Skip to main content

Analysis of the Carbohydrate and Lipid Components of Glycosylphosphatidylinos¡tol Structures

  • Protocol
  • 769 Accesses

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 76))

Abstract

Glycosylphosphatidylinositols (GPIs) are a family of structures that contain the structural motif: Manα1-4GlcNH2α1-6myo-Inositol-1-PO4-lipid. This common substructure suggests that this family of molecules are biosynthetically related and differentiates them from other glycosylated phosphoinositides, such as the glycosylated phosphatidylinositols of mycobacteria and the glycosylated inositol phosphoceramides of yeasts and plants. The GPI family can be conveniently divided into two groups based on structural homology and function. The first group (128) are the membrane protein anchors (Fig. 1) that are found covalently linked to the C-termini of a wide variety of externally disposed plasma membrane proteins throughout the eukaryotes These GPI anchors afford a stable attachment of proteins to the membrane and can be viewed as an alternative mechanism of membrane attachment to a single-pass hydrophobic transmembrane peptide domain. For recent reviews of GPI anchor structure, biosynthesis, and function see refs. 2931. The second group of GPI structures have only been found in protozoan organisms. These molecules exist as free glycophospholipids, such as the glycoinositol phospholipids (GIPLs) of the Leishmania, Trypanosoma cruzi, Leptomonas, Herpetomonas, and Phytomonas (29,3234), or attached to phosphorylated repeating units as in the lipophosphoglycans (LPGs) of the Leishmania (29,35). In this chapter protocols specifically designed to analyse the protein-linked GPI anchors, although they are also applicable to the GIPLs and, to some extent, the LPGs will be described.

GPI anchor structures All GPI anchors attached to protein contain the conserved structure shown above with various substituents (R1–R5) and lipids, as indicated. Some structures contain an additional fatty acyl chain attached to the 2-position of the myoinositol ring. All metazoan organisms contain at least one, and sometimes two, extra ethanolamine phosphate (EtNPO4) substituents in addition to the one used as a bridge to the protein C-terminal amino acid. When a substituent is known to be attached to a certain sugar residue but the linkage position is unknown, this is indicated by a question mark. Square brackets are used to show substituents for which the site of attachment has not been determined. The ± symbol indicates that the associated residue is found on only a proportion of the structures AEP is 2-amino-ethyl-phosphonate.

This is a preview of subscription content, log in via an institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Ferguson, M. A. J., Homans, S. W., Dwek, R. A., and Rademacher, T. W. (1988) Glycosylphosphatidylinositol moiety that anchors Trypanosoma brucei Variant surface glycoprotein to the membrane. Science 239, 753–759.

    Article  CAS  Google Scholar 

  2. Field, M. C., Menon, A. K., and Cross, G. C. (1991) A glycosylphosphatidylinositol protein anchor from procyclic stage Trypanosoma brucei lipid structure and biosynthesis EMBOJ 10, 2731–2739.

    CAS  Google Scholar 

  3. Ferguson, M. A. J., Murray, P., Rutherford, H., and McConville, M. (1993) A simple purification of procyclic acidic repetitive protein and demonstration of a sialylated glycosylphosphatidylinositol membrane anchor Biochem J 291, 51–55.

    CAS  Google Scholar 

  4. Guther, M. L. S., Almeida, M. L. C. D., Yoshida, N., and Ferguson, M. A. J. (1992) Structural studies on the glycosylphosphatidylinositol membrane anchor of Trypanosoma cruzi 1G7-Antigen. The structure of the glycan core. J Biol Chem 267, 6820–6828.

    CAS  Google Scholar 

  5. Heise, N., Almeida, M. L. C. D., and Ferguson, M. A. J. (1995) Characterisation of the lipid moiety of the glycosylphosphatidylinositol anchor of Trypanosoma cruzi 1G7-antigen. Mol. Biochem Parasitol 70, 71–84.

    Article  CAS  Google Scholar 

  6. Couto, A. S., Lederkremer, R. M. D., Colli, W., and Alves, M. J. M. (1993) The glycosylphosphatidylinositol anchor of the trypomastigote-specific Tc-85 glycoprotein from Trypanosoma cruzi. Metabolic labeling and structural studies. Eur J. Biochem 217, 597–602.

    Article  CAS  Google Scholar 

  7. Abuin, G., Couto, A. S., Lederkremer, R. M., Casal, O. L., Galli, C., Colli, W., and Alves, M. J. (1996) Trypanosoma cruzi. the Tc85 surface glycoprotein shed by trypomastigotes bears a modified glycosylphosphatidylinositol anchor. Exp Parasitol. 82, 290–297.

