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Abnormalities
of
g
I
ycosphingoli pids in
mucopolysaccharidosis Type
111
6
Atsushi
Hara,
Nobuko
Kitazawa, and Tamotsu Taketomi
Department
of
Biochemistry, Institute of Adaptation Medicine, Shinshu University School of Medicine,
Matsumoto 390, Japan
Abstract Glycosphingolipids from brain, liver, and spleen
of
a patient with mucopolysaccharidosis type 111
B
were quanti-
tatively analyzed. Neutral glycosphingolipids containing glu-
cosylceramide, lactosylceramide, globotriaosylceramide, glo-
botetraosylceramide, and gangliotriaosylceramide were increased
in
the
brain, while the contents
of
galactosylceramide and galac-
tosylceramide Is-sulfate were decreased. The total ganglioside
levels were
low
in
the grey matter (522 pg N-acetylneuraminic
acid/g) and high
in
the white matter (342 pg N-acetylneuraminic
acid/g), when compared
with
the
normal values (744-918 pg/
g
in
grey matter and 80-180 pg/g
in
white matter). The gan-
glioside compositions were characterized by a high proportion
of
11’-N-acetylneuraminosylgangliotriaosylceramide
(GM2),
IIs-
N-acetylneuraminosyllactosylceramide
(GM3), and II’-(N-ace-
tylneuraminosyl)21actosylceramide
(GD3).
An
unusual band
of
protein
in
place of an ordinary band of Wolfgram protein was
detected
as
a
major band by sodium
dodecylsulfate-polyacryl-
amide gel electrophoresis. The low levels
of
4-eicosasphingenine
in
the brain gangliosides indicated that the disturbance of
the
sphingolipid metabolism already began
at
age 3 at
the
latest and
that
the brain remained immature. These abnormal glycosphin-
golipids and protein
as
well
as
the accumulation of heparan
sulfate explain
in
part the severe progressive mental retardation
which
is
most characteristic of
the
mucopolysaccharidosis
I11
B.
Abnormalities
of
glycosphingolipids
in
the
liver and spleen are
also found.-Hara,
A.,
N.
Kitazawa, and
T.
Taketomi. Ab-
normalities of glycosphingolipids
in
mucopolysaccharidosis
Type
I11
B.J.
Lipid
RPS.
1984.
25:
175-184.
Supplementary
key
words
glycosphingolipids gangliosides sphin-
gosine
globosides
Mucopolysaccharidosis
111
B
(Sanfilippo syndrome
type
B)
is a metabolic disease in which a-N-acetylglucosamin-
idase is hereditarily defective, and is characterized by
severe progressive mental retardation and relatively mild
somatic features (1). While the large amount of heparan
sulfate accumulated in various organs, including brain,
in mucopolysaccharidosis
type
I11
B
is due to the defect
of
a-N-acetylglucosaminidase,
some abnormal patterns
of
glycosphingolipids determined by thin-layer chroma-
tography were reported in the brain (2). Glycosphingo-
lipids are intrinsic constituents especially in central ner-
vous system. It is well known that sphingolipidosis, in
which a certain glycosphingolipid accumulates in the cen-
tral nervous system due to the defect of the corresponding
glycosphingolipid hydrolase, also shows severe progressive
mental retardation which
is
characteristic
of
mucopoly-
saccharidosis type
111.
Thus, the abnormalities of
gly-
cosphingolipids in the mucopolysaccharidosis type
111
may
be responsible in part for this characteristic clinical feature
in the disease. The purpose of this report is to determine
the abnormal glycosphingolipids quantitatively in the
brain, liver, and spleen, and also to analyze sugar, sphin-
gosine, and fatty acid compositions
of
the glycosphin-
golipids.
MATERIALS
AND METHODS
Case report
The
patient was a female and
died
at 18 years and 10
months of age. She was one of seven siblings; two brothers
died at age 13 years and 15 years with similar symptoms.
She began to have ataxia at age
3.
Developmental retar-
dation was noticed at age
4.
