LIPID LINKED SACCHARIDES IN GLYCOPROTEIN SYNTHESIS

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A large number of eucaryotic proteins are anchored in the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor including the variable surface antigen of Trypanosomes, acetylcholinesterase, trehalase, 5'- nucleotidase, decay accelerating factor, etc., of animal cells, and various proteins in yeast and plants. Although the specific role of this anchoring mechanism has not been established, GPI anchors may be involved in 1). Targeting proteins to apical or basolateral surfaces, 2). Signal transduction, 3). Antigenic variation, 4). Facilitating the lateral movement of proteins in the membrane, 5). Providing a means to modulate the expression of proteins at the cell surface. At least one human disease, paroxysmal nocturnal hemoglobinuria (PNH) is due to an inability to synthesize the glycan portion of the GPI anchor which is essential for decay accelerating factor and CD59 to be anchored at the erythrocyte surface to protect these cells from the lytic action of complement. The biosynthetic pathway for the formation of the glycan portion of GPI anchors has been well established, but none of the glycosyltransferases has yet been purified or characterized. We are in an excellent position to purify the GlcNAc transferase that forms GlcNAc-PI since we have a rapid and reliable assay for this enzyme, and we have prepared several photoaffinity analogs of UDP-GlcNAc. Thus, we should be able to locate the specific catalytic protein on SDS gels. This is the enzyme proposed to be missing in PNH. Once we have this enzyme purified, we will prepare antibodies and oligonucleotide probes. In collaboration with Dr. Ed Yeh, we will use these probes to determine if the lesion in one of the lymphoma cell mutants that are unable to make GlcNAc-PI is in the GlcNAc transferase. We will also collaborate with Dr. Rosse to identify the missing enzyme in PNH cells. We also have a good assay for the first mannosyltransferase and expect to be able to purify this enzyme. Previously, we identified the site of inhibition of mannosamine in GPI glycan assembly at one of the mannosyltransferases. We have several new inhibitors that prevent mannose incorporation into the GPI intermediates and we will determine their site of action. We have also set up in vivo and in vitro screens to identify other inhibitors and to determine where and how they inhibit. Once we have purified transferases available, we will test any demonstrated inhibitors on these enzymes. The GPI anchored trehalase has been purified to homogeneity from pig kidney and we have antibody against this enzyme. Thus, we can also screen for inhibitors that block anchor assembly and therefore prevent attachment of trehalase to the cell surface. One of these inhibitors may mimic the effect of the PNH defect and allow us to set up a model system of this disease.