Biochimica et Biophysica Acta 1782 (2008) 408–413

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Biochimica et Biophysica Acta
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a d i s

Rescue of mutant α-galactosidase A in the endoplasmic reticulum by
1-deoxygalactonojirimycin leads to trafﬁcking to lysosomes
Ryoji Hamanaka a, Tetsuji Shinohara a, Shinji Yano b, Miki Nakamura a, Aiko Yasuda c, Shigeo Yokoyama b,
Jian-Qiang Fan d, Kunito Kawasaki e, Makoto Watanabe a, Satoshi Ishii e,⁎a

Department of Anatomy, Biology and Medicine, Faculty of Medicine, Oita University, Oita, Japan
Department of Oncological Science, Faculty of Medicine, Oita University, Oita, Japan
Institute of Scientiﬁc Research, Faculty of Medicine, Oita University, Oita, Japand
Department of Human Genetics, Mount Sinai School of Medicine, New York, USAe
Department of Agricultural and Life Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japanbca r t i c l e

i n f oArticle history:
Received 24 December 2007
Received in revised form 5 March 2008
Accepted 5 March 2008
Available online 12 March 2008
Keywords:
α-galactosidase A
Active-site-speciﬁc chaperone
Fabry diseaseIn vitro translation
Immunoelectron microscopya b s t r a c t
Active-site-speciﬁc chaperone therapy for Fabry disease is a genotype-speciﬁc therapy using a competitive
inhibitor, 1-deoxygalactonojirimycin (DGJ). To elucidate the mechanism of enhancing α-galactosidase A (α-GalA) activity by DGJ-treatment, we studied the degradation of a mutant protein and the effect of DGJ in theendoplasmic reticulum (ER). We ﬁrst established an in vitro translation and translocation system using rabbit
reticulocyte lysates and canine pancreas microsomal vesicles for a study on the stability of mutant α-Gal A
with an amino acid substitution (R301Q) in the ER. R301Q was rapidly degraded, but no degradation of wildtype α-Gal A was observed when microsomal vesicles containing wild-type or R301Q α-Gal A were isolated
and incubated. A pulse-chase experiment on R301Q-expressing TgM/KO mouse ﬁbroblasts showed rapid
degradation of R301Q, and its degradation was blocked by the addition of lactacystin, indicating that R301Q
was degraded by ER-associated degradation (ERAD). Rapid degradation of R301Q was also observed in TgM/
KO mouse ﬁbroblasts treated with brefeldin A, and the amount of R301Q enzyme markedly increased by
pretreatment with DGJ starting 12 h prior to addition of brefeldin A. The enhancement of α-Gal A activity andits protein level by DGJ-treatment was selectively observed in brefeldin A-treated COS-7 cells expressing
R301Q but not in cells expressing the wild-type α-Gal A. Observation by immunoelectron microscopy showedthat the localization of R301Q in COS-7 cells was in the lysosomes, not the ER. These data suggest that the
rescue of R301Q from ERAD is a key step for normalization of intracellular trafﬁcking of R301Q.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction
Fabry disease is a lysosomal storage disorder caused by deﬁcientlysosomal α-galactosidase A (α-Gal A, EC 3.2.1.22) activity, resulting inprogressive accumulation of neutral glycosphingolipids in vascularendothelial cells [1]. This disorder is classiﬁed into two major subtypes
based on clinical manifestations. Patients with classic early onset
Fabry disease show various clinical symptoms, such as angiokeratoma,
acroparathesis, hypohidrosis, corneal and lenticular opacities, and⁎ Corresponding author. Inada-cho, Obihiro, Hokkaido 080-8555, Japan. Fax: +81 155
49 5577.
E-mail address: ishiis@obihiro.ac.jp (S. Ishii).
Abbreviations: α-Gal A, α-galactosidase A; DGJ, 1-deoxygalactonojirimycin; ASSC,
active-site-speciﬁc chaperone; ER, endoplasmic reticulum; DTT, dithiothreitol; Endo H,
endoglycosidase H; ERAD, ER-associated degradation; ERT, enzyme replacement therapy;
TEA, triethanolamine; RM, rough microsomes; DMEM, Dulbecco's modiﬁed Eagle's
medium; FBS, fetal bovine serum; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gelelectrophoresis; PBS, phosphate buffered saline; BSA, bovine serum albumin; HRP,
horseradish peroxidase; EDEM, ER degradation enhancing α-mannosidase-like protein
0925-4439/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbadis.2008.03.001

