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 spirometosides
[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 spirometosidesforGSLshaving
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: nakam@kitasato-u.ac.jp
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) 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)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(Astands 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.
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/zof 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/z1362 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/zof 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)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/z1326.8 and m/z1354.9 (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/z1354.9), respectively. The
presence of ions due to elimination of one fucose (m/z
1208.9 and 1180.8) as well as one hexose (m/z1192.8 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/z1501.0 (Fig. 6D), in close accord with values
calculated from the structure of GalSEGLx [4] with
ceramides consisting of d18:0-C26:0 (m/z1472.9), and
d18:0-C28:0 (m/z1501.0), 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
¼3.9 Hz) and four b(J
1,2
¼5.9–7.8 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 Galbsignals,
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/zCeramides
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
FEBS 2002 Glycosphingolipids of Diphyllobothrium hottai (Eur. J. Biochem. 269) 3553