©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Unique Multifucosylated -3GalNAc14GlcNAc13Gal1- Motif Constitutes the Repeating Unit of the Complex O-Glycans Derived from the Cercarial Glycocalyx of Schistosoma mansoni(*)

(Received for publication, April 4, 1995)

Kay-Hooi Khoo (1)(§), Sunil Sarda (1), Xiaofei Xu (2), John P. Caulfield (2), Michael R. McNeil (3), Steven W. Homans (4)(¶), Howard R. Morris (1)(**), Anne Dell (1)(**)

From the  (1)Department of Biochemistry, Imperial College, London SW7 2AY, United Kingdom, (2)Syntex Discovery Research, Palo Alto, California 94304, (3)Department of Microbiology, Colorado State University, Ft. Collins, Colorado 80523, and (4)Institute of Biomolecular Sciences, University of St. Andrews, Fife KY16 9ST, Scotland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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, RGal13GalNAc, and type 2, RGal13(RGlcNAc16)GalNAc, core structures via repeat units of (3GalNAc14(Fuc12Fuc12Fuc13)GlcNAc13Gal), 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(14)--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.


INTRODUCTION

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)()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).

GCX has an apparent molecular mass of more than 5 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.


EXPERIMENTAL PROCEDURES

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 NHOH 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).


RESULTS

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), FucHexNAc (m/z 560, 790) and FucHexNAc (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)FucHexNAc. Bearing in mind the absence of FucHexNAc (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.


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)FucHexHexNAc, which is a Hex-HexNAc increment from m/z 978. The minor signal at m/z 1077 corresponds to FucHexNAc whereas that at m/z 1057 could be rationalized as the sodiated form of FucHexNAcOH. 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 FucHexNAc, 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 (FucHexNAc) or m/z 576 (FucHexNAc) 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 FucHexNAc 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 FucHexHexNAc (Fig. 2A). Both m/z 608 (FucHexNAc) and 1375 (FucHexNAc) 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 FucHexNAc 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 FucHexHexNAcHexNAcitol. 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.


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 FucHexNAc 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 FucHexNAc and (OH)FucHexNAc, respectively. Likewise, signals at m/z 1114, 1288, and 1462 corresponding to (OH)FucHexHexNAc were probably derived from the primary ions FucHexHexNAc. 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.

The high mass domain of Fig. 2B is characterized by a prominent A-type ion at m/z 3042 which is an increment of FucHexHexNAc 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 (Gal13GalNAcitol), the major portion of the glycan chains terminate with 3,6-GalNAcitol, and are thus likely to have a branched type 2 core (Gal13(GlcNAc16)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).

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.

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 HexHexNAc 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 (HexNacHex), as well as ring cleavage daughter ions at m/z 274 (HCO-HexNAc), m/z 478 (HCO-HexHexNAc), and m/z 723 (HCO-HexHexNAc), 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.


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 (HexHexNAc), 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 HexHexNAc higher could conceivably yield the weak ion at m/z 1403, corresponding to (HexHexNAc)-HexNAc-Hex. Signals at m/z 581 and 914 which correspond to (M + H) of HexNAc-C and HexHexNAc-C indicate the presence of minor O-glycan chains not containing the multifucosylated repeating units. Other signals are assigned in the text.




Figure Z2: Structure 2



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 HexHexNAc-C and HexHexNAc-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 HexHexNAc, 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 HexHexNAc 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 (HexHexNAc)-HexNAc and (HexHexNAc)-HexNAc-Hex-HexNAc, respectively.


Figure Z3: Structure 3



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 -GalNAc4--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.

To explore these possibilities a new batch of periodate-oxidized material was prepared and digested with -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 -GalNAc4-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.


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 -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 HexHexNAc, 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 (HexHexNAc), 1648 (HexHexNAc), 1893 (HexHexNAc), 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 (HexHexNAc), 741 (HexHexNAc-OMe + H), 1158 (HexHexNAc), 1190 (HexHexNAc-OMe + H), 1403 (HexHexNAc), 1435 (HexHexNAc-OMe + H), and 2130 (HexHexNAc-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.