Progressive mental retar-
dation was observed at age 12. Enzyme assay revealed
that
a-N-acetylglucosaminidase
was defective in both leu-
kocytes and cultured skin fibroblasts
of
the patient,
while
the other lysosomal enzymes were normal. Uronic acid
in urine was markedly increased (80.6-89.2 mg of uronic
acid/g of creatinine). Glycosaminoglycan in the urine
Abbreviations: GlcCer, glucosylceramide; GalCer, galactosylceramide;
LacCer, lactosylceramide; GbOsesCer, globotriaosylceramide;
GbOse,Cer, globotetraosylceramide; GgOsesCer, gangliotriaosylcera-
mide; GalCerls-sulfate, galactosylceramide 1’-sulfate; nLcOse4Cer,
neolactotetraosylceramide;
IISNeuAc-LacCer, 11’-N-acetylneurami-
nosyllactosylceramide; IIS(NeuAc)z-LacCer, 11’-di-N-acetylneurami-
nosyllactosylceramide; IISNeuAc-GgOseSCer, 11’-N-acetylneuraminos-
ylgdngliotriaosykeramide;
IISNeuAc-GgOse,Cer, 11’-N-acetylneurami-
nosylgangliotetraosylceramide;
II‘(NeuAc)&gOse4Cer, I15-di-N-
acetylneuraminosylgangliotetraosylceramide;
IVSNeuAc,IISNeuAc-
GgOse,Cer,
1VS,IIsdi-N-acetylneuraminosylgangliotetraosylce~mide;
IVSNeuAc,IIs(NeuAc)p-GgOse4Cer,
IVs-N-acetylneuraminosyl-Ils-di-
N-acetylneuraminosylgangliotetraosylceramide;
C-M, chloroform-
methanol; GLC, gas-liquid chromatography;
PAGE,
polyacrylamide
gel electrophoresis.
Journal
of
Lipid
Research
Volume
25, 1984
175
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consisted of heparan sulfate (92.5%) and chondroitin sul-
fate (7.5%). Thus, the patient was diagnosed as muco-
polysaccharidosis type
I I
I
B clinically and enzymatically.
Isolation
of
glycosphingolipids and
glycosaminoglycan
Brain, liver (104 g), and spleen (101 g) were obtained
at autopsy from the patient and kept frozen until use.
The brain was separated into grey matter (1 15 g) and
white matter (87 g). The procedure for isolation of gly-
cosphingolipids is described in detail elsewhere (3). The
tissue was homogenized with chloroform-methanol (C-
M)
according
to
Suzuki
(4).
The homogenate was filtered
and separated into total lipid extract and a protein residue
from which glycosaminoglycan was prepared as described
below. The total lipid was treated with mild alkali to
remove ester-lipids, then the alkali-stable lipids were sep-
arated into neutral and acidic sphingolipids
(3).
The neu-
tral glycosphingolipid fraction was acetylated with pyri-
dine-acetic anhydride 1:1 at 80°C for 2 hr. After evap-
oration of the solvent, the entire acetylated neutral
glycosphingolipid fraction was dissolved in hexane-tol-
uene 1:
1
and applied to the column of silica gel 60 (60
g, Merck, West Germany). The column was washed with
1500 ml of hexane-toluene 1:l to remove fatty acid
methyl esters, cholesterol acetate, and a part of the cho-
lesteryl esters. Acetylated glycosphingolipids
were
eluted
with
400
ml of C-M 8:2 and acetylated sphingomyelin
was eluted with 2,000 ml of C-M 4:6. The acetylated
glycosphingolipids were evaporated to dryness and de-
acetylated with 12% ammonia in methanol at room tem-
perature overnight. Particularly, for the isolation of brain
lipids, the deacetylated glycosphingolipids were applied
to a silica gel column and a large amount of cerebroside
was separated from other glycosphingolipids. The former
were eluted with C-M 9:
1,
while the latter
were
eluted
with C-M 4:6. Acidic glycolipids were separated into ga-
lactosylceramide I'-sulfate and ganglioside fractions ac-
cording to Ledeen, Yu, and Eng (5). Each sphingolipid
except brain cerebroside and sulfatide was separated by
preparative thin-layer chromatography (TLC) on plates
precoated with silica gel 60 (Merck, West Germany) and
developed in C-M-water 65:25:4 (by volume) for neutral
glycosphingolipids or with C-M-0.25% KCI 60:35:8 for
acidic glycosphingolipids. The scraped lipids from TLC
plates were further purified by silica gel column chro-
matography to remove nonlipid contaminants as follows.
The silica gel powder was suspended in chloroform and
applied to a column of silica gel 60 (1
0
g, 10
X
300 mm).
The column was eluted with
100
ml each of chloroform,
C-M 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and methanol.
The 10-ml fractions were separated and checked by TLC.
Purified lipids
were
subjected
to
chemical analyses.
Glycosaminoglycan was prepared as follows.
To
the
protein C-M residue was added hexane-isopropanol3:2
to obtain a fluffy powder (6). Hexane-isopropanol treat-
ment should be done while the residue is wet with C-M.
The mixture was filtered and the protein residue was
dried in vacuo. Glycosaminoglycan was isolated from the
residue according to Constantopoulos, McComb, and De-
kaban (7).
Analytical procedures
The amount of glycosaminoglycan was determined by
the carbazole borate method
(8).
Electrophoresis of gly-
cosaminoglycan was performed on cellulose acetate
membranes (Sartorius GmbH, West Germany) using 0.1
M
barium acetate at a constant current of
1
mA/cm for
3 hr
or
1
M
acetate-pyridine buffer, pH 3.5, at a constant
current of 0.5 mA/cm for 20 min
(9).
