Báo cáo Y học: Chemical structures and immunolocalization of glycosphingolipids isolated from Diphyllobothrium hottai adult worms and plerocercoids ppt

Chemical structures and immunolocalization of glycosphingolipids
isolated from
Diphyllobothrium hottai
adult worms and plerocercoids
Hideyuki Iriko
1
, Kazuo Nakamura
2
, Hisako Kojima
2
, Naoko Iida-Tanaka
3
, Takeshi Kasama
4
,
Yasushi Kawakami
1
, Ineo Ishizuka
3
, Akihiko Uchida
1
, Yoshihiko Murata
1
and Yoichi Tamai
5
1
Department of Medical Zoology, Azabu University, Sagamihara, Kanagawa, Japan;
2
Department of Biochemistry, Kitasato
University School of Medicine, Sagamihara, Kanagawa, Japan;
3

Department of Biochemistry, Teikyo University School of Medicine,
Itabashi-ku, Tokyo, Japan;
4
Instrumental Analysis Research Center for Life Science, Tokyo Medical and Dental University,
Bunkyo-ku, Tokyo, Japan;
5
University of Human Arts and Sciences, Iwatsuki, Saitama, Japan
Glycosphingolipids (GSLs) were purified from adults and
plerocercoids of the tapeworm Diphyllobothrium hottai,and
their chemical structures were determined. Total lipid frac-
tions prepared from chloroform/methanol extracts of whole
tissues were fractionated successively on ion-exchange
chromatography, silicic acid column chromatography, and
preparative TLC. The purified GSLs were characterized by
methylation analysis, TLC-immunostaining, liquid secon-
dary ion MS, MALDI-TOF MS, and
1
H-NMR. Ten GSLs
were isolated from adult worms and four from plerocercoids,
comprising mono-, di-, tri-, tetra-, and pentasaccharides. The
GSL Galb1–4(Fuca1–3)Glcb1–3Galb1-Cer was found in
adult worms but not in plerocercoids, whereas Galb1–4
(Fuca1–3)Glcb1–3(Galb1–6)Galb1-Cer was found in both
adult worms and plerocercoids. We previously found a
similar series of GSLs in plerocercoids of the cestode Spiro-
metra erinaceieuropaei, and termed them ÔspirometosidesÕ
[Kawakami, Y. et al. (1996) Eur J. Biochem. 239, 905–911].
The core structure of spirometosides, Galb1–4Glcb1–3
Galb1-Cer, may have taxonomic significance, being char-
acteristic of pseudophyllidean tapeworms. In the present

study, GSL compositions were significantly different
between adults and plerocercoids, and growth-dependent
changes in composition were documented. We found a novel
dihexosylceramide, Glcb1–3Galb1-Cer, which is a possible
precursor for spirometosides. Immunohistochemical exam-
ination showed that spirometoside GSLs are highly enriched
in the inner surface of bothria, the major point of contact
between the adult worm and the host’s intestine. Our findings
indicate that spirometosides are involved in host–parasite
interaction.
Keywords: bothrium; cestode; glycosphingolipids;
immunohistochemistry; parasites.
Glycosphingolipids (GSLs) as components of cell mem-
branes participate in many important events occurring on
the cell surface, including binding of viruses, bacterial
toxins, adhesion molecules, and antibodies to the plasma
membrane [1,2]. In this context, GSLs are involved in host–
parasite interaction and host immune response to parasites.
Biological functions of GSLs are borne by their specific core
saccharide structures, and modulated by the ceramide
moieties. Thus, structural characterization of membrane
GSLs is essential for understanding their functions.
However, our knowledge of GSLs in parasitic helminths
is fragmentary, although structural analysis has supported
their proposed role as antigens and species markers.
We previously found two novel GSLs, SEGLx [Galb1–4
(Fuca1–3)Glcb1–3Galb1-Cer] and GalSEGLx [Galb1–4
(Fuca1–3)Glcb1–3(Galb1–6)Galb1-Cer], in the cestode
Spirometra erinaceieuropaei (synonym, S. erinacei)[3,4],
and proposed the term ÔspirometosidesÕ forGSLshaving

the core carbohydrate structure Galb1–4Glcb1–3Galb1-Cer
[4]. We established a mAb AK97 which recognizes the
nonreducing terminal trisaccharide sequence, Galb1–4
(Fuca1–3)Glcb1-, of SEGLx [5]. Our studies using mAb
AK97 indicate that SEGLx and GalSEGLx have immuno-
logical properties similar to those of Le
x
, a key GSL molecule
defining the specificity of cell-to-cell interactions [6]. Our
preliminary experiments show that three other tapeworm
species, Diplogonoporus balaenopterae [7], Diphyllobothrium
nihonkaiense,andDiphyllobothrium hottai have GSLs that
react with mAb AK97, although these GSLs were not
structurally characterized. All four tapeworm species as
above belong to the order pseudophyllidea, and spirometo-
side GSLs may be characteristic of this order. We studied in
greater detail the distribution of GSLs in parasitic helminths,
to help elucidate their physiological roles and taxonomic
significance. Adults and plerocercoids of D. hottai contain
spirometosides, and GSL composition changed according to
Correspondence to K. Nakamura, Department of Biochemistry,
Kitasato University School of Medicine, Sagamihara, Kanagawa
228–8555, Japan.
Fax: +81 42 7788441, Tel. +81 42 7789117,
E-mail:
Abbreviations: C16:0, etc., hexadecanoic acid, etc. (number before
colon represents number of carbons in fatty acid and number after
colon represents number of double bonds); C18h:0, 2-hydroxyocta-
decanoic acid; CDH, dihexosylceramide; Cer, ceramide; CMH,
monohexosylceramide; CTH, trihexosylceramide; d18:0, sphinganine;

