J. Biochem. 2011;149(2):161–170

doi:10.1093/jb/mvq125

Increased globotriaosylceramide levels in a transgenic mouse
expressing human a1,4-galactosyltransferase and a mousemodel for treating Fabry disease
Received September 1, 2010; accepted October 14, 2010; published online October 19, 2010

1
Department of Agricultural and Life Sciences, Obihiro Universityof Agriculture and Veterinary Medicine, Obihiro, Hokkaido
080-8555; 2Department of Matrix Medicine, Faculty of Medicine,
Oita University, Yufu, Oita 879-5593; 3Laboratory of Animal
Models for Human Diseases, National Institute of Biomedical
Innovation, Ibaraki, Osaka 567-0085; 4Department of Cell Biology,
Faculty of Medicine, Oita University, Yufu, Oita 879-5593;
5Department of Diagnostic Pathology, Faculty of Medicine, Oita
University, Yufu, Oita 879-5593; 6Division of Laboratory Animal
Sciences, Research Promotion Project, Oita University, Yufu, Oita
879-5593, Japan; 7National Institute of Dental and Craniofacial
Research, Bethesda, MD 20892, USA; 8Department of Biochemistry
II, Nagoya University School of Medicine, Nagoya, Aichi 466-0065;
and 9Biochemical Laboratory, GlycoPharma Corporation, Oita
870-0822, Japan*Satoshi Ishii, Ph.D., Department of Matrix Medicine, Faculty of
Medicine, Oita University, Hasama-cho Idaigaoka 1-1, Yufu-shi,
Oita 879-5593, Japan, Tel: þ81 97 586 5661, Fax: þ81 97 546 2534,
email: ishiis01@oita-u.ac.jp

Fabry disease is a lysosomal storage disorder caused byan a-galactosidase A (a-Gal A) deﬁciency and resulting
in the accumulation of glycosphingolipids, predominantly globotriaosylceramide (Gb3). A transgenic
mouse expressing the human a-Gal A R301Q mutant
in an a-Gal A-knockout background (TgM/KO) should
be useful for studying active-site-speciﬁc chaperone(ASSC) therapy for Fabry disease. However, the Gb3
content in the heart tissue of this mouse was too low to
detect an ASSC-induced effect. To increase the Gb3
levels in mouse organs, we created transgenic mice
(TgG3S) expressing human a1,4-galactosyltransferase(Gb3 synthase). High levels of Gb3 were observed in
all major organs of the TgG3S mouse. A TgG3S(þ/)M(þ/)/KO mouse was prepared by crossbreeding the TgG3S and TgM/KO mice and the Gb3
content in the heart of the TgG3S(þ/)M(þ/)/KO
mouse was 1.4 mg/mg protein, higher than in the
TgM(þ/)/KO (50.1 mg/mg protein). Treatment
with an ASSC, 1-deoxygalactonojirimycin, caused amarked induction of a-Gal A activity and a concomitant reduction of the Gb3 content in the TgG3S(þ/)M(þ/)/KO mouse organs. These data indicated that
the TgG3S(þ/) M(þ/)/KO mouse was suitable forstudying ASSC therapy for Fabry disease, and that the
TgG3S mouse would be useful for studying the effect ofhigh Gb3 levels in mouse organs.

Keywords: active-site-speciﬁc chaperone therapy/
Fabry disease/globotriaosylceramide/mouse model.
Abbreviations: A4GalT, a1,4-galactosyltransferase;
ASSC, active-site-speciﬁc chaperone; a-Gal A,
a-galactosidase A; DGJ, 1-deoxygalactonojirimycin;
Gb3, globotriaosylceramide (Gala1,4Galb1,
4Glc-Ceramide); GLAko mouse, a-Gal A knockout
mouse; Gb4, globotetraosylceramide; G3Stg/GLAko
mouse, transgenic mouse expressing human Gb3
synthase in a-Gal A knockout background; G3S/
COS-7 cells, COS-7 cells expressing human Gb3
synthase; HPTLC, high-performance thin-layer
chromatography; LacCer, lactosylceramide; PBS,
phosphate-buffered saline; Stx1B, Shiga toxin 1
B-subunit; TgM/KO mouse, transgenic mouse
expressing human mutant R301Qa-Gal A in a-Gal A
knockout background; TgG3S mouse, transgenic
mouse expressing human Gb3 synthase; TgG3S(þ/)M(þ/)/KO mouse, transgenic mouse
expressing human Gb3 synthase and mutant
R301Qa-Gal A in a-Gal A knockout background.

Fabry disease is an X-linked recessive disease caused byinsufﬁcient activity of lysosomal a-galactosidase A(a-Gal A, EC 3.2.1.22), an enzyme responsible for the
catabolism of glycosphingolipids, predominantly globotriaosylceramide (Gb3) (1). Patients with Fabry
disease show diverse clinical manifestations caused bygeneralized vasculopathy—pain in the extremities,
hypohidrosis, angiokeratoma, corneal opacity, ischaemic heart disease, progressive nephropathy and cerebrovascular disease (2). Approximately 60% of themutations reported in patients with Fabry disease are
missense mutations in the a-Gal A gene. Althoughmany products encoded by these missense mutations
have normal catalytic properties, they also have low
thermostability and degrade rapidly after their synthesis in the endoplasmic reticulum (ER) (35). A novel
therapeutic strategy for the treatment of Fabry disease,which uses competitive inhibitors of a-Gal A as
active-site-speciﬁc chaperones (ASSCs), has been suggested (68). The normal folding of those missense
mutant enzymes can be restored by ASSC treatment,
thus preventing excessive ER-associated degradationand improving the transport of the mutant enzyme tothe lysosomes (4). Cultivation of patients’ cells with an
ASSC of a-Gal A [e.g. 1-deoxygalactonojirimycin(DGJ)] at low concentrations resulted in a substantial

