Department of Biochemistry, Imperial College, London, SW7 2AY, UK and 2Department of Clinical Veterinary Science, University of Bristol, Bristol BS40 5DU, UK
Received on June 16, 1999; revised on July 26, 1999; accepted on July 31, 1999.
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Abstract |
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Key words: Dictyocaulus viviparus/glycosylation/Lewis x/mass spectrometry/nematode/N-glycans
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Introduction |
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Our previous structural studies of the ovine parasite Haemonchus contortus provided information on N-glycosylation in H.contortus glycoproteins (Haslam et al., 1996). These studies uncovered highly unusual core modifications not previously observed in N-linked glycoproteins. Notably, we identified a new type of core fucosylation in which fucose is attached to the distal N-acetylglucosamine of the chitobiose moiety resulting in glycans which are substituted with up to three fucose residues on the core. The novel form of fucosylation of the distal N-acetylglucosamine was demonstrated to be stage specific since trifucosylated cores were only observed in the adult stage of the parasite and not in L3 glycoproteins (Haslam et al., 1998
). Therefore if stage-specific carbohydrates are present in D.viviparus, such molecules could provide a promising starting point for novel vaccine development.
Virtually nothing is known about glycosylation in D.viviparus. An exploratory study using lectins, PNGase F digestion and metabolic labelling has suggested that few of the excretory-secretory (ES) products have PNGase F-sensitive N-glycans (Britton et al., 1993). In another study monoclonal antibodies have been used to probe immunodominant antigens on the surface of D.viviparus larvae (Gilleard et al., 1995
). Significantly, this work has revealed an immunodominant antigen which migrates as a diffuse band between 29 and 40 kDa on SDS-PAGE. The poor resolution on SDS-PAGE is indicative of glycosylated molecules.
Rigorous structural studies of D.viviparus glycoproteins are an essential pre-requisite to exploring their possible involvement in protective immunity and their promise as vaccine candidates. To this end we have undertaken a systematic structural analysis of the oligosaccharides of the adult stage of D.viviparus. In the present paper we report structural studies based on fast atom bombardment mass spectrometry, which have provided information on N-glycosylation in D.viviparus glycoproteins. Importantly these studies have uncovered complex N-glycans in D.viviparus with Lewisx-type (Galb1-4(Fuca1-3)GlcNAc) antennae on bi-, tri-, and tetraantennary structures, the first ever description of the Lewisx structure in a nematode glycoconjugate.
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Results |
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It was necessary to use both enzymic and chemical methods to release fucose for the following reasons. Although bovine kidney -fucosidase is a useful enzyme because of its relatively broad specificity, it has a strong preference for
-16 linked fucose and only slowly releases
-13 linked residues from structures such as Lewisx. Therefore where complex mixtures are involved, especially if large amounts of
-13 linked fucose are present, incomplete digestions can occur which can be problematic for subsequent degradation. This problem is overcome by the use of HF which rapidly removes fucose residues attached to the 3-position while retaining the 6-linked fucose of the core (unpublished observations from our laboratory). Thus, the two methods are highly complementary: the fucosidase experiments are necessary for establishing the
-anomeric configuration of the fucose residues whilst the HF treatment produces fully defucosylated antennae for subsequent ß-galactosidase and ß-N-acetylhexosaminidase digestion.
Data from -fucosidase digestion are shown in Figure 2 (see Tables I and III for assignments). Comparison with Figure 1 reveals that all components whose compositions are consistent with the presence of fucose (Table I) are affected by the digestion. Thus monofucosylated components that are likely to be core fucosylated (e.g., m/z 1345) have disappeared and multi-fucosylated components (e.g., m/z 2591) have shifted to lower levels of fucosylation. These data establish the
-linkage of the fucose residues. Incubation with HF gave a less complex spectrum (Figure 3, Tables I and III) because of the almost complete removal of fucose from antennae. Thus fucosylated A-type fragment ions present in Figure 1 (m/z 638 and 679) are very minor in Figure 3 as are molecular ions containing more than one fucose residue.
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Discussion |
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Three major classes of N-glycan structures were observed: high mannose type structures (Man5-9GlcNAc2), substoichiometrically core fucosylated truncated structures (Fuc0-1Hex2-4HexNAc2), and complex-type structures (Fuc0-5Hex3-7HexNAc3-6). High mannose structures and truncated structures have been previously observed by us in parasitic nematodes namely H.contortus (Haslam et al., 1996, 1998), Acanthocheilonema viteae (Haslam et al., 1997
), Onchocerca volvulus, and O.gibsoni (Haslam et al., 1999
). Therefore their presence appears to be a common feature of nematode N-glycosylation. In contrast, the complex-type structures are unusual in that they have Lewisx (Galß1-4(Fuc
1-3)GlcNAc) antennae on bi-, tri-, and tetraantennary molecules (Figure 5). This discovery was unexpected since it represents the first description of the Lewisx structure in a nematode.
The Lewisx epitope is a major component of glycoconjugates of the human parasitic trematodes Schistosoma mansoni, S.japonicum, and S.haematobium (Cummings and Nyame, 1996; Khoo et al., 1997
; Nyame et al., 1998
). However, attempts to detect the Lewisx epitope in the parasitic nematodes H.contortus, Dirofilaria immitis, and the free living nematode Caenorhabditis elegans by use of specific monoclonal antibodies failed. In contrast, lectin binding experiments indicated that all the species of nematodes express the lacdiNAc equivalent of the Lewisx-epitope (GalNAcß1-4(Fuc
1-3)GlcNAc), a structure shared by other helminths such as the schistosomes (Nyame et al., 1998
). It is of interest that minor amounts of fucosylated lacdiNAc were also observed in this study (see Results), indicating that D.viviparus is unique among all nematodes studied to date in that it appears to have a functional ß1-4 galactosyl transferase as well as a ß1-4 N-acetylgalactosaminyl transferase.
Glycoconjugates containing the Lewisx-epitope have been proposed to play important immunological roles in schistosomiasis. Glycans containing the Lewisx-epitope have been implicated in promoting a Th2 immune response (humoral immunity) over a Th1 immune response (cellular immunity), thus potentially limiting the hosts cellular immune response to the parasite (Velupillai and Harn, 1994). Infection with schistosomes induces the production of antibodies to the Lewisx-epitope and as the epitope is normally expressed in many tissues of both rodents and humans including leucocytes (where it is called CD 15) this antibody production causes the development of autoimmunity and complement-dependent cytolysis of leukocytes (Nyame et al., 1996
, 1997). The Lewisx-epitope has been demonstrated on bovine glycoconjugates (Savage et al., 1990
; Siciliano et al., 1993
) so it is possible a similar autoimmune phenomenon is occurring during D.viviparus infection of cattle.
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Materials and methods |
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Detergent extraction of D.viviparus
Approximately 2 g of adult D.viviparus were homogenised on ice in an extraction buffer of 0.5% w/v cetyltrimethylammonium bromide (CTAB), in 0.1 M Tris (pH 7.4), and extracted for a further 24 h at 4°C. Solid debris were removed by centrifugation at 3000 rpm for 10 min. Detergent was removed by extensive dialysis against 50 mM ammonium bicarbonate buffer (pH 7.6).
Reduction and carboxymethylation
Reduction and protection of the disulphide bridges of the detergent extracted proteins of D.viviparus was carried out as described previously (Dell et al., 1994).
Tryptic digestion
The reduced carboxymethylated D.viviparus proteins were digested with L-1-tosylamide-2-phenylethylchloromethyl ketone (TPCK) bovine pancreas trypsin (EC 3.4.21.4, Sigma), for 5 h at 37°C in 50 mM ammonium bicarbonate buffer (pH 8.4). The products were purified by C18-Sep-Pak (Waters Ltd.) as described previously (Dell et al., 1994).
PNGase F digestion
PNGase F (EC 3.2.2.18, Boehringer Mannheim) digestion was carried out in ammonium bicarbonate buffer (50 mM, pH 8.4) for 16 h at 37°C using 0.6 U of the enzyme. The reaction was terminated by lyophilization and the products were purified on C18-Sep-Pak (Waters Ltd.) as described previously (Dell et al., 1994).
Exo-glycosidase digestions
These were carried out on released glycans using the following enzymes and conditions: -L-fucosidase (from bovine kidney, EC 3.2.1.51, Boehringer Mannheim): 0.2 U in 100 ml of 100 mM ammonium acetate buffer, pH 4.55.0, ß-galactosidase (from bovine testes, EC 3.2.1.23, Boehringer Mannheim) 10 mU in 100 ml of 50 mM ammonium formate pH 4.6, ß-N-acetylhexosaminidase (from bovine kidney, EC 3.2.1.30, Boehringer Mannheim) 0.2 U in 100 ml of 50 mM ammonium formate pH 4.6. All enzyme digestions were incubated at 37°C for 24 h with a fresh aliquot of enzyme being added after 12 h and terminated by lyophilization. An appropriate aliquot was taken after each digestion and permethylated for FAB-MS analysis after purification on a C18-Sep-Pak (Waters Ltd.).
Hydrogen fluoride treatment
Samples were incubated with 50 µl of 48% HF (Aldrich) at 0°C for 48 h after which the reagent was removed under a stream of nitrogen.
Chemical derivatization for FAB-MS and GC-MS analysis
Permethylation using the sodium hydroxide procedure was performed as described previously (Dell et al., 1994). After derivatization the reaction products were purified on C18-Sep-Pak (Waters Ltd.) as described (Dell et al., 1994
). Partially methylated alditol acetates were prepared from permethylated samples for GC-MS linkage analysis as described previously (Albersheim et al., 1967
).
GC-MS analysis
GC-MS analysis was carried out on a Fisons Instruments MD800 machine fitted with a DB-5 fused silica capillary column (30 m x 0.32 mm internal diameter, J &W Scientific). The partially methylated alditol acetates were dissolved in hexanes prior to on-column injection at 65°C. The GC oven was held at 65°C for 1 min before being increased to 290°C at a rate of 8°C/min.
FAB-MS analysis
FAB-MS spectra were acquired using a ZAB-2SE 2FPD mass spectrometer fitted with a caesium ion gun operated at 30 kV. Data acquisition and processing were performed using the VG Analytical Opus software. Solvents and matrices were as described previously (Dell et al., 1994).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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