Characterization of glycosphingolipids from Schistosoma mansonieggs carrying Fuc(a1±3)GalNAc-, GalNAc(b1±4)[Fuc(a1±3)]GlcNAc-and Gal(b1±4)[Fuc(a1±3)]GlcNAc- (Lewis X) terminal structures

Manfred Wuhrer1,*, Sven R. Kantelhardt1,*, Roger D. Dennis,1 Michael J. Doenhoff2, GuÈ nter Lochnit1

and Rudolf Geyer1

1Institute of Biochemistry, University of Giessen, Germany; 2School of Biological Sciences, University of Wales, Bangor, Wales, UK

The carbohydrate moieties of glycosphingolipids from eggsof the human parasite, Schistosoma mansoni, were enzy-matically released, labelled with 2-aminopyridine (PA),fractionated and analysed by linkage analysis, partialhydrolysis, enzymatic cleavage, matrix-assisted laserdesorption/ionization time-of-¯ight mass spectrometry andnano-electrospray ionization mass spectrometry. Apartfrom large, highly fucosylated structures with ®ve to sevenHexNAc residues, we found short, oligofucosylated speciescontaining three to four HexNAc residues. Their structureshave been determined as Fuc(a1±3)GalNAc(b1±4)[ � Fuc(a1±3)]GlcNAc(b1±3)GalNAc(b1±4)Glc-PA, GalNAc(b1±4)[Fuc(a1±3)]GlcNAc(b1±3)GlcNAc(b1±3)GalNAc(b1±4)Glc-PA, Fuc(a1±3)GalNAc(b1±4)[Fuc(a1±3)]GlcNAc(b1±4) GlcNAc(b1±3)GalNAc(b1±4)Glc-PA, and Fuc(a1±3)GalNAc(b1±4)[ � Fuc(a1±2) � Fuc(a1±2)Fuc(a1±3)]GlcNAc(b1±3)GlcNAc(b1±3)GalNAc(b1±4)Glc-PA. The last

structure exhibits a trifucosyl sidechain previously identi®edon the cercarial glycocalyx. These structures stress theimportance of 3-fucosylated GalNAc as a terminal epitopein schistosome glycoconjugates. To what degree these gly-cans contribute to thepronouncedantigenicityofS. mansoniegg glycolipids remains to be determined. In addition, wehave identi®ed the compounds GlcNAc(b1±3)GalNAc(b1±4)Glc-PA, Gal(b1±4)[Fuc(a1±3)]GlcNAc(b1±3) GalNAc(b1±4)Glc-PA, the latter of which is a Lewis X-pentasac-charide identical to that present on cercarial glycolipids, aswell as Gal(b1±3)GalNAc(1±4)Gal(1±4)Glc-PA, whichcorresponds to asialogangliotetraosylceramide and is mostprobably derived from the mammalian host.

Keywords: ceramide glycanase; internal fucose; oligo-saccharide structural analysis; Schistosoma mansoni eggglycolipids.

Schistosomiasis is caused by parasitic blood ¯ukes of thegenus Schistosoma and affects 200 million people world-wide. During infection, schistosomal glycoconjugates playimportant roles in host±parasite pathological interactions[1,2]. Schistosomes produce a variety of complex carbohy-drate structures, many of which are highly fucosylated [2].These glycans are conjugated to proteins and/or lipids indifferent life-cycle stages and vary in their recognition by theimmune system. For Schistosoma mansoni, the Lewis Xepitope has been found on egg and cercarial glycoproteins

[3±5] and cercarial glycolipids [6]. Although this epitope isshared with the mammalian host, S. mansoni infectionserum contains cytolytic antibodies directed against thisepitope [7]. Besides Lewis-X-carrying glycolipids, S. man-soni expresses highly antigenic glycolipids mainly in the eggand the cercarial stage, and to a lesser extent in the adultstage [8,9]. The stage-associated expression of carbohydratestructures on S. mansoni glycolipids is paralleled by changesin glycolipid ceramide structures during the life-cycle [10].

The highly antigenic glycolipids are speci®cally recog-nized by schistosome infection serum and not by otherhelminth infection sera, which makes them possible sero-diagnostic antigens [11]. They share a fucose-containingepitope with keyhole limpet haemocyanin (KLH) [12],which is consistent with the use of KLH for the diagnosis ofschistosomiasis [13]. The induction of signi®cant titres ofantibodies against these glycolipids is associated with theonset of patency [11], and the IgE-response against wormglycolipids may play a role in mediating resistance toS. mansoni reinfection after praziquantel treatment [14]. Allschistosome glycosphingolipids share the unique corestructure, GalNAcb4Glc1±1ceramide, which was ®rstdescribed by Makaaru et al. [15]. Cercarial glycolipids werefound to be dominated by short-chained carbohydratemoieties expressing Lewis X (Gal(b1±4)[Fuc(a1±3)]Glc-NAcb1-) and pseudo-Lewis Y (Fuc(a1±3)Gal(b1±4)[Fuc(a1±3)]GlcNAc(b1-)epitopes [6],whereasthestructuralchar-acterization of unfractionated complex glycosphingolipidsfrom S. mansoni eggs has revealed the terminal structure �

Correspondence to R. Geyer, Biochemisches Institut am Klinikum,

UniversitaÈ t Giessen, Friedrichstrasse 24, D-35392 Giessen, Germany.

Fax: + 49 641 99 47409, Tel.: + 49 641 99 47400,

E-mail: Rudolf.Geyer@biochemie.med.uni-giessen.de

Abbreviations: Cer, ceramide; dHex, deoxyhexose; ESI, electrospray-

ionization; Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactos-

amine; Glc, glucose; GlcNAc, N-acetylglucosamine; Hex, hexose;

HexNAc, N-acetylhexosamine; KLH, keyhole-limpet hemocyanin;

LSIMS, liquid secondary-ion mass spectrometry; mAb, monoclonal

antibody; Man, mannose; PA, 2-aminopyridine; PGC, porous

graphitic carbon.

