Department of Biochemistry, Imperial College, London, SW7 2AY, UK and 3James A.Baker Institute for Animal Health and 4Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
Received on February 4, 2000; revised on March 15, 2000; accepted on March 18, 2000.
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Abstract |
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Key words: fast atom bombardment mass spectrometry/nematode glycosylation/phosphorylcholine containing carbohydrates/structure analysis/Trichinella spiralis
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Introduction |
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The surface molecules and/or the excretory/secretory products of the parasite are of considerable interest since they provide the interface with the host. All mechanisms of parasite evasion and of host protection must principally concern these molecules. The surface molecules of T.spiralis are stage specific, with changes in stage frequently coinciding with a move to a new environment. Philipp et al. (1980) reported that the proteins displayed on the surface change with each moult. Jungery and Ogilvie (1982)
showed that the host antibody response mirrors the changes in antigen expression. The precise function of these stage specific molecules is undefined, but is likely to include protection against attack by the host. In addition to the antigenic variation observed in different life stages, the antibody response to the L1 larvae has been described as biphasic; antibodies against one set of antigens are detected 13 days after infection, and antibodies against a different set of antigens are detected 35 days after infection. Excretory/secretory and cuticle glycoproteins are recognized only by antibodies produced late in infection (Denkers et al., 1990a
). Protective antibodies bind specifically to the glycan moieties on these glycoproteins (Denkers et al., 1990a,b; Appleton and Usack, 1993
; Ellis et al., 1994
). The immunodominant glycans are tri- and tetra-antennary N-linked structures composed of GalNAcß14GlcNAc (lacdiNAc) antennae which are capped with tyvelose (3,6-dideoxy-D-arabino-hexose; Tyv). The majority of antennae are also fucosylated on the GlcNAc residues (Wisnewski et al., 1993
; Reason et al., 1994
; Ellis et al., 1997
). Thus, these glycans are characterized by clusters of very hydrophobic nonreducing structures and Tyv residues form the immunodominant epitope. In rats, this late antibody response prevents reinfection (Appleton and McGregor, 1987
; Appleton et al., 1988
). Rat pups suckling T.spiralis-infected dams acquire a protective immunity which eliminates up to 99% of a challenge dose of infective larvae (Culbertson, 1943
; Appleton and McGregor, 1984
). This dramatic protection is mediated by maternal antibodies specific for Tyv that force larvae from their intestinal epithelial niche (Appleton et al., 1988
; Ellis et al., 1994
).
The other phase of the antilarval response seems to be specific for phosphorylcholine-bearing glycoproteins (reviewed by Takahashi, 1997; Peters et al., 1999
). Phosphorylcholine (PC) is a haptenic molecule that is present in many organisms, including bacteria (Faro et al., 1985
), fungi (Claflin et al., 1972
), and helminths (Fletcher et al., 1980
; Maizels et al., 1987
; Ubeira et al., 1987
; Maizels and Selkirk, 1989
). A number of studies performed in vitro lead to the conclusion that PC has immunomodulatory effects in nematodes. A PC-bearing glycoprotein of the filarial nematode Acanthocheilonema viteae has been shown to inhibit B-cell activation (Harnett and Harnett, 1993
) and to interfere with the activation of the human T cell line Jurkat (Harnett et al., 1998
). It has also been demonstrated that PC-containing somatic antigens of Brugia malayi can inhibit phytohemagglutinin-induced T cell proliferation (Lal et al., 1990
). In addition, PC-containing filarial nematode-secreted products have been shown to modulate a number of signal transduction elements associated with the antigen receptor, including various isoforms of protein kinase C, several protein tyrosine kinases, phospholipase D, Ras, phosphoinositide 3-kinase, and mitogen-activated protein kinase in either or both murine B cells and the human T-cell line Jurkat (Harnett and Harnett, 1993
; Deehan et al., 1997
, 1998; Harnett et al., 1998
).
The presence of PC in T.spiralis was revealed from immune response studies in mice (Lim and Choy, 1986; Ubeira et al., 1987
). PC is widely distributed in tissues, including the cuticle, epidermis, hypodermis, hemolymph, and intestinal gland (Hernández et al., 1995
). The PC group occurs in several T.spiralis glycoproteins and is attached to the protein core via glycan side chains of varying length (Homan et al., 1993
). In addition muscle-stage larvae have been demonstrated to contain PC-substituted N-glycans by the loss of binding of an anti-PC monoclonal antibody (MoAb2), to a family of glycoprotein antigens (TSL-4 antigens), after N-glycanase treatment (Ortega-Pierres et al., 1996
). However, the precise structures of these PC containing N-glycans are not known.
In the work presented herein, we report the first rigorous structural analysis of PC containing glycans released from T.spiralis affinity-purified antigens by peptide N-glycosidase F digestion. We establish using fast atom bombardment mass spectrometry analysis that PC is attached to N-glycans whose sub-stoichiometrically fucosylated trimannosyl cores carry between one and eight N-acetylhexosamine residues.
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Results |
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Structural analysis strategy
Experimental strategies based on derivatization, chemical hydrolysis, exoglycosidase digestions, FAB-MS, and GC-MS, which have proven success in sequencing PC-substituted glycans (Haslam et al., 1997, 1999) were employed to characterize the glycans of affinity-purified T.spiralis antigens.
To facilitate the release of N-glycans without resorting to detergent denaturation, reduced/carboxymethylated T.spiralis affinity-purified antigens were first digested with trypsin. Glycans were then released from the resulting peptides/glycopeptides by digestion with peptide N-glycosidase F (PNGase F). PNGase F released oligosaccharides were separated from peptides and glycopeptides using a C18 Sep-Pak. Because of the availability of only limited amounts of material, the oligosaccharides were analyzed as mixtures. N-Glycans were subjected, in separate experiments, to the following three experimental protocols: (1) permethylation/FAB-MS; (2) mild hydrolysis with HF followed by permethylation and FAB-MS; (3) perdeuteroacetylation/FAB-MS. Protocol 1 provided information on all glycans other than those containing PC groups which were lost during the permethylation procedure. The initial HF treatment in protocol 2 removed all PC moieties by cleavage of phosphodiester linkages. Thus molecular ions that are observed only after HF treatment are indicative of components in the native sample that have HF-sensitive functional groups, such as PC. Finally, protocol 3, in which PC groups are preserved successfully afforded FAB-MS data on the smallest PC-containing glycans. In addition to the above FAB-MS experiments, HF-treated PNGase F released glycans were permethylated and analyzed by FAB-MS after sequential exoglycosidase digestions and by linkage analysis.
Monosaccharide composition of N-glycans released by PNGase F
The monosaccharide composition of the mixture of PNGase F released oligosaccharides from T.spiralis affinity-purified antigens was determined by GC/MS analysis of the trimethylsilylated derivatives of the methyl glycosides. Fucose, mannose, N-acetylgalactosamine, and N-acetylglucosamine were all detected at a molar ratio of approximately 1:6:3.5:7 (data not shown).
FAB-MS of permethylated N-glycans
Data from FAB-MS of permethylated PNGase F released glycans eluting in the 35% aqueous acetonitrile (v/v) fraction from a C18 Sep-Pak are shown in figure 1 and summarized in Table I. The data indicate that the parasite contains glycans having compositions consistent with high mannose structures (Hex5-9HexNAc2), truncated cores with and without fucose (Fuc0-1Hex3-4HexNAc2), and complex type structures (Fuc0-1Hex3HexNAc3-6) whose antenna contain HexNAc or HexNAc2 (Table I). Information on the types of antennae present is provided by A-type fragment ions. Fragment ions are present at m/z 260, 505 and 872 which correspond respectively to HexNAc+, HexNAc2+ and Hex3HexNAc+. No fragment ions of composition TyvHexNAc+ (m/z 404), TyvHexNAc2+ (m/z 649), FucHexNAc+ (m/z 434) and FucHexNAc2+ (m/z 679) are observed. These data suggest that antennae do not carry tyvelose and fucose residues. Since some molecular ions have compositions containing one fucose it appears that several complex structures are core fucosylated.
