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

Eur J Biochem 269, 481±493 (2002) Ó FEBS 2002 Characterization of glycosphingolipids from Schistosoma mansoni eggs carrying Fuc(a1±3)GalNAc-, GalNAc(b1±4)[Fuc(a1±3)]GlcNAcand Gal(b1±4)[Fuc(a1±3)]GlcNAc- (Lewis X) terminal structures Manfred Wuhrer1,*, Sven R Kantelhardt1,*, Roger D Dennis,1 Michael J Doenhoff2, Gunter Lochnit1 È and Rudolf Geyer1 Institute of Biochemistry, University of Giessen, Germany; 2School of Biological Sciences, University of Wales, Bangor, Wales, UK The carbohydrate moieties of glycosphingolipids from eggs of the human parasite, Schistosoma mansoni, were enzymatically released, labelled with 2-aminopyridine (PA), fractionated and analysed by linkage analysis, partial hydrolysis, enzymatic cleavage, matrix-assisted laser desorption/ionization time-of-¯ight mass spectrometry and nano-electrospray ionization mass spectrometry Apart from large, highly fucosylated structures with ®ve to seven HexNAc residues, we found short, oligofucosylated species containing three to four HexNAc residues Their structures have been determined as Fuc(a1±3)GalNAc(b1±4)[ ‹ Fuc (a1±3)]GlcNAc(b1±3)GalNAc(b1±4)Glc-PA, GalNAc(b1± 4)[Fuc(a1±3)]GlcNAc(b1±3)GlcNAc(b1±3)GalNAc(b1±4) Glc-PA, Fuc(a1±3)GalNAc(b1±4)[Fuc(a1±3)]GlcNAc(b1± 4) GlcNAc(b1±3)GalNAc(b1±4)Glc-PA, and Fuc(a1±3) GalNAc(b1±4)[ ‹ Fuc(a1±2) ‹ Fuc(a1±2)Fuc(a1±3)]Glc NAc(b1±3)GlcNAc(b1±3)GalNAc(b1±4)Glc-PA The last structure exhibits a trifucosyl sidechain previously identi®ed on the cercarial glycocalyx These structures stress the importance of 3-fucosylated GalNAc as a terminal epitope in schistosome glycoconjugates To what degree these glycans contribute to the pronounced antigenicity of S mansoni egg glycolipids remains to be determined In addition, we have identi®ed the compounds GlcNAc(b1±3)GalNAc(b1± 4)Glc-PA, Gal(b1±4)[Fuc(a1±3)]GlcNAc(b1±3) GalNAc (b1±4)Glc-PA, the latter of which is a Lewis X-pentasaccharide identical to that present on cercarial glycolipids, as well as Gal(b1±3)GalNAc(1±4)Gal(1±4)Glc-PA, which corresponds to asialogangliotetraosylceramide and is most probably derived from the mammalian host Schistosomiasis is caused by parasitic blood ¯ukes of the genus Schistosoma and affects 200 million people worldwide During infection, schistosomal glycoconjugates play important roles in host±parasite pathological interactions [1,2] Schistosomes produce a variety of complex carbohydrate structures, many of which are highly fucosylated [2] These glycans are conjugated to proteins and/or lipids in different life-cycle stages and vary in their recognition by the immune system For Schistosoma mansoni, the Lewis X epitope has been found on egg and cercarial glycoproteins [3±5] and cercarial glycolipids [6] Although this epitope is shared with the mammalian host, S mansoni infection serum contains cytolytic antibodies directed against this epitope [7] Besides Lewis-X-carrying glycolipids, S mansoni expresses highly antigenic glycolipids mainly in the egg and the cercarial stage, and to a lesser extent in the adult stage [8,9] The stage-associated expression of carbohydrate structures on S mansoni glycolipids is paralleled by changes in glycolipid ceramide structures during the life-cycle [10] The highly antigenic glycolipids are speci®cally recognized by schistosome infection serum and not by other helminth infection sera, which makes them possible serodiagnostic antigens [11] They share a fucose-containing epitope with keyhole limpet haemocyanin (KLH) [12], which is consistent with the use of KLH for the diagnosis of schistosomiasis [13] The induction of signi®cant titres of antibodies against these glycolipids is associated with the onset of patency [11], and the IgE-response against worm glycolipids may play a role in mediating resistance to S mansoni reinfection after praziquantel treatment [14] All schistosome glycosphingolipids share the unique core structure, GalNAcb4Glc1±1ceramide, which was ®rst described by Makaaru et al [15] Cercarial glycolipids were found to be dominated by short-chained carbohydrate moieties expressing Lewis X (Gal(b1±4)[Fuc(a1±3)]GlcNAcb1-) and pseudo-Lewis Y (Fuc(a1±3)Gal(b1±4)[Fuc (a1±3)]GlcNAc(b1-) epitopes [6], whereas the structural characterization of unfractionated complex glycosphingolipids from S mansoni eggs has revealed the terminal structure ‹ Correspondence to R Geyer, Biochemisches Institut am Klinikum, Universitat Giessen, Friedrichstrasse 24, D-35392 Giessen, Germany È 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, electrosprayionization; Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; 