Glycobiology vol. 16 no. 10 pp. 991–1006, 2006
doi:10.1093/glycob/cwl020
Advance Access publication on July 6, 2006
Gender-specific expression of complex-type N-glycans in schistosomes
Manfred Wuhrer1,2, Carolien A. M. Koeleman2,
Jennifer M. Fitzpatrick3, Karl F. Hoffmann3,
André M. Deelder2, and Cornelis H. Hokke2
2
Department of Parasitology, Leiden University Medical Center, PO Box
9600, 2300 RC Leiden, the Netherlands; and 3Department of Pathology,
University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK
Received on April 22, 2006; revised on June 28, 2006; accepted on July 4,
2006
Sex-specific gene expression by Schistosoma mansoni worms
has been demonstrated at the transcriptome as well as the
proteome levels. In view of the important role of glycans in
the biology of schistosomes and the interaction with their
human host, we have investigated the sex-specific protein glycosylation. Mass spectrometric profiling and structural characterization of PNGase F-released N-glycans revealed the
following gender-specific glycosylation patterns: Complextype N-glycans of females mainly carried Gal(b1-4)GlcNAc
(LacNAc) and Gal(b1-4)[Fuc(a1-3)]GlcNAc (Lewis x)
antennae structures, whereas GalNAc(b1-4)GlcNAc- (LacdiNAc; LDN) and GalNAc(b1-4)[Fuc(a1-3)]GlcNAc (LDN-F)
were prevalent in N-glycans from males. LDN(-F) motifs
were found to occur as repeats on the antennae of large Nglycans that contained up to seven LDN(-F) units. The
female complex-type glycans were mostly di-antennary and
tri-antennary, whereas male structures were predominantly of
the mono-antennary and di-antennary type. Oligomannosidic
N-glycans were expressed at similar levels in females and
males. The localization of the sex-biased glycan motifs was
studied by immunofluorescence microscopy using defined
anti-glycan monoclonal antibodies (mAbs). The Lewis x element was strongly expressed in the gut of both males and
females, but with respect to tegument localization, the
females expressed this structure, while Lewis x seemed to be
almost completely absent from the male tegument. The
expression of LDN-F was predominantly detected in the
parenchyma of both male and female worms as well as in the
tegument of the male ventral cavity facing the female. LDN
was detected in the tegument of male and female worms at
similar levels. The sex-specific expression and differential
localization of these antigenic glycan motifs in schistosomes
may play a role in male–female interactions during conjugal
biology and may lead to a differential immune reaction of the
host to the two sexes.
Key words: differential glycomics/mass spectrometry/
LacdiNAc/Lewis x/Schistosoma mansoni
1
To whom correspondence should be addressed;
e-mail: [email protected]
Introduction
Schistosomes are parasitic flatworms responsible for
200 million human infections worldwide. They exhibit a
complex life cycle with an intermediate snail host and
humans as the definitive host. In humans, the mature dioecious schistosomes dwell in the vascular system and form
permanent pairs of male and female worms. The females
produce hundreds of eggs per day, which may get trapped
in the liver as well as other organs, resulting in granuloma
formation, fibrosis, and damage to organ architecture.
Sexual maturation is a critical step in the life cycle of
schistosomes as the morphologically distinct male and
female worms depend on each other for successful survival.
To achieve a better understanding of the molecular details
of the sexual biology of schistosomes, several recent global
studies have been carried out to identify sex-associated
genes and gene products, based on the now elaborate Schistosoma sequence databases and tools such as DNA
microarrays and proteomics. Gene-profiling studies have
shown that, in line with their physiological and biological
characteristics, males specifically express several transcripts
and proteins associated with mechanical support, the
cytoskeleton and the tegument, whereas females specifically
express genes which are involved in egg shell formation and
metabolic processes (Hoffmann et al., 2002; Fitzpatrick
et al., 2004; Hoffmann and Fitzpatrick, 2004; Fitzpatrick
et al., 2005). At the proteomics level, gender-specific differences in Schistosoma japonicum relate to signal transduction, metabolism, immunity, and development (Cheng
et al., 2005).
In addition to genomic and proteomic profiling, the
mapping of protein glycosylation is an important step
toward a molecular understanding of schistosome biology.
Schistosome glycoconjugates are often located at the parasite surface or secreted into the circulation, and glycans of
each of the developmental stages play an important, if not
significant, role in host–parasite interactions (Cummings
and Nyame, 1999; Hokke and Deelder, 2001; Khoo, 2001;
Nyame et al., 2004). Glycans of schistosomes, either larvae,
adult worms, or eggs, are major targets of the humoral
immune response during schistosomiasis (Omer-Ali et al.,
1986; Eberl et al., 2001), and some have recently been
described as ligands for lectins of the human immune system (van den Berg et al., 2004; van Liempt et al., 2004;
Meyer et al., 2005; van Vliet et al., 2005). In addition, schistosome glycans, in particular of the eggs, are involved in the
induction of Th2-type immune responses and immunomodulation, characteristic of helminth infections (Okano et al.,
2001; Faveeuw et al., 2003; Pearce et al., 2004).
Glycans of schistosomes are typically complex fucosylated structures, as has been shown by nuclear magnetic
© The Author 2006. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]
991
M. Wuhrer et al.
resonance spectroscopy and mass spectrometry studies
(reviewed in Cummings and Nyame, 1999; Hokke and
Deelder, 2001; Khoo, 2001; Nyame et al., 2004). As indicated by studies using anti-carbohydrate antibodies, schistosome glycans exhibit marked changes in their expression
level during the life cycle (van Remoortere et al., 2000;
Robijn et al., 2005). For example, the structural motifs
Fuc(α1-3)GalNAc(β1-4)GlcNAc- (F-LDN) and Fuc(α13)GalNAc(β1-4)[Fuc(α1-3)]GlcNAc- (F-LDN-F) were
found at high levels in cercarial and egg (glyco)protein
extracts, but could hardly be detected on adult worm
(glyco)proteins (Robijn et al., 2005). Other determinants,
including Gal(β1-4)[Fuc(α1-3)]GlcNAc- (Lewis x), GalNAc(β1-4)[Fuc(α1-3)]GlcNAc- (LDN-F), and GalNAc(β14)[Fuc(α1-2)Fuc(α1-3)]GlcNAc- (LDN-DF), are particularly highly expressed on egg glycoproteins but also present
in (glyco)protein extracts from cercariae and adult worms,
albeit less abundantly.
