Mammalian glycosylphosphatidylinositol-anchored proteins and intracellular precursors.

Glycosylphosphatidylinositol-anchored proteins can be specifically identified by several methods. PI-PLC digestion analyses, the most widely used technique, can be performed more reliably when conducted with purified protein and phase partitioning to exclude steric effects and when combined with alkaline hydrolysis to control for inositol acylation. Reductive radiomethylation not only can definitively identify a candidate protein as being GPI anchored, but also can provide information on the number of amine components (GlcN, ethanolamine) in the anchor structure. Biosynthetic labeling with anchor precursors is relatively specific when performed with [3H]ethanolamine or [3H]inositol. Incorporation of the precursors additionally can be used to (1) document anchor transfer to primary translation products, (2) identify soluble derivatives of GPI-anchored proteins that have been released from cell surfaces, and (3) localize the site of GPI anchor attachment within a GPI-anchored protein. A pathway for mammalian GP anchor assembly is depicted in Fig. 12. Initially GlcNAc is transferred to PI. The resulting GlcNAc-PI is then deacetylated to yield GlcN-PI. After that step, several points of divergence are identifiable between the mammalian and T. brucei pathways: (1) all mammalian Man-containing intermediates are built on acylated inositol phospholipids; (2) a proximal phosphoethanolamine is found in mammalian GPI anchor intermediates and is added to Man 1 prior to incorporation of Man 2 and Man 3; (3) no Gal branching substituent is added to the mammalian core glycan; and (4) the most polar mammalian GPI contains a third phosphoethanolamine substituent linked to the 6 position of Man 2.