2 Institute of Biochemistry, Medical Faculty, University of Giessen, Friedrichstrasse 24, D-35392 Giessen, Germany
3 Division of Immunochemistry, Research Center Borstel, Center for Medicine and Biosciences, D-23845 Borstel, Germany
4 Department of Microbiology and Immunology, College of Medicine, Sultan Qaboos University, Muscat, Sultanate of Oman
Received on July 2, 2002; revised on August 29, 2002; accepted on August 30, 2002
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Abstract |
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Key words: electrospray mass spectrometry / liver fluke glycolipids / oligosaccharide structural analysis / parasitic trematode
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Introduction |
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F. hepatica displays antigenic cross-reactivity with the trematodes Schistosoma mansoni and S. bovis as well as the nematode Trichinella spiralis (Aronstein et al., 1986; Hillyer, 1984
; Rodriguez-Osorio et al., 1999
). In addition, cross-protection has been demonstrated for F. hepatica and S. mansoni infections by heterologous challenges with S. mansoni cercariae and F. hepatica metacercariae, respectively (Hillyer, 1984
). Glycoconjugates were believed to be the reason for these phenomena (Hillyer, 1984
), and some of the molecules responsible for the observed cross-reactivity between F. hepatica and S. mansoni have been identified as glycoproteins (Aronstein et al., 1985a
,b
, 1986
). The molecular basis of this phenomenon can be attributed at least in part to a shared fucose-containing glycanic determinant present on acidic glycoproteins of various tissues and organs, including the tegument glycocalyx of the adult F. hepatica fluke as well as other life-cycle developmental stages (Abdul-Salam and Mansour, 2000
).
In contrast to the glycoproteins, neutral glycolipids of F. hepatica apparently displayed no serological cross-reactivity with those of S. mansoni (Dennis et al., 1996). F. hepatica expresses the globo-series of glycosphingolipids (Gal(
1-4)Gal(ß1-4)Glc(ß1-1)Cer; Wuhrer et al., 2001
), whereas S. mansoni exhibits the schisto-series of glycosphingolipids based on GalNAc(ß1-4)Glc(ß1-1)Cer (Makaaru et al., 1992
) with the dominant structural determinants Fuc(
1-3)GalNAc-, Gal(ß1-4)[Fuc(
1-3)] GlcNAc- (Lewis X), GalNAc(ß1-4)GlcNAc, and -4[Fuc(
1-2)Fuc(
1-3)] GlcNAc- (Khoo et al., 1997
; Wuhrer et al., 2000b
, 2002
). Of particular interest are the charged glycolipids of parasitic helminths, as exemplified by the structurally conserved zwitterionic phosphocholine-containing glycolipids of both parasitic and free-living nematodes (Gerdt et al., 1999
; Lochnit et al., 1998
; Wuhrer et al., 2000c
), which are active in stimulating the release of proinflammatory cytokines from peripheral blood mononuclear cells (Lochnit et al., 1998
). We present here the structural characterization of F. hepatica acidic (glyco) lipids, one of which exhibits unique structural features, such as GlcNAc linked via a phosphodiester to ceramide monohexoside (CMH), and is apparently active in evoking a strong humoral immune response in both human and animal liver fluke infections.
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Results |
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Discussion |
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The GlcNAc1-HPO3 unit of AL-II is paralleled in various biomolecules. The lipid A structure of Gram-negative bacteria exhibits a similar -6GlcN
1-O-H2PO3 motif with acyl chains in 2- and 3-position of GlcN (Alexander and Zähringer, 2002
). It may be speculated as to whether this structural similarity might provide a basis for a related biological activity of F. hepatica AL-II and bacterial lipid A, of which the latter is specifically recognized by cell- surface toll-like receptors (TLR4 and TLR2) of various host cells and leads to the production of bioactive compounds as tumor necrosis factor
, various interleukins, oxygen radicals, and bioactive lipids (Alexander and Zähringer, 2002
; Ulmer et al., 2002
). GlcNAc
1-O-HPO3- has also been described as O-linked to a serine residue of the lysosomal proteinase I of Dictyostelium discoideum, where it might be involved in lysosomal targeting of the proteinase and influence its substrate specificity (Gustafson and Gander, 1984
). In addition to GlcNAc
1-O-HPO3-, the ß-anomeric variant has recently been described to occur on glycosylphosphatidylinositol (GPI) anchors from various vertebrates (Fukushima et al., 2001
).
Concerning the biosynthesis of AL-II, one might postulate an enzymatic transfer of GlcNAc1-O-HPO3- to the 6-position of galactosylceramide in analogy to the first step of the biosynthesis of the mannose-6-phosphate marker on N-glycans of lysosomal hydrolases which targets them to the lysosome. So far, however, the respon-sible enzyme, GlcNAc-phosphotransferase, has been only characterized from bovine origin (Bao et al., 1996a
,b
).
Though there are structural similarities of AL-II to other glycoconjugates as detailed, the GlcNAc1-HPO3-unit has not been described as an autonomous antigenic determinant in other infectious diseases. In fascioliasis, however, AL-II is a major target of the host humoral immune response against glycolipids, as evidenced by its intense recognition in HPTLC overlay (Figure 1). Anti-AL-II antibodies present in infection sera recognize the GlcNAc
1-HPO3-determinant (Figure 5) and might provide the basis for the usage of AL-II or structurally related neoglycoconjugates in the serodiagnosis of fascioliasis.
The second acidic lipid compound analyzed in this study, AL-I, has been structurally determined as 1-O-hexadecyl- (or octadecyl)-sn-glycerol-3-phosphoinositol, that is, the ether bond variants of lysophosphatidylinositol. These species might be derived from 1-alkyl-2-acyl-phosphatidylinositol after loss of the ester-linked fatty acid on saponification, which had been included in the work-up procedure. 1-Alkyl-2-acyl-phosphatidylinositol compounds are present in glycoinositol phospholipids of protozoan parasites as well as in many eukaryotic protein GPI anchors (Campbell, 2001; Treumann et al., 1998
). As for trematodes, in particular, GPIs have been shown to anchor various S. mansoni proteins in the parasite tegument, for example, acetylcholinesterase (Arnon et al., 1999
; Pearce and Sher, 1989
; Sauma and Strand, 1990
). In addition, GPI-specific phospholipase activities have been described for F. hepatica and S. mansoni adult worms and could provide an enzymatic mechanism for the release of GPI-anchored proteins (Hawn and Strand, 1993
).
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Materials and methods |
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HPTLC
HPTLC, orcinol/H2SO4 staining, and immunostaining were performed as described (Wuhrer et al., 1999). Sera from F. hepatica-infected humans (kindly provided by 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, St. Louis, MO) and humans (Dianova, Hamburg) were employed as secondary reagents. Visualization of binding was performed using a 5-bromo-4-chloro-3-indolyl phosphate/nitro-blue tetrazolium chloride substrate mixture (Wuhrer et al., 1999
). Porcine globo-series glycolipids were used as standards (Matreya, Pleasant Gap, PA) and stained by orcinol/H2SO4.
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 was performed with an Esquire 3000 ion-trap mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with an off-line nano-ESI source. A 25-µl aliquot of native or permethylated (glyco)lipids in chloroform:methanol:water (10:20:3) was loaded into a laboratory-made, gold-coated glass capillary and electrosprayed at 7001000 V using N2 as drying gas (100°C, 4 L/min). 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 MSn experiments were performed with He as collision gas.
HF treatment, constituent and linkage analysis
For cleavage of the phosphodiester linkages, dried samples were treated with 48% HF at 4°C overnight (Haslam et al., 2000). HF was removed by a stream of nitrogen at room temperature. The CMH generated by HF treatment of AL-II was purified by silica-gel chromatography (Dennis et al., 1995
). For constituent analyses, (glyco)lipids were hydrolyzed with 4 M TFA (4 h, 100°C) or 0.5 N H2SO4 in 85% aqueous acetic acid (by volume; 16 h, 80°C) and analyzed as alditol acetates by GC or GC-MS using flame-ionization or electron-impact detection, respectively (Geyer et al., 1982
). Besides monosaccharide derivatives, PAF (Calbiochem, Schwalbach, Germany) was used as a reference compound. For linkage analysis, (glyco)lipids were permethylated, treated with HF, 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 and Geyer, 1994
; Geyer et al., 1982
).
NMR spectroscopy
1H-NMR spectra were recorded on a 600 MHz spectrometer (Bruker Avance DRX 600) in microtubes (3 mm OD, Kontes, Vineland, NJ) with a 5-mm multinuclear probe head. About 25 µg of AL-II were dissolved in 200 µl of CDCl3-d1:MeOD-d4 7:3 (by volume), and the 1H NMR spectrum was recorded with 16,000 scans at 300°K with signals referenced to internal tetramethylsilane. Standard Bruker software was used to record and process all NMR data (XWINNMR 2.6).
Inhibition ELISA
Plates (Polysorb; Nunc, Wiesbaden, Germany) were coated with AL-II (20 ng in 20 µl n-propanol per well), air-dried, 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). For inhibition experiments, GlcNAc (Serva, Heidelberg, Germany), glucose--methylglycoside (Serva), GlcNAc
1-phosphate (Sigma), UDP-GlcNAc (ICN, Eschwege, Germany), and GlcNAc-methylglycoside (prepared as described in Kamerling et al., 1975
) were used. The inhibitors were first added in 50 µl TTBS-10-B (TBS 1:10 diluted, containing 0.05% Tween 20 and 0.25% bovine serum albumin) per well followed by rabbit F. hepatica infection serum in another 50 µl TTBS-10-B. Plates were thoroughly shaken and then incubated for 1 h at 37°C. 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 goat alkaline phosphataseconjugated anti-rabbit Ig (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.
Ceramide analysis
Purified AL-II (after HF treatment and purification by silica-gel chromatography) was treated with 100 µl 1 M HCl and 10 M H2O in methanol for 16 h at 100°C (Gaver and Sweeley, 1965). Fatty acids and sphingoid bases were sequentially extracted and analyzed as their methyl esters and pentafluoropropionic acid derivatives by both GC and GC-MS (Wuhrer et al., 2001
).
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Acknowledgements |
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Footnotes |
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1 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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