(Received for publication, April 4, 1995)
From the
The entire surface of the cercarial stage of the human blood fluke Schistosoma mansoni is covered by a 1-µm thick, highly immunogenic, fucose-rich glycocalyx (GCX). Using strategies based on enzymatic, chemical, and mass spectrometric analysis, we have defined the structures of the major glycans released by reductive elimination from GCX. They comprise a heterogeneous population of multifucosylated complex oligosaccharides with the following nonreducing terminal sequences:
Our structural data suggest
that these tri- to pentafucosylated epitopes are carried on type 1,
RGal
1
3GalNAc, and type 2,
R
Gal
1
3(R
GlcNAc
1
6)GalNAc, core
structures via repeat units of
(3GalNAc
1
4(Fuc
1
2Fuc
1
2Fuc
1
3)GlcNAc
1
3Gal
)
,
where n is mainly 0 and 1, and all sugars are in the pyranose
form. The proposed structure represents the first instance where an
-galactosylated
-GalNAc(1
4)-
-GlcNAc sequence
occurs as a repeating unit in a glycoprotein. It is also unique in
being substituted with oligofucosyl appendages. The unusual
oligosaccharide structures described here, particularly the potentially
immunodominant oligofucosyl moieties, are most likely responsible for
the known potency of GCX in modulating various immune responses
including complement activation, B cell mitogenesis, and delayed type
hypersensitivity in schistosomiasis.
Schistosomes are digenetic trematodes that infect approximately
200 million people worldwide. The parasite has a complex life cycle
that involves a snail intermediate host as well as man. Larval
parasites, called cercariae, develop in the snail, are released into
freshwater, and penetrate the mammalian hosts' skin.
Subsequently, the cercariae transform into schistosomula that
eventually leave the skin and migrate through the lungs and liver to
the intestinal blood vessels where they mate and lay eggs. In both mice
and humans, the early infection is characterized by a delayed type
hypersensitivity response that is switched to a predominantly humoral
response at the beginning of egg deposition.
Carbohydrates have been
increasingly implicated in the immune response in schistosomiasis.
Antibodies from infected mice and humans have been shown to react with
saccharide epitopes, particularly those on the surface of the invading
larva and the soluble egg antigens (Simpson, 1990; Dunne, 1990). One of
the major carbohydrate-containing surface antigens is the glycocalyx
(GCX)
GCX
has an apparent molecular mass of more than 5
Figure 1:
FAB spectrum of
peracetylated GCX O-glycans in the mass range m/z 250-2000. The major signals correspond to nonreducing terminal
A-type fragment ions, each accompanied by signals at 42 units lower as
well as 84 units higher. The former are derived from underacetylation
and double cleavages, while the latter are probably due to
overacetylation. The signal at m/z 1553 corresponds to
(OH)
Figure 2:
FAB spectra of permethylated GCX O-glycans in the mass range m/z 200-2000 (A)
and m/z 2000-6500 (B). The complex clusters of signals
associated with the Fuc
The high mass domain of Fig. 2B is characterized by
a prominent A-type ion at m/z 3042 which is an increment of
Fuc
In earlier work (Xu et al., 1994) automated
hydrazinolysis methodology was used to release glycans from GCX which
were then separated by electrophoresis after fluorescent tagging. Bands
co-migrating with standards containing 11, 12, 16, and 17 sugar
residues were detected. We now have FAB-MS evidence that suggests that
these bands correspond to core type 1 glycans which lack the reducing
GalNAc residue (data not shown). Thus it appears that highly complex
structures of the type found in GCX are very susceptible to
``peeling'' under alkaline elimination conditions and
hydrazinolysis procedures optimized for release of intact O-glycans from mammalian glycoproteins (Patel et al.,
1993) should be used with caution on molecules like GCX.
Figure 3:
FAB
spectra of the permethyl derivatives of periodate-oxidized GCX O-glycans in low (A) and high (B) mass
regions. The periodate-oxidized sample was reduced with borohydride and
permethylated directly. Remnants of oxidized Fuc residues were
presumably acid labile and lost during the work-up. The A-type ion at m/z 709 (Hex
Figure Z2:
Structure 2
Figure Z3:
Structure 3
To explore these possibilities a
new batch of periodate-oxidized material was prepared and digested with
Figure 4:
FAB
spectra in the mass range m/z 1000-2400 of the permethyl
derivatives of GCX O-glycans after Smith degradation (A) and further sequential digestions with
Figure 5:
Low
mass regions of the FAB spectra of the permethyl derivatives of GCX O-glycans after Smith degradation (A) and further
sequential digestions with
Figure Z4:
Structure 4
Some conclusions may
nonetheless be drawn with respect to the complete structures of the
intact O-glycans. First, a prominent A-type ion was observed
at m/z 3042 together with a weaker one at m/z 2868
which correspond to a Fuc
Second, if the core structures of the O-glycans are defined as shown in Fig. Z5, where R refers to the repeating motif
(
Figure Z5:
Structure 5
Figure Z6:
Structure 6
The GCX oligosaccharides characterized in this study are
among the most complex glycans determined to date. They range in size
from 12 to at least 60 glycosyl residues, and they exhibit the
following novel features: (i) multiple copies of
GalNAc
Until recently the
GalNAc
To date, several UDP-GalNAc:GlcNAc
Available structural data
suggest that capping and elongation of lacdiNAc antennae are frequently
analogous to lacNAc modifications, especially for lacdiNAc-containing
glycoproteins from higher animals e.g.