The bands of
glycosaminoglycan
were
detected by Alcian blue (Wako
Pure Chemical Industries, Japan). The cellulose acetate
membrane was dried and analyzed by densitometry at
570 nm.
Fifty mg of white matter
or
grey matter was homog-
enized with hexane-isopropanol3:2 containing 3% water
to delipidate the tissue (6). The homogenate was centri-
fuged briefly and the supernatant was discarded. The
pellet was dried under a stream of nitrogen and then
stored in a vacuum desiccator. The dried residue was
solubilized with 1
%
SDS and analyzed according to Lae-
mmli (1
0).
Electrophoresis was performed on 13% poly-
acrylamide gel containing
0.1
%
SDS (0%" thickness)
at 25 mA/14 cm for
2.5
hr using 0.02
M
Tris-glycine
buffer, pH 8.3. Bands were detected by Coomassie Bril-
liant Blue-R250. Molecular weight markers (BDH Co.,
England) were used as authentic samples.
Glycosphingolipid was methanolized with 3% HCI in
dry methanol at
80"
for
3
hr with
or
without mannitol
as
an internal standard. To the methanolyzate was added
3
X
3
ml of hexane and the two solvent layers (hexane
and methanol) were analyzed by gas-liquid chromatog-
raphy (GLC). The hexane layer which contained fatty
acid methyl esters was analyzed by GLC using a silicone
capillary column OV-101 (0.2 mm
X
25 m) at 250°C.
Methyl glycosides in the methanol layer were re-N-acet-
ylated (1 1) and analyzed as trimethylsilyl derivatives by
GLC (3). Sphingosine was analyzed
as
trimethylsilyl de-
rivative by GLC as reported elsewhere (12). For the
sphingosine composition of brain ganglioside, the ratio
of
4-eicosasphingenine to 4-sphingenine was determined
by ozonolysis (1 3). The amount of ganglioside was de-
termined with resorcinol reagent (14) or estimated on
TLC by densitometry at 570 nm. Cholesterol glucuronide
was determined as described elsewhere
(3).
Sugar se-
quence of
neolactotetraosylceramide
was determined ac-
cording
to
Svennerholm, Mansson, and Li (1 5) using jack
bean &galactosidase and jack bean /3-N-acetylhexosamin-
176
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of
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1984
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idase (Seikagaku
Kogyo
Co., Japan).
To
ascertain the
activity and specificity of each enzyme, gangliotetraosyl-
ceramide (GA
1)
prepared from bovine brain ganglioside
by formic acid hydrolysis, globotriaosylceramide and glo-
botetraosylceramide obtained from human erythrocytes,
and Forssman globoside isolated from canine stomach
were used as enzyme substrates.
RESULTS
Accumulation
of
glycosaminoglycan in brain and
visceral organs
Electrophoresis of glycosaminoglycan was performed
on cellulose acetate membranes. The electrophoresis pat-
terns were analyzed by densitometry and the accumulation
of heparan sulfate was confirmed
as
described below.
The samples from
liver
and spleen contained heparan
sulfate exclusively, while the brain showed multiple bands
which
were
comprised of heparan sulfate
(53%
in grey
matter,
49%
in white matter), a mixture of dermatan
sulfate and hyaluronic acid
(26%
in
grey
matter,
3
1
%
in
white matter), and unidentified materials
(22%
in grey
matter,
21%
in white matter).
As
the separation
of
der-
matan sulfate and hyaluronic acid was
poor
in this buffer
system, another buffer system,
1
M
acetate-pyridine buffer,
pH
3.5,
was used to separate and identify dermatan sulfate
and hyaluronic acid. The contents of uronic acid in various
organs were determined by the carbazole method (7,
8).
The following values were obtained: liver,
2.12%;
spleen,
1.29%;
grey matter,
0.40%;
white matter, 0.48% (values
are expressed as percent of C-M residue). Accumulation
of glycosaminoglycan (mostly heparan sulfate) was
ob-
served in all the organs tested. Those values well agree
with the data of Constantopoulos, Eiben, and Schafer
(2).
Abnormal protein in brain
The cerebral protein fraction of the patient was an-
alyzed by slab-SDS-PAGE. The strange PAGE pattern
was obtained in the white matter. One major band which
is not originally present in normal brain appeared as
shown in
Fig.
1,
while the protein which has
the
same
mobility as a high molecular weight protein, the so-called
Wolfgram protein, was almost missing. The molecular
mass of the abnormal protein was calculated to be ap
proximately
54,000
based on molecular weight marker
proteins. In addition, the relative content of myelin basic
protein in the white matter was somewhat lower than
normal. The grey matter showed a pattern similar to that
of the white matter, but contained less abnormal protein.
The abnormal SDS-PAGE pattern of brain protein may
be
related to the defect of
a-N-acetylglucosaminidase,
Ham,
Kitnzaun,
avd
Fig.
