www.biochemj.org
Biochem. J. (2007) 406, 285–295 (Printed in Great Britain)285

doi:10.1042/BJ20070479

Mutant α-galactosidase A enzymes identified in Fabry disease patients withresidual enzyme activity: biochemical characterization and restoration of
normal intracellular processing by 1-deoxygalactonojirimycin
Satoshi ISHII*†, Hui-Hwa CHANG*, Kunito KAWASAKI†, Kayo YASUDA*, Hui-Li WU*, Scott C. GARMAN‡ and Jian-Qiang FAN*1

Fabry disease is a lysosomal storage disorder caused by the
deficiency of α-Gal A (α-galactosidase A) activity. In orderto understand the molecular mechanism underlying α-Gal A
deficiency in Fabry disease patients with residual enzyme activity,
enzymes with different missense mutations were purified fromtransfected COS-7 cells and the biochemical properties werecharacterized. The mutant enzymes detected in variant patients(A20P, E66Q, M72V, I91T, R112H, F113L, N215S, Q279E,
M296I, M296V and R301Q), and those found mostly in mildclassic patients (A97V, A156V, L166V and R356W) appeared
to have normal K m and V max values. The degradation of all
mutants (except E59K) was partially inhibited by treatment withkifunensine, a selective inhibitor of ER (endoplasmic reticulum) αmannosidase I. Metabolic labelling and subcellular fractionation
studies in COS-7 cells expressing the L166V and R301Q α-Gal A
mutants indicated that the mutant protein was retained in the ER
and degraded without processing. Addition of DGJ (1-deoxygalactonojirimycin) to the culture medium of COS-7 cells
transfected with a large set of missense mutant α-Gal A cDNAs

effectively increased both enzyme activity and protein yield. DGJ
was capable of normalizing intracellular processing of mutant
α-Gal A found in both classic (L166V) and variant (R301Q)
Fabry disease patients. In addition, the residual enzyme activity
in fibroblasts or lymphoblasts from both classic and variant
hemizygous Fabry disease patients carrying a variety of missense
mutations could be substantially increased by cultivation of thecells with DGJ. These results indicate that a large proportionof mutant enzymes in patients with residual enzyme activity
are kinetically active. Excessive degradation in the ER could be
responsible for the deficiency of enzyme activity in vivo, and the
DGJ approach may be broadly applicable to Fabry disease patientswith missense mutations.

INTRODUCTION

heart failure or end-stage renal failure in the fifth or sixth decadeof life [8].
To date, more than 400 mutations have been identified in the
α-Gal A gene (termed GLA; Human Gene Mutation Database),and more than 57 % of mutations are missense. The majority ofmutations are private, occurring in only one or a few families.
The correlation between genotype and residual enzyme activity(measured primarily in leucocytes) is not strong, and presumably
depends upon the nature of the mutation and additional genetic
or non-genetic factors. However, it has been clearly demonstrated
that a higher level of residual enzyme activity results in a milder
disease phenotype, typically involving a few or monosymptomaticclinical manifestations, and are less likely to cause the classic formof the disease [9].
The mature α-Gal A enzyme contains 398 amino acid residues
once the signal peptide at the 31st amino acid residue (an alanine
residue) has been cleaved [10]. From the X-ray crystal structural
information [11], 13 amino acid residues were predicated to be
directly involved in the interaction with α-galactose; mutations
occurring at any of these amino acids result in the severeclassic phenotype of Fabry disease [11,12]. Structural studies
also revealed that the majority of amino acids within the mutant
proteins do not directly contribute to the catalytic function of the

Fabry disease is an X-linked inherited lysosomal storage disorderresulting from the deficient activity of α-Gal A (α-galactosidaseA), an enzyme responsible for the catabolism of neutral glycosphingolipids that have an α-galactose residue at their nonreducing terminus [1]. Deficient enzyme activity results inthe progressive deposition of glycosphingolipids, predominantly
globotriaosylceramide, in the lysosomes of vascular endothelial
cells. The disease is classified into two major subtypes based
upon clinical manifestations. Patients with little or no detectable
enzyme activity have early onset, or classic, Fabry disease.
Clinical symptoms are severe and range from angiokeratomas,
acroparesthesia, hypohidrosis, corneal opacity in the early teens,and progressive vascular disease of the heart, kidneys and central
nervous system [2]. Without medical intervention, death typicallyoccurs in the fourth or fifth decade of life, as a result ofrenal failure, cardiac disease or cerebrovascular disease [3,4].
In contrast, patients with residual enzyme activity have a lateonset phenotype, and include atypical cardiac or renal variants.
These patients are usually asymptomatic until their late thirties,
and their clinical manifestations are often limited to the heart [5,6]or kidneys [7]. Without treatment, patients eventually suffer from

Key words: active-site-specific chaperone (ASSC), 1-deoxygalactonojirimycin (DGJ), endoplasmic reticulum-associateddegradation (ERAD), Fabry disease, α-galactosidase A, protein
misfolding.

Abbreviations used: ASSC, active-site-specific chaperone; DGJ, 1-deoxygalactonojirimycin; DMEM, Dulbecco’s modified Eagle’s medium; ER,
endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; FCS, fetal calf serum; α-Gal A, α-galactosidase A; GLA, α-Gal A gene;
4MU, 4-methylumbelliferyl; 4MU-α-Gal, 4MU α-D-galactopyranoside.
1
To whom correspondence should be addressed (email jian-qiang.fan@mssm.edu).
c The Authors Journal compilation 
c 2007 Biochemical Society

Biochemical Journal*Department of Human Genetics, Mount Sinai School of Medicine, Fifth Avenue at 100th Street, New York, NY 10029, U.S.A., †Department of Agricultural and Life Sciences,
Obihiro University of Agriculture and Veterinary Medicine, Obihiro, 080-8555, Japan, and ‡Department of Biochemistry and Molecular Biology, University of Massachusetts,
710 North Pleasant Street, Amherst, MA 01003, U.S.A.