d20:0, icosasphinganine; Fuc, fucose; Gal, galactose; Glc, glucose;
GlcNAc, N-acetylglucosamine; GSL, glycosphingolipid; HOHAHA,
homonuclear Hartmann-Hahn spectroscopy; LSIMS, liquid secon-
dary ion mass spectrometry; t18:0, 4-hydroxysphinganine; t20:0,
4-hydroxyicosasphinganine.
(Received 6 March 2002, revised 29 May 2002, accepted 11 June 2002)
Eur. J. Biochem. 269, 3549–3559 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03041.x
developmental stage. We also studied immunohistological
localization of GSLs in this species.
MATERIALS AND METHODS
Plerocercoids and adult worms of
D. hottai
Plerocercoids of D. hottai were collected from Japanese surf
smelts, Hypomesus pretiosus japonicus. Some plerocercoids
were stored at )20 °C until use for chemical analysis of
glycolipids. Others were used for infection of golden
hamsters, Mesocricetus auratus, by oral administration.
Twenty to 30 days after infection, adult D. hottai were
obtained from the hamster’s intestine and stored at )20 °C
until chemical analysis. For immunohistochemical studies,
some adult worms were fixed with 4% formaldehyde in
75 m
M
phosphate buffer.
Glycolipids and antibodies
A mixture of authentic GSLs comprising galactosyl-
ceramide, lactosylceramide, globotriaosylceramide, and
globotetraosylceramide was purchased from Matreya. A
standard mixture of partially methylated alditol acetates
was from BioCarb. Anti-paramyosin mAb PM was donated

by T. Nakamura (Kitasato University School of Medicine)
[8]. Anti-H mAb 92FR-A2 and anti-Le
x
mAb 73–30 were
from Seikagaku Corporation. Anti-SEGLx mAb (AK97)
was established previously in our laboratory [5].
Purification of glycolipids
Total lipids were extracted from adults (about 28 g) and
plerocercoids (about 1.4 g) of D. hottai using successive
mixtures of chloroform/methanol (2 : 1, v/v) and chloro-
form/methanol/water (1 : 1 : 0.1, v/v/v). Neutral GSLs
were separated through a column of DEAE-Toyopearl
(Tosoh Co.) and purified on an Iatrobeads 6RS-8060
column (Iatron Laboratories) as described previously [3,4].
Final purification was achieved by preparative TLC.
TLC and TLC-immunostaining
GSLs were separated on a silica-gel 60 HPTLC plate
(Merck) using chloroform/methanol/water (60 : 35 : 8 or
65 : 25 : 4, v/v/v) as the developing solvent, and were
detected by orcinol-H
2
SO
4
reagent followed by heating. For
TLC-immunostaining, the developed TLC plate was soaked
with 0.4% polyisobutylmethacrylate (in 10% CHCl
3
/90%
hexane) for 1 min, dried, overlaid with mAb AK97 diluted
in NaCl/P

i
containing 1% BSA for 1 h at room tempera-
ture, washed with NaCl/P
i
containing 0.05% (w/v) Tween
20, and incubated with horseradish peroxidase-conjugated
sheep anti-mouse immunoglobulin F(ab¢)
2
fragment (Amer-
sham Pharmacia Biotech) for 1 h at room temperature. The
plate was washed again with NaCl/P
i
, and antigen-bound
secondary antibody was visualized with Konica Immuno-
stain HRP-1000 (Konica Co.).
GLC and GC/MS
GLC analysis was performed with a 5890-A gas chroma-
tograph (Hewlett-Packard) using a SPB-1 fused-silica
capillary column (Supelco) with a cool-on column injector.
GC/MS analysis was performed with a QP1100-EX mass
spectrometer (Shimadzu) equipped with SPB-1 column.
Chemical analysis of glycolipids
Sugar compositions of purified GSLs were determined by
GLC as trimethylsilyl derivatives. Analysis of fatty acid
composition was performed by GC/MS after conversion of
samples to methyl esters. For determination of sphingoid,
materials were hydrolyzed with aqueous methanolic HCl,
and components were analysed as trimethylsilyl derivatives
by GC/MS. For methylation analysis, partially methylated
alditol acetates were prepared from purified GSLs and

analysed by GC/MS. Detailed analytical procedures and
conditions were described previously [3].
Liquid secondary ion MS (LSIMS) and MALDI-TOF MS
Intact GSLs were analysed by negative LSIMS using a TSQ
70 triple quadrupole mass spectrometer (Thermo Finnigan
MA, USA). The primary cesium ion was accelerated at
20 kV, and diethanolamine was used as the matrix. GSLs
were also analysed by MALDI-TOF MS using a Voyager
DE-Pro (Applied Biosystems). GSL samples (about
200 ngÆlL
)1
) dissolved in chloroform/methanol (2 : 1, v/v)
were mixed with matrix solution (10 mg 2,5-dihydroxyben-
zoic acid in 1 mL water) (1 : 1, v/v) and the suspensions were
loaded on a sample plate. Positive mass spectra were
measured in reflector mode with 100 nsec delayed extraction.
1
H-NMR analysis
Purified GSLs were dissolved in 0.5 mL (CD
3
)
2
SO/D
2
O
(98 : 2) containing tetramethylsilane as the internal stand-
ard. Final GSL concentration was 10–20 l
M
. NMR spectra
of GSLs were recorded on a Jeol GX-400 spectrometer at