progressive vasculopathy of the kidney, heart, and central nervous
system. In contrast, atypical variants with residual α-Gal A activity
show late onset cardiomyopathy with left ventricular hypertrophy but
not the classic symptoms [2].
Currently, enzyme replacement therapy (ERT) is the only approved
therapy for Fabry disease [3,4]. ERT has been well-tolerated bypatients, as characterized by improvement in gastrointestinal andneurological manifestations (acroparaesthesia, hypohidrosis, vasomotion) and in quality of life [5,6]. However, results from treating variant
Fabry disease patients have been mixed, suggesting that ERT may be
inefﬁcient for treating severe late-stage patients, presumably as aresult of insufﬁcient delivery of the enzyme to particular tissue [7,8].
ERT is expensive, and it could therefore be an economic burden forpatients, so it is necessary to develop alternative affordable therapies
for treating patients with Fabry disease.
We have shown that residual enzyme activity in lymphoblasts
established from atypical Fabry patients with R301Q or Q279E
mutations can be increased by cultivating cells with 1-deoxygalactonojirimycin (DGJ), a potent competitive inhibitor of α-Gal A [9]. We
designated our therapeutic strategy as active-site-speciﬁc chaperone

R. Hamanaka et al. / Biochimica et Biophysica Acta 1782 (2008) 408–413(ASSC) therapy because we hypothesized that DGJ binds to the active
site of the enzyme, works as a folding template of misfold proteins,and enhances the enzyme activity [10,11]. Our recent study showedthat the majority of missense mutant enzymes, identiﬁed in patientswith residual enzyme activity, retain the catalytic activity and fragile
conformation resulting in excessive degradation of affected mutant
protein in endoplasmic reticulum-associated degradation (ERAD).
This is the main molecular cause for the deﬁciency of the protein andbroadness of the DGJ effect in the treatment of both classic and variant
Fabry patients with residual enzyme activity [12]. We proposed that
DGJ acts for those mutant proteins with a fragile conformation tofacilitate proper folding, thus avoiding retardation and excessive
degradation of these mutant enzymes in the endoplasmic reticulum(ER) [13].
To prove our hypothesis, in this study we focused on the behavior ofmutant α-Gal A in the ER of mammalian cells and provided the evidence
that DGJ acts in the ER. To determine the manner of degradation ofmutant α-Gal A in the ER and the enhancement of enzyme activity ofmutant α-Gal A by the treatment of DGJ in the ER, we used in vitrotranslation, translocation, and cells cultured in the presence of brefeldin
A, which is an inhibitor of transport from the ER to the cis-Golgi [14,15].
We also provided the evidence that the majority of mutant α-Gal A wasretained in the ER when it was expressed in COS-7 cells, whereas, incontrast, the rescued mutant α-Gal A by the treatment of DGJ was mainly
located in lysosomes.