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S. Ishii and others

enzyme, but rather to the maintenance of the tertiary structure.
Despite the fact that many mutations have been identified in Fabry
disease patients, with a few exceptions [13–15], the biochemical
and molecular defects of mutant enzymes in patients who retain
residual enzyme activity are not well understood.
Protein misfolding has been recognized as an important
pathophysiological cause in many inherited disorders, including
cystic fibrosis, α1-antitrypsin deficiency, familial hypercholesterolaemia and Alzheimer’s disease, in which mutant proteins
are not properly folded [16–18]. Improperly folded proteins are
retained in the ER (endoplasmic reticulum) and subject to ERAD
(ER-associated degradation), resulting in a protein deficiency.
Despite the broadness of protein misfolding, which has been
supported by the fact that individual cases of misfolding exist ina variety of diseases, the significant impact of protein misfoldingin each genetic disorder has not yet been well-addressed except
in a few examples, such as cystic fibrosis, in which one
mutation (F508) causing misfolding of CFTR (cystic fibrosis
transmembrane conductance regulator), is responsible for the
majority of patients [19]. Although powerful computer modelling
techniques can predict the folding status of many mutations oncethe crystal structure is available, direct experimental evidence
would still be important. Therefore it is important to examine
protein misfolding in a variety of affected mutations within clearly
defined subsets of patients; such a study can provide evidence
that protein misfolding can be pathogenetically significant, notonly in individual mutations, but also in clinical subsets of
diseases.
Previously we have shown that addition of DGJ (1-deoxygalactonojirimycin), a competitive inhibitor of α-Gal A, at subinhibitory concentrations, to cultured lymphoblasts establishedfrom Fabry disease patients with the R301Q or Q279E mutation,
substantially increases residual enzyme activity [20]. A reductionof large-size lysosomes and loss of characteristic multilamellar
lysosomal inclusions were demonstrated upon DGJ treatment[21]. The proposed mechanism for this observation is that DGJ isan ASSC (active-site-specific chaperone) and serves as a folding
template by which the mutant protein is induced to attain theproper conformation, thus preventing excessive degradation by
ERAD [22]. Administration of DGJ to transgenic mice that
express human R301Q α-Gal A in a null background results
in a substantial increase in enzyme activity in various tissuesof the heart, kidneys, liver and spleen, suggesting that DGJ could
have a therapeutic effect in treating certain Fabry disease patients,
particularly those with a cardiac phenotype [23]. Currently, DGJ
is under phase II clinical evaluation for Fabry disease.
In the present study, we generated 19 mutant α-Gal A cDNA
constructs with various missense mutations found in Fabry disease
patients with residual enzyme activity, including nine variant
mutations, five classic mutations, one presymptomatic mutationand four mutations causing both variant and classic phenotypes.
Sixteen mutant enzymes were efficiently purified from transfected
COS-7 cells, and their enzymatic and biochemical properties
were characterized. These present studies demonstrated that asignificant number of missense mutations give rise to enzymes
which are defective with respect to conformational stability,
but which retain full or partial catalytic activity. These findings
apply to a relatively large set of missense mutations identified
in both classic and variant Fabry disease patients, and suggestthat protein misfolding may be more widespread than previously
thought. The results also provide a molecular understanding of
the therapeutic effect of ASSCs in Fabry disease patients andindicate that DGJ treatment may be effective for a large numberof Fabry disease patients with missense mutations regardless of
their clinical phenotypes.
c The Authors Journal compilation 
c 2007 Biochemical Society

MATERIALS AND METHODS
Cells from patients

Fabry disease lymphoblasts and fibroblasts were supplied by Dr S.
Nakao (Kagoshima Prefectural Kanoya Hospital, Japan), Dr R.O.
Brady of the NIH (National Institutes of Health, Bethesda, MD,
U.S.A.) and Dr R.J. Desnick (Mount Sinai School of Medicine,
New York, NY, U.S.A.). GM1 -gangliosidosis fibroblasts were
established from an adult patient with the homozygous I51T
mutation, and Gaucher fibroblasts were obtained from a type I
patient with the homozygous N370S mutation (purchased fromthe Coriell Institute, Camden, NJ, U.S.A.).
Cell culture

Human lymphoblasts were maintained in RPMI-1640 (Mediatech) supplemented with 10 % FCS (fetal calf serum; Mediatech)and 1 % penicillin/streptomycin (Invitrogen). Human fibroblasts and COS-7 cells were cultured in DMEM (Dulbecco’s
modified Eagle’s medium; Mediatech) supplemented with 10 %FCS and 1 % penicillin/streptomycin. All cells were incubated ina water-jacket incubator at 37 ◦C under 5 % CO2 . DGJ (Toronto
Research Chemicals) was added to the culture medium whenneeded to increase residual enzyme activity.
Site-directed mutagenesis of α-Gal A

Expression vectors containing α-Gal A mutations were generated
by site-directed PCR mutagenesis [24] using the QuikChange®
Site-Directed Mutagenesis Kit (Stratagene). Nineteen amino acidsubstitutions were individually introduced by PCR amplification
using PfuTurbo DNA polymerase, the wild-type α-Gal A cDNA
sequence as a template and a 35-mer primer set, with sense andantisense primers harbouring one of the nucleotide substitutionsin the middle of their sequence. Each mutant cDNA was verified
by DNA sequencing before being subcloned into the expression
vector pCXN2 [25].
Transient expression of mutant enzymes in COS-7 cells

Transient expression of mutant enzymes in COS-7 cells was
carried out using FuGENETM 6 transfection reagent (Roche
Molecular Biochemicals) or LipofectamineTM 2000 reagent(Invitrogen), according to the manufacturer’s protocol. Typically,prior to transfection, a mixture of plasmid DNA (0.5 µg) andFuGENETM 6 transfection reagent (1.5 µl) in 100 µl of serum-free
DMEM medium was incubated at room temperature (25 ◦C) for
20 min, followed by mixing with 400 µl of the complete medium.
The transfection mixture was added to COS-7 cells grown in sixwell culture plates, and the cells were then incubated at 37 ◦C.
Additional complete medium (1 ml) was added to each well thefollowing day, and cells were further incubated until harvest.
Purification of mutant α-Gal A enzymes expressed in COS-7 cells

Mutant α-Gal A enzymes were typically harvested from homogenates of transfected COS-7 cells grown on 10-cm diameter
culture dishes, and purified as described previously [26]. The
purified protein appeared as a single band after SDS/PAGE andvisualization using a silver stain kit (Bio-Rad Laboratories).
Enzyme assay and protein determination

Cell pellets obtained from cell cultures were homogenized inwater using a micro homogenizer. The supernatant collected after
centrifugation of the homogenate at 10 000 g for 5 min was usedin enzyme assays.