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 (EC 3.2.1.23); ceramide glycanase (oligoglycosylceramideglycohydrolase; EC 3.2.1.23) *Note: these authors contributed equally to this work (Received 31 August 2001, revised November 2001, accepted 14 November 2001) Keywords: ceramide glycanase; internal fucose; oligosaccharide structural analysis; Schistosoma mansoni egg glycolipids 482 M Wuhrer et al (Eur J Biochem 269) Fuc(1±2)Fuc(1±3)GalNAc (b1- attached to a series of up to three )4)[Fuc(1±2)Fuc(1±3)]GlcNAc(b1-repeats and one )4)[ ‹ Fuc(1±2) ‹ Fuc(1±3)]GlcNAc(b1-unit, which is linked to )3)GalNAc(b1±4)Glc(1±1)ceramide, i.e the Schisto-core [16] In the present study, we have chosen an approach that differs from the one employed in the aforementioned and other investigations of glycolipid structures from S mansoni eggs [16,17] in which complex mixtures of glycosphingolipids have been analysed mainly by liquid secondary-ion mass spectrometry (LSIMS) using various chemical derivatizations Similar to previous studies on glycolipids from Caenorhabditis elegans [18] and S mansoni cercariae [6], we have enzymatically removed the ceramide moieties and fractionated the released oligosaccharide chains before structural characterization by mass spectrometry, chemical and enzymatic degradation and linkage analysis Using this strategy, structural information on the carbohydrate moieties of individual glycolipids from S mansoni eggs is obtained MATERIALS AND METHODS Glycolipid puri®cation and fractionation S mansoni egg glycolipids were puri®ed by organic solvent extraction, saponi®cation, desalting and anion-exchange chromatography as described previously [6] Neutral glycolipids were fractionated on a silica-gel cartridge (Waters, Eschborn, Germany) as outlined elsewhere [19] by step-wise elution with chloroform/methanol (80 : 20, v/v), chloroform/methanol/water (65 : 25 : 4) and chloroform/methanol/water (10 : 70 : 20), and analysed by HPTLC orcinol/ H2SO4-staining and HPTLC-immunostaining as described previously [6] The ®rst fraction contained ceramide monohexoside and dihexoside and was not further analysed The second fraction was positive in HPTLCimmunostaining and was used for preparation of PAoligosaccharides For HPTLC-immunostaining, the mAbs M2D3H, G11P and C1C7 were provided by Q Bickle, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, England, while the mAb 290-2E6 [20] was provided by A M Deelder, Leiden University Medical Center, the Netherlands Preparation and separation of PA-oligosaccharides The glycan moieties were released from glycolipids using recombinant ceramide glycanase (endoglycoceramidase II from Rhodococcus sp.; Takara Shuzu Co., Otsu, Shiga, Japan) and separated from uncleaved glycolipids and free ceramides on a reverse-phase cartridge [6] Released oligosaccharides were labelled with 2-aminopyridine (PA) and excess reagent was partitioned with chloroform [21] PA-oligosaccharides were fractionated on an amino-phase HPLC column (4.6 ´ 250 mm, Nucleosil-Carbohydrate; Macherey and Nagel, Duren, Germany) at a ¯ow rate of È mLámin)1 at room temperature and detected by ¯uorescence (310/380 nm) [21] The column was equilibrated with 200 mM aqueous triethylamine/acetic acid, pH 7.3 : acetonitrile (25 : 75, v/v) A gradient of 25±60% aqueous triethylamine/acetic acid buffer was applied within a 60-min period and the column was run isocratically for a Ó FEBS 2002 further 10 Peak fractions were collected and lyophilized Heterogeneous fractions were resolved further on a porous graphitic carbon (PGC)-column (4.6 ´ 100 mm, Hypercarb; Hypersil, Runcorn, UK) at a ¯ow rate of mLámin)1 at room temperature with ¯uorescence detection (310/380 nm) The column was equilibrated with 20 mM triethylamine/acetic acid, pH 5.0 A gradient from to 30% acetonitrile was applied in 50 Individual peak fractions were collected and lyophilized MALDI-TOF MS and ESI MS MALDI-TOF MS was performed on a Vision 2000 (ThermoFinnigan, Egelsbach, Germany) equipped with a UV nitrogen laser (337 nm) as described previously [6] The instrument was operated in the positive-ion re¯ectron mode throughout using 6-aza-2-thiothymine (Sigma) as matrix ESI MS was performed with an Esquire 3000 ion-trap mass spectrometer (Bruker Daltoniks, Bremen, Germany) equipped with an off-line nano-ESI source A 2±5 lL aliquot of PA-oligosaccharides in methanol/water (1 : 1, v/v) or glycolipids in chloroform/methanol/water (10 : 20 : 3) was loaded into a laboratory-made, gold-coated glass capillary and electrosprayed at a voltage of 700±1000 V using N2 as dry-gas (120 °C, Lámin)1) The skimmer voltage was set to 30 V, except for Fig A and B, where 