The parasitic trematode Fasciola hepatica exhibits mammalian-type glycolipids as well as Gal(ß1-6)Gal-terminating glycolipids that account for cestode serological cross-reactivity

Manfred Wuhrer1,3, Christiane Grimm3, Roger D. Dennis3, Mohamed A. Idris4 and Rudolf Geyer2,3

3 Institute of Biochemistry, Medical Faculty, University of Giessen, D-35392 Giessen, Germany; and 4 Department of Microbiology and Immunology, College of Medicine, Sultan Qaboos University, Muscat, Sultanate of Oman

Received on June 6, 2003; revised on July 7, 2003; accepted on October 24, 2003


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Neutral glycosphingolipids from sheep-derived Fasciola hepatica liver flukes were isolated and characterized both structurally and serologically. After HPLC fractionation, glycolipids were analyzed by linkage analysis, enzymatic cleavage, and MALDI-TOF as well as electrospray ionization mass spectrometry. Obtained results revealed the presence of two types of neutral glycolipids. The first group represented mammalian-type species comprising globo- and isoglobotriaosylceramides (Gal({alpha}1-4)Gal(ß1-4)Glc(1-1)ceramide and Gal({alpha}1-3)Gal(ß1-4)Glc(1-1)ceramide, respectively) as well as Forssman antigen (GalNAc({alpha}1-3)GalNAc(ß1-3/4)Gal({alpha}1-4/3)Gal(ß1-4)Glc(1-1)ceramide). Applying Helix pomatia agglutinin, recognizing terminal {alpha}-linked GalNAc, to cryosections of adult flukes, the latter glycolipid could be localized to the F. hepatica gut. As Forssman antigen from the parasite and sheep host led to identical MALDI-TOF MS profiles, this glycolipid might be acquired from the definitive host. As a second group, highly antigenic glycolipids were structurally characterized as Gal(ß1-6)Gal(ß1-4)Glc(1-1)ceramide, Gal(ß1-6)Gal({alpha}1-3/4)Gal(ß1-4)Glc(1-1)ceramide and Gal(ß1-6)Gal(ß1-6)Gal({alpha}1-3/4)Gal(ß1-4)Glc(1-1)ceramide, the latter two structures of which exhibited both isoglobo- or globo-series core structures. Terminal Gal(ß1-6)Gal1-motifs have previously been shown to represent antigenic epitopes of neogala-series glycosphingolipids from tape worms. Using human Echinococcus granulosus infection sera, Gal(ß1-6)Gal-terminating glycolipids could be allocated to the gut in adult liver fluke cryosections. Corresponding neogala-reactive antibodies in F. hepatica infection serum were detected by their binding to E. granulosus and Taenia crassiceps neogala-glycosphingolipids. These antibodies might contribute to the known serological cross-reactivity between F. hepatica and parasitic cestode infections.

Key words: Forssman antigen / liver-fluke glycolipids / neogala-series glycolipids / oligosaccharide structural analysis / parasitic trematode


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Fascioliasis is a disease caused by the parasitic trematodes Fasciola hepatica and F. gigantica, which affects sheep, cattle, various other animal species, and humans. The F. hepatica life cycle resembles that of schistosomes: For both species, eggs are excreted by infected mammals, miracidia hatch from the eggs and infect snail intermediate hosts, where asexual reproduction by sporocysts leads to the production of emerging cercariae. Cercariae—in the case of F. hepatica via encysted metacercariae—complete the life cycle by infecting mammalian definitive hosts. For F. hepatica, larval flukes emerge from ingested metacercariae. Flukes then migrate from the digestive tract via the peritoneum to the liver, where they finally enter the bile ducts and, after a few months, start egg excretion.

Parasitic helminths express various immunogenic glycoconjugates, which have been reviewed for nematodes (Dell et al., 1999Go) and schistosomes (Hokke and Deelder, 2001Go). In the case of the best-studied parasitic trematode, Schistosoma mansoni, parasite glycans have been shown to modulate the host's immune response (Palanivel et al., 1996Go; van der Kleij et al., 2002Go). Glycan moieties of S. mansoni egg antigens, for example, trigger the Th2-shift associated with the beginning of patency (Faveeuw et al., 2002Go). In addition, important roles of S. mansoni glycoconjugates have been indicated, for example, by their secretion into the circulation (Deelder and Kornelis, 1980Go; Deelder et al., 1996Go; Hokke and Deelder, 2001Go), their expression in the parasite's gut or tegument (Bogers et al., 1994Go; Khoo et al., 1995Go; van Remoortere et al., 2000Go) and their pronounced regulation during the life cycle of the parasite (Wuhrer et al., 1999Go).

Compared to the rather detailed picture of S. mansoni glycobiology, little is known on the expression and role of parasitic glycoconjugates in other trematode infections. To address this imbalance, we have started to investigate F. hepatica glycoconjugates and compare them to schistosome glycans. This approach has already revealed pronounced differences in glycosylation: Whereas S. mansoni exhibits Glc(1-1)ceramide, Gal(1-1)ceramide and glycosphingolipids making up the unique schisto-core structure GalNAc(ß1-4)Glc(1-1)ceramide in all life cycle stages studied so far (Makaaru et al., 1992Go; Wuhrer et al., 2000aGo, 2002Go), adult F. hepatica flukes contained glucosylceramide, galactosylceramide, lactosylceramide, and globotriaosylceramide, known to represent mammalian-type glycosphingolipid structures (Wuhrer et al., 2001Go). In contrast to the differences in the glycan moieties, glycolipid ceramide moieties from the two trematode species consistently exhibited mainly phytosphingosines and {alpha}-hydroxlated fatty acids (Wuhrer et al., 2000aGo, 2001Go). A further distinction is the expression of a highly antigenic acidic glycolipid by F. hepatica (Wuhrer et al., 2003Go), while acidic glycolipids have not been described for schistosomes so far.

