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. 2003 Jan 1;369(Pt 1):89–102. doi: 10.1042/BJ20021074

Structural elucidation of zwitterionic carbohydrates derived from glycosphingolipids of the porcine parasitic nematode Ascaris suum.

Claudia H Friedl 1, Günter Lochnit 1, Ulrich Zähringer 1, Ute Bahr 1, Rudolf Geyer 1
PMCID: PMC1223059  PMID: 12234251

Abstract

Carbohydrates substituted with phosphocholine (PC) and phosphoethanolamine (PE) were released from zwitterionic glycosphingolipids of the pig parasitic nematode Ascaris suum by treatment with endoglycoceramidase. Individual glycans were obtained by HPLC on porous graphitic carbon followed by high-pH anion-exchange chromatography. In addition to the known pentasaccharides Gal alpha 3GalNAc beta 4[PC6]GlcNAc beta 3Man beta 4Glc and Gal alpha 3GalNAc beta 4[PC6]GlcNAc beta 3[PE6]Man beta 4Glc, the corresponding tri- and tetra-saccharides, as well as components with elongated structures, could be identified by matrix-assisted laser-desorption ionization-time-of-flight MS, methylation analysis, 1H- and 13C-NMR spectroscopy, exoglycosidase cleavage and electrospray ionization ion-trap MS. The extended components comprised novel structural motifs such as di-substituted alpha-galactose carrying two beta-linked galactosyl residues, which were found to bear, in part, further fucose, galactose, N -acetylgalactosamine and/or N -acetylglucosamine moieties. Furthermore, additional fucosylation of the PC-substituted N -acetylglucosamine and a non-terminal fucosyl motif were detected. In conclusion, this study contributes significant new information on the glycome of nematodes.

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Selected References

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  1. Altmann F., Fabini G., Ahorn H., Wilson I. B. Genetic model organisms in the study of N-glycans. Biochimie. 2001 Aug;83(8):703–712. doi: 10.1016/s0300-9084(01)01297-4. [DOI] [PubMed] [Google Scholar]
  2. Anumula K. R. Quantitative determination of monosaccharides in glycoproteins by high-performance liquid chromatography with highly sensitive fluorescence detection. Anal Biochem. 1994 Aug 1;220(2):275–283. doi: 10.1006/abio.1994.1338. [DOI] [PubMed] [Google Scholar]
  3. Bulik D. A., Wei G., Toyoda H., Kinoshita-Toyoda A., Waldrip W. R., Esko J. D., Robbins P. W., Selleck S. B. sqv-3, -7, and -8, a set of genes affecting morphogenesis in Caenorhabditis elegans, encode enzymes required for glycosaminoglycan biosynthesis. Proc Natl Acad Sci U S A. 2000 Sep 26;97(20):10838–10843. doi: 10.1073/pnas.97.20.10838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen S., Zhou S., Sarkar M., Spence A. M., Schachter H. Expression of three Caenorhabditis elegans N-acetylglucosaminyltransferase I genes during development. J Biol Chem. 1999 Jan 1;274(1):288–297. doi: 10.1074/jbc.274.1.288. [DOI] [PubMed] [Google Scholar]
  5. Dennis R. D., Lochnit G., Geyer R. Strategies for preliminary characterization of novel amphoteric glycosphingolipids. Methods Mol Biol. 1998;76:197–212. doi: 10.1385/0-89603-355-4:197. [DOI] [PubMed] [Google Scholar]
  6. Fletcher T. C., White A., Baldo B. A. Isolation of a phosphorylcholine-containing component from the turbot tapeworm, Bothriocephalus scorpii (Müller), and its reaction with C-reactive protein. Parasite Immunol. 1980 Winter;2(4):237–248. doi: 10.1111/j.1365-3024.1980.tb00056.x. [DOI] [PubMed] [Google Scholar]
  7. Friedl C. H., Lochnit G., Geyer R., Karas M., Bahr U. Structural elucidation of zwitterionic sugar cores from glycosphingolipids by nanoelectrospray ionization-ion-trap mass spectrometry. Anal Biochem. 2000 Sep 10;284(2):279–287. doi: 10.1006/abio.2000.4681. [DOI] [PubMed] [Google Scholar]
  8. Gerdt S., Dennis R. D., Borgonie G., Schnabel R., Geyer R. Isolation, characterization and immunolocalization of phosphorylcholine-substituted glycolipids in developmental stages of Caenorhabditis elegans. Eur J Biochem. 1999 Dec;266(3):952–963. doi: 10.1046/j.1432-1327.1999.00937.x. [DOI] [PubMed] [Google Scholar]
  9. Gerdt S., Lochnit G., Dennis R. D., Geyer R. Isolation and structural analysis of three neutral glycosphingolipids from a mixed population of Caenorhabditis elegans (Nematoda:Rhabditida). Glycobiology. 1997 Mar;7(2):265–275. doi: 10.1093/glycob/7.2.265. [DOI] [PubMed] [Google Scholar]
  10. Geyer H., Schmitt S., Wuhrer M., Geyer R. Structural analysis of glycoconjugates by on-target enzymatic digestion and MALDI-TOF-MS. Anal Chem. 1999 Jan 15;71(2):476–482. doi: 10.1021/ac980712w. [DOI] [PubMed] [Google Scholar]
  11. Geyer R., Geyer H., Kühnhardt S., Mink W., Stirm S. Capillary gas chromatography of methylhexitol acetates obtained upon methylation of N-glycosidically linked glycoprotein oligosaccharides. Anal Biochem. 1982 Apr;121(2):263–274. doi: 10.1016/0003-2697(82)90478-x. [DOI] [PubMed] [Google Scholar]
  12. Geyer R., Geyer H. Saccharide linkage analysis using methylation and other techniques. Methods Enzymol. 1994;230:86–108. doi: 10.1016/0076-6879(94)30009-7. [DOI] [PubMed] [Google Scholar]
  13. Goodridge H. S., Wilson E. H., Harnett W., Campbell C. C., Harnett M. M., Liew F. Y. Modulation of macrophage cytokine production by ES-62, a secreted product of the filarial nematode Acanthocheilonema viteae. J Immunol. 2001 Jul 15;167(2):940–945. doi: 10.4049/jimmunol.167.2.940. [DOI] [PubMed] [Google Scholar]
  14. Gualzata M., Weiss N., Heusser C. H. Dipetalonema viteae: phosphorylcholine and non-phosphorylcholine antigenic determinants in infective larvae and adult worms. Exp Parasitol. 1986 Feb;61(1):95–102. doi: 10.1016/0014-4894(86)90139-6. [DOI] [PubMed] [Google Scholar]
  15. Gutman G. A., Mitchell G. F. Ascaris suum: location of phosphorylcholine in lung larvae. Exp Parasitol. 1977 Oct;43(1):161–168. doi: 10.1016/0014-4894(77)90019-4. [DOI] [PubMed] [Google Scholar]
  16. Guérardel Y., Balanzino L., Maes E., Leroy Y., Coddeville B., Oriol R., Strecker G. The nematode Caenorhabditis elegans synthesizes unusual O-linked glycans: identification of glucose-substituted mucin-type O-glycans and short chondroitin-like oligosaccharides. Biochem J. 2001 Jul 1;357(Pt 1):167–182. doi: 10.1042/0264-6021:3570167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Harnett W., Harnett M. M. Modulation of the host immune system by phosphorylcholine-containing glycoproteins secreted by parasitic filarial nematodes. Biochim Biophys Acta. 2001 May 28;1539(1-2):7–15. doi: 10.1016/s0167-4889(01)00101-x. [DOI] [PubMed] [Google Scholar]
  18. Haslam S. M., Coles G. C., Munn E. A., Smith T. S., Smith H. F., Morris H. R., Dell A. Haemonchus contortus glycoproteins contain N-linked oligosaccharides with novel highly fucosylated core structures. J Biol Chem. 1996 Nov 29;271(48):30561–30570. doi: 10.1074/jbc.271.48.30561. [DOI] [PubMed] [Google Scholar]
  19. Haslam S. M., Houston K. M., Harnett W., Reason A. J., Morris H. R., Dell A. Structural studies of N-glycans of filarial parasites. Conservation of phosphorylcholine-substituted glycans among species and discovery of novel chito-oligomers. J Biol Chem. 1999 Jul 23;274(30):20953–20960. doi: 10.1074/jbc.274.30.20953. [DOI] [PubMed] [Google Scholar]
  20. Haslam S. M., Khoo K. H., Houston K. M., Harnett W., Morris H. R., Dell A. Characterisation of the phosphorylcholine-containing N-linked oligosaccharides in the excretory-secretory 62 kDa glycoprotein of Acanthocheilonema viteae. Mol Biochem Parasitol. 1997 Mar;85(1):53–66. doi: 10.1016/s0166-6851(96)02807-1. [DOI] [PubMed] [Google Scholar]
  21. Herman T., Hartwieg E., Horvitz H. R. sqv mutants of Caenorhabditis elegans are defective in vulval epithelial invagination. Proc Natl Acad Sci U S A. 1999 Feb 2;96(3):968–973. doi: 10.1073/pnas.96.3.968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Herman T., Horvitz H. R. Three proteins involved in Caenorhabditis elegans vulval invagination are similar to components of a glycosylation pathway. Proc Natl Acad Sci U S A. 1999 Feb 2;96(3):974–979. doi: 10.1073/pnas.96.3.974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Houston K. M., Wilson E. H., Eyres L., Brombacher F., Harnett M. M., Alexander J., Harnett W. Presence of phosphorylcholine on a filarial nematode protein influences immunoglobulin G subclass response to the molecule by an interleukin-10-dependent mechanism. Infect Immun. 2000 Sep;68(9):5466–5468. doi: 10.1128/iai.68.9.5466-5468.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Khoo K. H., Chatterjee D., Caulfield J. P., Morris H. R., Dell A. Structural characterization of glycophingolipids from the eggs of Schistosoma mansoni and Schistosoma japonicum. Glycobiology. 1997 Jul;7(5):653–661. doi: 10.1093/glycob/7.5.653. [DOI] [PubMed] [Google Scholar]
  25. Khoo K. H., Maizels R. M., Page A. P., Taylor G. W., Rendell N. B., Dell A. Characterization of nematode glycoproteins: the major O-glycans of Toxocara excretory-secretory antigens are O-methylated trisaccharides. Glycobiology. 1991 Mar;1(2):163–171. doi: 10.1093/glycob/1.2.163. [DOI] [PubMed] [Google Scholar]
  26. Khoo K. H., Sarda S., Xu X., Caulfield J. P., McNeil M. R., Homans S. W., Morris H. R., Dell A. A unique multifucosylated -3GalNAc beta 1-->4GlcNAc beta 1-->3Gal alpha 1- motif constitutes the repeating unit of the complex O-glycans derived from the cercarial glycocalyx of Schistosoma mansoni. J Biol Chem. 1995 Jul 21;270(29):17114–17123. doi: 10.1074/jbc.270.29.17114. [DOI] [PubMed] [Google Scholar]
  27. Kornfeld R., Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. doi: 10.1146/annurev.bi.54.070185.003215. [DOI] [PubMed] [Google Scholar]
  28. Lal R. B., Paranjape R. S., Briles D. E., Nutman T. B., Ottesen E. A. Circulating parasite antigen(s) in lymphatic filariasis: use of monoclonal antibodies to phosphocholine for immunodiagnosis. J Immunol. 1987 May 15;138(10):3454–3460. [PubMed] [Google Scholar]
  29. Lochnit G., Dennis R. D., Geyer R. Phosphorylcholine substituents in nematodes: structures, occurrence and biological implications. Biol Chem. 2000 Sep-Oct;381(9-10):839–847. doi: 10.1515/BC.2000.106. [DOI] [PubMed] [Google Scholar]
  30. Lochnit G., Dennis R. D., Ulmer A. J., Geyer R. Structural elucidation and monokine-inducing activity of two biologically active zwitterionic glycosphingolipids derived from the porcine parasitic nematode Ascaris suum. J Biol Chem. 1998 Jan 2;273(1):466–474. doi: 10.1074/jbc.273.1.466. [DOI] [PubMed] [Google Scholar]
  31. Lochnit G., Dennis R. D., Zähringer U., Geyer R. Structural analysis of neutral glycosphingolipids from Ascaris suum adults (Nematoda:Ascaridida). Glycoconj J. 1997 Apr;14(3):389–399. doi: 10.1023/a:1018530914067. [DOI] [PubMed] [Google Scholar]
  32. Morelle W., Haslam S. M., Olivier V., Appleton J. A., Morris H. R., Dell A. Phosphorylcholine-containing N-glycans of Trichinella spiralis: identification of multiantennary lacdiNAc structures. Glycobiology. 2000 Sep;10(9):941–950. doi: 10.1093/glycob/10.9.941. [DOI] [PubMed] [Google Scholar]
  33. Okajima T., Yoshida K., Kondo T., Furukawa K. Human homolog of Caenorhabditis elegans sqv-3 gene is galactosyltransferase I involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans. J Biol Chem. 1999 Aug 13;274(33):22915–22918. doi: 10.1074/jbc.274.33.22915. [DOI] [PubMed] [Google Scholar]
  34. Parente J. P., Cardon P., Leroy Y., Montreuil J., Fournet B., Ricart G. A convenient method for methylation of glycoprotein glycans in small amounts by using lithium methylsulfinyl carbanion. Carbohydr Res. 1985 Aug 15;141(1):41–47. doi: 10.1016/s0008-6215(00)90753-5. [DOI] [PubMed] [Google Scholar]
  35. Pfeiffer G., Geyer H., Geyer R., Kalsner I., Wendorf P. Separation of glycoprotein-N-glycans by high-pH anion-exchange chromatography. Biomed Chromatogr. 1990 Sep;4(5):193–199. doi: 10.1002/bmc.1130040505. [DOI] [PubMed] [Google Scholar]
  36. Péry P., Luffau G., Charley J., Petit A., Rouze P., Bernard S. Phosphorylcholine antigens from Nippostrongylus brasiliensis. II.--Isolation and partial characterization of phosphorylcholine antigens from adult worm. Ann Immunol (Paris) 1979 Nov-Dec;130(6):889–900. [PubMed] [Google Scholar]
  37. Péry P., Petit A., Poulain J., Luffau G. Phosphorylcholine-bearing components in homogenates of nematodes. Eur J Immunol. 1974 Sep;4(9):637–639. doi: 10.1002/eji.1830040914. [DOI] [PubMed] [Google Scholar]
  38. Reason A. J., Ellis L. A., Appleton J. A., Wisnewski N., Grieve R. B., McNeil M., Wassom D. L., Morris H. R., Dell A. Novel tyvelose-containing tri- and tetra-antennary N-glycans in the immunodominant antigens of the intracellular parasite Trichinella spiralis. Glycobiology. 1994 Oct;4(5):593–603. doi: 10.1093/glycob/4.5.593. [DOI] [PubMed] [Google Scholar]
  39. Schachter H. The joys of HexNAc. The synthesis and function of N- and O-glycan branches. Glycoconj J. 2000 Jul-Sep;17(7-9):465–483. doi: 10.1023/a:1011010206774. [DOI] [PubMed] [Google Scholar]
  40. Toyoda H., Kinoshita-Toyoda A., Selleck S. B. Structural analysis of glycosaminoglycans in Drosophila and Caenorhabditis elegans and demonstration that tout-velu, a Drosophila gene related to EXT tumor suppressors, affects heparan sulfate in vivo. J Biol Chem. 2000 Jan 28;275(4):2269–2275. doi: 10.1074/jbc.275.4.2269. [DOI] [PubMed] [Google Scholar]
  41. Whelan M., Harnett M. M., Houston K. M., Patel V., Harnett W., Rigley K. P. A filarial nematode-secreted product signals dendritic cells to acquire a phenotype that drives development of Th2 cells. J Immunol. 2000 Jun 15;164(12):6453–6460. doi: 10.4049/jimmunol.164.12.6453. [DOI] [PubMed] [Google Scholar]
  42. Wuhrer M., Rickhoff S., Dennis R. D., Lochnit G., Soboslay P. T., Baumeister S., Geyer R. Phosphocholine-containing, zwitterionic glycosphingolipids of adult Onchocerca volvulus as highly conserved antigenic structures of parasitic nematodes. Biochem J. 2000 Jun 1;348(Pt 2):417–423. [PMC free article] [PubMed] [Google Scholar]
  43. Wuhrer Manfred, Kantelhardt Sven R., Dennis Roger D., Doenhoff Michael J., Lochnit Günter, Geyer Rudolf. Characterization of glycosphingolipids from Schistosoma mansoni eggs carrying Fuc(alpha1-3)GalNAc-, GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc- and Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc- (Lewis X) terminal structures. Eur J Biochem. 2002 Jan;269(2):481–493. doi: 10.1046/j.0014-2956.2001.02673.x. [DOI] [PubMed] [Google Scholar]
  44. Yamada S., Van Die I., Van den Eijnden D. H., Yokota A., Kitagawa H., Sugahara K. Demonstration of glycosaminoglycans in Caenorhabditis elegans. FEBS Lett. 1999 Oct 15;459(3):327–331. doi: 10.1016/s0014-5793(99)01286-7. [DOI] [PubMed] [Google Scholar]

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