1.
Slab SDS-polyacrylamide gel electrophoresis of the brain pro-
tein. Electrophoresis was performed on 13% polyacrylamide gel con-
taining
0.1%
SDS (0.8-mm thickness) at 25 mA/14 cm for 2.5 hr
using
0.02
M
Tris-glycine buffer, pH 8.3. Bands were detected by
Coomassie Brilliant Blue-R250. A. Molecular weight markers (from
top
to
bottom: ovotransferrin, bovine serum albumin, ovalbumin,
bo-
vine chymotrypsinogen A, equine myoglobin, and equine cytochrome
C);
B, porcine spinal cord (control);
C.
white matter (patient); D, grey
matter (patient);
E,
white matter (normal control); and
F,
grey matter
(normal control).
but the abnormality in brain protein is now under
in-
vestigation.
Abnormal glycosphingolipids in brain
Neutral glycosphingolipids, galactosylceramide 13-sulfnte, and
sphingomyelin.
Fig.
2
shows the TLC of neutral glyco-
sphingolipids from which most of the galactosylceramide
was first removed by silica gel column chromatography.
The grey matter contained many bands, indicated by
A,
B,
C, D,
E
and
F
in Fig.
2.
Some of these bands were
composed of double bands due to differences in the cer-
amide portions. Analytical results by GLC of trimethylsilyl
(TMS) derivatives of methyl glycosides together with the
R,value of each glycosphingolipid band on TLC revealed
that band
B
was
lactosylceramide(g1ucose-galactose
1
:
1.1);
band C was
globotriaosylceramide(g1ucose-galac-
tose
1
:
1.9);
band
D
was a mixture of gangliotriaosylcer-
amide(74%) and
globotriaosylceramide(26%)
which con-
tained hydroxy fatty acid, as
will
be discussed later;
and band
E
was
globotetraosykeramide(g1ucose
-
galac-
tose
-
N
-
acetylgalactosamine
1: 1.8:O.g).
Band
F
was
separated into two bands when the TLC plate was de-
veloped in C-M-water
60:35:8.
Band F could not be
determined due to its low sample concentration, although
fucose, galactose, N-acetylgalactosamine, and glucose
Takelomi
Clycosphingolipids in mucopolysaccharidosis
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-
IB
Jc
D
F
-E
7
123
Fig. 2.
Thin-layer chromatogram of neutral glycolipid fraction in
brain. The plate was developed with chloroform-methanol-water
(65:25:4). Bands were detected by cupric-phosphoric acid charring
spray (28).
1,
Glycosphingolipids from goat erythrocytes;
2,
grey matter
(patient); 3, white matter (patient).
were found as sugar constituents by GLC analysis when
the
two
bands were analyzed as a mixture. The cerebro-
side fraction separated by silica gel column chromatog-
raphy contained glucose and galactose at the ratio of
9.2% and 90.8%. respectively. The structures of glycos-
phingolipids in the grey matter
were
also confirmed by
GLC of the partially methylated alditol acetates of sugars
(16, 17).
It
should be noted that the brain of the patient
contained not only galactosylceramide and galactosyl-
ceramide 1'-sulfate, but also glucosylceramide, lactosyl-
ceramide,
gangliotriaosylceramide,
globotriaosylceram-
ide, and globotetraosylceramide. AI1 of these glycolipids
except galactosylceramide and galactosylceramide I'-sul-
fate are usually undetectable in normal brain. The white
matter showed a similar TLC pattern of glycosphingolip-
ids. However, the cerebroside fraction contained only
galactose, and gangliotriaosylceramide was present as a
trace amount. Although lactosylceramide in extra-neural
organ usually shows
two
bands on TLC due to the dif-
ference of fatty acid chain length, the lactosylceramide
in the brain of the patient showed a high content
of
upper
band in the white matter and, on the other hand, a high
content of
lower
band in the grey matter. This difference
of fatty acid chain length was confirmed by the analysis
of fatty acid composition of lactosylceramide in both
grey
and white matter. This result suggested that the synthesis
of lactosylceramide in the grey matter was somehow dif-
ferent from that in the white matter. The content of each
glycosphingolipid in both the grey and white matter is
shown in
Table
1.
The content of galactosylceramide in
the grey matter (0.15% of fresh tissue) and the white
matter (0.95% of fresh tissue) and the content of gal-
actosylceramide Is-sulfate in the grey matter (0.07% of
fresh tissue) and the white matter (0.42% of fresh tissue)
were
markedly lower than the established values
(1
8)
in
normal brain (0.3%, 3.1%, O.l%,and 0.9%, respectively).
The sphingosine compositions of neutral glycosphingo-
lipids, galactosylceramide 1'-sulfate, and sphingomyelin
were determined by GLC of the trimethylsilyl (TMS)
derivatives of sphingosine
(Table
2).