ß The Authors 2010. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

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Chikara Shiozuka1, Atsumi Taguchi1,2,
Junichiro Matsuda3, Yoko Noguchi3,
Takanori Kunieda3, Kozue Uchio-Yamada3,
Hidekatsu Yoshioka2, Ryoji Hamanaka4,
Shinji Yano5, Shigeo Yokoyama5,
Kazuaki Mannen6, Ashok B. Kulkarni7,
Koichi Furukawa8 and Satoshi Ishii1,2,9,*

C. Shiozuka et al.

Materials and Methods
Establishment of a stable transformant expressing A4GalT
in COS-7 cells and A4GalT-overexpressing
transgenic mice (TgG3S)
The human Gb3 synthase (G3S) cDNA containing the full-length
coding sequence and an EcoRI site at both ends was prepared bypolymerase chain reaction (PCR) using a PhusionTM High-Fidelity
PCR Kit [New England BioLabs (NEB), Ipswich, MA, USA]. The
cDNA for a1,4Gal-TpVTR1 (19) was the template, and the primer
sequences were 50 -TGGGAATTCCATGTCCAAGCC-30 and
50 -GGGGAATTCACAAGTACATTTTCATGGC-30 . The 1.1-kb
PCR product was puriﬁed with a PCR Puriﬁcation Kit (QIAGEN
K.K., Japan) and digested with EcoRI (NEB). The digested cDNA
was subcloned into the EcoRI site of expression vector pCXN2 (20),and the product was designated as pCXN2-G3S. For the preparation of the stably transformed G3S/COS-7 cells, pCXN2-G3S
was linearized with HindIII (NEB) and then transfected into
COS-7 cells with the FuGENETM6 transfection reagent (Roche
Molecular Biochemicals, Basel, Switzerland), according to the
manufacturer’s protocol. Stably transformed G3S/COS-7 cells wereselected by growing them in culture medium containing 400 mg/ml of
G418 (Sigma, St Louis, MO, USA) for 4 weeks.

162

A DNA fragment comprising a mammalian expression unit andthe human Gb3 synthase cDNA was prepared by digesting thepCXN2-G3S with SalI and BamHI (NEB). A fragment that was3.5 kb was isolated by agarose gel electrophoresis and puriﬁed bya Gel Extraction Kit (QIAGEN). A transgenic mouse (TgG3S) expressing human Gb3 synthase was generated by injecting the DNA
fragments into the pronuclei of fertilized eggs taken from superovulated C57BL/6J Jms female mice, and the embryos were implanted
into pseudopregnant Jcl:MCH mice, as described previously (21).
Transgenic founder mice were identiﬁed by PCR with a primer set(50 -ATTGTTCTCAAGAACCTGCG-30 and 50 -ATTTGTGAGCC
AGGGCATTG-30 ). A transgene fragment was conﬁrmed as asingle 548-bp band on agarose gel electrophoresis. To generate an
animal model exclusively expressing both human G3S and humanmutant a-Gal A in an endogenous GLA knockout background[TgG3S(þ/)M(þ/)/KO mice], we ﬁrst prepared a mouse line
overexpressing the human G3S transgene in an a-Gal A knockout
background (G3Stg/GLAko) by crossbreeding male TgG3S miceand homozygous female GLAko mice (since GLA is located on the
X chromosome). After the G3Stg/GLAko mouse line was established, TgM/KO and G3Stg/GLAko mice were crossbred to obtain
TgG3S(þ/)M(þ/)/KO mice.
Determination of A4GalT mRNA expression in mouse tissues
The A4GalT mRNA expression of the transgene (human A4GalT)
was determined by a reverse transcriptase (RT)-PCR using Takara
RNA PCR Kit (AMV) Ver.3.0 (Takara Bio Inc., Shiga, Japan). The
total RNA samples from tissues of wild-type and transgenic mice
were prepared with RNAiso (Takara Bio Inc.), and the RNA concentrations were determined by absorbance at 260 nm. The RT
reaction was performed at 30 C for 10 min, and then at 42 C for
15 min, followed by incubation at 99 C for 5 min. PCR ampliﬁcation
was performed using the following conditions: initial denaturation(94 C, 5 min) followed by 30 cycles of denaturation at 94 C for 30 s,
annealing at 58 C for 30 s, elongation at 72 C for 30 s and further
elongation at 72 C for 2 min. The DNA fragment (409 bp), whichcontained a sequence that is highly conserved between the mouseand human A4GalT genes, was ampliﬁed with primers 50 -GGCATC
TC(A/T)CTTCTGAGCTG-30 and 50 -GGATGGAACACCACTTC
TTG-30 . PCR products were run on a 2% agarose gel, stainedwith ethidium bromide and photographed. The glyceraldehyde3-phosphate dehydrogenase (GAPDH) gene was used as an expression control gene as described previously (22).
Administration of DGJ to TgG3S(þ/)M(þ/)/KO mice
TgG3S(þ/)M(þ/)/KO mice were supplied with fresh tap water
ad libitum and rodent pellets. DGJ (Toronto Research Chemicals,
Toronto, Canada) was administrated to one group of female
TgG3S(þ/)M(þ/)/KO mice in tap water as a DGJ aqueous
solution without any other substances. After 4 weeks, the animals
were sacriﬁced and the organs were quickly removed and rinsed withphosphate-buffered saline (PBS). Tissue homogenates were subjected
to enzyme assays and lipid extraction.
Assay of enzyme activity and protein content
All samples were kept on ice and processed as rapidly as possible.
Approximately 10% (w/v) tissue homogenates were prepared inwater using a micro-homogenizer (Physcotron, Niti-on Inc., Chiba,
Japan). The supernatant obtained from the homogenate after centrifugation at 10,000g for 5 min was used in the enzyme assays. The
a-Gal A activity was assayed with 4-methylumbelliferyl a-D-galactoside (Sigma) as the substrate and N-acetyl-D-galactosamine (75 mM)
as the inhibitor for a-N-acetylgalactosaminidase in 0.1 M sodium
citrate buffer (pH 4.6) as described previously (16). The protein concentration was determined using a DC Protein Assay kit (Bio-Rad
Laboratories, Hercules, CA, USA) with bovine serum albumin as
the standard.
Detection of neutral and acidic glycosphingolipids bythin-layer chromatography analysis
Glycosphingolipids were extracted from cultured cells and mouse
tissues and were analysed as described previously (14), with somemodiﬁcations. Crude lipids were extracted from cultured cells(eq. to 1 mg of protein) and tissue homogenates (eq. to 5 mg ofprotein), using a mixture of chloroform : methanol (2 : 1, v/v).
Glycosphingolipids were dried under a stream of nitrogen.