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 -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 HexHexNAc 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 HexNAcHex-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.


Figure Z4: Structure 4



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.

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 FucHexHexNAc 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 (Fuc2Fuc2Fuc3)4GlcNAc3Gal3GalNAc 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.

Second, if the core structures of the O-glycans are defined as shown in Fig. Z5, where R refers to the repeating motif (3GalNAc4GlcNAc3Gal), 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.


Figure Z5: Structure 5



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).


Figure Z6: Structure 6



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.


DISCUSSION

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 GalNAc14GlcNAc 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.

Until recently the GalNAc14GlcNAc 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 Gal14GlcNAc (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 GalNAcGlcNAcMan-R (Kang et al., 1993). Another nematode Trichinella spiralis was found to synthesize exclusively tri- and tetra-antennary N-glycans terminating with tyvelose13GalNAc14(Fuc13)GlcNAcMan (Reason et al., 1994). The adult worms of S. mansoni appear to be capable of synthesizing both lacNAc and lacdiNAc type chains with the GalNAc14(±Fuc13)GlcNAcMan 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).

To date, several UDP-GalNAc:GlcNAc-R 14 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 14-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.

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. 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.

The discovery of in-chain -Gal residues was unexpected because -galactosylation is normally associated with termination, not elongation, of glycan chains. Commonly found structures capped with -Gal include Gal13Gal14GlcNAc, the blood group P1 antigen Gal14Gal1 and the blood group B antigen Gal13(Fuc12)Gal1. It has also been identified in O-glycans from the jelly coat of amphibian eggs (Strecker et al., 1992, 1995) which terminate with Gal14(Fuc12)Gal1; in O-glycans from cobra venom which have -galactosylated Le and Le epitopes (Gowda and Davidson, 1994); and as Gal13Gal14GlcNAc 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 Fuc13GalNAc13Gal13Gal14GlcNAc1Man in the penta-antennary N-glycans isolated from the eggs of flounder, Paralichthys olivaceus (Seko et al., 1989).

Although epitopes carrying several separately linked fucosyl residues, such as poly-Le 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 Fuc13Fuc1 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 FucGlcNAc 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.

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 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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant AI-23083 (to J. P. C.); a Medical Research Council Programme Grant, a Biotechnology and Biological Sciences Research Council Project Grant, and a Wellcome Trust Grant 030826 (to H. R. M. and A. D.); a Wellcome Trust Prize Fellowship 036485/Z/92/Z (to K. H. K.); and a BBSRC Student'ship (to S. S.). The CAD MS-MS work was supported by the EC Large Scale Installation Plan (EC/CNR Contract ERBGEI CT920045 REP457) located in Consiglio Nazionale delle Ricerche-Napoli (Italy). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Heiser Research Fellow at the Dept. of Microbiology, Colorado State University, Ft. Collins, Colorado 80523.

Lister Institute Centenary Research Fellow.

**
To whom correspondence should be addressed: Tel: 44-171-594-5218; Fax: 44-171-225-0458; a.dell{at}ic.ac.uk

The abbreviations used are: GCX, glycocalyx preparation used in this work; Fuc etc., indicates the presence of an unsaturated bond as a consequence of eliminating a moiety of MeOH from the permethyl derivatives, or AcOH from peracetyl derivatives; ABEE, ethyl p-aminobenzoate; AcOH, acetic acid; CAD, collision-activated dissociation; FAB, fast atom bombardment; GC, gas chromatography; lacdiNAc, N,N`-diacetyllactosediamine; lacNAc, N-acetyllactosamine; Le, Lewis X; Le, Lewis Y; -OMe, methoxy-; MeOH, methanol; MS, mass spectrometry; mAb, monoclonal antibody; Th-1, helper T cells (subclass 1); TMS, trimethylsilyl.