Lactosylceramide
contained not only 4-sphingenine and sphinganine but
also 4-eicosasphingenine and eicosasphinganine. The 4-
eicosasphingenine and eicosasphinganine are usually
found only in gangliosides in normal brain. These
ei-
cosasphingosines
were
demonstrated in gangliosides of
the patient's brain as described below in the section about
the gangliosides. Thus,
it
was concluded that the lacto-
sylceramide, which has 4-eicosasphingenine or eicosas-
phinganine, was at least an intermediate in the metabolic
pathway
of
gangliosides, and, therefore, the metabolism
of ganglioside might have been disturbed in the brain of
the patient. Fatty acid compositions of the neutral gly-
cosphingolipids, galactosylceramide Is-sulfate, and sphin-
gomyelin are shown in
Table
3.
The neutral glycosphin-
golipids in the globo-series contained a relatively high
proportion of stearic acid. Another characteristic is the
presence of hydroxy fatty acids in the neutral glycos-
phingolipids, although the amount is not large. Band
D
in Fig.
2
also contained hydroxy fatty acids, which in-
dicated that band
D
was a mixture of nonhydroxy fatty
acidcontaining gangliotriaosylceramide and hydroxy fatty
acid-containing globotriaosylceramide.
No
abnormalities
were observed in the fatty acid compositions of cerebro-
side, galactosylceramide 1'-sulfate, and sphingomyelin.
Gnngliosida.
Gangliosides in the grey and white matter
showed abnormal patterns of TLC
(Fig.
3).
The relative
amounts of
1I'"N-acetylneuraminosylgangliotriaosylcer-
TABLE
1.
Content of neutral glycosphingolipids and galactosylceramide 19-sulfate in grey and white matter
GalCer GlcCer LacCer CbOsesCer GgOsesCer GbOse4Cer GalCer I'-sulfa~e
paollgJrtsh
tisrw
Grey matter 1.884 0.192
0.142
0.048
0.010
0.020 0.753
White matter
1
1.656 0.171
0.025
2
0.051 4.692
178
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1984
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TABLE
2.
Sphingosine composition
of
neutral glycosphingolipids, galactosylceramide
1'-sulfate, and sphingomyelin
Grey
Matter
White
Matter
d18:I" d18:Ob d20:Ir d20:Od
d18:I
d18:O d201
d200
~
Ir
offofnl
sph,l,gosrlze
-
89
11
-
-
-
GalCer
99
1
LacCer
67
9
22
2 84
10
5
1
GbOses Cer
75 25
GgOsesCer 64
36
GbOse4 Cer
79
21
GalCer 1'-sulfate
89
11
Sohingomyelin
90
10
-
78
22
-
- -
-
-
-
85
15
-
- -
- -
98
2-
-
- -
98
2-
-
4-Sphingenine.
Sphinganine.
4-Eicosasphingenine
Eicosasphinganine.
amide (GM2-ganglioside),
11'-N-acetylneuraminosyllac-
tosylceramide (GM3-ganglioside), and 11'-(N-acetylneu-
raminosy1)Jactosylceramide
(GD3-ganglioside) were in-
creased (Fig. 3 and
Table
4).
Total gangliosides in the
grey and white matter were determined with resorcinol
reagent. The white matter contained 341.7 pg of lipid-
bound N-acetylneuraminic acid per gram wet tissue [cf.
normal value:
80-1
80
pg NANA/g wet tissue
(1
8)], while
the grey matter contained 522.3 pg of lipid-bound
N-
acetylneuraminic acid per gram of
wet
tissue [cf. normal
value: 744-918 fig NANA/g wet tissue (18)]. The in-
creased amount of ganglioside in the white matter may
partially explain the contamination of the grey matter in
the white matter fraction, because complete separation
of white matter and grey matter is almost impossible.
However, such contamination would be too small to ex-
plain the high value obtained in the white matter. This
increase may be explained by the histological observation
that revealed the presence of the demyelination and
marked gliosis in the white matter, if the proliferated glia
cell contained much more ganglioside than the myelin.
On the other hand, the ganglioside content was low
in
the grey matter. This result was consistent with the his-
tological data where a pseudolaminar spongy state was
observed. The fatty acids of each ganglioside in the grey
and white matter were analyzed.
A
small but significant
increase in longer chain fatty acids (longer than C20) was
observed in comparison with the established values ob-
tained from normal brain, although the major fatty acid
was stearic acid as usual.
The sphingosine composition was determined by ozon-
olysis. The ratio of 4-eicosasphingenine to 4-sphingenine
in the total ganglioside was determined as 0.51 and 0.33
in grey and white matter, respectively.
It
is
well known
that the ratio of 4-eicosasphingenine to 4-sphingenine
rapidly increases just after birth and approaches
1.0
around age
10
(1
9,20).