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increase in both residual enzyme activity and theamount of intracellular enzyme protein (4, 6, 9).
A mouse line deﬁcient in a-Gal A (GLAko mice)was established in 1997 by disruption of the murine
a-Gal A gene (10). Despite the accumulation of Gb3
in Fabry disease-relevant organs of the GLAko mice,the mice were clinically normal and had a typical life
span. These mice have served as an excellent model forstudies on enzyme replacement therapy (11), genetherapy (1214) and substrate reduction therapy (15),wherein the increased a-Gal A activity and reduced
Gb3 accumulation were primary objectives. However,this mouse model is not suitable for studying ASSC
therapy, which requires the expression of a humanmutant enzyme. In a previous report (16), we established a homozygous transgenic mouse in a murine
a-Gal A knockout background (TgM/KO mouse) by
crossbreeding a TgM mouse expressing the humana-Gal A R301Q mutant (17) in GLAko mice. Like
the GLAko mouse, the TgM/KO mouse does notexhibit a disease phenotype, however these mice do
express human mutant a-Gal A and serve as an excellent biochemical model for studying ASSC (18).
Heterozygous TgM/KO mice show a lower a-Gal A
activity than the homozygotes, and Gb3 accumulates
in the kidney of the heterozygous mouse, but not in theheart, which is affected in Fabry disease (16).
The purpose of this study was to establish a transgenic mouse that expressed high levels of Gb3 as wellas the human mutant a-Gal A enzyme in heart tissue,
which would permit us to assess the effect of ASSC
(enhancement of a-Gal A activity and decrease in
Gb3 accumulation) in the heart. Here, in order to increase cardiac Gb3 production, we simply overexpressed human a1,4-galactosyltransferase [A4GalT,
Gb3 synthase (G3S, EC 2.4.1.228)] to obtain the
TgG3S mouse line. We conﬁrmed that these mice expressed high levels of Gb3 in their major organs, andthen bred them with the TgM/KO mice to obtain
TgG3S(þ/)M(þ/)/KO mice. Here we report thecharacterization and potential usefulness of this new
mouse line.

a1,4-galactosyltransferase transgenic mouse

Binding assay
The Stx1B binding assay was performed as described previously (23),with some modiﬁcations. After glycosphingolipids were separated by
TLC as described above, a TLC plate was sunk in a 0.4% polyisobutylmethacrylate (GlycoTech, Rockville, MD, USA) solution(2.5% polyisobutylmethacrylate in chloroform was diluted to 0.4%
with hexane) and then blocked with 1% bovine serum albumin in
PBS (BSA-PBS). The plate was incubated with 2.5 mg/ml of Stx1B in
BSA-PBS at room temperature for 20 min and washed with PBS.
After incubation with 7.0 mg/ml of anti-Stx1B polyclonal antibody(produced in rabbits with puriﬁed Stx1B) for 20 min the plate waswashed with PBS. After further incubation with horseradish
peroxidase-conjugated anti-rabbit IgG (Pierce Chemical, Rockford,
IL, USA) at room temperature for 20 min and the following
ﬁnal washing, Stx1B-binding was visualized with an enhanced
chemiluminescent substrate (Pierce).

markedly higher than those in the parental COS-7
cells or in a control line transformed with only theempty vector. The content of lactosylceramide(LacCer) in the G3S/COS-7 cells was lower than it
was in the COS-7 cells, indicating that the expression
product from the A4GalT gene in our construct catalysed the construction of Gb3 from LacCer.
A mouse line expressing the A4GalT gene (TgG3S)
was generated by injecting the DNA fragment into thepronuclei of fertilized mouse ova taken from C57BL/
6J female mice. A marked increase in the expression of
the A4GalT gene in heart, kidney, liver, spleen, small
intestine, brain, lungs and muscle of the TgG3S micecompared with wild-type C57BL/6J mice was observedby RT-PCR (Fig. 2A). High levels of Gb3 synthase
expression resulted in an increase in Gb3 levels of allorgans examined in this study (Fig. 2B). A glycosphingolipid band (possibly galabiosylceramide) with an Rf
value similar to that of LacCer by HPTLC was also
increased in the kidney and brain of the TgG3S mouse.
No change was observed in the content of acidic glycosphingolipids including gangliosides in organs of
TgG3S mice (data not shown). The TgG3S mouseline did not show any clinical phenotype and had atypical life span.
Preparation of TgG3S(þ/)M(þ/)/KO mouse line asa model mouse for ASSC therapy

To study the effect of ASSC on Gb3 content in theheart, we tried to increase the cardiac Gb3 content of
the TgM/KO mouse by introducing the G3Soverexpressing transgene. The TgG3S(þ/)M(þ/)/KO mouse was created by crossbreeding the G3Stg/
GLAko mouse and the TgM/KO mouse. The Gb3
content in the TgG3S(þ/)M(þ/)/KO heart andkidney was determined and compared with that in

Immunoelectron microscopy
The G3S/COS-7 cells were cultured with or without 10 mM DGJ for
4 days, then ﬁxed in 2% glutaraldehyde in 0.2 M phosphate buffer(pH 7.4) for 3 h; they were then further incubated in 1% osmium
tetraoxide for 2 h, dehydrated in ethanol and embedded in Epok 812.
Immunoelectron microscopy was performed as described previously(24). In summary, each thin section was brieﬂy microwaved inTarget Retrieval Solution, pH 10 (DAKO, Carpinteria, CA, USA)and then incubated for 30 min at room temperature with Stx1B(2.5 mg/ml). After being washed with Wash Buffer [50 mM
TrisHCl (pH 7.6) containing 0.8% NaCl and 0.1% BSA], the ultrathin section was incubated with anti-Stx1B antibody (7 mg/ml). After
another wash, the section was incubated with gold-conjugated goatanti-rabbit IgG. The section was then washed again, stained withuranyl acetate and lead citrate, and examined using a transmission
electron microscope (JEM-1200EXII, JEOL, Akishima, Tokyo,
Japan).

