Glycobiology Advance Access originally published online on May 15, 2006
Glycobiology 2006 16(9):854-862; doi:10.1093/glycob/cwl001
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Protein glycosylation in Parelaphostrongylus tenuis—first description of the Gal
1-3Gal sequence in a nematode
2 James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853; and 3 Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, UK
1 To whom correspondence should be addressed; e-mail: s.haslam{at}imperial.ac.uk
Received on March 6, 2006; revised on May 9, 2006; accepted on May 10, 2006
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Abstract |
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The white-tailed deer is the definitive host of the parasitic nematode Parelaphostrongylus tenuis. This parasite also infects a wide variety of domesticated livestock, causing a debilitating neurologic disease. Glycoconjugates are becoming increasingly implicated in nematode strategies to maintain persistent infections in immunologically competent hosts. In this study, we have carried out detailed mass spectrometric analysis together with classical biochemical techniques, including western blotting and immunohistochemical staining with anticarbohydrate monoclonal antibodies and have shown that P. tenuis contains complex-type N-glycans with the antennae capped with Gal
1-3Galß1-4GlcNAc sequence. By mimicking a vertebrate glycan, Gal1-3Gal may aid the parasite in evading immunological detection by the host. This is the first report of the Gal1-3Gal sequence in a nematode. Key words: glycosylation / Parelaphostrongylus tenuis / mass spectrometry / nematode
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementAcknowledgmentsReferences |
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Parelaphostrongylus tenuis is a parasitic nematode common in white-tailed deer (Odocoileus virginianus) in eastern North America (Anderson, 1992
). Deer acquire P. tenuis by ingestion of infective third-stage larvae (L3) that develop within gastropods. The neurotropic L3 penetrate the alimentary canal, migrate to the posterior spinal cord, and then migrate within the subarachnoid space to the cranium (Anderson, 1992). Adult worms occupy the cerebrospinal fluid or associated blood vessels and blood sinuses of the cranium. Adult P. tenuis are long-lived, persisting up to 6 years in deer (Duffy, Greaves, et al., 2004). Infection of atypical hosts, including ovids, camelids, and cervids, causes a debilitating neurologic disease (Anderson, 1992). Deer infected with adult P. tenuis resist challenge infection with L3 (Duffy, Greaves, et al., 2004). Whereas this and other evidence (Slomke et al., 1995) indicate that acquired immunity develops in infected deer, the specificity and mechanism of protection are not known.
Nematode glycans are known to drive potent immune responses or, alternatively, to modulate immunity (Denkers et al., 1990; Terrazas et al., 2001; Goodridge et al., 2004). Tremendous progress has been made in recent years to define the structure of the glycan component of glycoconjugates from a variety of nematode species. In terms of protein glycosylation, the majority of detailed structural work has been concentrated on N-linked glycans. It is clear that structural features such as an abundance of high mannose (Man) glycans and the presence of both 1-6 and 1-3 core fucosylation are common to most nematode species (Haslam et al., 2001; Haslam and Dell, 2003). However, several examples of species-specific features such as the presence of Lewis x glycans in Dictyocaulus viviparus and tyvelose-containing glycans in Trichinella spiralis have also been reported (Reason et al., 1994; Haslam et al., 2000)
In order to identify targets of immunity to P. tenuis, we investigated the structure and composition of N-linked glycans in P. tenuis adult worms. We report here that the parasite synthesizes and secretes glycans that likely evade rather than stimulate the host’s immune system.