55 V were applied For each spectrum 20±100 repetitive scans were averaged The accumulation time was between and 50 ms All MS/MS experiments were performed with helium as collision gas Enzymatic and chemical degradation PA-oligosaccharides were treated with either a-fucosidase from bovine kidney (4 mUálL)1; Roche Diagnostics, Mannheim, Germany) or with b-galactosidase from bovine testes (4 mUálL)1; Roche Diagnostics) on the MALDITOF MS target [22] Enzymes were dialysed for h against 25 mM ammonium acetate solution adjusted to pH 5.0 for a-fucosidase and pH 4.0 for b-galactosidase After measurement of the educts by MALDI-TOF MS using 6-aza-2-thiothymine matrix, dialysed enzymes (2 lL) were added undiluted to sample aliquots on the target and spots were analysed again by MALDI-TOF MS after incubation overnight at 37 °C For chemical defucosylation, dried samples were treated with 48% HF at °C overnight (modi®ed from [23]) HF was removed by a stream of nitrogen Monosaccharide composition and linkage analysis After hydrolysis in M aqueous tri¯uoroacetic acid at 100 °C for h and labelling with anthranilic acid, monosaccharides were determined by HPLC and ¯uorescence detection [9,24] For linkage analysis, PA-oligosaccharides were permethylated with methyl iodide after deprotonation with lithium methylsul®nyl carbanion [25] and hydrolysed (4 M aqueous tri¯uoroacetic acid, 100 °C, h) Partially methylated alditol acetates obtained after sodium borohydride reduction and peracetylation were analysed by capillary GC followed by ¯ame ionization detection or chemical ionization mass spectrometry (single ion monitoring), using a moving needle injector, fused silica bonded phase capillary columns of different polarity (60-m DB-1 Ó FEBS 2002 Schistosoma mansoni egg glycolipids (Eur J Biochem 269) 483 and 30-m DB-210; ICT, Bad Homburg, Germany) and helium as carrier gas as detailed elsewhere [26] RESULTS Preparation of glycolipids Glycolipids were isolated from S mansoni eggs analogously to the study performed on S mansoni cercarial glycolipids [6] and analysed by HPTLC (Fig 1) Orcinol/H2SO4staining (lane 1) revealed some major, slow-migrating compounds and a weaker staining for smaller, minor -CTH -CTetH components Both murine S mansoni infection serum (lane 2) and four monoclonal antibodies (lanes 3±6) visualized antigenic glycolipids Murine infection serum and the mAbs M2D3H, G11P and C1C7 exhibited a similar pattern, whereas the mAb 290±2E6, which is known to recognize mono- and difucosylated structures like GalNAc(b1±4) [ ‹ Fuc(a1±2)Fuc(a1±3)]GlcNAc(1- [20], displayed a completely different pattern The murine infection serum and mAb M2D3H were especially ef®cient in detecting the fastmigrating glycolipids (lanes and 3), which orcinol/H2SO4staining revealed to be present in only low amounts We expected these minor, fast-migrating, antigenic glycolipids not to be covered by the structural characterization of whole mixtures of complex glycolipids [16,17] and therefore decided to fractionate these compounds as their corresponding PA-oligosaccharides and to structurally characterize the individual species Preparation and separation of PA-oligosaccharides Fig HPTLC of S mansoni egg glycolipids S mansoni egg glycolipids 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 the mAbs M2D3H (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 globotetraosylceramide standards, respectively Glycans were released from the ceramide moieties by ceramide glycanase treatment of complex egg glycolipids For the separation of uncleaved glycolipids and ceramides from the released oligosaccharides, the sample was fractionated on a reverse-phase cartridge Released oligosaccharides were collected as the combined ¯ow-through and wash fractions, while the uncleaved glycolipids and ceramide moieties were obtained by elution with organic solvents Released glycans and uncleaved glycolipids were quantitated by monosaccharide composition analysis (Table 1), showing an overall ef®cacy of over 95% glycan release Released oligosaccharides were analysed by MALDI-TOF MS (Fig 2C) and the obtained pattern was very similar to the patterns observed for the intact glycolipids in ESI- and MALDI-TOF MS (Fig 2A,B) This indicated that the released oligosaccharides were representative for the glycans of the major complex egg glycolipids The released oligosaccharides were pooled and labelled with the ¯uorescent tag, 2-aminopyridine (PA) PA-oligosaccharides were fractionated by amino-phase HPLC (Fig 3A) Collected fractions (1 to 25; hereafter, fractions denoted by number only) were screened by MALDI-TOF MS and assessed for monosaccharide content