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The resulting FAB spectrum of permethylated N-glycans eluting in the 35% aqueous acetonitrile (v/v) fraction from a C18 Sep-Pak was remarkably different from Figure 1, indicating that the majority of glycans are not amenable to the permethylation/FAB-MS strategy without prior HF treatment. Thus the spectrum of HF treated material (Figure 2, Table II) is dominated by molecular ions corresponding to a family of N-glycans of composition Fuc0-1Hex3HexNAc2-10, while components observed prior to HF treatment are of relatively low abundance except for oligosaccharides which have compositions Fuc0-1Hex3HexNAc2. The abundant signals are assignable to oligosaccharides which have compositions consistent with sub-stoichiometrically fucosylated trimannosyl cores to which are attached between one and eight N-acetylhexosamine residues (Figure 2, Table II). Since these oligosaccharides were not observed or were much less abundant before HF treatment (Figure 1, Table I), it can be concluded that they originally contained one or more PC moieties. The low mass fragment ion region of the FAB spectrum was also different from Figure 1. The same A-type fragment ions are still present at m/z 260, 505, and 872 but the ions at m/z 260 and 505 are much more abundant after HF treatment. The most abundant A-type ion occurs at m/z 260 (HexNAc+) and indicates that the majority of antennae terminate with GalNAc or GlcNAc. The signal at m/z 505 corresponds to an A-type ion of composition HexNAc2+. Based on current knowledge of helminth glycans the fragment ion HexNAc2+ is likely to be the GalNAc-GlcNAc moiety which is a common building block in lower animals (Khoo et al., 1991; Kang et al., 1993
; Reason et al., 1994
; Khoo et al., 1995
; Haslam et al., 1996
). Moreover, it is known that this HF treatment rapidly removes fucose residues attached to the 3-position whilst retaining the 6-linked fucose of the core (Haslam et al., 1999
). Since the fucose-containing N-glycans were not affected by this treatment, this result corroborates FAB-MS evidence for core fucosylation of complex and truncated glycans.
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Linkage analysis of HF treated glycans
GC-MS analysis of partially methylated alditol acetates generated from the permethylated glycans gave the data shown in Table III. Key features of these data are as follows. (1) 3,6-Man and 4-GlcNAc are components of the core of all N-glycans; the absence of detectable levels of 3,4,6-Man indicates that bisecting GlcNAc is unlikely to be present; (2) the presence of comparable components of 2-Man, 2,4-Man, and 2,6-Man is consistent with bi-, tri-, and tetraantennary complex type glycans being dominant components of the glycan mixture; (3) Fuc, Man and GalNAc are the major nonreducing sugars; (4) the observation of terminal mannose is in accord with the existence of high mannose and truncated structures suggested by the FAB-MS data; (5) the presence of terminal GalNAc as a major component is convincing evidence for the majority of complex-type glycans having this sugar at their non-reducing termini and corroborates the tentative assignment of GalNAc-GlcNAc to the A-type fragment ion at m/z 505 (Figure 2, Table II); (6) FAB-MS evidence for core fucosylation of complex-type and truncated glycans is corroborated by 4,6-GlcNAc and terminal Fuc.