    Article  CAS  Google Scholar 

  8. Previato, J. O., Jones, C., Xavier, M. T., Wait, R., Travassos, L. R., Parodi, A. J., and Mendoça-Previato, L. (1995) Structural characterisation of the major glycosylphosphatidylinositol membrane anchored glycoprotein from epimastigote forms of Trypanosoma cruzi strains. J Biol Chem 270, 7241–7250.

    Article  CAS  Google Scholar 

  9. Serrano, A. A., Schenkman, S., Yoshida, N., Mehlert, A., Richardson, J. M., and Ferguson, M. A. J. (1995) The lipid structure of the glycosylphosphatidylinositol-anchored mucin-like sialic acid acceptors of Trypanosoma cruzi changes during parasite differentiation from epimastigotes to infective metacyclic trypomastigote forms. J. Biol. Chem. 270, 27,244–27,253.

    Article  CAS  Google Scholar 

  10. Schneider, P., Ferguson, M. A. J., McConville, M. J., Mehlert, A., Homans, S. W., and Bordier, C. (1990) Structure of the glycosylphosphatidylinositol membrane anchor of the Leishmania major promastigote surface protease. J Biol Chem 265, 16,955–16,964.

    CAS  Google Scholar 

  11. McConville, M. J., Collidge, T. A. C., Ferguson, M. A J., and Schneider, P. (1993) The glycoinositol phospholipids of Leishmania mexicana promastigotes. Evidence for the presence of three distinct pathways of glycolipid biosynthesis J Biol Chem 268, 15,595–15,604.

    CAS  Google Scholar 

  12. Tomavo, S., Dubremetz, J.-F., and Schwarz, R. T. (1993) Structural analysis of glycosylphosphatidylinositol membrane anchor of the Toxoplasma gondu tachyzoite surface glycoprotein gp23. Biol. Cell 78, 155–162.

    Article  CAS  Google Scholar 

  13. Azzouz, N., Striepen, B., Gerold, P., Capdeville, Y., and Schwarz, R. T. (1995) Glycosylinositol-phophoceramide in the free-living protozoan Paramecium primaurelia modification of core glycans by mannosyl phosphate. EMBO J 14, 4422–4433.

    CAS  Google Scholar 

  14. Gerold, P., Schofield, L., Blackman, M. J., Holder, A. A., and Schwarz, R. T. (1996) Structural analysis of the glycosylphosphatidylinositol membrane anchor of the merozoite surface proteins-1 and-2 of Plasmodium falciparum. Mol Biochem Parasitol, in press.

    Google Scholar 

  15. Fankhauser, C., Homans, S. W., Thomas-Oates, J. E., McConville, M. J., Desponds, C., Conzelmann, A., and Ferguson, M. A. J. (1993) Structures of glycosylphosphatidylinositol membrane anchors from Saccharomyces cerevisiae. J Biol Chem 268, 26,365–26,374.

    CAS  Google Scholar 

  16. Haynes, P. A., Gooley, A. A., Ferguson, M. A. J., Redmond, J. W., and Williams, K. L. (1993) Post-translational modifications of the Dictyostelium discoideum glycoprotein PsA Glycosylphosphatidylinositol membrane anchor and composition of O-linked oligosaccharides. Eur J Biochem 216, 729–737.

    Article  CAS  Google Scholar 

  17. Butikofer, P., Kuypers, F. A., Shackleton, C., Brodbeck, U., and Streger, S. (1990) Molecular species analysis of the glycosylphosphatidylinositol anchor of Torpedo marmorata acetylcholinesterase. J Biol Chem 265, 18,983–18,987.

    CAS  Google Scholar 

  18. Mehlert, A., Varon, L., Silman, I., Homans, S. W., and Ferguson, M. A. J. (1993) Structure of the glycosylphosphatidylinositol membrane anchor of acetylcholmesterase from the electric organ of the electric fish, Torpedo californica. Biochem J 296, 473–479.

    CAS  Google Scholar 

  19. Homans, S. W., Ferguson, M. A. J., Dwek, R. A., Rademacher, T. W., Anand, R., and Williams, A. F. (1988) Complete structure of the glycosylphosphatidylinositol membrane anchor of rat brain Thy-1 glycoprotein. Nature 333, 269–272.

    Article  CAS  Google Scholar 

  20. Stahl, N., Baldwin, M. A., Hecker, R., Pan, K.-M., Burlingame, A. L., and Prusiner, S. B. (1992) Glycosylinositol phospholipid anchors of the scrapie and cellular prion proteins contain sialic acid. Biochemistry 31, 5043–5053.

    Article  CAS  Google Scholar 

  21. Mukasa, R., Umeda, M., Endo, T., Kobata, A., and Inoue, K. (1995) Characterisation of glycosylphosphatidylinositol (GPl)-anchored NCAM on mouse skeletal muscle cell line C2C12 the structure of the GPl glycan and release during myogenesis. Arch Biochem Biophys 318, 182–190.