60 °C. HOHAHA spectra were measured with a mixing
time of 100 ms. Spectra were recorded with 64 (t
1
) · 512 (t
2
)
data points. A total of 920 scans were accumulated for
each t
1
, with a spectral width of 1500 Hz. After zero-filling
in the t
1
dimensions, the digital resolutions were 23 and
5.9 HzÆpoint
)1
in w
1
and w
2
dimensions, respectively.
Immunohistochemical examination of adult
D. hottai
Adult D. hottai from experimentally infected hamsters as
described above were fixed with 4% formaldehyde in
75 m
M
phosphate buffer pH 7.4, and then washed with
aqueous solution containing 15% sucrose (w/v), 0.5%
Arabic gum (w/v), and 0.01% thymol (w/v) for 3 days, with
daily renewal of solution. Fixed worms were embedded in

O.C.T. compound (Miles) and rapidly frozen in liquid N
2
.
Transverse sections (7 lm) were cut by cryostat and
collected on poly
D
-lysine treated glass slides. Sections were
rehydrated for 5 min with NaCl/P
i
, treated with 5% (w/v)
BSA in NaCl/P
i
for 10 min at room temperature for
blocking, incubated with primary antibody (AK97, 97FR-
A2, or 73-30) for 1 h at room temperature, washed three
times with NaCl/P
i
, and incubated for 30 min at room
temperature with fluorescein isothiocyanate-conjugated
anti-mouse immunoglobulin antibody diluted with 1%
BSA in NaCl/P
i
at 1 : 40. For paramyosin staining, sections
3550 H. Iriko et al. (Eur. J. Biochem. 269) Ó FEBS 2002
were incubated with tetramethylrhodamine isothiocyanate-
conjugated anti-paramyosin antibody after blocking. After
washing with NaCl/P
i
, sections were mounted with glycerol
buffer, observed by fluorescence microscopy, and photo-

graphed. To confirm the presence of lipid-bound epitopes,
fixed sections were treated for 1 h with chloroform/meth-
anol (2 : 1, v/v) before incubation with antibodies.
RESULTS
Purification and TLC-immunostaining of GSLs
Neutral GSLs of adult worms and plerocercoids of
D. hottai were separated into several fractions ranging
from the region corresponding to monohexosylceramide
(CMH) to that lower than tetrahexosylceramide on a
TLC plate, each fraction giving double or triple bands
(Fig. 1A, lanes 4 and 5). TLC profiles of GSLs differed
between adults and plerocercoids: a GSL fraction migra-
ting slightly faster than authentic SEGLx was detected
only in adults. GSLs corresponding to CMH and
GalSEGLx also showed different migration rates between
adults and plerocercoids. TLC-immunostaining using
mAb AK97 showed that both adults and plerocercoids
contained GSLs having Galb1–4(Fuca1–3)Glcb-sequence
(Fig. 1B).
Purified GSLs are shown in Fig. 2 (adults) and Fig. 3
(plerocercoids). Ten GSLs were isolated from adults (less
polar ones shown in Fig. 2A; more polar ones in Fig. 2B)
anddesignatedasA-1throughA-10(ÔAÕ stands for adult).
Five GSLs were isolated from plerocercoids (Fig. 3) and
designated as P-1 through P-5. mAb AK97 bound to A-6, 7,
8, 9 and 10 (Fig. 2C), and to P-5 (Fig. 1B, lane 5), indicating
that these GSLs contain Galb1–4(Fuca1–3)Glcb1-
sequence.
Structural determination of GSLs
Monohexosylceramides. GSLs corresponding to CMH

were purified as three fractions from adult worms (Fig. 2A).
GLC analysis showed that all three fractions contained
galactose and glucose: 75.5% and 24.5% in A-1, 68.8% and
31.2% in A-2, and 70.5% and 29.5% in A-3. MALDI-TOF
MS spectra (Fig. 4) proved that three fractions were CMH,
and each of them was found to be a mixture of
galactosylceramide and glucosylceramide comprising sever-
al ceramide species as discussed later (see Table 1 for m/z-
values and corresponding ceramide species; see also
Table 2).
From plerocercoids, GSLs corresponding to CMH were
isolated as four fractions (Fig. 3), each containing galac-
tose and glucose: 66.6% and 33.4% in P-1, 79.6% and
20.4% in P-2, 90.0% and 10.0% in P-3, and 83.5% and
16.5% in P-4. MALDI-TOF MS spectra proved that four
GSL fractions were CMH (Fig. 4), a mixture of galact-
osylceramide and glucosylceramide, and their ceramide
Fig. 1. TLC and TLC-immunostaining of total GSLs from D. hottai
plerocercoids and adult worms. GSLsweredevelopedonanHPTLC
plate (Merck) with a solvent system of chloroform/methanol/water
(60 : 35 : 8, v/v/v). (A) Orcinol-H
2
SO
4
staining. (B) TLC immuno-
staining with mAb AK97 (1 : 1000). Lane 1, authentic GSLs, GalCer,
galactosylceramide (CMH); LacCer, lactosylceramide (CDH);
Gb
3
Cer, globotriaosylceramide (CTH); Gb