The possible gender-specific expression of these and
other schistosome glycans, however, has not yet been investigated. Triggered by the observations of clear genderassociated gene expression in Schistosoma mansoni by
DNA micro arrays, we performed a differential mass spectrometric profiling of protein glycosylation in male and
female S. mansoni worms, aiming to determine if schistosomes produce glycans or glycan elements in a sex-specific
manner. We found marked differences with LDN (GalNAc
(β1-4)GlcNAc-) and LDN-F as major motifs of male complex-type N-glycans, while Lewis x-containing N-glycans
seemed to occur predominantly in females.
Results
Glycan release and profiling
To screen for sex-specific features of protein glycosylation,
glycans were released from glycoproteins of female and
male S. mansoni worms by consecutive treatment with
PNGase F, PNGase A, and alkaline borohydride. The
MALDI-TOF-MS profiles of the PNGase F-released Nglycans from female worms and male worms showed major
differences (Figure 1), indicating that gender-related glycans or glycan elements occur. These results were the same
for S. mansoni worms obtained from hamsters and mice,
and the following experiments were performed on glycans
from hamster-derived worms. Pools of glycans from
females and males obtained after PNGase A treatment and
reductive β-elimination were virtually identical to those of
the preceding PNGase F release, as judged from the
obtained MALDI-TOF-MS profiles (not shown). This suggested that the PNGase F digestion was incomplete and
also that the non-released glycan pool is identical to the initial PNGase F-released pool. As we did not detect O-glycans
or PNGase F resistant, core-1–3-fucosylated N-glycans in
either of the glycoprotein preparations, we considered the
PNGase F pool to be characteristic for the overall
protein glycosylation in adult schistosomes. Therefore, we
focused in this study on the glycans released by PNGase F
treatment.
N-glycans from male and female worms were profiled by
MALDI-TOF-MS (Figure 1). Compositions were deduced
992
in terms of hexose (H), N-acetylhexosamine (N), and deoxyhexose (F), as listed in Table I. Groups of N-glycan species
differing in fucose content were labeled with a common
letter giving the number of fucoses in subscript. In the case
of oligomannosidic N-glycans with (b2–4) and without (a2–10)
core fucosylation, the subscript indicates the number of H
units. To facilitate comparison of relative signal intensities,
the intensity for the hexamannosidic glycan (a6) was set to
1.00 in each of the MALDI-TOF mass spectra. The comparison of the N-glycan profiles from male and female worms
showed major differences: significant female-associated Nglycan species were H5N5F1 (peak x1 in Figure 1), H6N5F1,
and H6N5F2 (peaks α1, α2, respectively, in Figure 1), each
difficult to detect in male schistosomes. In contrast, the Nglycans of composition H3N4F1 (d1) and H3N6F1 (f1),
which were major species in the male sample (Figure 1B),
were less prominent in female schistosomes (Figure 1A).
More differences between the N-glycan patterns of the two
sexes were observed in the high-mass range, with for example H7N6F1–6 (δ1–6) being found in the female sample
exclusively, whilst glycans of composition H3N8F1–4 (h1–4),
H3N10F1–5 (j1–5), H3N12F2–6 (l2–6), H3N14F1–7 (n1–7), and
H3N16F1–8 (o1–8) were more abundant in the male sample
(Figure 1C–F).
Indications for sex-specific glycosylation differences were
also obtained by reverse-phase (RP) chromatography of
the female and male N-glycans after labeling with 2aminobenzamide (2AB). As many glycan species could only
be partially resolved chromatographically, quantification
of individual glycans via fluorescence detection was not
possible. To additionally employ the resolving power of
MS, further analyses of the 2AB-glycans were performed
by RP-nano-LC-MS (Figure 2). The species α1 (H6N5F1),
α2 (H6N5F2) and x1 (H5N5F1), for example, were associated
with the female worms, confirming the MALDI-TOF MS
data in Figure 1. The N-glycans d1 (H3N4F1), f1 (H3N6F1),
p1 (H4N3F1), and b2 (H2N2F1) showed a higher relative
expression in the male worms, which was again consistent
with the sex-specific expression pattern obtained by
MALDI-TOF-MS.
Structural characterization
To structurally characterize the differentially expressed Nglycans from S. mansoni females and males, 2AB-labeled
glycans were analyzed by RP nano-LC-ESI-IT-MS/MS. In
addition, N-glycans obtained from mixed male and female
worms were fractionated by normal-phase high-performance
liquid chromatography (HPLC) and analyzed by RP nanoLC-ESI-IT-MS/MS. To define specific structural differences, several glycans were subjected to linkage analysis
(Table I), if sufficient amounts were available. On the basis
of these data, various groups of glycans with common
structural features were defined (Table I; Figures 1 and 2).
Structures together with expression information are summarized in Figure 3.
N-glycans enriched in females
Female N-glycans were found to comprise large amounts of
core-(α1-6)-fucosylated tri-antennary structures (α1). Fragmentation of both the disodium adduct and the diproton
Sex-specific glycosylation in schistosomes
α
Δ
α
α
Δ
Δ
Δ
Δ
m/z
Fig. 1. Sex-specific glycosylation in Schistosoma mansoni worms: MALDI-TOF-MS of N-glycans. PNGase F-released N-glycans from glycoproteins of
S. mansoni female (A, C, E) and male (B, D, F) worms were registered as sodium adducts by MALDI-TOF-MS. Low (A, B), intermediate (C, D), and high
(E, F) mass ranges are given separately. To facilitate comparison, the intensity of the hexamannosidic N-glycans (a6) is set to 1.00 for both sexes. Peaks are
labeled with low-case letters indexed with numbers, which refer to Table I. Schematic structures were assigned based on the structural data presented in
this work. Dark circle, mannose; light circle, galactose; light square, N-acetylgalactosamine; dark square, N-acetylglucosamine; dark triangle, fucose; 䉭,
hexose polymer.
adduct clearly indicated the tri-antennary nature of this
N-glycan and gave no indications for the occurrence of diLacNAc units (Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)GlcNAc
(β1-) (Figure 4). The α1 N-glycans were purified by twodimensional HPLC (not shown) and subjected to linkage
analysis (Table I). This indicated α1 to be a core-fucosylated 3′-tri-antennary N-glycan with a 2,6-disubstituted
branching mannose, as schematically represented in Figure
3. This N-glycan structure has been detected before in
mixed male/female S. mansoni worms (Srivatsan et al.,
1992b; Cummings and Nyame, 1999). The core-fucosylated
3′-tri-antennary N-glycan may in addition carry a fucose on
one of the antennae resulting in a Lewis x-type antenna
structure (α2) (see also Figure 3). Truncated versions of
the tri-antennary species α1 and α2 were likewise observed
(x1–2, see Figure 3). Truncated structures carrying an
additional LacNAc [Gal(β1-4)GlcNAc] antenna are likewise associated with females (β1–2 structures, Figure 1C).