The discovery of
in-chain
Although epitopes carrying several separately linked fucosyl
residues, such as poly-Le
The developmental stage-specific
expression of the highly complex and unusual oligosaccharide structures
described here for the cercarial GCX may play a role in the mechanical
stabilization of the surface of schistosome larvae as they progress
from the snail intermediate host, through freshwater and into their
definitive mammalian host. In addition, these multifucosylated
saccharide moieties are likely to be involved in GCX's action as
a very potent immunological modulator. It is known that the GCX
activates complement by the alternative pathway and will stimulate
strong serological responses after immunization. It is also strongly
recognized by sera from schistosomiasis patients. GCX, obtained as
described by Xu et al.(1994) and used for the analysis here,
stimulates splenocytes from naive mice (Xu et al., 1993). In
particular, it causes proliferation of purified B cells in the absence
of T cells or antigen presenting cells in several strains of mice
including C3H/HeJ, a strain resistant to the proliferatory stimulation
by lipopolysaccharide.
While the analysis of the immunostimulatory
role of this molecule is still in progress, it is possible that the GCX
or epitopes on other molecules similar to the GCX are responsible for
the delayed type hypersensitivity responses seen early in the
infection. Further, the large nonspecific IgE responses seen after egg
laying may be related to the nonspecific stimulation of B cells by
related epitopes released by the eggs. It remains to be established
whether the uniquely fucosylated epitopes in GCX contribute to the
known Th-1 down-regulation in schistosomiasis, through a mechanism
similar to that described for the Le
(
)which covers the entire surface of the
cercariae and extends approximately 1 µm from it. The GCX is known
to be an extremely potent immunogen and is recognized by monoclonal
antibodies (mAb) that are protective in passive transfer experiments
(Grzych et al., 1982; Harn et al., 1984), by
antibodies found during natural infection (Kemp, 1970; Standen, 1952),
and by mAb raised against egg antigens (Harn et al., 1984).
Conversely, a mAb (128C3/3) prepared against cercarial glycoproteins
associated with the GCX was shown to recognize an epitope present on
both glycolipid and glycoprotein fractions of the egg antigens (Weiss
and Strand, 1985; Weiss et al., 1986; Dalton et al.,
1987). Monosaccharide inhibition studies indicated that this epitope
contains L-fucose in a unique structure which is distinct from
the Lewis X epitope (Levery et al., 1992). The latter is
present on the surface of the parasite, in the soluble materials
released from the eggs, and in the gut-associated antigens excreted
into the host circulation, as well as being a mammalian host epitope
(Ko et al., 1990; Srivatsan et al., 1992b;
Köster and Strand, 1994; van Dam et al., 1994).
10
Da
with an unusually high fucose content (Samuelson and Caulfield, 1985;
Caulfield et al., 1987). Preliminary reductive
-elimination experiments on GCX purified by lectin affinity
chromatography indicated the presence of fucose-rich complex
oligosaccharides O-linked via GalNAc (Nanduri et al.,
1991). We report here detailed structural analysis on O-glycans similarly released from Anguilla lectin-purified material and demonstrate that cercarial GCX
contains a heterogeneous population of highly fucosylated
oligosaccharides distinct from, but related to, the provisional
structures of the egg glycolipids (Levery et al., 1992). In
particular, we have defined unique multifucosylated nonreducing
terminal sequences which may constitute the immunodominant epitopes
that cross-react with mAbs against egg antigens.
Parasite Origin and Preparation of GCX
O-Glycans
Schistosoma mansoni (Puerto Rican
strain)-infected Biomphalaria glabrata snails were obtained
from Dr. Fred Lewis, Bethesda, MD, and Dr. Tag Mansour, Palo Alto, CA.
Cercariae were shed by exposing the snails to light for 2 h, collected
by centrifugation, and stored at -20 °C. The preparation of
GCX sample from frozen cercariae and its subsequent purification by
Sepharose 2B-CL column chromatography followed by Anguilla anguilla lectin affinity chromatography were performed as described
previously (Xu et al., 1994). Free fucose was removed by
Bio-Gel P-6 or dialysis against distilled water. Typically, O-glycans were released from 1-mg aliquots (by carbohydrate
content) of GCX by reductive elimination (1 M NaBH in 0.05 M NaOH at 45 °C for 16 h) and desalted
through a Dowex (50W-X 8(H)) column. Excess borates were then removed
by co-evaporation with 10% acetic acid in methanol under a stream of
nitrogen.
Periodate Oxidation and Smith Degradation
The
released O-glycans were incubated with 50 µl of 50 mM sodium m-periodate in 100 mM ammonium acetate
buffer (pH 5.5) at room temperature for 4 h in the dark. The reaction
was then quenched with 2 µl of ethylene glycol and left at room
temperature for a further 30 min. The oxidized glycans were lyophilized
and then reduced with 10 mg/ml NaBH in 2 M NH
OH at room temperature for 2 h after which the
reaction mixture was neutralized with a few drops of glacial acetic
acid, desalted on a Dowex (50W X-8(H)) column and the borates removed
by co-evaporation with 10% acetic acid in methanol under N
.
The oxidized sample was permethylated directly for FAB-MS analysis.
Alternatively, the sample was subjected to Smith degradation using 40
mM trifluoroacetic acid at 100 °C for 20 min. After drying
down under a stream of nitrogen, the partially degraded sample was
eluted off a Bio-Gel P-2 column and pooled for sequential enzyme
digestions/FAB-MS analysis.
Sequential Exo-glycosidase
Digestions
--N-Acetylhexosaminidase (from bovine
kidney, EC 3.2.1.52; Boehringer Mannheim): 0.2 unit in 100 µl of
50 mM sodium-citrate-phosphate buffer, pH 4.6, initially for
18 h and then for a further 18 h with another aliquot of fresh enzyme;
-galactosidase (from bovine testes, EC 3.2.1.23; Boehringer
Mannheim): 10 milliunits in 100 µl of 50 mM sodium-citrate-phosphate buffer, pH 4.6, for 24 h;
-galactosidase (from green coffee bean, EC 3.2.1.22; Boehringer
Mannheim): 0.5 unit in 100 µl of 50 mM sodium-citrate-phosphate buffer, pH 6.0, for 24 h;
-L-fucosidase (from bovine kidney, EC 3.2.1.51;
Boehringer Mannheim): 0.2 unit in 200 µl of 100 mM sodium
acetate buffer, pH 4.5-5.0, for 24 h. All enzyme digestions were
incubated at 37 °C and terminated by boiling for 3 min before
lyophilization. For sequential enzyme digestions, an appropriate
aliquot was taken after each digestion and permethylated for FAB-MS
analysis.
