PRODUCTION OF A NESTED SET OF GLYCOSYLPHOSPHATIDYLINOSITOL STRUCTURES FROM A GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED PROTEIN

Glycosylphosphatidylinositol (GPI) membrane anchors are synthesized in the endoplasmic reticulum of eukaryotic cells, Synthesis of the core GPI structure is achieved by the sequential transfer of monosaccharides and phosphoethanolamine to phosphatidylinositol. The assembly process can be reproduced in vitro using membrane preparations supplemented with sugar nucleotides, With one exception, however, none of the biosynthetic enzymes involved have been isolated. One impediment to progress in the isolation of these enzymes is the nonavailability of adequate amounts of partially assembled GPI structures for use as assay substrates, In this paper we present procedures to prepare these structures from a GPI-anchored protein, The methods described include selective dephosphorylation of the GPI-anchored variant surface glycoprotein from Trypanosoma brucei variant 118 to generate Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6-myo-inositol-P-dimyristoylglycerol (Man(3)GlcN-PI), followed by exoglycosidase treatments and N-acetylation to produce Man(2)GlcN-PI, Man(1)GlcN-PI, GlcN-PI, and GlcNAc-PI. Procedures are also described for the stabilization and purification of these structures. It is anticipated that the convenient preparation of this range of partially assembled GPIs will be useful not only in developing assays for the eventual purification of the GPI biosynthetic enzymes but will also contribute to evaluating the specificity of the phospholipases that hydrolyze GPI anchors, (C) 1995 Academic Press, Inc.