Mutant α-galactosidase A enzymes identified in Fabry disease patients

All 4MU (4-methylumbelliferyl) substrates were obtained from
Sigma. The α-Gal A activity was assayed with a mixture (60 µl)
of 4MU-α-D-galactopyranoside (4MU-α-Gal, 5 mM) and Nacetyl-D-galactosamine (75 mM) in 0.1 M sodium citrate buffer(pH 4.6) as described previously [20]. β-Hexosaminidase activity
was determined with 5 mM 4MU-N-acetyl-β-D-glucosaminide as
substrate in the same buffer. β-Galactosidase activity was assayedwith 1 mM 4MU-β-D-galactopyranoside in 0.1 M sodium citrateand 0.2 M disodium hydrogen phosphate buffer (pH 4.6). The
activity of glucocerebrosidase was determined with 3 mM 4MUβ-D-glucopyranoside in the presence of 0.25 % (w/v) sodium
taurocholate and 0.1 % (v/v) Triton X-100 in McIlvaine buffer(0.1 M sodium citrate and 0.2 M disodium hydrogen phosphatebuffer, pH 5.2). All enzyme reactions were terminated by theaddition of 1.2 ml of 0.1 M glycine buffer (pH 10.7), preparedby adjusting the pH using 0.1 M NaOH. The released 4MU wasdetermined by fluorescence measurement at 360 and 450 nm as
the excitation and emission wavelengths respectively. One unit ofenzyme activity is defined as the amount of enzyme that releases
1 nmol of 4MU per h. The protein concentration was determinedby using a DC Protein Assay kit (Bio-Rad Laboratories), using
BSA as a standard.
Kinetic properties of mutant enzymes

The kinetic properties were determined with 4MU-α-Gal atvarious concentrations as substrate in 0.1 M sodium citrate buffer(pH 4.6) at 37 ◦C for 10 min. The K m and V max values of eachmutant enzyme were obtained from Lineweaver–Burk plots.
Measurement of optimal pH and pH stability of purified mutant
α-Gal A enzymes

The optimal pH of mutant enzymes was determined by assaying the enzyme activity in 0.1 M McIlvaine buffer at various
pHs (pH 3.5–5.5). The pH stability of purified enzymes was
assayed as described previously [13]. In order to determine therelative stability of the mutant enzymes at neutral pH, the enzyme
was pre-incubated in 2.5 mM phosphate buffer (pH 7.5; preparedby mixing 2.5 mM sodium dihydrogen phosphate and 2.5 mM
disodium hydrogen phosphate) at 37 ◦C for various times. Afterincubation, an aliquot was diluted with a 4-fold volume of 0.2 M
sodium citrate buffer (pH 4.6), and α-Gal A activity was assayed
immediately.
Western blot analysis

Western blot analysis for the detection of α-Gal A protein was
performed using an anti-α-Gal A polyclonal antibody produced
in rabbit and a HRP (horseradish peroxidase)-conjugated antirabbit IgG antibody produced in goat (Pierce Biothechnology).
An anti-Bip monoclonal antibody purchased from StressGen
Biotechnologies and a HRP-conjugated anti-mouse IgG antibody(Pierce Biotechnology) were used for the detection of Bip. After
SDS/PAGE, proteins were transferred electorophoretically to a
PVDF (Immobilon P) membrane (Millipore). The membrane
was blocked with 5 % (w/v) non-fat dried skimmed 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 primary
antibody diluted in a milk/blot solution [1 % (w/v) non-fat driedskimmed milk in blot solution] for 1 h at room temperature
with mild shaking. After washing with an excess volume of themilk/blot solution, the membrane was treated for 1 h at room
temperature with a secondary antibody diluted in the milk/blot
solution. Following extensive washing with the milk/blot solu-

287tion, protein bands were visualized with SuperSignal® Chemiluminescent Substrate (Pierce Biotechnology).
Subcellular fractionation of expressed mutant α-Gal A enzymes
in COS-7 cells

COS-7 cells plated on to four 10-cm diameter dishes weretransfected with plasmid DNA containing mutant α-Gal A cDNA.
Cells were harvested in PBS on the third day after transfection,and cell pellets were resuspended in Suc Buffer (0.25 M sucrose,
10 mM Hepes and 1 mM EDTA, pH 7.4) on ice. Cells were
homogenized in the same buffer using a Potter–Elvehjem Teflonglass homogenizer. Following centrifugation at 1000 g for 5 min
at 4 ◦C, the supernatant (1 ml) was pooled as a postnuclear
fraction, and layered on top of a 9 ml 40 % Percoll solution in Suc
Buffer. Subcellular fractionation was performed by centrifugation
at 25 000 g for 1 h. Each fraction containing approx. 0.5 ml wascollected from the bottom of the centrifugation tube and used forthe enzyme assay and Western blot analysis.
Metabolic labelling of mutant α-Gal A enzymes expressedin COS-7 cells

COS-7 cells transfected with the wild-type or mutant α-Gal
A cDNAs were washed with PBS and incubated in 0.4 ml ofmethionine- and cystine-free DMEM containing 10 % dialysed
FCS for 30 min. A 4 µl portion of EXPRE35 S35 S[35 S]Proteinlabelling mix (293 MBq/ml; DuPont) was added to each well,and the cells further incubated for 2 h. After washing the cellswith PBS, complete culture medium (2 ml DMEM with 10 %FCS) was added, and the cells further incubated at 37 ◦C, 5 %CO2 for various times as the chasing period. After washing with
PBS, the cell pellets were resuspended in 10 mM Tris/HCl buffer(pH 7.5) containing 40 mM KCl, 0.1 % Triton X-100, 62.5 µM
EDTA, and 2.5 µg/ml leupeptin, and incubated at 4 ◦C for 30 min.
Final cellular samples were prepared by centrifugation at 10 000 g
for 5 min.
Immunoprecipitation of α-Gal A enzymes

A polyclonal anti-α-Gal A antibody was added to the sample
and incubated at 4 ◦C overnight. Protein A Sepharose CL-4B(Amersham Biosciences) suspended in 10 mM Tris/HCl buffer(pH 7.5) containing 40 mM KCl, 0.1 % Triton X-100, 62.5 µM
EDTA and 2.5 µg/ml leupeptin was added to each sample whichwere then incubated at 4 ◦C for 1 h. The precipitate was collectedby centrifugation at 10 000 g for 5 min and washed three times
with 25 mM Tris/HCl buffer (pH 7.5) containing 0.5 M NaCl,
0.5 % Triton X-100, 0.1 % SDS and 1 mM EDTA. After resuspending the sample in 50 mM Tris/HCl buffer (pH 6.8) containing
2 % SDS, 10 % glycerol, 0.01 % Bromophenol Blue and 5 % 2mercaptoethanol, the protein was recovered by boiling thesample for 5 min, followed by SDS/PAGE and visualization byautoradiography.
RESULTS
Expression and purification of mutant α-Gal A enzymes
in COS-7 cells