Release of Oligofucose Units and Subsequent Tagging with
ABEE
Fuc pieces were released from GCX by
incubating in 50 µl of 0.1 M aqueous trifluoroacetic acid
at 80 °C for 40 min. The reagents were evaporated to dryness under
a stream of nitrogen. For tagging with ABEE, the hydrolysate was
redissolved in 10 µl of water, followed by addition of 80 µl of
a reagent mixture containing 4 mg of sodium cyanoborohydride and 35 mg
of ABEE in a solution of methanol (350 µl) andacetic acid (45
µl), and incubated for 60-90 min at 80 °C. The reaction
was terminated by the addition of 1 ml of water, and excess ABEE was
extracted into 5
1 ml of diethyl ether. The aqueous layer
containing the tagged sample was then lyophilized, redissolved in
methanol:water, 1:1 (v/v), loaded on to a reverse phase Sep-Pak
C
cartridge (Waters), and eluted with an
increasing step-gradient of acetonitrile (15, 35, 50, 75, and 100%)
after initial washes with water. ABEE-tagged glycans were normally
recovered in the 35% acetonitrile fraction.
Chemical Derivatization for FAB-MS and GC-MS
Analysis
Peracetylation was carried out using 100 µl of
pyridine:acetic anhydride (1:1, v/v) at 80 °C for 2 h.
Permethylation using the sodium hydroxide procedure was performed as
described by Dell et al.(1994). For sugar analysis, TMS
derivatives of methyl glycosides were prepared from methanolysates (1 M methanolic-HCl at 80 °C for 16 h) of both intact GCX and
the released O-glycans using the Tri-Sil Z
derivatizing reagent (Pierce, 15 min at room temperature) after initial
re-N-acetylation with 500 µl of methanol, 10 µl of
pyridine, and 50 µl of acetic anhydride at room temperature for 15
min. The absolute configurations of glycosyl residues were determined
by GC-MS analysis of the TMS derivatives of (R)-(-)-
and (S)-(+)-2-butanol essentially as described by Gerwig et al.(1978). Partially methylated alditol acetates were
prepared from permethylated samples for GC-MS linkage analysis
according to Albersheim et al.(1967).
GC-MS Analysis
GC-MS analysis was carried out on a
Fisons Instruments MD800 machine fitted with a DB-5 fused silica
capillary column (30 m 0.32 mm internal diameter, J&
Scientific). The TMS derivatives and the partially methylated alditol
acetates were dissolved in hexanes prior to on-column injection at 65
°C. For sugar analysis, the oven was held at 65 °C for 1 min
before increasing to 140 °C at 25 °C/min and then to 200 °C
at 5 °C/min, and finally to 300 °C at 10 °C/min. For
linkage analysis, the oven was held at 65 °C for 1 min before being
increased to 290 °C at a rate of 8 °C/min.
FAB-MS Analysis
FAB-MS spectra were acquired using
a ZAB-2SE FPD mass spectrometer fitted with a cesium ion gun operated
at 20-25 kV. Data acquisition and processing were performed using
the VG Analytical Opus software. The derivatized O-glycans were aliquoted in methanol and loaded in a
1-monothioglycerol matrix. CAD MS-MS collisionally activated
decomposition spectra were recorded using a Fisons VG Analytical four
sector ZAB-T mass spectrometer in the array detector mode. The array
was calibrated using daughters of CsI clusters, and all experiments
were carried out at 4-kV collision cell voltage (8-kV primary ion beam)
using an argon gas pressure set to reduce the
C(M +
H)
of a substance P standard to two thirds of the
normal peak height prior to gas introduction. Spectra were recorded by
FAB ionization of samples in a monothioglycerol matrix (cesium ion gun)
and transmission of the
C parent ion of interest from MS1
into the collision cell. Array daughter spectra produced from the B/E
scan of MS2 were summed over the period of ionization of the sample.
NMR Analysis
H-
H COSY
spectra were acquired with a spectral width of 2900 Hz in each
dimension, and a total of 2048 complex points in F
and 512
complex points in F
. Prior to two-dimensional Fourier
transformation, data were apodized with a sine-bell weighting function.
Chemical shifts are referenced indirectly to acetone (
=
2.225 at 27 degrees Celsius).
Monosaccharide Composition of GCX
After
separation from glycogen-like material, the Anguilla lectin
affinity chromatography purified GCX was previously determined to
consist mainly of carbohydrates containing predominantly fucose,
galactose, galactosamine and glucosamine (Xu et al., 1994).
Similar data were obtained from the studies reported herein; these same
four sugars were consistently found to be the major constituents at a
molar ratio of approximately 5:1:1:1 in all batches of GCX examined, as
well as in the glycans released by reductive elimination. In addition,
GalNAcitol was the only alditol detected in the latter, suggesting that
the released glycans were originally O-linked to the protein
backbone via a GalNAc residue at the reducing end. The absolute
configurations of Gal, GalNAc, and GlcNAc were all shown to be D- while Fuc was shown to be in the L-configuration.
Nonreducing Terminal Sequence of GCX
The unusual
monosaccharide composition of the O-glycans from GCX
implicates unique multifucosylated structures. Preliminary information
pertaining to the structures of the multifucosylated epitopes was
obtained by FAB-MS analysis of permethyl and peracetyl derivatives of
reductively eliminated material. Although no molecular ions were
afforded by either derivative when analyzed in the mass range below m/z 2000, numerous strong signals corresponding to A-type
fragment ions of unusual composition were present throughout this mass
range. For the peracetyl derivatives, the major ions observed
correspond to Fuc (m/z 273,
503), Fuc
HexNAc
(m/z 560, 790) and Fuc
HexNAc
(m/z 1307, 1537, 1767) (Fig. 1). Significantly,
the presence of Fuc
indicates that some of
the fucoses of GCX must be directly linked to one another. The strong
signal at m/z 978 can be assigned to a
-cleavage ion of
composition
(OH)
Fuc
HexNAc
.