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Results |
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MALDI-MS analysis of N-glycans
The glycans identified from P. tenuis somatic extracts and excretory/secretory (E/S) products were very similar; so results from somatic extracts only will be presented. N-glycans from P. tenuis glycoproteins were released with peptide N-glycosidase F (PNGase F), permethylated, and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The spectrum (Figure 1A, Table I) revealed three major classes of N-glycan structures: high Man-type structures (Hex5-9HexNAc2); truncated structures with substoichiometric fucosylation (Fuc0 1Hex2-4 HexNAc2); and complex structures, with and without core fucosylation (Fuc0-1Hex3-9HexNAc3-5). The compositions of the complex structures are consistent with mono-, bi-, and tri-antennary glycans with Hex2HexNAc antennae. It was considered likely that these antennae have the sequence Gal-Gal-GlcNAc. As we have previously demonstrated in the characterization of N-glycans from Haemonchus contortus, the use of sequential digestion of extracted glycopeptides with PNGase F and PNGase A allows a partial fractionation of released N-glycans on the basis of whether they have a fucose (Fuc) attached to the 3-position of the Asn-linked N-acetylglucosamine (GlcNAc) residue (Haslam et al., 1996
). Such glycans are resistant to PNGase F digestion. Analysis of the PNGase A digest did reveal minor amounts of new glycan species with compositions of Fuc2Hex1-3HexNAc2 (data not shown). Experiments of the type described below for the PNGase F-digestion products revealed that these paucimannosidic glycans have di-fucosylated core structures that are common in invertebrates (Paschinger et al., 2005). Hence they were not examined further.
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Q-TOF-MS/MS of PNGase F-sensitive N-glycans
To provide additional evidence for the proposed N-glycan structures, the [M+2Na]2+ molecular ion (m/z 1338.0), which is consistent with a composition of FucHex7HexNAc4, was subjected to (Q-TOF)-MS/MS analysis (Figure 2). Key sequence informative fragment ions were observed at m/z 690, consistent with a Hex2HexNAc nonreducing terminal antennae structure and at m/z 1985 (10042+) and m/z 1317 which are consistent with loss of one and two Hex2HexNAc nonreducing terminal antennae structures from the molecular ion. These data are fully consistent with a core fucosylated bi-antennary complex glycan with Gal-Gal-GlcNAc antennae.
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Linkage analysis of PNGase F-sensitive N-glycans
To facilitate the identification of the constituent monosaccharides and to identify the position of glycosidic bonds, the PNGase F-released N-glycans were subjected to linkage analysis (Table II). A number of conclusions could be drawn from the linkage data: (1) high levels of 3,6-linked Man and 4-linked GlcNAc were observed, in accordance with their being essential constituents of the core of the majority of N-glycans; (2) the observed high levels of terminal Man corroborated the assignments of abundant high Man and paucimannose structures from the MALDI data; (3) galactose (Gal) is the other major terminal sugar; (4) the presence of high levels of 2-linked Man with lower levels of 2,4- and 2,6-linked Man is consistent with the majority of the complex glycans being bi-antennary with lesser amounts of tri- and possibly tetra-antennary glycans; (5) fucosylation of the cores was supported by the presence of 4,6-linked GlcNAc and terminal Fuc; and (6) high levels of 3-linked Gal suggest a Gal1-3Gal linkage in the putative Gal-Gal-GlcNAc antenna of the complex glycans.
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Sequential exoglycosidase digestions of PNGase F-sensitive N-glycans
In order to define anomeric configuration as well as to provide additional structural information, the PNGase F-released glycans were subjected to sequential digestion with green coffee bean -galactosidase and bovine testes ß-galactosidase. After each enzyme digestion, an aliquot was removed, methylated, and screened by MALDI-MS and linkage analysis. After -galactosidase digestion (Figure 1B, Table I), the mono-, bi-, and tri-antennary complex N-glycans (m/z 3306, 2653, and 2000) lost 1, 2, and 3 hexose (Hex) units, respectively (m/z 2693, 2245, and 1795). Linkage analysis showed the loss of the 3-linked Gal signal (data not shown). After subsequent ß-galactosidase digestion (Figure 1C, Table I), again the mono-, bi-, and tri-antennary complex N-glycans lost 1, 2, and 3 Hex units, respectively, leaving complex glycans with a single N-acetylhexosamine (HexNAc) in their antenna (m/z 2082, 1836, and 1591). A new signal at m/z 2327 is also observed, which is consistent with a composition of FucHex3HexNAc6, indicating the presence of minor amounts of tetra-antennary glycans. In accordance with the MALDI data, the linkage analysis showed a loss of the terminal Gal and an increase in the abundance of the terminal GlcNAc (data not shown). These data are fully consistent with the complex glycans containing Gal1-3Galß1-4GlcNAc antennae.
Assignment of oligosaccharide structures
Taken together, these data indicate that the complex N-glycans from adult P. tenuis are substoichiometricly core fucosylated mono-, bi-, tri-, and tetra-antennary structures with Gal1-3Galß1-4GlcNAc antennae. Paucimannosidic structures with mono- and di-fucosylated cores and high Man structures are also present (Figure 3).
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Detection of Gal in adult E/S products and somatic extracts of P. tenuis
Worm extracts were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and blotted to nitrocellulose. Anti-Gal monoclonal antibody (mAb) 86 bound to several molecules from adult P. tenuis. There was a smear of reactivity from 48–109 kDa in adult male E/S products and recognition of a discrete 37 kDa band (Figure 4A). Five proteins with apparent molecular masses of 50–118 kDa were detected in E/S products from adult female worms (Figure 4A), and three discrete proteins of 50–91 kDa were detected in mixed adult somatic extracts (Figure 4A). Anti-Gal mAb 86 did not bind proteins from P. tenuis L3 or first-stage larvae (L1) (Figure 4A), or T. spiralis larvae (not shown). Influenza virus-specific antibody [anti-equine influenza virus (EIV)], which was used as a negative control, did not bind parasite antigens. Binding of anti-Gal to blotted P. tenuis proteins was inhibited completely by 10 mM Gal1-3Gal (Figure 4B). Binding in the presence of 0.5 mM Gal1-3Gal was enhanced in two separate experiments, a result that is unexplained.
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Localization of Gal in adult P. tenuis
Immunohistochemical staining detected Gal in adult male and female parasites (Figure 5) but not in L3 or L1 (not shown). The epitope was detected in the uterus (Figure 5A and C), the excretory glands (Figure 5E), and the intestine of female worms (Figure 5A). Gal was restricted to the excretory glands (Figure 5G and I), the intestine (Figure 5K), and luminal contents of male worms (Figure 5I). Absence of reactivity in the alimentary canal of some individual worms was observed and might reflect differences in worm age (ranging from several months to several years) or their location within the host (subdural versus intravascular) (Duffy, Greaves, et al., 2004). These parameters were not known for individual worms used in this study. Tissue sections from the central nervous system (CNS) of a sheep and gibbon were included as controls. Gal is synthesized by all mammals, with the exception of primates (Galili, 2005). Consistent with this, sheep endothelium was bound by anti-Gal, and gibbon tissues were not (not shown). Anti-EIV failed to bind with any tissues (Figure 5B, D, F, H, J, and L).
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Discussion |
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Here we report the discovery of Gal
1-3Galß1-4GlcNAc as the dominant antenna of complex type N-glycan of adult stage P. tenuis. Adult P. tenuis feed on host tissues, and deer, like most mammals, also synthesize Gal1-3Gal (Galili, 2001, 2005). Two observations provide evidence that Gal1-3Gal recovered from P. tenuis was not host-derived. First, we detected Gal in reproductive and secretory organs of the parasite rather than strictly within the gut lumen, as would be expected if the glycan were ingested. Second, we failed to detect the expected host-derived sialic acid in P. tenuis extracts. Thus, this report constitutes the first description of Gal1-3Galß1-4GlcNAc in a metazoan parasite.
Evasion of the immune response is a necessary strategy for parasites that require prolonged association with their hosts. Vertebrate hosts of P. tenuis produce Gal1-3Gal and would not recognize this glycan as a foreign antigen. The restriction of Gal to adults of P. tenuis suggests that it may be a form of molecular mimicry (Damian, 1997) that specifically enables evasion of the immune response of the definitive host. Other nematodes also demonstrate life-stage restricted synthesis of glycans (Haslam et al., 1998; Cipollo et al., 2005). Studies of parasites across the phylum have revealed that N-glycans of nematodes are diverse; however, in none of the species characterized to date has the Gal1-3Gal sequence been observed (Haslam et al., 2001; Haslam and Dell, 2003). It appears that among related metastrongyloid nematodes, synthesis of Gal is not a common strategy, as we did not detect Gal in Parastrongylus (=Angiostrongylus) cantonensis and P. costaricensis (Duffy and Appleton, unpublished data). The Gal1-3Gal sequence appears to be restricted to N-linked glycoprotein glycans. MALDI-MS screening of O-linked glycans revealed two major species with compositions of HexHexNAc and FucHexHexNAc. Additional Q-TOF MS-MS and gas chromatography (GC)-MS linkage data are consistent with a core type 1 (Galß1-3GalNAc) and fucosylated core type 1 (Galß1-3(Fuc1-2)GalNAc) structures (data not shown). Experiments are underway to assess whether the Gal1-3Gal sequence might also be present on glycolipids.
A role in facilitating immune evasion is supported by the finding that modification with Gal appeared to be common among P. tenuis proteins targeted for export. Glycoproteins in E/S products that bear Gal may originate in the intestine, secretory gland, or female gonad. The apparent difference in the number and masses of glycoproteins bearing Gal in male versus female E/S products may be attributed to molecules that are unique to the female gonad. Although the identities of Gal-bearing glycoproteins were not determined, the sugar was abundant in the matrix contained within the uterus. The glycan might coat egg surfaces prior to their delivery into the bloodstream, facilitating immune evasion during their intravenous passage from the CNS and development in the lungs. Within the intestine, Gal was localized exclusively to the luminal contents and cells of the anterior intestine of both male and female worms. The filarial nematode, Acanthocheilonema viteae, synthesizes an immunomodulatory E/S product, ES-62, in the anterior regions of the alimentary canal (Stepek et al., 2002). Like Gal in P. tenuis, ES-62 of A. viteae is restricted to stages resident in the definitive host (Stepek et al., 2002). The modulatory properties of ES-62 are attributed to N-glycans that are modified with phosphorylcholine (Haslam et al., 1997; Houston et al., 2000). We have determined that P. tenuis also contains paucimannosidic N-glycans with Fuc-attached 1-3 to the Asn-linked GlcNAc residue. Such structures have been shown to be immunogenic in mammals (van Die et al., 1999). We have not determined whether animals infected with P. tenuis mount antibody responses against 1-3-linked Fuc. Such paucimannosidic N-glycans can also be naturally O-methylated in nematodes (Haslam and Dell, 2003; Haslam et al., 2001). Deuteromethylation experiments will be undertaken to assess whether this is also the case in P. tenuis.
While the gut is known to be a target of immunity and an important source of E/S products in parasitic nematodes, much less is known of the excretory gland in these contexts. The P. tenuis excretory gland is comprised of two large (5–10% of body length) granular cells within the pseudocoelom (Anderson, 1956; Mobarak and Ryan, 1999). In other nematodes, the excretory system has been implicated in osmoregulation (Nelson and Riddle, 1984), secretion of enzymes (Romanowski et al., 1973), and production of protease inhibitors (Rhoads et al., 1978). The function of the excretory gland in P. tenuis is not known; however, modifying its products with glycans that would be invisible to the immune system may facilitate their distribution and longevity in the host.
To our knowledge, this is the first description of such a glycan structure in a nematode or in any other helminth. Display of the Gal1-3Gal sequence by P. tenuis would equate with presentation of "self" glycans to the host immune system. Therefore, this form of molecular mimicry could represent a strategy utilized by the parasite to evade immune defense in order to persist for such great lengths of time in the host.
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Materials and Methods |
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Nematode extracts
Adult P. tenuis were recovered from the craniums of white-tailed deer. Worms were disrupted in Dulbecco’s phosphate-buffered saline (DPBS) containing detergents (1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS) and protease inhibitors (1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 25 µg/mL TLCK, and 25 µg/mL L-1-tosylamido-2-phenylethyl chloromethyl ketone [TPCK]). The somatic extract was clarified by centrifugation at 600 x g for 10 min. Adult worm E/S antigens were collected previously (Duffy and Burt, 2002
). Protein concentrations of E/S antigens and somatic extracts were determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). Proteins were stored at –20°C until use.