ACKNOWLEDGEMENTS

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.


REFERENCES
  1. Albersheim, P., Nevins, D. J., English, P. D., and Karr, A.(1967)Carbohydr. Res. 5, 340-345 [CrossRef]
  2. Almeida, I. C., Ferguson, M. A. J., Schenkman, S., and Travassos, L. R.(1994) Biochem. J. 304, 793-802 [Medline] [Order article via Infotrieve]
  3. Caulfield, J. P., Cianci, C. M. L., McDiarmid, S. S., Suyemitsu, T., and Schmid, K.(1987) J. Parasitol. 73, 514-522 [Medline] [Order article via Infotrieve]
  4. Couto, A. S., Gonalves, M. F., Colli, W., and de Lederkremer, R. M.(1990) Mol. Biochem. Parasitol. 39, 100-108
  5. Dalton, J. P., Lewis, S. A., Aronstein, W. S., and Strand, M.(1987)Exp. Parasitol. 63, 215-226 [Medline] [Order article via Infotrieve]
  6. Dell, A.(1987) Adv. Carbohydr. Chem. Biochem. 45, 19-72 [Medline] [Order article via Infotrieve]
  7. Dell, A., and Khoo, K.-H. (1993)Curr. Opin. Struct. Biol. 3, 687-693 [CrossRef]
  8. Dell, A., Reason, A. J., Khoo, K.-H., Panico, M., McDowell, R. A., and Morris, H. R. (1994)Methods Enzymol. 230, 108-132 [Medline] [Order article via Infotrieve]
  9. Dunne, D.(1990) Parasitol. Today 6, 45-48
  10. Gerwig, G. J., Kamerling, J. P., and Vliegenthart, J. F. G.(1978) Carbohydr. Res. 62, 349-357 [CrossRef]
  11. Gowda, D. C., and Davidson, E. A.(1994)J. Biol. Chem. 269, 20031-20039 [Abstract/Free Full Text]
  12. Grzych, J. M., Capron, M., Bazin, H., and Capron, A.(1982)J. Immunol. 129, 2739-2743 [Abstract/Free Full Text]
  13. Harn, D. A., Mitsuyama, M., and David, J. R.(1984)J. Exp. Med. 159, 1371-1387 [Abstract]
  14. Kang, S., Cummings, R. D., and McCall, J. W.(1993)J. Parasitol. 79, 815-828 [Medline] [Order article via Infotrieve]
  15. Kemp, W. M.(1970) J. Parasitol. 56, 713-723 [Medline] [Order article via Infotrieve]
  16. Ko, A. I., Dräger, U. C., and Harn, D. A.(1990)Proc. Natl. Acad. Sci. U. S. A. 87, 4159-4163 [Abstract]
  17. Köster, B., and Strand, M.(1994)Parasitology 108, 433-446 [Medline] [Order article via Infotrieve]
  18. Levery, S. B., Weiss, J. B., Salyan, M. E., Roberts, C. E., Hakomori, S.-I., Magnani, J. L., and Strand, M.(1992)J. Biol. Chem. 267, 5542-5551 [Abstract/Free Full Text]
  19. McConville, M. J., and Ferguson, M. A. J.(1993)Biochem. J. 294, 305-324 [Medline] [Order article via Infotrieve]
  20. Mulder, H., Spronk, B. A., Schachter, H., Neeleman, A. P., Van den Eijnden, D. H., de Jong-Brink, M., Kamerling, J. P., and Vliegenthart, J. F. G. (1995)Eur. J. Biochem. 227, 175-185 [Abstract]
  21. Nanduri, J., Dennis, J. E., Rosenberry, T. L., Mahmoud, A. A. F., and Tartakoff, A. M. (1991)J. Biol. Chem. 266, 1341-1347 [Abstract/Free Full Text]
  22. Neeleman, A. P., Van der Knaap, W. P. W., and van den Eijnden, D. H.(1994) Glycobiology 4, 641-652 [Abstract]