Another variant of the tri-antennary species is y1, which
exhibits two LacNAc antennae and one LDN antenna
(Figure 3).
In contrast to the structures α1 and α2, the N-glycans α3
and α4, which differed only in fucose content, were found
to exhibit fucosylated di-LacNAc units, resulting in a diLewis x unit for α4 (Figure 5). Again, these species were
more highly expressed in females than in males, as summarized in Figure 3. Likewise associated with females were Nglycans carrying a total of four LacNAc units, which could
be tetra-antennary structures or N-glycans exhibiting partially fucosylated LacNAc tandem repeats, similar to the
α-series glycans.
The higher fucosylated species of the y-series, y3 and y4,
were found to represent other variants of di-antennary
structures with one elongated antenna. In the case of y4,
two isomers were detected and partially resolved by a first
dimension normal-phase separation and the second dimension RP nano-LC-MS/MS analysis. Isomer-1 was found to
be di-antennary with a Lewis x antenna and a second
antenna showing a Lewis x trisaccharide on top of a
LDN-F unit (Figure 6). Isomer-2 was likewise found to be
di-antennary and exhibited a LDN-F antenna as well as a
di-Lewis x antenna (Figure 7). In contrast to α3 and α4,
993
M. Wuhrer et al.
α
β
δ
α
β
α
δ
γ
δ
δ
δ
γ
Δ
Δ
α
Δ
δ
α
α
Δ
Δ
Δ
m/z
Fig. 1. continued
the species y3 and y4 which contain mixed LacNAc/LDN
antennae occurred at similar expression level in male and
female N-glycan preparations (summarized in Figure 3), as
judged by MALDI-TOF-MS.
Another group of N-glycans, which were more prevalent
in females than in males, were di-antennary structures with
two LacNAc/Lewis x antennae (w1–3). The structure of w3
was supported by linkage analysis, indicating two Lewis x
antennae and core fucosylation (Table I). Moreover, negativemode MALDI-TOF/TOF-MS of w3 clearly showed the
strict monofucosylation of the antennae (Wuhrer and
Deelder, 2005), excluding the occurrence of Lewis y
(Fuc(α1-2)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-) or pseudoLewis y (Fuc(α1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-) (Wuhrer
et al., 2000) antenna structures.
N-glycans enriched in males
The dominant group of glycans found in the male schistosomes featured LDN and LDN-F moieties, which were
found at lower relative abundance in the females. These Nglycans were mostly core-(α1-6)-fucosylated, mono-antennary
and/or di-antennary structures exhibiting the following
variation in the number of LDN(-F) units: 1 unit (d 0–2),
2 units (f1–5), 3 units (h1–7), 4 units (j1–9), 5 units (l1–9), 6
994
units (n1–8), and 7 units (p3–8) (see Figures 1 and 3 as well
as Table I). Of these N-glycans, the di-antennary species f1–
3 have been observed in mixed schistosome male and female
populations before (Srivatsan et al., 1992a; Cummings and
Nyame, 1999) (Figure 3). The mono-antennary species d1
obtained by normal-phase HPLC purification was subjected to linkage analysis, in intact form as well as after
α-mannosidase treatment (see Table I). This allowed the
assignment of the LDN antenna to the 6-linked mannose,
as shown in Figure 3. For the group of N-glycans containing two and more LDN units we have recently shown that
they exhibit di-LDN units and, when fucosylated, di-LDN-F
units (GalNAc(β1-4)[±Fuc(α1-3)]GlcNAc(β1-3)GalNAc(β1-4)
[±Fuc(α1-3)]GlcNAc(β1-) (Wuhrer, Koeleman, Deelder
et al., 2006). These structures were found to occur at
higher levels in males than in females, as summarized in
Figure 3.
Several more N-glycan species were found at higher relative levels in males than in females. These include N-glycans
with a tandem repeat of LDN-F as one antenna and a
Lewis x motif as second antenna (t4 Figure 3) as well as a
variant thereof carrying one fucose less (t3); mono-antennary
glycans with a LacNAc or a Lewis x antenna (p0–2); a
di-antennary core-(α1-6)-fucosylated glycan with a LDN
and a truncated antenna (e1); mono-antennary truncated
Sex-specific glycosylation in schistosomes
Δ
Δ
Δ
Δ
Δ
Δ
Δ
Δ
Δ
Δ
Δ
Δ
Δ
Δ
Δ
Δ
Δ
Δ
Δ
m/z
Fig. 1. continued
structures with or without core-(α1-6)-fucosylation (c0–1);
paucimannosidic core-(α1-6)-fucosylated N-glycans (b2–4).
N-glycans without sex bias
Besides the above-mentioned y3–4 structures, oligomannosidic N-glycans, which have been described for adult schistosomes before (Nyame and Damian, 1988; Cummings and
Nyame, 1999), were found for females and males at similar
levels. They varied in H content between 2 and 10 residues
(a2–10) with the tenth H presumably being glucose (Figures
1–3). An exception was the tetramannosidic species (a4),
which showed a slightly higher expression in females
(Figures 2 and 3). For the abundant trimannosyl species
(a3), two structural isomers were resolved by preparative
HPLC (peaks 8 and 9, Figure 3 of Wuhrer, Koeleman,
Deelder et al., 2006), permethylated and characterized by
linkage analysis and MALDI in-source decay combined
with TOF/TOF-MS (Wuhrer and Deelder, 2006). The
major species exhibited a branched trimannosyl core, whilst
the minor isomer was characterized as Man(1-3)Man(16)Man(1-4)GlcNAc(1-4)GlcNAc- (see Figure 3).
Other glycans found at similar levels in males and
females were a bi-antennary core-(α1-6)-fucosylated structure with a LacNAc and a LDN antenna (r1) and a biantennary, optionally core-(α1-6)-fucosylated glycan with a
LacNAc/Lewis x antenna and a truncated antenna (q0–2).
monoclonal antibodies (mAbs) that bind to Lewis x (Figure
8A), LDN (B), and LDN-F (C). These mAbs have been
previously characterized by surface plasmon resonance
spectroscopy using defined neoglycoconjugates (van
Remoortere et al., 2000). The natural antigens and the
exact context in which the epitopes of the mAbs occur in
the schistosome have not been absolutely defined so far.