Bearing in mind the absence of
Fuc
HexNAc
(m/z 1020),
these data are consistent with the major nonreducing moiety being
((Fuc
)HexNAc)((Fuc
)HexNAc). Less abundant
components having fewer fucoses on the second HexNAc also appear to be
present.
Fuc
Hex
HexNAc
,
which is a Hex-HexNAc increment from m/z 978. The minor signal
at m/z 1077 corresponds to
Fuc
HexNAc
whereas that at m/z 1057 could be rationalized as the sodiated form of
Fuc
HexNAc
OH. Other signals are assigned in
the text.
These conclusions were supported by data from the permethyl
derivative (Fig. 2). Signals at m/z 434 and 608
correspond to FucHexNAc
and
Fuc
HexNAc
, respectively, while
an ion at m/z 331 is consistent with loss of methanol from a
Fuc
A-type ion (Fig. 2A).
The presence of a strong signal at m/z 228
(
HexNAc
) but not at m/z 260
(HexNAc
), m/z 402
(
Fuc
HexNAc
) or m/z 576
(
Fuc
HexNAc
) is consistent
with the (Fuc
) moiety being 3-linked to the terminal
HexNAc (Dell, 1987). Collision-activated dissociation (CAD) on the m/z 608 ion in tandem MS/MS analysis yielded daughter ions at m/z 157 (
Fuc
), 189
(Fuc
), 228 (
HexNAc
),
and 331 (
Fuc
), thus firmly
establishing the nonreducing terminal sequence as
±Fuc-Fuc-3HexNAc (data not shown). Signals corresponding to
Fuc
HexNAc
were present at m/z 1027, 1201, and 1375, respectively, while fragment ions
having an additional Hex-HexNAc moiety at the reducing end of this
structure afforded signals at m/z 1476, 1650, and 1824,
respectively, corresponding to
Fuc
Hex
HexNAc
(Fig. 2A). Both m/z 608
(Fuc
HexNAc
) and 1375
(Fuc
HexNAc
) were obtained
after CAD on the ion at m/z 1824 (data not shown). Taken
together, the low mass fragment ion data from the permethyl and
peracetyl derivatives defined the nonreducing terminal sequence of GCX
as
((Fuc
)HexNAc)-((Fuc
)HexNAc)-(Hex-HexNAc)-.
Since m/z 1824 is significantly more abundant than m/z 1650, which is in turn more abundant than m/z 1476, the
major glycoforms carry the full complement of fucoses on the
nonreducing HexNAc-HexNAc moiety, i.e. five in total.
Heterogeneity in fucosylation is primarily associated with the
nonreducing residue, as judged by the m/z 434:608 ratio.
Above m/z 1824 (Fig. 2B), the next major
A-type ions are m/z 2070 and 2244, corresponding to a
Fuc
HexNAc
increment from m/z 1650 and
1824, respectively. A further extension of Hex plus HexNAcitol at the
reducing end of these fragments provides an explanation for the
smallest (M + H)
molecular ions which were
observed at m/z 2568 and 2742, corresponding to
Fuc
Hex
HexNAc
HexNAcitol.
Incorporating the established partial sequence and taking into
consideration that Gal and GalNAcitol were the only Hex and HexNAcitol
found, the structures of the smallest O-glycans of GCX may
thus be defined as
((Fuc
)HexNAc)-((Fuc
)HexNAc)-((Gal-HexNAc))-((Fuc
)
HexNAc)-Gal-GalNAcitol with the major pair containing a total of 5 and
6 fucoses.
HexNAc
fragment ions are a consequence of both undermethylation and
multiple modes of glycosidic cleavages. Secondary cleavages of primary
ions would yield, for example, ions at m/z 821 and 839 which
correspond to
Fuc
HexNAc
and
(OH)
Fuc
HexNAc
,
respectively. Likewise, signals at m/z 1114, 1288, and 1462
corresponding to
(OH)
Fuc
Hex
HexNAc
were probably derived from the primary ions
Fuc
Hex
HexNAc
.
Above m/z 2000, five major (M + H)
molecular ion clusters were observed, the compositions of which
are assigned in Table 1. Other signals are assigned in the
text.
In an additional experiment, the GCX oligosaccharides
were digested with -fucosidase, permethylated, and analyzed by
FAB-MS. This resulted in a significant reduction in intensity of the
signals at m/z 434, m/z 1650 and m/z 2568
with a concomitant increase in m/z 260, 1476, and 2394,
respectively (data not shown). The remainder of the spectrum below m/z 3000 was largely unaffected, indicating first, that the
oligofucosyl residues are resistant to digestion in the intact
oligosaccharide and second, that the fucosyl heterogeneity suggested by
the molecular ion masses largely resides in the nonreducing moiety.
Thus the most abundant short glycans have the structure
((Fuc
)HexNAc)-((Fuc
)HexNAc)-((Gal-HexNAc))-((Fuc
)HexNAc)-Gal-GalNAcitol.
HexHexNAc
above m/z 1824 and by a
series of molecular ion clusters continuing to at least m/z 6000 (Fig. 2B). Linkage analysis on the permethyl
derivatives yielded predominantly t-Fucp,
2-Fucp, and an approximately equimolar amount of
3-Galp, 3-GalNAcp, and 3,4-GlcNAcp. Lesser
amounts of 4GlcNAcp, 3,6-GalNAcitol, and 3-GalNAcitol were
also detected, with the last two being present in approximately 2:1
ratio. Thus, although the smaller O-glycans from GCX are
probably linked via a type 1 core (
Gal1
3GalNAcitol), the
major portion of the glycan chains terminate with 3,6-GalNAcitol, and
are thus likely to have a branched type 2 core
(
Gal1
3(
GlcNAc1
6)GalNAcitol). The molecular ion
clusters observed at masses in excess of 3000 Da (Fig. 2B) are consistent with this conclusion. To
facilitate interpretation of these high mass data additional structural
information from chemical and enzymatic degradations was sought (see
below).