409

CaCl2) and incubated with or without 20 μM DGJ at 37 °C for 0.5, 1, 2, and 4 h. Eachvesicle was subjected to SDS-PAGE and quantiﬁed by a BAS 2000 phosphoimage
analyzer (Fuji Film, Tokyo, Japan).
2.4. Pulse-chase experiments and immunoprecipitation
TNK and TMK2 cells were preincubated in serum/Met/Cys-free DMEM (Sigma
Chemicals) for 30 min and then radiolabeled for 30 min by the addition of [35S]-Met/Cys(ICN Biomedicals Inc., Costa Mesa, CA). Following the labeling period, the cells were
cultured in complete DMEM containing excessive amounts of cold methionine/cysteine,
10% FBS, and 2 μM lactacystin or 5 μg/ml brefeldin A with or without 20 μM DGJ. At 1 and
2 h, the cells were washed with phosphate buffered saline (PBS) and lysed in cold buffer(100 mM Tris–HCl, pH 7.5, 0.4 M KCl, 1% Triton X-100, 1 mM EDTA, 0.1 mg/ml leupeptin).
Cell debris was removed by sedimentation at top speed in a microfuge for 15 min. The
indicated antibodies absorbed by protein A-sepharose CL-4B (GE Healthcare BioSciences) were then added to the supernatants from medium and cell lysates for
immunoprecipitation, which was carried out with a slight modiﬁcation as previously
reported [20]. The immunoprecipitates were brieﬂy washed ﬁve times in a washingbuffer (25 mM Tris–HCl, pH 7.5, 0.5 M NaCl, 0.1% Triton X-100 and 0.1% SDS) to remove
nonspeciﬁcally absorbed protein. Bound antigen was eluted from the beads by boilingin SDS-PAGE sample buffer and then analyzed by SDS-PAGE. The gels were quantiﬁed in
the BAS 2000 phosphoimage analyzer.
2.5. Enzyme assay and protein determination
α-Gal A activity was assayed with 4-methylumbelliferyl α-D-galactopyranoside (4MUα-Gal, Sigma Chemicals) as a substrate, as described previously [18]. One unit of enzyme
activity was deﬁned as the amount of enzyme that releases 1 nmol of 4-methylumbelliferone per hour. Protein concentration was determined using a DC Protein Assay kit (Bio-Rad
Laboratories, Hercules, CA) with bovine serum albumin (BSA, Sigma Chemicals) as astandard.

2. Materials and methods
2.1. Cell culture and transient expression
Murine ﬁbroblasts (TNK and TMK2 cells) had been established from heterozygoustransgenic mice expressing human wild-type and mutant α-Gal A (R301Q), respectively,
in an endogenous murine α-Gal A knock-out background [16]. TNK, TMK2, and COS-7
cells were grown in Dulbecco's modiﬁed Eagle's medium (DMEM, Sigma Chemicals, St.
Louis, MO) supplemented with 10% fetal bovine serum (FBS, Invitrogen Corp., Carlsbad,
CA) and antibiotics (penicillin and streptomycin) at 37 °C under 5% CO2. For the
preparation of COS-7 cells expressing wild-type or mutant α-Gal A, 2 μg of plasmid DNA(pCXN2-GLA or pCXN2-GLA-R301Q, respectively) [17] were transfected into the COS-7
cells with Lipofectamine 2000 (Invitrogen Corp.) according to the manufacturer's
instructions. DGJ (Toronto Research Chemicals, North York, ON, Canada) was added tothe culture medium.
2.2. Antibodies and inhibitors
Polyclonal antibody against human α-Gal A [18] was puriﬁed by ImmunoPure
Immobilized Protein A Gel (Pierce Biotechnology, Rockford, IL) for immunoprecipitationand immunoelectron microscopic study. Horseradish peroxidase (HRP)-conjugatedgoat anti-rabbit IgG antibody (Pierce Biotechnology), gold-conjugated goat anti-rabbit
IgG (GE Healthcare Bio-Sciences Corp., Piscataway, NJ), lactacystin (kindly provided by
Dr. S. Omura, The Kitasato Institute, Tokyo, Japan) and brefeldin A (Sigma Chemicals)
were obtained from indicated sources.
2.3. In vitro translation and translocation, and degradation assay
The pGEM-9Zf(−)9 vector containing either human wild-type or R301Q α-Gal A
cDNA was linearized beyond the 3' end of cDNA using Xba I (New England Biolabs,
Beverly, MA). Capped mRNAs were prepared using mMESSAGE mMACHINE kit(Ambion, Austin, TX). Wild-type and R301Q α-Gal A polypeptides were synthesizedfrom 0.2 μg mRNA and labeled with [35S]-methionine in the rabbit reticulocyte lysate
cell-free system (Promega, Madison, WI) with canine pancreas microsomal vesicles
prepared as previously described by Walter and Blobel [19], according to the
manufacturer's protocol. Rough microsomes (RM) containing synthesized proteins
were sedimented by centrifugation for 5 min at 100,000 ×g through a cushion of 50 mM
triethanolamine (TEA) (pH 7.4) buffer containing 0.5 M sucrose and 1 mM dithiothreitol(DTT). Resulting vesicles were subjected to trypsin-digestion or endoglycosidase H(Endo H, New England Biolabs) digestion as follows. Trypsin (25 μg/ml) was added to
RM in the presence of 1% Triton X-100. Following incubation for 10 min on ice, thereaction was terminated by the addition of sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) sample buffer (50 mM Tris–HCl buffer, pH 6.8, containing
2% SDS, 10% glycerol, 0.01% bromophenol blue, and 3% 2-mercaptoethanol). Endo H
digestion was carried out according to manufacturer's protocol (CALBIOCHEM, San
Diego, CA). For the degradation assay, the vesicles were washed with a high-salt buffer(0.5 M potassium acetate, 5 mM magnesium acetate) and subjected to ultracentrifugation through a 0.5 M sucrose cushion to remove the reticulocyte lysates. The isolated
vesicles were resuspended in a chase buffer (20 mM HEPES, pH 7.5, 50 mM KCl, 1 mM