The Lewis x binding mAb bound to the lining of the gut of
both male and female worms, where Lewis x is present as a
repetitive element of the circulating cathodic antigen (CCA)
(van Dam et al., 1993; van Dam et al., 1994). Intense
staining of this determinant in the tegument, however,
was observed exclusively for female worms. The tegument
of male worms stained only faintly with the Lewis x-binding
mAb. In contrast, the LDN-F-directed mAb bound to the
parenchyma of both male and female worms. In addition,
most parts of the male and female tegument were only
very weakly detected by the anti-LDN-F antibody, but
the parts of the tegument of the male along the gynaecophoric groove showed a much more intense staining
(Figure 8C). The LDN-binding mAb recognized the teguments of male and female worms equally well (Figure 8B).
The guts were negative, whilst the parenchyma showed
weak staining, in line with a previous report (van
Remoortere et al., 2000).
Immunolocalization
Discussion
To determine the localization of glycans that contain
the differentially expressed carbohydrate determinants,
frozen sections of S. mansoni worms were probed using
The gender-specific glycosylation of S. mansoni adults was
studied by mass spectrometric profiling and sequencing of
PNGase F-released glycoprotein glycans. Two different MS
995
996
IT: 510 (N1F1A), 632/778 (N3F0–1)
IT: 632/778 (N3F0–1), 364/510 (N1F0–1A)
ND
ND
1136.4, 1282.6
1339.6, 1485.7, 1631.7
1688.8; LA of p1: t-Fuc, t-GlcNAc, t-GalNAc,
2-Man, 3,6-Man, 4-GlcNAc
1891.8, 2037.9, 2183.9, 2330.1, 2476.2
2241.0, 2387.1
2298.1, 2444.1, 2590.2, 2736.3, 2882.4,
3028.5, 3174.6
2501.2, 2647.3, 2793.4, 2939.4, 3085.5
2704.3, 2850.4, 2996.5, 3142.5, 3288.7,
3434.8, 3582.5a, 3728.8a, 3875.0a
2907.4, 3053.5, 3199.6, 3343.7, 3493.4a, 3637.9a,
3785.4a, 3931.6a
3110.5, 3256.6, 3402.7, 3550.5a, 3696.6a, 3842.7a,
3988.6a, 4134.6, 4280.8a
3461.8a, 3608.1a, 3754.3a, 3900.5a, 4046a
3518.9a, 3664.9a, 3811.3a, 3957.1a, 4103.3a, 4249.5a,
a, 4541.0a
1298.4, 1444.7, 1590.7
1501.7, 1647.8, 1793.7
1704.8, 1850.8, 1996.8, 2143.0
2054.0, 2200.0, 2346.1
2257.0, 2403.1, 2549.2, 2695.3
H3N3F0–1
H3N4F0–2
H3N5F1
H3N6F1–5
H3N7F2–3
H3N8F1–7
H3N9F1–5
H3N10F1–9
H3N11F1–6
H3N12F1–9
H3N13F2–6
H3N14F1–8
H3N16F3–8
H4N3F0–2
H4N4F0–2
H4N5F0–3
H4N6F1–3
H4N7F1–4
c0–1
d0–2
e1
f1–5
g2–3
h1–7
i1–5
j1–9
k1–8
l1–9
m2–6
n1–8
o3–8
p0–2
q0–2
r0–3
s1–3
t1–4
4217.5a, 4364.0a, 4509.5a, 4657.0a, 4802.1a, 4948.1a
4395.6
IT: 632/778 (N3F0–1), 981 (N4F1), 510 (N1F1A)
917.3, 1079.4, 1241.6
H2–4N2F1
b2–4
–
Monofucosylated species: 1 LDN-LDN; 1 LacNAc;
core-α1-6-fucosylation
IT: 813b (N4), 366b (N1H1), 488b (N1F1A)
1 LacNAc/Lewis x, 1 LDN/LDNF; core fucosylation
1 LacNAc or Lewis x, core-α1-6-fucosylation;
antenna, 1 single HexNAc
1 LacNAc/Lewis x; core fucosylation
–
–
–
2 LDN(F)-LDN(F) antennae; 1LDN(F) antenna;
core-α1-6-fucosylation
At least 1 LDN(F)-LDN(F); core-α1-6-fucosylation
2 LDN(F)-LDN(F) antennae, core-α1-6-fucosylation
Monofucosylated species: 1 LDN-LDN; core-α1-6fucosylation
ND
IT: 364/510 (N1F0–1A), 429/575 (N2F0–1),
388/534 (N1H1F0–1)
IT: 388/534 (N1H1F0–1), 364/510 (N1F0–1A)
IT: 364/510 (N1F0–1A), 388/534 (N1H1F0–1)
ND
IT: 510 (N1F1A), 632/778 (N3F0–1), 981 (N4F1),
Core-α1-6-fucosylation; 1 LDN(F) antenna, 1
LDN(F)-LDN(F) antenna; additional fucoses
Monofucosylated species: 1 LDN–LDN; single
HexNAc antenna; core-α1-6-fucosylation
IT: 813b (N4), 488b (N1F1A), 632 (N3), 835 (N4)
IT: 510 (N1F1A), 429/575 (N2F0–1), 835 (N4),
778 (N3F1)
Two LDN(F) antennae; alternatively 1
LDN(F)LDN(F) antenna
1 LDN; 1 single HexNAc; core-α1-6-fucosylation
1 LDN(F); core-α1-6-fucosylation
Monofucosylated species: single HexNAc antenna;
core-α1-6-fucosylation
Core-α1-6-fucosylated paucimannosidic
N-glycans
Mannosidic N-glycans, H10N2 probably with a
terminal glucose
Structural characteristics based on MS/MS data
IT: 429/575 (N2F0–1), 835 (N4); 364/510 (N1F0–1A);
IT: 429 (N2), 510 (N1F1A),
IT: 429/575 (N2F0–1), 364/510 (N1F0–1A); LA of d1:
t-Fuc, t-Man, 2-Man, 3,6-Man, t-GalNAc,
4-GlcNAc; LA of d1 after α-mannosidase treatment: t-Fuc, 2-Man, 6-Man, t-GalNAc, 4-GlcNAc
IT: 510 (N1F1A); 1199 (H3N2F1A); LA of c1:
t-Fuc, t-Man, t-GlcNAc, 2-Man, 3,6-Man,
4-GlcNAc
IT: 510 (N1F1A); LA of b2: t-Fuc, t-Man, 6-Man,
4-GlcNAc
IT: 364 (N1A), 567 (N2A); LA of a3(isomer 1):
t-Man, 3,6-Man, 4-GlcNAc; LA of a3(isomer 2):
t-Man, 3-Man, 6-Man, 4-GlcNAc; LA of a8:
t-Man, 2-Man, 3,6-Man
771.3, 933.3, 1095.5, 1257.6, 1419.7, 1581.7, 1743.8,
1905.8, 2067.9
H2–10N2
a2–10
Key structural data
Registered masses (Figure 1)
Detected
species
Labels used
in Figures 1–3
Table I. Differential expression of N-glycans in Schistosoma mansoni adult male and female flukes