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After ß-N-acetylhexosaminidase treatment, the A-type ions at m/z 260 (HexNAc+) and m/z 505 (HexNAc2+) disappeared while that at m/z 872 (Hex3HexNAc+) was unaffected. A major molecular ion was observed which corresponds to Hex3HexNAc2 (m/z 1171) and is consistent with the trimannosyl core without core fucosylation. A very minor molecular ion was also observed at m/z 1345 which corresponds to the trimannosyl core with core fucosylation. Other than Hex3HexNAc2, the major structures present in PNGase F pool after this treatment were Hex5HexNAc2, Hex6HexNAc2, Hex7HexNAc2, Hex8HexNAc2, and Hex9HexNAc2 (Figure 3A). This result indicates that the components with a trimannosyl core, with and without core fucosylation, carrying between one and eight additional N-acetylhexosamine residues (Fuc0-1Hex3HexNAc3-10) were efficiently digested to the main product Hex3HexNAc2. It can therefore be concluded that the N-acetylhexosamine residues of the N-glycan antenna are ß-linked. Since the ß-N-Acetyl-hexosaminidase is contaminated by an -fucosidase activity (< 2%), the removal of terminal HexNAc residues is accompanied by the removal of terminal
-Fuc residues. Comparison of linkage data before and after ß-N-acetylhexosaminidase treatment indicated that loss of terminal ß-GlcNAc residues, ß-GalNAc residues and
-Fuc residues is accompanied by a decrease in 2-linked Man, 2,4-linked Man and 2,6-linked Man, a concomitant increase in terminal Man, and loss of 4,6-linked GlcNAc and a increase of 4-linked GlcNAc. The signals at m/z 1580, 1784, 1988, 2193, and 2397 were unaffected by this treatment, a result that is consistent with the assignment of high mannose structures to these ions. Linkage data corroborated the assignment since terminal Man and 2-linked Man were the major residues.
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The glycan pool was then subjected to digestion with ß-mannosidase and the product was examined by FAB-MS after Sep-Pak purification. The FAB data indicated that, as expected, HexHexNAc2 (m/z 763) has shifted to HexNAc2 (m/z 559) and FucHexHexNAc2 (m/z 937) has shifted to FucHexNAc2 (m/z 733) (Figure 3C).
Assignment of oligosaccharide structures
Taking into consideration the FAB-MS, linkage, and exoglycosidase data, we conclude that the major N-glycans released by PNGase F fall into three classes, namely high mannose (Hex5-9HexNAc2) (Figure 4, structures I), truncated (Fuc0-1Hex3-4HexNAc2) (Figure 4, structures II, III), and complex (Fuc0-1Hex3HexNAc3-10) (Figure 4, structures IVXIX). The major non-reducing epitopes in the complex type glycans are: GlcNAc and GalNAcß14GlcNAc (lacdiNAc). The oligosaccharides which were undetectable prior to HF treatment, are composed of partially fucosylated trimannosyl cores, to which are attached up to six HexNAc residues to give mono-, bi-, tri-, and tetra-antennary structures as shown in Figure 4.
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Discussion |
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The results presented here have identified the structures shown in Figure 4 as the major N-linked glycans to be released from T.spiralis PC-bearing affinity-purified antigens by PNGase F. Three major classes of N-glycan structures were observed: high mannose type structures (Hex5-9HexNAc2), truncated structures (Fuc0-1Hex3-4HexNAc2), and those with a trimanosyl core, with and without core fucosylation, carrying between one and eight N-acetylhexosamine residues (Fuc0-1Hex3HexNAc3-10). The major non reducing epitopes in the complex type glycans are GlcNAc and GalNAcß14GlcNAc (lacdiNAc). Of the three classes of glycans, only the complex type glycans were found to be substituted with detectable levels of PC. In this study we have convincing evidence for the presence of a single PC group on structures of composition Fuc0-1Hex3HexNAc4 (Figure 4, structures V-VII), and Fuc1Hex3HexNAc5 (Figure 4, structures VIIIX). However, the glycans Fuc1Hex3HexNAc4-5 are also present in minor forms without a PC group since their molecular ions (m/z 1836 and 2081) were observed in the FAB-MS spectrum of permethylated glycans before HF treatment (Figure 1, Table I). The fact that the complex glycans (Fuc0-1Hex3HexNAc6-10) (Figure 4, structures XI-XIX) were only observed in the FAB-MS spectrum of permethylated glycans after HF treatment (Figure 2, Table II) and that perdeuteroacetylated forms of these oligosaccharides were not detected, suggest that they carry several PC groups resulting in multiple permanent charges which pose difficulties for FAB-MS.