Nineteen α-Gal A cDNAs that encode various missense mutations
found in Fabry disease patients with residual enzyme activity
were generated to study the biochemical and molecular propertiesof the mutant proteins. Among them, nine mutations (A20P,
E66Q, M72V, I91T, F113L, N215S, Q279E, M296I and M296V)
were found in variant Fabry disease patients [6,7,27–31] and fivemutations (E59K, A156V, L166V, R356W and G373S) [32–34]
c The Authors Journal compilation 
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S. Ishii and others

Table 1 Kinetic properties of human mutant α-Gal A enzymes expressed in
COS-7 cells.
Human α-Gal A enzymes with various missense mutations were purified by affinity column
chromatography of the homogenates of COS-7 cells. The kinetic properties of mutant enzymes
were determined using 4MU-α-Gal at variable concentrations. The K m and V max values of eachmutant enzyme were obtained from Lineweaver–Burk plots.

MutationKm(mM)

V max (mmol/h
per mg of protein)

Wild-type
A20P
E59K
E66Q
M72V
I91T
A97V
R112H
F113L
A156V
L166V
N215S
Q279E
M296I
M296V
R301Q
R356W

2.8
2.8
16.3
3.1
3.2
3.4
3.7
5.8
3.6
3.4
2.6
2.8
1.8
2.7
2.4
1.9
2.0

2.56
2.67
6.13
4.10
4.56
0.99
4.80
1.52
2.57
3.93
6.80
5.29
2.84
3.14
4.06
2.56
2.46

were identified in classic Fabry disease patients. Four mutations(A97V, R112H, R301Q and G373D) [7,28,32,35–37] were foundin both mild classic and variant patients, and P146S was found ina presymptomatic patients [38].
COS-7 cells were transfected with expression vectors encoding
various mutant α-Gal A cDNAs and cultivated in the presenceof DGJ (to ensure maximum production of the mutant enzymes).
The expression of variant mutant enzymes was generally higher(median value of 3870 units/mg) than that of classic mutant
enzymes (median value of 2090 units/mg), in line with observations that variant patients have higher residual enzyme activity.
Since the purification strategy for these enzymes may not permit
total separation of expressed exogenous human mutant enzyme
from the endogenous enzyme in COS-7 cells, only those mutant
enzymes with activities at least 10-fold higher than that ofendogenous enzyme activity were used for further characterization. The expression of the P146S, G373D and G373S enzyme
mutants was relatively low (2- to 5-fold above the endogenouslevel), therefore these mutant enzymes were excluded from further
studies. This precaution ensured that the kinetic and biochemical
results generated from COS-7 cell-derived enzymes are comparable with those of the human mutant enzyme, and contamination by endogenous enzymes from COS-7 cells was kept to aminimum.
Kinetic properties of human mutant α-Gal A enzymes purified fromtransfected COS-7 cells

K m and V max values of purified mutant α-Gal A enzymes were
determined (Table 1). The K m and V max values of the wildtype enzyme were 2.8 mM and 2.56 mmol/h per mg of protein
respectively, in agreement with earlier findings [39]. Except for
the E59K mutant, most mutant enzymes had K m and V max values
similar to those of the wild-type enzyme (K m of 1.8–5.8 mM,
and V max of 0.99–6.80 mmol/h/mg of protein). The K m and V max
values for the E59K mutant were 16.3 mM and 6.13 mmol/h/mg ofprotein respectively, indicating that this mutation may cause
impaired kinetic ability. These results suggest that the majorityof missense mutant enzymes studied here are catalytically
c The Authors Journal compilation 
c 2007 Biochemical Society

Figure 1

pH Stability of wild-type and mutant α-Gal A enzymes

The enzymes were purified from cell lysates of COS-7 cells transfected with expression plasmids
encoding various mutant α-Gal A enzymes. The stable pH range is defined as the pH in whichmore than 50 % enzyme activity remained after incubation at 37 ◦C for 1 h in the absence (darkgrey bar) or presence (light grey bar) of 1 µM DGJ.

active, regardless of the clinical phenotype with which they areassociated.
Optimal pH for activity and pH stability of human missense mutant
α-Gal A enzymes

The optimal pH for enzyme activity was found to be the same
(pH 4.0–4.5) for both the wild-type enzyme and all purified
mutant enzymes. This indicated that these mutant enzymes werelikely to be enzymatically and physically active within the lysosomes. Compared with the wild-type enzyme, which is relatively
stable between pH 3.0 and 7.5, most mutant enzymes were
only stable over a narrow pH range, typically pH 3.5–6.5(Figure 1). Since the A20P mutation occurs within the signal
peptide, the purified A20P enzyme was expected to be identicalto the wild-type enzyme. Therefore it was not surprising that its
stability profile was the same as that of the wild-type protein. The
pH stability of almost all of the mutant enzymes was restored tonormal by the addition of 1 µM DGJ to the pre-incubation buffer.
The exception was with the Q279E mutant, the stability of which
nevertheless improved.
Because the environment of the ER has a neutral pH, thestability of the mutant enzymes at pH 7.5 was monitored in a timedependent fashion. The wild-type enzyme retained more than halfof its activity after pre-incubation at pH 7.5 and 37 ◦C for 90 min(Table 2), indicating that the protein is relatively stable within the
ER. However, all mutants lost more than 50 % of their enzyme
activity within a 45-min pre-incubation period. More strikingly,
five variant mutants (M72V, A97V, R112H, F113L and Q279E)
completely lost their enzyme activity within 15 min under thesame conditions, clearly indicating that these mutants are notphysically stable at neutral pH, which resembles the condition
within the ER. The stability of the mutant enzymes could bepartially or completely restored with the addition of 1 µM DGJ
to the pre-incubation buffer, indicating that DGJ is effective at
stabilizing the conformation of these missense mutant enzymes
at neutral pH.