M. Wuhrer et al.
2955.4, 3101.5
1460.7, 1606.7
1866.8, 2012.9, 2158.9
2216.0, 2362.1, 2508.2, 2654.3
1622.7
H4N9F3–4
H5N3F0–1
H5N4F0–3
H5N5F0–2
H5N6F1–4
H6N3
H6N5F0–4
H6N6F1–2
H6N7F1–2
H7N6F1–6
u3–4
v0–1
w0–3
x0–2
y1–4
z0
α0−4
β1–2
γ1–2
δ1–6
ND
ND
–
–
–
see Figures 4 and 5
ND
–
IT: see Figures 4 and 5; LA of α1: t-Fuc, t-Gal,
2-Man, 2,6-Man, 3,6-Man, 4-GlcNAc
Monofucosylated species: core-α1-6-fucosylation;
2 LacNAc antennae, 1LDN antenna for
tetrafucosylated species see Figures 6 and 7
2 LacNAc or 1LacNAc/1Lewis x; 1 single HexNAc;
core-α1-6-fucosylation
2 LacNAc/Lewis x, core-α1-6-fucosylation
Hybrid-type structure; 1 LacNAc
–
Structural characteristics based on MS/MS data
ND
IT: 510 (N1F1A), 388 (N1H1), 429 (N2); Figures 6
and 7
IT: 388/534 (N1H1F0–1), 364/510 (N1F0–1A)
IT: 388/534 (N1H1F0–1); LA of x3: t-Fuc, t-Gal,
2-Man, 3,6-Man, 4-GlcNAc, 3,4-GlcNAc
IT: 388 (N1H1)
ND
Key structural data
A, 2-aminobenzamide; F, deoxyhexose; H, hexose; LA, linkage analysis; N, N-acetylhexosamine; ND, not done.
Glycans without the 2AB-tag were measured by MALDI-TOF-MS in the positive-ion reflectron mode. Monoisotopic masses of sodium adducts were registered (see Figure 1). Fragment ions were
determined as monosodiated or disodiated species using a two-dimensional LC approach with RP-nano-LC-MS/MS using an ion-trap mass spectrometer (IT). Linkage analysis data (LA) are given in
terms of t-Fuc (terminal fucose), 2-Man (2-substituted mannose), 3,4-GlcNAc (3,4-disubstituted N-acetylglucosamine), etc.
aAverage mass.
bProtonated species.
2540.1, 2686.2, 2832.3, 2978.4, 3124.5, 3270.6
2581.2, 2727.3
2378.1, 2524.2
2028.9, 2174.9, 2321.1, 2467.3, 2613.2,
1663.5, 1809.8, 1955.8, 2101.9
Registered masses (Figure 1)
Detected
species
Labels used
in Figures 1–3
Table I. continued
Sex-specific glycosylation in schistosomes
997
M. Wuhrer et al.
α
Fig. 2. Sex-specific glycosylation in Schistosoma mansoni worms: nano-LC-MS of 2-aminobenzamide (2AB)-labeled N-glycans. PNGase F-released N-glycans
of S. mansoni females (continued lines) and males (dotted lines) were labeled with 2AB and analyzed by nano-LC-MS. Glycans of specific compositions
were monitored by extracted ion chromatograms (EIC) representing the doubly sodiated or, in the case of glycan b2, the singly sodiated species. Mass spectrometric signal intensities for individual glycans of both males and females were normalized so that the hexamannosidic N-glycans (a6) corresponded to
signal intensity of 1.0 in all LC-MS runs. Based on this normalization, one group of glycans exhibited a higher expression in females than in males (shown
for w1, x1, and α1), whilst a second group of glycans (shown for p1, d1, f1 and b2) was more highly expressed in males.
techniques, namely, MALDI-TOF-MS of native glycans
and nano-LC-IT-MS of their 2AB-labeled equivalents,
gave similar results indicating marked differences between
the N-glycans populations of the two sexes (summarized in
Figure 3). The structures of the differentially expressed glycans were characterized by nano-LC-IT-MS/MS and linkage analysis, which revealed that Lewis x and LacNAc
termini mainly occur on female N-glycans, whereas elevated levels of LDN and fucosylated-LDN are present on
N-glycans of the males. Furthermore, the female complextype glycans are mainly based on di- and tri-antennary core
types, and the males display a high proportion of monoantennary and di-antennary, in part truncated glycans. It is
not clear whether the latter structures are immature glycan
species due to decreased glycosyltransferase action or
rather are the result of increased exoglycosidase activities.
998
Next to N-glycans, the N-glycan pools of both females
and males did contain a H polymer, which was sensitive to
α-amyloglucosidase treatment (not shown), indicating that
this polymer was derived from glycogen.
In our LC-MS analyses, fragmentation of proton
adducts of fucosylated N-glycans repeatedly resulted in
fragment ions containing an excess of fucose residues (see
lower panels in Figures 5–7). As discussed in previous studies on schistosome glycans and glycopeptides (Wuhrer,
Koeleman, Hokke et al., 2006; Wuhrer, Balog, Catalina
et al., 2006; and references therein), these ions arise from
rearrangement events which are generally known to occur
upon fragmentation of proton adducts of fucosylated
glycoconjugates.
Comparative transcriptomic studies of male and female
schistosomes have indicated that major differences between
Sex-specific glycosylation in schistosomes
α
H6N5F3-4
δ
H7N6F1-6
H4N2
H5N4F1-3
≈
H3N2
H4N3F0-2
≈
H3,5-9 N2
H3N5F1
≈
H4N5F1
H4N7F4
H3N3F0-1
≈
H4N4F0-2
H3N8F1-4
H2-3N2F1
α
H6N5F0-2
H3N10F1-5
≈
H5N6F3-4
H5N5F0-2
H3N6F1-3
H5N6F1
H3N4F0-2
H3N12, 14 F1-7
Fig. 3. Scheme of N-glycan structures derived from Schistosoma mansoni worms with sex-related expression profile. H, hexose; F, fucose; N, N-acetylhexosamine. Low-case letters with subscript numbers refer to Table I.
the two genders occur in terms of gene expression and gene
products (Hoffmann et al., 2002; Fitzpatrick et al., 2004;
Hoffmann and Fitzpatrick, 2004; Fitzpatrick et al., 2005),
which is not surprising regarding the very different appearance and physiological characteristics of the two sexes.
Post-genomic comparative analyses of male and female
schistosomes are limited. A proteomic analysis of differentially expressed proteins between male and female S. japonicum
worms after pairing has been performed (Cheng et al.,
2005), and in the framework of DNA microarray profiling
studies, enzyme activity measurements have been carried
out to correlate the gene expression differences. For example, extracellular superoxide dismutase and tyrosinase
activity are both elevated in female S. japonicum, in agreement with the gender association of the corresponding transcripts (Fitzpatrick et al., 2004). A larger number of more
focused studies performed at the level of single genes or
proteins in the pre-genomics era have also indicated that
numerous proteins are differentially expressed in male and
female schistosomes (Hoffmann, 2004).
Sex-specific glycosylation, however, has not been
described before in schistosomes, although it has been
noted that some female specifically expressed and released
proteins are glycoproteins (Aronstein and Strand, 1984). The
only defined example of any pronounced sex-specific glycosylation is to our knowledge that of the human glycoprotein
glycodelin. In this case, the glycosylation of two identical
proteins (in terms of the polypeptide) was studied. Glycodelin A from amniotic fluid (Dell et al., 1995) was found to
carry complex-type glycans with sialyl-LacNAc, LDN,
sialyl-LDN, and LDN-F as terminal structures that do not
occur on glycodelin S from seminal fluid (Morris et al.,
1996). LacNAc and Lewis x terminal motifs are found on
both glycodelin A and glycodelin S, but half of the glycans
of glycodelin S carry one or two Lewis y units, which are
not found on glycodelin A. Importantly, the two different
glycodelins also differ in the sense that glycodelin A has a
contraceptive activity and inhibits sperm binding to zona
pellucida in in vitro assays, whilst glycodelin S does not
have this activity but instead has been proposed to have a
glycosylation-dependent immunosuppressive activity (Morris
et al., 1996).
Although the current study did not address the glycosylation of specific proteins or gender-specific organs but rather
monitored overall N-glycosylation, clear specific differences were found. This indicates that large differences in the
glycosylation machinery of male and female worms must
occur, either in general or in specific organs or cells that
contribute significantly to the overall profile.
So far, the only example of a gender-associated glycosyltransferase transcript that appeared in the DNA microarray studies was that of the putative GlcNAcT-II gene
999
M. Wuhrer et al.
m/z
Fig. 4. Nano-LC-MS/MS analysis of the 2-aminobenzamide (2AB)-labeled oligosaccharide α1. Both the disodium adduct ([M+2Na]2+, upper panel) and
the diproton adduct ([M+2H]2+, lower panel) of the glycan species k1 (see Figures 1 and 3; Table I) obtained from mixed-sex worms were fragmented.
Double-headed arrows indicate differences in fucose content. The deduced structure is shown in the box. A, 2-aminobenzamide.
(contig 1475), which is up-regulated in S. mansoni females
(Fitzpatrick et al., 2005). GlcNAcT-II is the N-acetylglucosaminyl transferase that catalyzes the formation of the
GlcNAcβ1-2 linkage to the α1-6-linked Man residue in an
N-glycan trimannosyl core structure, which is required for
any further extension and branching of complex-type glycans (Brockhausen et al., 1988; Schachter, 2000). Although
no clear differences in the presence of this particular
GlcNAc residue were observed between the males and
females (Figure 3), the relatively high expression of this
gene in females might be related to the fact that the females
contain less truncated and mono-antennary glycans than the
males. On the basis of the differential glycomics data, it
could be hypothesized that in particular a gender-specific difference in the expression of a β4GalT and a β4GalNAcT
responsible for the formation of LacNAc and LDN (Neeleman
et al., 1994; van den Eijnden et al., 1995a; van den Eijnden
et al., 1995b), respectively, should occur. Indications for
this have hitherto not been observed at the genomic or
1000
proteomic level. At least four different putative β4GalT/
GalNAcT genes (SmEA714357.1/SmEA605488.1, Sm07779/
SmEA604548.1, SmEA603871.1, and SmEA609849.1),
which were retrieved from http://verjo18.iq.usp.br/schisto/
(S. mansoni EST genome project) and http://www.genedb.
org/genedb/smansoni/ (Sanger), are present in the schistosome genome, and reverse transcription–polymerase
chain reaction (RT–PCR) indicates that the first two are
each significantly differentially expressed in male and
female schistosomes (not shown). We have measured the
ratio of GalT and GalNAcT activities with GlcNAc as
general acceptor structure in male and female schistosome extracts. The activity data (not shown) did not
provide a clear correlation with the structural data. More
work is needed to find out which of the apparently several
GalT and GalNAcT variants are responsible for the
synthesis of the LacNAc and LDN elements in N-glycans,
and how specific activities of these transferases can be
determined.
Sex-specific glycosylation in schistosomes
m/z
Fig. 5. Nano-LC-MS/MS analysis of the 2-aminobenzamide (2AB)-labeled oligosaccharide α4. Both the disodium adduct ([M+2Na]2+, upper panel) and
the diproton adduct ([M+2H]2+, lower panel) of the glycan species k4 (see Figures 1 and 3; Table I) obtained from mixed-sex worms were fragmented. –F,
fragment ion arising from neutral loss of a fucose residue.
It should be mentioned that the gut of both the male and
female worms express large amounts of polymeric Lewis xcontaining glycans on CCA (van Dam et al., 1993; van
Dam et al., 1994; Figure 8A), indicating that although Lewis
x is minor in the antennae of the male N-glycans found in
this study, male worms are capable of synthesizing Lewis xcontaining glycoconjugates. Most probably due to their
high mass (average mass above 10 kDa) and pronounced
heterogeneity, the poly-Lewis x-carrying O-glycans of CCA
were not detected in the present study.