by composition analysis (Tables and 3) Fractions to were not found to contain carbohydrate Starting with 8, MALDI-TOF MS revealed several compounds for most of the fractions (Fig 4) In Table 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 ˆ 2.0 in parentheses Monosaccharide GalNAc GlcNAc Gal Glc Fuc S Egg glycolipid fraction (2.0) (3.1) (0.3) (2.1) (3.8) Released monosaccharide (lg) Water fraction Organic solvent fraction Released monosaccharide (%) 235 (2.0) 375 (3.2) 27 (0.2) 329 (2.8) 522 (4.5) 1488 (2.0) 18 (8.6) (4.2) 35 (16.6) 10 (4.6) 75 98 95 76 90 98 95 484 M Wuhrer et al (Eur J Biochem 269) Ó FEBS 2002 Fig Mass spectrometry of unfractionated complex glycolipids and derived oligosaccharides from S mansoni eggs (A) ESI MS and (B) MALDI-TOF MS of complex S mansoni egg glycosphingolipids (C) MALDI-TOF MS of oligosaccharides released from S mansoni egg glycosphingolipids order to reduce peak heterogeneity and obtain as far as possible pure compounds, the amino-phase fractions to 14 were subfractionated by PGC-HPLC (Fig 3B,C) Subfractions (designated, for example, 12-5 for subfraction of fraction 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 analysis, linkage analysis, ESI MS/MS, as well as chemical and enzymatic degradation followed by a second linkage analysis ESI MS fragments were assigned according to the nomenclature introduced by Domon & Costello [27] Anomeric con®gurations were in some cases determined enzymatically (Fig 5D,G), but generally assigned based on the results of egg glycolipid CrO3 oxidation, which indicated b-anomeric linkages for GlcNAc and GalNAc and a-anomeric linkage for fucose as described recently [9] The major compound in as judged from MALDI-TOF MS (Fig 4) was Hex3HexNAc1PA After rechromatography by PGC-HPLC, it was detected in 8-11 by MALDI- Ó FEBS 2002 Schistosoma mansoni egg glycolipids (Eur J Biochem 269) 485 Fig HPLC separation of egg glycolipidderived PA-oligosaccharides (A) Separation on an amino-phase column Elution positions of the PA-labelled dextran hydrolysate standards of di€erent chain-length are indicated by arrows Fractions devoid of carbohydratepositive material are marked by asterisks (*) Rechomatography of fractions 11 (B) and 12 (C) an a PGC-column and analysis of the major peaks by MALDI-TOF MS (insets) n, minor compound identical in mass with 10-10 TOF MS ESI MS2 of this compound revealed the sequence Hex-HexNAc-Hex-Hex-PA and thus indicated a nonSchisto-core (Fig 6A) Linkage analysis showed major proportions of 3-substituted GalNAc, 4-substituted galactose and terminal galactose (Table 2), and the anomeric con®guration of the latter was determined by MALDI-TOF MS/on-target enzymatic cleavage with b-galactosidase from bovine testes (data not shown) Taken together, the structure was found to be Gal(b1±3)GalNAc(1±4)Gal(1±4) Glc-PA (Table 4), which probably corresponds to the hostderived glycolipid asialogangliotetraosylceramide As a minor component in 8-11, the proton adduct of Hex1HexNAc2PA was detected by ESI MS at m/z 664.9, and its sequence was determined by ESI MS2 to be HexNAcHexNAc-Hex-PA (data not shown) Linkage analysis of fraction 8-11 indicated minor amounts of terminal GlcNAc Based on the assumption of a Schisto-core structure, this leads to the structure GlcNAc(b1±3)GalNAc(b1±4)Glc-PA (Table 4) for this minor compound Fraction was not analysed further, but its major compound HexNAc2Hex2PA ([M + Na]+ at 849.5; Fig 4) might be identical with the PA-tetrasaccharide Galb3GlcNAcb3GalNAcb4Glc-PA derived from S mansoni cercarial glycolipids [6] For 10-10, linkage analysis before and after fucose removal by HF-treatment allowed the localization of the fucose at the 3-position of GalNAc and the assignment of the structure (Tables and 4) In the case of 11-7, terminal fucose and galactose (Table 2) had a- and b-anomeric con®guration, respectively, as determined by on-target enzymatic cleavage with bovine kidney a-fucosidase and b-galactosidase from bovine testes (data not shown) Linkage analyses before and after preparative removal of fucose by HF-treatment (Table 2) and ESI MS/MS (Fig 6B,C) indicated 11-7 to have the Lewis X-containing structure Gal(b1±4)[Fuc(a1±3)]GlcNAc(b1±3)GalNAc (b1±4)Glc-PA 12-5 was found to contain fucose both in the 3-position of GalNAc and in the 3-position of GlcNAc (cf linkage analysis, Table 2) ESI MS2 (Fig 7B) showed the two fucosylated HexNAc residues to be adjacent to each other (B3 ion at m/z 721.5) One of the fucoses of 12-5 could be removed enzymatically, while the second was only removed by HF-treatment (Fig 5B,E,H) These accumulated data for 12-5 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 