Two main conclusions can be drawn from our mass spectrometric screening experiments on T.spiralis PC-bearing antigens. Firstly, the presence of lacdiNAc as a major non reducing epitope in the complex type glycans indicates that T.spiralis has PC-glycans that are different from those observed among filarial nematodes (Haslam et al., 1997, 1999). The PC attachment site(s) on these complex type glycans remains to be established. However, it is probable that, as with the PC-containing glycans characterized to date, attachment of PC is likely to be to GlcNAc (Haslam et al., 1997
, 1999; Lochnit et al., 1998
). Secondly, it was suggested in earlier studies of filarial nematodes (Houston et al., 1997
) that addition of PC might act as a termination signal and prevent the action of Gal and GalNAc transferases on the antennae "stubs." The present study provides evidence to the contrary. Thus, the presence of Hex3HexNAc10 (Figure 4, structure XIX) which is only observed after HF treatment, suggests that PC attachment does not prevent the action of GalNAc transferase on the antennae "stubs" in T.spiralis, since every antennae of this glycan carries lacdiNAc. Further work is required to establish whether PC attachment occurs before or after the action of GalNAc transferase to produce the lacdiNAc motif.
Phosphorylcholine bearing glycoproteins induce high levels of specific antibodies during the intestinal phase of T.spiralis infection (Peters et al., 1999). In contrast to the highly immunogenic tyvelose-bearing glycoproteins, PC-glycoproteins are not found in larval secreted products nor are they present on the surfaces of larvae (Ortega-Pierres et al., 1996
; Appleton, unpublished observations). Most likely, they are released by larvae during ecdysis. PC antigens in the filarial nematode, Brugia malayi, have been implicated in the induction of IL-10 production by B-1 lymphocytes (Palanivel et al., 1996
). The potent immunogenicity of T.spiralis PC-antigens suggests that they may influence the nature of the immune response despite the fact that PC-specific antibodies do not directly affect the survival of the parasite (Peters et al., 1999
).
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Materials and methods |
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Reduction and carboxymethylation
Reduction and protection of the disulfide bridges of the proteins of T.spiralis was carried out as described previously (Dell et al., 1994).
Tryptic digestion
The reduced carboxymethylated T.spiralis proteins were digested with L-1-tosylamide-2-phenylethylchloromethyl ketone (TPCK) bovine pancreas trypsin (EC 3.4.21.4, Sigma), for 5h at 37°C in 50mM 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, Roche Molecular Biochemicals) 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).
Sequential exo-glycosidase digestions
These were carried out on released glycans using the following enzymes and conditions: ß-N-acetylhexosaminidase (from bovine kidney, EC 3.2.1.30, Roche Molecular Biochemicals) 0.2 U in 200µl of 50mM ammonium formate pH 4.6, -mannosidase (from jack bean, EC 3.2.1.24, Roche Molecular Biochemicals) 0.5 U in 200 µl of 50mM ammonium acetate buffer, pH 4.5, ß-mannosidase (from Helix pomatia, EC 3.2.1.25, Oxford GlycoSciences) 0.2 U in 200 µl of 50 mM ammonium acetate buffer, pH 4. All enzyme digestions were incubated at 37°C for 48 h with a fresh aliquot of enzyme being added after 24 h and terminated by boiling for 3 min before 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 (Schneider and Ferguson, 1995)
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 previously (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 hexane 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.
FAB-MS analysis
FAB-MS spectra were acquired using a ZAB-2SE 2FPD mass spectrometer fitted with a cesium ion gun operated at 30kV. 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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2 To whom correspondence should be addressed
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References |
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