Fig. 1. In vitro translation, translocation, and degradation of R301Q by ERAD. (A) R301Q
mRNA was translated in rabbit reticulocyte lysates with RM (lanes 2–7) or without RM(lane 1) as described in Materials and methods. After the reaction, the mixture wasseparated into supernatant (lane 3) and pellet (lane 4) by centrifugation, and aliquots ofthe mixtures were treated with trypsin in the absence (lane 5) or presence (lane 6) of 1%
Triton X-100, and treated with Endo H in the presence of 1% Triton X-100 (lane 7). All the
products were subjected to SDS-PAGE. Closed and open arrowheads indicate theglycosylated precursor form (50 kDa) and non-glycosylated precursor form (46 kDa),
respectively. (B) Stability of in vitro translated and translocated wild-type or R301Q α-Gal
A. The vesicles were washed with a high-salt buffer and subjected to sedimentation, thenresuspended in a chase buffer and incubated at 37 °C for indicated times. Same amount ofeach sample was applied onto a single gel and determined by the autoradiography.
(C) Quantitative analysis of 50-kDa band detected in (B). The amounts of α-Gal A
remaining in the vesicles were plotted. The radioactivity of each band for wild-type(closed circle) and R301Q (open circle) was quantiﬁed by a phosphoimage analyzer. The
mean and standard deviation of three independent experiments are shown.
(D) Degradation of R301Q in murine ﬁbroblasts and the effect of lactacystin (LC) addition
into culture medium. TMK2 cells were pulse-labeled and treated with 2 μM lactacystin as
described in Materials and methods.

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R. Hamanaka et al. / Biochimica et Biophysica Acta 1782 (2008) 408–413

Fig. 2. Effect of DGJ on the degradation of R301Q in cells treated with brefeldin A.
(A) Murine ﬁbroblasts expressing wild-type and R301Q α-Gal A were labeled in thepresence of 5 μg/ml brefeldin A with or without 20 μM DGJ. The treatment of brefeldin A
was started 30 min before the radiolabeling period and continued until the end of thechase period. DGJ was applied in two ways (one way was adding DGJ at same time as
brefeldin A-treatment, and the other was pretreatment starting from 12 h prior toradiolabeling). After 30-min radiolabeling and chase period, cell lysates were preparedand subjected to immunoprecipitation with an anti-α-Gal A antibody, followed by SDSPAGE and ﬂuorography. (B) Quantitative analysis of (A). The mean and standard
deviation of three independent experiments are shown. (Closed circle), wild-type;(open circle), R301Q; (closed triangle), R301Q (DGJ added at 0 h); and (open triangle),
R301Q (DGJ-pretreated).