The brain
of
this patient showed
low values which corresponded to that observed at about
3 years of age. This result strongly suggested that gan-
glioside metabolism in the brain of this patient was se-
verely disturbed at an early age.
Glycosphingolipids in liver and spleen
Fig.
4
shows the TLC pattern of neutral glycosphin-
golipids in the liver and spleen
of
the patient. In both
organs, bands corresponding to monohexosylceramide,
dihexosylceramide, trihexosylceramide, and tetrahexo-
sylceramide were detected. For the acidic lipids
(Fig.
5),
TLC showed one major band which corresponded to 11'-
N-acetylneuraminosyllactosylceramide
(GM3), and several
minor bands. One of the minor bands had the same
Rf
value as that of cholesterol glucuronide. The TMS de-
rivatives
of
methyl glycosides of each glycolipid were an-
alyzed by GLC. The sugar compositions and the con-
centrations of glycosphingolipids in the liver and spleen
are shown in
Table
5.
The tetrahexosylceramide in the
spleen contained glucose, galactose, N-acetylgalactos-
amine, and N-acetylglucosamine. This indicated the pres-
ence of a glucosamine-containing glycolipid in addition
to globotetraosylceramide in the tetrahexosylceramide
fraction.
It
was difficult to separate these two glycolipids
by TLC using the solvent system of C-M-water 65:25:4
or
C-M-water 60:35:8. Thus, this fraction was acetylated
by the method described in Materials and Methods. Pre-
parative TLC was performed with the solvent system of
C-M-water 95:5:0.3 and complete separation of two
bands was obtained. Each band was scraped from the
TLC plate, eluted from the silica powder, and deacety-
lated. The upper band was identified as globotetraosyl-
ceramide by GLC analysis of TMS derivatives, while the
lower band
was
identified as
lactoneotetraosylceramide
by GLC analysis and enzymatic sequential degradation
according to Svennerholm et al.
(I
5).
Concentrations of glycolipids in the liver are shown in
Hara,
Kitazauu,
and
Taketomi
Glycosphingolipids in mucopolysaccharidosis
179
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Journal
of
Lipid
Research
Volume
25,
1984
by guest, on July 14, 2011www.jlr.orgDownloaded from
“Y-ruL
GM4
GM3
GM2
A
B
C
-
GDla
-
m
GDlb
-
GTlb
-
-
-
”-
-
I
2
3
Fig.
3.
Thin-layer chromatogram of gangliosides
in
brain. Developed
with
chloroform-methanol-0.25%
KC1
(60:35:8). Detected by cupric-
phosphoric acid charring spray.
1,
Control (from top to bottom, CM
I,
GDla. GDlb. and GTIb); 2, white matter; 3. grey matter. Abbre-
viations for gangliosides are as follows: GM4, I’NeuAc-GalCer; GM3.
II’NeuAc-LacCer; GM2. ll’NeuAc-GgOseSCer; GM
1,
Il’NeuAc-
GgOse,Cer; GD3, II’(NeuAc)2-LacCer; GDla, lV’NeuAc,ll’NeuAc-
GgOse,Cer;
GDI
b. Il’(NeuAc)p-CgOse.Cer; CTl b, 1V’NeuAc.
lI’((NeuA~)~-GgOse~Cer.
Table
5.
The
levels of monohexosylceramide and lac-
tosylceramide were
in
the normal range, while those of
globotriaosylceramide and globotetraosylceramide were
three times higher than normal values estabhed by oth-
ers
(2
1,
22).
The spleen also showed a high level of
glo-
botetraosylceramide when compared
with
the normal
level
(23).
Acidic lipids were composed
90.8%
of 1I’”N-
D
I23
Fig.
4.
Thin-layer chromatogram of neutral glycolipids
in
liver and
spleen. Developed with chloroform-methanol-water (65:25:4). De-
tected by cupric-phosphoric acid charring spray.
l,
Goat erythrocyte
membrane (control);
2,
liver; 3, spleen.
A.
B,
C,
and D represent
mono-. di-, tri-, and tetra-hexosylceramides, respectively.
acetylneuraminosyllactosylceramide,
3.1
%
of 11’-(N-ace-
tylneuraminosyl)21actosylceramide,
and
6%
of four other
minor bands
in
the liver. In the spleen, the major gan-
glioside was
I13”N-acetylneuraminosyllactosylceramide
(76.1
%).
II’-(N-acetylneuraminosyl)21actosylceramide
was
9.9%
of the total gangliosides and the six minor bands
together were
14.0%.
The minor gangliosides could not
be determined because of low sample concentration. Lev-
els of
11’-N-acetylneuraminosyllactosylceramide
were in
the normal range in both the liver and spleen.