To provide additional information on the glycobiology of this parasite, it was the aim of this study to structurally and serologically characterize as well as histochemically localize F. hepatica neutral glycolipids with elongated carbohydrate chains and compare them with known glycan structures from other parasitic helminths.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Neutral glycolipids were isolated from F. hepatica flukes and analyzed by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Figure 1A). Besides the intense signals for ceramide monohexoside (CMH), ceramide dihexoside (CDH) and ceramide trihexoside (CTH), that is, hexose1–3ceramide (Hex1–3Cer), ions corresponding to Hex4–5Cer and Hex3HexNAc2–3Cer were detected. Linkage analysis further revealed terminal galactose; 3-substituted, 4-substituted, and 6-substituted Gal; 4-substituted Glc; 3,6-disubstituted Gal; terminal GlcNAc; terminal GalNAc; 4-substituted GlcNAc; as well as minor amounts of 3-substituted GalNAc (Figure 2). Because the most abundant Hex1–3Cer species had already been characterized (Wuhrer et al., 2001Go), we have focused here on less abundant Hex3Cer species and more complex structures, which were purified by Iatrobeads–high-performance liquid chromatography (HPLC) for structural and serological characterization. Fractions were screened by MALDI-TOF MS (Figure 1B–G) as well as electrospray ionization ion-trap (ESI-IT) tandem MS (MS-MS; Table I), and combined accordingly. Resulting glycolipid pools made up preponderantly Hex3Cer (fractions 20–25), Hex4Cer (26–27), Hex3HexNAc2Cer (28), Hex5Cer (29–30) and Hex3HexNAc3Cer (31–32) species.



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Fig. 1. MALDI-TOF MS of F. hepatica neutral glycolipids. Glycolipids were analyzed as a pool (A) or after HPLC fractionation (B–G). Sodium adducts were registered in the positive-ion mode. Glycolipid compositions were deduced by ESI-IT MS (Table I) and are given in terms of HexnHexNAcmCer, where n and m indicate the numbers of hexoses and N-acetylhexosamines, respectively. Hex3Cer-containing HPLC fractions 20–25 (B) were treated on-target with {alpha}-galactosidase (C). Alternatively, fractions 20–25 were treated with ß-galactosidase, leading to Hex1Cer species (inset in C). HPLC fractions 26–27, 28, 29–30, and 31–32 comprised glycolipids with compositions of Hex4Cer (D), Hex3HexNAc2Cer (E), Hex5Cer (F), and Hex3HexNAc3Cer (G), respectively. The inset in (E) shows the analysis of Forssman glycolipid species isolated from sheep erythrocytes which revealed an identical MALDI-TOF MS profile. Cer, ceramide; *, contaminant.

 


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Fig. 2. Linkage analysis of F. hepatica neutral glycolipids. The partially methylated sugar derivatives obtained after permethylation, hydrolysis, reduction, and peracetylation were analyzed by capillary GC-MS using chemical ionization with ammonia. t-Gal, terminal galactose (2,3,4,6-tetra-O-methylgalactitol as derivative); 3-Gal, 3-substituted galactose (2,4,6-tri-O-methylgalactitol); t-GlcNAc, terminal GlcNAc (2-deoxy-2-(N-methyl)acetamido-3,4,6-tri-O-methylglucitol), and so on. Signals in the amino sugar region (53–62 min) are enlarged as indicated.

 

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Table I. Analysis of glycolipids by MALDI-TOF MS, ESI-MS-MS, and linkage analysis

 
Carbohydrate constituent analysis of the pooled Hex3Cer species (20–25) revealed Gal and Glc in a ratio of 2:1. On-target enzymatic cleavage indicated major species to contain terminal {alpha}-linked Gal residues (Figure 1C). Linkage analysis of Hex3Cer (Table I) revealed terminal Gal, 4-substituted Glc, as well as 4-substituted, 3-substituted, and 6-substituted Gal, which occurred in a ratio of 0.75:0.15:0.10. {alpha}-Galactosidase treatment prior to linkage analysis depleted 4-substituted Gal and 3-substituted Gal, leaving 6-substituted Gal unimpaired (Table I). Based on the assumption of a mammalian-type lactosyl ceramide core structure, this indicated the presence of Gal({alpha}1-3)Gal(ß1-4) Glc(1-1)ceramide (isoglobotriaosylceramide; iGb3) in addition to the previously described Gal({alpha}1-4)Gal(ß1-4) Glc(1-1)ceramide (globotriaosylceramide; Gb3) (Wuhrer et al., 2001Go). An alternative approach using glycolipid-derived oligosaccharides labelled with 2-aminopyridine (PA) corroborated these results. PA-labeled glycans were fractioned by a 2D-HPLC system (Figure 3). For the two dominant PA-oligosaccharides, that is, PA6–2 and PA6–3, MALDI-TOF MS indicated a Hex3PA composition (Figure 4A, C). Linkage analysis revealed terminal Gal, 4-substituted Gal, and 4-substituted Glc for PA6–2, whereas PA6–3 exhibited terminal Gal, 3-substituted Gal, and 4-substituted Glc (not shown). Together with on-target {alpha}-galactosidase treatment (Figure 4B, D), the structures of PA6–2 and PA6–3 were determined as Gal({alpha}1-4)Gal(ß1-4) Glc1-PA and Gal({alpha}1-3)Gal(ß1-4)Glc1-PA, respectively, confirming the structures deduced for the corresponding glycolipids.