4
Cer, globotetraosylcera-
mide). Lane 2, authentic SEGLx. Lane 3, authentic GalSEGLx. Lane 4,
total GSLs from adult worms. Lane 5, total GSLs from plerocercoids.
Fig. 2. TLC and TLC-immunostaining of isolated GSLs from D. hottai adult. GSLsweredevelopedonanHPTLCplatewithasolventsystemof
chloroform/methanol/water (65 : 25 : 4, v/v/v for A; 60 : 35 : 8, v/v for B and C). (A) Less polar GSLs. (B) and (C) More polar GSLs. (A) and (B)
Orcinol-H
2
SO
4
staining. (C) TLC-immunostaining with mAb AK97 (1 : 1000). (A) Lane 1, authentic GSLs (GalCer, galactosylceramides, three
bands; LacCer, lactosylceramide, two bands; Gb3Cer, globotriaosylceramide, two bands; Gb4Cer, globotetraosylceramide, two bands). Lane 2,
total GSLs from adults. Lane 3, A-1. Lane 4, A-2. Lane 5, A-3. Lane 6, A-4. Lane 7, A-5. (B) and (C): Lane 1, authentic GSLs. Lane 2, total GSLs
from adults. Lane 3, A-6. Lane 4, A-7. Lane 5, A-8. Lane 6, A-9. Lane 7, A-10.
Ó FEBS 2002 Glycosphingolipids of Diphyllobothrium hottai (Eur. J. Biochem. 269) 3551
compositions were assigned as discussed later (Table 1; see
also Table 3).
Di- and tri-hexosylceramides. Partially methylated alditol
acetates derived from A-4 were analysed by GC-MS
(Fig. 5A). Two major ion peaks, 1 and 3, were identified
as 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylglucitol and
1,3,5-tri-O-acetyl-2,4,6-tri-O-methylgalactitol, respectively;
a small amount of 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-
galactitol (Fig. 5A, peak 2) was also detected. MALDI-TOF
MS spectrum of A-4 (Fig. 5C) showed an ion peak at m/z
886 which is in accord with the calculated m/z of sodium
adducted molecular ion [M + Na]
+
of dihexosylceramide
(CDH), comprising sphinganine (d18:0) and hexadecanoic

acid (C16:0) as the ceramide composition. From these
results, Glc1–3Gal1-Cer was determined as a major com-
ponent of A-4, with Gal1–3Gal1-Cer as a minor component.
On methylation analysis of A-5, five components,
1,5-di-O-acetyl-2,3,4,6-tetra-O-methylgalactitol (peak 1),
1,5-di-O-acetyl-2,3,4,6-tetra-O-methylglucitol (peak 2), 1,3,
5,6-tetra-O-acetyl-2,4-di-O-methylgalactitol (peak 3), 1,3,5-
tri-O-acetyl-2,4,6-tri-O-methylgalactitol (peak 4), and
1,3,4,5-tetra-O-acetyl-2,6-di-O-methylglucitol (peak 5) were
detected (Fig. 5B). This result indicates that A-5 was a
mixture of more than one structure. MALDI-TOF MS
spectrum (Fig. 5D) shows that predominant components of
A-5 are trihexosylceramide (CTH): there are two ions at m/z
1188 and 1216, corresponding, respectively, to calculated m/z
of sodium adducted molecular ions of CTH with cera-
mides comprising sphinganine and hexacosanoic acid
(d18:0-C:26:0) and d18:0-C:28:0 as sphingoid-fatty acid
combination. Considering that D. hottai contains Gal1–
4(Fuc1–3)Glc1–3(Gal1–6)Gal1-Cer as shown below and
that the structure of CDH is Glc1–3Gal-Cer as described
above, the most likely structure of CTH which is compatible
with results of methylation analysis (Fig. 5B, peaks 1, 2 and
5) is Glc1–3(Gal1–6)Gal-Cer (see Discussion).
Methylation analysis of CTH also showed the presence of
1,3,5-tri-O-acetyl-2,4,6-tri-O-methylgalactitol and 1,3,4,5-
tetra-O-acetyl-2,6-di-O-methylglucitol (Fig. 5B, peaks 3
and 4, respectively). These components may be attributed
to contamination of SEGLx, as supported by the presence
of a molecularly related ion at m/z 1362 in MALDI-TOF
MS spectrum (Fig. 5D), corresponding to SEGLx with

ceramide consisting of d18:0-C28:0.
In plerocercoids CDH was not detected on TLC. CTH
may be present in trace amounts; a faintly stained band was
observed on TLC with orcinol-H
2
SO
4
detection, but was
not analysed further because the quantity was so small.
Fig. 3. TLC of isolated GSLs from D. hottai plerocercoids. GSLs were
developed on an HPTLC plate with a solvent system of chloroform/
methanol/water (65 : 25 : 4, v/v/v). GSLs were detected with orcinol-
H
2
SO
4
reagent followed by heating. Lane 1, authentic GSLs (GalCer;
LacCer; Gb
3
Cer; Gb
4
Cer). Lane 2, total GSLs from plerocercoids.
Lane 3, P-1. Lane 4, P-2. Lane 5, P-3. Lane 6, P-4. Lane 7, P-5.
Fig. 4. MALDI-TOF MS spectra of monohexosylceramides in
D. hottai adults and plerocercoids. Intact GSLs were analysed by pos-
itive mode MALDI-TOF MS. Values indicate m/z of sodium adducted
molecular ions, [M + Na]
+
, in nominal mass. Possible ceramide
species are listed in Table 1.

3552 H. Iriko et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Fucosyl tri- and tetra-hexosylceramides. GLC analysis of
trimethylsilyl derivatives showed that sugar components of
A-6 to A-9 were galactose, glucose and fucose, the molar
ratios being 2 : 1 : 1 in A-6, A-7, and A-8, and 3 : 1 : 1 in
A-9 (ratios were compensated by authentic standard).
Methylation analysis revealed that A-6, A-7, and A-8
gave rise to four components, which were identified as
1,5-di-O-acetyl-2,3,4-tri-O-methylfucitol (peak 1), 1,5-di-
O-acetyl-2,3,4,6-tetra-O-methylgalactitol (peak 2), 1,3,4,5-
tetra-O-acetyl-2,6-di-O-methylglucitol (peak 3), and
1,3,5-tri-O-acetyl-2,4,6-tri-O-methylgalactitol (peak 4)
(GLC chromatogram of A-7 is shown as an example in
Fig. 6A).
The LSIMS spectrum of A-7 showed deprotonated
molecules, [M-H]
–
,atm/z 1326.8 and m/z 1354.9 (Fig. 6B),
which correspond to calculated molecular masses of GSL
SEGLx [3] with ceramides comprising t18:0-C26:0 (m/z
1326.9) and t18:0-C28:0 (m/z 1354.9), respectively. The
presence of ions due to elimination of one fucose (m/z
1208.9 and 1180.8) as well as one hexose (m/z 1192.8 and
1164.8) confirmed a branched carbohydrate structure in
which fucose is linked to penultimate glucose, in accord with
results of methylation analysis. Fragment ions generated on
sequential elimination of hexoses were detected. Based on
these results in combination with TLC-immunostaining
(Figs 1 and 2), the structure of A-6, A-7, and A-8 was
concluded to be Gal1–4(Fuc1–3)Glc1–3Gal1-Cer. The

