Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1993 May 1;291(Pt 3):713–721. doi: 10.1042/bj2910713

Evidence for N-linked glycosylation in Toxoplasma gondii.

M Odenthal-Schnittler 1, S Tomavo 1, D Becker 1, J F Dubremetz 1, R T Schwarz 1
PMCID: PMC1132427  PMID: 8489499

Abstract

In this paper we report experiments demonstrating the presence of N-linked oligosaccharide structures in Toxoplasma gondii tachyzoites, providing the first direct biochemical evidence that this sporozoan parasite is capable of synthesizing N-linked glycans. The tachyzoite surface glycoprotein gp23 was metabolically labelled with [3H]glucosamine and [3H]mannose. Gel-filtration chromatography on Bio-Gel P4 columns produced four radiolabelled N-linked glycopeptides which were sensitive to peptidase-N-glycanase F, but resistant to endoglycosidases H and F. Using chemical analysis and exoglycosidase digestions followed by Dionex-high-pH anion-exchange chromatography and size fractionation on Bio-Gel P4 we show that gp23 has N-linked glycans in the hybrid- or complex-type structure composed of N-acetylgalactosamine, N-acetylglucosamine and mannose and devoid of sialic acid and fucose residues. In addition, the sensitivity of glycopeptides from glycoprotein extracts to endoglycosidases H and F revealed the in vivo synthesis of oligomannose-type structures by T. gondii tachyzoites. We have extended these findings by demonstrating the ability of T. gondii microsomes to synthesize in vitro a glucosylated lipid-bound high-mannose structure (Glc3Man9GlcNAc2) that is assumed to be identical with the common precursor for N-glycosylation in eukaryotes.

Full text

PDF

Images in this article

715Figure 1
on p.715
717Figure 3
on p.717
718Figure 4
on p.718
719Figure 6
on p.719
720Figure 7
on p.720