Results
Establishment of TgG3S miceFirst, we prepared a stable transformant COS-7 cellline expressing Gb3 synthase (G3S/COS-7 cells) to
conﬁrm that the expression construct prepared in ourpresent study could increase intracellular Gb3 content.
We extracted neutral glycosphingolipids from the cells
and quantitated them by high performance TLC(HPTLC) (Fig. 1). The Gb3 and globotetraosylceramide (Gb4) levels in the G3S/COS-7 cells were

Fig. 1 Neutral glycosphingolipids in stably transformed G3S/COS-7
cells. Neutral glycosphingolipids were extracted from cell homogenates (containing 1 mg protein), as described in ‘Materials andMethods’ section. Glycosphingolipids were visualized withorcinol-sulphuric acid reagent. Lane 1, intact COS-7 cells; lane 2,
mock transfection; lane 3, stable transformant G3S/COS-7 cells.

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The dried samples were dissolved in chloroform : methanol : water(30 : 60 : 8, v/v) and applied to a TOYOPEARL DEAE-650 column(Tosoh Corporation, Tokyo, Japan). The column was washed withchloroform : methanol : water (30 : 60 : 8, v/v). The pass-through fraction was pooled for the preparation of neutral glycosphingolipids.
The acidic glycosphingolipids including gangliosides were eluted
with chloroform : methanol : 0.8 M sodium acetate (30 : 60 : 8, v/v),
and they were desalted with Sep-Pak C18 reverse-phase cartridge(Waters, Milford, MA, USA) and then analysed by thin-layer chromatography (TLC). The neutral glycosphingolipids fraction wasdried, and then subjected to mild alkaline treatment with 1 ml of 0.1
N NaOH in methanol at 40 C for 2 h. After neutralizing the solution
with glacial acetic acid, glycosphingolipids were further subjected to
the Folch’s partition (choloroform : methanol : H2O, 8 : 4 : 3 in v/v/v),
recovered in the lower phase, and then quantitatively applied to TLC
plates. TLC analyses were performed with HPTLC-Silica gel 60 plates(Merck & Co., Inc., Whitehouse Station, NJ, USA) using a solvent
system of chloroform : methanol : water (60 : 35 : 8, v/v/v) and
chloroform : methanol : 0.2% CaCl2 (60 : 35 : 8, v/v/v) for the
separation of neutral and acidic glycosphingolipids, respectively.
Glycosphingolipids were detected by spraying the plate withorcinol-sulphuric acid reagent, and heating the plate at 110 C. The
Gb3 from porcine erythrocytes was purchased from Nakalai Tesque(Kyoto, Japan), and other glycosphingolipid standards were purchased from Wako Pure Chemicals (Osaka, Japan). The gangliosidestandards from bovine brain (GM1, GM2 and GM3) were purchasedfrom Wako.

C. Shiozuka et al.

kidney by the expression of Gb3 synthase, however noabnormality was observed in the histological examination of both organs from TgG3S(þ/)M(þ/)/KO
mouse (data not shown).
Age-related increase in the kidney Gb3 content in
TgG3S(þ/)M(þ/)/KO mice

Effect of 0.05 mM DGJ treatment on Gb3 content inheart and kidney of TgG3S(þ/)M(þ/)/KO mice

Fig. 2 Expression of A4GalT gene and glycosphingolipids in tissues ofTgG3S mice. A human G3S-overexpressing mouse line was prepared
as described in ‘Materials and Methods’ section. (A) The expressionof A4GalT mRNA was determined by RTPCR analysis with aprimer set that can amplify both mouse and human genes. The
glyceraldehyde phosphate dehydrogenase (GAPDH) gene wasampliﬁed as an internal control. (B) Neutral glycosphingolipids
were extracted from tissue homogenates (containing 5 mg protein)prepared from age-matched TgG3S and wild-type mice andapplied to HPTLC, as described in the legend to Fig. 1.

wild-type C57BL/6J, TgG3S(þ/) and TgM(þ/)/KO
mice (Fig. 3). A Gb3 level as high as that of the TgG3S
mice (1.3  0.3 mg/mg protein) was observed in
the heart of the TgG3S(þ/)M(þ/)/KO mice(1.1  0.3 mg/mg protein) (Fig. 3A). Furthermore,when we probed the HPTLC plate with Shiga toxin 1
B-subunit (Stx1B), which selectively binds Gb3, we did
not detect Gb3 in the heart tissue of mouse lines that
did not express the A4GalT transgene (i.e. wild-type
and TgM(þ/)/KO mice) (Fig. 3C). In contrast, Gb3
was detectable in kidney tissue of all the mouse lines,
and a slight increase was observed in the lines expressing the A4GalT transgene (Fig. 3B, D and F). The
A4GalT transgene did not change the a-Gal A activity
in either the heart or kidney (Fig. 3G and H). Marked
increase in Gb3 content was detected in the heart and164