difference in TLC mobility between A-6 and A-7 was
assumed to reflect different ceramide composition, as
discussed later.
Four peaks obtained from methylation analysis of
A-9 were identified as 1,5-di-O-acetyl-2,3,4-tri-O-methylfuc-
itol (peak 1), 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylgalact-
itol (peak 2), 1,3,4,5-tetra-O-acetyl-2,6-di-O-methylglucitol
(peak 4), and 1,3,5,6-tetra-O-acetyl-2,4-di-O-methylgalacti-
tol (peak 5) (Fig. 6C). In the LSIMS spectrum of A-9,
deprotonated molecules, [M-H]
–
, were detected at m/z
1472.9 and m/z 1501.0 (Fig. 6D), in close accord with values
calculated from the structure of GalSEGLx [4] with
ceramides consisting of d18:0-C26:0 (m/z 1472.9), and
d18:0-C28:0 (m/z 1501.0), respectively. Fragment ions
produced by sequential elimination of fucose and/or hexoses
were also detected, as in the case of A-7 described above.
The structure of A-9 was concluded to be Gal1–4(Fuc1–
3)Glc1–3(Gal1–6)Gal-Cer. A-10 (from adults) and P-5
(from plerocercoids) were not analysed chemically because
quantities were insufficient. However, several lines of
evidences including TLC mobility (Fig. 1A), mAb AK97
binding (Fig. 1B), and MALDI-TOF MS analysis (data not
shown), suggested that the structure of these components
was the same as that of A-9: Gal1–4(Fuc1–3)Glc1–3(Gal1–
6)Gal1-Cer.
1
H NMR spectroscopy
In order to determine anomeric configuration and confirm

linkage sequence of carbohydrates, A-7 and A-9 were
subjected to proton NMR spectroscopy, and showed four
and five anomeric protons, respectively. Chemical shifts and
coupling constants, summarized in Table 4, are in good
agreement with those of SEGLx [3] and GalSEGLx
[4]. One-dimensional spectrum and two-dimensional
HOHAHA spectrum of A-9 are presented in Fig. 7. The
one-dimensional spectrum in the low-field region of A-9
showed a fucose H-5 resonance and five anomeric protons,
one a (J
1,2
¼ 3.9 Hz) and four b (J
1,2
¼ 5.9–7.8 Hz)
(Fig. 7A). Signal resolution at around 4.15 p.p.m. was poor
in one-dimensional spectrum, but two-dimensional
HOHAHA spectrum (Fig. 7B) showed two Galb signals,
i.e. 3,6Galb (I) at 4.18 p.p.m and Galb (IV) at 4.16 p.p.m.
Based on these results, we concluded that the structure of
A-7 was Galb1–4(Fuca1–3)Glcb1–3Galb1-Cer (SEGLx)
and that of A-9 was Galb1–4(Fuca1–3)Glcb1–3(Galb1–
6)Galb1-Cer (GalSEGLx).
Ceramide species of glycosphingolipids
To examine the combinations of sphingoid and fatty acids
comprising ceramide moieties of GSLs, sphingoids were
chemically analysed, and sphingoid-fatty acid combinations
were deduced from MALDI-TOF MS spectra. The results
are summarized in Table 2 (adults) and Table 3 (plerocerc-
oids). They explain reasonably the order of migration rate
of each CMH: hydrophobicity of ceramide moieties was

highest in A-1 and lowest in A-3, and similar trends are seen
for A-6 to A-8 and P-1 to P-4. Sphingoid of A-10 and P-5
(both are GalSEGLx) were not chemically analysed;
however, MALDI-TOF MS spectrum (not shown) showed
possible ceramide species as indicated in Tables 3 and 4. As
the proportion of fatty acids analysed by GLC as methy-
lesters was not always identical to that analysed by
MALDI-TOF MS, data from the latter method were
adopted to determine ceramide species, as described above.
Immunohistochemical localization of GSLs
in adult
D. hottai
To investigate localization of spirometosides (SEGLx and
GalSEGLx) in adult D. hottai, transverse sections (7 lm)
of scolex were incubated with anti-SEGLx mAb AK97,
and bound antibodies were detected by fluorescence
Table 1. Mass numbers and possible sphingoid–fatty acid combinations
of ceramides in CMH. Molecular related ions, [M + Na]
+
,detectedin
CMH (see Fig. 4) are listed. Values are expressed as nominal mass.
Listed ceramide species were deduced from chemical analysis of
sphingoid and MALDI-TOF MS spectra (see also Tables 3 and 4).
m/z Ceramides
696 d18:0-C14:0 d20:0-C12:0
712 t18:0-C14:0 t20:0-C12:0
724 d18:0-C16:0 d20:0-C14:0
740 t18:0-C16:0 t20:0-C14:0
752 d18:0-C18:0 d20:0-C16:0
768 t18:0-C18:0 t20:0-C16:0