and terminal fucose (Table 2) Loss of HexNAc in ESI MS2 (Y4a at 1036.1; Fig 8B) corroborated the presence of a terminal HexNAc Loss of a further HexNAc residue appeared only together with the loss of fucose (Y3 at m/z 687.0 but no signal at m/z 833), which indicated fucose to be linked to the subterminal HexNAc residue Bovine kidney a-fucosidase 486 M Wuhrer et al (Eur J Biochem 269) Ó FEBS 2002 Table Analysis of PA-oligosaccharides by MALDI-TOF MS, ESI MS, composition analysis (CA) and linkage analysis (LA) Masses were determined by MALDI-TOF MS and ESI MS (*) and were rounded to the ®rst 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 Hex3 HexNAc1 PA Hex1 HexNAc2 PA LA: t-Gal, 4-Gal, 3-GalNAc, t-GlcNAc 10-10 808.0 (808.3) [M + Na]+; 404.5* (404.7) [M + H + Na]2+ 664.9 (665.3) [M + H]+ 1036.1 (1036.4) [M + Na]+ Hex1 HexNAc3 dHex1 PA 10-10HF 11-7 890.9 (890.4) [M + Na]+ 995.6 (995.4) [M + Na]+ Hex1 HexNAc3 PA Hex2 HexNAc2 dHex1 PA 11-7HF 12-5 Hex2 HexNAc2 PA Hex1 HexNAc3 dHex2 PA 13-4HF 827.2* (827.4) [M + H]+ 1182.5 (1182.5) [M + Na]+; 591.6* (591.7) [M + H + Na]2+ 445.6* (445.7) [M + H + Na]2+ 1240.6 (1239.6) [M + Na]+; 620.5* (620.3) [M + H + Na]2+ 547.3* (547.2) [M + H + Na]2+ CA: GlcN:GalN (1.1 : 2.0) LA: t-Fuc, 4-GlcNAc, 3-GalNAc LA: t-GalNAc, 4-GlcNAc, 3-GalNAc CA: GlcN:GalN:Gal:Fuc (1.1 : 1.2 : 0.9 : 1.0) LA: t-Fuc, t-Gal; 3-GalNAc; 3,4-GlcNAc LA: t-Gal; 4-GlcNAc; 3-GalNAc LA: t-Fuc; 3-GalNAc; 3,4-GlcNAc 14-2 1384.4 (1385.5) [M + Na]+ Hex1 HexNAc4 dHex2 PA 14-3 1384.4 (1385.5) [M + Na]+ Hex1 HexNAc4 dHex2 PA 14-3HF 547.0* (547.2) [M + H + Na]2+ Hex1 HexNAc4 PA 14-4 1530.8 (1531.6) [M + Na]+; 766.5* (766.3) [M + H + Na]2+ Hex1 HexNAc4 dHex3 PA 14-5 839.1* (839.3) [M + H + Na]2+ Hex1 HexNAc4 dHex4 PA 14-5HF 1094.5 (1093.5) [M + Na]+ Hex1 HexNAc4 PA 15 1385.8 (1385.5) [M + Na]+ Hex1 HexNAc4 dHex2 PA 15HF 1094.6 (1093.5) [M + Na]+ 1071.1* (1071.4) [M + H]+ 1587.4 (1588.6) [M + Na]+ 1876.7 (1880.7) [M + Na]+ Hex1 HexNAc4 PA 8-11 12-5HF 13-4 16-4 16-5 Hex1 HexNAc3 PA Hex1 HexNAc4 dHex1 PA Hex1 HexNAc4 PA Hex1 HexNAc5 dHex2 PA Hex1 HexNAc5 dHex4 PA did not act on 13-4, but the fucose could be removed by HFtreatment (Fig 5) Taken together, the data showed 13-4 to represent the structure shown in Table Subfractionation of 14 led to the resolution of difucosylated 14-2 and 14-3, trifucosylated 14-4 and tetrafucosylated 14-5 species ESI MS2 analysis of 14-5 (Fig 9B) showed the four fucose residues to be linked to the two outermost GalNAc and/orGlcNAc residues (B4 ion at m/z 1013.2) Three of the fucose residues are attached to one of these HexNAc residues (Y4bB4 at m/z 664.9) to form a Fuc(a1±2) Fuc(a1±2)Fuc(a1±3)GlcNAc unit (see linkage analysis, Table 2), and this ion could lose one or two fucose residues on further fragmentation (Fig 9C) The HexNAc, which carried this oligofucosyl chain is not outermost, as indicated by the Y6aY4b ion at m/z 1182.8 (Fig 9B) An ion at m/z 1328, which would have indicated the loss of one fucose and LA: t-GalNAc; 4-GlcNAc; 3-GalNAc LA: t-Fuc; t-GalNAc; 3-GlcNAc; 3-GalNAc; 3,4-GlcNAc LA: t-GalNAc; 4-GlcNAc, 3-GlcNAc; 3-GalNAc LA: t-Fuc; 4-GlcNAc; 3-GalNAc; 3,4-GlcNAc CA: GlcN:GalN:Fuc (2.1 : 2.0 : 1.9) LA: t-Fuc; 3-GlcNAc; 3-GalNAc; 3,4-GlcNAc LA: t-GalNAc; 4-GlcNAc; 3-GlcNAc; 3-GalNAc CA: GlcN:GalN:Fuc (2.1 : 2.0 : 2.3) LA: t-Fuc; 2-Fuc; 3-GlcNAc; 3-GalNAc; 3,4-GlcNAc CA: GlcN:GalN:Fuc (2.2 : 2.0 : 3.2) LA: t-Fuc; 2-Fuc; 3-GlcNAc; 3-GalNAc; 3,4-GlcNAc LA: t-GalNAc; 4-GlcNAc; 3-GlcNAc; 3-GalNAc CA: GlcN:GalN:Fuc (2.1 : 1.8 : 2.0) LA: t-Fuc; 4-GlcNAc; 3-GlcNAc; 3-GalNAc; 3,4-GlcNAc LA: t-GalNAc; 4-GlcNAc; 3-GlcNAc; 3-GalNAc CA: GlcN:GalN : Fuc (2.9:2 : 2.8) CA: GlcN:GalN : Fuc (3.2:2 : 4.3) 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 corroborating the location of the trifucosyl chain on the second and not on the outermost HexNAc Taken together with the linkage analysis data (Table 2), the structure of 14-5 could be elucidated as Fuc(a1±3)GalNAc(b1±4)[Fuc(a1±2) Fuc (a1±2)Fuc(a1±3)]GlcNAc(b1±3)GlcNAc(b1±3) GalNAc (b1±4) Glc-PA (Table 4) The simultaneous presence of isobaric structural isomers in this fraction could not be excluded Similarly, ESI MS2 of the 14-4 [M + Na + H]2+ ion at m/z 766 (data not shown) yielded a [HexNAc2dHex3 + Na]+ ion at 867.0 Da, indicating the three fucoses to be linked to adjacent HexNAc residues For 14-4, the loss of one fucose and one HexNAc resulted in an intense fragment ion at m/z 1182.0, which showed that the Ó FEBS 2002 Schistosoma mansoni egg glycolipids (Eur J Biochem 269) 487 Table 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 ˆ 2.0 were determined after hydrolysis and