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Fig. 3. HPLC separation of F. hepatica glycolipid-derived PA-oligosaccharides. PA-oligosaccharides were fractionated on an amino-phase column (A). Elution positions of the PA-labeled dextran hydrolysate standards of different chain length are indicated by arrows. Fractions devoid of carbohydrate-positive material are marked by asterisks (*). Fractions PA6 (B) and PA9 (C) were rechromatographed on a reverse-phase column. Subfraction PA9-4 was reanalyzed after ß-N-acetylhexosaminidase treatment (D) verifying its isoglobo-type core-structure. Elution positions of Gal({alpha}1-4)Gal(ß1-4)Glc-PA (Hex3PA-1) and Gal({alpha}1-3)Gal(ß1-4)Glc-PA (Hex3PA-2) are marked by arrows (in D).

 


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Fig. 4. On-target enzymatic cleavage and MALDI-TOF MS analysis of PA-oligosaccharides. PA-oligosaccharides PA6-2 (A) and PA6-3 (C) were measured by MALDI-TOF MS. Then sample spots were incubated with {alpha}-galactosidase overnight and cleavage products of PA6-2 (B) and PA6-3 (D) were registered. Pseudomolecular ions are given in accurate, monoisotopic mass values rounded up to the first decimal place. *, contaminant.

 
{alpha}-Galactosidase resistant Hex3Cer species exhibited signals at m/z 1136.9 and 1174.9 (Figure 1C) and thus seemed to differ in part from {alpha}-galactosidase-sensitive Hex3Cer species in ceramide composition. As mentioned, linkage analysis of Hex3Cer after {alpha}-galactosidase treatment led to the reduction of 3-substituted and 4-substituted Gal, and 6-substituted Gal was retained (Table I). ß-Galactosidase treatment of total Hex3Cer, however, specifically removed 6-substituted Gal and yielded, in addition to uncleaved glycolipids, small amounts of Hex1Cer, which was separated and purified by Folch partition followed by silica gel chromatography. MALDI-TOF MS of the isolated ceramide monohexoside revealed major signals at m/z 812.6 and 850.5 (see inset in Figure 1C), which corresponded to the signals at m/z 1136.9 and 1174.9 in Figure 1C. Together with linkage analysis of this Hex1Cer sample, which revealed only terminal Glc (data not shown), this minor Hex3Cer species was structurally determined as Gal(ß1-6)Gal(ß1-4)Glc(1-1)Cer. This compound shares the Gal(ß1-6)Gal-terminal unit with neogala-series glycolipids from parasitic cestodes (Dennis et al., 1992Go; Persat et al., 1992Go), which are antigenic and recognized by various tapeworm infection sera (Dennis et al., 1993Go).

Accordingly, high-performance thin-layer chromatography (HPTLC) immunostaining revealed antigenic F. hepatica Hex3Cer species, which were weakly recognized by a sheep F. hepatica infection serum and intensely stained by a rabbit F. hepatica infection serum (Figure 5A–C). Serological recognition by both F. hepatica and Echinococcus granulosus infection sera (Figure 5D, E) was not based on the {alpha}-galactosidase-sensitive globotriaosyl- and isoglobotriaosylceramides, but ß-galactosidase treatment revealed the Gal(ß1-6)Gal-motif to be the target structure recognized.



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Fig. 5. Characterization of F. hepatica antigenic ceramide trihexosides by HPTLC immunostaining and enzymatic degradation. Glycolipids were resolved by HPTLC using the running solvent chloroform:methanol:0.25% aqueous KCl (50:40:10, by volume) and detected by orcinol/H2SO4 staining (A) or immunostaining with F. hepatica infection sera from sheep (B) and rabbit (C). F. hepatica combined Hex3Cer fractions 20–25 before (-) and after enzyme treatments with {alpha}-galactosidase ({alpha}-Gal) and ß-galactosidase (ß-Gal) were probed with rabbit F. hepatica infection serum (diluted 1:100; D) and human E. granulosus infection serum (diluted 1:200; E). Glycolipid amounts in µg carbohydrate per lane are given in parentheses. The standard (S) comprising CMH-CTetH corresponded to globo-series ceramide mono-, di-, tri-, and tetrasaccharides, respectively. H3Cer, combined F. hepatica Hex3Cer glycolipid fractions 20–25.

 
Human E. granulosus infection serum visualized a wide range of F. hepatica Hex3Cer species eluting predominantly in fractions 20 to 25. The dispersion of immune reactivity over all these fractions is in accordance with the ceramide heterogeneity of Gal(ß1-6)Gal-terminating Hex3Cer species as evidenced by MALDI-TOF MS after ß-galactosidase treatment (inset in Figure 1C). In addition, human E. granulosus infection serum recognized other glycolipids present in HPLC fractions 26 to 33 (Figure 6). MALDI-TOF MS (Figure 1D, F) and ESI-MS-MS (Table I) of fractions 26–27 and 29–30 revealed Hex4Cer and Hex5Cer to occur as the dominant glycolipids, respectively, whereas fraction 32 displayed minor amounts of Hex6Cer in MALDI-TOF MS (not shown). Linkage analysis of 26–27 and 29–30 before and after ß-galactosidase treatment revealed the occurrence of terminal Gal(ß1-6)Gal- and Gal(ß1-6)Gal(ß1-6)Gal- structural motifs, respectively (Table I). {alpha}-Galactosidase treatment of Hex4Cer (26–27) and Hex5Cer (29–30) did not result in hexose removal, as judged from MALDI-TOF MS (data not shown), thus ruling out the occurrence of significant amounts of {alpha}-Gal-terminating Hex4Cer or Hex5Cer species in these fractions. For 26–27 and 29–30, the simultaneous presence of 3-subsituted Gal and 4-substituted Gal in linkage analysis both before and after ß-galactosidase treatment indicated a heterogeneity in the Hex3Cer core structure, with iGb3 dominating over Gb3. The deduced structures are presented in Table II. The Hex6Cer species registered in 31–32 might have a similar Gal(ß1-6)Gal-containing structure, because these compounds are also recognized by E. granulosus infection serum (Figure 6), yet they could not be analyzed further due to the limited amounts of material. The various Gal(ß1-6)Gal-terminating F. hepatica glycolipids were further shown to account for corresponding antibodies in F. hepatica infection serum, which were demonstrated to strongly recognize tapeworm glycolipids (Figure 7).