Enzymes: a-L-fucosidase (EC 3.2.1.51); b-D-galactosidase (EC3.2.1.23); ceramide glycanase (oligoglycosylceramideglycohydrolase;

EC 3.2.1.23).

*Note: these authors contributed equally to this work.

(Received 31 August 2001, revised 8 November 2001, accepted 14

November 2001)

Eur. J. Biochem. 269, 481±493 (2002) Ó FEBS 2002

Fuc(1±2)Fuc(1±3)GalNAc (b1- attached to a series ofup to three )4)[Fuc(1±2)Fuc(1±3)]GlcNAc(b1-repeats andone )4)[ � Fuc(1±2) � Fuc(1±3)]GlcNAc(b1-unit, whichis linked to )3)GalNAc(b1±4)Glc(1±1)ceramide, i.e. theSchisto-core [16].

In the present study, we have chosen an approach thatdiffers from the one employed in the aforementioned andother investigations of glycolipid structures from S. mansonieggs [16,17] in which complex mixtures of glycosphingo-lipids have been analysed mainly by liquid secondary-ionmass spectrometry (LSIMS) using various chemical deri-vatizations. Similar to previous studies on glycolipids fromCaenorhabditis elegans [18] and S. mansoni cercariae [6], wehave enzymatically removed the ceramide moieties andfractionated the released oligosaccharide chains beforestructural characterization by mass spectrometry, chemicaland enzymatic degradation and linkage analysis. Using thisstrategy, structural information on the carbohydrate moi-eties of individual glycolipids from S. mansoni eggs isobtained.

M A T E R I A L S A N D M E T H O D S

Glycolipid puri®cation and fractionation

S. mansoni egg glycolipids were puri®ed by organic solventextraction, saponi®cation, desalting and anion-exchangechromatography as described previously [6]. Neutral gly-colipids were fractionated on a silica-gel cartridge (Waters,Eschborn, Germany) as outlined elsewhere [19] by step-wiseelution with chloroform/methanol (80 : 20, v/v), chloro-form/methanol/water (65 : 25 : 4) and chloroform/metha-nol/water (10 : 70 : 20), and analysed by HPTLC orcinol/H2SO4-staining and HPTLC-immunostaining as describedpreviously [6]. The ®rst fraction contained ceramidemonohexoside and dihexoside and was not furtheranalysed. The second fraction was positive in HPTLC-immunostaining and was used for preparation of PA-oligosaccharides. For HPTLC-immunostaining, the mAbsM2D3H, G11P and C1C7 were provided by Q. Bickle,Department of Infectious and Tropical Diseases, LondonSchool of Hygiene and Tropical Medicine, England, whilethe mAb 290-2E6 [20] was provided by A. M. Deelder,Leiden University Medical Center, the Netherlands.

Preparation and separation of PA-oligosaccharides

The glycan moieties were released from glycolipids usingrecombinant ceramide glycanase (endoglycoceramidase IIfrom Rhodococcus sp.; Takara Shuzu Co., Otsu, Shiga,Japan) and separated from uncleaved glycolipids and freeceramides on a reverse-phase cartridge [6]. Released oligo-saccharides were labelled with 2-aminopyridine (PA) andexcess reagent was partitioned with chloroform [21].PA-oligosaccharides were fractionated on an amino-phaseHPLC column (4.6 ´ 250 mm, Nucleosil-Carbohydrate;Macherey and Nagel, DuÈ ren, Germany) at a ¯ow rate of1 mLámin)1 at room temperature and detected by ¯uores-cence (310/380 nm) [21]. The column was equilibrated with200 mM aqueous triethylamine/acetic acid, pH 7.3 : aceto-nitrile (25 : 75, v/v). A gradient of 25±60% aqueoustriethylamine/acetic acid buffer was applied within a60-min period and the column was run isocratically for a

further 10 min. Peak fractions were collected and lyophil-ized. Heterogeneous fractions were resolved further on aporous graphitic carbon (PGC)-column (4.6 ´ 100 mm,Hypercarb; Hypersil, Runcorn, UK) at a ¯ow rate of1 mLámin)1 at room temperature with ¯uorescence detec-tion (310/380 nm). The column was equilibrated with20 mM triethylamine/acetic acid, pH 5.0. A gradient from0 to 30%acetonitrile was applied in 50 min. Individual peakfractions were collected and lyophilized.

MALDI-TOF MS and ESI MS

MALDI-TOF MS was performed on a Vision 2000 (Ther-moFinnigan, Egelsbach, Germany) equipped with a UVnitrogen laser (337 nm) as described previously [6]. Theinstrument was operated in the positive-ion re¯ectron modethroughout using 6-aza-2-thiothymine (Sigma) as matrix.ESI MS was performed with an Esquire 3000 ion-trap massspectrometer (Bruker Daltoniks, Bremen, Germany)equippedwithanoff-linenano-ESI source.A2±5 lLaliquotof PA-oligosaccharides in methanol/water (1 : 1, v/v) orglycolipids in chloroform/methanol/water (10 : 20 : 3) wasloaded into a laboratory-made, gold-coated glass capillaryand electrosprayed at a voltage of 700±1000 V using N2 asdry-gas (120 °C, 4 Lámin)1). The skimmer voltage was set to30 V,except forFig. 7AandB,where55Vwereapplied.Foreach spectrum 20±100 repetitive scans were averaged. Theaccumulation time was between 5 and 50 ms. All MS/MSexperiments were performed with helium as collision gas.

Enzymatic and chemical degradation

PA-oligosaccharides were treated with either a-fucosidasefrom bovine kidney (4 mUálL)1; Roche Diagnostics,Mannheim, Germany) or with b-galactosidase from bovinetestes (4 mUálL)1; Roche Diagnostics) on the MALDI-TOFMS target [22]. Enzymes were dialysed for 4 h against25 mM ammonium acetate solution adjusted to pH 5.0 fora-fucosidase and pH 4.0 for b-galactosidase. Aftermeasurement of the educts by MALDI-TOF MS using6-aza-2-thiothymine matrix, dialysed enzymes (2 lL) wereadded undiluted to sample aliquots on the target and spotswere analysed again by MALDI-TOFMS after incubationovernight at 37 °C. For chemical defucosylation, driedsamples were treated with 48% HF at 4 °C overnight(modi®ed from [23]). HF was removed by a stream ofnitrogen.

Monosaccharide composition and linkage analysis

After hydrolysis in 4 M aqueous tri¯uoroacetic acid at100 °C for 4 h and labelling with anthranilic acid, mono-saccharides were determined by HPLC and ¯uorescencedetection [9,24]. For linkage analysis, PA-oligosaccharideswere permethylated with methyl iodide after deprotonationwith lithium methylsul®nyl carbanion [25] and hydrolysed(4 M aqueous tri¯uoroacetic acid, 100 °C, 4 h). Partiallymethylated alditol acetates obtained after sodium boro-hydride reduction and peracetylation were analysed bycapillary GC followed by ¯ame ionization detection orchemical ionization mass spectrometry (single ion monitor-ing), using a moving needle injector, fused silica bondedphase capillary columns of different polarity (60-m DB-1

482 M. Wuhrer et al. (Eur. J. Biochem. 269) Ó FEBS 2002

and 30-m DB-210; ICT, Bad Homburg, Germany) andhelium as carrier gas as detailed elsewhere [26].

R E S U L T S

Preparation of glycolipids

Glycolipids were isolated from S. mansoni eggs analogouslyto the study performed on S. mansoni cercarial glycolipids[6] and analysed by HPTLC (Fig. 1). Orcinol/H2SO4-staining (lane 1) revealed some major, slow-migratingcompounds and a weaker staining for smaller, minor

components. Both murine S. mansoni infection serum (lane2) and four monoclonal antibodies (lanes 3±6) visualizedantigenic glycolipids. Murine infection serum and the mAbsM2D3H, G11P and C1C7 exhibited a similar pattern,whereas the mAb 290±2E6, which is known to recognizemono- and difucosylated structures like GalNAc(b1±4)[ � Fuc(a1±2)Fuc(a1±3)]GlcNAc(1- [20], displayed a com-pletely different pattern. The murine infection serum andmAb M2D3H were especially ef®cient in detecting the fast-migrating glycolipids (lanes 2 and 3), which orcinol/H2SO4-staining revealed to be present in only low amounts. Weexpected these minor, fast-migrating, antigenic glycolipidsnot to be covered by the structural characterization of wholemixtures of complex glycolipids [16,17] and thereforedecided to fractionate these compounds as their corre-sponding PA-oligosaccharides and to structurally charac-terize the individual species.

Preparation and separation of PA-oligosaccharides

Glycans were released from the ceramide moieties byceramide glycanase treatment of complex egg glycolipids.For the separation of uncleaved glycolipids and ceramidesfrom the released oligosaccharides, the sample was frac-tionated on a reverse-phase cartridge. Released oligosac-charides were collected as the combined ¯ow-through andwash fractions, while the uncleaved glycolipids and cera-mide moieties were obtained by elution with organicsolvents. Released glycans and uncleaved glycolipids werequantitated by monosaccharide composition analysis(Table 1), showing an overall ef®cacy of over 95% glycanrelease. Released oligosaccharides were analysed by MAL-DI-TOF MS (Fig. 2C) and the obtained pattern was verysimilar to the patterns observed for the intact glycolipids inESI- and MALDI-TOF MS (Fig. 2A,B). This indicatedthat the released oligosaccharides were representative for theglycans of the major complex egg glycolipids. The releasedoligosaccharides were pooled and labelled with the ¯uores-cent tag, 2-aminopyridine (PA). PA-oligosaccharides werefractionated by amino-phase HPLC (Fig. 3A). Collectedfractions (1 to 25; hereafter, fractions denoted by numberonly) were screened by MALDI-TOF MS and assessed formonosaccharide content by composition analysis (Tables 2and 3). Fractions 1 to 7 were not found to containcarbohydrate. Starting with 8, MALDI-TOF MS revealedseveral compounds for most of the fractions (Fig. 4). In

-CTH

-CTetH

1 2 3 4 5 6

Fig. 1. HPTLC of S. mansoni egg glycolipids. S. mansoni egg glycoli-

pids were resolved with chloroform/methanol/0.25% aqueous KCl

(50 : 40 : 10, v/v/v) and visualized by orcinol/H2SO4 staining (lane 1)

or immunostaining using a pool of eight murine S. mansoni infection

sera (lane 2) and themAbsM2D3H (1 : 20 000; lane 3), G11P (1 : 200;

lane 4), C1C7 (1 : 200; lane 5) and 290±2E6 (1 : 50; lane 6). CTH and

CtetH mark the migration positions of globotriaosyl- and globotet-

raosylceramide standards, respectively.

Table 1. E�cacy of ceramide glycanase cleavage of the complex egg glycolipids shown by monosaccharide composition analysis. Complex egg

glycolipids were cleaved with ceramide glycanase and fractionated on a reverse-phase cartridge. The aqueous fractions of two experiments were

combined (water fraction) as well as the organic solvent-eluted fractions (organic solvent fraction) and compared by composition analysis to the

starting complex egg glycolipids. The amounts of monosaccharides are given in micrograms and their relative ratios are normalized to GalNAc �2.0 in parentheses.

Monosaccharide

Egg glycolipid

fraction

Released monosaccharide (lg)

Water fraction Organic solvent fraction Released monosaccharide (%)

GalNAc (2.0) 235 (2.0) 4 (2.0) 98

GlcNAc (3.1) 375 (3.2) 18 (8.6) 95

Gal (0.3) 27 (0.2) 9 (4.2) 76

Glc (2.1) 329 (2.8) 35 (16.6) 90

Fuc (3.8) 522 (4.5) 10 (4.6) 98

S 1488 75 95

Ó FEBS 2002 Schistosoma mansoni egg glycolipids (Eur. J. Biochem. 269) 483

order to reduce peak heterogeneity and obtain as far aspossible pure compounds, the amino-phase fractions 8 to 14were subfractionated by PGC-HPLC (Fig. 3B,C). Subfrac-tions (designated, for example, 12-5 for subfraction 5 offraction 12) were again screened by MALDI-TOF MS (cf.insets in Fig. 3B,C and Table 3).

Structural elucidation of individual PA-glycans

Individual PA-glycans were analysed by composition anal-ysis, linkage analysis, ESI MS/MS, as well as chemical and

enzymatic degradation followed by a second linkageanalysis. ESI MS fragments were assigned according tothe nomenclature introduced by Domon & Costello [27].Anomeric con®gurations were in some cases determinedenzymatically (Fig. 5D,G), but generally assigned based onthe results of egg glycolipid CrO3 oxidation, which indicatedb-anomeric linkages for GlcNAc and GalNAc anda-anomeric linkage for fucose as described recently [9].

The major compound in 8 as judged fromMALDI-TOFMS (Fig. 4) was Hex3HexNAc1PA. After rechromatogra-phy by PGC-HPLC, it was detected in 8-11 by MALDI-

Fig. 2. Mass spectrometry of unfractionated

complex glycolipids and derived oligosaccha-

rides from S. mansoni eggs. (A) ESI MS and

(B) MALDI-TOF MS of complex S. mansoni

egg glycosphingolipids. (C)MALDI-TOFMS

of oligosaccharides released from S. mansoni

egg glycosphingolipids.

484 M. Wuhrer et al. (Eur. J. Biochem. 269) Ó FEBS 2002

TOFMS. ESIMS2 of this compound revealed the sequenceHex-HexNAc-Hex-Hex-PA and thus indicated a non-Schisto-core (Fig. 6A). Linkage analysis showed majorproportions of 3-substituted GalNAc, 4-substituted galac-tose and terminal galactose (Table 2), and the anomericcon®guration of the latter was determined byMALDI-TOFMS/on-target enzymatic cleavage with b-galactosidase frombovine testes (data not shown). Taken together, thestructure was found to be Gal(b1±3)GalNAc(1±4)Gal(1±4)Glc-PA (Table 4), which probably corresponds to the host-derived glycolipid asialogangliotetraosylceramide. As aminor component in 8-11, the proton adduct of Hex1Hex-NAc2PA was detected by ESI MS at m/z 664.9, and itssequence was determined by ESI MS2 to be HexNAc-HexNAc-Hex-PA (data not shown). Linkage analysis offraction 8-11 indicated minor amounts of terminal GlcNAc.Based on the assumption of a Schisto-core structure, thisleads to the structure GlcNAc(b1±3)GalNAc(b1±4)Glc-PA(Table 4) for this minor compound. Fraction 9 was notanalysed further, but its major compound Hex-NAc2Hex2PA ([M + Na]+ at 849.5; Fig. 4) might beidentical with the PA-tetrasaccharide Galb3GlcNAcb3Gal-NAcb4Glc-PA derived from S. mansoni cercarial glycoli-pids [6]. For 10-10, linkage analysis before and after fucoseremoval by HF-treatment allowed the localization of thefucose at the 3-position of GalNAc and the assignment of

the structure (Tables 2 and 4). In the case of 11-7, terminalfucose and galactose (Table 2) had a- and b-anomericcon®guration, respectively, as determined by on-targetenzymatic cleavage with bovine kidney a-fucosidase andb-galactosidase from bovine testes (data not shown).Linkage analyses before and after preparative removal offucose by HF-treatment (Table 2) and ESI MS/MS(Fig. 6B,C) indicated 11-7 to have the Lewis X-containingstructure Gal(b1±4)[Fuc(a1±3)]GlcNAc(b1±3)GalNAc(b1±4)Glc-PA.

12-5was found to contain fucose both in the 3-position ofGalNAc and in the 3-position of GlcNAc (cf. linkageanalysis, Table 2). ESI MS2 (Fig. 7B) showed the twofucosylated HexNAc residues to be adjacent to each other(B3 ion at m/z 721.5). One of the fucoses of 12-5 could beremoved enzymatically, while the second was only removedbyHF-treatment (Fig. 5B,E,H).These accumulateddata for12-5 resulted in the structure Fuc(a1±3)GalNAc(b1±4)[Fuc(a1±3)]GlcNAc(b1±3)GalNAc(b1±4)Glc-PA (Table 4).The species 13-4 exhibited both terminal GalNAc andterminal fucose (Table 2). Loss ofHexNAc inESIMS2 (Y4a

at 1036.1; Fig. 8B) corroborated the presence of a terminalHexNAc. Loss of a further HexNAc residue appeared onlytogetherwith the loss of fucose (Y3 atm/z 687.0 but no signalat m/z 833), which indicated fucose to be linked to thesubterminal HexNAc residue. Bovine kidney a-fucosidase

Fig. 3. HPLC separation of egg glycolipid-

derived PA-oligosaccharides. (A) Separation

on an amino-phase column. Elution positions

of the PA-labelled dextran hydrolysate stan-

dards of di�erent chain-length are indicated by

arrows. Fractions devoid of carbohydrate-

positive material are marked by asterisks (*).

Rechomatography of fractions 11 (B) and 12

(C) an a PGC-column and analysis of the

major peaks byMALDI-TOFMS (insets). n,

minor compound identical in mass with

10-10.

Ó FEBS 2002 Schistosoma mansoni egg glycolipids (Eur. J. Biochem. 269) 485

did not act on 13-4, but the fucose could be removed byHF-treatment (Fig. 5). Taken together, the data showed 13-4 torepresent the structure shown in Table 4.

Subfractionation of 14 led to the resolution of difucosy-lated 14-2 and 14-3, trifucosylated 14-4 and tetrafucosylated14-5 species. ESI MS2 analysis of 14-5 (Fig. 9B) showed thefour fucose residues to be linked to the two outermostGalNAc and/orGlcNAc residues (B4 ion at m/z 1013.2).Three of the fucose residues are attached to one of theseHexNAc residues (Y4bB4 atm/z 664.9) to form a Fuc(a1±2)Fuc(a1±2)Fuc(a1±3)GlcNAc unit (see linkage analysis,Table 2), and this ion could lose one or two fucose residueson further fragmentation (Fig. 9C). The HexNAc, whichcarried this oligofucosyl chain is not outermost, as indicatedby the Y6aY4b ion at m/z 1182.8 (Fig. 9B). An ion at m/z1328, which would have indicated the loss of one fucose and

one HexNAc (Y4b) was not registered in the 14-5 MS2

shown in Fig. 9B, but was detected in similar MS2

experiments as a minor ion (not shown), thus corroboratingthe location of the trifucosyl chain on the second and not onthe outermost HexNAc. Taken together with the linkageanalysis data (Table 2), the structure of 14-5 couldbe elucidated as Fuc(a1±3)GalNAc(b1±4)[Fuc(a1±2) Fuc(a1±2)Fuc(a1±3)]GlcNAc(b1±3)GlcNAc(b1±3) GalNAc(b1±4) Glc-PA (Table 4). The simultaneous presence ofisobaric structural isomers in this fraction could not beexcluded. Similarly, ESIMS2 of the 14-4 [M +Na + H]2+

ion at m/z 766 (data not shown) yielded a [Hex-NAc2dHex3 + Na]+ ion at 867.0 Da, indicating the threefucoses to be linked to adjacent HexNAc residues. For 14-4,the loss of one fucose and one HexNAc resulted in anintense fragment ion at m/z 1182.0, which showed that the

Table 2. Analysis of PA-oligosaccharides by MALDI-TOF MS, ESI MS, composition analysis (CA) and linkage analysis (LA). Masses were

determined byMALDI-TOFMS and ESIMS (*) and were rounded to the ®rst decimal place. The type of pseudomolecular ion is given in brackets

and the calculated, monoisotopic masses in parentheses. 10-10HF, fraction 10-10 after HF-treatment, etc. t-Fuc, terminal fucose; 4-Gal,

4-substituted galactose, etc.

Fraction

Measured mass (Theoretical mass)

[Pseudomolecular ion] Deduced composition Key structural data

8-11 808.0 (808.3) [M + Na]+; 404.5*

(404.7) [M + H + Na]2+Hex3 HexNAc1 PA

Hex1 HexNAc2 PA

LA: t-Gal, 4-Gal, 3-GalNAc, t-GlcNAc

664.9 (665.3) [M + H]+

10-10 1036.1 (1036.4) [M + Na]+ Hex1 HexNAc3 dHex1 PA CA: GlcN:GalN (1.1 : 2.0)

LA: t-Fuc, 4-GlcNAc, 3-GalNAc

10-10HF 890.9 (890.4) [M + Na]+ Hex1 HexNAc3 PA LA: t-GalNAc, 4-GlcNAc, 3-GalNAc

11-7 995.6 (995.4) [M + Na]+ Hex2 HexNAc2 dHex1 PA CA: GlcN:GalN:Gal:Fuc

(1.1 : 1.2 : 0.9 : 1.0)

LA: t-Fuc, t-Gal; 3-GalNAc; 3,4-GlcNAc

11-7HF 827.2* (827.4) [M + H]+ Hex2 HexNAc2 PA LA: t-Gal; 4-GlcNAc; 3-GalNAc

12-5 1182.5 (1182.5) [M + Na]+;

591.6* (591.7) [M + H + Na]2+Hex1 HexNAc3 dHex2 PA LA: t-Fuc; 3-GalNAc; 3,4-GlcNAc

12-5HF 445.6* (445.7) [M + H + Na]2+ Hex1 HexNAc3 PA LA: t-GalNAc; 4-GlcNAc; 3-GalNAc

13-4 1240.6 (1239.6) [M + Na]+;

620.5* (620.3) [M + H + Na]2+Hex1 HexNAc4 dHex1 PA LA: t-Fuc; t-GalNAc; 3-GlcNAc;

3-GalNAc; 3,4-GlcNAc

13-4HF 547.3* (547.2) [M + H + Na]2+ Hex1 HexNAc4 PA LA: t-GalNAc; 4-GlcNAc, 3-GlcNAc;

3-GalNAc

14-2 1384.4 (1385.5) [M + Na]+ Hex1 HexNAc4 dHex2 PA LA: t-Fuc; 4-GlcNAc; 3-GalNAc;

3,4-GlcNAc

14-3 1384.4 (1385.5) [M + Na]+ Hex1 HexNAc4 dHex2 PA CA: GlcN:GalN:Fuc (2.1 : 2.0 : 1.9)

LA: t-Fuc; 3-GlcNAc; 3-GalNAc;

3,4-GlcNAc

14-3HF 547.0* (547.2) [M + H + Na]2+ Hex1 HexNAc4 PA LA: t-GalNAc; 4-GlcNAc; 3-GlcNAc;

3-GalNAc

14-4 1530.8 (1531.6) [M + Na]+;

766.5* (766.3) [M + H + Na]2+Hex1 HexNAc4 dHex3 PA CA: GlcN:GalN:Fuc (2.1 : 2.0 : 2.3)

LA: t-Fuc; 2-Fuc; 3-GlcNAc;

3-GalNAc; 3,4-GlcNAc

14-5 839.1* (839.3) [M + H + Na]2+ Hex1 HexNAc4 dHex4 PA CA: GlcN:GalN:Fuc (2.2 : 2.0 : 3.2)

LA: t-Fuc; 2-Fuc; 3-GlcNAc;

3-GalNAc; 3,4-GlcNAc

14-5HF 1094.5 (1093.5) [M + Na]+ Hex1 HexNAc4 PA LA: t-GalNAc; 4-GlcNAc; 3-GlcNAc;

3-GalNAc

15 1385.8 (1385.5) [M + Na]+ Hex1 HexNAc4 dHex2 PA CA: GlcN:GalN:Fuc (2.1 : 1.8 : 2.0)

LA: t-Fuc; 4-GlcNAc; 3-GlcNAc;

3-GalNAc; 3,4-GlcNAc

15HF 1094.6 (1093.5) [M + Na]+ Hex1 HexNAc4 PA LA: t-GalNAc; 4-GlcNAc; 3-GlcNAc;

1071.1* (1071.4) [M + H]+ 3-GalNAc

16-4 1587.4 (1588.6) [M + Na]+ Hex1 HexNAc5 dHex2 PA CA: GlcN:GalN : Fuc (2.9:2 : 2.8)

16-5 1876.7 (1880.7) [M + Na]+ Hex1 HexNAc5 dHex4 PA CA: GlcN:GalN : Fuc (3.2:2 : 4.3)

486 M. Wuhrer et al. (Eur. J. Biochem. 269) Ó FEBS 2002

outermost HexNAc carried only one fucose residue.Together with the linkage analysis data, this allowed thededuction of the structure shown in Table 4. For 14-2 and14-3, MALDI-TOF MS indicated similar compositions asfor 15. Linkage analysis, however, revealed a difference inthe substitution positions at the monosubstituted GlcNAc.While 14-2 contained only 4-substituted and 14-3 only3-substituted GlcNAc, 15 exhibited approximately equalamounts of 3- and 4-substitutedGlcNAc (Table 2). Linkageanalysis of 15 before and after HF showed fucose to belinked to the 3-position of GalNAc and to the 3-position of3,4-disubstituted GlcNAc. ESIMS2 of 15 (Fig. 7D) showedaB3 ion atm/z 721.0, whichwas the same branched terminalgroup as 12-5 (Table 4). As for 12-5, also in the case of 15, a-fucosidase from bovine kidney could remove only onefucose from this difucosylated terminal group (Fig. 5G).Taken together the structure of 15 was shown to be Fuc-(a1±3)GalNAc(b1±4)[Fuc(a1±3)]GlcNAc(b1±3/4)Glc NAc(b1±3)GalNAc(b1±4)Glc-PA, containing either a 3-substi-tuted or 4-substituted GlcNAc unit (Table 4).

Characterization of large PA-glycans

Due to the pronounced heterogeneity and increasingcomplexity of fractions 16 to 25, we could only partiallycharacterize these compounds by MALDI-TOF MS(Fig. 4), carbohydrate composition and linkage analysis(Table 3).MALDI-TOFMS revealed compositions with anincreasing number of HexNAc residues. The highest peak inthis region, 21 (Fig. 3), contained as its major compoundHex1HexNAc6dHex7PA, which is consistent with the resultsof MALDI-TOF MS and ESI MS analyses of total eggglycosphingolipids and released glycans (Fig. 2). While 19to 22 contained almost exclusively PA-oligosaccharides withsix HexNAc residues, species with ®ve HexNAc were mostintense in 16 to 18. Likewise, 23 to 25 were dominated byPA-oligosaccharides with seven HexNAc residues, whichhave not been described in previous studies [16,17]. Linkageanalyses of the intact PA-oligosaccharides revealed3-substituted GalNAc as the only GalNAc species through-

out and fucose as the only terminal sugar (Table 3). Linkageanalysis of 24 after fucose removal by HF-treatmentresulted in the loss of all fucose species and the appearanceof 3-substituted and terminal GalNAc in similar amounts,while all GlcNAc residues were converted to 4-substitutedGlcNAc (data not shown). This shows GalNAc to be theoutermost HexNAc in 24. The composition analyses of 18to 25 imply that all complex PA-oligosaccharides contain anaverage of 2 GalNAc residues. Of the ®ve HexNAc residuesin 18, approximately two are GalNAc and three areGlcNAc. Of the six HexNAc residues in 19 to 22,approximately two are GalNAc and four are GlcNAc,and for the species in 24, the average HexNAc compositionis twoGalNAc and ®ve GlcNAc residues. This supports thehypothesis, that there is one GalNAc residue both at thereducing and at the nonreducing end of the HexNAc chain,the ®rst being involved in the Schisto-core structure, whilethe core of the HexNAc chain would appear to consist ofGlcNAc residues throughout. This is consistent with thestructures proposed by Khoo et al. [16]. Concerningfucosylation, there exists a vast heterogeneity, as exempli®edby the detection of Hex1HexNAc6dHex2)8 in 21. Severalfractions showed a 2-substituted fucose/terminal fucoseratio of approximately 1 : 1 (Table 3), which could beexplained by the occurrence of difucosylated chainsthroughout. However, based on the observation of atrifucosylated HexNAc in 14-5 and odd fucose numberspermolecule, e.g. in 21, it can be assumed that trifucosylatedHexNAc residues may also occur in some of these complexPA-oligosaccharides. Taken together, the partial character-ization of these larger PA-oligosaccharides after amino-phase fractionation gave a detailed overview of theHexNAcchain-lengths and fucosylation heterogeneity, whichwas notobtained by FAB-MS analyses of the entire mixture ofglycolipids [16,17].

D I S C U S S I O N

In this study, individual S. mansoni egg glycolipidstructures have been elucidated. Seven of the determined

Table 3. Composition and linkage analyses of the large PA-oligosaccharides derived from complex glycolipids of S. mansoni eggs.As for composition

analysis, molar ratios based on GalNAc � 2.0 were determined after hydrolysis and reverse-phase chromatography of the anthranilic acid-

derivatized components. The components were quanti®ed by application of a standard mixture for determination of the individual detection

response factors. PA-Glc conjugates were not registered. For linkage analysis, the partially methylated monosacharide derivatives obtained after

hydrolysis, reduction and peracetylationwere analysed byGC/MS andGCusing chemical ionization in conjunctionwith single-ionmonitoring and

¯ame-ionization detection, respectively. Results are expressed as peak ratios of the alditol acetates after ¯ame-ionization detection on the basis of

3-GalNAc � 2.0 forHexNAc species and t-Fuc � 1 for fucose species. For fraction 25, peak ratios were determined byGC/MS due to the limited

amount of material. 4-GlcNAc, 4-substituted GlcNAc, etc.