780 d20:0-C18:0 d18:0-C20:0
784 t18:0-C18h:0 t20:0-C16h:0
796 t20:0-C18:0 t18:0-C20:0
812 t20:0-C18h:0
864 d18:0-C26:0 d20:0-C24:0
880 t18:0-C26:0 t20:0-C24:0
892 d18:0-C28:0 d20:0-C26:0
896 t18:0-C26h:0
908 t18:0-C28:0 t20:0-C26:0
Ó FEBS 2002 Glycosphingolipids of Diphyllobothrium hottai (Eur. J. Biochem. 269) 3553
Table 2. Ceramide composition in GSLs of D. hottai adult worms. Sphingoids were determined by GLC as trimethylsilyl derivatives. Sphingoid–fatty acid combinations were deduced from MALDI-TOF MS
spectra. Bold type indicates dominant ions. ND, Not determined.
CMH SEGLx GalSEGLx
Sph
a
FA
d
A-1 A-2 A-3 CDH A-4 CTH A-5 A-6 A-7 A-8 A-9 A-10
d18:0 100
e
71.7 – 100 100 100 4.3 100 100 ND
d20:0 – 20.3 – – – – 18.6 – – ND
t18:0 – 8.0 17.3 – – – 77.1 – – ND
t20:0 – – 82.7 – – – – – – ND
d
b
> C24:0 d18:0-C26:0 d18:0-C26:0 d18:0-C28:0 d18:0-C26:0
d18:0-C28:0 d18:0-C28:0 d18:0-C28:0
< C24:0 d18:0-C18:0 d18:0-C16:0 d18:0-C18:0 d18:0-C16:0 d18:0-C16:0
d20:0-C16:0 d20:0-C16:0 d18:0-C18:0

d20:0-C18:0 d18:0-C20:0
d20:0-C14:0
d20:0-C16:0
d20:0-C18:0
t
c
> C24:0 t18:0-C26:0 t18:0-C26:0
t18:0-C28:0 t18:0-C28:0
t20:0-C24:0
t20:0-C26:0
< C24:0 t18:0-C18:0 t18:0-C18:0
t20:0-C18:0 t18:0-C20:0
t20:0-C16:0
a
Sphingoid.
b
Dihydroxy.
c
Trihydroxy.
d
Fatty acid.
e
Values are expressed as percentages of the total component.
3554 H. Iriko et al. (Eur. J. Biochem. 269) Ó FEBS 2002
(Fig. 8A). To visualize the tissue structure of scolex,
muscle fibers of the same section were stained with anti-
paramyosin antibody, PM, detected with rhodamine
(Fig. 8B). A double staining picture is shown in Fig. 8C.
SEGLx and/or GalSEGLx were concentrated mainly at
the inner surface of bothria, and distributed diffusely in

parenchyma. Staining patterns of both antibodies showed
restricted distribution within scolex tissue; only marginal
overlapping fluorescence was observed by double staining.
Although AK97 is known to cross-react with Le
x
antigen,
Galb1–4(Fuca1–3)GlcNAcb1-R [5], the presence of Le
x
(either glycoprotein or glycolipids) in scolex tissue was
ruled out because no staining was observed with anti-Le
x
mAb 73–30 (data not shown). Anti-H mAb 92FR-A2,
used as a control IgM antibody, did not stain any scolex
tissue (data not shown). Chloroform/methanol treatment
abolished AK97 binding in bothria and parenchyma, but
PM binding was not affected (data not shown). These
findings indicate that AK97 recognized only lipid-bound
antigen(s).
Table 3. Ceramide composition in GSLs of D. hottai plerocercoids. Sphingoids were determined by GLC as trimethylsilyl derivatives. Sphingoid–
fatty acid combinations were deduced from MALDI-TOF MS spectra. Boldface indicates dominant ions. ND, Not determined.
CMH GalSEGLx
Sph
a
FA
d
P-1 P-2 P-3 P-4 P-5
d18:0 23.4
f
–––ND
d20:0 – 47.0 – – ND

t18:0 76.6 – 100 71.1 ND
t20:0 – 53.0 – 28.9 ND
d
b
> C24:0
< C24:0 d18:0-C14:0
d18:0-C16:0
d18:0-C18:0
t
c
> C24:0 t18:0-C26:0 t18:0-C28:0
t18:0-C28:0 t20:0-C26:0
t20:0-C26:0
< C24:0 t20:0-C18:0 t18:0-C18:0
t20:0-C16:0
t20:0-C18:0
t18:0-C14:0
t18:0-C16:0
t18:0-C18:0
tOH
e
t18:0-C26h:0
> C24:0
OH t18:0-C18h:0
< C24:0 t20:0-C18h:0
a
Sphingoid.
b
Dihydroxy.
c

Trihydroxy.
d
Fatty acid.
e
Hydroxy fatty acid.
f
Values are expressed as percentages of the total component.
Fig. 5. Methylation analysis and MALDI-
TOF MS analysis of A-4 and A-5. (A) GLC
chromatogram of partially methylated alditol
acetates derived from A-4. (B) GLC chroma-
togram of A-5. Peaks designated 1, 2, 3, 4, and
5are1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-
glucitol, 1,5-di-O-acetyl-2,3,4,6-tetra-O-
methylgalactitol, 1,3,5-tri-O-acetyl-2,4,6-tri-
O-methylgalactitol, 1,3,4,5-tetra-O-acetyl-2,6-
di-O-methylglucitol, 1,3,5,6-tetra-O-acetyl-
2,4-di-O-methylgalactitol, respectively. Aster-
isks denote contaminants of nonsugar origin.
(C) MALDI-TOF MS spectrum of intact A-4.
(D) MALDI-TOF MS spectrum of intact A-5.
Values in (C) and (D) indicate m/z of sodium
adducted molecular related ions, [M + Na]
+
,
in nominal mass.
Ó FEBS 2002 Glycosphingolipids of Diphyllobothrium hottai (Eur. J. Biochem. 269) 3555
DISCUSSION
The mechanism of parasitism in platyhelminth parasites
includes processes such as host infection, encounter with