reverse-phase chromatography of the anthranilic acidderivatized 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 peracetylation were analysed by GC/MS and GC using chemical ionization in conjunction with single-ion monitoring 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 ˆ 2.0 for HexNAc species and t-Fuc ˆ for fucose species For fraction 25, peak ratios were determined by GC/MS due to the limited amount of material 4-GlcNAc, 4-substituted GlcNAc, etc Composition analysis Linkage analysis Fraction GalNAc GlcNAc Fuc 2-Fuc: t-Fuc 4-GlcNAc: 3,4-GlcNAc: 3-GalNAc 18 19 20 21 22 23 24 25 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.84 3.23 3.64 3.62 3.67 4.04 4.51 3.88 3.16 2.48 2.85 4.65 4.00 3.33 4.09 4.63 0.9 : 1.0 0.95 : 1.0 0.8 : 1.0 1.05 : 1.0 0.8 : 1.0 0.95 : 1.0 0.5 : 1.0 0.65 : 1.0 1.4 : 3.3 : 2.0 1.55 : 3.55 : 2.0 1.55 : 4.0 : 2.0 1.2 : 5.15 : 2.0 1.25 : 4.6 : 2.0 1.45 : 3.25 : 2.0 2.0 : 4.15 : 2.0 0.7 : 6.45 : 2.0 outermost HexNAc carried only one fucose residue Together with the linkage analysis data, this allowed the deduction of the structure shown in Table For 14-2 and 14-3, MALDI-TOF MS indicated similar compositions as for 15 Linkage analysis, however, revealed a difference in the substitution positions at the monosubstituted GlcNAc While 14-2 contained only 4-substituted and 14-3 only 3-substituted GlcNAc, 15 exhibited approximately equal amounts of 3- and 4-substituted GlcNAc (Table 2) Linkage analysis of 15 before and after HF showed fucose to be linked to the 3-position of GalNAc and to the 3-position of 3,4-disubstituted GlcNAc ESI MS2 of 15 (Fig 7D) showed a B3 ion at m/z 721.0, which was the same branched terminal group as 12-5 (Table 4) As for 12-5, also in the case of 15, afucosidase from bovine kidney could remove only one fucose 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-substituted or 4-substituted GlcNAc unit (Table 4) Characterization of large PA-glycans Due to the pronounced heterogeneity and increasing complexity of fractions 16 to 25, we could only partially characterize these compounds by MALDI-TOF MS (Fig 4), carbohydrate composition and linkage analysis (Table 3) MALDI-TOF MS revealed compositions with an increasing number of HexNAc residues The highest peak in this region, 21 (Fig 3), contained as its major compound Hex1HexNAc6dHex7PA, which is consistent with the results of MALDI-TOF MS and ESI MS analyses of total egg glycosphingolipids and released glycans (Fig 2) While 19 to 22 contained almost exclusively PA-oligosaccharides with six HexNAc residues, species with ®ve HexNAc were most intense in 16 to 18 Likewise, 23 to 25 were dominated by PA-oligosaccharides with seven HexNAc residues, which have not been described in previous studies [16,17] Linkage analyses of the intact PA-oligosaccharides revealed 3-substituted GalNAc as the only GalNAc species through- out and fucose as the only terminal sugar (Table 3) Linkage analysis of 24 after fucose removal by HF-treatment resulted in the loss of all fucose species and the appearance of 3-substituted and terminal GalNAc in similar amounts, while all GlcNAc residues were converted to 4-substituted GlcNAc (data not shown) This shows GalNAc to be the outermost HexNAc in 24 The composition analyses of 18 to 25 imply that all complex PA-oligosaccharides contain an average of GalNAc residues Of the ®ve HexNAc residues in 18, approximately two are GalNAc and three are GlcNAc Of the six HexNAc residues in 19 to 22, approximately two are GalNAc and four are GlcNAc, and for the species in 24, the average HexNAc composition is two GalNAc and ®ve GlcNAc residues This supports the hypothesis, that there is one GalNAc residue both at the reducing and at the nonreducing end of the HexNAc chain, the ®rst being involved in the Schisto-core structure, while the core of the HexNAc chain would appear to consist of GlcNAc residues throughout This is consistent with the structures proposed by Khoo et al [16] Concerning fucosylation, there exists a vast heterogeneity, as exempli®ed by the detection of Hex1HexNAc6dHex2)8 in 21 Several fractions showed a 2-substituted fucose/terminal fucose ratio of approximately : (Table 3), which could be explained by the occurrence of difucosylated chains throughout However, based on the observation of a trifucosylated HexNAc in 14-5 and odd fucose numbers per molecule, e.g in 21, it can be assumed that trifucosylated HexNAc residues may also occur in some of these complex PA-oligosaccharides Taken together, the partial characterization of these larger PA-oligosaccharides after aminophase fractionation gave a detailed overview of the HexNAc chain-lengths and