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Analysis of the Carbohydrate Components of Glycosylphosphatidylinositol Structures Using Fluorescent Labeling

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Book cover Protein Lipidation Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 116))

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 is biosynthetically related, and it 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 (1–34) is the membrane protein anchors (Fig. 1) which 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 singlepass hydrophobic transmembrane peptide domain. For recent reviews of GPI anchor structure, biosynthesis, and function, see refs. 35–40. 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, Phytomonas, and Toxoplasma (35,41–45), or are attached to phosphorylated repeating units, as in the lipophosphoglycans (LPGs) of the Leishmania (35,46). This chapter describes protocols specifically designed to analyze the carbohydrate components of protein-linked GPI anchors, although they are also applicable to the GIPLs and, to some extent, the LPGs.

GPI anchor structures. All GPI anchors attached to protein contain the conserved structure shown above, with various substituents (R1–R7) and lipids, as indicated. Some structures contain an additional fatty acyl chain attached to the 2-position of the myo-inositol 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-aminoethylphosphonate.

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Author information

Authors and Affiliations

  1. Department of Biochemistry, Oxford Glycobiology Institute, Oxford University, Oxford, UK

    Nicole Zitzmann

  2. Department of Biochemistry, University of Dundee, Dundee, Scotland

    Michael A. J. Ferguson

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  1. Nicole Zitzmann
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  2. Michael A. J. Ferguson
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Editors and Affiliations

  1. University of Washington, Seattle, WA

    Michael H. Gelb

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Zitzmann, N., Ferguson, M.A.J. (1998). Analysis of the Carbohydrate Components of Glycosylphosphatidylinositol Structures Using Fluorescent Labeling. In: Gelb, M.H. (eds) Protein Lipidation Protocols. Methods in Molecular Biology, vol 116. Humana Press. https://doi.org/10.1385/1-59259-264-3:73

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  • DOI: https://doi.org/10.1385/1-59259-264-3:73

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-534-8

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