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Burg J. L., Perelman D., Kasper L. H., Ware P. L., Boothroyd J. C. Molecular analysis of the gene encoding the major surface antigen of Toxoplasma gondii. J Immunol. 1988 Nov 15;141(10):3584–3591. [PubMed] [Google Scholar]
  2. Burke D., Keegstra K. Carbohydrate structure of Sindbis virus glycoprotein E2 from virus grown in hamster and chicken cells. J Virol. 1979 Feb;29(2):546–554. doi: 10.1128/jvi.29.2.546-554.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Couvreur G., Sadak A., Fortier B., Dubremetz J. F. Surface antigens of Toxoplasma gondii. Parasitology. 1988 Aug;97(Pt 1):1–10. doi: 10.1017/s0031182000066695. [DOI] [PubMed] [Google Scholar]
  4. Dieckmann-Schuppert A., Bender S., Odenthal-Schnittler M., Bause E., Schwarz R. T. Apparent lack of N-glycosylation in the asexual intraerythrocytic stage of Plasmodium falciparum. Eur J Biochem. 1992 Apr 15;205(2):815–825. doi: 10.1111/j.1432-1033.1992.tb16846.x. [DOI] [PubMed] [Google Scholar]
  5. Donald A. S., Feeney J. Oligosaccharides obtained from a blood-group-Sd(a+) Tamm-Horsfall glycoprotein. An n.m.r. study. Biochem J. 1986 Jun 15;236(3):821–828. doi: 10.1042/bj2360821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dubé S., Fisher J. W., Powell J. S. Glycosylation at specific sites of erythropoietin is essential for biosynthesis, secretion, and biological function. J Biol Chem. 1988 Nov 25;263(33):17516–17521. [PubMed] [Google Scholar]
  7. Green E. D., Boime I., Baenziger J. U. Differential processing of Asn-linked oligosaccharides on pituitary glycoprotein hormones: implications for biologic function. Mol Cell Biochem. 1986 Nov-Dec;72(1-2):81–100. doi: 10.1007/BF00230637. [DOI] [PubMed] [Google Scholar]
  8. Grimwood B. G., Hechemy K., Stevens R. W. Toxoplasma gondii: purification of trophozoites propagated in cell culture. Exp Parasitol. 1979 Oct;48(2):282–286. doi: 10.1016/0014-4894(79)90110-3. [DOI] [PubMed] [Google Scholar]
  9. Handman E., Goding J. W., Remington J. S. Detection and characterization of membrane antigens of Toxoplasma gondii. J Immunol. 1980 Jun;124(6):2578–2583. [PubMed] [Google Scholar]
  10. Hobman T. C., Qiu Z. Y., Chaye H., Gillam S. Analysis of rubella virus E1 glycosylation mutants expressed in COS cells. Virology. 1991 Apr;181(2):768–772. doi: 10.1016/0042-6822(91)90915-x. [DOI] [PubMed] [Google Scholar]
  11. Johnson A. M., McDonald P. J., Neoh S. H. Molecular weight analysis of the major polypeptides and glycopeptides of Toxoplasma gondii. Biochem Biophys Res Commun. 1981 Jun 16;100(3):934–943. doi: 10.1016/0006-291x(81)91913-6. [DOI] [PubMed] [Google Scholar]
  12. Kasper L. H., Crabb J. H., Pfefferkorn E. R. Isolation and characterization of a monoclonal antibody-resistant antigenic mutant of Toxoplasma gondii. J Immunol. 1982 Oct;129(4):1694–1699. [PubMed] [Google Scholar]
  13. Kasper L. H., Crabb J. H., Pfefferkorn E. R. Purification of a major membrane protein of Toxoplasma gondii by immunoabsorption with a monoclonal antibody. J Immunol. 1983 May;130(5):2407–2412. [PubMed] [Google Scholar]
  14. 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]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Machamer C. E., Rose J. K. Influence of new glycosylation sites on expression of the vesicular stomatitis virus G protein at the plasma membrane. J Biol Chem. 1988 Apr 25;263(12):5948–5954. [PubMed] [Google Scholar]
  17. Matzuk M. M., Boime I. The role of the asparagine-linked oligosaccharides of the alpha subunit in the secretion and assembly of human chorionic gonadotrophin. J Cell Biol. 1988 Apr;106(4):1049–1059. doi: 10.1083/jcb.106.4.1049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Matzuk M. M., Keene J. L., Boime I. Site specificity of the chorionic gonadotropin N-linked oligosaccharides in signal transduction. J Biol Chem. 1989 Feb 15;264(5):2409–2414. [PubMed] [Google Scholar]
  19. Mauras G., Dodeur M., Laget P., Senet J. M., Bourrillon R. Partial resolution of the sugar content of Toxoplasma gondii membrane. Biochem Biophys Res Commun. 1980 Dec 16;97(3):906–912. doi: 10.1016/0006-291x(80)91462-x. [DOI] [PubMed] [Google Scholar]
  20. Mills J. Pneumocystis carinii and Toxoplasma gondii infections in patients with AIDS. Rev Infect Dis. 1986 Nov-Dec;8(6):1001–1011. doi: 10.1093/clinids/8.6.1001. [DOI] [PubMed] [Google Scholar]
  21. Nagel S. D., Boothroyd J. C. The major surface antigen, P30, of Toxoplasma gondii is anchored by a glycolipid. J Biol Chem. 1989 Apr 5;264(10):5569–5574. [PubMed] [Google Scholar]
  22. Ng D. T., Hiebert S. W., Lamb R. A. Different roles of individual N-linked oligosaccharide chains in folding, assembly, and transport of the simian virus 5 hemagglutinin-neuraminidase. Mol Cell Biol. 1990 May;10(5):1989–2001. doi: 10.1128/mcb.10.5.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nyame K., Smith D. F., Damian R. T., Cummings R. D. Complex-type asparagine-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni adult males contain terminal beta-linked N-acetylgalactosamine. J Biol Chem. 1989 Feb 25;264(6):3235–3243. [PubMed] [Google Scholar]
  24. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  25. Pierce J. G., Parsons T. F. Glycoprotein hormones: structure and function. Annu Rev Biochem. 1981;50:465–495. doi: 10.1146/annurev.bi.50.070181.002341. [DOI] [PubMed] [Google Scholar]
  26. Pizer L. I., Cohen G. H., Eisenberg R. J. Effect of tunicamycin on herpes simplex virus glycoproteins and infectious virus production. J Virol. 1980 Apr;34(1):142–153. doi: 10.1128/jvi.34.1.142-153.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ruskin J., Remington J. S. Toxoplasmosis in the compromised host. Ann Intern Med. 1976 Feb;84(2):193–199. doi: 10.7326/0003-4819-84-2-193. [DOI] [PubMed] [Google Scholar]
  28. Semenkovich C. F., Luo C. C., Nakanishi M. K., Chen S. H., Smith L. C., Chan L. In vitro expression and site-specific mutagenesis of the cloned human lipoprotein lipase gene. Potential N-linked glycosylation site asparagine 43 is important for both enzyme activity and secretion. J Biol Chem. 1990 Apr 5;265(10):5429–5433. [PubMed] [Google Scholar]
  29. Sharma S. D., Mullenax J., Araujo F. G., Erlich H. A., Remington J. S. Western Blot analysis of the antigens of Toxoplasma gondii recognized by human IgM and IgG antibodies. J Immunol. 1983 Aug;131(2):977–983. [PubMed] [Google Scholar]
  30. Tai T., Yamashita K., Kobata A. The substrate specificities of endo-beta-N-acetylglucosaminidases CII and H. Biochem Biophys Res Commun. 1977 Sep 9;78(1):434–441. doi: 10.1016/0006-291x(77)91273-6. [DOI] [PubMed] [Google Scholar]
  31. Tao M. H., Morrison S. L. Studies of aglycosylated chimeric mouse-human IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region. J Immunol. 1989 Oct 15;143(8):2595–2601. [PubMed] [Google Scholar]
  32. Tarentino A. L., Gómez C. M., Plummer T. H., Jr Deglycosylation of asparagine-linked glycans by peptide:N-glycosidase F. Biochemistry. 1985 Aug 13;24(17):4665–4671. doi: 10.1021/bi00338a028. [DOI] [PubMed] [Google Scholar]
  33. Tomavo S., Dubremetz J. F., Schwarz R. T. A family of glycolipids from Toxoplasma gondii. Identification of candidate glycolipid precursor(s) for Toxoplasma gondii glycosylphosphatidylinositol membrane anchors. J Biol Chem. 1992 Jun 15;267(17):11721–11728. [PubMed] [Google Scholar]
  34. Tomavo S., Schwarz R. T., Dubremetz J. F. Evidence for glycosyl-phosphatidylinositol anchoring of Toxoplasma gondii major surface antigens. Mol Cell Biol. 1989 Oct;9(10):4576–4580. doi: 10.1128/mcb.9.10.4576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wright K. E., Salvato M. S., Buchmeier M. J. Neutralizing epitopes of lymphocytic choriomeningitis virus are conformational and require both glycosylation and disulfide bonds for expression. Virology. 1989 Aug;171(2):417–426. doi: 10.1016/0042-6822(89)90610-7. [DOI] [PubMed] [Google Scholar]
  36. de Carvalho L., Souto-Padrón T., de Souza W. Localization of lectin-binding sites and sugar-binding proteins in tachyzoites of Toxoplasma gondii. J Parasitol. 1991 Feb;77(1):156–161. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES