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Structural Studies of N-Glycans of Filarial Parasites

1999; Elsevier BV; Volume: 274; Issue: 30 Linguagem: Inglês

10.1074/jbc.274.30.20953

1083-351X

Stuart M. Haslam, Katrina M. Houston, William Harnett, Andrew J. Reason, Howard R. Morris, Anne Dell,

Nematode management and characterization studies

N-Type glycans containing phosphorylcholine (PC-glycans), unusual structures found in the important human pathogens filarial nematodes, represent a novel target for chemotherapy. Previous work in our laboratories produced compositional information on the PC-glycan of ES-62, a secreted protein of the rodent parasite Acanthocheilonema viteae. In particular, we established using fast atom bombardment mass spectrometry (MS) analysis that PC was attached to a glycan with a trimannosyl core, with and without core fucosylation, carrying between one and four additional N-acetylglucosamine residues. In the present study, we demonstrate that this structure is conserved among filarial nematodes, including the parasite of humans,Onchocerca volvulus, for which new drugs are most urgently sought. Furthermore, by employing a variety of procedures, including collision-activated dissociation MS-MS analysis and matrix-assisted laser desorption MS analysis, we reveal that surprisingly, filarial nematodes also contain N-linked glycans, the antennae of which are composed of chito-oligomers. To our knowledge, this is the first report of such structures in a eukaryotic glycoprotein. N-Type glycans containing phosphorylcholine (PC-glycans), unusual structures found in the important human pathogens filarial nematodes, represent a novel target for chemotherapy. Previous work in our laboratories produced compositional information on the PC-glycan of ES-62, a secreted protein of the rodent parasite Acanthocheilonema viteae. In particular, we established using fast atom bombardment mass spectrometry (MS) analysis that PC was attached to a glycan with a trimannosyl core, with and without core fucosylation, carrying between one and four additional N-acetylglucosamine residues. In the present study, we demonstrate that this structure is conserved among filarial nematodes, including the parasite of humans,Onchocerca volvulus, for which new drugs are most urgently sought. Furthermore, by employing a variety of procedures, including collision-activated dissociation MS-MS analysis and matrix-assisted laser desorption MS analysis, we reveal that surprisingly, filarial nematodes also contain N-linked glycans, the antennae of which are composed of chito-oligomers. To our knowledge, this is the first report of such structures in a eukaryotic glycoprotein. Adult filarial nematodes secrete phosphorylcholine (PC) 1The abbreviations used are: PC, phosphorylcholine; CAD, collision-activated dissociation; FAB, fast atom bombardment; Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; GC, gas chromatography; GlcNAc, N-acetylglucosamine; Hex, hexose; HexNAc, N-acetylhexosamine; HF, hyrdrofluoric acid; MALDI, matrix-assisted laser desorption; Man, mannose; MS, mass spectrometry; PNGase F, peptide N-glycosidase F-containing glycoproteins during parasitism of their vertebrate hosts (1Harnett W. Parkhouse R.M.E. Sood M.L. Kapur J. Perspectives in Nematode Physiology and Biochemistry. Narendra Publishing House, Delhi, India1995: 207-242Google Scholar). A number of studies performed in vitro lead to the conclusion that this PC may have an immunomodulatory function. Thus, PC-containing filarial products have been shown to (i) inhibit proliferation of human T-cells induced by phytoheamagglutinin (2Lal R.B. Kumaraswami V. Steel C. Nutman T.B. Am. J. Trop. Med. Hyg. 1990; 42: 56-64Crossref PubMed Scopus (78) Google Scholar) and also murine B cells induced via the antigen receptor (3Harnett W. Harnett M.M. J. Immunol. 1993; 151: 4829-4837PubMed Google Scholar), and (ii) 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 of murine B cells and the human T-cell line Jurkat (3Harnett W. Harnett M.M. J. Immunol. 1993; 151: 4829-4837PubMed Google Scholar, 4Deehan M.R. Harnett M.M. Harnett W. J. Immunol. 1997; 159: 6105-6111PubMed Google Scholar, 5Deehan M.R. Frame M.J. Parkhouse R.M.E. Seatter S.D. Reid S.D. Harnett M.M. Harnett W. J. Immunol. 1998; 160: 2692-2699PubMed Google Scholar, 6Harnett M.M. Deehan M.R. Williams D. Harnett W. Parasite Immunol. 1998; 20: 551-564Crossref PubMed Scopus (61) Google Scholar). As these effects appear to be largely due to the PC moiety of the molecules, we are interested in the possibility of developing novel anti-filarial drugs that prevent PC attachment during biosynthesis. Structural and biosynthetic information on PC-containing filarial biopolymers is an essential prerequisite to rational design of such drugs. ES-62 is the major PC-containing protein secreted byAcanthocheilonema viteae, a rodent filarial nematode (7Harnett W. Worms M.J. Kapil A. Grainger M. Parkhouse R.M.E. Parasitology. 1989; 99: 229-239Crossref PubMed Scopus (71) Google Scholar). We have recently shown that PC is attached to the N-linked glycans in ES-62 (8Harnett W. Houston K.M. Amess R. Worms M.J. Exp. Parasitol. 1993; 77: 498-502Crossref PubMed Scopus (58) Google Scholar, 9Houston K.M. Harnett W. J. Parasitol. 1996; 82: 320-324Crossref PubMed Scopus (22) Google Scholar), almost certainly being added in the Golgi during intracellular trafficking following the generation of an appropriate glycan substrate (10Houston K.M. Cushley W.C. Harnett W. J. Biol. Chem. 1997; 272: 1527-1533Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Mass spectrometric structural analyses have defined the types of N-glycan in ES-62 that carry PC substitution (11Haslam S.M. Khoo K.-H. Houston K.M. Harnett W. Morris H.R. Dell A. Mol. Biochem. Parasitol. 1997; 85: 53-66Crossref PubMed Scopus (93) Google Scholar). They all have a trimannosyl core, which is characteristic of eukaryotic N-glycans, and a portion are core-fucosylated. In addition they carry between one and fourN-acetylglucosamine (GlcNAc) residues that are separately attached as antenna “stubs” to the core. It is likely that PC is attached directly to one or more of these GlcNAc residues (11Haslam S.M. Khoo K.-H. Houston K.M. Harnett W. Morris H.R. Dell A. Mol. Biochem. Parasitol. 1997; 85: 53-66Crossref PubMed Scopus (93) Google Scholar). Although such information on structure/biosynthesis may be employed in the design of inhibitors of PC attachment to ES-62, it is unknown whether PC is attached to similar glycan structures on all filarial nematodes, and hence the relevance to drug development for human filarial parasites is uncertain. To address this issue, we have begun to screen a range of filarial parasites for their N-glycan content. In this study, we compared the major N-glycans present in extracts of adult A. viteae with similar extracts from Onchocerca volvulus, the human filarial nematode for which safe and effective drugs against adult stages is most urgently sought (12World Health Organization Tropical Disease Research, Progress 1995–96, Thirteenth Programme Report UNDP/World Bank/WHO Special Programme for Research & Training in Tropical Diseases. WHO, Geneva1997: 86-99Google Scholar), and with secreted material from the closely related (13Copeman D.B. Acta Trop. 1993; 53: 307-317Crossref PubMed Scopus (3) Google Scholar) bovine parasite Onchocerca gibsoni, which has been employed for drug screening (14Copeman D.B. Trop. Med. Parasitol. 1979; 30: 469-474Google Scholar). We show that the two Onchocercaspecies have PC-containing glycans of the type identified in A. viteae, indicating that PC-glycans could be a target for the development of wide-spectrum anti-filarial drugs. Furthermore, we report the unexpected discovery of highly unusual N-linked glycans, the antennae of which are composed of chito-oligomers. All three nematodes show remarkable similarity in their chito-oligomer content. Adult A. viteae (mixed males and females) were recovered from jirds (Meriones libycus) infected eight weeks previously according to the method of Wormset al. (15Worms M.J. Terry R.J. Terry A. J. Parasitol. 1961; 47: 963-970Crossref Google Scholar). The worms were washed several times in sterile RPMI 1640 medium before being homogenized in phosphate-buffered saline (pH 7.2) containing proteolytic enzyme inhibitors as described previously (16Harnett W. Patterson M. Copeman D.B. Parkhouse R.M.E. Int. J. Parasitol. 1994; 24: 543-550Crossref PubMed Scopus (8) Google Scholar). The extract was centrifuged (20,000 ×g for 25 min), and the supernatant was recovered and stored at −20 °C until used. Adult O. volvulus were recovered from nodules (frozen) surgically removed from Guatemalan patients. A phosphate-buffered saline extract was prepared according to the method employed with A. viteae. Adult female O. gibsoniwere recovered from nodules excised from infected Queensland cattle, washed thoroughly with RPMI 1640 medium, and then incubated for 48 h at 37 °C. The spent medium was recovered, passed through a 0.2 mm membrane, and excretory secretory material was prepared as described previously (17Macdonald M. Copeman D.B. Harnett W. Int. J. Parasitol. 1996; 26: 1075-1080Crossref PubMed Scopus (17) Google Scholar). Reduction and protection of the disulfide bridges of the filarial proteins was carried out as described (18Dell A. Reason A.J. Khoo K.-H. Panico M. McDowell R.A. Morris H.R. Methods Enzymol. 1994; 230: 108-132Crossref PubMed Scopus (240) Google Scholar, 19Haslam S.M. Coles G.C. Munn E.A. Smith T.S. Smith H.F. Morris H.R. Dell A. J. Biol. Chem. 1996; 271: 30561-30570Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The reduced carboxymethylated filarial proteins were digested withl-1-tosylamide-2-phenylethylchloromethyl ketone-treated bovine pancreas trypsin (EC 3.4.21.4) (Sigma) for 5 h at 37 °C in 50 mm ammonium bicarbonate buffer (pH 8.4). PNGase F (EC3.2.2.18) (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 units of the enzyme. The reaction was terminated by lyophilization and the products were purified on Sep-Pak C18 (Waters Corp.) as described (18Dell A. Reason A.J. Khoo K.-H. Panico M. McDowell R.A. Morris H.R. Methods Enzymol. 1994; 230: 108-132Crossref PubMed Scopus (240) Google Scholar, 19Haslam S.M. Coles G.C. Munn E.A. Smith T.S. Smith H.F. Morris H.R. Dell A. J. Biol. Chem. 1996; 271: 30561-30570Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Samples were incubated with 50 μl of 48% HF (Aldrich) at 0 °C for 48 h (11Haslam S.M. Khoo K.-H. Houston K.M. Harnett W. Morris H.R. Dell A. Mol. Biochem. Parasitol. 1997; 85: 53-66Crossref PubMed Scopus (93) Google Scholar). The reagent was removed under a stream of nitrogen. Released glycans were digested with N-acetyl-β-d-hexosaminidase (from bovine kidney) (EC 3.2.1.30) (Roche Molecular Biochemicals): 0.2 units in 100 μl of 50 mm sodium citrate phosphate buffer at pH 4.6 for 18 h at 37 °C. The reaction was terminated by lyophilization, and the reaction products were permethylated for FAB-MS analysis (18Dell A. Reason A.J. Khoo K.-H. Panico M. McDowell R.A. Morris H.R. Methods Enzymol. 1994; 230: 108-132Crossref PubMed Scopus (240) Google Scholar,19Haslam S.M. Coles G.C. Munn E.A. Smith T.S. Smith H.F. Morris H.R. Dell A. J. Biol. Chem. 1996; 271: 30561-30570Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Permethylation using the sodium hydroxide procedure was performed as described (18Dell A. Reason A.J. Khoo K.-H. Panico M. McDowell R.A. Morris H.R. Methods Enzymol. 1994; 230: 108-132Crossref PubMed Scopus (240) Google Scholar, 19Haslam S.M. Coles G.C. Munn E.A. Smith T.S. Smith H.F. Morris H.R. Dell A. J. Biol. Chem. 1996; 271: 30561-30570Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Perdeuteroacetylation was performed with 100 μl pyridine:d 6-acetic anhydride (1:1, v/v) at 80 °C for 2 h, after which the reagents were removed under a stream of nitrogen. After both derivatization procedures the reaction products were purified on Sep-Pak C18 (Waters Corp.) as described (18Dell A. Reason A.J. Khoo K.-H. Panico M. McDowell R.A. Morris H.R. Methods Enzymol. 1994; 230: 108-132Crossref PubMed Scopus (240) Google Scholar, 19Haslam S.M. Coles G.C. Munn E.A. Smith T.S. Smith H.F. Morris H.R. Dell A. J. Biol. Chem. 1996; 271: 30561-30570Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Partially methylated alditol acetates were prepared from permethylated samples for GC-MS linkage analysis as described (20Albersheim P. Nevins D.J. English P.D. Karr A. Carbohydr. Res. 1967; 5: 340-345Crossref Scopus (1522) Google Scholar). 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 spectra were acquired using a ZAB-2S.E. 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 (18Dell A. Reason A.J. Khoo K.-H. Panico M. McDowell R.A. Morris H.R. Methods Enzymol. 1994; 230: 108-132Crossref PubMed Scopus (240) Google Scholar,19Haslam S.M. Coles G.C. Munn E.A. Smith T.S. Smith H.F. Morris H.R. Dell A. J. Biol. Chem. 1996; 271: 30561-30570Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 21Dell A. Adv. Carbohydr. Chem. Biochem. 1987; 45: 19-72Crossref PubMed Scopus (333) Google Scholar). CAD MS-MS collision-activated decomposition spectra were recorded using a Fisons VG Analytical four sector ZAB-T mass spectrometer in the array detector mode as described (19Haslam S.M. Coles G.C. Munn E.A. Smith T.S. Smith H.F. Morris H.R. Dell A. J. Biol. Chem. 1996; 271: 30561-30570Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). MALDI-MS was performed in the Reflectron mode using a Perseptive Biosystems Voyager Elite mass spectrometer with delayed extraction. Samples were dissolved in 20 μl of 80:20 (v/v) methanol:water, and aliquots (0.5 μl) of the resulting solutions were analyzed using a matrix of 2,5-dihydrobenzoic acid. Angiotensin and insulin B chain were employed as external calibrants. In our previous work on ES-62, we showed that FAB-MS of perdeuteroacetylated derivatives provides a sensitive means of detecting low molecular weight glycans substituted with PC (11Haslam S.M. Khoo K.-H. Houston K.M. Harnett W. Morris H.R. Dell A. Mol. Biochem. Parasitol. 1997; 85: 53-66Crossref PubMed Scopus (93) Google Scholar). We have used a similar strategy in the present work to examine each of the parasite samples for PC-substituted glycans. N-Glycans were released by PNGase F digestion of reduced/carboxymethylated trypsinized material. The glycans were separated from more hydrophobic components by passing the digest through a Sep-Pak cartridge and were analyzed by FAB-MS after perdeuteroacetylation and Sep-Pak purification. All samples gave molecular ions analogous to those observed previously in ES-62 (Fig.1 and TableI). The major signals in Fig.1 A correspond to protonated molecular ions, whereas those in Fig. 1 B are sodiated; Fig. 1 C shows similar amounts of protonated and sodiated species. The data are consistent with compositions PC1HexNAc3–6Hex3Fuc0–1(Table I) corresponding to the structures shown in Fig.2. It is notable that the most abundant PC-containing glycans from A. viteae and O. gibsoni are core-fucosylated, whereas O. volvulusglycans are predominantly nonfucosylated. Signals corresponding to neutral N-glycans were observed in all three samples (Fig. 1and Table I). Once again, these were consistent with data previously obtained from ES-62 (11Haslam S.M. Khoo K.-H. Houston K.M. Harnett W. Morris H.R. Dell A. Mol. Biochem. Parasitol. 1997; 85: 53-66Crossref PubMed Scopus (93) Google Scholar).Table IAssignments of molecular ions observed in FAB spectra of perdeuteroacetylated N-glycans of A. viteae, O. gibsoni, and O. volvulus eluting in 50% aqueous acetonitrile (v/v) fraction from a Sep-Pak C18Signal (m/z)Assignment1311Hex2HexNAc2+Na+1525FucHex2HexNAc2+H+1547FucHex2HexNAc2+Na+1586Hex3HexNAc2+H+1608Hex3HexNAc2+Na+1822FucHex3HexNAc2+H+1844FucHex3HexNAc2+Na+1879Hex3HexNAc3+H+1901Hex3HexNAc3+Na+1905Hex4HexNAc2+Na+1999PCHex3HexNAc3+H+2021PCHex3HexNAc3+Na+2115FucHex3HexNAc3+H+2137FucHex3HexNAc3+Na+2172Hex3HexNAc4+H+2180Hex5HexNAc2+H+2194Hex3HexNAc4+Na+2202Hex5HexNAc2+Na+2235PCFucHex3HexNAc3+H+2257PCFucHex3HexNAc3+Na+2292PCHex3HexNAc4+H+2314PCHex3HexNAc4+Na+2408FucHex3HexNAc4+H+2430FucHex3HexNAc4+Na+2465Hex3HexNAc5+H+2487Hex3HexNAc5+Na+2528PCFucHex3HexNAc4+H+2550PCFucHex3HexNAc4+Na+2585PCFucHex3HexNAc5+H+2607PCHex3HexNAc5+Na+2723FucHex3HexNAc5+H+2821PCFucHex3HexNAc5+H+2843PCFucHex3HexNAc5+Na+2878PCHex3HexNAc6+H+2900PCHex3HexNAc6+Na+3114PCFucHex3HexNAc6+H+3136PCFucHex3HexNAc6+Na+The signals at 45 mass unit intervals below major signals in Fig. 1correspond to under-deuteroacetylation. Open table in a new tab Figure 2Proposed structures of the PC containingN-glycans of ES-62 (11Haslam S.M. Khoo K.-H. Houston K.M. Harnett W. Morris H.R. Dell A. Mol. Biochem. Parasitol. 1997; 85: 53-66Crossref PubMed Scopus (93) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The signals at 45 mass unit intervals below major signals in Fig. 1correspond to under-deuteroacetylation. Our earlier studies of ES-62 indicated that although glycans containing several PC substituents are rather intractable to direct MS analysis, putative glycan scaffolds for multiple PC substitution could be readily detected after PC release. We proposed the following simple strategy for screening for large and/or multiply PC-substituted glycans (11Haslam S.M. Khoo K.-H. Houston K.M. Harnett W. Morris H.R. Dell A. Mol. Biochem. Parasitol. 1997; 85: 53-66Crossref PubMed Scopus (93) Google Scholar). A portion of the sample is initially screened for the presence of components lacking PC moieties. This is achieved by MS analyses of permethylated derivatives using experimental procedures involving chloroform extraction that do not allow the recovery of charged material from the permethylation procedure. A second portion is then treated with aqueous hydrofluoric acid using conditions that are known to cleave phosphodiester linkages. The products are permethylated and analyzed by MS. 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. When we applied this strategy to the phosphate-buffered saline and excretory secretory extracts, we made the unexpected discovery that N-glycans with highly unusual antennae are present in all three nematodes. FAB-MS of PNGase-F released permethylated glycans from the three species of filarial nematodes gave data corresponding to high mannose (Hex5–9HexNAc2) and truncated structures (Fuc0–1Hex2–4 HexNAc2) consistent with those previously observed in our studies of ES-62 (11Haslam S.M. Khoo K.-H. Houston K.M. Harnett W. Morris H.R. Dell A. Mol. Biochem. Parasitol. 1997; 85: 53-66Crossref PubMed Scopus (93) Google Scholar) and were not investigated further (data not shown). In contrast, spectra from samples that had been treated with HF after the PNGase F digestion showed abundant new signals, the compositions of which suggested novel structures (Fig. 3). The spectra are dominated by a series of glycans with composition HexNAc3–11Hex3Fuc0–1 (TableII). These compositions are consistent with substoichiometrically fucosylated trimannosyl cores to which are added between one and eight N-acetylhexosamine residues. As previously observed (Fig. 1 and Table I), the N-glycans ofO. volvulus are core-fucosylated to a much lesser degree than those of A. viteae and O. gibsoni.Table IIAssignments of molecular and fragment ions observed in FAB and MALDI spectra of permethylated N-glycans of A. viteae, O. gibsoni, and O. volvulus after treatment with HF eluting in the 50% aqueous acetonitrile (v/v) fraction from a Sep-Pak C18Signal (m/z)Assignment260HexNAc+464HexHexNAc+505HexNAc2+679FucHexNAc2+750HexNAc3+872Hex3HexNAc+967Hex2HexNAc2+Na+995HexNAc4+1117Hex3HexNAc2+1141FucHex2HexNAc2+Na+1171Hex3HexNAc2+Na+1240HexNAc5+1280Hex5HexNAc+1416Hex3HexNAc3+Na+1579Hex5HexNAc2+Na+1590FucHex3HexNAc3+Na+1661Hex3HexNAc4+Na+1783Hex6HexNAc2+Na+1835FucHex3HexNAc4+Na+1906Hex3HexNAc5+Na+1987Hex7HexNAc2+Na+2080FucHex3HexNAc5+Na+2151Hex3HexNAc6+Na+2325FucHex3HexNAc6+Na+2396Hex3HexNAc7+Na+2570FucHex3HexNAc7+Na+2641Hex3HexNAc8+Na+2815FucHex3HexNAc8+Na+2886Hex3HexNAc9+Na+3060FucHex3HexNAc9+Na+3131Hex3HexNAc10+Na+3305FucHex3HexNAc10+Na+3376Hex3HexNAc11+Na+3550FucHex3HexNAc11+Na+3621Hex3HexNAc12+Na+3795FucHex3HexNAc12+Na+3866Hex3HexNAc13+Na+4040FucHex3HexNAc13+Na+4111Hex3HexNAc14+Na+4285FucHex3HexNAc14+Na+4356Hex3HexNAc15+Na+ Open table in a new tab In order to ensure that the data acquired after HF treatment were associated with N-glycans released by PNGase F digestion, rather than being derived from saccharides present in the original sample, the following series of experiments was carried out. Worm material was reduced, carboxymethylated, and digested with trypsin, and the product mixture was fractionated on reverse phase Sep-Pak C18. The aqueous eluent, which would contain oligo- and polysaccharide components, was analyzed in the same way as described above for PNGase F-released glycans, whereas the included fractions containing peptides and glycopeptides was subjected to PNGase F digestion as before. These experiments (data not shown) confirmed that the chito-oligomeric components were not present in the aqueous eluent and were only observed after PNGase F digestion of the glycopeptide fraction. Corroborative evidence for the existence of a family ofN-acetylhexosamine-rich N-glycans was provided by MALDI-MS, which yields molecular ions at very high sensitivity (Fig.4). In addition to the molecular ions consistent with compositions HexNAc3–11Hex3Fuc0–1, the added sensitivity of the instrumentation allows the detection of larger structures in the series up to a maximum composition of HexNAc15Hex3. An important feature of FAB-MS is that it yields abundant fragment ions (A-type fragment ions), which are formed by cleavages at HexNAc residues. These provide information on the types and lengths of antennae (23.Deleted in proof.Google Scholar, 22Peter-Katalinic J. Egge H. Mass Spectrom. Rev. 1987; 6: 331-393Crossref Scopus (237) Google Scholar). The low mass fragment ion region of the FAB spectra were very similar for all three filarial nematodes; the A. viteae spectrum is shown as a representative example (Fig.5 and Table II). The most abundant A-type ions occur at m/z 260, 505, 750, 995, and 1240, which correspond to HexNAc1–5+. This indicates the presence of N-glycans with highly unusual antennae comprising up to five N-acetylhexosamine residues; in some experiments, an additional A-type ion at m/z 1485 was observed, indicating the probable presence of HexNAc6 as a minor constituent. To further explore the nature of the unusual HexNAc-rich antenna, the A-type fragment ions were subjected to CAD MS-MS experiments. The CAD MS-MS spectrum of HexNAc5+ (m/z 1240 (Fig.6)) contains three series of daughter ions (m/z 995, 750, 505, and 260; m/z 1009, 764, 519, and 274; and m/z 1178, 933, 688, 443, and 198), the derivation of which is shown in Fig. 6, inset. These fragment ions are consistent with an unbranched sequence of fiveN-acetylhexosamine residues in 1–4 linkage.Figure 6Positive ion CAD mass spectrum of theA. viteae HexNAc 5+ A-type fragment ion. Structurally informative fragment ions are assigned on the inset. The fragment ions at m/z 154 and 196 are a ketene increment apart. The latter has a mass equivalent to loss of two methanol groups from m/z 260 and is probably formed by extrusion of HexNAc moieties from the parent ion by a combination of elimination and glycosidic cleavage.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Linkage analysis data for the HF-treated N-glycans of A. viteae are shown in Table III. The other two species of filarial nematode gave similar data. From these data we can conclude that the presence of high levels of 2-Man, 2,4-Man, and 2,6-Man is consistent with bi-, tri-, and tetraantennnary complex type glycans being the dominant structures. However, the most striking feature of the linkage data is the abundance of the peak corresponding to 4-linked GlcNAc (Fig. 7). Its size cannot be explained purely by core derived 4-linked GlcNAc, and the lack of other major signals corresponding to linked HexNAc residues suggests that the unusual HexNAc-rich N-glycan antennae are composed of 4-linked GlcNAc. GlcNAc is also the most abundant terminal sugar indicating that the majority of complex type structures have this residue at there nonreducing termini, although some antennae are likely to be capped with GalNAc because this is present as a minor terminal residue in the linkage data (Fig. 7). The presence of terminal mannose is consistent with the high mannose and truncated structures observed in the FAB experiments both before and after HF treatment (see above). The presence of terminal fucose and 4,6-GlcNAc, both of which are of lower abundance in O. volvulus, are in accord with core fucosylation. The identification of high levels of 3,6-Man without detectable levels of 3,4,6-Man indicates the lack of bisecting GlcNAc.Table IIIGC-MS analysis of partially methylated alditol acetates obtained from the PNGase F released N-glycans of A. viteae after HF treatmentElution timeCharacteristic fragment ionsAssignmentRelative abundancemin16.95115, 118, 131, 162, 175Terminal fucose0.2118.55102, 118, 129, 145, 161, 205Terminal mannose0.4418.83102, 118, 129, 145, 161, 205Terminal galactose0.0119.78129, 130, 161, 1902-Linked mannose0.6520.23102, 118, 129, 162, 189, 2336-Linked mannose0.0721.02130, 190, 2332,4-Linked mannose0.1721.45129, 130, 189, 1902,6-Linked mannose0.0921.62118, 129, 189, 2343,6-Linked mannose0.5122.62117, 159, 203, 205Terminal GlcNAc0.2823.10117, 159, 203, 205Terminal GalNAc0.0423.57117, 159, 2334-Linked GlcNAc1.0024.92117, 159, 2614,6-Linked GlcNAc0.06 Open table in a new tab To ascertain the anomeric configuration of the N-acetylhexosamine residues that make up the highly unusual N-glycan antennae, HF-treated A. viteae glycans were digested withN-acetyl-β-d-hexosaminidase prior to permethylation and FAB-MS screening. The spectra revealed that large HexNAc-rich molecular ions had been digested to two main products atm/z 1171 and 1345, which are consistent with trimannosyl cores with and without core fucosylation (data not shown). It can therefore be concluded that the N-acetylhexosamine residues of the N-glycan antenna are β-linked. Taking into account the FAB-MS, MALDI-MS, CAD MS-MS, linkage, and exoglycosydase data, we conclude that the three species of filarial nematodes share a major family ofN-glycans. The glycans are composed of substoichiometrically fucosylated trimannosyl cores to which are attached up to 13N-acetylhexosamine residues, the majority of which are GlcNAc, to give mono-, bi, tri-, and tetra-antennary structures. These structures contain highly unusual chito-oligomeric antennae comprising up to five β1–4-linked GlcNAc residues capped with GlcNAc or GalNAc (Fig. 8).