DGJ was administrated to TgG3S(þ/)M(þ/)/KO
mice in their water as a 0.05 mM solution, available
ad libitum, for 4 weeks. Based on the daily water consumption, the DGJ dosage was calculated to be3 mg/kg body weight/day. In the heart tissues ofTgG3S(þ/)M(þ/)/KO mice after treatment with
DGJ, a 0.69-fold decrease in Gb3 content(1.44  0.38 and 0.99  0.40 mg/mg protein in control
and DGJ-treated mice, respectively) was observed,
along with a 5.4-fold increase in a-Gal A activity(24.7  7.6 and 133.0  24.9 unit/mg protein in control
and DGJ-treated mice, respectively) (Fig. 5). Likewise,
the Gb3 content of the kidney tissue decreased
0.61-fold (6.56  1.33 and 3.97  0.57 mg/mg protein
in control and DGJ-treated mice, respectively), andthe a-Gal A activity in the kidney increased 4.4-fold(6.4  3.1 and 28.0  2.8 unit/mg protein in control and
DGJ-treated mice, respectively) after treatment. These
data indicate that the enhancement of the mutant
a-Gal A activity by treatment with DGJ reduced theGb3 content of the heart as well as the kidney, and that
the TgG3S(þ/)M(þ/)/KO mouse line is a usefulmouse model for the study of ASSC treatment inFabry disease-relevant organs.
Inhibitory effect of high-concentration DGJ on a-Gal A
activity causes Gb3 accumulation in G3S/COS-7 cells

To determine the critical concentration of DGJ
required to reduce the Gb3 accumulation of mammalian cells, G3S/COS-7 cells were cultured in DGJ(010 mM)-supplemented medium for 4 days.
Signiﬁcant Gb3 accumulation was observed in cases
where DGJ dosage was 41 mM (Fig. 6A and B). As
an explanation, we found that DGJ decreased theintracellular a-Gal A activity at these higher concentrations, although it slightly increased the a-Gal A
activity at 10 mM DGJ, by the ASSC effect (Fig. 6C).
To conﬁrm that the Gb3 accumulated in the lysosomes of the G3S/COS-7 cells, an immunoelectron
microscopic study was conducted using the Stx1B
and anti-Stx1B polyclonal antibodies to label the

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The Gb3 content in the kidney of both the TgM(þ/)/KO and the TgG3S(þ/)M(þ/)/KO mice increased
3-fold between 5 and 10 weeks of age (Fig. 4A and
B). In contrast, neither the Gb3 content in the heart
nor the a-Gal A activity in the heart or kidney changed
during this time (Fig. 4BD). These data showed that
we needed to use age-matched TgG3S(þ/)M(þ/)/KO mice to determine the effect of DGJ on Gb3 content in the kidney. Because the change in the Gb3 content in the kidney changed relatively little between
10 and 15 weeks of age, we used mice in this age
range to study the effects of DGJ treatment.

a1,4-galactosyltransferase transgenic mouse

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Fig. 3 Characterization of TgG3S(þ/)M(þ/)/KO mouse line. Gb3 content and a-Gal A activity in heart (A, C, E, and G) and kidney (B, D,
F, and H) tissue from 10-week-old TgG3S(þ/)M(þ/)/KO mice were compared to those from age-matched mice from other lines. In (AH),
the lanes and columns show results from the following genotypes: 1, wild-type; 2, TgG3S(þ/); 3, TgM(þ/)/KO; 4, TgG3S(þ/)M(þ/)/KO.
The neutral glycosphingolipids were extracted and visualized with orcinol-sulphuric acid reagent (A and B) or by Stx1B-binding (C and D). The
Gb3 content (E and F) and a-Gal A activity (G and H) were determined as described in ‘Materials and Methods’ section.

Gb3 (Fig. 6D and E). The number of gold particles ineach lysosome was markedly increased in the samples
treated with 10-mM DGJ, indicating that the Gb3 hadaccumulated in them.

Administration of DGJ at high concentrations to
TgG3S(þ/)M(þ/)/KO mice

To elucidate whether high concentrations of DGJ
would also cause Gb3 accumulation in mouse tissues,165

C. Shiozuka et al.

Fig. 5 Effect of DGJ treatment on Gb3 content and a-Gal A activity in TgG3S(þ/)M(þ/)/KO mice. DGJ (0.05 mM) was administrated to
10-week-old TgG3S(þ/)M(þ/)/KO mice in their drinking water for 4 weeks. The dosage of DGJ was estimated to be 3 mg/kg/day. Six female
mice each were used in the DGJ-treated (dark-coloured bar) and control (light-coloured bar) groups. The Gb3 content (A) and a-Gal A activity(B) in the heart and kidney were determined as described in ‘Materials and Methods’ section. The statistical signiﬁcance of the difference wasdetermined by Student’s t test. *P50.05, **P50.01.

TgG3S(þ/)M(þ/)/KO mice (three 10-week-old
female mice for each group) were treated with DGJ
at 5-fold (0.25 mM) and 25-fold (1.25 mM) higher concentrations than in the original experiment, for
4 weeks. No signiﬁcant increase in Gb3 content
either in heart or kidney tissue was observed after
treatment with DGJ at high concentrations, and thelow level of Gb3 in the TgG3S(þ/)M(þ/)/KO
mouse tissues (1.0 and 3.7 mg/mg protein in heart
and kidney, respectively) was maintained (Fig. 7A
166

and B), compared to that of age-matched
G3Stg/GLAko mice (14 and 38 mg/mg protein inheart and kidney, respectively), which have no a-Gal
A activity in their organs. Likewise, DGJ increased
a-Gal A activity dose-dependently in the heart and kidney of the TgG3S(þ/)M(þ/)/KO mice (Fig. 7C and
D). These data indicated that at least a 25-fold higher
DGJ concentration (75 mg/kg body weight/day) may
be safe for the treatment of patients with Fabry
disease.