2.6. Western blot analysis
Western blot analysis for the detection of α-Gal A protein was performed using
polyclonal anti-α-Gal A antibody and HRP-conjugated anti-rabbit IgG antibody as
described previously [18]. Cell homogenate containing approximately 30 μg protein wasapplied to a 10% polyacrylamide gel. After SDS-PAGE, proteins were electorophoreticallytransferred to a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). The
membrane was blocked with 5% skim milk in BLOT solution (10 mM Tris–HCl, pH 7.5, with
0.25 M NaCl and 0.05% Tween 20) at 4 °C overnight, and then treated with a rabbit
polyclonal anti-α-Gal A antibody diluted in a milk/BLOT solution (1% skim milk in BLOT
solution) for 1 h at room temperature with mild shaking. After washing with an excess
amount of the milk/BLOT solution, the membrane was treated for 1 h at room temperature
with HRP-conjugated anti-rabbit IgG antibody diluted in the milk/BLOT solution. Afterextensive washing with the milk/BLOT solution, protein bands were visualized with
SuperSignal® Chemiluminescent Substrate (Pierce Biotechnology).
2.7. Immunoelectron microsopy
The COS-7 cells expressing wild-type or R301Q α-Gal A were cultured with or without
20 μM DGJ for 48 h after transfection, then ﬁxed in 2% glutaraldehyde in 0.2 M Tris–HCl (pH7.4) for 3 h; and further incubated in 1% osmium tetraoxide for 2 h, dehydrated in ethanol,and embedded in Epok 812. Immunoelectron microscopy was performed as described
previously [21]. Thin sections were brieﬂy microwaved in Target Retrieval Solution, pH 10(DAKO, Carpinteria, CA), and then incubated for 30 min at room temperature with afﬁnity
puriﬁed anti-α-Gal A antibody diluted 1:50. After washing with 50 mM Tris–HCl (pH 7.5)
containing 0.8% NaCl, 0.1% BSA, and 0.1% Tween 20, the ultrathin sections were incubatedwith gold-conjugated goat anti-rabbit IgG. After washing, the ultrathin sections were
stained with uranyl acetate and lead citrate, and examined using a transmission electron
microscope (JEM-1200EXII, JEOL, Japan).

demonstrated that this in vitro system is well functioning, and suggested that α-Gal A is properly translocated and processed as previously described for other proteins [22,23]. There was no difference
between wild-type and R301Q α-Gal A on the proﬁles and in the
efﬁciency of the translation, translocation, and glycosylation from thesame amount of transcripts. The amount of 50-kDa polypeptide ofwild-type and R301Q α-Gal A was 33% and 36%, respectively, of thetotal product. To study the stability of the protein product from an invitro translation and translocation system, the microsomal vesicles
containing R301Q or wild-type α-Gal A were pooled and further
incubated. Two minor bands other than the 50-kDa band weredecreased within 4-h chase in both wild-type and R301Q. Although
the 50-kDa band was remained in wild-type during 4-h chase, the 50kDa band of R301Q was decreased in this treatment (Fig. 1B and C).
These data suggest that R301Q is unstable in the microsomal vesicles,
and this instability may cause low enzyme activity in patients' cells.
The addition of DGJ had no effect on the degradation of R301Q in thiscell-free system (data not shown). To conﬁrm further that R301Q was
degraded by ERAD, the effect of lactacystin, which is an inhibitor forproteasome (ERAD) [24] on the degradation of R301Q in TMK2 cells
was studied (Fig. 1D). Pulse-labeled R301Q was markedly degradedwithin a 2-h chase period, but it was blocked by the treatment withlactacystin.
To identify the cellular compartment in which the effect of DGJ on
R301Q occurred, the pulse-chase experiment using TMK2 cells was
carried out in the presence of brefeldin A, which is a transport
inhibitor causing retention of newly synthesized membrane proteinsand secretory proteins in the ER [25,26]. As shown in Fig. 2, R301Q
pulse-labeled with [35S]-Met/Cys was substantially reduced within a
2-h chase period in the presence of brefeldin A, whereas the wild-typeα-Gal A was not changed during this time. The pulse-labeled R301Q in
the cells, which were pretreated with DGJ for 12 h prior to thetreatment of brefeldin A, was stable during this chase period. The
simultaneous addition of DGJ with brefeldin A did not protect thedegradation of R301Q at all, presumably due to the inhibitory effect ofbrefeldin A on the DGJ import into the ER. These data suggest that
R301Q is degraded by ERAD and that DGJ acts in the ER to restore
R301Q.
To elucidate the change in enzyme activity of R301Q in the ER, weused a transient expression system because stable transformants suchas TMK2 cells are not suitable when we selectively determine theactivity of newly synthesized protein. Wild-type or R301Q α-Gal A was

3. Results
In order to examine the stability of R301Q in the ER, we establishedthe in vitro translation and translocation system using rabbit
reticulocyte lysates and canine pancreas RM. The α-Gal A wassynthesized as a single product 46-kDa polypeptide by translation
with reticulocyte lysates (Fig. 1A, lane 1). An approximate 50-kDa
band appeared with the addition of RM (upper band in Fig. 1A, lane 2).
Evidence of translocation into RM is recovery of material in this upperband in the vesicular precipitates (Fig. 1A, lanes 3 and 4), resistance totrypsin-digestion when it was not made permeable with Triton X-100(Fig. 1A, lane 5) and disappearance following trypsin-digestion in thepresence of Triton X-100 (Fig. 1A, lane 6). Two minor bands (approximately 48 kDa and 46 kDa) other than the 50-kDa band wereobserved in the incorporated products into RM. All bands incorporated into RM were sensitive to Endo H, which removes high-mannose
type oligosaccharides from glycoproteins; and as a result, were shiftedto an approximate 43-kDa band after Endo H treatment (Fig. 1A, lane
7). From the trypsin-treatment with or without Triton X-100, we

Fig. 3. Enzyme activity and the amount of R301Q in brefeldin A-treated COS-7 cells.
COS-7 cells were transfected with pCXN2-GLA, pCXN2-GLA-R301Q, or pCXN2 vector[49] (mock transfection) and brefeldin A (5 μg/ml) was present in culture medium
during 24–48 h after transfection. Before brefeldin A addition, at 6 h after transfection,some cells were treated with DGJ. At 48 h after transfection, cells were harvested andhomogenized in water using a micro-homogenizer (Physcotron, Niti-on, Inc., Chiba,
Japan). The supernatant collected after centrifugation of the homogenate (10,000 ×g for
5 min) was used in enzyme assays. The assay of α-Gal A activity and Western blot
analysis with an anti-α-Gal A antibody was performed as described in Materials andmethods.

R. Hamanaka et al. / Biochimica et Biophysica Acta 1782 (2008) 408–413

transiently expressed in COS-7 cells and kept in their ER by treatment
with brefeldin A for 24–48 h after transfection. In the expression
system using COS-7 cells, recombinant α-Gal A was transiently
synthesized after 24 h of transfection, and the enzyme activity andamount rapidly increased during this time. Therefore, newly synthesized enzymes were retained in the ER by the treatment with
brefeldin A, and the effect of DGJ on the activity of newly synthesized
proteins could be examined. The enzyme activity in COS-7 cells
expressing R301Q (202 ± 12 nmol/h/mg protein) was almost the samelevel as mock transfected COS-7 cells (168 ± 5 nmol/h/mg protein) andwas much lower than cells expressing wild-type α-Gal A (4380± 160 nmol/h/mg protein) (Fig. 3). The pretreatment of those cells with
DGJ allowed a signiﬁcant increase to be selectively observed in cells
expressing R301Q (1740 ± 176 nmol/h/mg protein), which wasapproximately 50% of the level in cells expressing wild-type α-Gal A
treated with DGJ and brefeldin A (3700 ± 765 nmol/h/mg protein). The
enzyme amount detected by Western blot analysis was high in cells
expressing wild-type α-Gal A and DGJ-treated cells expressing R301Q.
These data indicated that the degradation of R301Q was related to its
enzyme activity; and enzymes that lost their activity could berecognized by the ERAD in mammalian cells.
To verify the distribution of R301Q in transfected COS-7 cells,
immunoelectron microscopic study was conducted with anti-α-Gal A
antibody (Fig. 4). Although it is a little hard to distinguish ribosomes(20 nm) from gold particles (15 nm, clear dots) at lower magniﬁcation,
it is much easier at higher magniﬁcation. After we applied themodiﬁed technique [21] to prepare the specimens, the COS-7 cells
expressing R301Q revealed gold particles predominantly in the ER
surrounding the nucleus (Fig. 4A–C). In contrast, gold particles wereobserved mainly in the lysosomes but could not be detected in the ER
in cells expressing R301Q treated with DGJ for 48 h (Fig. 4D–F), andthere was a similar pattern for cells expressing wild-type α-Gal A (datanot shown). To determine the relative amount of R301Q localized in
the lysosomes, we counted the whole number of gold particles in 50
independent cells and calculated a percentage of gold particles locatedin the lysosomes. Almost of all gold particles (represents α-Gal A) were411