TABLE 4. Ganelioside comoosition in brain
Grey Matter White Matter
NANA
(%)
nmol Ganglioside/g Fresh
NANA
(%)
nmol Canglioside/g Fresh
GM4
4.3h 47.6
GM3 15.0
(<1)R
253.3 9.6
(<1)
106.1
GM2 12.2 (1.5-2)
206.1 8.9 (0.6-2.0) 98.4
GMI 17.7 (13.0-15.6)
299.0 18.8
(1
4.6-2 1.2) 207.8
GD3
10.1
(1.0-2.8)
85.5 13.0 (1.2-5.0) 71.9
GDla 19.4 (29.1-43.7)
163.9 18.9 (30.0-38.2) 104.6
CD2
0.7 (1.2-4.2)
6.0 2.9 (1.2-3.1) 16.0
CDlb
12.6 (14.3-19.9)
106.5 10.8 (12.2-18.1) 59.7
GTlb
10.6 (15.8-25.7)
59.8
11.0
(14.1-21.2) 40.6
GQ
1.7 (3.2-4.8)
7.2 1.9 (2.8-6.1) 5.3
Abbreviations for gangliosides are shown in Fig. 3.
’
Nine to eleven percent of total sialic acid (5).
Values in parentheses express normal levels of ganglioside in brain
(1
8).
Hnm,
Kilnznrcln,
nnd
Tnkrkmi
Glycosphingolipids in mucopolysaccharidosis
181
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GM3
12
34
Fig.
5.
Thin-layer chromatogram
of
acidic lipids in liver and spleen.
Developed with chloroform-methanol-0.25% KC1 (60:35:8). Detected
by cupric-phosphoric acid charring spray.
1,
Brain ganglioside (control);
2, liver;
3,
spleen;
4,
cholesterol glucuronide.
Cholesterol glucuronide contents
were
15.9 nmol/g
in the
liver
and 6.0 nmol/g in the spleen. No increase
of cholesterol glucuronide was observed in the
liver.
On
the other hand, a small amount
of
cholesterol glucuronide
was detected in the spleen of this patient, although
we
could not detect any in the spleen of the patient with
GM 1-gangliosidosis
type
I1
(3).
The fatty acid composition
of each glycolipid in the
liver
and spleen was analyzed.
All
the glycolipids
were
composed
of
nonhydroxy fatty
acids and their major chain lengths
were
C16, C22, and
C24. The fatty acid composition of lactoneotetraosylcer-
amide was C16(47%), C18(6%), C22:1(1%), C22(7%),
C23(
1
%),
C24: 1 (30%), and C24(9%). The composition
was different from that
of
other glycosphingolipids and
was characterized by a large proportion of C16. The fatty
acid composition of
IV'-N-acetylneuraminosyllacto-
neotetraosylceramide from normal human erythrocytes
was also determined for comparison. The values obtained
were C16(
1
%),
C22(
1 1
%), C22:
1 (1
%),
C23(2%),
C24(4
1
%),
C24: 1(37%), C25(
1
%),
C26(2%), and
C26: 1(3%). The
lactoneotetraosylceramide
in the spleen
is thought
to
originate probably from erythrocytes which
contain a large amount
of
1V'-N-acetylneuramino-
syllactoneotetraosylceramide.
However, lactoneotetrao-
sylceramide in the spleen of this patient contained 47%
of C 16, while
1V'"N-acetylneuraminosyllactoneotetra-
osylceramide had only 1%
of
C16. When glucosylcer-
amide obtained by enzymatic hydrolysis
of
the lactoneo-
tetraosylceramide from the patient was analyzed by TLC,
it
corresponded
to
the
lower
half
of
the control gluco-
sylceramide which was obtained from the spleen
of
the
patient with Gaucher's disease and had a large amount
of
C24 fatty acid. This supports the analytical result of
the fatty acid composition.
Sphingosine compositions of each glycolipid are shown
in
Table
6.
Relatively high contents of sphinganine
were
observed in all the glycosphingolipids except for 1I'"N-
acetylneuraminosyllactosylceramide
in the spleen.