PAGE and western blot
Antigen preparations included adult P. tenuis E/S products, somatic extracts from mixed-sex adult P. tenuis, and somatic extracts from L1 and L3. T. spiralis glycoproteins (L1 somatic extract and E/S products) were included as negative control antigens (Beiting et al., 2004). Proteins (20 µg) were resolved by reducing SDS–PAGE (Laemmli, 1970) in 11% analytical gels and were blotted to nitrocellulose membranes (Towbin et al., 1979). Western blots were incubated with monoclonal antibody (mAb) M86 specific for Gal (Gal1-3Gal1-4GlcNAc, IgM isotype; Alexis Biochemicals, San Diego, CA) or an irrelevant isotype control mAb specific for equine influenza virus (anti-EIV clone 1; Appleton et al., 1987). Antibody binding was detected with HRP-conjugated goat anti-mouse IgG (2.5 µg/mL; ICN/Cappel, Aurora, OH), developed with a chemiluminescent substrate (ECL Reagent; Amersham Pharmacia Biotech, Piscataway, NJ), and detected by autoradiography.
To confirm the specificity of binding, each mAb was incubated for 2 h in 5% bovine serum albumin (BSA) containing 0–20 mM Gal1-3Gal (V-Laboratories Inc., Covington, LA). Antibody mixtures were added to the strips cut from western blots of adult female P. tenuis E/S products (9 µg/strip). Binding was detected as described above.
Immunohistochemistry
P. tenuis larvae and adult worms were fixed in formalin and embedded in paraffin as described elsewhere (Duffy et al., 2006). Negative control tissues included mouse diaphragm infected with T. spiralis larvae (Beiting et al., 2004), adult T. spiralis, Heligmosomoides polygyrus L3, and CNS tissues from an Old World primate [Hylobates lar (gibbon) CNS (Duffy, Miller, et al., 2004)]. Sheep spinal cord served as a positive control for Gal. Five micron sections were cut, mounted on glass slides, and deparaffinized. Antigens were unmasked, and immunohistochemistry was performed as described previously (Duffy et al., 2006). Briefly, antibodies were diluted in 5% BSA and detected with HRP-conjugated goat anti-mouse IgG (20 µg/mL) using 3-amino-9-ethyl-carbazole (AEC) (Sigma, St. Louis, MO) as substrate. Sections were counter-stained with Harris’ modified hematoxylin (Fisher Scientific, Pittsburgh, PA), and coverslips were mounted with Glycergel (Dako Corporation, Carpinteria, CA). Sections were examined, and the images were captured using an Olympus BX51 microscope fitted with a DP12 digital camera. Resolution was adjusted to 300 dpi (Adobe Photoshop).
Nematode N-glycan preparation
Nematode extracts were reduced in 1 mL of 50 mM Tris–HCl buffer (pH 8.5) containing 2 mg/mL dithiothreitol. Reduction was performed under a nitrogen atmosphere at 37°C for 1 h. Carboxymethylation was carried out by the addition of iodoacetic acid (5-fold molar excess over dithiothreitol), and the reaction was allowed to proceed under a nitrogen atmosphere at 37°C for 1 h. Carboxymethylation was terminated by dialysis against 4 x 2.5 L of 50 mM ammonium bicarbonate (pH 8.5) at 4°C for 48 h. After dialysis, the sample was lyophilized. The reduced carboxymethylated extracted proteins were digested with TPCK treated bovine pancreas trypsin (EC 3.4.21.4
[EC]
, 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 Corp., Milford, MA) as described (Dell et al., 1994). PNGase F (EC 3.5.1.52
[EC]
, Roche Molecular Biochemicals, Mannheim, Germany) 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 digested sample is loaded on a pre-conditioned C18-Sep-Pak (Waters Corp.) and eluted with 5 mL of 5% (v/v) acetic acid followed by elution with 4 mL of 20% (v/v) propanol in 5% (v/v) acetic acid. Glycopeptides remaining after PNGase F digestion were further digested with PNGase A (EC 3.5.1.52
[EC]
, Roche Molecular Biochemicals), in ammonium acetate buffer (50 mM, pH 5.0), for 16 h at 37°C using 0.2 mU of the enzyme. The released N-glycans were purified on a C18-Sep-Pak (Waters Corp.) as described above.
Sequential exoglycosidase digestion
The PNGase F-released glycans were incubated with -galactosidase (from green coffee beans, EC 3.2.1.22
[EC]
, Glyko, San Leandro, CA): 0.5 U in 100 µL of 50 mM ammonium formate buffer (pH 6.0) for 24 h and then for an additional 24 h with a second aliquot of enzyme; ß-galactosidase (from bovine testes, EC 3.2.1.23
[EC]
, Roche Molecular Biochemicals): 10 mU in 100 µL of 50 mM ammonium formate buffer (pH 4.6) for 48 h. All enzyme digestions were incubated at 37°C and terminated by boiling for 3 min before lyophilization.
Chemical derivatization for MALDI-MS and GC-MS
Permethylation using the sodium hydroxide procedure was performed as described (Dell et al., 1994). After derivatization, the reaction products were purified on C18-Sep-Pak (Waters Corp.). MALDI data were acquired using a PerSeptive Biosystems (Framingham, MA). Voyager-DETM STR mass spectrometer in the reflectron mode with delayed extraction. Derivatized glycans were dissolved in 10 µL of methanol, and 1 µL of dissolved sample was premixed with 1 µL of matrix (2,5-dihydroxy benzoic acid) before loading onto a target plate.
CAD-ES-MS/MS analysis
Collisionally activated dissociation (CAD)-ES-MS/MS spectra were acquired using a Q-TOF (Micromass, Manchester, UK) instruments. The permethylated glycans were dissolved in methanol before loading into a spray capillary coated with a thin layer of gold/palladium, inner diameter 2 µL (Proxeon, Odense, Denmark). A potential of 1.5 kV was applied to a nanoflow tip to produce a flow rate of 10–30 nL/min. The drying gas used was N2, and the collision gas was argon, with the collision gas pressure maintained at 10–4 mb. Collision energies varied depending on the size of the carbohydrate, typically between 30 and 90 eV.
GC-MS linkage analysis
Partially methylated alditol acetates were prepared from permethylated glycans. Briefly, the permethylated glycans were hydrolyzed with 2 M trifluoroacetic acid for 2 h at 121°C, reduced with 10 mg/mL sodium borodeuteride in 2 M aqueous ammonium hydroxide at room temperature for 2 h, and then acetylated with acetic anhydride at 100°C for 1 h. Linkage analysis was carried out on a Perkin Elmer Clarus 500 instrument fitted with an RTX-5 fused silica capillary column (30 m x 0.32 mm internal diameter; Restek Corp., Bellefonte, PA). The sample was dissolved in hexanes and injected onto the column at 65°C. The column was maintained at this temperature for 1 min and then heated to 290°C at a rate of 8°C/min.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementAcknowledgmentsReferences |
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None declared.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementAcknowledgmentsReferences |
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We thank the following individuals at Cornell University: S. Stehman, M. Stefanski-Seymour, and M. Smith for assistance with worm recovery from infected deer; R. Mo and L. Gagliardo for preparation of worm extracts; A. de Lahunta and D. Schlafer for gibbon and baboon tissues; and D. Beiting and S. Thrasher for materials from T. spiralis and H. polygyrus. We thank C. Miller (Miami MetroZoo) and J.M. Kinsella (HelmWest Laboratories) for materials from P. cantonensis and P. costaricensis. This work was supported by a grant from the Morris Animal Foundation (D01LA-13; M.S.D. and J.A.A.) and a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada (MSD) and by the Biotechnology and Biological Sciences Research Council (BBSRC) and the Wellcome Trust (grants to A.D., H.R.M., and S.M.H.). A.D. is a BBSRC Professorial Fellow. Funding to pay the Open Access publication charges for this article was provided by the BBSRC.
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Abbreviations |
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CAD, collisionally activated dissociation; CNS, central nervous system; E/S, excretory/secretory; EIV, equine influenza virus; Fuc, fucose; Gal, galactose; GC, gas chromatography; GlcNAc, N-acetylglucosamine; Hex, hexose; HexNAc, N-acetylhexosamine; mAb, monoclonal antibody; MALDI, matrix-assisted laser desorption/ionization; Man, mannose; MS, mass spectrometry; PNGase A, peptide N-glycosidase A; PNGase F, peptide N-glycosidase F; Q-TOF, Q-STAR Pulsar quadrupole time-of-flight; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone
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References |
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