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Fig. 4 Developmental changes in Gb3 content and a-Gal A activity in mouse heart and kidney. The Gb3 content and a-Gal A activity in the heart
and kidney of TgM(þ/)/KO (A and C) and TgG3S(þ/)M(þ/)/KO mice (B and D) were determined at the indicated ages, as described in‘Materials and Methods’ section. Each bar represents the means  SD of data from three or four mice.

a1,4-galactosyltransferase transgenic mouse

Discussion
To increase the Gb3 content in mouse tissues, we tried
overexpressing human Gb3 synthase in them. Sincehigh Gb3 content was observed in tissues of the
TgG3S mouse compared with wild-type mouseorgans (Fig. 2), Gb3 synthase may be a critical
enzyme for the synthesis of Gb3 in mouse organs, particularly in the heart and small intestine, where theincrease was greatest. The increase in the tissue Gb3
level was determined by the binding of Stx1B (Fig. 3),
which selectively binds Gb3 (25, 26). The effect of
G3S-overexpression on Gb3 content was greater inthe heart than in the kidney, indicating that Gb3 synthase is expressed at low levels in wild-type mouseheart. The absence of Gb3 in the TgM/KO mouseheart was caused not only by its low expression of
the Gb3 synthase gene, but also by the high activityof the mutant a-Gal A. However, the G3S-stimulated
Gb3 production outstripped Gb3’s decomposition
by a-Gal A activity in the heart of the TgG3S(þ/)M(þ/)/KO mouse, leading to the accumulationof Gb3 (even though the Gb3 content was still lower
than it was in the kidney).
We did not identify the neutral glycolipid, which has
a similar Rf value to that of LacCer on HPTLC andwhich increased in the kidney and brain tissues in
response to G3S-overexpression. However, it may
be galabiosylceramide (Gala1,4Gal-Cer), since this
glycolipid can be made by Gb3 synthase (19), andgalactosylceramide (Gal-Cer) is produced in thekidney as well as in the brain (27).
The kidney Gb3 level increased with age in bothTgM(þ/)/KO and TgG3S(þ/)M(þ/)/KO mice
(Fig. 4). However, the a-Gal A activity was not changed
in kidney tissue by the additional expression of
the Gb3 synthase gene, and an increase in the kidney
Gb3 level with age was also reported in GLAko mice(28), indicating that the age-related Gb3 accumulationin the kidney is not caused by a decrease in the catabolism of Gb3. Although the mechanism by which
kidney Gb3 levels increase with age is unknown, theseﬁndings meant that we had to use age-matched mice
to determine the ASSC effect on the kidney Gb3 level.
After successfully increasing the heart Gb3 level inmice, we were able to examine the ASSC effect of DGJ
on Gb3 content in the heart tissue of TgG3S(þ/)M(þ/)/KO mice. A signiﬁcant reduction of Gb3
in the heart was observed following treatment with
0.05 mM DGJ for 4 weeks, indicating that TgG3S(þ/)M(þ/)/KO mice are a useful animal model for
determining the effect of ASSC therapy. A recent
report on ASSC by Khanna et al. (29) using model
mice also demonstrated that treatment with DGJ
decreases the Gb3 level in the mouse heart. Although
they used the same human mutant a-Gal A (R301Q)
cDNA for the preparation of a model mouse, theirstrategy was very different from ours. Their mousemodel, which shows Gb3 accumulation in heart
167

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Fig. 6 Treatment of G3S/COS-7 cells with high-concentration DGJ. G3S/COS-7 cells were cultured for 4 days in high-glucose DMEM medium
containing 10% foetal bovine serum with different concentrations of DGJ. After cells were rinsed with PBS, cells were harvested with PBS by aplastic scraper, and collected by centrifugation (3000g, 5 min). The cell pellet was homogenized with water and used for the determination of Gb3
content and a-Gal A activity. In (A), HPTLC analysis of Gb3 and Gb4 was visualized by orcinol-sulphuric acid reagent. The values of Gb3
content (B) and a-Gal A activity (C) are the means  SD from three cultures. The statistical signiﬁcance of the difference was determined byStudent’s t test; *P50.05 versus DGJ-free culture. In (A, B and C), lanes and columns are the same, and 1, DGJ-free control culture; 2, 1 mM
DGJ; 3, 10 mM DGJ; 4, 100 mM DGJ; 5, 1 mM DGJ; 6, 10 mM DGJ. The accumulation of Gb3 in lysosomes was determined by Stx1B-binding
immunoelectron microscopic study in DGJ-free (D) and 10 mM DGJ-treated G3S/COS-7 cells (E). The ultrathin sections were incubated with
Stx1B, then with anti-Stx1B polyclonal antibody, followed by immunogold labeling. Typical gold particles are pointed out by arrows.

C. Shiozuka et al.

tissues, was prepared by reducing the expression levelof the mutant a-Gal A gene by using the human GLA
promoter—which is a very weak promoter— instead of
the stronger CAG (cytomegalovirus immediate-earlyenhancer/chicken b-actin hybrid) promoter. In contrast, we prepared a mouse model overexpressing
both Gb3 synthase and the mutant a-Gal A genes.
Our study conﬁrmed the work of Khanna et al. (29)by reproducing their ﬁndings on the effect of DGJ in
the heart, in a different mouse model. Although theeffect of ASSC on Gb3 content can be determined in
our mice and mice reported by Khanna et al. (29), thebeneﬁt of our mice is to be able to determine the localization and content of mutant enzyme in mice tissue
with its antibody (16, 18) as well as the localization of
Gb3. Our mice will be useful for the elucidation of themechanism of ASSC effect in detail by the detection ofboth mutant enzyme and Gb3 at the same time.
Although treatment with DGJ at a low concentration (5100 mM) can increase mutant a-Gal A activity,
at high concentrations (41 mM) DGJ signiﬁcantly
inhibits a-Gal A (30), leading to a corresponding increase in Gb3 levels in G3S/COS-7 cells (Fig. 6). Here,the increase in Gb3 content correlated with a decreasein the intracellular a-Gal A activity, and we used
immunoelectron microscopy to show that Gb3 accumulated in the lysosomes of G3S/COS-7 cells treatedwith 10 mM DGJ. These data demonstrate the importance of determining a safe and effective concentrationof DGJ for use in clinical therapy. In our present
168

study, we used a 25-fold higher concentration(75 mg/kg/day treatment) of DGJ than the usual
dose (3 mg/kg/day treatment), which did not inhibit
a-Gal A activity nor increase the tissue Gb3 level(Fig. 7), indicating that the tissue concentration of
DGJ is below the inhibitory level at oral dosage.
From no further reduction of Gb3 content either inheart or kidney tissue was observed by the treatment
with DGJ at higher concentrations, the usual dose
(3 mg/kg/day treatment) may be a suitable dose for
the ASSC effect in our mouse model.
In conclusion, the Gb3-synthase-overexpressing
mouse (TgG3S) shows an increased Gb3 level in its
organs, and was useful in generating a mouse line
(TgG3S(þ/)M(þ/)/KO) that can be used as an animal model for studying the efﬁcacy of ASSC therapy
for Fabry disease.
Funding
Grant-in-Aid for Scientiﬁc Research (C) [KAKENHI (20590300)]
from Japan Society for the Promotion of Science.