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Fig. 6. HPTLC immunostaining of HPLC-separated F. hepatica neutral fraction glycolipids with E. granulosus infection serum. Glycolipids were separated by HPTLC using the running solvent chloroform:methanol:0.25% aqueous KCl (50:40:10, by volume) and detected by immunostaining with human E. granulosus infection serum (diluted 1:200). Fractions were resolved individually, except for 26 and 27 as well as 36 to 38, which were combined. The standard (S) of CMH-CTetH corresponded to globo-series ceramide mono-, di-, tri-, and tetrasaccharides, respectively, and was detected by orcinol/H2SO4 staining.

 

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Table II. Proposed structures of F. hepatica adult worm neutral glycolipids

 


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Fig. 7. Recognition of cestode glycolipids by F. hepatica infection serum. Glycolipids of E. granulosus (Eg) and Taenia crassiceps (Tc) were resolved by HPTLC using the running solvent chloroform:methanol:0.25% aqueous KCl (50:40:10, by volume) and detected by orcinol/H2SO4 staining (A), immunostaining with human E. granulosus infection serum (diluted 1:200; B) and immunostaining with rabbit F. hepatica infection serum (diluted 1:200; C). Glycolipid amounts in µg carbohydrate per lane are given in parentheses. The standard of CDH-CTetH corresponded to globo-series ceramide di-, tri-, and tetrasaccharides, respectively.

 
E. granulosus infection serum was additionally used as a tool to detect glycolipids with Gal(ß1-6)Gal-epitopes in cryosections of F. hepatica liver flukes. Respective neogala-type glycolipids could be localized to the parasite's gut as well as to some until now unidentified subtegumental structures (Figure 8A, B). Specific glycolipid detection was demonstrated by organic solvent extraction of cryosections that abolished staining of gut and subtegumental entities (Figure 8C, D).



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Fig. 8. Histochemical localization of Gal(ß1-6)Gal-terminating glycolipids and Forssman antigen. Cryosections (7 µm) of adult F. hepatica worms were incubated with human E. granulosus infection serum (A–D; dilution 1:50) and horseradish peroxidase–conjugated goat anti-human Ig antibodies (dilution 1:100). 3-Amino-9-ethylcarbamazol was used as substrate, resulting in red staining. In (C, D), sections were pretreated with organic solvent to extract (glyco)lipids. (E–H) Forssman antigen was detected by biotinylated H. pomatia lectin followed by alkaline phosphatase–conjugated avidin and the chromogenic substrate system resulting in purple staining. In (G, H) sections were pretreated with organic solvent to extract (glyco)lipids. The scale bar represents 100 µm. C, gut content; G, gut; L, gut lumen; S, subtegumental structures; T, testes; Tg, tegument; V, vitelline glands.

 
Besides the neogala-cross-reactive glycolipids, HexNAc-containing species were revealed by MALDI-TOF MS of glycolipids (Figure 1) and PA-oligosaccharides (Figure 3A), as well as linkage analysis (Figure 2). Hex3HexNAc2Cer was recovered in fraction 28 and analyzed by MALDI-TOF MS (Figure 1E), ESI-IT-MS-MS (Table I), and {alpha}-N-acetylgalactosaminidase treatment combined with linkage analysis (Table I). As analytical results did not allow a distinction between a 4-substituted iGb3 and a 3-substituted Gb3 core, its structure may be only proposed as GalNAc ({alpha}1-3)GalNAc(ß1-3)Gal({alpha}1-3/4)Gal(ß1-4/3)Glc(1-1)Cer, that is, a glycolipid carrying the Forssman antigenic determinant.

An anti-Forssman monoclonal antibody and Helix pomatia agglutinin were used to detect the Forssman antigen isolated from the parasite both in microtiter-plate and HPTLC-overlay assays (Figure 9). When rabbit F. hepatica infection serum was used as a control (Figure 9E), several glycolipid species were picked up that clearly differed in their migration properties from Forssman antigen (compare staining pattern of fraction 28 glycolipids in Figure 9C–E). Together with the detection of minor amounts of terminal and 6-substituted Gal by linkage analysis of fraction 28 compounds, this suggested the presence of additional neogala-type structures, which most likely account for the immunoreactivity not only with F. hepatica infection serum (Figure 9E) but also with E. granulosus infection serum (Figure 6).



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Fig. 9. Detection of F. hepatica Forssman antigen by ELISA and HPTLC immunostaining. HPLC fractions of F. hepatica glycolipids were probed in ELISA with an anti-Forssman mAb (A; diluted 1:100) and H. pomatia agglutinin (B; 5 µg/ml). Forssman antigen (FA; amounts in ng per well are given in parentheses) was used as a positive control. (C–E) Glycolipids were resolved by HPTLC using the running solvent chloroform:methanol:0.25% aqueous KCl (50:40:10, by volume) and detected by immunostaining with an anti-Forssman mAb (C; diluted 1:20), H. pomatia agglutinin (D; 5 µg/ml) and rabbit F. hepatica infection serum (E; diluted 1:200) as well as orcinol/H2SO4 staining (F). The amounts (in µg) of Forssman antigen (FA) applied per lane are given in parentheses. The standard (S) of CMH-CTetH corresponded to globo-series ceramide mono-, di-, tri-, and tetrasaccharides, respectively.