    Article  CAS  Google Scholar 

  22. Taguchi, R., Hamakawa, N., Harada-Nishida, M., Fukui, T., Nojima, K., and Ikezawa, H. (1994) Microheterogeneity in glycosylphosphatidylinositol anchor structures of bovine liver 5′ nucleotidase. Biochemistry 33, 1017–1022.

    Article  CAS  Google Scholar 

  23. Brewis, I. A., Ferguson, M. A. J., Mehlert, A., Turner, A. J., and Hooper, N. M. (1995) Structures of the glycosylphosphatidylinositol anchors of porcine and human renal membrane dipeptidase. Comprehensive structural studies on the porcine anchor and interspecies comparison of the glycan core structures. J Biol Chem 270, 22,946–22,956.

    Article  CAS  Google Scholar 

  24. Deeg, M. A., Humphrey, D. R., Yang, S. H., Ferguson, T. R., Reinhold, V. N., and Rosenberry, T. L. (1992) Glycan components in the glycoinositol phospholipid anchor of human erythrocyte acetylcholinesterase. J Biol. Chem 267, 18,573–18,580.

    CAS  Google Scholar 

  25. Redman, C. A., Thomas-Oates, J. E., Ogata, S., Ikehara, Y., and Ferguson, M. A. J. (1994) Structure of the glycosylphosphatidylinositol membrane anchor of human placental alkaline phosphatase. Biochem. J 302, 861–865.

    CAS  Google Scholar 

  26. Treumann, A., Lifely, M. R., Schneider, P., and Ferguson, M. A. J. (1995) Primary structure of CD52. J Biol Chem 270, 6088–6099.

    Article  CAS  Google Scholar 

  27. Nakano, Y., Noda, K., Endo, T., Kobata, A., and Tomita, M. (1994) Structural study on the glycosylphosphatidylinositol anchor and the asparagine-linked sugar chain of a soluble form of CD59 in human urine. Arch Biochem Biophys 311, 117–126.

    Article  CAS  Google Scholar 

  28. Sugita, Y., Nakano, Y., Oda, E., Noda, K., Tobe, T., Miura, N.-H., and Tomita, M. (1993) Determination of carboxyl-terminal residue and disulfide bonds of MACIF (CD59), a glycosylphosphatidylinositol-anchored membrane protein. J Biochem 114, 473–477.

    CAS  Google Scholar 

  29. McConville, M. J. and Ferguson, M. A. J. (1993) The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes. Biochem J 294, 305–324.

    CAS  Google Scholar 

  30. Brown, D. A. (1992) Interactions between GPI-anchored proteins and membrane lipids. Trends Cell Biol 2, 338–343.

    Article  CAS  Google Scholar 

  31. Stevens, V. L. (1995) Biosynthesis of glycosylphosphatidylinositol membrane anchors. Biochem J 310, 361–370.

    CAS  Google Scholar 

  32. Redman, C. A., Schneider, P., Mehiert, A., and Ferguson, M. A. J. (1995) The glycoinositol-phospholipids of Phytomonas. Biochem J 311, 495–503.

    CAS  Google Scholar 

  33. Previato, J. O., Mendonça-Previato, L., Jones, C., and Fournet, B. (1992) Structural characterisation of a novel class of glycophosphosphingolipids from the protozoan Leptomonas-samueli. J Blol Chem 267, 24,279–24,286.

    CAS  Google Scholar 

  34. Routier, F. H., da Silvena, E. X., Wait, R., Jones, C., Previato, J. O., and Mendonça-Previato, L. (1995) Chemical characterisation of glycosylinositolphospholipids of Herpetomonas samuelpessoai. Mol Biochem Parasitol 69, 61–69.

    Article  Google Scholar 

  35. McConville, M. J., Schnur, L. F., Jaffe, C., and Schneider, P. (1995) Structure of Leishmania liposphosphoglycan: inter-and intra-specific polymorphism in Old World species. Biochem J 310, 807–818.

    CAS  Google Scholar 

  36. Schneider, P. and Ferguson, M. A. J. (1995) Microscale analysis of glycosylphosphatidylinositol structures. Meth Enzymol. 250, 614–630.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 1998 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Treumann, A., Güther, M.L.S., Schneider, P., Ferguson, M.A.J. (1998). Analysis of the Carbohydrate and Lipid Components of Glycosylphosphatidylinos¡tol Structures. In: Hounsell, E.F. (eds) Glycoanalysis Protocols. Methods in Molecular Biology™, vol 76. Humana Press. https://doi.org/10.1385/0-89603-355-4:213

Download citation

  • DOI: https://doi.org/10.1385/0-89603-355-4:213

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-355-9

  • Online ISBN: 978-1-59259-562-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics