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
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Abstract |
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Key words: Forssman antigen / liver-fluke glycolipids / neogala-series glycolipids / oligosaccharide structural analysis / parasitic trematode
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Introduction |
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Parasitic helminths express various immunogenic glycoconjugates, which have been reviewed for nematodes (Dell et al., 1999) and schistosomes (Hokke and Deelder, 2001
). 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., 1996
; van der Kleij et al., 2002
). Glycan moieties of S. mansoni egg antigens, for example, trigger the Th2-shift associated with the beginning of patency (Faveeuw et al., 2002
). In addition, important roles of S. mansoni glycoconjugates have been indicated, for example, by their secretion into the circulation (Deelder and Kornelis, 1980
; Deelder et al., 1996
; Hokke and Deelder, 2001
), their expression in the parasite's gut or tegument (Bogers et al., 1994
; Khoo et al., 1995
; van Remoortere et al., 2000
) and their pronounced regulation during the life cycle of the parasite (Wuhrer et al., 1999
).
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., 1992; Wuhrer et al., 2000a
, 2002
), adult F. hepatica flukes contained glucosylceramide, galactosylceramide, lactosylceramide, and globotriaosylceramide, known to represent mammalian-type glycosphingolipid structures (Wuhrer et al., 2001
). In contrast to the differences in the glycan moieties, glycolipid ceramide moieties from the two trematode species consistently exhibited mainly phytosphingosines and
-hydroxlated fatty acids (Wuhrer et al., 2000a
, 2001
). A further distinction is the expression of a highly antigenic acidic glycolipid by F. hepatica (Wuhrer et al., 2003
), 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.
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Results |
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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 5AC). Serological recognition by both F. hepatica and Echinococcus granulosus infection sera (Figure 5D, E) was not based on the -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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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 9CE). 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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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-HPO36Gal(1-1)Cer (Wuhrer et al., 2003) as a carry-over into the neutral glycolipid fraction (data not shown). The Hex3HexNAc3Cer species present in fractions 3132 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 3132 (Table I indicated that elongation of the Hex3Cer core may occur via two branches. Composition analysis of 3132 as well as PA9-4 revealed GalNAc and GlcNAc in a ratio of approximately 2:1. Together with the linkage analysis of 3132, 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 Hex34HexNAc37 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 3438 revealed a similar monosaccharide pattern as obtained for 3132 (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 -hydroxyoctadecanoic acid, which has been analyzed in detail in a previous study (Wuhrer et al., 2001
). 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 2627, 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).
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Discussion |
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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., 1993) with that of F. hepatica acidic glycolipids (Wuhrer et al., 2003
) revealed striking differences. Hence, cross-reactivity of E. granulosus infection serum with the F. hepatica acidic glycolipid GlcNAc
1-HPO36 Gal(11)Cer (Wuhrer et al., 2003
), 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., 2001), 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., 2001
). 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., 2000a
). 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 hostparasite 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., 1992; Persat et al., 1992
), but not with schistosomes, which express unique N-acetylhexosamine chains with oligofucosyl side chains based on a schisto-core (Khoo et al., 1997
; Wuhrer et al., 2000b
, 2002
). 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., 2000
; Schantz et al., 1975
; Sturchler et al., 1986
).
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 -hydroxy fatty acids and phytosphingosines resembling that of F. hepatica and F. gigantica ceramide monohexosides (Wuhrer et al., 2001
), thus supporting their parasitic origin. In the case of Forssman antigen, however, MALDI-TOF MS profiles of respective F. hepatic and sheep erythrocytederived 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., 1971
; Goldring et al., 1976
). 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 (1964
, 1989
).
A third group of Hex34HexNAc37Cer 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)]03GlcNAc(ß1-3/4)GlcNAc (ß1-3)GalNAc(ß1-3)Glc(1-1)Cer backbone structures (Khoo et al., 1997; Wuhrer et al., 2002
). 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, 1999
; Hokke and Deelder, 2001
; van Remoortere et al., 2000
) and demonstrated to be targets of the humoral immune response (Nyame et al., 1999
; Eberl et al., 2001
; van Remoortere et al., 2001
). Possibly, these glycolipids represent a structural link between F. hepatica and S. mansoni glycoconjugates.
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Materials and methods |
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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, 1994). 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 2560% 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., 1999). Sera from F. hepatica or E. granulosusinfected 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 phosphataseconjugated 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., 1999
). 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 08120 (Bethke et al., 1987
; 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 phosphataseconjugated 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., 2002, 2003
). Electrospray was achieved at 7001000 V using laboratory-made, gold-coated glass capillaries. The skimmer voltage was set to 30 V. For each spectrum 20100 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 -galactosidase from green coffee beans (Roche Diagnostics, Mannheim, Germany; buffer pH 6.0), ß-galactosidase form jack beans (Sigma; buffer pH 4),
-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., 1999
; Wuhrer et al., 2000b
).
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., 1982; Geyer and Geyer, 1994
). Fatty acids were converted to fatty acid methyl esters and analysed by GC-MS as described previously (Wuhrer et al., 2001
).
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 peroxidaseconjugated 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 phosphataseconjugated 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.
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Acknowledgements |
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Footnotes |
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2 To whom correspondence should be addressed; e-mail: rudolf.geyer{at}biochemie.med.uni-giessen.de
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Abbreviations |
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References |
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