Báo cáo khoa học: Characterization of glycosphingolipids fromSchistosoma mansoni eggs carrying Fuc(a1±3)GalNAc-, GalNAc(b1±4)[Fuc(a1±3)]GlcNAc-and Gal(b1±4)[Fuc(a1±3)]GlcNAc- (Lewis X) terminal structures pot

Characterization of glycosphingolipids from
Schistosoma mansoni
eggs carrying Fuc(a1±3)GalNAc-, GalNAc(b1±4)[Fuc(a1±3)]GlcNAc-
and Gal(b1±4)[Fuc(a1±3)]GlcNAc- (Lewis X) terminal structures
Manfred Wuhrer
1,
*, Sven R. Kantelhardt
1,
*, Roger D. Dennis,
1
Michael J. Doenhoff
2
,GuÈ nter Lochnit
1
and Rudolf Geyer
1
1
Institute of Biochemistry, University of Giessen, Germany;
2
School of Biological Sciences, University of Wales, Bangor, Wales, UK
The carbohydrate moieties of glycosphingolipids f rom eggs
of the human parasite, Schistosoma mansoni,wereenzy-
matically released, labelled with 2-aminopyridine (PA),
fractionated and analysed by linkage analys is, partial
hydrolysis, enzymatic cleavage, matrix-assisted laser
desorption/ionization time-of-¯ight m ass spectrometry a nd
nano-electrospray ionization mass spectrometry. Apart
from large, highly fuc osylated structures w ith ® ve to seven
HexNAc residues, we found short, oligofucosylated species
containing three to f our He xNAc residues. Their s tructures
have been determined as Fuc(a1±3)GalNAc(b1±4)[  Fuc

(a1±3)]GlcNAc(b1±3)GalNAc(b1±4)Glc-PA, GalNAc(b1±
4)[Fuc(a1±3)]GlcNAc(b1±3)GlcNAc(b1±3)GalNAc(b1±4)
Glc-PA, Fuc(a1±3)GalNAc(b1±4)[Fuc(a1±3)]GlcNAc(b1±
4) GlcNAc(b1±3)GalNAc(b1±4)Glc-PA, and Fuc(a1±3)
GalNAc(b1±4)[  Fuc(a1±2)  Fuc(a1±2)Fuc(a1±3)]Glc
NAc(b1±3)GlcNAc(b1±3)GalNAc(b1±4)Glc-PA. The last
structure e xhibits a trifucos yl sidechain p reviously identi®ed
on the cercarial glycocalyx. These structures stress the
importance of 3-fucosylated GalN Ac as a terminal epitope
in schistosome glycoconjugates. To w hat degree t hese gly-
cans contribute to the pronounced antigen icity of S. mansoni
egg glycolipids remains to be determined. In addition, we
have identi®ed the compounds GlcNAc(b1±3)GalNAc(b1±
4)Glc-PA, Gal(b1±4)[Fuc(a1±3)]GlcNAc(b1±3) GalNAc
(b1±4)Glc-PA, the latter of which is a Lewis X -pentasac-
charide identical to that present on cercarial glycolipids, as
well as Gal(b1±3)GalNAc(1±4)Gal(1±4)Glc-PA, which
corresponds to asialo gangliotetraosylceramide a nd is most
probably derived from the mammalian host.
Keywords: ceramide glycanase; internal fucose; oligo-
saccharide structural analysis; Schistosoma mansoni egg
glycolipids.
Schistosomiasis is caused b y parasitic blood ¯ukes of the
genus Schistosoma and affects 200 million p eople w orld-
wide. During infection, schistosomal glycoconjugates play
important ro les in host±parasite pathological interactions
[1,2]. Schistosomes p roduce a variety of complex carbohy-
drate s tructures, many of which a re highly fucosylated [2].
These glycans are conjugated to pr oteins and/or lipids i n
different life-cycle stages and vary in their recognition by the

immune system. For Schistosoma mansoni,theLewisX
epitope has been found on egg a nd cercarial g lycoproteins
[3±5] and cercarial glycolipids [6]. Although this e pitope is
shared with the mammalian host, S. mansoni infection
serum contains cytolytic antibodies directed against this
epitope [7]. Besides L ewis-X-carrying glycolipids, S. ma n-
soni expresses highly a ntigenic glyco lipids mainly i n t he egg
and the cercarial stage, and to a lesser extent in the adult
stage [ 8,9]. T he stage-associated expression of carbohydrate
structures on S. mansoni glycolipids i s paralleled by changes
in glycolipid ceramide s tructures during the life -cycle [10].
The highly antigenic glycolipids are speci®cally recog-
nized by schistosome in fection serum and not by other
helminth infection s era, which m akes them possible s ero-
diagnostic antigens [ 11]. They share a fucose-containing
epitope with keyhole limpet haemocyanin (KLH) [12],
which is c onsistent with the u se of KL H for the d iagnosis of
schistosomiasis [ 13]. The induction of signi®cant titres of
antibodies against these glycolipids is a ssociated with the
onset of patency [11], and the IgE-response against worm
glycolipids may play a role in mediating resistance to
S. mansoni reinfection after praziquan tel treatment [14]. All
schistosome glycosphingolipids share the unique core
structure, GalNAcb4Glc1±1ceramide, which was ®rst
described by M akaaru et al. [15]. C ercarial glycolipids were
found to be dominated by short-chained carbohydrate
moieties expressing Lewis X (Gal(b1±4)[Fuc(a1±3)]Glc-
NAcb1-) and pseudo -Lewis Y (Fuc(a1±3)Gal(b1±4)[Fuc
(a1±3)]GlcNAc(b1-) epitopes [ 6], whereas the structural char-
acterization of unfractionated complex glycosphingolipids