Previous to the male–female differences found in the current study, it has clearly been shown that a stage-specific
expression of N-glycans occurs between the larval, adult,
and egg stages of S. mansoni. In particular, core decoration
shows stage-specific features: in this study, we have only
found core α1-6-fucosylation and could not detect core
xylosylation. The occurrence of core-α1-3-fucosylation as
well as core xylosylation of N-glycans from S. mansoni
adults has been indicated previously by western blot analysis
(van Die et al., 1999). Based on our results, these glycans
must be present in relatively low amounts as they have not
been detected in the PNGase A-released or the β-elimination
cleaved glycan pools, which both appeared virtually identical
to the PNGase F-released material. In contrast, significant
portions of egg N-glycans are (1) core α1-6-fucosylated and
core xylosylated (PNGase F-sensitive portion) and (2) core
α1-3/α1-6-difucosylated (PNGase F-resistant portion)
(Khoo et al., 1997 as well as unpublished results). S. mansoni females are known to harbor immature eggs in the
reproductive system, but we did not detect any core-xylosylated N-glycans of the egg-type in the PNGase F-released
N-glycan pool (Figures 1 and 3). This may indicate that glycosylation changes during egg maturation and that core
xylosylation possibly occurs on N-glycans in mature eggs
but not in immature eggs present in the female flukes.
Alternatively, the contribution of N-glycans of these immature eggs to the total female N-glycosylation profile may be
too small to allow their detection.
1001
M. Wuhrer et al.
m/z
Fig. 6. Nano-LC-MS/MS analysis of the 2-aminobenzamide (2AB)-labeled oligosaccharide y4 (isomer-1). Both the disodium adduct ([M+2Na]2+, upper
panel) and the diproton adduct ([M+2H]2+, lower panel) of isomer-1 of species y4 (see Fig. 1) obtained from mixed-sex worms were fragmented. –F,
fragment ion arising from neutral loss of a fucose residue.
An earlier study has shown that sera from only male,
only female, or mixed-sex S. mansoni-infected mice exhibited pronounced differences with respect to immunoprecipitation activity toward adult worm glycoproteins (Aronstein
and Strand, 1984). Such gender-related differences in schistosome immunogenicity are in line with our finding of the
differential (surface) expression of the terminal glycan
motifs Lewis x (female) and LDN-(F) (males) by S. mansoni
adult worms: whilst the females were found to express
Lewis x in the tegument, the tegument of the males was
richer in LDN-F. In terms of humoral host response, it
seems that in infection, serum antibody levels to LDN(-F)
are generally higher than to Lewis x (Eberl et al., 2001; van
Remoortere et al., 2001). On the other hand, Lewis x has
been described as molecular element acting on immune cells
to modulate the immune response (Okano et al., 1999;
Velupillai et al., 2000; Okano et al., 2001), but this activity
seems to be mainly egg associated. It would be interesting
to evaluate differential immune responses to male- and
female-expressed glycans by studying single-sex and mixedsex infection sera with respect to anti-LDN, anti-LDN-F,
and anti-Lewis x antibodies.
1002
Finally, the location of Lewis x, LDN, and LDN-F at the
contact site of male and female worms may indicate a
potential role of these glycans in female/male contact and
mating. For example, the LDN-F motifs expressed by the
male in the gynecophoric canal could serve as ligands of a
female surface receptor. Future work will have to demonstrate if the differentially expressed glycan motifs of female
and male schistosomes play a role in their interaction.
Materials and methods
(Glyco)protein preparation
Five different preparations of S. mansoni adult flukes were
used: a mixture of female and male worms obtained by perfusion of infected hamsters (i); male worms separated manually after perfusion of S. mansoni mixed-sex infected
hamsters (ii) and mice (iii); female worms separated manually after perfusion of S. mansoni mixed-sex infected hamsters (iv) and mice (v). All worm preparations were first
delipidized. To this end, worms were homogenized in water
[1 mL (1 volume) per 100 mg wet weight of worms] and
Sex-specific glycosylation in schistosomes
m/z
Fig. 7. Nano-LC-MS/MS analysis of the 2-aminobenzamide (2AB)-labeled oligosaccharide y4 (isomer-2). Both the disodium adduct ([M+2Na]2+, upper
panel) and the diproton adduct ([M+2H]2+, lower panel) of isomer-2 of species i4 (see Figure 1) obtained from mixed-sex worms were fragmented. –F,
fragment ion arising from neutral loss of a fucose residue. *Fragment ions derived from isomer-1, which are observed due to incomplete separation of the
two isomers.
sequentially methanol and chloroform were added (5 volumes
each). The supernatants were removed after centrifugation,
and each extraction was repeated. To extract (glyco) proteins,
the pellets were resuspended in phosphate-buffered saline
(PBS) (35 mM sodium phosphate, pH 7.6, 0.85% NaCl).
Sodium dodecyl sulfate (SDS) and 2-mercaptoethanol were
added to final concentrations of 1% (weight/volume) and
0.5%, respectively. The samples were incubated for 10 min at
100°C and allowed to cool to room temperature, and CHAPS
(Fluka, Schnelldorf, Germany) was added to a final concentration of 1% (weight/volume). Samples were centrifuged, and
supernatants were used for N-glycan release by PNGase F.
Glycan release
(Glyco)protein samples were incubated with PNGase F
(2 mU per 100 mg wet weight; Roche Diagnostics, Mannheim,
Germany) overnight at 37°C. For the purification of the
released glycans, samples were first applied to a RP cartridge
(500 mg of Bakerbond octadecyl; Baker, Phillipsburg, NJ).
Flow-through and wash (5 ml of water) were then applied
to a carbon cartridge (150 mg Carbograph; Alltech, Deerfield, IL). After a wash with water (5 ml), glycans were
eluted with 25% aqueous acetonitrile (5 ml) and detected by
MALDI-TOF-MS. To ensure maximum deglycosylation,
(glyco)protein samples were subjected to the glycan-release
procedure for a second time. For this purpose, (glyco)proteins as well as detergents were retrieved from the RP cartridge by sequential elution with 20, 40, and 60%
acetonitrile containing 0.1% trifluoroacetic acid (5 ml
each). Eluates were pooled, the acetonitrile content was
reduced under a stream of N2, and the samples were lyophilized. Samples were taken up in PBS containing 0.5%
2-mercaptoethanol. PNGase F treatment was repeated, and
glycans were purified as described above. Eluted glycans
were detected by MALDI-TOF-MS.
For subsequent PNGase A treatment, (glyco)proteins
were again retrieved from the RP cartridge, taken up in
PBS, and trypsinized. Trypsin was inactivated by incubation
of the samples at 100°C for 5 min. The samples were
1003
M. Wuhrer et al.
Fig. 8. Immunolocalization of Lewis x, LDN, and LDN-F carbohydrate structures. Frozen sections of Schistosoma mansoni worms were immunostained
using Lewis x-binding (A), LDN-binding (B) and LDN-F-binding (C) monoclonal antibodies (lines 128–4F9, 273–3F2-A, and 114–4E8-A, respectively).