detected in lysosomes and the ER, and the ratio of R301Q localized inlysosomes and the ER was 6:94 in the absence of DGJ, but it waschanged to 79:21 in the presence of DGJ, and it became similar to thatof wild-type α-Gal A (82:18), implying that R301Q rescued by DGJ in
the ER was normally processed and sent to its ﬁnal destination, thelysosomes.
4. Discussion
In this study, we established an in vitro translation and translocation assay for studying human α-Gal A and observed somecharacteristics of R301Q in the ER. The in vitro translated product(approximately 46-kDa) had a similar size as that predicted from the429-amino acid precursor sequence of α-Gal A [27]. The translocatedprotein (50-kDa band) appeared when in vitro translation was
performed in the presence of canine pancreas RM. This protein,
recovered in RM sedimented by centrifugation, had a resistance totrypsin-digestion, and it was characterized as the precursor-form of αGal A translocated into RM. The size of precursor-form α-Gal A was
identical to that observed in mammalian cells [12,28]. The Endo Hdigestion of the 50-kDa protein strongly suggested that it was
glycosylated. The difference in size between the 50-kDa precursor
protein and digested product (43 kDa) was approximately 7 kDa,
which is equal in size to 3 U of N-glycan (about 2.4 kDa). The productof our in vitro translation and translocation system may be fully
glycosylated because 3 sites were glycosylated with a recombinanthuman α-Gal A expressed in mammalian cells [29]. Furthermore, the
3-kDa difference between the in vitro translated product (46-kDa) andEndo H-digested product (43-kDa) was equal to the size predictedfrom the signal sequence containing 31 amino acids [27]. Two minor
bands (48-kDa and 46-kDa bands) translocated into RM may be 2 U
and single unit, respectively, of N-glycan-binding products, and theseminor bands of both wild-type and R301Q were unstable in the
microsomal vesicles. We feel that all of the above data indicate thatmutant α-Gal A, as well as wild-type protein, was normally translated,translocated, and glycosylated in our cell-free system.

Fig. 4. Intracellular localization of R301Q. The intracellular localization of R301Q in COS-7 cells in the absence (A–C) or presence (D–F) of DGJ. COS-7 cells were transfected with pCXN2GLA-R301Q and treated with 20 μM DGJ for 48 h, then cells were ﬁxed and embedded as described in Materials and methods. The ultrathin sections were incubated with an anti-α-Gal
A antibody followed by immunogold labeling. Each thin section was examined with a transmission electron microscope. (B) and (E) show the higher magniﬁcations of the lysosome.
(C) and (F) show the higher magniﬁcations of the ER. Scale bars represent 500 nm. Nu, nucleus; Mt, mitochondria; Ly, lysosomes. Typical gold particles are pointed by arrowhead.