fucosylation heterogeneity, which was not obtained by FAB-MS analyses of the entire mixture of glycolipids [16,17] DISCUSSION In this study, individual S mansoni egg glycolipid structures have been elucidated Seven of the determined 488 M Wuhrer et al (Eur J Biochem 269) Ó FEBS 2002 Fig MALDI-TOF MS analysis of the HPLC-fractionated PA-oligosaccharides Fractions 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 Ó FEBS 2002 Schistosoma mansoni egg glycolipids (Eur J Biochem 269) 489 Fig 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) structures consist of fucosylated HexNAc chains based on the Schisto-core structure They are in agreement with the structure proposed by Khoo 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 the major structural motif at the nonreducing end of the HexNAc chain [16] is supported by our analyses of individual PA-oligosaccharides (10-10, 12-5, 15, 14-4 and 14-5) and by the linkage analyses of larger PA-oligosaccharides, in particular 24, before and after HF-treatment Recently, the terminal motif GalNAc(b1±4)GlcNAc- has been identi®ed as a good acceptor for fucosylation by S mansoni egg extracts, leading to a heterogeneity of fucosylated products, which parallels our ®nding of various oligofucosylated terminal structures [28] Our data, however, not support the proposed Fuc(a1± 4)[Fuc(a1±3)]GlcNAc-terminal motif, or the interdigitation of the HexNAc chain by fucose residues [17], because terminal GlcNAc was never generated by defucosylation in any of the PA-oligosacchride fractions studied, and MS analyses of HF-treated individual PA-oligosaccharides only revealed the 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] comprises HexNAc4-HexNAc6 chains, the structures 10-10 and 12-5 show that fucosylated glycolipids with HexNAc3-chains also exist Together with the Hex1HexNAc2PA compound found in 8-11, these carbohydrate chains obviously represent the Ômissing linkÕ between the Schisto-core and the extended structures described [16] Likewise, 23 to 25 are dominated by HexNAc7 species, which also have not been detected in the FAB-MS analyses [16,17] Secondly, we have found trifucosyl sidechains on 14-5, which have so far only been described for O-glycans derived from the cercarial glycocalyx [29] and not for egg glycolipids [16] The high relative amount of 2-Fuc in the larger PA-oligosaccharides further indicates that trifucosyl sidechains are also likely to occur in the glycolipids with HexNAc5-HexNAc7 chains Thirdly, 13-4 shows that incomplete fucosylation may also occur, leading in low amounts to terminal GalNAc residues involved in GalNAc(b1±4)[Fuc(a1±3)]GlcNAc(b1- units, as have been described for N-glycans of adult worm glycoproteins [30] Fourthly, the characterization of the HexNAc backbone revealed in some cases a deviation from the GalNAc(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 elucidated structures (14-2, which is identical to one compound in 15), while four structures (13-4, 14-3, which is identical to a second compound in 15, 14-4 and 14-5) showed the motif±GalNAc(1±4)GlcNAc(1±3)GlcNAc(1±3)GalNAc(1± 4) Glc(1±.The presence of internal 3-substituted GlcNAc residues is in agreement with previous studies on the carbohydrate structure of a cercarial Lewis-X-containing ceramide hexahexoside [6] 490 M Wuhrer et al (Eur J Biochem 269) Ó FEBS 2002 Fig 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 at m/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 Apart from the dominant glycolipids with fucosylated HexNAc chains, we characterized in this study the glycan of a Lewis X-containing glycolipid (11-7), which we previously have shown to be the dominating glycolipid of S mansoni cercariae [6] Though this result shows that Lewis X glycolipids are not absolutely restricted to the cercarial and schistosomular life-cycle stage, they are drastically downreguated in the egg and thus have not been detected by HPTLC-overlay [9] The ®nding of a PA-oligosaccharide corresponding to asialogangliotetraosylceramide (8-11) structurally identi®es a glycolipid which the parasite most probably has taken up from the host MALDI-TOF MS of the corresponding intact glycolipid after silica-gel puri®cation showed that its ceramides contain more than 40 carbon atoms (data not shown), which indicated the presence of long-chain fatty acids and thus differed from the typical ceramides of complex egg glycolipids, which are dominated by C20- Table 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 GlcNAc(b1±3)GalNAc(b1±4)Glc-PA Gal(b1±4)[Fuc(a1±3)]GlcNAc(b1±3)GalNAc(b1±4)Glc-PA Fuc(a1±3)GalNAc(b1±4)GlcNAc(b1±3)GalNAc(b1±4)Glc-PA 