Periodate Oxidation Defines the Backbone of GCX
O-Glycans
From the linkage analysis data, it was predicted that
the 3-Gal, 3-GalNAc, and 3,4-GlcNAc residues would be resistant to
periodate oxidation, whereas the t-Fuc and 2-Fuc would not.
This provided a simple way to defucosylate without cleaving other
residues. The periodate oxidized GCX O-glycans were
permethylated and analyzed directly by FAB-MS. In the low mass region (Fig. 3A), the three dominant A-type ions at m/z 260, 505, and 954 correspond, respectively, to
HexNAc,
HexNAc
, and
Hex
HexNAc
which are consistent
with defucosylated counterparts of the major A-type ions in Fig. 2A. Importantly, the presence of
HexNAc-HexNAc
after defucosylation rules out an
alternative structure having the fucoses in chain, in between the two
HexNAcs. CAD on the ion at m/z 954 yielded strong A-type ions
at m/z 260 (HexNAc
), m/z 473 (
HexNAc
), m/z 505
(HexNAc
) and m/z 709
(HexNac
Hex
), as well as ring
cleavage daughter ions at m/z 274
(HCO-HexNAc
), m/z 478
(HCO-Hex
HexNAc
), and m/z 723 (HCO-Hex
HexNAc
), thus
unambiguously defining a terminal linear sequence of
HexNAc-HexNAc-Hex-HexNAc- (data not shown). Linkage analysis on the
periodate-oxidized sample yielded predominantly
3-Gal:4-GlcNAc:t-GalNAc:3-GalNAc at a molar ratio of
approximately 1.0:1.4:0.6:0.6, suggesting that, after oxidation, (i) a
significant portion of the 3-GalNAc in the intact sample was converted
to t-GalNAc and (ii) 3,4-GlcNAc was completely oxidized to
4-GlcNAc. It may thus be concluded that the fucoses were originally
3-linked to terminal GalNAc and to 4-linked GlcNAc, resulting in the
nonreducing terminal sequence and A-type ions as shown in Fig. Z2.
HexNAc
),
which is relatively less abundant than m/z 505 and 954, was
shown by CAD to be a mixture of both HexNAc-Hex-HexNAc and
HexNAc
-Hex sequences. Analogous A-type cleavage at
Hex
HexNAc
higher could conceivably yield the
weak ion at m/z 1403, corresponding to
(Hex
HexNAc
)-HexNAc-Hex
.
Signals at m/z 581 and 914 which correspond to (M +
H)
of HexNAc
-C
and
Hex
HexNAc
-C
indicate the presence
of minor O-glycan chains not containing the multifucosylated
repeating units. Other signals are assigned in the
text.
Periodate oxidation on the 3-GalNAcitol and
3,6-GalNacitol was expected to cleave between carbons 3 and 4 which
upon further reduction would give the reducing end structures (I) and (II), denoted as C and C
,
respectively (Fig. Z3). In accordance with this prediction,
neither 3-GalNAcitol nor 3,6-GalNAcitol was detected in the linkage
analysis of the oxidized sample. In the FAB-spectrum of the permethyl
derivatives (Fig. 3A), the smallest molecular ions were
observed at m/z 1275 and 1608, corresponding to (M +
H)
of Hex
HexNAc
-C
and Hex
HexNAc
-C
,
respectively. Both ions were shifted one mass unit higher when
borodeuteride was used to reduce the periodate-oxidized sample. CAD on
both ions yielded strong daughter ions at m/z 260, 505, and
954 corresponding to the A-type ions of
HexNAc
,
HexNAc
, and
Hex
HexNAc
, respectively (data
not shown). Elimination of a MeOH moiety (from position 3) was observed
only for the first two ions and not m/z 954, consistent with
the deduced sequence of GalNAc-4GlcNAc-3Gal-3GalNAc constituting the
nonreducing termini of these two molecular ions. Accordingly, it was
concluded that defucosylated backbones of
(GalNAc-4GlcNAc-3Gal-3GalNAc)-HexNAc- and
(GalNAc-4GlcNAc-3Gal-3GalNAc)-HexNAc-3Gal were originally attached to
C-6 and C-3 of GalNAcitol, respectively. The latter when attached to a
nonbranched 3-GalNAcitol constitutes the non-fucosylated backbone of
the aforementioned smallest intact O-glycans observed (Fig. 2). At higher mass (Fig. 3B), pairs of
molecular ions were detected at a regular increment of a
Hex
HexNAc
unit from m/z 1275/1608 to m/z 1970/2303, m/z 2665/2998, and m/z 3360/3694. Since Gal, GalNAc, and GlcNAc are present in
approximately equimolar amounts in the GCX O-glycans and,
apart from t-GalNAc, 3-Gal, 3-GalNAc, and 4-GlcNAc, are the
only residues detected in the periodate-oxidized sample, it is likely
that a linear sequence of -4GlcNAc-3Gal-3GalNAc constitutes a regular
repeating unit in the larger O-glycan chains with the 4GlcNAc
being further substituted by fucoses at position 3 in intact GCX. In
support of this tentative assignment, strong A-type ions were detected
at m/z 1199 and 1648 in the FAB spectrum of the
periodate-oxidized sample (Fig. 3A) corresponding to
(Hex
HexNAc
)-HexNAc
and
(Hex
HexNAc
)-HexNAc-Hex-HexNAc
,
respectively.