 
Because H. pomatia lectin solely recognized Forssman antigen among F. hepatica–derived glycolipids, it was further used for probing cryosections of adult flukes. Besides the parasite's gut, both vitelline glands (Figure 8E) and testes (Figure 8F) were recognized by the lectin. To establish the glycolipid and/or glycoprotein nature of the lectin ligands, sections were pretreated with organic solvent, which abolished the reactivity of the lectin with the gut, while vitelline glands (Figure 8G) and testes (Figure 8H) remained positive. As organic solvent extraction indicated the occurrence of a glycolipid type of ligand of H. pomatia lectin in the parasite's gut, and Forssman antigen was the only parasite-derived glycolipid reacting with this lectin (Figure 9), it may be concluded that the staining of the parasite's gut is at least in part due to the presence of the Forssman glycolipid.

Besides the Forssman antigen, other HexNAc-containing glycolipids were detected. The terminal GlcNAc moieties found in linkage analysis of total glycolipids (Figure 2) correlated with the negative-ion mode MALDI-TOF MS registration of the F. hepatica acidic glycolipid GlcNAca1-HPO3–6Gal(1-1)Cer (Wuhrer et al., 2003Go) as a carry-over into the neutral glycolipid fraction (data not shown). The Hex3HexNAc3Cer species present in fractions 31–32 were analyzed by MALDI-TOF MS (Figure 1G) and ESI-IT-MS-MS (Table I), the latter of which indicated a Hex3Cer core. The observation of terminal Gal and 6-substituted Gal in linkage analysis (Table 1) as well as the weak reaction with E. granulosus infection serum (Figure 6) indicated 31 and 32 to additionally contain minor amounts of neogala-type structures with five or more sugar residues, which, however, were not studied in detail. ß-N-acetylhexosaminidase treatment of the corresponding PA-oligosaccharide, PA9-4, allowed the chromatographic identification of this core as isoglobotriaosylceramide (Figure 3C, D). The occurrence of 3,6-disubstituted galactose in 31–32 (Table I indicated that elongation of the Hex3Cer core may occur via two branches. Composition analysis of 31–32 as well as PA9-4 revealed GalNAc and GlcNAc in a ratio of approximately 2:1. Together with the linkage analysis of 31–32, which exhibited terminal GalNAc and 4-substituted GlcNAc as the only HexNAc species (Table I), these data may lead to a model for Hex3HexNAc3Cer with GalNAc(ß1- and GalNAc(ß1-4)GlcNAc(ß1- units attached separately to the iGb3 core. The Hex3HexNAc3Cer species was the main representative of a group of more complex neutral F. hepatica glycolipids having Hex3–4HexNAc3–7 Cer compositions as evidenced by MALDI-TOF MS (not shown) of PA-glycans recovered in amino-phase HPLC fractions PA10 to PA12 (Figure 3A). Linkage analyses of fraction 33 glycolipids as well as glycolipid pool 34–38 revealed a similar monosaccharide pattern as obtained for 31–32 (Table I). A complete structural elucidation of these more complex structures could not be achieved due to limited amounts of material.

Ceramide compositions of the various glycolipid species were deduced from MS data (Table I). First, fragment ion analysis revealed the composition of the oligosaccharide moiety, with the Y-type ion of lowest molecular mass corresponding to the putative biosynthetic CMH precursor. Comparison of determined masses before and after permethylation revealed the number of accessible hydroxyl and amide functions. Acquired methyl groups were then assigned to the oligosaccharide and ceramide moieties. Incorporation of four methyl groups into the ceramide moiety of the Hex3Cer species at m/z 1136 thus indicated the occurrance of phytosphingosine and a hydroxy fatty acid. The Y-type ion at m/z 812 corresponded to CMH with C20-phytosphingosine and {alpha}-hydroxyoctadecanoic acid, which has been analyzed in detail in a previous study (Wuhrer et al., 2001Go). Likewise, the set of MS data was interpreted for the other characterized glycolipid compounds. Additional information by gas chromatography (GC) MS analysis of fatty acid methyl esters was obtained for a Forssman antigen standard as well as F. hepatica fractions 28 and 26–27, which revealed mainly an unsaturated fatty acid with 24 carbon atoms (tetracosenoic acid), in addition to minor amounts of tetracosanoic acid. Combined data provided the basis for the proposed compositions for the major ceramide species of each compound (Table I).


    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
In this study, neutral glycolipids from F. hepatica flukes have been structurally characterized and partly histochemically located. Most of the glycolipids were susceptible to ceramide glycanase verifying that these compounds represented glycosphingolipids. Yet ceramide glycanase discriminated the various glycolipid species in that cleavage efficacy was higher for isoglobotriaosylceramide than for globotriaosylceramide, whereas no cleavage products could be detected in the case of neogala-type of structures. Thus the established analysis of enzymatically released and fluorescently tagged glycolipid-derived oligosaccharides (Wuhrer et al., 2000bGo, 2002Go) had to be accompanied by the investigation of intact F. hepatica glycolipids, which revealed the presence of different biosynthetic series. A first group of antigenic glycolipids exhibited terminal Gal(ß1-6)Gal-units and was recognized by various F. hepatica (Wuhrer et al., 2003Go) and cestode infection sera. As glycolipids isolated from F. hepatica were found to be a heterogeneous mixture, HPLC fractionation combined with ß-galactosidase treatment was necessary to identify the Gal(ß1-6)Gal-determinants as cross-reacting target structures of antibodies from E. granulosus infection serum. This assumption is corroborated by earlier observations demonstrating that (1) the parasitic cestodes Taenia crassiceps (Dennis et al., 1992Go) and Echinococcus multilocularis (Persat et al., 1992Go) express mainly glycosphingolipids with neogala-series core structures, that is, [Gal(ß1-6)]1–3Gal(ß1-1)ceramide, which are (2) recognized by the respective murine infection sera. Furthermore, T. crassiceps infection serum–derived, monospecific, polyclonal antibodies directed to neogala-type ceramide trihexoside have been shown to cross-react also with glycolipids from E. granulosus reflecting the presence of similar carbohydrate epitopes in this parasite (Dennis and Wiegandt, 1993Go; Dennis et al., 1993Go). Hence, recognition of glycolipids from T. crassiceps and E. granulosus by F. hepatica infection serum verifies the presence of antibodies interacting with neogala-type Gal(ß1-6)Gal-units. Consequently, this determinant may be addressed as the basis of the observed cross-reactivity.