Conflict of interest
None declared.

References
1. Brady, O.R., Gal, A.E., Bradley, R.M., Martensson, E.,
Warshaw, A.L., and Laster, L. (1967) Enzymatic defectDownloaded from jb.oxfordjournals.org at Oita University Library, Medical Branch on February 2, 2011

Fig. 7 Administration of high concentrations of DGJ to TgG3S(þ/)M(þ/)/KO mice. High concentrations of DGJ were administered to
TgG3S(þ/)M(þ/)/KO mice (three or four 11-week-old female mice in each group) for 4 weeks. The Gb3 content and a-Gal A activity in theheart (A and C, respectively) and in the kidney (B and D, respectively) were determined as described in ‘Materials and Methods’ section. Lanes inAD were the same, and lane 1, 0.05 mM DGJ treated; lane 2, 0.25 mM DGJ treated; and lane 3, 1.25 mM DGJ treated TgG3S(þ/)M(þ/)/KO mice, and lane 4, age-matched TgG3S(þ/)/KO mice.

a1,4-galactosyltransferase transgenic mouse

15. Abe, A., Gregoory, S., Lee, L., Killen, P.D., Brady,
R.O., Kulkarni, A., and Shayman, J.A. (2000)
Reduction of globotriaosylceramide in Fabry disease
mice by substrate deprivation. J. Clin. Invest. 105,15631571
16. Ishii, S., Yoshioka, H., Mannen, K., Kulkarni, A.B., andFan, J.-Q. (2004) Transgenic mouse expressing humanmutant a-galactosidase A in an endogenous enzyme
deﬁcient background: a biochemical animal model forstudying active-site speciﬁc chaperone therapy for
Fabry disease. Biochim. Biophys. Acta 1690, 250257
17. Shimmoto, M., Kase, R., Itoh, K., Utsumi, K., Ishii, S.,
Taya, C., Yonekawa, H., and Sakuraba, H. (1997)
Generation and characterization of transgenic mice expressing a human mutant a-galactosidase with an R301Q
substitution causing a variant form of Fabry disease.
FEBS Lett. 417, 8991
18. Ishii, S., Chang, H.-H., Yoshioka, H., Shimada, T.,
Mannen, K., Higuchi, Y., Taguchi, A., and Fan, J.-Q.
(2009) Preclinical efﬁcacy and safety of 1-deoxygalactonojirimycin in mice for Fabry disease. J. Pharmacol. Exp.
Ther. 328, 723731
19. Kojima, Y., Fukumoto, S., Furukawa, K., Okajima, T.,
Wiels, J., Yokoyama, K., Suzuki, Y., Urano, T., Ohta,
M., and Furukawa, K. (2000) Molecular cloning of globotriaosylceramide/CD77 synthase, a glycosyltransferasethat initiates the synthesis of globo series glycosphingolipids. J. Biol. Chem. 275, 1515215156
20. Niwa, H., Yamamura, K., and Miyazaki, J. (1991)
Efﬁcient selection for high-expression transfectants witha novel eukaryotic vector. Gene 108, 193200
21. Matsuda, J., Suzuki, O., Oshima, A., Yamamoto, Y.,
Noguchi, A., Takimoto, K., Itoh, M., Matsuzaki, Y.,
Yasuda, Y., Ogawa, S., Sakata, Y., Nanba, E., Higaki,K., Ogawa, Y., Tominaga, L., Ohno, K., Iwasaki, H.,
Watanabe, H., Brady, R.O., and Suzuki, Y. (2003)
Chemical chaperone therapy for brain pathology inG(M1)-gangliosidosis. Proc. Natl Acad. Sci. USA 100,
1591215917
22. Ishii, S., Katsumura, T., Shiozuka, C., Ooyauchi, K.,
Kawasaki, K., Takigawa, S., Fukushima, T., Tokuji,Y., Kinoshita, M., Ohnishi, M., Kawahara, M., andOhba, K. (2008) Anti-inﬂammatory effect of buckwheat
sprout in lipopolysaccharide-activated human coloncancer cells and mice. Biosci. Biotechnol. Biochem. 72,
31483157
23. Shin, I.-S., Nishikawa, K., Maruyama, H., and Ishii, S.
(2006) Histidine-tagged Shiga toxin B subunit
binding assay: simple and speciﬁc determination ofGb3 content in mammalian cells. Chem. Pharm. Bull.
54, 522527
24. Yano, S., Kashima, K., Daa, T., Urabe, S., Tsuji, K.,
Nakayama, I., and Yokoyama, S. (2003) An antigen
retrieval method using an alkaline solution allows
immunoelectron microscopic identiﬁcation of secretory
granules in conventional epoxy-embedded tissue
sections. J. Histochem. Cytochem. 51, 199204
25. Jacewicz, M., Clausen, H., Nudelman, E., DonohueRolfe, A., and Keusch, G.T. (1986) Pathogenesis of shigella diarrhea. XI. Isolation of a shigella toxin-binding
glycolipid from rabbit jejunum and HeLa cells and its
identiﬁcation as globotriaosylceramide. J. Exp. Med.
163, 13911404
26. Lingwood, C.A., Law, H., Richardson, S., Petric, M.,
Brunton, J.L., De Grandis, S., and Karmali, M. (1987)
Glycolipid binding of puriﬁed and recombinant
Escherichia coli produced verotoxin in vitro. J. Biol.
Chem. 262, 88348839
169