Mutant α-galactosidase A enzymes identified in Fabry disease patients
Table 2

Effect of DGJ on mutant α-Gal A enzymes

Stability of mutant α-Gal A at pH 7.5

The enzyme was pre-incubated in 2.5 mM phosphate buffer (pH 7.5) in the presence or absenceof DGJ (1 µM) for various times, and the remaining enzyme activity determined in 0.2 M citratebuffer (pH 4.5) using 4MU-α-Gal as a substrate. Enzyme stability was measured in terms of thelength of incubation time that results in more than 50 % remaining enzyme activity.
Time 50 %, pH 7.5 (min)Mutation

−DGJ

+DGJ

Wild-type
A20P
E59K
E66Q
M72V
I91T
A97V
R112H
F113L
A156V
L166V
N215S
Q279E
M296I
M296V
R301Q
R356W

90
90
15
30
< 15
30
< 15
< 15
< 15
30
45
45
< 15
30
15
30
30

90
90
90
90
90
60
90
90
90
90
90
90
45
90
90
90
90

Effects of ERAD inhibitors on mutant α-Gal A

To investigate the intracellular degradation of mutant α-Gal A,
the effects of lactacystin (a proteasome inhibitor) and kifunensine(an inhibitor of ER α-mannosidase I) on the processing ofmutant α-Gal A were determined (Figure 2). After treatment
with kifunensine, a substantial increase in the enzyme protein
was observed in M72V, I91T, A97V, R112H, F113L, L166V,
N215S, Q279E and R301Q mutations, and a modest increase
was also found in A20P, E66Q, A156V, M296I, M296V, R356W,
G373D and G373S mutations. The amount of E59K protein was
not affected by this treatment. The protein amount of P146S
was too low to be detectable by Western blot analysis, evenin the presence of kifunensine (results not shown). In contrast
with the kifunensine-effect, the enzyme amount was increased
by treatment with lactacystin in E66Q, F113L, N215S, M296I
and R301Q mutations, but not in other mutations. This result
suggested that the final degradation of mutant α-Gal A protein
may involve different proteases.

Figure 2289

In order to assess whether the enzyme activity of mutant enzymes
could effectively be increased by DGJ, DGJ was added to theculture medium of transfected COS-7 cells. The intracellularenzyme activities of all mutant enzymes were substantially higher(1.7- to 10-fold) in cells incubated with DGJ, as compared withthose of transfected cells not treated with DGJ (Figure 3). Western
blotting determined that the amount of mature α-Gal A increased
accordingly in cells treated with DGJ. These results indicate thatthe mutant enzymes studied here are conformational amendable,
and that DGJ is an effective agent for the rescue of these mutations.
Intracellular processing and trafficking of mutant α-Gal A enzymes
found in classic Fabry disease patients

It has been demonstrated that abnormal processing and maturationof α-Gal A occurred in mutations causing the Fabry disease cardiac phenotype [20]. To confirm that abnormal protein folding that
affects trafficking and processing can occur in mutant enzymes
in classic Fabry disease patients, the E59K and L166V mutants
were subjected to further cellular processing studies.
In order to assess the intracellular processing of mutant proteins,metabolic labelling of proteins was performed in COS-7 cells
transfected with vectors expressing mutant α-Gal A cDNA
(E59K, L166V or R301Q), followed by immunoprecipitation
with an anti-α-Gal A antibody. The wild-type enzyme showed
partial maturation after 6 h, and complete maturation after 12 h
(Figure 4), evidenced by the transition of the band from 50 kDa(ER and Golgi forms) to 46 kDa (mature lysosomal form) [40].
Maturation of the E59K mutant was also complete after 12 h,suggesting that this mutation does not perturb processing andmaturation of the protein. On the other hand, both the L166V
and R301Q mutant α-Gal A enzymes remained in the nonprocessed 50 kDa form, even after a 24 h chase, and showed rapid
decay after 6 h, indicating that enzyme processing was defective.
Upon incubation of the cells with DGJ, the mutant R301Q αGal A enzyme was processed after 12 h, and the processing wascomplete after 24 h. Processing of the L166V mutant α-Gal A
progressed slowly over a 24 h period following incubation of cellswith DGJ, suggesting that DGJ treatment also partially corrects
this mutation, and improves processing during the maturation ofmutant proteins.
To examine the intracellular trafficking of the E59K and L166V
mutant α-Gal A enzymes, subcellular fractions of both mutant
enzymes cultured in the absence or presence of DGJ were

Effects of ERAD inhibitors on the amount of mutant α-Gal A expressed in COS-7 cells

Wild-type or mutant α-Gal A enzymes were transiently expressed with FuGENETM 6 transfection reagent in COS-7 cells. At 5 h after transfection, cells were treated with 2 µM lactacystin (LC), 0.2 mM
kifunensine (KFN) or nothing as a control (C), and harvested at 48 h. Western blot analyses of cell lysates from transfected COS-7 cells were performed.
c The Authors Journal compilation 
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Figure 3

S. Ishii and others

Effect of DGJ treatment on various mutant α-Gal A enzymes expressed in COS-7 cells

COS-7 cells were re-plated on six-well plates and transfected with expression constructs containing various mutant α-Gal A cDNAs using LipofectamineTM 2000 reagent. Cells were cultured incomplete medium for 3 days with or without 20 µM DGJ. α-Gal A activity and Western blot analyses of cell lysates from transfected COS-7 cells were performed as described in the Materials andmethods section. Lane 1, Mock transfection; lane 2, wild-type α-Gal A; lane 3, A20P; lane 4, E59K; lane 5, E66Q; lane 6, M72V; lane 7, I91T; lane 8, A97V; lane 9, R112H; lane 10, F113L; lane 11,
P146S; lane 12, A156V; lane 13, L166V; lane 14, N215S; lane 15, Q279E; lane 16, M296I; lane 17, M296V; lane 18, R301Q; lane 19, R356W; lane 20, G373D; and lane 21, G373S.

examined by Percoll density gradient centrifugation of COS-7
cells transfected with the mutant enzyme expression plasmids.
The mature form of the wild-type α-Gal A was recovered inboth high-density (#1–5) and low-density (#14–18) fractions(Figure 5A) that comprised the lysosomal and ER/endosomal
fractions respectively [41], in line with the pattern of βhexosaminidase as a lysosomal marker enzyme. Mutant E59K
enzyme activity was also recovered in both high- and low-density
fractions, although at a reduced level, indicating that the mutation
does not compromise normal trafficking of the mutant enzyme.
The mutant protein recovered from the lysosomal fractions
showed a reduced size (Figure 5B), indicating that it is processed
normally to the mature form. In contrast, neither enzyme activity
nor protein could be detected in the lysosomal fractions of COS-7
cells transfected with the L166V or R301Q expression plasmid.
Only trace amounts of L166V or R301Q enzyme activity andprotein were detected in the ER/endosomal fractions, although
the protein remained unprocessed. The results clearly indicatethat the E59K mutation alters the catalytic capability of themutant enzyme, and has little effect on processing, maturationand trafficking, whereas disruption of maturation and trafficking
occurred in the L166V and R301Q mutations. The results also
indicate that the abnormal processing and trafficking can be a
primary cause for the protein deficiency in mutations associatedwith classic Fabry disease. After incubation of the cells with
c The Authors Journal compilation 
c 2007 Biochemical Society