Although the acidic glycolipids of E. granulosus, partly comprising sialic acid, have not yet been structurally elucidated, comparison of their HPTLC migration pattern (Dennis et al., 1993Go) with that of F. hepatica acidic glycolipids (Wuhrer et al., 2003Go) revealed striking differences. Hence, cross-reactivity of E. granulosus infection serum with the F. hepatica acidic glycolipid GlcNAc{alpha}1-HPO3–6 Gal(1–1)Cer (Wuhrer et al., 2003Go), which would interfere with the immunohistochemical localization of neogala-type glycolipids (Figure 8), appears to be unlikely.

The major species of Gal(ß1-6)Gal-terminating Hex3Cer (MALDI-TOF MS signal at m/z 1174), Hex4Cer (m/z 1336), and Hex5Cer (m/z 1498) of F. hepatica seem to contain identical ceramide moieties (Table I). The Hex3Cer species is expected to be formed from lactosylceramide by the action of a parasitic ß1-6-galactosyltransferase. Although lactosylceramide species isolated from F. hepatica are characterized by mainly C18- and C20-phytosphingosine and hydroxyoctadecanoic acid (Wuhrer et al., 2001Go), the deduced Gal(ß1-6)Gal-terminating Hex3Cer, Hex4Cer, and Hex5Cer species are assumed to contain predominantly C18-phytosphingosine and tetracosenoic acid, whereas the presumed globo- and isoglobotriaosylceramide precursors comprised mainly C20-phytosphingosine and hydroxyoctadecanoic acid (compare Figure 1B and Wuhrer et al., 2001Go). This selective elongation of glycolipid precursors might be explained by tissue or subcellular distribution, with the majority of glycolipids not being available as substrates for the involved ß1-6-galactosyltransferase. Alternatively, it might be due to the substrate specificity of this enzyme. A similar shift in ceramide patterns is seen in S. mansoni cercarial glycolipids, where CMH contains mainly hydroxyhexadecanoic acid, whereas CDH and elongated species are dominated by fatty acids of more than 20 carbon atoms (Wuhrer et al., 2000aGo). Intriguingly, only Forssman glycolipid species were found to contain C18-sphingosine.

The location of neogala-cross-reactive glycolipids in F. hepatica gut might indicate a role for these glycolipids in host–parasite interaction that remains to be defined. It is remarkable that this F. hepatica glycolipid motif is shared with the neogala-series glycolipids of cestodes, (Dennis et al., 1992Go; Persat et al., 1992Go), but not with schistosomes, which express unique N-acetylhexosamine chains with oligofucosyl side chains based on a schisto-core (Khoo et al., 1997Go; Wuhrer et al., 2000bGo, 2002Go). The occurrence of both glycolipids with Gal(ß1-6)Gal-epitopes and corresponding antibodies in F. hepatica as well as parasitic cestode infection sera contributes to the described serological cross-reactivity observed in these infections (Bossaert et al., 2000Go; Schantz et al., 1975Go; Sturchler et al., 1986Go).

A second group of neutral glycolipids isolated from F. hepatica was made up from globotriaosylceramide, isoglobotriaosylceramide, and Forssman antigen. Because these are mammalian-type glycolipids, the question arises as to whether they were acquired from the host or expressed by the parasite itself. Metabolic labeling experiments would be helpful to address this question, but ceramide structural features can also provide information in this respect. As for F. hepatica globotriaosylceramide and isoglobotriaosylceramide, the MALDI-TOF MS pattern indicated a ceramide composition of mainly {alpha}-hydroxy fatty acids and phytosphingosines resembling that of F. hepatica and F. gigantica ceramide monohexosides (Wuhrer et al., 2001Go), thus supporting their parasitic origin. In the case of Forssman antigen, however, MALDI-TOF MS profiles of respective F. hepatic– and sheep erythrocyte–derived glycolipids were identical, pointing in this case in the direction of host origin. This assumption is supported by its divergent ceramide structure and the fact that Forssman antigen is found in the parasite's gut. Uptake of host glycolipid antigens has so far been best studied for schistosomes, where immunological studies revealed the uptake of blood group antigens by the trematode (Clegg et al., 1971Go; Goldring et al., 1976Go). Both surface presentation of host-derived antigens and self-made glycolipids mimicking host antigens may contribute to parasite camouflage or induce autoimmune processes, two effects that are circumscribed by the term molecular mimicry introduced by Damian (1964Go, 1989Go).

