Specificities of enzymes of glycosylphosphatidylinositol biosynthesis in Trypanosoma brucei and HeLa cells.
A series of synthetic analogues of d-GlcN alpha 1-6-d-myo-inositol-1-HPO(4)-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-GlcN alpha 1-6-d-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol, proved to be an inhibitor of both the trypanosomal and HeLa pathways, whereas 4-O-methyl-d-GlcN alpha 1-6-d-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol and the 4'-epimer, d-GalN-alpha1-6-d-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol, were neither substrates nor inhibitors. The results with other analogues showed that the 6-OH of the alpha-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 beta-linked analogue d-GlcN beta 1-6-d-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol is striking. This suggests that, like the GlcNAc-PI de-N-acetylase, the trypanosomal glycosylphosphatidylinositol alpha-mannosyltransferases, inositol acyltransferse and ethanolamine phosphate transferase, do not recognize the 2-, 3-, 4-, and 5-OH groups of the d-myo-inositol residue, whereas the human inositol acyltransferase and/or first alpha-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 of sn-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.
Smith
Terry K
TK
Division of Biological Chemistry & Molecular Microbiology, The School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
Crossman
Arthur
A
Paterson
Michael J
MJ
Borissow
Charles N
CN
Brimacombe
John S
JS
Ferguson
Michael A J
MA
D000117:Acetylglucosamine / Q000378:metabolism; D000217:Acyltransferases / Q000378:metabolism; D000818:Animals; D017261:Glycosylphosphatidylinositols / Q000096:biosynthesis*; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008364:Mannosyltransferases / Q000378:metabolism; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D017261:Glycosylphosphatidylinositols; D006155:Guanosine Diphosphate Mannose; D000217:Acyltransferases; D008364:Mannosyltransferases; D000117:Acetylglucosamine