Sequential Exo-glycosidase Digestions
To define
the anomeric configurations as well as to confirm tentative sequences,
the periodate-oxidized sample was subjected to sequential
-N-acetylhexosaminidase and
-galactosidase
digestions. Aliquots were taken after each digestion and permethylated
prior to FAB-MS analysis. After
-N-acetylhexosaminidase
treatment, A-type ions at m/z 505 and 954 (cf. Fig. 3A) were significantly reduced concomitant with
the appearance of m/z 464 attributable to
Hex-HexNAc
(data not shown). In addition, the
molecular ions at m/z 1275 and 1608 were shifted to m/z 785 and 1118, respectively, consistent with the removal of the
terminal
-GalNAc
4-
-GlcNAc moiety. Other molecular ion
pairs were observed at m/z 1479/1812 and 2174/2508, consistent
with the analogous loss of two
-HexNAcs from the original pairs at m/z 1970/2303 and 2665/2978. Unexpectedly, subsequent
digestions with
-galactosidases from a variety of commercially
available sources failed to remove the putative nonreducing Gal thus
exposed, suggesting the possibility of an
-Gal, or an unusual
resistance to
-galactosidases.
-N-acetylhexosaminidase followed by
- or
-galactosidase. In these experiments the oxidized sample was
partially hydrolyzed with mild acid in order to maximize the abundance
of nonreducing termini including residues originally located in chain.
The mild acid hydrolysate was chromatographed on a Bio-Gel P-2 column
to remove salts and other contaminants. The permethyl derivative of an
aliquot of the resulting sample yielded a similar FAB spectrum to the
nonhydrolyzed, periodate-oxidized sample (Fig. 3), but
additional signals attributable to partially hydrolyzed fragments were
also present (Fig. 4A). Thus additional C
-
and C
-containing molecular ions were observed at m/z 1030 and m/z 1363, respectively (minus HexNAc from m/z 1275 and 1608, respectively), probably resulting from a
single cleavage at the particularly labile
-GalNAc
4
-GlcNAc glycosyl bond (Reason et al.,
1994). All these HexNAc-terminated
C
/C
-containing molecular ions (m/z 1030, 1275, 1363, 1608, 1970, 2303) disappeared following
-N-acetylhexosaminidase treatment, yielding new signals
at m/z 1118, 1479, and 1812, and their sodiated counterparts
at 22 units higher (Fig. 4B), consistent with the
trimming of one or two terminal
-HexNAc residues, as described
above.
-N-acetylhexosaminidase (B) followed by
-galactosidase (C). All protonated molecular ions in B also desorbed as sodiated ions at 22 units higher. The ions
at m/z 1884/1906 in B correspond to (M +
H)
/(M + Na)
of
Hex
HexNAc
, resulting from the removal of one
and two
-HexNAc moieties from m/z 2130 and 2375 in A, respectively. Other signals are assigned in the
text.
Assuming the absence of three consecutive HexNAc residues in
the sequence, for which there was no experimental evidence from any of
the experiments described above, the ions at m/z 954
(HexHexNAc
), 1199
(Hex
HexNAc
), 1648
(Hex
HexNAc
), 1893
(Hex
HexNAc
), and their
corresponding methyl glycosides 32 units higher (Fig. 4A and 5A) would necessarily terminate with a HexNAc at the
nonreducing end. This was confirmed by the
-N-acetylhexosaminidase treatment, upon which all these
ions were abolished, or greatly reduced in intensity. In contrast, ions
at m/z 709
(Hex
HexNAc
), 741
(Hex
HexNAc
-OMe + H
), 1158
(Hex
HexNAc
), 1190
(Hex
HexNAc
-OMe + H
), 1403
(Hex
HexNAc
), 1435
(Hex
HexNAc
-OMe + H
), and
2130 (Hex
HexNAc
-OMe + H
)
were retained (Fig. 4B and 5B), reflecting the
presence of Gal at their nonreducing end. Importantly, at the low mass
end of the
-N-acetylhexosaminidase treated sample (Fig. 5B), a very strong signal was present at m/z 464, corresponding to Hex-HexNAc
while the ion at m/z 505 (HexNAc
) had disappeared.
The HexNAc
ion at m/z 260 was also
significantly reduced and was largely derived from the enzyme-released
HexNAc which gave a protonated molecular ion at m/z 292.
-N-acetylhexosaminidase (B) followed by
-galactosidase (C). Signals are
assigned in the text.
As
expected from the experiments described earlier, further treatment with
a variety of -galactosidases did not result in any significant
change, indicating the absence of terminal
-Gal. In contrast,
after
-galactosidase digestion the ion at m/z 464
disappeared concomitant with the appearance of m/z 505
(HexNAc
) and a significant diminution in
the abundance of Hex
HexNAc
at m/z 709 (Fig. 5C). Significantly, all
molecular ions including the C
/C
-containing
molecular ions at m/z 1479 and 1812 (Fig. 4B)
were shifted by 204 units to lower mass (Fig. 4C),
consistent with the removal of a terminal
-Gal. The resulting
spectrum (Fig. 4C) was thus very similar to the one
prior to digestions (Fig. 4A). In summary, it is
apparent from the FAB-MS data that Smith degradation had resulted in
fragments terminating with either
-Gal- or
±
-HexNAc
-HexNAc-, the latter being trimmed down
to
-Gal termini by initial
-N-acetylhexosaminidase
digestion. Subsequent
-galactosidase treatment removed the
terminal
-Gal, giving products which could be further sequentially
digested with
-N-acetylhexosaminidase and
-galactosidase (data not shown). Focussing on the
C
/C
-containing molecular ion pair of m/z 1970/2303, sequential
-N-acetylhexosaminidase and
-galactosidase digestions firmly established that a nonreducing
terminal sequence of (
-HexNAc)
-Gal extends
from the smallest pair of m/z 1275/1608. Assuming an identical
repeating unit throughout the sequence, the backbone structures of the
GCX O-glycans may thus be defined as shown in Fig. Z4,
where n is 1, 2, 3, or 4. The failure to detect the
-galactosidase-digested products of HexNAc
-C
(m/z 581) and HexNAc
Hex
-C
(m/z 914) precluded further trimming into the reducing
end. Hence the anomeric configurations of these residues were not
defined, but they are most likely to be
-linked. Finally, the
undefined HexNAc can be either 3-GalNAc or 4-GlcNAc, although we
consider the latter to be more likely, thus corresponding to the
conventional core 1 and core 2 type structures for O-glycans.