DISCUSSION
Glycosphingolipids in the brain, liver, and spleen of a
patient with mucopolysaccharidosis type
111
B
were
an-
alyzed. In the neutral glycolipid fraction, the presence
of glycosphingolipids in the globo-series (globotriaosyl-
ceramide and globotetraosylceramide) and ganglio-series
(gangliotriaosylceramide)
in addition
to
glucosylceramide
and lactosylceramide
were
identified. Constantopoulos
et
al. (2) reported the presence of bands which had the same
mobilities with ceramidedihexoside, gal-gal-glc-cer, and
galNAc-gal-glc-cer on the TLC plate. In the acidic gly-
colipid fraction, the proportion of 11'-N-acetylneura-
minosylgangliotriaosylceramide,
I13-N-acetylneuramino-
syllactosylceramide and
II'-(N-acetylneuraminosyl)21ac-
tosylceramide
were
also increased in the brain as observed
by Constantopoulos
et
al. (2). They concluded that the
increase of glycolipids which
were
the minor components
TABLE 5. Sugar composition and content
of
glycolipids in liver and spleen
Liver
Spleen
tz1nollg
Jrr.<h
tisrur
GlcCer 50.7 (glc only)
102.6 (glc only)
LacCer 78.6
(gkgal
=
1:0.98)
175.0 (glc:gal
=
1:l.O)
GbOsesCer 78.0 (&:gal
=
1:2.1)
50.7 (&:gal
=
1
:
1.9)
GbOserCer 44.8 (g1c:gal:galNAc
=
1:1.7:0.9)
96.1 (g1c:gal:galNAc
=
1:
I
.88:0.99)
n LcOse4Cer
30.3
(g1c:gal:glcNAc
=
1:
1.80:
I
.OO)
GM3 284.0 (&:gal
=
1:0.8)
235.2 (gkrdl
=
1:o.g)
182
Journal
of
Lipid
Research
Volume
25,
1984
by guest, on July 14, 2011www.jlr.orgDownloaded from
TABLE 6. Sphingosine composition
of
glycosphingolipids
in liver and spleen
Liver
Spleen
d18:I" d18:Ob d18:l d18:O
'2
of
total
sphiizgosiiir
bosr
GlcCer 86.4 13.6 85.1 14.9
LacCer 77.8 22.2 89.9 10.1
GbOsesCer 84.3 15.7 85.4 14.6
GbOse4Cer 73.8 26.2 84.3 15.7
nLcOse4Cer 73.0 27.0
GM3 80.9 19.1 98.2 1.8
4-Sphingenine.
Sphinganine.
in the brain was due to the low glycosidase activities as
a result of the accumulation of heparan sulfate. In our
report, some of the neutral glycosphingolipids in the brain
of the patient were identified as those in globo-series.
Apart from the pathogenesis of the disease, these gly-
cosphingolipids may be present in normal brain, although
the contents of those glycosphingolipids are too low to
detect.
Ganglioside compositions in fetal brain are absolutely
different from those of adult brain as reported by Irwin,
Michael, and Irwin (24). They observed that 11'-(N-ace-
tylneuraminosyl),lactosylceramide
and 11'-N-acetyl-
neuraminosyllactosylceramide
comprised
77%
of resor-
cinol-positive bands in the brain at 15 days of gestation,
although their amounts decrease with age. Thus, the
high proportion of
II'-(N-acetylneuraminosyl)21actosyl-
ceramide and
11'-N-acetylneuraminosyllactosylceramide
may in part account for the immaturity of the brain of
the patient with mucopolysaccharidosis I11
B.
It is possible
to conclude that ganglioside metabolism, particularly the
biosynthetic pathway, in the brain of the patient was al-
ready disturbed at an early stage of life. This conclusion
can be supported by the abnormal sphingosine compo-
sition of gangliosides.
As
a matter of fact, the ratio of 4-
eicosasphingenine
to
4-sphingenine of gangliosides in the
brain (0.51 in grey matter and
0.33
in white matter)
corresponded to that at age
3
or
less. The
lower
level
of
the ratio indicated that the ganglioside synthesis in the
brain of the patient was already disturbed at age
3
at the
latest. The immaturity of the brain may account for the
severe progressive mental retardation in mucopolysac-
charidosis type 111
B.
The contents of galactosylceramide and galactosylcer-
amide 1'-sulfate
were
significantly decreased. This result
was consistent with histological observations which showed
demyelination and marked gliosis in the white matter and
pseudolaminar spongy state in the
grey
matter. The ab-
normal pattern of brain protein on slab SDS-PAGE may
be related to the defect of
a-N-acetylglucosaminidase
in
the brain.
The spleen
of
the patient contained a relatively large
amount of
lactoneotetraosylceramide
which comprised
one-fourth of the tetrahexosylceramide fraction. Lacto-
neotetraosylceramide in the spleen may
be
derived from
erythrocytes which are trapped by the spleen, since human
erythrocytes contain
IV3-N-acetylneuraminosyllactoneo-
tetraosylceramide as a major ganglioside (25).
The analysis of monohexosylceramide in the liver
showed that the sugar component is only glucose and
that only nonhydroxy fatty acids are present. Recently
Nilsson and Svennerholm (21) reported that the human
liver contained a considerable amount of galactosylcer-
amide. In our analyses of human
liver
from Japanese (26,
27), only glucosylceramide as monohexosylceramide was
found. Recently,
we
found only glucosylceramide in the
liver
of a Japanese patient with GM 1 -gangliosidosis type
I1
as monohexosylceramide (unpublished data). The
composition of monohexosylceramide may vary with
race.l
We are grateful to Dr. K. Yoshida, Research Division, Seikagaku
Kogyo Co. Ltd. Tokyo, for supplying the samples of heparan
sulfate.
Manuscript received
12
August
1983.
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