| Abstrakt: |
A series of synthetic analogues ofd-GlcNα1–6-d-myo-inositol-1-HPO4-sn-1,2-dipalmitoylglycerol, consisting of 22 variants of the d-GlcN or lipid components, were tested in trypanosomal and human (HeLa) cell-free systems. The assays measured the abilities of the analogues to act as substrates or inhibitors of the enzymes of glycosylphosphatidylinositol biosynthesis downstream of GlcNAc-phosphatidylinositol (GlcNAc-PI) de-N-acetylase. One compound, 4-deoxy-d-GlcNα1–6-d-myo-inositol-1-HPO4-sn-1,2-dipalmitoylglycerol, proved to be an inhibitor of both the trypanosomal and HeLa pathways, whereas 4-O-methyl-d-GlcNα1–6-d-myo-inositol-1-HPO4-sn-1,2-dipalmitoylglycerol and the 4′-epimer,d-GalN-α1–6-d-myo-inositol-1-HPO4-sn-1,2-dipalmitoylglycerol, were neither substrates nor inhibitors. The results with other analogues showed that the 6-OH of the α-d-GlcN residue is not required for substrate recognition in the trypanosomal and human pathways, whereas the 3-OH group is essential for both. Parasite-specific recognition of the β-linked analogued-GlcNβ1–6-d-myo-inositol-1-HPO4-sn-1,2-dipalmitoylglycerol is striking. This suggests that, like the GlcNAc-PI de-N-acetylase, the trypanosomal glycosylphosphatidylinositol α-mannosyltransferases, inositol acyltransferse and ethanolamine phosphate transferase, do not recognize the 2-, 3-, 4-, and 5-OH groups of thed-myo-inositol residue, whereas the human inositol acyltransferase and/or first α-mannosyltransferase recognizes one or more of these groups. All of the various lipid analogues tested served as substrates in both the trypanosomal and HeLa cell-free systems, suggesting that a precise lipid structure and stereochemistry are not essential for substrate recognition. However, an analogue containing a single C18:0 alkyl chain in place ofsn-1,2-dipalmitoylglycerol proved to be a better substrate in the trypanosomal than in the HeLa cell-free system. These findings should have a bearing on the design of future generations of specific inhibitors of the trypanosomal glycosylphosphatidylinositol biosynthetic pathway. |