from S. mansoni eggs has revealed the t erminal str ucture 
Correspondence to R. Geyer, Biochemisches I nstitut am Klinikum,
Universita
È
t Giessen, Friedrichstrasse 24, D-35392 Giessen, Germany.
Fax: + 49 641 99 47409, Tel.: + 49 641 99 47400,
E-mail:
Abbreviations: Cer, ceramide; dH ex, deoxyhexose; E SI, electrospray-
ionization; Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactos-
amine; Glc, glucose; GlcNAc, N-acetylglucosamine; Hex, hexose;
HexNAc, N-acetylhexosamine; KLH, keyhole-limpet hemocyanin;
LSIMS, liquid secondary-ion mass spectrometry; mAb, monoclonal
antibody; Man, m annose; PA, 2- a minopyridine; PGC, porou s
graphitic carbon.
Enzymes: a-
L
-fucosidase (E C 3.2.1.51); b-
D
-galactosidase (EC
3.2.1.23); ceramide glycanase (oligoglycosylceramideglycohydrolase;
EC 3.2.1.23).
*Note: these authors c ontributed e qually t o th is work.
(Received 31 August 2001, r evised 8 November 2001, accepted 14
November 2001)
Eur. J. Biochem. 269, 481±493 (2002) Ó FEBS 2002
Fuc(1±2)Fuc(1±3)GalNAc (b1- attached to a s eries of
up to three )4)[Fuc(1±2)Fuc(1±3)]GlcNAc(b1-repeats and
one )4)[  Fuc(1±2)  Fuc(1 ±3)]GlcNAc(b1-unit, which
is linked to )3)GalNAc(b1±4)Glc(1±1)ceramide, i.e. the
Schisto-core [16].

In the present study, we have chosen an approach that
differs from the one employed in the aforementioned and
other investigations of glycolipid structures from S. mansoni
eggs [16,17] i n w hich complex m ixtures o f g lycosphingo-
lipids have been analysed mainly by liquid secondary-ion
mass spectrometry (L SIMS) u sing various chemical deri-
vatizations. Similar t o previous studies on glycolipids from
Caenorhabditis elegans [18] and S. mansoni cercaria e [6], w e
have enzymatically removed the ceramide moieties and
fractionated the released oligosaccharide chains before
structural c haracterization by mass spectrometry, chemical
and e nzymatic degradation and linkage analysis. Using this
strategy, structural information on the carbohydrate moi-
eties of individual glycolipids from S. m ansoni eggs is
obtained.
MATERIALS AND METHODS
Glycolipid puri®cation and fractionation
S. mansoni egg glycolipids were puri®ed by organic solvent
extraction, saponi®cation, desalting and anion-exchange
chromatography as described previously [6]. Neutral gly-
colipids were f ractionated on a silica-gel cartridge (Waters,
Eschborn, G ermany) as outlined elsewhere [ 19] by s tep-wise
elution w ith chloroform/methanol (80 : 20, v/v), chloro-
form/methanol/water (65 : 25 : 4 ) and chlorof orm/metha -
nol/water (10 : 70 : 20), a nd analysed by HPTLC orcinol/
H
2
SO
4
-staining and HPTLC-immunostaining a s d escribed

previously [6]. The ®rst fraction contained ceramide
monohexoside and dihexoside an d was not further
analysed. Th e second fraction was positive in HPTLC-
immunostaining and was used for preparation of PA-
oligosaccharides. For HPTLC-immunostaining, the mAbs
M2D3H, G11P and C1C7 were provided by Q. Bickle,
Department of Infectious and Tropical Diseases, London
School of Hygiene and Tropical Medicine, England, while
the mAb 290-2E6 [20] was provided by A. M. Deelder,
Leiden University Medical C enter, the Netherlands.
Preparation and separation of PA-oligosaccharides
The g lycan m oieties w ere released from glycolipids using
recombinant ceramide glycanase (endoglycoceramidase I I
from Rhodococcus sp.; Takara S huzu Co., Otsu, Shiga,
Japan) and separated from uncleaved glycolipids and free
ceramides on a reverse-phase cartridge [6]. Released o ligo-
saccharides were labelled with 2-aminopyridine (PA) and
excess reagent was partitioned with chloroform [21].
PA-oligosaccharides were fractionated on an amino-phase
HPLC column (4.6 ´ 250 mm, N ucleosil-Carbohydrate;
Macherey and Nagel, Du
È
ren, Germany) at a ¯ow rate of
1mLámin
)1
at room temperature and detected by ¯uores-
cence (310/380 nm) [ 21]. The c olumn was equilibrated with
200 m
M
aqueous triethylamine/ac etic acid, pH 7.3 : aceto-

nitrile (25 : 75, v/v). A gradient of 25±60% aqueous
triethylamine/acetic acid buffer was applied within a
60-min period and the column was run isocratically for a
further 10 min. Peak fractions were collected and lyophil-
ized. H eterogeneous fractions were re so lved furthe r o n a
porous graphitic carbon (PGC)-column (4.6 ´ 100 mm,
Hypercarb; Hypersil, Runcorn, UK) at a ¯ow rate of
1mLámin
)1
at room temperature with ¯uorescence detec-
tion (310/380 nm). The column was equilibrated w ith
20 m
M
triethylamine/acetic acid, pH 5.0. A gradient from
0 to 30% acetonitrile was applied in 50 min. Individual peak
fractions were collected and lyoph ilized.
MALDI-TOF MS and ESI MS
MALDI-TOF MS was performed on a Vision 2000 (Ther-
moFinnigan, Egelsbach, Germany) equipped with a UV
nitrogen laser (337 nm) as d escribed previously [6]. The
instrument was operated i n the positive-io n re¯ectron mode
throughout using 6 -aza-2-thiothymine (Sigma) a s matrix.
ESI M S was performed with a n Esquire 3000 ion-trap mass
spectrometer (Bruker Daltoniks, Bremen, Germany)
equipped with an off-line nano-ESI source. A 2±5 lL aliquot
of PA-oligosaccharides in methanol/water (1 : 1, v/v) or
glycolipids i n c hloroform/methanol/water (10 : 20 : 3) w as
loaded into a laboratory-made, go ld-coated glass capillary
and electrosprayed at a voltage of 700±1000 V u sing N
2

as
dry-gas (120 °C, 4 L ámin
)1
). The skimmer voltage was set to
30 V, except for Fig. 7 A an d B , where 55 V were applied. For
each spectrum 20±100 repetitive scans were a veraged. The
accumulation time was between 5 and 50 ms. All MS/MS
experiments w ere p erformed with helium as c ollision g as.
Enzymatic and chemical degradation
PA-oligosaccharides were treated with either a-fucosidase
from bovine kidney (4 m UálL
)1
; Roche Diagnostics,
Mannheim, Germany) or with b-galactosidase from bovine
testes (4 mUálL
)1
; Roche Diagnostics) on the MALDI-
TOF MS t arget [22]. Enzymes were d ialysed for 4 h against
25 m
M
ammonium acetate solution adjusted to pH 5.0 for
a-fucosidase and pH 4.0 for b-galactosidase. A fter
measurement of the educts by MALDI-TOF M S using
6-aza-2-thiothymine matrix, dialysed enzymes (2 lL) were
added undiluted to sample aliquots on t he target and spots
were analysed a gain by M ALDI-TOF MS aft er incubation
overnight at 37 °C. For chemical defucosylation, dried
samples were treated with 48% HF at 4 °Covernight
(modi®ed from [23]). HF was removed by a stream of
nitrogen.

Monosaccharide composition and linkage analysis
After hydrolysis in 4
M
aqueous tri¯uoroacetic acid at
100 °C for 4 h and labelling with anthranilic acid, mono-
saccharides were determined by HPLC and ¯uorescence
detection [9,24]. For linkage analysis, PA-oligosaccharides
were permethylated w ith m ethyl iodide after deprotonation
with lithium methylsul®nyl carbanion [25] and hydrolysed
(4
M
aqueous tri¯uoroacetic acid, 100 °C, 4 h). Partially
methylated alditol acetates obtained after sodium boro-
hydride reduction and peracetylation were analysed by
capillary GC followed by ¯ ame i onization detection or
chemical ionization mass spectrometry (single ion monitor-
ing), using a moving needle injector, f used silica bonded
phase capillary columns of different po larity (60-m D B-1
482 M. Wuhrer et al. (Eur. J. Biochem. 269) Ó FEBS 2002
and 30-m DB-210; ICT, Bad Homburg, Germany) and
helium as carrier gas as detailed elsewhere [26].
RESULTS
Preparation of glycolipids
Glycolipids were i solated from S. mansoni eggs analogously
to the study performed o n S. mansoni cercarial glycolipids
[6] a nd analysed by HPTLC (Fig. 1). Orcinol/H
2
SO
4
-

staining (lan e 1) revealed some major, slow-migrating
compounds and a weaker s taining for s maller, minor
components. Both murine S. mansoni infection s erum (lane
2) and four monoclonal antibodies (lanes 3±6) visualized
antigenic glycolipids. Murine infection serum and the mAbs
M2D3H, G11P and C1C7 exhibited a similar pattern,
whereas the mAb 290±2E6, which is known t o recognize
mono- and difucosylated structures like GalNAc(b1±4)
[Fuc(a1±2)Fuc(a1±3)]GlcNAc(1- [20], displayed a c om-
pletely different pattern. The m urine infection serum and
mAb M2D3H w ere especially ef®cient in detecting the fast-
migrating g lycolipids ( lanes 2 and 3 ), which orcinol/H
2
SO
4
-
staining revealed to be present in only low amounts. We
expected these minor, fast-migrating, antigenic glycolipids
not to be covered by the structural characterization of whole
mixtures of complex glycolipids [16,17] and therefore
decided to fractionate these compounds as their corre-
sponding PA-oligosaccharides and to structurally charac-
terize the i ndividual species.
Preparation and separation of PA-oligosaccharides
Glycans were released from the ceramide moieties by
ceramide glycanase treatment of complex e gg glycolipids.
For the separation of uncleaved glycolipids and ceramides
from the released oligosaccharides, the sample was frac-
tionated on a reverse-phase cartridge. Released oligosac-
charides were collected as the combined ¯ow-through and

wash fractions, while the uncleaved glycolipids and cera-
mide moieties were obtained by elution with organic
solvents. Released glycans and uncleaved glycolipids were
quantitated by monosaccharide composition analysis
(Table 1), s howing an o verall ef®cacy o f over 9 5% glycan
release. Released oligosaccharides were ana lysed by MAL-
DI-TOF MS (Fig. 2 C) and t he ob tained pattern w as very
similar to the patterns observed f or th e intact glycolipids i n
ESI- and MALDI-TOF MS (Fig. 2 A,B). This indicated
that the released oligosaccharides were representative for the
glycans of t he major c omplex egg glycolipids. The released
oligosaccharides were pooled and l abelled with the ¯uores-
cent tag, 2-aminopyridine (PA). PA-oligosaccharides were
fractionated by amino-phase HPLC (Fig. 3 A). Collected
fractions (1 to 25; hereafter, fractions denoted by number
only) we re scre ened by MA LDI-T OF MS a nd as sessed f or
monosaccharide content by composition analysis (Tables 2
and 3). Fractions 1 to 7 were not found to contain
carbohydrate. Starting with 8, MALDI-TOF MS revealed
several compounds for m ost o f the fractions (Fig. 4). In
-CTH
-CTetH
12345
6
Fig. 1. HPTLC of S. mansoni egg glycolipids. S. man soni egg g lyco li-
pids were resolved with chloroform/methanol/0.25% aqueous KC l
(50 : 40 : 10, v/v/v) and v isualized by orcinol/H
2
SO
4

staining (lane 1)
or im munostaining using a pool of eight murine S. man soni infection
sera (lane 2) and the mAbs M2D3H (1 : 20 000; lane 3), G11P (1 : 200 ;
lane 4), C1C7 (1 : 200; lane 5 ) and 290±2E6 (1 : 50; l ane 6). CTH and
CtetH m ark the migration positions of globotriaosyl- and globotet-
raosylceramide standards, respectively.
Table 1. E cacy of ceramide glycanase cleavage of the complex egg glycolipids shown by monosaccharide composition analysis. Complex egg
glycolipids w ere cleaved with ceramide glyc anase a n d fraction ated on a reverse-phase cartridge. The aqueous fractions of two experiments were
combined (water fraction) as w ell as the organic solvent -eluted fractions ( organic solvent fraction) and c ompared by c omposition analysis to the
starting complex egg glycolipids. The amounts of monosaccharides are given in micrograms and their relative ratios are normalized to GalNAc 
2.0 i n p arentheses.
Monosaccharide
Egg glycolipid
fraction
Released monosaccharide (lg)
Water fraction Organic solvent fraction Released monosaccharide (%)
GalNAc (2.0) 235 (2.0) 4 (2.0) 98
GlcNAc (3.1) 375 (3.2) 18 (8.6) 95
Gal (0.3) 27 (0.2) 9 (4.2) 76
Glc (2.1) 329 (2.8) 35 (16.6) 90
Fuc (3.8) 522 (4.5) 10 (4.6) 98
S 1488 75 95
Ó FEBS 2002 Schistosoma mansoni egg glycolipids (Eur. J. Biochem. 269) 483
order to r educe p eak heterogeneity and o btain a s f ar as
possible pure compounds, the amino-phase fractions 8 to 14
were subfractionated by PGC-HPLC (Fig. 3B,C). Subfrac-
tions (designated, for e xample, 12-5 for s ubfraction 5 of
fraction 12) were a gain screened by MALDI-TOF MS (cf.
insets in Fig. 3B,C and Table 3).
Structural elucidation of individual PA-glycans

Individual PA-glycans were analysed by c omposition anal-
ysis, linkage analysis, ESI MS/MS, as well as chemical and
enzymatic degradation followed by a second linkage
analysis. ESI MS fragments were assigned according to
the nomenclature introduced by Domon & Costello [27].
Anomeric con®gurations were in some cases determined
enzymatically (Fig. 5 D,G), but generally assigned based on
the results of egg glycolipid CrO
3
oxidation, which indicated
b-anomeric linkages for GlcNAc and GalNAc and
a-anomeric linkage for fucose a s described r ecently [9].
The m ajor compound in 8 as judged from MALDI-TOF
MS (Fig. 4) was Hex
3
HexNAc
1
PA. After rechromatogra-
phy by PGC-HPLC, it was detected in 8-11 by MALDI-
Fig. 2. Mass spectrometry of unfractionated
complex glycolipids and derived oligosaccha-
rides from S. mansoni eggs. (A) ESI MS and
(B) MALDI-TOF MS of complex S. mansoni
egg glycosphingo lipids. (C) MALDI-TOF MS
of olig osaccharides re leased from S. mansoni
egg g lycosphingolipids.
484 M. Wuhrer et al. (Eur. J. Biochem. 269) Ó FEBS 2002
TOF MS. ESI MS
2
of this compound revealed the s equence

Hex-HexNAc-Hex-Hex-PA and thus indicated a non-
Schisto-core (Fig. 6A). Linkage analysis showed major
proportions of 3-substituted GalNAc, 4-substituted galac-
tose and terminal galactose (Table 2), and the anomeric
con®guration of the latter was determined by MALDI-TOF
MS/on-target enzym atic cleavage with b-galactosidase from
bovine testes (data not shown). Taken together, the
structure was found to be Gal(b1±3)GalNAc(1±4)Gal(1±4)
Glc-PA (Table 4), which probably corresponds to the host-
derived glycolipid asialogangliotetraosylceramide. As a
minor component in 8-11, the proton adduct of H ex
1
Hex-
NAc
2
PA was d etected by ESI MS at m/z 664.9, and its
sequence w as determined by ESI MS
2
to be HexNAc-
HexNAc-Hex-PA (data not shown). Linkage analysis of
fraction 8-11 indicated minor amounts of terminal G lcNAc.
Based on the assumption of a Schisto-core structure, this
leads to the structure G lcNAc(b1±3)GalNAc(b1±4)Glc-PA
(Table 4) for t his minor compound. Fraction 9 was not
analysed further, but its major compound Hex-
NAc
2
Hex
2
PA ([M + Na]

+
at 849.5; Fig. 4) might be
identical with the P A-tetrasaccharide Gal b3GlcNAcb3Gal-
NAcb4Glc-PA d erived from S. mansoni cercarial glycoli-
pids [6]. For 10-10, linkage analysis before and after f ucose
removal by HF-treatment allowed the loc alization of t he
fucose at the 3-position of GalNAc a nd the assignment o f
the structure (Tables 2 and 4). In the case of 11-7,terminal
fucose and galactose (Table 2) had a-andb-anomeric
con®guration, r espectively, as determined by on-target
enzymatic cleavage with bovine kidney a-fucosidase and
b-galactosidase from bovine testes (data not shown).
Linkage analyses before and after preparative removal of
fucose by HF-treatment (Table 2) and ESI MS/MS
(Fig. 6B,C) indica ted 11-7 to have the Lewis X-containing
structure Gal(b1±4)[Fuc(a1±3)]GlcNAc(b1±3)GalNAc
(b1±4)Glc-PA.
12-5 was found to contain fucose both in the 3-position of
GalNAc and in the 3-position of GlcNAc (cf. linkage
analysis, Table 2). ESI MS
2
(Fig. 7 B) showed the two
fucosylated H exNAc r esidues to be adjacent to each other
(B
3
ion at m/z 721.5). One of the fucoses of 12- 5 could be
removed e nzymatically, w hile the second was only r emoved
by HF-treatment (Fig. 5B,E,H). These accumulated data for
12-5 re sulted in the structure Fuc(a1±3)GalNAc(b1±4)
[Fuc(a1±3)]GlcNAc(b1±3)GalNAc(b1±4)Glc-PA (Table 4).

The species 13-4 exhibited both terminal GalNAc and
terminal fucose (Table 2). Loss of HexNAc in ESI MS
2
(Y
4a
at 1036.1; Fig. 8B) c orroborated the presence of a terminal
HexNAc. Loss o f a further HexNAc r esidue appeared only
together with the loss of fucose (Y
3
at m/z 687.0 but no signal
at m/z 833), which indicated fucose to be linked to the
subterminal HexNAc residue. Bovine kidney a-fucosidase
Fig. 3. HPLC separation of egg g lycolipid-
derived PA-oligosaccharides. (A) Separation
on an am ino-phase column. Elution p osit ions
of the PA-labelled dextran hydrolysate stan-
dards of dierent chain-length are indicated by
arrows. F rac tions devoid of carbohydrate-
positive material are m arked by asterisks (*).
Rechomatography of fractions 11 (B) a nd 12
(C)anaPGC-columnandanalysisofthe
major peaks by MALDI-TOF MS (insets). n,
minor compound i dentical in mass with
10-10.
Ó FEBS 2002 Schistosoma mansoni egg glycolipids (Eur. J. Biochem. 269) 485
did not act on 13-4, but the fucose could be removed by HF-
treatment (Fig. 5) . Taken together, the data showed 13-4 to
represent the structure shown i n T able 4.
Subfractionation of 14 led to the r esolution of difucosy-
lated 14-2 and 14-3, trifucosylated 14 -4 and tetrafucosylated

14-5 species. ESI M S
2
analysis of 14-5 (Fig. 9B) showed the
four fucose residues to b e linked to the two outermost
GalNAc and/orGlcNAc residues (B
4
ion at m/z 1013.2).
Three of the fucose residues are attached to one of these
HexNAc residues (Y
4b
B
4
at m/z 664.9) to form a Fuc(a1±2)
Fuc(a1±2)Fuc(a1±3)GlcNAc unit (see linkage analysis,
Table 2), and this ion could lose one or two fucose r esidues
on further fragmentation (Fig. 9C). The HexNAc, w hich
carried this oligofucosyl chain is not outermost, a s indicated
by the Y
6a
Y
4b
ion at m/z 1182.8 (Fig. 9B). An ion at m/z
1328, which w ould have indicated the loss of one fucose and
one HexNAc (Y
4b
) was not registered in the 14-5 MS
2
shown in Fig. 9 B, but was detected in similar MS
2
experiments as a minor ion (not shown), thus corroborating

the location of the trifucosyl chain o n the second and not on
the out ermost HexNAc. T aken together with the linkage
analysis data (Table 2 ), the structure of 14-5 could
be elucidated as Fuc(a1±3)GalNAc(b1±4)[Fuc(a1±2) Fuc
(a1±2)Fuc(a1±3)]GlcNAc(b1±3)GlcNAc(b1±3) GalNAc
(b1±4) Glc-PA (Table 4). The simultaneous presence of
isobaric structural isomers in this fraction could not be
excluded. Similarly, ESI MS
2
of the 14-4 [M + Na + H]
2+
ion at m/z 766 (data not shown) yielded a [Hex-
NAc
2
dHex
3
+Na]
+
ion at 867.0 Da, indicat ing the three
fucoses to be linked t o adjacent H exNAc residues. For 14-4,
the loss of one fucose and one HexNAc resulted in an
intense f ragment ion at m/z 1182.0, which showed that the
Table 2. A nalysis of PA-oligosaccharides by MALDI-TOF MS, ESI MS, composition analysis (CA) and linkage analysis (LA). Masses were
determined by MALDI-TOF MS and ESI MS (*) and were rounded to the ®rst decim al place. The type of pseudomolecular ion is given in bracke ts
and t he calculated, mo noisotopic masses in parentheses. 10-10H F, f ractio n 10-10 after HF-treatment, etc. t-Fuc, terminal fucose; 4-Gal,
4-substituted g alact ose, etc.
Fraction
Measured mass (Theoretical mass)
[Pseudomolecular ion]
Deduced composition Key structural data

8-11 808.0 (808.3) [M + Na]
+
; 404.5*
(404.7) [M + H + Na]
2+
Hex
3
HexNAc
1
PA
Hex
1
HexNAc
2
PA
LA: t-Gal, 4-Gal, 3-GalNAc, t-GlcNAc
664.9 (665.3) [M + H]
+
10-10 1036.1 (1036.4) [M + Na]
+
Hex
1
HexNAc
3
dHex
1
PA CA: GlcN:GalN (1.1 : 2.0)
LA: t-Fuc, 4-GlcNAc, 3-GalNAc
10-10HF 890.9 (890.4) [M + Na]
+

Hex
1
HexNAc
3
PA LA: t-GalNAc, 4-GlcNAc, 3-GalNAc
11-7 995.6 (995.4) [M + Na]
+
Hex
2
HexNAc
2
dHex
1
PA CA: GlcN:GalN:Gal:Fuc
(1.1 : 1.2 : 0.9 : 1.0)
LA: t-Fuc, t-Gal; 3-GalNAc; 3,4-GlcNAc
11-7HF 827.2* (827.4) [M + H]
+
Hex
2
HexNAc
2
PA LA: t-Gal; 4-GlcNAc; 3-GalNAc
12-5 1182.5 (1182.5) [M + Na]
+
;
591.6* (591.7) [M + H + Na]
2+
Hex
1

HexNAc
3
dHex
2
PA LA: t-Fuc; 3-GalNAc; 3,4-GlcNAc
12-5HF 445.6* (445.7) [M + H + Na]
2+
Hex
1
HexNAc
3
PA LA: t-GalNAc; 4-GlcNAc; 3-GalNAc
13-4 1240.6 (1239.6) [M + Na]
+
;
620.5* (620.3) [M + H + Na]
2+
Hex
1
HexNAc
4
dHex
1
PA LA: t-Fuc; t-GalNAc; 3-GlcNAc;
3-GalNAc; 3,4-GlcNAc
13-4HF 547.3* (547.2) [M + H + Na]
2+
Hex
1
HexNAc

4
PA LA: t-GalNAc; 4-GlcNAc, 3-GlcNAc;
3-GalNAc
14-2 1384.4 (1385.5) [M + Na]
+
Hex
1
HexNAc
4
dHex
2
PA LA: t-Fuc; 4-GlcNAc; 3-GalNAc;
3,4-GlcNAc
14-3 1384.4 (1385.5) [M + Na]
+
Hex
1
HexNAc
4
dHex
2
PA CA: GlcN:GalN:Fuc (2.1 : 2.0 : 1.9)
LA: t-Fuc; 3-GlcNAc; 3-GalNAc;
3,4-GlcNAc
14-3HF 547.0* (547.2) [M + H + Na]
2+
Hex
1
HexNAc
4

PA LA: t-GalNAc; 4-GlcNAc; 3-GlcNAc;
3-GalNAc
14-4 1530.8 (1531.6) [M + Na]
+
;
766.5* (766.3) [M + H + Na]
2+
Hex
1
HexNAc
4
dHex
3
PA CA: GlcN:GalN:Fuc (2.1 : 2.0 : 2.3)
LA: t-Fuc; 2-Fuc; 3-GlcNAc;
3-GalNAc; 3,4-GlcNAc
14-5 839.1* (839.3) [M + H + Na]
2+
Hex
1
HexNAc
4
dHex
4
PA CA: GlcN:GalN:Fuc (2.2 : 2.0 : 3.2)
LA: t-Fuc; 2-Fuc; 3-GlcNAc;
3-GalNAc; 3,4-GlcNAc
14-5HF 1094.5 (1093.5) [M + Na]
+
Hex

1
HexNAc
4
PA LA: t-GalNAc; 4-GlcNAc; 3-GlcNAc;
3-GalNAc
15 1385.8 (1385.5) [M + Na]
+
Hex
1
HexNAc
4
dHex
2
PA CA: GlcN:GalN:Fuc (2.1 : 1.8 : 2.0)
LA: t-Fuc; 4-GlcNAc; 3-GlcNAc;
3-GalNAc; 3,4-GlcNAc
15HF 1094.6 (1093.5) [M + Na]
+
Hex
1
HexNAc
4
PA LA: t-GalNAc; 4-GlcNAc; 3-GlcNAc;
1071.1* (1071.4) [M + H]
+
3-GalNAc
16-4 1587.4 (1588.6) [M + Na]
+
Hex
1

HexNAc
5
dHex
2
PA CA: GlcN:GalN : Fuc (2.9:2 : 2.8)
16-5 1876.7 (1880.7) [M + Na]
+
Hex
1
HexNAc
5
dHex
4
PA CA: GlcN:GalN : Fuc (3.2:2 : 4.3)
486 M. Wuhrer et al. (Eur. J. Biochem. 269) Ó FEBS 2002
outermost HexNAc carried only one fucose residue.
Together with the linkage analysis data, this allowed the
deduction of t he structure shown i n Table 4. For 14- 2 and
14-3, MALDI-TOF MS indicated similar compositions as
for 15. L inkage analysis, however, revealed a difference in
the s ubstitution positions at the monosubstituted G lcNAc.
While 14-2 contained only 4-substituted and 14-3 only
3-substituted GlcNAc, 15 exhibited approximately equal
amounts of 3- and 4-substituted GlcNAc (Table 2). Linkage
analysis of 15 before and after HF showed fucose to be
linked t o t he 3-position of G alNAc and to the 3 -position o f
3,4-disubstituted GlcNAc. ESI MS
2
of 15 (Fig. 7D) showed
aB

3
ion at m/z 721.0, which was the same branched terminal
group as 12-5 (Table 4). As for 12-5, also in the case of 15, a-
fucosidase from bovine kidney could remove only one
fucose from this difucosylated terminal group (Fig. 5 G).
Taken together the structure of 15 was shown to be Fuc-
(a1±3)GalNAc(b1±4)[Fuc(a1±3)]GlcNAc(b1±3/4)Glc NAc
(b1±3)GalNAc(b1±4)Glc-PA, containing either a 3-substi-
tuted or 4 -substituted GlcNAc unit ( Table 4 ).
Characterization of large PA-glycans
Due to the pronounced heterogeneity and increasing
complexity of fractions 16 to 25 , we could only p artially
characterize thes e compounds by MALDI-TOF M S
(Fig. 4), carbohydrate composition and linkage analysis
(Table 3 ). MALDI-TOF MS revealed compositions with an
increasing number of HexNAc residues. The highest peak in
this region, 21 (Fig. 3), contained as its major compound
Hex
1
HexNAc
6
dHex
7
PA, which is consistent with the results
of MALDI-TOF MS and ESI MS analyses of total egg
glycosphingolipids and released glycans (Fig. 2). While 19
to 22 contained almost exclusively PA-oligosaccharides with
six HexNAc residues, species with ®ve HexNAc were most
intense in 16 to 18. L ikewise, 23 to 25 were dominated by
PA-oligosaccharides with seven HexNAc residues, which

have not been described in p revious studies [16,17]. Linkage
analyses of the intact PA-oligosaccharides revealed
3-substituted GalNAc as the o nly GalNAc species through-
out and fucose as the only terminal sugar (Table 3). Linkage
analysis of 24 after f ucose r emoval by HF-treatment
resulted in the loss of all fucose species and the appearance
of 3-substituted and terminal GalN Ac in similar amounts,
while all GlcNAc residues were converted to 4 -substituted
GlcNAc (data not shown). This shows GalNAc to b e the
outermost HexNAc in 24. The composition analyses of 18
to 25 imply that all complex PA-oligosaccharides contain an
average of 2 G alNAc residues. Of the ® ve HexNAc residues
in 18, a pproximately two are G alNAc and three a re
GlcNAc. Of the six HexNAc residues in 19 to 22,
approximately two are GalNAc and four are GlcNAc,
and for the species in 24, the avera ge HexNA c composition
is two G alNAc and ®ve GlcNAc r esidues. This supports the
hypothesis, that there is one GalNAc residue both at t he
reducing a nd at the nonreducing end of th e H exNAc chain,
the ® rst b eing involved in the Schisto-core structure, w hile
the core o f the HexNAc chain would appear to consist of
GlcNAc residues throughout. T his i s consistent with the
structures proposed by Khoo et al. [16]. Concerning
fucosylation, there exists a vast heterogeneity, as exempli®ed
by the detection of Hex
1
HexNAc
6
dHex
2)8

in 21 . Several
fractions showed a 2-substituted fucose/terminal fucose
ratio of approximately 1 : 1 (Table 3), which could be
explained by the occur rence of difucosylated chains
throughout. H owever, based on the observation of a
trifucosylated HexNAc in 14-5 and odd fucose numbers
per molecule, e.g. in 21, it can be assumed that trifucosylated
HexNAc residues m ay also occur in some of t hese complex
PA-oligosaccharides. Taken together, the partial character-
ization of these larger PA-oligosaccharides after amino-
phase fractionation gave a detailed overview of the HexNAc
chain-lengths and fucosylation heterogeneity, which was not
obtained by FAB-MS analyses of the entire mixture of
glycolipids [16,17].
DISCUSSION
In this study, individual S. man soni egg glycolipid
structures have been elucidated. Seven of the determined
Table 3. C omposition and linkage analyses of the large PA-oligosaccharides derived from complex glycolipids of S. mansoni eggs. As for composition
analysis, m olar ratios base d on G alNAc  2.0 w ere determined a fter hydrolysis a nd reverse-phase c hromatography of the anthranilic acid-
derivatized components. The c omponen ts were qu anti®e d by application o f a standard mixture fo r determination of t he individual detection
response f actors. PA-Glc conjugates were not registered. For linkage analysis, the partially methylated monosacharide derivatives obtained after
hydrolysis, reduction and peracetylation were a nalysed by G C/MS and GC using chemical ionization in conjunction with single-ion monitoring and
¯ame-ionization detection, respectively. Results are expressed as peak ratios o f the aldito l acetates after ¯ame-ionization detection on the basisof
3-GalNAc  2.0 for HexNAc species and t-Fuc  1 for fucose species. For fraction 25, peak ratios were determined b y GC/MS d ue to the limited
amount of material. 4 -GlcN Ac, 4-sub s tituted G lcNAc, etc.
Fraction
Composition analysis Linkage analysis
GalNAc GlcNAc Fuc 2-Fuc: t-Fuc
4-GlcNAc: 3,4-Glc-
NAc: 3-GalNAc

18 2.0 2.84 3.16 0.9 : 1.0 1.4 : 3.3 : 2.0
19 2.0 3.23 2.48 0.95 : 1.0 1.55 : 3.55 : 2.0
20 2.0 3.64 2.85 0.8 : 1.0 1.55 : 4.0 : 2.0
21 2.0 3.62 4.65 1.05 : 1.0 1.2 : 5.15 : 2.0
22 2.0 3.67 4.00 0.8 : 1.0 1.25 : 4.6 : 2.0
23 2.0 4.04 3.33 0.95 : 1.0 1.45 : 3.25 : 2.0
24 2.0 4.51 4.09 0.5 : 1.0 2.0 : 4.15 : 2.0
25 2.0 3.88 4.63 0.65 : 1.0 0.7 : 6.45 : 2.0
Ó FEBS 2002 Schistosoma mansoni egg glycolipids (Eur. J. Biochem. 269) 487
Fig. 4. MALDI-TOF M S a nalysis of the HPLC-fractionated PA-oligosaccharides. Fractions 8 to 25 (8 to 25 ) of the a mino-phase separated
PA-oligosaccharides (Fig. 3) w ere analysed b y MALDI-TOF MS as their sodium ( 8 to 19 ) or lithium adducts ( 20 to 25). Fucose increments are
indicated by a rrows. #, proton adduct; s, p otassium adduct; *, contaminant.
488 M. Wuhrer et al. (Eur. J. Biochem. 269) Ó FEBS 2002
structures consist of fucosylated HexNAc chains based on
the Schisto-core s tructure. They a re in agreement with the
structure proposed by Khoo et al.[16],whichisFuc(1±2)
Fuc(1±3)GalNAc(1±4)([Fuc(1±2)Fuc(1±)3]GlcNAc (1±4))
1)3
[  Fuc(1±2)  Fuc(1±3)]GlcNAc(1±3)GalNAc (1±4) Glc-
(1±1)Cer. The reported role of 3-fucosylated GalNAc a s the
major structural m otif at the nonreducing end of the
HexNAc chain [16] is supported by our analyses of
individual PA-oligosaccharides (10-10, 12-5 , 15, 14-4 and
14-5) and by the linkage analyses of larger PA-oligosac-
charides, i n p articular 24, b efore and after HF-treatment.
Recently, the terminal motif GalNAc(b1±4)GlcNAc- has
been identi®ed as a good acceptor for fucosyl ation by
S. mansoni egg extracts, leading t o a heterogeneity of
fucosylated products, which parallels our ®nding of various
oligofucosylated terminal structures [28].

Our data, however, do not support the proposed Fuc(a1±
4)[Fuc(a1±3)]GlcNAc-terminal motif, or the interdigitation
of the HexNAc chain by fucose residues [17], because
terminal GlcNAc was never generated by defucosylation in
any of the PA-oligosacchride fractions studied, and MS
analyses of HF-treated individual PA-oligosaccharides only
revealed the l oss of fucose a nd not of HexNAc residues.
Our data require an extension of the existing picture.
While the structure outlined by Khoo et al.[16]comprises
HexNAc
4
-HexNAc
6
chains, the structures 10-10 and 12-5
show that fucosylated glycolipids with HexNAc
3
-chains
also ex ist. Together with the Hex
1
HexNAc
2
PA com pound
found in 8-11, these carbohydrate chains obviously repre-
sent the Ômissing linkÕ between the Schisto-core and the
extended structures described [16]. Likewise, 23 to 25 are
dominated by HexNAc
7
species, w hich also have not be en
detected in the FAB-MS analyses [16,17]. Secondly, we have
found trifucosyl sidechains on 14-5, which have so far only

been described f or O-glycans derived from the cercarial
glycocalyx [29] and not for egg glycolipids [16]. The high
relative amount of 2-Fuc in the larger PA-oligosaccharides
further indicates that trifucosyl sidechains are a lso likely to
occur i n t he glycolipids with HexNAc
5
-HexNAc
7
chains.
Thirdly, 13-4 shows that incomplete f ucosylation may also
occur, leading in low amounts t o terminal GalNAc residues
involved in GalNAc(b1±4)[Fuc(a1±3)]GlcNAc(b1- units, a s
have been described for N-glycans of adult worm glyco-
proteins [30]. Fourthly, the characterization of t he HexNAc
backbone revealed in some cases a deviation from the
GalNAc(1±4)(GlcNAc(1±4))
1)3
GlcNAc(1±3)GalNAc(1±4)
Glc(1±1)ceramide basic structure described by Khoo et al .
[16], as we found this motif only in one of the elucidated
structures (14-2, w hich is identical to one compou nd in 15),
while four structures (13-4, 14-3, which is identical to a
second compound in 15, 14-4 and 14-5) s howed the
motif±GalNAc(1±4)GlcNAc(1±3)GlcNAc(1±3)GalNAc(1±
4) Glc(1±.The presence o f internal 3 -substituted GlcNAc
residues is in agreement with previous studies on the
carbohydrate structure of a cercarial Lewis-X-containing
ceramide hexahexoside [ 6].
Fig. 5. Fu cose removal from individual PA-oligosaccharides by a-fucosidase or H F-treatment. MALDI-TO F MS o f 13-4 b efore (A) a nd after
(B) HF-treatment, intact 12-5 (C), 12-5 after a-fucosidase treatment (D), 12-5 after HF-treatment (E), intact 15 (F), 15 after a-fucosidase treatment

(G), an d 15 after H F-treatm ent (H).
Ó FEBS 2002 Schistosoma mansoni egg glycolipids (Eur. J. Biochem. 269) 489
Apart from the dominant glycolipids with fucosylated
HexNAc chains, we c haracterized in this study the glycan of
a Lewis X-containing glycolipid (11-7), which we previously
have shown to b e t he dominating glycolipid of S. mansoni
cercariae [6]. Though this result s hows that Lewis X
glycolipids are not absolutely restricted to the cercarial
and schistosomular life-cycle stage, they are drastically
downreguated in the egg and thus have not been detected by
HPTLC-overlay [9].
The ®nding of a PA-oligosaccharide corresponding to
asialogangliotetraosylceramide (8-11) structurally iden ti®es
a glycolipid which the parasite most probably has taken up
from the host. MALDI-TOF MS of the corresponding
intact glycolipid after silica-gel puri®cation showed th at its
ceramides contain more t han 40 c arbon atoms (data not
shown), w hich indicated the presence of long-chain fatty
acids and thus differed from the typical ceramides of
complex egg glycolipids, which are dominated by C20-
Fig. 6. ESI MS/MS of 8-11 and 11-7. (A) ESI
MS
2
of the 8-11 [M + Na + H]
2+
precursor
ion a t m/z 404.5. ( B) ESI MS
2
of th e 11-7
[M + H]