A third group of Hex3–4HexNAc3–7Cer glycolipids exhibited two HexNAc species, 4-substituted GlcNAc and terminal GalNAc, which would suggest some structural similarity to S. mansoni egg glycosphingolipids, exhibiting GalNAc(ß1-4)[GlcNAc(ß1-4)]0–3GlcNAc(ß1-3/4)GlcNAc (ß1-3)GalNAc(ß1-3)Glc(1-1)Cer backbone structures (Khoo et al., 1997Go; Wuhrer et al., 2002Go). In S. mansoni these chains are heavily substituted by oligofucosyl side chains, and the lack of fucosylation in F. hepatica complex glycolipids suggests the presentation of terminal LacdiNAc motifs (GalNAc(ß1-4)GlcNAc(ß1-), which have been found on schistosome glycoproteins (Cummings and Nyame, 1999Go; Hokke and Deelder, 2001Go; van Remoortere et al., 2000Go) and demonstrated to be targets of the humoral immune response (Nyame et al., 1999Go; Eberl et al., 2001Go; van Remoortere et al., 2001Go). Possibly, these glycolipids represent a structural link between F. hepatica and S. mansoni glycoconjugates.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Isolation and purification of neutral glycolipids
F. hepatica adult worms were collected from infected sheep at abattoirs in Muscat and Salalah, Sultanate of Oman, and stored in 10% formaldehyde at 4°C until use. Glycolipids were isolated by consecutive extractions as described previously (Wuhrer et al., 1999Go). Raw extracts were saponified in 50 ml methanolic 0.1 M NaOH for 2 h at 37°C. Salt and hydrophilic contaminants were removed using a reverse-phase (RP)-cartridge (Chromabond C18ec, Macherey & Nagel, Düren, Germany) as described (Dennis et al., 1998Go). Glycolipids were fractionated on a DEAE-Sephadex-A25 column (Dennis et al., 1998Go). Neutral glycolipids were collected as the flow-through, purified by Florisil chromatography (Waters, Eschborn, Germany; Dennis et al., 1998Go) and fractionated by HPLC (Iatrobeads 6RS-8010, 10 µm, 4.6 x 500 mm; Macherey & Nagel) at a flow rate of 1 ml/min, using a linear gradient from 100% solvent A (chloroform:methanol:water 83:16:1, by volume) in 60 min to 60% solvent B (chloroform:methanol:water 10:70:20, by volume), followed by a 20-min elution with 100% solvent B. Isolation of Forssman glycolipid from sheep erythrocytes (Sigma, St. Louis, MO) was performed by organic solvent extraction and Folch partitioning (Schnaar, 1994Go). The Folch upper phase was analyzed by MALDI-TOF MS.

Preparation and separation of PA-oligosaccharides
As an alternative to Iatrobeads HPLC, glycolipids were treated after Florisil chromatography with recombinant ceramide glycanase (endoglycoceramidase II from Rhodococcus spp.; Takara Shuzu, Otsu, Shiga, Japan). Released glycan moieties were separated from uncleaved glycolipids and free ceramides by an RP-cartridge (Wuhrer et al., 2000 and labeled with 2-PA. Excess reagent was partitioned with chloroform (Hase, 1994Go). PA-oligosaccharides were purified by gel filtration on a TSK-HW-40(F) column (TosoHaas, Stuttgart, Germany) using fluorescence detection (320/400 nm). PA-oligosaccharides were fractionated on an amino-phase HPLC column (4.6 x 250 mm, Nucleosil-Carbohydrate; Machery & Nagel) at a flow rate of 1 ml/min at room temperature and detected by fluorescence (310/380 nm). The column was equilibrated with 200 mM aqueous triethylamine/acetic acid pH 7.3:acetonitrile (25:75, by volume). 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 further 10 min. Peak fractions were collected and lyophilized. Heterogeneous fractions were resolved further on an ODS-Hypersil HPLC column (4.6 x 250 mm; 3 µm; Shandon) at 1 ml/min and room temperature. Solvent A was 50 mM acetic acid-triethylamine, pH 5.0, solvent B was solvent A containing 0.5% n-butanol. The column was equilibrated with solvent A for 20 min. After injection of the sample, the proportion of solvent B was linearly increased up to 50% within 50 min and kept constant for 30 min.

HPTLC and binding assays
HPTLC, orcinol/H2SO4 staining, and overlay detection assay were performed as described (Wuhrer et al., 1999Go). Sera from F. hepatica or E. granulosus–infected humans (kindly provided by Dr. John Dalton, Dublin, Ireland, and Prof. Egbert Geyer, Marburg, Germany) and rabbits (infected with 100 metacercariae, 40 weeks postinfection) were diluted 1:100 and used as primary antibodies. Goat, alkaline phosphatase–conjugated antibodies directed against immunoglobulins from rabbit (Sigma) and humans (Dianova, Hamburg, Germany) were employed as secondary reagents. Visualization of binding was performed using a 5-bromo-4-chloro-3-indolylphosphate/nitrobluetetrazolium chloride substrate mixture (Wuhrer et al., 1999Go). Porcine globo-series glycolipids were used as standards (Matreya, Pleasant Gap, PA) and stained by orcinol/H2SO4. For enzyme-linked immunosorbent assay (ELISA), plates (Polysorb; Nunc, Wiesbaden, Germany) were coated with glycolipids (added in 20 µl n-propanol per well), air-dried, and blocked by a 1-h incubation in 250 µl per well of 0.5% bovine serum albumin in Tris-buffered saline (TBS; 25 mM Tris-HCl, pH 7.5, 100 mM NaCl). Anti-Forssman rat monoclonal antibody (mAb) ICB 08–120 (Bethke et al., 1987Go; diluted 1:100) was added in 100 µl TTBS-10-B (TBS 1:10 diluted, containing 0.05% Tween 20 and 0.25% bovine serum albumin) per well. After multiple washes with TBS, diluted 1:10 and containing 0.05% Tween 20, binding was detected following a 60-min incubation with 100 µl per well of alkaline phosphatase-conjugated goat anti-rat IgG (1:1000; Sigma) secondary antibody in TTBS-10-B. Staining was performed with 100 µl per well of 0.1% p-nitrophenylphosphate in 100 mM glycine, 1 mM ZnCl2, 1 mM MgCl2. Absorption was measured at 405 nm. Alternatively to HPTLC immunostaining or ELISA, incubations with biotinylated H. pomatia lectin (5 µg/ml; Sigma) followed by alkaline phosphatase–conjugated avidin (dilution 1:4000; Sigma) were used for detection.