mechanical and chemical stress of the host’s internal
environment, defence against immune attack, and specific
uptake of nutrients. These mechanisms seem to depend on
specialized and possibly unusual biological properties of cell
Table 4. Chemical shifts (p.p.m) and coupling constants (
3
J
1,2
, Hz) for A-7 and A-9.
Compound Proton
V
Fuca1
IV
Galb1
III
Galb1-
II
4Glcb1-
I
3(,6)Galb1-Cer
A-7 H1 (
3
J
1,2
) 5.20 (3.9) 4.30 (> 6.0) 4.52 (6.8) 4.16 (> 6.0)
H2 3.31
a
3.37 3.55
a
H3 3.31

a
3.57 3.55
a
H4 3.69 3.69 3.93
H5 4.60
A-9 H1 (
3
J
1,2
) 5.20 (3.9) 4.16 (> 6.0) 4.30 (> 6.0) 4.53 (7.8) 4.18 (> 6.0)
H2 3.33
a
3.30
a
3.37 3.55
a
H3 3.33
a
3.30
a
3.55 3.55
a
H4 3.66 3.69 3.66 3.93
H5 4.60
a
H2 and H3 of the galactosyl residues are strongly coupled.
Fig. 6. Methylation analysis and LSIMS analysis of A-7 and A-9. (A) GLC chromatogram of partially methylated alditol acetates derived from A-7.
(B) Negative LSIMS spectrum of intact A-7. (C) GLC chromatogram of partially methylated alditol acetates derived from A-9. (D) Negative
LSIMS spectrum of intact A-9. In (A) and (C) peaks designated as 1, 2, 3, 4, and 5 are 1,5-di-O-acetyl-2,3,4-tri-O-methylfucitol, 1,5-di-O-acetyl-
2,3,4,6-tetra-O-methylgalactitol, 1,3,5-tri-O-acetyl-2,4,6-tri-O-methylgalactitol, 1,3,4,5-tetra-O-acetyl-2,6-di-O-methylglucitol, 1,3,5,6-tetra-O-

acetyl-2,4-di-O-methylgalactitol, respectively. In (D) the fragment ions with cleavage of glycosidic linkages were observed in LSIMS spectrum.
3556 H. Iriko et al. (Eur. J. Biochem. 269) Ó FEBS 2002
surface membranes of the parasite’s outer tegument.
Membrane-linked GSLs are the most likely candidate
molecules for functional participation in such mechanisms,
as they anchor their hydrophobic ceramide moieties in the
outer leaflet of the cell surface membrane and orient their
hydrophilic carbohydrate moieties toward the external
medium. This characteristic topological distribution of
GSL molecules in cell membranes implies functional
significance of the carbohydrate structures.
We previously characterized novel GSLs, SEGLx and
GalSEGLx, later termed ÔspirometosidesÕ, from plero-
cercoids of S. erinaceieuropaei [3,4]. In the present study
we found SEGLx and GalSEGLx in adults and GalS-
EGLx in plerocercoids of D. hottai, and showed changes
of GSL composition as a function of developmental
stage. CMH, CDH, and CTH were found in adults,
whereas neither CDH nor CTH was detected in plero-
cercoids.
Fig. 7. NMR spectra of A-9. (A)
1
H-NMR
spectrum in the low field region. (B) Two-
dimensional HOHAHA spectrum in the cross-
peak region between resonances of anomeric
protons and other protons in three galactosyl
residues. The abbreviation labelling each
cross-peak corresponds to sugar residue
numbering (Roman numerals), followed by

the proton assignment (Arabic numerals).
Fig. 8. Immunohistochemical staining of
D. hottai adults. Transverse sections of adult
scolex were double stained with anti-SEGLx
mAb AK97 (1 : 1000) and anti-paramyosin
antibody PM (1 : 50). (A) SEGLx and/or
GalSEGLx (green). (B) Paramyosin (red).
(C) Merge image of both types of fluorescence.
Ó FEBS 2002 Glycosphingolipids of Diphyllobothrium hottai (Eur. J. Biochem. 269) 3557
The major species of CDH found in adults was Glc1–
3Gal1-Cer, a novel GSL first characterized in this study,
and a possible precursor of SEGLx (Fig. 9). It was difficult
to isolate CTH as a single species chromatographically, and
further purification was not possible because of insufficient
amount of material. Methylation analysis, however, sug-
gested that one of the structures of CTH is Glc1–3(Gal1–6)
Gal1-Cer, a possible precursor of GalSEGLx (Fig. 9).
Considering possible biosynthetic pathways of SEGLx, one
would predict occurrence of an intermediate having Gal1–4
Glc1–3Gal1-Cer structure (structure C in Fig. 9). However,
CTH with such structure was not detected by methylation
analysis. GalSEGLx can be synthesized by two reaction
steps from Glc1–3(Gal1–6)Gal1-Cer, to which one galac-
tose residue and then one fucose residue are added.
However, a galactose-added intermediate (structure F in
Fig. 9) was not detected. It is interesting that plerocercoids
contained only CMH and GalSEGLx; possible biosynthetic
intermediates were not detected in significant amounts.
Pseudophyllidean tapeworms have five distinct growth
stages in their life cycle, i.e. egg, coracidium, procercoid,