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Although there was no difference between wild-type and R301Q αGal A in the process of translation, translocation and glycosylation,
they could be distinguished on the basis of their stability. R301Q was
rapidly degraded in microsomal vesicles (Fig. 1B), while α-Gal A wasfound to be stable. The stability of R301Q in the ER may be the crucial
factor which causes its low activity in patients' cells.
The addition of DGJ in this cell-free system did not show any
protective effect on this degradation, which may be due to theinaccessibility of DGJ to R301Q in this system. Although we do not haveany data on the transport of DGJ, we predicted that DGJ would be taken
up by cells through other iminosugar derivatives like 1-doxymannojirimycin (dMM) because of their structural similarity. Neefjes et al.
[30] have reported that uptake of dMM by human erythroleukemic
cells was not inhibited by glucose, and it (and other iminosugars) may
not be carried into cells by hexose transporters [31]. They also
discussed the presence of the translocation system for dMM in cells
because passive diffusion of dMM cannot explain the difference of theKi value (80 μM) of dMM for isolated mannosidase and theconcentration (1 mM) of dMM necessary to inhibit mannosidase incells. The presence of two distinct mannose transporters which are
dMM-sensitive and insensitive had been reported in intestinal
epithelial cells [32,33]. If dMM were incorporated into cells throughglucose transporter GLUT4, it might be affected by the treatment with
brefeldin A [34]. The data in our present study supported the presenceof a translocation system for DGJ into the ER; pretreatment of DGJ prior
to brefeldin A-treatment was necessary for showing the protective
effect against R301Q degradation. We used a pulse-chase technique on
TNK and TMK2 mouse ﬁbroblasts cultured in the presence of brefeldin
A (Fig. 2), so that newly synthesized enzymes were retained in the ER
and their stability in the ER could be observed. The pulse-labeled
R301Q quickly degraded within a 2-h chase period, but wild-type αGal A did not change. The pretreatment of DGJ for 12 h prior to
brefeldin A-treatment was necessary for the protective effect of DGJ on
the degradation of R301Q. Therefore, transport of DGJ into the ER must
be required to have an effect on R301Q. DGJ protected against not only
the degradation of R301Q, but also the loss of enzyme activity (Fig. 3).
In our previous paper [12], we described that majority of enzyme
mutants, which were detected in Fabry patients who display residual
activity, were thermolabile proteins in a solution at neutral pH, anddegraded in ERAD because the amount of protein was increased bytreatment with kifunensine, a selective inhibitor of ER α-mannosidase
I and lactacystin (a proteasome inhibitor) [35]. In the present study,
we conﬁrmed the effect of lactacystin on R301Q-degradation by using
R301Q-expressing murine ﬁbroblasts (Fig. 1D). Its effect had previously been observed in some mutant α-Gal A's such as E66Q, F113L,
N215S, M296I, and R301Q, which were transiently expressed in COS-7
cells [12].
Several chaperones such as BiP and calnexin are known to exist in
the ER [36], and BiP might be a strong candidate as a molecular sensor,
since it is associated with only R301Q but not wild-type proteins [37]or other misfolded proteins [38]. Recently, it has been postulated thatER degradation enhancing α-mannosidase-like protein (EDEM) or the
EDEM family sequesters ERAD candidates released from calnexin,
thereby interfering with the UDP-Glc:glycoprotein glucosyltransferasethat reglucosylates misfolded glycoproteins released from the calnexin cycle [39,40]. Although the molecular mechanism of recognizing misfolded α-Gal A's to be degraded in the ER is still unclear, we
may be able to shed some light on this area by using mutant enzyme
R301Q as a good model of misfolded proteins.
The addition of DGJ changed the localization of R301Q from the ER
to lysosomes (Fig. 4). Although Yam et al. [37] reported that rescued
R301Q by DGJ-treatment was located in the lysosomes in ﬁbroblasts
established from transgenic mice [16] (as observed by immunoelectron microscopy), we further demonstrated its localization inlysosomes in COS-7 cells expressing R301Q in the presence of DGJ.
COS-7 cells were established from monkey kidney cells [41], and thekidney is one of the target organs for the treatment of Fabry disease. In
our present study, we demonstrated using immunoelectron microscopy that mutant α-Gal A (R301Q) was mainly located in the ER.
These data strongly suggested that mutant α-Gal A was degraded by
ERAD, and the enhancement of enzyme activity in ASSC therapy using
DGJ was caused by restoration of R301Q in the ER and normalization ofits movement to the lysosomes. It has been reported that lysosomal
glycosphingolipids accumulation in ﬁbroblasts from patients with
Fabry disease was diminished by the treatment with DGJ [42], and thedata demonstrated that the rescued mutant protein could work after
reached to the lysosomes. Our therapeutic strategy may be useful formany diseases, and positive results using our concept of therapeuticstrategy have been reported for Gaucher disease [43,44], GM1-gangliosidosis [45], Tay–Sachs disease [46], retinitis pigmentosa 17 [47], andPompe disease [48].
In our present study, we demonstrated that mutant α-Gal A(R301Q) was retained and degraded in the ER; DGJ acted as a folding
template in the ER and rescued R301Q from the loss of enzyme activity
and degradation. Although the details of the degradation process of
R301Q in the ER still remain to be clariﬁed, our in vitro translation andtranslocation technique and use of cells treated with brefeldin A will
be useful tools for studying the degradation process of misfolded
proteins.
Acknowledgements
We would like to thank Mr. Glen Hill for his critical reading of thismanuscript, Dr. T. Shimada and Mr. H. Kawazato for technical help, and
Dr. K. Kashima and Mr. K. Kuroki for editorial help. This work was
supported in part by a research grant from the Ministry of Health,Labour and Welfare of Japan (S.I.).
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