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) using 6-aza-2-thiothymine (Sigma) as matrix. ESI-MS of glycolipids (dissolved in chloroform:methanol, 1:1; dry-gas 80°C, 4 L/min) and PA-oligosaccharides (dissolved in methanol:water, 1:1; dry-gas 120°C, 4 L/min) was performed with an Esquire 3000 ion-trap mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with an offline nano-ESI source (Wuhrer et al., 2002Go, 2003Go). Electrospray was achieved at 700–1000 V using laboratory-made, gold-coated glass capillaries. The skimmer voltage was set to 30 V. For each spectrum 20–100 repetitive scans were recorded and averaged. The accumulation time was between 5 and 50 ms. All MS-MS experiments were performed with He as collision gas.

Exoglycosidase treatment
Glycolipids were taken up in 50 µl of 50 mM sodium citrate buffer of the appropriate pH, containing 0.1% sodium taurodeoxycholate and incubated at 37°C for 48 h with {alpha}-galactosidase from green coffee beans (Roche Diagnostics, Mannheim, Germany; buffer pH 6.0), ß-galactosidase form jack beans (Sigma; buffer pH 4), {alpha}-N-acetylgalactosaminidase from chicken (Sigma; buffer pH 6.0), or ß-N-acetylhexosaminidase from jack beans (Sigma; buffer pH 4). To 100 µl enzymatic digest 1.35 ml of chloroform:methanol (1:1, by voume.) and 100 µl water were added, which resulted in a phase partitioning. The lower phase was applied to DEAE-chromatography, and the flow-through was analyzed by linkage analysis. PA-oligosaccharides were similarly enzymatically degraded in the absence of taurodeoxycholate. On-target enzymatic cleavage was performed as described previously (Geyer et al., 1999Go; Wuhrer et al., 2000bGo).

Constituent, linkage, and fatty acid analysis
For constituent analyses, aliquots were hydrolyzed with 4 M trifluoroacetic acid (TFA; 4 h, 100°C) and analyzed as alditol acetates by GC or GC-MS using flame-ionization detection or chemical ionization with ammonia, respectively. For linkage analysis, samples were permethylated, hydrolyzed (4 M TFA, 4 h, 100°C), reduced (NaBH4) and peracetylated. The resultant partially methylated alditol acetates were analyzed by GC-MS after electron-impact or chemical ionization (Geyer et al., 1982Go; Geyer and Geyer, 1994Go). Fatty acids were converted to fatty acid methyl esters and analysed by GC-MS as described previously (Wuhrer et al., 2001Go).

Immunohistochemistry
F. hepatica adult worms, which had been stored at 4°C in 10% formaldehyde, were embedded in Tissue-Tek OCT-Compound (Sakura Finetek, Zoeterwoude, Netherlands) and longitudinal cryosections (7 µm) were prepared with a Jung Frigocut 2800E cryotome (Leica, Wetzlar, Germany) at -32°C. The cryosections were adhered to SuperFrost/Plus glass slides (Menzel, Braunschweig, Germany), were fixed with acetone for 5 min at -35°C and air-dried. The sections were rehydrated and blocked with phosphate buffered saline (PBS) containing 1% bovine serum albumine (PBS-B; 100 mM sodium phosphate, pH 7.2, 150 mM sodium chloride) for 30 min, followed by a 1-h incubation with human E. granulosus infection serum (diluted 1:50 in PBS-B). After 5 PBS washes the sections were incubated with horseraddish peroxidase–conjugated goat anti-human Ig (diluted 1:100, Sigma). Antibody binding was visualized with the 3-amino-9-ethylcarbamazol staining kit (Sigma). Likewise, Forssman antigen was detected with biotinylated H. pomatia lectin (5 µg/ml; Sigma). After five PBS washes, sections were incubated for 30 min with alkaline phosphatase–conjugated avidin (diluted 1:400; Sigma). After washing 2x with PBS and 3x with 100 mM glycine buffer, pH 10.4, 1 mM ZnCl2, and 1 mM MgCl2, visualization was obtained with 10 mg 5-bromo-4-chloro-3-indolylphosphate (Biomol, Hamburg) and 5 mg nitrobluetetrazolium chloride (Sigma) in 10 ml glycine buffer. To remove (antigenic) lipids, sections were incubated for 30 min with chloroform:methanol (1:1, by volume) before blocking.


    Acknowledgements
 
We wish to acknowledge the expert technical assistance of Peter Käse, Werner Mink, and Siegfried Kühnhardt in GC and GC-MS analysis. This study was supported by the Deutsche Forschungsgemeinschaft (SFB 535, projects A15 and Z1; GE 386/3). It is in partial fulfilment of the requirements of C. Grimm for the degree of doctor of medicine at Giessen University.


    Footnotes
 
1 Present address: Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, The Netherlands. Back

2 To whom correspondence should be addressed; e-mail: rudolf.geyer{at}biochemie.med.uni-giessen.de Back


    Abbreviations
 
CDH, ceramide dihexoside; CMH, ceramide monohexoside; CTetH, ceramide tetrahexoside; CTH, ceramide trihexoside; ELISA, enzyme-linked immunosorbent assay; ESI, electrospray ionization; Gb3, globotriaosylceramide; GC, gas chromatography; HPTLC, high-performance thin-layer chromatography; iGb3, isoglobotriaosylceramide; IT, ion-trap; mAb, monoclonal antibody; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; MS-MS, tandem mass spectrometry; PA, 2-aminopyridine; PBS, phosphate buffered saline; RP, reverse-phase; TBS, Tris-buffered saline; TFA, trifluoroacetic acid; TOF, time-of-flight


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 Introduction
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