEYLLKAYVLLGDDSFLERFNTHY 320

DAIMRYISQPPLLLDVHIHKPMLNARTWMDALLAFFPGLQVLKGDIRPAIETHEMLYQVIKKHNFLPEAFTTDFRVHWAQ 400

HPLRPEFAESTYFLYKATGDPYYLEVGKTLIENLNKYARVPCGFAAMKDVRTGSHEDRMDSFFLAEMFKYLYLLFADKED 480

IIFDIEDYIFTTEAHLLPLWLSTTNRSISKKNTTSEYTELDDSNFDWTCPNTQILFPNDPLYAQSIREPLKNVVDKSCPR 560

GIIRVEESFRSGAKPPLRARDFMATNPEHLEILKKMGVSLIHLKDGRVQLVQHAIQAASSIDAEDGLRFMQEMIELSSQQ 640

QKEQQLPPRAVQIISHPFFGRVVLTAGPAQFGLDLSKHKETRGFVASSKPYNGCSELTNPEAVMGKIALIQRGQCMFAEK 720

ARNIQNAGAIGGIVIDDNEGSSSDTAPLFQMAGDGKDTDDIKIPMLFLFSKEGSIILDAIREHKQVEVLLSDKARDRDPE 800

MENEDQPSSENDSQNQSAEQMLSLSQTVDLADKESPEHPADSHSEASPSDSEEAAGFAPSEQISGSTENHETTSLDGECT 880

DLDNQVQEQSETEEDSSPNVSWGTKAQPIDSILADWNEDIEAFEMMEKDEL 931.

..

..

.

.A

B

glycosyl hydrolase family 47 domain

transmembrane region putative N-glycans

protease associated domain

ER ManI

EDEM

EDEM3ERretrievalsignal

signalsequence

Figure 1


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9.5

4.4

7.5

1.35

2.4

kb

hear

tbr

ainsp

leen

lung

liver

skele

tal m

uscle

kidne

yte

stis

EDEMEDEM3

9.57.5

4.4

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leen

lung

liver

skele

tal m

uscle

kidne

yte

stis

A

B

BiP

EDEM3

CO

S7

HEK

293

HeL

aS3

- + - + - + - + - +Tu

PC12

h

BALB

/c3T

3

2.4k

5.8k

EDEM

β-actin

EDEM3

BiP

β-actin

HSP70

cont

rol

tuni

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ycin

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seni

tehe

atsh

ock

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C

Figure 2 by guest on A

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PDIEDEM3-HA merge

B

M SEDEM-HA

82.6 kDa

M S

Hsp47M S

EDEM3-HA

130 kDa

A

C

- + - +

EDEM3-HA

EndoH PNGaseF

deglycosylatedEDEM3-HA

82.6 kDa

130 kDa

Figure 3


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100

50

010

1 2

(%)

(h)Chase

mockEDEM3

Rel

ativ

era

dioa

ctiv

ity

NHK

EDEM3150

250

100

50

75

0 1 2 0 1 2 0 1 2 (h)Chase

EDEM3 - ++α-α1AT AbIP α-HA Ab

lane 1 2 3 4 5 6 7 8 9

0 45 90 0 45 90 (min)

- +

+EDEM3

Lactacystin

Chase

NHK

lane 1 2 3 4 5 6

NHK

P C P C P C P CPulse/Chase

Kifunensine

EDEM3

+ +- -- +

lane 1 2 3 4 5 6 7 8

C D

BA

Figure 4

E

0 1 2 -

+

+EDEM3

Lactacystin

Chase (h)

NHK

-

NHK-CHO

+ PNGaseF

-

50 kDa

37 kDa

lane 1 2 3 4 5


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50

010

1 2

(%)

(h)Chase

mockEDEM3

0 1 2 0 1 2 (h)Chase

EDEM3 - +

NHK-QQQ40.8 kDa

lane 1 2 3 4 5 6

BA

Figure 5

0 45 90 0 45 90 0 45 90 0 45 90 (min)

KIF

plasmid mock

α-FLAG AbIP

EDEM3

- + - +chase

TCRα-FLAG

lane 1 2 3 4 5 6 7 8 9 10 11 12

82.6

130

200

31.5

40.8

100

50

01

1 2

(%)

(h)Chase

10

5

mockEDEM3

D

C


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MOCKEDEM3E147Q

chase

0

10

20

0 1 2

M6

(h)

(%)

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G1M9

0

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0 1 2

M7

0

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0 1 2

M8

0

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0 1 2

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A

100

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(%)

(h)Chase

EDEM3

E147Qmock

Rel

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era

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plasmid mock

α-α1AT AbIP

EDEM3 E147Q

NHK

B

C

M9

G1M9M8M7

M6

M5

27.5 30.0 32.5

10

0

20

M9

G1M9

M8

M7

M6M5

27.5 30.0 32.5 35.0

40

0

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Flu

ores

cenc

ein

tens

ity

Retention time (min)

EDEM3-HepG2

HepG2

D

EDEM3

E147Q

Mock

Rad

ioac

tivity

(dpm

)

Chase 0 h Chase 1 h Chase 2 h

Fraction Number

Figure 6 by guest on A

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Nagata and Nobuko HosokawaNatsuka, Pedro Romero, Barry Sleno, Linda O. Tremblay, Annette Herscovics, Kazuhiro

Kazuyoshi Hirao, Yuko Natsuka, Taku Tamura, Ikuo Wada, Daisuke Morito, Shunjitrimming

EDEM3, a soluble EDEM homolog, enhances glycoprotein ERAD and mannose

published online January 23, 2006J. Biol. Chem.

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edem3, a soluble edem homolog, enhances glycoprotein erad

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