Fraction

Composition analysis Linkage analysis

GalNAc GlcNAc Fuc 2-Fuc: t-Fuc

4-GlcNAc: 3,4-Glc-

NAc: 3-GalNAc

18 2.0 2.84 3.16 0.9 : 1.0 1.4 : 3.3 : 2.0

19 2.0 3.23 2.48 0.95 : 1.0 1.55 : 3.55 : 2.0

20 2.0 3.64 2.85 0.8 : 1.0 1.55 : 4.0 : 2.0

21 2.0 3.62 4.65 1.05 : 1.0 1.2 : 5.15 : 2.0

22 2.0 3.67 4.00 0.8 : 1.0 1.25 : 4.6 : 2.0

23 2.0 4.04 3.33 0.95 : 1.0 1.45 : 3.25 : 2.0

24 2.0 4.51 4.09 0.5 : 1.0 2.0 : 4.15 : 2.0

25 2.0 3.88 4.63 0.65 : 1.0 0.7 : 6.45 : 2.0

Ó FEBS 2002 Schistosoma mansoni egg glycolipids (Eur. J. Biochem. 269) 487

Fig. 4. MALDI-TOF MS analysis of the HPLC-fractionated PA-oligosaccharides. Fractions 8 to 25 (8 to 25) of the amino-phase separated

PA-oligosaccharides (Fig. 3) were analysed by MALDI-TOF MS as their sodium (8 to 19) or lithium adducts (20 to 25). Fucose increments are

indicated by arrows. #, proton adduct; s, potassium adduct; *, contaminant.

488 M. Wuhrer et al. (Eur. J. Biochem. 269) Ó FEBS 2002

structures consist of fucosylated HexNAc chains based onthe Schisto-core structure. They are in agreement with thestructure proposed byKhoo et al. [16], which is�Fuc(1±2)Fuc(1±3)GalNAc(1±4)([Fuc(1±2)Fuc(1±)3]GlcNAc (1±4))1)3[ � Fuc(1±2) � Fuc(1±3)]GlcNAc(1±3)GalNAc (1±4) Glc-(1±1)Cer. The reported role of 3-fucosylated GalNAc as themajor structural motif at the nonreducing end of theHexNAc chain [16] is supported by our analyses ofindividual PA-oligosaccharides (10-10, 12-5, 15, 14-4 and14-5) and by the linkage analyses of larger PA-oligosac-charides, in particular 24, before and after HF-treatment.Recently, the terminal motif GalNAc(b1±4)GlcNAc- hasbeen identi®ed as a good acceptor for fucosylation byS. mansoni egg extracts, leading to a heterogeneity offucosylated products, which parallels our ®nding of variousoligofucosylated terminal structures [28].

Our data, however, do not support the proposed Fuc(a1±4)[Fuc(a1±3)]GlcNAc-terminal motif, or the interdigitationof the HexNAc chain by fucose residues [17], becauseterminal GlcNAc was never generated by defucosylation inany of the PA-oligosacchride fractions studied, and MSanalyses of HF-treated individual PA-oligosaccharides onlyrevealed the loss of fucose and not of HexNAc residues.

Our data require an extension of the existing picture.While the structure outlined by Khoo et al. [16] comprisesHexNAc4-HexNAc6 chains, the structures 10-10 and 12-5show that fucosylated glycolipids with HexNAc3-chainsalso exist. Together with the Hex1HexNAc2PA compound

found in 8-11, these carbohydrate chains obviously repre-sent the Ômissing linkÕ between the Schisto-core and theextended structures described [16]. Likewise, 23 to 25 aredominated by HexNAc7 species, which also have not beendetected in the FAB-MS analyses [16,17]. Secondly, we havefound trifucosyl sidechains on 14-5, which have so far onlybeen described for O-glycans derived from the cercarialglycocalyx [29] and not for egg glycolipids [16]. The highrelative amount of 2-Fuc in the larger PA-oligosaccharidesfurther indicates that trifucosyl sidechains are also likely tooccur in the glycolipids with HexNAc5-HexNAc7 chains.Thirdly, 13-4 shows that incomplete fucosylation may alsooccur, leading in low amounts to terminal GalNAc residuesinvolved in GalNAc(b1±4)[Fuc(a1±3)]GlcNAc(b1- units, ashave been described for N-glycans of adult worm glyco-proteins [30]. Fourthly, the characterization of the HexNAcbackbone revealed in some cases a deviation from theGalNAc(1±4)(GlcNAc(1±4))1)3GlcNAc(1±3)GalNAc(1±4)Glc(1±1)ceramide basic structure described by Khoo et al.[16], as we found this motif only in one of the elucidatedstructures (14-2, which is identical to one compound in 15),while four structures (13-4, 14-3, which is identical to asecond compound in 15, 14-4 and 14-5) showed themotif±GalNAc(1±4)GlcNAc(1±3)GlcNAc(1±3)GalNAc(1±4) Glc(1±.The presence of internal 3-substituted GlcNAcresidues is in agreement with previous studies on thecarbohydrate structure of a cercarial Lewis-X-containingceramide hexahexoside [6].

Fig. 5. Fucose removal from individual PA-oligosaccharides by a-fucosidase or HF-treatment. MALDI-TOF MS of 13-4 before (A) and after

(B) HF-treatment, intact 12-5 (C), 12-5 after a-fucosidase treatment (D), 12-5 after HF-treatment (E), intact 15 (F), 15 after a-fucosidase treatment

(G), and 15 after HF-treatment (H).

Ó FEBS 2002 Schistosoma mansoni egg glycolipids (Eur. J. Biochem. 269) 489

Apart from the dominant glycolipids with fucosylatedHexNAc chains, we characterized in this study the glycan ofa Lewis X-containing glycolipid (11-7), which we previouslyhave shown to be the dominating glycolipid of S. mansonicercariae [6]. Though this result shows that Lewis Xglycolipids are not absolutely restricted to the cercarialand schistosomular life-cycle stage, they are drasticallydownreguated in the egg and thus have not been detected byHPTLC-overlay [9].