Release of Oligofucoses and Fucosidase
Digestion
The presence of
Fuc[2Fuc
]
3-linked to the
GlcNAc in the intact GCX O-glycans was evident from the data
presented above. Mild acid hydrolysis of the intact sample released
Fuc
, Fuc
, and Fuc
, which were
readily detected as their peracetyl derivatives by FAB-MS (data not
shown). After tagging the reducing end with ABEE by reductive
amination, Fuc
-ABEE, Fuc
-ABEE, and
Fuc
-ABEE were successfully purified from the nonhydrolyzed O-glycans by reverse phase Sep-Pak C
and identified by FAB-MS (data not shown). Treatment with
-fucosidase converted both Fuc
-ABEE and
Fuc
-ABEE into Fuc
-ABEE, suggesting that the
additional fucoses are all
-linked. The anomeric configuration of
the reducing end Fuc which was originally 3-linked to GlcNAc was not
defined by this experiment although additional NMR data (see below)
indicates that it too is
-linked.
Intact Multifucosylated O-Glycans from GCX
The
successful recovery of intact Fuc and Fuc
(but
not Fuc
or larger oligomers) from mild acid hydrolysates of
GCX corroborated earlier conclusions that such moieties are present in
the intact GCX O-glycans as side chains. In addition, the
periodate oxidation data firmly established a linear backbone sequence
comprising only Gal, GalNAc, and GlcNAc residues. The extremely
heterogeneous nature of the O-glycans which constitute the
main bulk of GCX is reflected by the molecular ion clusters observed in
the FAB spectrum of their permethyl derivatives (Fig. 2B). The heterogeneity is apparently a
consequence of (i) the multifucosylated nature where the total number
of fucose residues attached varies within a defined range, (ii) the
varying number of repeating units contained within each O-glycan, and (iii) the incomplete substitution of the
``6-arm'' of the reducing end GalNAc resulting in either
linear or branched core structures. Further complexity as observed in
the spectrum was imparted by undermethylation and by the presence of
fragment ions among the molecular ion clusters.
Hex
HexNAc
increment from m/z 1824 and 1650, respectively (Fig. 2). This is consistent with the derived sequence for m/z 1824 (see above) being extended at the reducing end with
an additional
(Fuc
2Fuc
2Fuc
3)4GlcNAc
3Gal
3GalNAc
unit. In agreement with the periodate oxidation data (Fig. 4B) which indicates that the major glycan chains
contain one or two repeats of the GlcNAc-Gal-GalNAc motif, higher mass
A-type ions were not detected.
3GalNAc
4GlcNAc
3Gal), with x and y defining the number of such repeating units on each arm, then the
overall composition of the molecular ion clusters observed in Fig. 2B may be assigned as in Table 1. These
compositions suggest that each additional repeating motif carries two
or three additional fucoses.
Finally, a composite structure for the
GCX O-glycans which is consistent with all the data presented
may be schematically drawn as shown in Fig. Z6. As described
above, the number of repeating units, n, is mainly 1 or 2
although up to 4 were detected after periodate oxidation. The maximum
number of fucoses are carried when all the Fuc residues indicated by a
``±'' sign are present. Thus for example, with n = 1 on each arm, the maximum number of fucoses is 12 (see
also Table 1).
Preliminary data from high resolution H-
H NMR measurements are consistent with the
structure proposed above. The
H-
H COSY spectrum
at 500 MHz contains a variety of anomeric protons downfield of solvent
HOD in the region
= 4.7-5.5 (data not shown). The
magnitudes of the spin coupling constants
J
, which are all
3.5 Hz, and
the characteristic resonance positions suggest the presence of
-Gal and
-Fuc residues. Further evidence for
-Fuc
residues derives from a series of cross-peaks correlating the fucose
methyl groups (
1.2) with their C-5 protons. These
correlations form two distinct groups due to differences in resonance
position of their C-5 protons. In particular, a group of cross-peaks at
F
1.2 ppm and F
4.75 ppm are highly
characteristic of fucose in
(1-3) linkage, whereas a group
at F
1.2 ppm and F
4.2 ppm are
characteristic of fucose in either
(1-2) or
(1-6)
linkage (Vliegenthart et al., 1983). Hence the NMR data are
consistent with the linkage positions proposed above, and further
define the anomeric configurations as
. A variety of anomeric
proton resonances are observable at
4.4 ppm, corresponding to
-HexNAc, and while these data are not inconsistent with the
proposed structure, it is not possible from these data alone to
determine whether the HexNAc residues proximal to GalNAcitol are
3-GalNAc or 4-GlcNAc. Clarification of this structural feature will
require more detailed NMR analysis which is in progress.
1
4GlcNAc in each antenna, (ii) repeating units which
contain an
-linked galactose, (iii) di- and trifucosyl appendages,
(iv) a nonreducing epitope which is more heavily fucosylated than any
known glycoprotein-derived oligosaccharide.
1
4GlcNAc structural element, coined
``lacdiNAc,'' was believed to be restricted to a limited set
of glycoproteins. However, it now appears that this building block
might be as common as the ubiquitous Gal
1
4GlcNAc (lacNAc)
motif (Neeleman et al.(1994) and references therein). While
lacNAc-based oligosaccharide chains are prevalent on glycoproteins of
higher organisms, the reverse may be true for lower animals where
lacdiNAc appears to be preferentially synthesized as the backbone motif
of many N-glycan antennae. In parasitic helminths, for
example, the complex type N-glycans of the microfilariae Dirofilaria immitis were shown to be devoid of Gal, containing
instead the terminal sequence GalNAc
GlcNAc
Man-R (Kang et
al., 1993). Another nematode Trichinella spiralis was
found to synthesize exclusively tri- and tetra-antennary N-glycans terminating with
tyvelose1
3GalNAc
1
4(Fuc
1
3)GlcNAc
Man
(Reason et al., 1994). The adult worms of S. mansoni appear to be capable of synthesizing both lacNAc and lacdiNAc type
chains with the
GalNAc
1
4(±Fuc
1
3)GlcNAc
Man sequence
constituting the major antennae of the bi- and triantennary complex
type N-glycans (Nyame et al., 1989; Srivatsan et
al., 1992a), along with some poly-lacNAc chains carrying Le
epitopes (Srivatsan et al., 1992b). The poly-Le
motif was also found to constitute the repeating unit of the O-glycans from the circulating cathodic antigen released by
the worm into the circulation of the mammalian host (van Dam et
al., 1994).
-R
1
4 GalNAc-transferases (
4-GalNAc-T) have been
characterized (Mulder et al. (1995) and references therein)
including one from the cercariae of the avian schistosome Trichobilharzia ocellata (Neeleman et al., 1994) and
another from the albumen gland and connective tissue of its
intermediate host, the freshwater snail Lymnaea stagnalis (Mulder et al., 1995). Similar
4-GalNAc-T enzyme
activity was also demonstrated in extracts of adult S. mansoni (Srivatsan et al., 1994), supporting the presence of
lacdiNAc structures. Although the
4-GalNAc-T from the cercariae of T. ocellata can clearly be distinguished from the
1
4-galactosyltransferase (
4-Gal-T) of higher animals,
their acceptor specificities are strikingly similar (Neeleman et
al., 1994). Thus, it appears that
4-Gal-T and
4-GalNAc-T
may compete for the same
-GlcNAc-terminating substrate and that
their relative level of expression which may be developmentally
regulated and tissue-specific will determine the synthesis of either
lacNAc or lacdiNAc-type glycans, or both.
3-fucosylation of
GlcNAc and
3- or
6-sialylation of GalNAc (reviewed in Dell
and Khoo(1993)). In lower animals, however, there appears to be greater
structural diversity and, although
3-fucosylation remains common,
capping groups are frequently more esoteric, e.g. tyvelose in T. spiralis (Reason et al., 1994) and
-linked
3-MeO-Gal in hemocyanin from L. stagnalis (van Kuik et
al., 1987). The heterogeneous polyfucosylation found in GCX adds
an extra dimension to this structural diversity. It is noteworthy that
no evidence for the existence of poly-lacdiNAc structures analogous to
poly-N-acetyllactosamine has yet been found. Indeed, as far as
we are aware, all known lacdiNAc-containing glycans have only a single
lacdiNAc moiety per antenna. GCX is therefore unique among known
lacdiNAc molecules in having lacdiNAc repeats, albeit separated by
-Gal and thus not in a poly-lacdiNAc motif.
-Gal residues was unexpected because
-galactosylation is normally associated with termination, not
elongation, of glycan chains. Commonly found structures capped with
-Gal include Gal
1
3Gal
1
4GlcNAc, the blood
group P1 antigen Gal
1
4Gal
1
and the blood group B
antigen Gal
1
3(Fuc
1
2)Gal
1
. It has also
been identified in O-glycans from the jelly coat of amphibian
eggs (Strecker et al., 1992, 1995) which terminate with
Gal
1
4(Fuc
1
2)Gal
1
; in O-glycans from cobra venom which have
-galactosylated
Le
and Le
epitopes (Gowda and Davidson, 1994);
and as Gal
1
3Gal
1
4GlcNAc in N-glycans
from the bloodstream form of Trypanosoma brucei (Zamze et
al., 1991) and in N- and O-glycans of Trypanosoma cruzi trypomastigotes (Cuoto et al.,
1990; Almeida et al., 1994). In contrast, in eukaryotic
glycoconjugates, the presence of in-chain
-Gal is largely confined
to glycosphingolipids (Stults et al., 1989) and various
glycosyl-phosphatidylinositol anchored glycolipids (McConville and
Ferguson, 1993). To our knowledge, there is only one example of a
glycoprotein where in-chain
-Gal was reported to occur viz Fuc
1
3GalNAc
1
3Gal
1
3Gal
1
4GlcNAc
1
Man
in the penta-antennary N-glycans isolated from the eggs of
flounder, Paralichthys olivaceus (Seko et al., 1989).
and Le
are not
uncommon in N- and O-glycans, oligofucosyl
substituents are rare. Indeed the only such terminal sequences
previously reported are those on the O-glycans from the jelly
coat of the Mexican axolotl where Fuc
1
3Fuc
1
is
4-linked to a KDN residue (Strecker et al., 1992).
Interestingly, the cross-reacting egg antigens of S. mansoni are also extensively fucosylated. In particular, the epitope
recognized by mAb 128C3/3 (raised against cercarial glycoproteins) is
reported to contain Fuc
GlcNAc repeating units carried on a
series of novel egg glycosphingolipids (Levery et al., 1992).
However, the structure of GCX differs significantly from that proposed
for the egg glycolipids. Most strikingly, neither oligofucose nor
3-linked Gal is present in the latter, for which a backbone containing
in-chain fucose residues was proposed. However, alternative structures
were not rigorously excluded and it is possible that the nonreducing
epitope in the egg antigens could be identical to that proposed for
GCX. Alternatively mAb 128C3/3 might recognize a common
fucose-dominated topology despite differences in the primary structure
of the egg antigens and GCX.
epitope (Velupillai
and Harn, 1994). The role of the various domains of the GCX in
immunological stimulation must await testing with neoglycoconjugates
prepared with portions of the GCX.
We gratefully acknowledge the co-operation of Prof. A. Malorni and S. Howe in provision of the CAD MS/MS facility in Consiglio Nazionale delle Ricerche-Napoli (Italy), and Laura Kassotakis at Syntex for maintaining the schistosome life cycle and preparing the cercarial glycocalyx.