PRODUCTION OF A NESTED SET OF GLYCOSYLPHOSPHATIDYLINOSITOL STRUCTURESFROM 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 monosaccharide s and phosphoethanolamine to phosphatidylinositol. The assembly proces s can be reproduced in vitro using membrane preparations supplemented with sugar nucleotides, With one exception, however, none of the biosy nthetic enzymes involved have been isolated. One impediment to progres s in the isolation of these enzymes is the nonavailability of adequate amounts of partially assembled GPI structures for use as assay substr ates, In this paper we present procedures to prepare these structures from a GPI-anchored protein, The methods described include selective d ephosphorylation of the GPI-anchored variant surface glycoprotein from Trypanosoma brucei variant 118 to generate Man alpha 1-2Man alpha 1-6 Man alpha 1-4GlcN alpha 1-6-myo-inositol-P-dimyristoylglycerol (Man(3) GlcN-PI), followed by exoglycosidase treatments and N-acetylation to p roduce Man(2)GlcN-PI, Man(1)GlcN-PI, GlcN-PI, and GlcNAc-PI. Procedure s are also described for the stabilization and purification of these s tructures. It is anticipated that the convenient preparation of this r ange 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 phosphol ipases that hydrolyze GPI anchors, (C) 1995 Academic Press, Inc.