  23. Nyame, K., Smith, D. F., Damian, R. T., and Cummings, R. D.(1989)J. Biol. Chem. 264, 3235-3243 [Abstract/Free Full Text]
  24. Patel, T., Bruce, J., Merry, A., Bigge, C., Wormald, M., Jaques, A., and Parekh, R. (1993)Biochemistry 32, 679-693 [Medline] [Order article via Infotrieve]
  25. Reason, A. J., Ellis, L. A., Appleton, J. A., Wisnewski, N., Grieve, R. B., McNeil, M., Wassom, D. L., Morris, H. R., and Dell, A.(1994)Glycobiology 4, 593-604 [Abstract]
  26. Samuelson, J. C., and Caulfield, J. P.(1985)J. Cell Biol. 100, 1423-1434 [Abstract]
  27. Seko, A., Kitajima, K., Iwasaki, M., Inoue, S., and Inoue, Y.(1989)J. Biol. Chem. 264, 15922-15929 [Abstract/Free Full Text]
  28. Simpson, A. J. G. (1990)Parasitol. Today 6, 40-45
  29. Srivatsan, J., Smith, D. F., and Cummings, R. D.(1992a)Glycobiology 2, 445-452 [Abstract]
  30. Srivatsan, J., Smith, D. F., and Cummings, R. D.(1992b)J. Biol. Chem. 267, 20196-20203 [Abstract/Free Full Text]
  31. Srivatsan, J., Smith, D. F., and Cummings, R. D.(1994)J. Parasitol. 80, 884-890 [Medline] [Order article via Infotrieve]
  32. Standen, O. D. (1952)J. Helminthol. 26, 25-42
  33. Strecker, G., Wieruszeski, J.-M., Michalski, J.-C., Alonso, C., Leroy, Y., Boilly, B., and Montreuil, J.(1992)Eur. J. Biochem. 207, 995-1002 [Abstract]
  34. Strecker, G., Wieruszeski, J.-M., Plancke, Y., and Boilly, B.(1995) Glycobiology 5, 137-146 [Abstract]
  35. Stults, C. L. M., Sweeley, C. C., and Macher, B. A.(1989)Methods Enzymol. 179, 167-214 [Medline] [Order article via Infotrieve]
  36. van Dam, G. J., Bergwerff, A. A., Thomas-Oates, J. E., Rotmans, J. P., Kamerling, J. P., Vliegenthart, J. F. G., and Deelder, A. M.(1994) Eur. J. Biochem. 225, 467-482 [Abstract]
  37. van Kuik, J. A., Sijbesma, R. P., Kamerling, J. P., Vliegenthart, J. F. G., and Wood, E. J.(1987)Eur. J. Biochem. 169, 399-411 [Abstract]
  38. Velupillai, P., and Harn, D. A.(1994)Proc. Natl. Acad. Sci. U. S. A. 91, 18-22 [Abstract]
  39. Vliegenthart, J. F. G., Dorland, L., and van Halbeek, H.(1983)Adv. Carbohydr. Chem. Biochem. 41, 209-375
  40. Weiss, J. B., and Strand, M.(1985)J. Immunol. 135, 1421-1429 [Abstract/Free Full Text]
  41. Weiss, J. B., Magnani, J. L., and Strand, M.(1986)J. Immunol. 136, 4275-4282 [Abstract/Free Full Text]
  42. Xu, X. F., Holm, M. J., Chiu, L., Devens, B. H., and Caulfield, J. P.(1993) Mol. Biol. Cell4,455a
  43. Xu, X., Stack, R. J., Rao, N., and Caulfield, J. P.(1994)Exp. Parasitol. 79, 399-409 [CrossRef][Medline] [Order article via Infotrieve]
  44. Zamze, S. E., Ashford, D. A., Wooten, E. W., Rademacher, T. W., and Dwek, R. A.(1991) J. Biol. Chem. 266, 20244-20261 [Abstract/Free Full Text]

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