Structural characterization of the N-glycans of Dictyocaulus viviparus: discovery of the Lewisx structure in a nematode

Stuart M. Haslam, Gerald C. Coles2, Howard R. Morris and Anne Dell1

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.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This paper reports the first rigorous evidence for the existence of N-linked oligosaccharides in Dictyocaulus viviparus, an economically important nematode that parasitises cattle. Structural strategies based upon fast atom bombardment mass spectrometry were employed to examine detergent extracts of homogenised adult D.viviparus for their N-glycan content. These revealed that detergent-soluble material is rich in high mannose, truncated and complex-type families of N-linked oligosaccharides. Importantly, the most abundant antennae in the complex-type structures were shown to carry the Lewisx epitope (Galß1-4(Fuc{alpha}1-3)GlcNAc). Although the Lewisx moiety occurs in other helminths such as schistosomes, nematodes have previously been thought to lack this epitope. The Lewisx epitopes in D.viviparus are carried on bi-, tri-, and tetraantennary glycans and are therefore candidates for recognition events requiring multivalent ligands. There is compelling evidence from schistosome research that glycoconjugates containing Lewisx structures are immunomodulators. We propose that the Lewisx-rich glycans identified in this study might similarly be involved in D.viviparus host interactions.

Key words: Dictyocaulus viviparus/glycosylation/Lewis x/mass spectrometry/nematode/N-glycans


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Dictyocaulus viviparus, commonly referred to as cattle lungworm, is a major pathogen of cattle with heavy infections being fatal. Prevention is important since once damage has been caused to the lungs the animals may not fully recover. Good immunity can develop after an infection and control is now frequently undertaken with vaccination using two doses of irradiated larvae given 4 weeks apart (Peacock and Poynter, 1980Go). However, for this protocol to be fully effective there must be a pasture larval challenge. With the widespread adoption of long acting anthelmintics many farmers have abandoned vaccination. The result has been a marked increase in outbreaks of lungworm in adult dairy cattle (David, 1993Go). There is thus a need for an effective molecular based vaccine that does not suffer from the short shelf life of irradiated larvae.

Our previous structural studies of the ovine parasite Haemonchus contortus provided information on N-glycosylation in H.contortus glycoproteins (Haslam et al., 1996Go). 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., 1998Go). 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., 1993Go). In another study monoclonal antibodies have been used to probe immunodominant antigens on the surface of D.viviparus larvae (Gilleard et al., 1995Go). 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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
FAB-MS of N-glycans released from detergent extracts by PNGase F
Detergent extracts of adult D.viviparus were prepared as described in Materials and methods. The extracts were reduced/carboxymethylated and tryptically digested to facilitate de-glycosylation with peptide N-glycosidase F (PNGase F). The released glycans were separated from peptides and glycopeptides and were analyzed by FAB-MS after permethylation and Sep-Pak purification (Figure 1, Table I). The spectra indicate that the parasite contains glycans having compositions consistent with high mannose structures (Hex5-9HexNAc2), truncated cores with and without fucose (Fuc0-1Hex2-4HexNAc2) and complex type glycans (Fuc0-5Hex3-7HexNAc3-6). Information on the type of antennae present on the complex glycans were obtained from the low molecular weight A-type fragment ions observed in the FAB-mass spectra (Figure 1, Table I). Thus, A-type fragment ions indicate the following nonreducing structures: HexNAc- (m/z 260), HexHexNAc- (m/z 464), HexNAc2- (m/z 505), FucHexHexNAc- (m/z 638), Hex2HexNAc- (m/z 668), and FucHexNAc2- (m/z 679).



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Fig. 1. FAB mass spectra of permethylated N-glycans from D.viviparus. The N-glycans of D.viviparus were released from tryptic glycopeptides by digestion with PNGase F, separated from peptides by Sep-Pak purification and permethylated. The derivatized glycans were purified by Sep-Pak and the 50% (v/v) aq. acetonitrile fraction was screened by FAB-MS. Assignments of the major molecular ions are given in Table I.

 

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Table I. Assignments of molecular and fragment ions observed in FAB spectra of permethylated N-glycans of D.viviparus eluting in the 50% aqueous acetonitrile (v:v) fraction from a C18 Sep-Pak
 
Linkage analysis of PNGase F released glycans
Data are summarized in Table II. The complexity of the mixture precludes allocation of individual components to particular oligosaccharides. Nevertheless a number of important conclusions can be drawn from these data: (1) the high levels of 3,6-linked Man and 4-linked GlcNAc are in accordance with their being essential constituents of the core of the majority of N-glycans; (2) the minor amount of 3,4,6-linked Man indicates that some cores contain a bisecting GlcNAc; (3) the high levels of terminal mannose is in accordance with the abundant high mannose and truncated structures suggested by the FAB-MS data; (4) fucose, galactose and GlcNAc are the other major terminal sugars with GalNAc being present as a minor terminal constituent; this is fully consistent with the A-type fragment ion data reported above; (5) the presence of high levels of 2-linked Man with lower levels of 2,4-linked Man and 2,6-linked Man is consistent with the majority of the complex glycans being bi-antennary with lesser amounts of tri-and tetra-antennary glycans; (6) the 3,4-linked and 4,6-linked GlcNAc residues are consistent with the presence of fucosylated antennae and fucosylated cores, respectively; (7) the presence of 6-linked Man indicates that the 3-linked Man is removed from the trimannosyl core in the truncated structures Fuc0-1Hex2HexNAc2..


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Table II. GC-MS analysis of partially methylated alditol acetates obtained from the PNGase F released N-glycans of D.viviparus
 
Exoglycosidase and chemical digestions
In order to facilitate anomeric assignments and sugar sequencing, glycan pools were subjected to treatment with {alpha}-fucosidase or aqueous hydrofluoric acid (HF), followed by ß-galactosidase and ß-N-acetylhexosaminidase digestion. The products were examined by FAB-MS and linkage analysis after permethylation and Sep-Pak purification.

It was necessary to use both enzymic and chemical methods to release fucose for the following reasons. Although bovine kidney {alpha}-fucosidase is a useful enzyme because of its relatively broad specificity, it has a strong preference for {alpha}-1–6 linked fucose and only slowly releases {alpha}-1–3 linked residues from structures such as Lewisx. Therefore where complex mixtures are involved, especially if large amounts of {alpha}-1–3 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 {alpha}-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 {alpha}-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 {alpha}-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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Fig. 2. FAB-mass spectrum of permethylated N-glycans from D.viviparus after {alpha}-fucosidase digestion. The N-glycans of D.viviparus were released from tryptic glycopeptides by digestion with PNGase F, separated from peptides by Sep-Pak purification, digested with {alpha}-fucosidase and permethylated. The derivatized glycans were purified by Sep-Pak and the 50% (v/v) aq. acetonitrile fraction was screened by FAB-MS.

 


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Fig. 3. FAB-mass spectrum of HF treated permethylated N-glycans from D.viviparus. The N-glycans of D.viviparus were released from tryptic glycopeptides by digestion with PNGase F, separated from peptides by Sep-Pak purification, treated with HF, and permethylated. The derivatized glycans were purified by Sep-Pak and the 50% (v/v) aq. acetonitrile fraction was screened by FAB-MS.

 

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Table III. Assignments of new molecular ions observed in FAB spectra of permethylated N-glycans of D.viviparus eluting in the 50% aqueous acetonitrile (v:v) fraction from a C18 Sep-Pak after HF treatment, exoglycosidase digestion or both HF treatment and exoglycosidase digestion
 
Linkage analysis of the HF treated glycans showed a significant reduction in the 3,4-GlcNAc:4,6-GlcNAc ratio (0.06) compared with the ratio for the untreated pool (0.87), consistent with the removal of fucose from the antennae (data not shown). Subsequent sequential digestion with ß-galacto­sidase followed by ß-N-acetylhexosaminidase yielded a major peak at m/z 1171 (Hex3HexNAc2), accompanied by its fucosylated counterpart at m/z 1345 (Figure 4), consistent with truncation to the trimannosyl core. Taken together, these data are consistent with fucose being {alpha}-linked and Gal, GalNAc, and GlcNAc residues all being ß-linked. The signals at m/z 1579, m/z 1783, m/z 1987, m/z 2191, and m/z 2395 (Figure 1, Figure 4) were unaffected by the above exoglycosidase digestions, a result that is consistent with their assignment as high mannose structures.



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Fig. 4. Partial FAB-mass spectrum of permethylated N-glycans from D.viviparus after HF treatment, ß-galactosidase and ß-N-acetylhexosaminidase digestion. The N-glycans of D.viviparus were released from tryptic glycopeptides by digestion with PNGase F, separated from peptides by Sep-Pak purification, treated with HF, then sequentially digested with ß-galactosidase, ß-N-acetylhexosaminidase, and permethylated. The derivatized glycans were purified by Sep-Pak and the 50% (v/v) aq. acetonitrile fraction was screened by FAB-MS.

 
Assignment of Lewisx oligosaccharide structures
Taking account of the data described above we conclude that Lewisx (Galß1-4(Fuc{alpha}1-3)GlcNAc) containing N-glycans are the major complex-type structures in the detergent extracts of adult D.viviparus (Figure 5). The following pieces of evidence support these conclusions. (1) Major molecular ions at m/z 2591 (Fuc3Hex5HexNAc4), 3214 (Fuc4Hex6HexNAc5) and 3837(Fuc5Hex7HexNAc6) (Figure 1, Table I) have compositions consistent with the structures in Figure 5. (2) The major A-type ion in Figure 1 at m/z 638 (FucHexHexNAc+) supports the presence of fucosylated lacNAc antennae. The absence of a secondary fragment ion at m/z 606 and the presence of an abundant ion at m/z 432 indicates that fucose is attached at the 3-position of the GlcNAc in the lacNAc antennae. (3) Confirmation of the position of attachment of the fucose was provided by the linkage data before and after removal of fucose by HF treatment.



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Fig. 5. Proposed structures of the Lewisx-containing complex-type glycans from D.viviparus. Some glycans have only a single Lewisx antenna (m/z 1968, m/z 2039, m/z 2213 and m/z 2243), but it is not known whether this is on the 6- or the 3-arm.

 
General structural conclusions
The combined data presented above show that adult D.viviparus contains three classes of N-glycans, namely high mannose (Man5-9GlcNAc2), truncated (Fuc0-1Man2-4GlcNAc2) and Lewisx-containing complex-type structures (Figure 5 and related structures whose compositions are given in Table I). Further, there is evidence that minor constituents of the N-glycan population have lacdiNAc and fucosylated lacdiNAc antennae which afford the A-type ions observed at m/z 505 and 679 (Figure 1, Table I) and give terminal GalNAc in the linkage analysis (Table II).


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The data presented in this paper represent the first rigorous structural investigation of glycosylation in D.viviparus. We show that homogenates of this parasite are rich in N-linked glycans. This is in contrast with earlier lectin and metabolic labeling studies of adult excretory–secretory products that showed little evidence for N-glycosylation (Britton et al., 1993Go).

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., 1996Go, 1998), Acanthocheilonema viteae (Haslam et al., 1997Go), Onchocerca volvulus, and O.gibsoni (Haslam et al., 1999Go). 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{alpha}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, 1996Go; Khoo et al., 1997Go; Nyame et al., 1998Go). 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{alpha}1-3)GlcNAc), a structure shared by other helminths such as the schistosomes (Nyame et al., 1998Go). 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 host’s cellular immune response to the parasite (Velupillai and Harn, 1994Go). 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., 1996Go, 1997). The Lewisx-epitope has been demonstrated on bovine glycoconjugates (Savage et al., 1990Go; Siciliano et al., 1993Go) so it is possible a similar autoimmune phenomenon is occurring during D.viviparus infection of cattle.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Production of Dictyocaulus viviparus
Worm free Fresian male calves were infected with 25 L3 larvae of D.viviparus per kg body weight. Animals were humanely slaughtered 28 days after infection and the adult worms collected manually from the lungs. Worms were thoroughly rinsed in Earle’s solution and stored at –80°C until used.

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., 1994Go).

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., 1994Go).

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., 1994Go).

Exo-glycosidase digestions
These were carried out on released glycans using the following enzymes and conditions: {alpha}-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 4.5–5.0, 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., 1994Go). After derivatization the reaction products were purified on C18-Sep-Pak (Waters Ltd.) as described (Dell et al., 1994Go). Partially methylated alditol acetates were prepared from permethylated samples for GC-MS linkage analysis as described previously (Albersheim et al., 1967Go).

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., 1994Go).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This work was supported by the Biotechnology and Biological Sciences Research Council and the Wellcome Trust (Grants 030825 and 046294). We would like to thank Intervet for a gift of D.viviparus larvae.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
FAB, fast atom bombardment; Hex, hexose; HexNAc, N-acetylhexosamine; lacNAc, Galß1-4GlcNAc; lacdiNAc, GalNAcß1-4GlcNAc; MS, mass spectrometry; PNGase F, peptide N-glycosidase F; u, mass unit.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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Haslam,S.M., Coles,G.C., Munn,E.A., Smith,T.S., Smith,H.F., Morris,H.R. and Dell,A. (1996) Haemonchus contortus glycoproteins contain N-linked oligosaccharides with novel highly fucosylated core structures. J. Biol. Chem., 271, 30561–30570.[Abstract/Free Full Text]

Haslam,S.M., Khoo, K.-H., Houston,K.M., Harnett,W., Morris,H.R. and Dell,A. (1997) Characterisation of the phosphorylcholine-containing N-linked oligosaccharides in the ES-62 glycoprotein of Acanthocheilonema viteae. Mol. Biochem. Parasitol., 85, 53–66.[ISI][Medline]

Haslam,S.M., Coles,G.C., Reason,A.J., Morris,H.R. and Dell,A. (1998) The novel core fucosylation of Haemonchus contortus N-glycans is stage specific. Mol. Biochem. Parasitol., 93, 143–147.[ISI][Medline]

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