The ®nding of a PA-oligosaccharide corresponding toasialogangliotetraosylceramide (8-11) structurally identi®esa glycolipid which the parasite most probably has taken upfrom the host. MALDI-TOF MS of the correspondingintact glycolipid after silica-gel puri®cation showed that itsceramides contain more than 40 carbon atoms (data notshown), which indicated the presence of long-chain fattyacids and thus differed from the typical ceramides ofcomplex egg glycolipids, which are dominated by C20-

Fig. 6. ESI MS/MS of 8-11 and 11-7. (A) ESI

MS2 of the 8-11 [M + Na + H]2+ precursor

ion at m/z 404.5. (B) ESI MS2 of the 11-7

[M + H]+ precursor ion atm/z 973.5 and (C)

subsequent ESI MS3 fragmentation of the ion

at m/z 511.9, which corresponds to the Lewis

X trisaccharide unit. ,, sodium adduct; #,

proton adduct.

Table 4. Proposed structures of S. mansoni egg glycolipid-derived PA-oligosaccharides.

PA-Oligosaccharide structure Fraction

Gal(b1±3)GalNAc(1±4)Gal(1±4)Glc-PA 8-11

GlcNAc(b1±3)GalNAc(b1±4)Glc-PA 8-11

Gal(b1±4)[Fuc(a1±3)]GlcNAc(b1±3)GalNAc(b1±4)Glc-PA 11-7

Fuc(a1±3)GalNAc(b1±4)GlcNAc(b1±3)GalNAc(b1±4)Glc-PA 10-10

Fuc(a1±3)GalNAc(b1±4)[Fuc(a1±3)]GlcNAc(b1±3)GalNAc(b1±4)Glc-PA 12-5

GalNAc(b1±4)[Fuc(a1±3)]GlcNAc(b1±3)GlcNAc(b1±3)GalNAc(b1±4)Glc-PA 13-4

Fuc(a1±3)GalNAc(b1±4)[Fuc(a1±3)]GlcNAc(b1±3)GlcNAc(b1±3)GalNAc(b1±4)Glc-PA 15 + 14-3

Fuc(a1±3)GalNAc(b1±4)[Fuc(a1±3)]GlcNAc(b1±4)GlcNAc(b1±3)GalNAc(b1±4)Glc-PA 15 + 14-2

Fuc(a1±3)GalNAc(b1±4)[Fuc(a1±2)Fuc(a1±3)]GlcNAc(b1±3)GlcNAc(b1±3)GalNAc(b1±4)Glc-PA 14-4

Fuc(a1±3)GalNAc(b1±4)[Fuc(a1±2)Fuc(a1±2)Fuc(a1±3)]GlcNAc(b1±3)GlcNAc(b1±3)GalNAc(b1±4)Glc-PA 14-5

490 M. Wuhrer et al. (Eur. J. Biochem. 269) Ó FEBS 2002

phytosphingosine and C16:0 fatty acid (Fig. 2 and[3,10,17]). It is not known whether this host glycolipid istaken up directly by the egg or if it is acquired by the adultfemale and then transferred to the growing egg. Our ®ndingparallels the described acquisition of glycolipid blood-groupantigens by the adult schistosome [31,32] and ®ts in with thegeneral pattern of aquisition and imitation of host antigensby the parasite leading to immunosuppression and/orautoimmunity [33].

Concerning the elucidation of anomeric con®gurations inschistosome glycoconjugates, their unique structures largelyhamper the use of exoglycosidases. Alternatives are theusage of NMR, which requires quite large amounts ofmaterial, or CrO3 oxidation, which we have used tocharacterize anomeric linkages in egg glycosphingolipids[9]. While a-fucosidase treatment of the highly fucosylatedstructures could only remove some fucoses from the fucose

chains (Fig. 5), the Lewis-X-containing PA-oligosaccha-rides derived from cercarial glycolipids could be completelysequenced by exoglycosidases [6].

The amino-phase HPLC fractionation of PA-glycans didnot show a clear relationship between retention time andmonosaccharide composition similar to the additivity ruledescribed for N-glycans [21]. In 19 to 22, for example, whichare dominated by Hex1HexNAc6dHex3)7PA species, thedegree of fucosylation does not correlate with the retentiontime (Fig. 4), as, e.g. the heptafucosylated species in 21 elutesearlier than the tetrafucosylated species in 22. Likewise, theelution sequence of Hex1HexNAc7dHex4)8PA species in23 to 25 shows that the fucoses in these unique highlyfucosylated glycans vary considerably in their effect onthe retention time in amino-phase separation, and that theattachment of some of the fucoses might reduce theretention time.

Fig. 7. ESI MS/MS of 12-5 and 15. (A) ESI

MS of 12-5 and (B) subsequent ESI MS2

fragmentation of the sodium adduct at m/z

1182.0. Of all the pseudomolecular ions

detected in (A), [M + Na]+ gave the clearest

fragmentation pattern (B). In (A), a skimmer

voltage of 55 V was used instead of the rou-

tinely applied 30 V, which led to the detection

of the [M + Na]+ signal and resulted in

fragment ions already in the basic MS spec-

trum. (C) ESI MS of 15 and (D) subsequent

ESIMS2 fragmentation of the double-charged

species at m/z 693.0. ,, sodium adduct; #,

proton adduct.

Ó FEBS 2002 Schistosoma mansoni egg glycolipids (Eur. J. Biochem. 269) 491

Fig. 8. ESI MS/MS of 13-4. (A) ESI MS of

13-4 and (B) subsequent ESI MS2 fragmen-

tation of the double-charged species at m/z

631.0.Ñ, sodium adduct; #, proton adduct.

Fig. 9. ESI MS/MS of 14-5. (A) ESI MS of

14-5 and (B) subsequent ESI MS2 fragmen-

tation of the double-charged species at m/z

839.8 and (C) ESI MS3 fragmentation of the

species at m/z 664.9. ,, sodium adduct; #,

proton adduct.

492 M. Wuhrer et al. (Eur. J. Biochem. 269) Ó FEBS 2002

A C K N O W L E D G E M E N T S

We acknowledge the expert technical assistance of Peter Kaese,Werner

Mink and Siegfried KuÈ hnhardt. The authors thank Dr Quentin Bickle,

Department of Infectious and Tropical Diseases, London School of

Hygiene and Tropical Medicine, England, and Dr Andre M. Deelder,

Leiden University Medical Center, the Netherlands, for supply of

mAbs. This study was supported by the German Research Council

(SFB 535, Teilprojekt Z1).

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