plerocercoid, and adult worms. In this study we compared
the GSL composition of plerocercoids with that of adults.
These two stages are characterized by totally different
morphology and different, specific hosts. We found that
each stage also shows distinct, specific GSL composition.
Even for CMH, different molecular species were found in
the two stages. In contrast, we observed only one pattern of
CMH for S. erinaceieuropaei despite the entirely different
environmental conditions between plerocercoids and adults
[9]. In D. hottai, only A-3 and P-1 were identical, having the
same ceramide composition with sphingoid–fatty acid
combinations of t18:0-C18:0, t20:0-C16:0, and t20:0-C18:0
(see Fig. 4 and Table 3). Most of the other ceramide
compositions except d18:0-C18:0 species are specific for
either plerocercoids or adults. In considering the overall
ceramide composition of D. hottai GSLs, we noticed that
adults have no hydroxy fatty acid, plerocercoids have
predominantly trihydroxy sphingoids, and hydroxy fatty
acids are always bound with trihydroxy sphingoids.
SEGLx and GalSEGLx of D. hottai contained C16:0,
C18:0, C26:0, and C28:0 as major fatty acid components,
lacking hydroxy fatty acids. These findings contrast with
those of S. erinaceieuropaei, which contain a significant
amount of 2-hydroxy octadecanoic acid (18h:0) [3]. The
major sphingoids of D. hottai GSLs were d18:0, t18:0,
d20:0, and t20:0, whereas S. erinaceieuropaei GSLs did not
contain d20:0 or t20:0 [3]. To elucidate the different GSL
compositions, including ceramide species, the influence of
hosts on GSL composition of tapeworms must be consid-
ered. Studies of GSL composition of adult worms from

different hosts (e.g. cats) are in progress.
We previously determined the three-dimensional struc-
ture of SEGLx through computer simulation and showed
that SEGLx forms two kinds of stable conformation
depending on ceramide species [10]. SEGLx having cera-
mide comprised of sphinganine and nonhydroxy fatty acid
has an almost horizontal conformation, whereas one with
trihydroxysphinganine and nonhydroxy fatty acid has a
right-angled shape. SEGLx A-6 and A-8 in this study are
considered to be the former type, and A-7 to be the latter.
We presume that these two types of SEGLx present their
unique carbohydrate chain differently on the cell mem-
brane, and hypothesize that interaction of SEGLx and host
molecule differ depending on their ceramide species.
The carbohydrate structures of spirometosides are char-
acterized by the presence of an intervening (penultimate)
glucose residue, and a b1–3 linkage between the reducing
end galactose and the penultimate glucose (Galb1–4Glcb1–
3Galb1-Cer). GSLs with these structural features had not
been found in any other organism. There are two major
orders of tapeworms which infect with human: Cyclophyll-
idea and Pseudophyllidea. Other GSLs so far characterized
in Cyclophyllidea are galactosylceramides from S. manso-
noides [11], Taenia crassiceps [12], Taenia solium [13],
Echinococcus multilocularis [14,15], and Metroliasthes cotur-
nix [16], and di-, tri- and tetragalactosylceramides from
M. cortunix [16], T. crassiceps [12], and E. multilocularis
[14,15]. Fucosylated di- and tetragalactosylceramides have
also been found in E. multilocularis [14,15]. The core
carbohydrate structure of these GSLs is mainly Galb1–6

Gal,andtoalesserextentGala1–4Gal. Another GSL with
a unique carbohydrate linkage, GalNAcb1–4Glc, was
found in Schistosoma mansoni, a trematode [17,18]. Our
preliminary experiments show that SEGLx and GalSEGLx
occur in another tapeworm, D. nihonkaiense. As S. erina-
ceieuropaei, D. hottai,andD. nihonkaiense are all Pseudo-
phillidea, GSLs in spirometosides may have taxonomic
significance.
Adult cestodes inhabit the intestines of their hosts, and
are anchored to the intestinal wall by specific holdfast
organs in the scolex [19]. The organs of pseudophyllidean
tapeworms are deep dorsal and ventral bothria. As the
bothria comprise the major point of contact between adults
and host tissue, elucidation of their structure and function at
the molecular level is important for understanding host–
parasite interaction. In this study, immunohistochemical
analysis showed that spirometosides are present at the inner
surface of bothria of D. hottai. Chemical analysis revealed
that the spirometoside SEGLx is found in adults but not in
plerocercoids. These findings suggest that spirometosides
play physiological roles in host–parasite interaction, e.g.
recognition of and attachment to host intestine. We showed
Fig. 9. GSLs in D. hottai adults and their putative biosynthetic path-
way. Structures shown in bold are GSLs that have been identified also
in ploerocercoids. Structures C and F are putative biosynthetic inter-
mediates which have not yet been detected.
3558 H. Iriko et al. (Eur. J. Biochem. 269) Ó FEBS 2002
previously that mAb AK97 cross-reacts with stage-specific
embryonic antigen-1 (SSEA-1 or Le
x

) [5]. There is structural
similarity between Le
x
and SEGLx; glucose of SEGLx is
replaced by GlcNAc in the Le
x
epitope saccharide sequence.
Cummings and Nyame [20] proposed that Le
x
is important
in host–schistosome interactions, e.g. elicitation of antibod-
ies to Le
x
,eggadhesiontovesselwalls,andmovement
through tissues. Le
x
has also been implicated as a key
molecule defining specificity of cell-to-cell interactions
including Le
x
-Le
x
(carbohydrate–carbohydrate) adhesion
[6]. Le
x
antigens are also found in dog small intestine [21],
and in human intestine and colon [22]. Considering the
structural similarity between Le
x
and SEGLx, we suspect

that SEGLx and related compounds contribute to the
infection mechanism in small intestine. Findings from this
and our previous studies indicate that spirometoside GSLs
play significant roles in parasitic infection.
ACKNOWLEDGEMENTS
We thank T. Nakamura (Kitasato University School of Medicine) for
supplying us with anti-paramyosin mAb.
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