Downloaded from jb.oxfordjournals.org at Oita University Library, Medical Branch on February 2, 2011

in Fabry’s disease: ceramidetrihexosidase deﬁciency.
N. Engl. J. Med. 276, 11631167
2. Desnick, R.J., Ioannou, Y.A., and Eng, C.M. (2001) in
The Metabolic and Molecular Bases of Inherited Disease(Scriver, C.R., Beaudet, A.L., Sly, W.S., and Valle, D.,eds.), pp. 37333774, McGraw-Hill, New York
3. Ishii, S., Kase, R., Sakuraba, H., and Suzuki, Y. (1993)
Characterization of a mutant a-galactosidase gene product for the late-onset cardiac form of Fabry disease.
Biochem. Biophys. Res. Comm. 197, 15851589
4. Ishii, S., Chang, H.-H., Kawasaki, K., Yasuda, K., Wu,
H.-L., Garman, S.C., and Fan, J.-Q. (2007) Mutant
a-galactosidase A enzymes identiﬁed in Fabry patientswith residual enzyme activity: biochemical characterization and restoration of normal intracellular processingby 1-deoxygalactonojirimycin. Biochem. J. 406, 285295
5. Hamanaka, R., Shinohara, T., Yano, S., Nakamura, M.,
Yasuda, A., Yokoyama, S., Fan, J.-Q., Kawasaki, K.,
Watanabe, M., and Ishii, S. (2008) Rescue of mutant
a-galactosidase A in the endoplasmic reticulum by
1-deoxygalactonojirimycin leads to trafﬁcking to lysosomes. Biochim. Biophys, Acta 1782, 408413
6. Fan, J.-Q., Ishii, S., Asano, N., and Suzuki, Y. (1999)
Accelerated transport and maturation of lysosomal
a-galactosidase A in Fabry lymphoblasts by an enzyme
inhibitor. Nat. Med. 5, 112115
7. Fan, J.-Q. and Ishii, S. (2003) Cell-based screening ofactive site speciﬁc chaperone for the treatment ofFabry disease. Methods Enzymol. 363, 412420
8. Fan, J.-Q. and Ishii, S. (2007) Active-site-speciﬁc chaperone therapy for Fabry disease: Yin and Yang of
enzyme inhibitors. FEBS J. 274, 49624971
9. Benjamin, E.R., Flanagan, J.J., Schilling, A., Chang,
H.H., Agarwal, L., Katz, E., Wu, X., Pine, C.,
Wustman, B., Desnick, R.J., Lockhart, D.J., andValenzano, K.J. (2009) The pharmacological chaperone
1-deoxygalactonojirimycin increases alpha-galactosidase
A levels in Fabry patient cell lines. J. Inherit. Metab. Dis.
32, 424440
10. Ohshima, T., Murray, G.J., Swaim, W.D., Longenecker,
G., Quirk, J.M., Cardarelli, C.O., Sugimoto, Y., Pastan,I., Gottesman, M.M., Brady, R.O., and Kulkarni, A.B.
(1997) a-Galactosidase A deﬁcient mice: a model of
Fabry disease. Proc. Natl Acad. Sci. USA 94, 25402544
11. Ioannou, Y.A., Zeidner, K.M., Gordon, R.E., andDesnick, R.J. (2001) Fabry disease: preclinical studies
demonstrate the effectiveness of a-galactosidase A replacement in enzyme-deﬁcient mice. Am. J. Hum.
Genet. 68, 1425
12. Ziegler, R.J., Yew, N.S., Li, C., Cherry, M., Berthlette,
P., Romanczuk, H., Ioannou, Y.A., Zeidner, K.M.,
Desnick, R.J., and Cheng, S.H. (1999) Correction of enzymatic and lysosomal storage defects in Fabry mice byadenovirus-mediated gene transfer. Hum. Gene Ther. 10,
16671682
13. Takenaka, T., Murray, G.J., Qin, G., Quirk, J.M.,
Ohshima, T., Qasba, P., Clark, K., Kurkarni, A.B.,
Brady, R.O., and Medin, J.A. (2000) Long-term
enzyme correction and lipid reduction in multiple
organs of primary and secondary transplanted Fabry
mice receiving transduced bone marrow cells. Proc.
Natl Acad. Sci. USA 97, 75157520
14. Nakamura, G., Maruyama, H., Ishii, S., Shimotori, M.,
Kameda, S., Kono, T., Miyazaki, J., Kulkarni, A.B., andGejyo, F. (2008) Naked plasmid DNA-basedalpha-galactosidase A gene transfer partially reduces systemic accumulation of globotriaosylceramide in Fabry
mice. Mol. Biotechnol. 38, 109119

C. Shiozuka et al.

27. McCluer, R.H. and Gross, S.K. (1985) Biosynthesis ofneutral glycosphingolipids in kidney slices from male andfemale mice. J. Lipid Res. 26, 593599
28. Ohshima, T., Schiffmann, R., Murray, G.J., Kopp, J.,
Quirk, J.M., Stahl, S., Chan, C.C., Zerfas, P.,
Tao-Cheng, J.H., Ward, J.M., Brady, R.O., andKulkarni, A.B. (1999) Aging accentuates and bonemarrow transplantation ameliorates metabolic defects
in Fabry disease mice. Proc. Natl Acad. Sci. USA 96,
64236427
29. Khanna, R., Soska, R., Lun, Y., Feng, J., Franscella, M.,
Young, B., Brignol, N., Pellegrino, L., Sitaraman, S.,

Desnick, R.J., Benjamin, E.R., Lockhart, D.J., andValenzano, K.J. (2010) The pharmacological chaperone
1-deoxygalactonojirimycin reduces tissue globotriaosylceramide levels in a mouse model of Fabry disease.
Mol. Ther. 18, 2333
30. Asano, N., Ishii, S., Kizu, H., Ikeda, K., Yasuda, K.,
Kato, A., Martin, O.R., and Fan, J.-Q. (2000) In vitro
inhibition and intracellular enhancement of lysosomal
a-galactosidase A activity in Fabry lymphoblasts by
1-deoxygalactonojirimycin and its derivatives. Eur. J.
Biochem. 267, 41794186

Downloaded from jb.oxfordjournals.org at Oita University Library, Medical Branch on February 2, 2011170