DGJ for 3 days, the fully mature lysosomal form of both mutant
enzymes (46 kDa) could be recovered in the lysosomal fractions(Figures 5A and 5B). These results indicate that DGJ treatment
can at least partially correct for the trafficking defect caused byboth mutations.
Lysosomal enzyme activities in the cells of patients with lysosomal
storage diseases cultured with DGJ

To rule out the possibility that DGJ may effect ERAD by some
unknown mechanism, we investigated the effect of DGJ on humanfibroblasts established from patients with GM1 -gangliosidosis or
Gaucher disease, in which there is a deficiency of lysosomal
β-galactosidase or glucocerebrosidase (acid β-glucosidase) respectively. The mutations identified in the GM1 -gangliosidosis andGaucher fibroblasts were homozygous I51T in the β-galactosidase gene (GLB1) and N370S in the glucocerebrosidase gene
(GBA) respectively. Both mutations are known to result in protein
trafficking defects [42,43], and residual enzyme activities of thesemutant enzymes can be partially restored by other small molecules[44].
After cultivation of Fabry disease lymphoblasts with DGJ at
20 µM for 5 days, a 3.8-fold increase in α-Gal A activity wasobserved, whereas the activities of β-galactosidase and glucocerebrosidase, which were normal in the cultured lymphoblasts,

Mutant α-galactosidase A enzymes identified in Fabry disease patients

Figure 4

Metabolic labelling of mutant α-Gal A expressed in COS-7 cells

COS-7 cells were transfected in six-well plates with expression plasmids coding for the wild-type,
E59K, L166V or R301Q α-Gal A enzymes respectively, and cultured in the absence or presenceof 20 µM DGJ. The cells were exposed to 4 µl of [35 S]Protein labelling mix for 2 h. Afterwashing the cells with PBS, the labelled proteins were chased by replacement of medium forthe indicated period. Following immunoprecipitation with a polyclonal anti-α-Gal A antibody,an aliquot (one-fifth of the sample) was analysed by SDS/PAGE (10 % gels) and visualized byfluorography.

were not significantly affected (Table 3). The activities of both mutant β-galactosidase in GM1 -gangliosidosis fibroblasts and mutant
glucocerebrosidase in Gaucher fibroblasts did not increase after
DGJ-treatment, indicating that DGJ at such concentrations had
little or no effect on these mutant proteins. Our result indicatesthat DGJ does not directly affect the ERAD machinery, and at
20 µM concentrations, its effect is limited specifically to mutant
α-Gal A.
Increase in residual enzyme activity in the cells of Fabry disease
patients by DGJ

Human lymphoblasts and fibroblasts established from hemizygous Fabry disease patients with a variety of disease-causing GLA
mutations were incubated with DGJ. Residual enzyme activity
increased substantially in cells incubated for 5 days with DGJ at
20 µM, regardless of the clinical phenotype that the mutation
conferred (Table 4). Both lymphoblasts and fibroblasts thatharboured the same mutation (e.g. A97V and R301Q) responded
to DGJ treatment, though the response in lymphoblasts was higher
than those in fibroblasts, which could be attributed to differencesin the permeability of DGJ. Noticeably, residual enzyme activity in all cells following DGJ treatment was increased by over
20 % of those in normal subjects, a level of enzyme activity
that would be expected to have a significant impact upon diseaseprogression [45], suggesting that the DGJ treatment would betherapeutically beneficial for patients with these genotypes.
DISCUSSION

The consequence of genetic errors that lead to the dysfunctionof coding proteins involved in genetic disorders can be various.

291

Nonsense, frame-shift mutations, splicing mutations and missense
mutations involving the substitution of critical amino acids
often result in the biosynthesis of mutant proteins that are nonfunctional. In other cases, missense mutations or small in-framedeletions/insertions could have little or no impact on the biologicalactivity of the mutant protein, but may cause misfolding and analtered tertiary structure of the protein. The ER lumen is a cellularcompartment where newly synthesized proteins fold into theirtertiary structure to gain biological functionality. To maintainthe integrity of each synthesized protein, cells have evolved
an efficient ‘quality control’ system, termed ERAD, in which
only properly folded and assembled proteins are transported to
the Golgi complex for further maturation, and those improperly
folded proteins are retained by molecular chaperones in the ER for
subsequent degradation [46]. Although this process is essential
for normal cellular function, the process may also contributesignificantly to protein deficiency in many inherited disorders.
It is clear that the correction of the conformation of a mutant
protein could be a therapeutic option for protein deficiencies.
Since protein folding is a thermodynamic process, even a slightshift in favour of proper folding could have a significant impact
on the increase of enzyme activity in treating the disease.
A significant number of patients with missense mutations haveresidual α-Gal A activity ranging from 1 to 10 % of normal individuals, and they are usually associated with less severe phenotypes,
either a milder classic phenotype or an atypical cardiac or renalvariant. The low level of residual enzyme activity could be theresult of either normal processing of mutant enzyme with analtered catalytic site, producing an enzyme with reduced catalytic
activity, or a small amount of correctly folded mutant enzymethat escapes ERAD. To assess the direct cause responsible for
deficient enzyme activity in Fabry disease, we studied mutations
that have been identified in patients who present a significant levelof residual enzyme activity, regardless of clinical phenotypes. In
most cases, patient’s fibroblasts or lymphoblasts harbouring thesame mutations were available to us for studies at an endogenouslevel. Based on the X-ray crystallography structure of α-Gal
A [11], twelve mutated amino acids were used in the present
study (Glu66 , Met72 , Ile91 , Ala97 , Arg112 , Phe113 , Pro146 , Ala156 ,Leu166 , Gln279 , Met296 and Gly373 ) which are normally buried
inside the tertiary structure and maintain the gross folding stateof the molecule. Three amino acids (Glu59 , Arg301 and Arg356 )are involved in the formation of ion pairs with other amino acids
that could contribute to protein folding and/or catalytic function.
The Arg20 residue is located in the signal peptide, and the Asn215
residue is one of the N-linked carbohydrate-attachment sites.
Because mutant proteins with a misfolded conformation would
be subject to rapid degradation by ERAD [47], the conformation of residual enzyme in lysosomes is considered to be
fully folded. The conformation of the mutant proteins purified
in the present study are expected to be fully folded and havea conformation similar to that of the residual enzyme in thephysiological condition. In order to assess the conformational
stability of these purified mutant enzymes, they were subjected tothermo- and pH-denaturation. A protein with a stable conformation typically resists denaturation, whereas those proteins with afragile conformational structure are often intolerant to thermo- or
pH-denaturation. The wild-type enzyme is relatively stable overa wider range of pH and particularly at neutral pH (Figure 1
and Table 2), indicating that its structure is well-folded andmaintained. Most mutant proteins were found to be stable only
over a narrower pH range (typically pH 4–6.5). Noticeably, thesemutant proteins were stable at pH 4.5–5.0, the pH similar tothat of the environment in the lysosomes, suggesting that thefolded conformation of mutant proteins is stable in lysosomes.
c The Authors Journal compilation 
c 2007 Biochemical Society

