Analysis of the carbohydrate components of glycosylphosphatidylinositol structures using fluorescent labeling

Glycosylphosphatidylinositols (GPIs) are a family of structures that contain the structural motif: Mana1-4GlcNH2a1-6myo-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