Green fluorescence was registered using fluorescein isothiocyanate (FITC)-labeled secondary antibodies. (D) buffer control (no primary antibody); G, gut;
T, tegument; , female worm; , male worm.
adjusted to pH 5 by addition of HCl and subjected to
PNGase A treatment (10 mU, Roche Diagnostics)
overnight at 37°C. Released glycans were purified
following the procedure as outlined above for
PNGase F treatment.
After the sequential PNGase F and PNGase A treatments, the remaining (glyco)peptides were again
retrieved from the RP cartridges as described above and
subjected to reductive β-elimination. The dry samples
were taken up in 100 μl of 1 M NaBH4, 0.1 M NaOH at
40°C for 24 h, and neutralized with 4 M acetic acid. Four
hundred microliter of 1% acetic acid in methanol was
added, and the sample was dried down in a vacuum centrifuge. This step was repeated three times. Released glycans were purified by sequential RP cartridge and
carbon cartridge extraction, and were detected by
MALDI-TOF-MS.
MALDI-TOF-MS
Glycan samples were analyzed by MALDI-TOF-MS with
an Ultraflex II mass spectrometer (Bruker Daltonics, Bremen,
Germany) in the reflectron mode using 6-aza-2-thiothymine
(5 mg/ml; Sigma) as matrix.
1004
Labeling and fractionation
Glycans released with PNGase F were tagged with 2AB by
reductive amination as outlined previously (Wuhrer et al.,
2004). The reaction mixture was applied to a carbon cartridge
(Alltech), and the 2AB-labeled glycans were eluted with 5 ml
of 25% acetonitrile. The acetonitrile content was reduced
under a stream of N2, and the samples were lyophilized.
Fractionation by normal-phase HPLC
2AB-labeled glycans were fractionated by normal-phase
HPLC on a TSK-Amide 80 column (4 mm × 250 mm;
Tosohaas, Montgomeryville, PA) at 0.4 mL/min. Solvent A
was 5 mM formic acid adjusted to pH 4.4 with ammonia
solution. The salt concentration was thus 10 times lower
than in the published separation system (Garner et al.,
2001). Solvent B was 20% of solvent A in acetonitrile. The
following gradient conditions were used: at time t = 0 min,
100% solvent B; t = 152 min, 52.5% solvent B; t = 155 min,
0% solvent B; t = 162 min, 0% solvent B; and t = 163 min,
100% solvent B. The total run time was 180 min. Samples
were injected in 80% acetonitrile. Because of the large
amounts of material injected, fluorescence was detected at
280 nm/500 nm instead of the routinely used 360 nm/425 nm
Sex-specific glycosylation in schistosomes
to avoid saturation of the detector. Fractions were collected
manually and analyzed by MALDI-TOF-MS.
Laidlaw for assistance with the separation of male and
female worms, and Dr Alexandra van Remoortere for critically reading the manuscript.
Permethylation and linkage analysis
2AB-labeled glycans were permethylated (Ciucanu and
Kerek, 1984) and hydrolyzed (4 M trifluoroacetic acid, 4 h,
100°C). Partially methylated alditol acetates obtained after
sodium borohydride reduction and peracetylation were
analyzed by capillary GLC-MS using electron-impact ionization (Geyer and Geyer, 1994).
nano-LC-MS/MS
2AB-labeled glycans were separated on a PepMap column
(75 μm x 150 mm; Dionex/LC Packings, Amsterdam, the
Netherlands) using an Ultimate nano-LC system (Dionex/
LC Packings) equipped with a Switchos guard column system (Pepmap guard column, 300 μm x 10 mm). The system
was equilibrated with eluent A (H2O/acetonitrile 95:5, v/v,
0.1% formic acid) at a flow rate of 150 nl/min. After injecting the sample, a linear gradient to 50% eluent B (H2O/acetonitrile 5:95, v/v, containing 0.1% formic acid) in 30 min
was applied, followed by a final wash with 100% eluent B
for 5 min. The system was directly coupled to an Esquire
High Capacity Trap (HCT) ESI-IT-MS (Bruker) equipped
with an online nano-spray source operating in the positiveion mode. For electrospray (900–1200 V), capillaries (360
μm OD, 20 μm ID with 10 μm opening) from New Objective (Cambridge, MA) were used. The solvent was evaporated at 165°C with a nitrogen stream of 5 L/min. Ions from
m/z 50 to m/z 2500 were registered. Automatic fragment ion
analysis was enabled, resulting in MS/MS spectra of the
most abundant peaks. To register predominantly sodium
adducts by MS, part of the analyses was performed after
addition of 0.8 mM NaOH to solvent A. For the determination of relative glycan amounts, the system was run in the
MS mode.
Immunolocalization
Immunofluorescence assays were performed on frozen gut
sections of S. mansoni-infected hamsters as reported previously (van Dam et al., 1993). Briefly, slides were fixed in
acetone and incubated with hybridoma cell culture supernatant in a humid atmosphere at 37°C for 30 min. The following mAbs were used: 128-4F9 (anti-Lewis x) (van
Remoortere et al., 2000), 273-3F2-A (anti-LDN) (van
Remoortere et al., 2000), and 114-4E8-A (anti-LDN-F,
unpublished data). Subsequently, the slides were washed
and incubated with fluorescein isothiocyanate (FITC)labeled goat anti-mouse Ig (1:40) in PBS containing 0.1 mg/mL
Evans blue. Para-phenylenediamine was used as anti-fading
agent, and slides were observed with a Leica DM-RB
fluorescence microscope with the appropriate filter combination for FITC fluorescence. Slides incubated with PBS
were used as negative control.
Acknowledgments
The authors thank Dieuwke Kornelis for performing the
immunolocalization experiments, Frances Jones and Maureen
Conflict of interest statement
None declared.
Abbreviations
2AB, 2-aminobenzamide; CCA, circulating cathodic antigen;
H, hexose; LacNAc, Gal(β1-4)GlcNAcβ1-; LC, liquid chromatography; LDN or LacdiNAc, GalNAc(β1-4)GlcNAcβ1-;
LDN-F, GalNAc(β1-4)[Fuc(α1-3)]GlcNAc(β1-; Lewis x,
Gal(β1-4)[Fuc(α1-3)]GlcNAc; HPLC, high-performance liquid chromatography; mAb, monoclonal antibody; RP, reverse
phase; PBS, phosphate-buffered saline.
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