292

Figure 5

S. Ishii and others

Subcellular fractionation of the wild-type or mutant α-Gal A expressed in COS-7 cells

COS-7 cells were transfected with expression plasmids pCXN2-GLA, pCXN2-GLA-E59K, pCXN2-GLA-L166V or pCNX2-GLA-R301Q respectively. Subcellular fractionation was performed by Percoll
gradient centrifugation at 25 000 g for 1 h. Each fraction was collected, and the density determined using density markers (Fraction 2, 1.11 g/ml; fraction 4, 1.07 g/ml; fraction 7, 1.056 g/ml; fraction
12, 1.053 g/ml; and fraction 16, 1.041 g/ml). (A) α-Gal A (䊊) and β-hexosaminidase (∇) activities in each fraction of wild type α-Gal A. α-Gal A activities of E59K, L166V and R301Q in cells
treated with 20 µM DGJ (䊉) or without (䊊) in each fraction. (B) Western blot analyses with an antibody against human α-Gal A. Bip was used as an ER marker protein.

Table 3 Residual enzyme activity in the cells of patients with various
lysosomal storage diseases after DGJ treatment
Residual enzyme activities in respective cells of patients were in the range of 3–8 % of normal.
All other enzyme activities in the cells of patients were within a normal range. All cells were
cultivated with DGJ at 20 µM for 5 days prior to the enzyme assay. Enzyme activities in cells
treated with DGJ were compared with those without DGJ treatment. All values are the average ofthree parallel assays and have a S.D. of less than 15 %.
Fold increase in enzyme activity
Patient cells

α-Gal A

β-Galactosidase

Glucocerebrosidase

Fabry lymphoblasts (R301Q)
GM1 -gangliosidosis fibroblasts (I51T)
Gaucher fibroblasts (N370S)

3.8
1.3
1.1

1.1
1.2
1.3

1.3
1.1
1.0


c The Authors Journal compilation 
c 2007 Biochemical Society

All mutant proteins showed less stability compared with the wildtype enzyme at pH 7.0–7.5, a value similar to that in the ER,
indicating that they do not conform well at neutral pH. In thepresence of DGJ at pH 7.5, the instability of the mutant proteins
was alleviated to a certain degree, suggesting that DGJ helps tostabilize the conformation of the protein molecule. These results
indicate that the substitution of an amino acid residue in missense
mutant α-Gal A enzymes alters conformational stability, creating
a more fragile molecular structure under extreme pH conditions.
Folding and refolding of glycoproteins in the ER involves thecalnexin/calreticulin system and glucosidases I and II, whereas
ER α-mannosidases and EDEM (ER degradation enhancing αmannosidase I-like protein) are involved in retrotranslocationand degradation of misfolded proteins in the process of ERAD
[48]. Removal of a mannose residue from Man9 N-linked

Mutant α-galactosidase A enzymes identified in Fabry disease patients

293

Table 4 Increase in residual enzyme activity in cells from Fabry disease
patients cultivated with DGJ
Fibroblasts and lymphoblasts were obtained from hemizygous Fabry disease patients with oneof the mutations listed. All cells were cultivated in the presence or absence of DGJ at 20 µM for
5 days before their residual enzyme activity was determined. The values are the average of three
independent assays and have a S.D. of less than 10 %.
Residual enzyme activity(% of normal)
Mutation

Cell type

−DGJ

+DGJ

E59K
E66Q
I91T
A97V

Fibroblasts
Lymphoblasts
Fibroblasts
Lymphoblasts
Fibroblasts
Fibroblasts
Fibroblasts
Fibroblasts
Lymphoblasts
Lymphoblasts
Fibroblasts
Lymphoblasts
Fibroblasts

6.0
29.9
6.9
7.5
11.1
5.2
1.8
3.7
3.6
11.9
11.5
4.0
6.3

47.6
43.2
22.6
61.5
32.2
31.9
35.0
21.2
45.0
62.6
71.9
48.0
26.3

R112H
F113L
N215S
Q279E
M296I
M296V
R301Q

oligosaccharides by the ER α-mannosidase I is a critical luminal
event for preventing proteins re-entering the refolding process andserves as a signal for targeted ERAD. Therefore inhibition of the
ER α-mannosidase I often delays the degradation of glycoproteinsin ERAD in favour of protein refolding. When kifunensin, aselective inhibitor of the ER α-mannosidase I, was added to theculture medium, the amount of all mutant proteins (except E59K)
appeared to be increased (Figure 2), suggesting the degradationof mutants was partially inhibited. This result indicates that
degradation of mutant α-Gal A enzymes occurred within the
ERAD as the result of misfolding of the mutant proteins. Protein
misfolding has been recognized as having an important role in theprotein deficiency in various inherited disorders [16]. The results
obtained from a large set of missense mutant proteins further
provide evidence that protein misfolding as a primary cause ofprotein deficiency is not only limited in a few mutations, but israther a generalized cause that exists in many missense mutations
in a single genetic disorder.
Fabry disease patients with an atypical variant cardiac phenotype often retain a measurable level of residual enzyme activity,
and the main cause of the depletion of enzyme activity has been
demonstrated to be caused by impaired processing and transport
from the ER, as exemplified by the R301Q mutation [20]. Certain
milder classic patients also present residual enzyme activity, often
at a lower level. In the present study, five mutations (E59K, A97V,
A156V, L166V and R356W) causing classic Fabry disease were
examined for their effect on the kinetic properties of α-Gal A. Allof the mutant enzymes, except for the E59K mutant, retained
kinetic properties similar to those of the wild-type enzyme,
suggesting that the primary reason for the disruption in enzyme
activity does not involve reduced catalytic ability. To further
characterize the direct cause for the deficiency in enzyme activity
in classic Fabry disease patients, the E59K and L166V mutant
enzymes were chosen as examples to study their processing
and trafficking. The E59K mutant protein demonstrated normal
processing and trafficking, suggesting that the primary cause
for the deficiency in enzyme activity is the alteration in kinetic
activity. On the other hand, subcellular fractionation (Figure 5)
and metabolic labelling (Figure 4) studies showed that the L166V
mutant has perturbed processing and stalled trafficking profiles,