+
precursor ion at m/z 973.5 and (C)
subsequent ESI MS
3
fragmentation of t he ion
at m/z 511.9, which corresponds to the Lewis
X t risaccharide unit. ,, sodium a dduc t; #,
proton adduct.
Table 4. P roposed s tructures of S. mansoni egg glycolipid-derived PA-oligosaccharides.
PA-Oligosaccharide structure Fraction
Gal(b1±3)GalNAc(1±4)Gal(1±4)Glc-PA 8-11
GlcNAc(b1±3)GalNAc(b1±4)Glc-PA 8-11
Gal(b1±4)[Fuc(a1±3)]GlcNAc(b1±3)GalNAc(b1±4)Glc-PA 11-7
Fuc(a1±3)GalNAc(b1±4)GlcNAc(b1±3)GalNAc(b1±4)Glc-PA 10-10
Fuc(a1±3)GalNAc(b1±4)[Fuc(a1±3)]GlcNAc(b1±3)GalNAc(b1±4)Glc-PA 12-5
GalNAc(b1±4)[Fuc(a1±3)]GlcNAc(b1±3)GlcNAc(b1±3)GalNAc(b1±4)Glc-PA 13-4
Fuc(a1±3)GalNAc(b1±4)[Fuc(a1±3)]GlcNAc(b1±3)GlcNAc(b1±3)GalNAc(b1±4)Glc-PA 15 + 14-3
Fuc(a1±3)GalNAc(b1±4)[Fuc(a1±3)]GlcNAc(b1±4)GlcNAc(b1±3)GalNAc(b1±4)Glc-PA 15 + 14-2
Fuc(a1±3)GalNAc(b1±4)[Fuc(a1±2)Fuc(a1±3)]GlcNAc(b1±3)GlcNAc(b1±3)GalNAc(b1±4)Glc-PA 14-4
Fuc(a1±3)GalNAc(b1±4)[Fuc(a1±2)Fuc(a1±2)Fuc(a1±3)]GlcNAc(b1±3)GlcNAc(b1±3)GalNAc(b1±4)Glc-PA 14-5
490 M. Wuhrer et al. (Eur. J. Biochem. 269) Ó FEBS 2002
phytosphingosine and C16:0 fatty acid (Fig. 2 and
[3,10,17]). It is not known whether this host glycolipid is
taken up directly by the egg or if it is acquired by the adult
female and then t ransferred to t he growing e gg. Our ®nding
parallels the described acquisition of glycolipid blood-group
antigens by t he adult s chistosome [31,32] and ®ts in with th e
general pattern of aquisition and i mitation of host antigens
by the parasite l eading to immunosuppression and/or
autoimmunity [33].

Concerning the e lucidation of anomeric con®gurations in
schistosome g lycoconjugates, t heir unique structures largely
hamper the use of exoglycosidases. Alternatives are the
usage of NMR, which requires quite large amounts of
material, o r CrO
3
oxidation, which we have used to
characterize anomeric linkages in egg glycosphingolipids
[9]. While a-fucosidase t reatment of the highly fucosylated
structures could only remove some f ucoses from the fucose
chains (Fig. 5), the Lewis-X-containing PA-oligosaccha-
rides derived from cercarial glycolipids could be c ompletely
sequenced by exoglycosidases [ 6].
The a mino-phase HPLC fractionation of PA-glycans did
not show a clear relationship between retention time and
monosaccharide composition similar to the additivity rule
described for N-glycans [21]. In 19 to 22, for example, which
are dominated by Hex
1
HexNAc
6
dHex
3)7
PA species, the
degree of fucosylation does not co rrelate with the retention
time (Fig. 4), as, e.g. the heptafucosylated species in 21 elutes
earlier than the tetrafucosylated species in 22. Likewise, the
elution sequence of Hex
1
HexNAc

7
dHex
4)8
PA species in
23 to 25 shows that the fucoses in t hese unique highly
fucosylated glycans vary considerably in their effect on
the r etention time in amino-phase sep aration, and that the
attachment of some of the f ucoses might reduce the
retention time.
Fig. 7. ESI MS/MS of 12-5 and 15. (A) E SI
MS of 12-5 an d (B)subsequentESIMS
2
fragmentation of t he sodium addu ct at m/z
1182.0. Of all the pseudom olecular i ons
detected i n (A), [ M + N a]
+
gave the clearest
fragmentation p att ern (B). In (A), a skimmer
voltage of 55 V was use d instead of the rou-
tinely applied 30 V, which led to the detection
of the [M + Na]
+
signal and resulted in
fragment ions already in the basic MS spec-
trum. ( C) ESI MS of 15 and ( D) subsequent
ESI MS
2
fragmentation of the double-charged
species a t m/z 693.0. ,, sodium adduct; # ,
proton adduct.

Ó FEBS 2002 Schistosoma mansoni egg glycolipids (Eur. J. Biochem. 269) 491
Fig. 8. ESI MS/MS of 13-4. (A) ESI MS of
13-4 and (B) subsequent ESI M S
2
fragmen-
tation o f the double- charged species at m/z
631.0.
Ñ
, sodium adduct; #, pro ton adduct.
Fig. 9. ESI MS/MS of 14-5. (A) ESI MS of
14-5 and (B) subsequent ESI M S
2
fragmen-
tation o f the double- charged species at m/z
839.8 and (C) ESI MS
3
fragmentation of th e
species a t m/z 664.9. ,, sodium adduct; # ,
proton adduct.
492 M. Wuhrer et al. (Eur. J. Biochem. 269) Ó FEBS 2002
ACKNOWLEDGEMENTS
We acknowledge the expert technical assistance of Peter Kaese, Werner
Mink and Siegfried Ku
È
hnhardt. The authors thank Dr Quentin Bickle,
Department of Infe ctious and T ropical Diseases, London School of
Hygiene a nd Tropical Medicine, England, and D r Andre
Â
M. Deelder,
Leiden University Medical Center, the Netherlan ds, f or su pply o f

mAbs. This study was supported by t he German Research Co uncil
(SFB 53 5, Teilprojekt Z 1).
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