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±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 Fuc(a1±3)GalNAc(b1±4)[Fuc(a1±2)Fuc(a1±3)]GlcNAc(b1±3)GlcNAc(b1±3)GalNAc(b1±4)Glc-PA 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 8-11 8-11 11-7 10-10 12-5 13-4 15 + 14-3 15 + 14-2 14-4 14-5 Ó FEBS 2002 Schistosoma mansoni egg glycolipids (Eur J Biochem 269) 491 Fig 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 routinely applied 30 V, which led to the detection of the [M + Na]+ signal and resulted in fragment ions already in the basic MS spectrum (C) ESI MS of 15 and (D) subsequent ESI MS2 fragmentation of the double-charged species at m/z 693.0 ,, sodium adduct; #, proton adduct phytosphingosine and C16:0 fatty acid (Fig and [3,10,17]) It is not known whether this host glycolipid is taken up directly by the egg or if it is acquired by the adult female and then transferred to the growing egg Our ®nding parallels the described acquisition of glycolipid blood-group antigens by the adult schistosome [31,32] and ®ts in with the general pattern of aquisition and imitation of host antigens by the parasite leading to immunosuppression and/or autoimmunity [33] Concerning the elucidation of anomeric con®gurations in schistosome glycoconjugates, their unique structures largely hamper the use of exoglycosidases Alternatives are the usage of NMR, which requires quite large amounts of material, or CrO3 oxidation, which we have used to characterize anomeric linkages in egg glycosphingolipids [9] While a-fucosidase treatment of the highly fucosylated structures could only remove some fucoses from the fucose chains (Fig 5), the Lewis-X-containing PA-oligosaccharides derived from cercarial glycolipids could be completely sequenced by exoglycosidases [6] The amino-phase HPLC fractionation of PA-glycans did not show a clear relationship between retention time and monosaccharide composition similar to the additivity rule described for N-glycans [21] In 19 to 22, for example, which are dominated by Hex1HexNAc6dHex3)7PA species, the degree of fucosylation does not correlate with the retention time (Fig 4), as, e.g the heptafucosylated species in 21 elutes earlier than the tetrafucosylated species in 22 Likewise, the elution sequence of Hex1HexNAc7dHex4)8PA species in 23 to 25 shows that the fucoses in these unique highly fucosylated glycans vary considerably in their effect on the retention time in amino-phase separation, and that the attachment of some of the fucoses might reduce the retention time 492 M Wuhrer et al (Eur J Biochem 269) Ó FEBS 2002 Fig ESI MS/MS of 13-4 (A) ESI MS of 13-4 and (B) subsequent ESI MS2 fragmentation of the double-charged species at m/z 631.0.Ñ, sodium adduct; #, proton adduct Fig ESI MS/MS of 14-5 (A) ESI MS of 14-5 and (B) subsequent ESI MS2 fragmentation 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 Ó FEBS 2002 Schistosoma mansoni egg glycolipids (Eur J Biochem 269) 493 ACKNOWLEDGEMENTS We acknowledge the expert technical assistance of Peter Kaese, Werner Mink and Siegfried Kuhnhardt The authors thank Dr Quentin Bickle, È 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) REFERENCES van Dam, G.J & Deelder, A.M (1996) Glycoproteins of parasites ± Schistosome glycoconjugates and their role in host±parasite pathological interactions In Glycoproteins and Disease (Montreuil, J., Vliegenthart, J.F.G & Schachter, H., eds), pp 123±142 Elsevier Science, Amsterdam Cummings, R.D & Nyame, A.K (1996) Glycobiology of schistosomiasis FASEB J 10, 838±848 Khoo, K.H., Chatterjee, D., Caul®eld, J.P., Morris, H.R & Dell, A (1997) Structural mapping of the glycans from the egg glycoproteins of Schistosoma mansoni and Schistosoma japonicum: identi®cation of novel core structures and terminal sequences Glycobiology 7, 663±677 Huang, H.H., Tsai, P.L & Khoo, K.H (2001) Selective expression of di€erent fucosylated epitopes on two distinct sets of Schistosoma mansoni cercarial O-glycans: identi®cation of a novel core type and Lewis X 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spectrometry of unfractionated complex glycolipids and derived oligosaccharides from S mansoni eggs (A) ESI MS and (B) MALDI-TOF MS of complex S mansoni egg glycosphingolipids (C) MALDI-TOF MS of oligosaccharides... employed in the aforementioned and other investigations of glycolipid structures from S mansoni eggs [16,17] in which complex mixtures of glycosphingolipids have been analysed mainly by liquid... species 13-4 exhibited both terminal GalNAc and terminal fucose (Table 2) Loss of HexNAc in ESI MS2 (Y4a at 1036.1; Fig 8B) corroborated the presence of a terminal HexNAc Loss of a further HexNAc residue