Figure 6 Predicted interactions between DGJ and the active siteof α-Gal A
Based upon the crystal structure of α-Gal A with bound α-galactose, we modelled the interactionsof α-Gal A with DGJ. The DGJ is shown bound to the active-site of the enzyme, in a mannervery similar to α-galactose binding. The key interactions to the 2, 3, 4, and 6 hydroxy groups
on the ligand are maintained when either α-galactose or DGJ binds to the active-site. One key
interaction between Glu231 on the enzyme and the 1 hydroxy group of α-galactose is lost when
DGJ binds, because DGJ lacks a functional group at the 1 position. At acidic pH, DGJ becomes
protonated and positively charged, which may increase its affinity for the highly negatively
charged enzyme.

comparable with those of the R301Q mutant enzyme, indicatingthat the primary biochemical defect responsible for diminishedenzyme activity is abnormal processing and trafficking. Since
four of the five classic mutations retained the normal catalytic
capability similar to the L166V mutation, deficiencies in enzyme
activity caused by abnormal processing and trafficking may be
more widespread in Fabry disease patients with residual enzyme
activity than those previously thought to be limited to the variant
phenotype.
Human α-Gal A is a homodimeric glycoprotein with eachmonomer composed of two domains, a (β/α)8 domain formed withamino acid residues 32–330 and a C-terminal domain (amino acidresidues 331–429) containing eight antiparallel β-strands on two
sheets in a β-sandwich [11]. The first domain contains the active
site formed by the C-terminal ends of the β-strands at the centre
of a barrel. Thirty residues from loops β1–α1, β6–α6, β7–α7,
β8–α8, β11–β12 and β15–β16 of each monomer contributeto the dimer interface. DGJ is an ASSC specifically serving
as a folding template for mutant enzymes that have a fragile
conformational structure. Based upon active-site interactions seenin the crystal structure of α-galactose bound to α-Gal A, a modelof DGJ binding to α-Gal A shows many favourable interactions.
The imino group on DGJ is expected to interact with Asp170 , thehydroxy groups of DGJ form hydrogen bonds with Asp92 , Asp93 ,Lys168 , Glu203 , Arg227 and Glu231 , and a hydrophobic surface on
DGJ makes van der Waals interactions with Trp47 (Figure 6). A
binding between DGJ and the protein would fix the active site
involving five loops β1–α1, β2–α2, β4–α4, β5–α5 and β6–α6.
c The Authors Journal compilation 
c 2007 Biochemical Society

294

S. Ishii and others

Mutations at the amino acid residues involving the interaction
resulted in severe classic phenotypes [11] and are predicted tobe reluctant to the DGJ rescue. On the other hand, all of themutations investigated in the present study are not involved withthe proposed interactions between the protein and DGJ, thus
permitting the rescue effect.
The E59K mutant enzyme was found to have compromised
kinetic properties, and abnormal trafficking was not a major
obstacle for expression of this mutation. Nevertheless, residual
enzyme activity in cultured fibroblasts expressing the E59K
mutant α-Gal A enzyme increased 8-fold in the presence of DGJ,
and reached a level approx. 48 % of normal enzyme activity(Table 4). It has been proposed that retention and degradationof misfolded proteins entering the secretory pathway may not be
restricted to mutant proteins [49]. Protein folding is not a perfectprocess even with wild-type proteins. A large fraction of newly
synthesized proteins never attain their native structure, and are
ubiquitinylated before being degraded by cytosolic proteasomes.
In the present study we have demonstrated that α-Gal A activity inboth normal human lymphoblasts and COS-7 cells transfectedwith wild-type GLA can be raised (approx. 10–20 %) by DGJ
treatment [20]. Small molecular ligands termed pharmacologicalchaperones have also been shown to be effective at increasing
maturation of the wild-type δ-opioid receptor [50]. Evidence
obtained from the E59K mutant enzyme indicate that chaperonelike enhancement may be effective at correcting a broad rangeof mutations beyond those mutations that mainly cause protein
misfolding.
Currently, enzyme replacment therapy is the only FDA (Foodand Drug Administration) approved therapy for Fabry disease.
The effect of infusion with exogenous wild-type α-Gal A on
patients has been well-documented. Compared with protein
macromolecules that could be difficult to deliver to tissues, DGJ
is a small molecule and therefore has the advantage of being able
to be delivered to various tissues. In the present study we also
demonstrated that residual enzyme activity could be increased by
DGJ treatment in a variety of fibroblast and lymphoblast cell lines
established from Fabry disease patients with missense mutations(Table 4). Since a large proportion of mutant forms of α-Gal A
that are found in patients with missense mutations may havefolding defects, DGJ could conceivably be effective at treating abroad number of Fabry disease patients with missense mutations
regardless of the clinical phenotype of the patients.
We thank Dr S. Nakao (Kagoshima Prefectural Kanoya Hospital, Japan) Dr R. O. Brady(National Institutes of Health, Bethesda, MD, U.S.A.) and Dr R. J. Desnick (Mount Sinai
School of Medicine, New York, NY, U.S.A.) for providing patient cell lines. We are also
grateful to Dr Dominique Germain of Hôpital Européen Georges Pompidou, Paris, France
for valuable discussion. This work was supported in part by research grants from the
Ministry of Education, Science and Culture of Japan (to S. I. and J. Q. F.), the Ministry of
Health, Labour and Welfare of Japan (to S. I.), the Mizutani Glycoscience Foundation, Irma
T. Hirschl Foundation and American Heart Association (to J. Q. F.). Dr Fan and Dr Ishii areco-inventors of patents related to the ASSC technology which is now licensed to Amicus
Therapeutics, Inc, and declares competing financial interests.

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Received 5 April 2007/24 May 2007; accepted 8 June 2007
Published as BJ Immediate Publication 8 June 2007, doi:10.1042/BJ20070479


c The Authors Journal compilation 
c 2007 Biochemical Society