Regulation of glycosylphosphatidylinositol biosynthesis by GTP. Stimulation of N-acetylglucosamine-phosphatidylinositol deacetylation.
Glycosylphosphatidylinositol (GPI) is biosynthesized by the sequential addition of carbohydrates to phosphatidylinositol (PI). In the first two reactions, GlcNAc is transferred from UDP-GlcNAc to PI and then deacetylated to form GlcNAc-PI and GlcN-PI, respectively. In this paper, stimulation of GlcNAc-PI deacetylation by GTP, is reported. Addition of this nucleotide triphosphate to incubations in which GPI precursors were synthesized from UDP-[6-3H]GlcNAc by microsomes prepared from the lymphoma cell line EL4 resulted in a shift in the relative amount of each intermediate formed such that [6-3H]GlcN-PI was the predominant product. GTP also increased the total synthesis of the first GPI intermediate, GlcNAc-PI, by inhibiting reactions that metabolize UDP-[6-3H]GlcNAc into non-GPI-related products. However, unlike the stimulation of GlcNAc-PI deacetylation, ATP was equally effective in increasing the formation of GlcNAc-PI. An additional product, tentatively identified as [6-3H]GlcN-PI(acyl-inositol), was also detected when GTP was present in the incubation. The synthesis of this GPI precursor, which is proposed to be the third intermediate in GPI biosynthesis in mammals, was increased by GTP because the level of GlcN-PI, the substrate for acylation, was elevated. To isolate the effects of GTP on the GlcNAc-PI deacetylation, this reaction was studied directly by using [6-3H]GlcNAc-PI as the substrate. The stimulation was found to be specific for the guanosine-containing nucleotide triphosphate and optimal with approximately 1 mM GTP. Both the reaction rate at early time points and the total amount of deacetylated product formed in 60 min were increased by GTP. The effect on the second reaction of the pathway does not appear to be coupled to the first reaction because GlcNAc-PI deacetylation was increased by GTP in microsomes from cells defective in the GlcNAc-PI synthesis. Finally, 0.5 mM GTP gamma S (guanosine 5'-O-(thiotriphosphate)) completely inhibited the stimulation of GlcNAc-PI deacetylation caused by 1 mM GTP, indicating that hydrolysis of the nucleotide triphosphate was required for this effect. Although the mechanism and role of the GTP stimulation of GlcNAc-PI deacetylation is not clear, this regulation could influence the biosynthesis of mature GPI precursors and the subsequent expression of GPI-anchored proteins.
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322.
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D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D017261:Glycosylphosphatidylinositols / Q000096:biosynthesis*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology*; D007700:Kinetics; D008223:Lymphoma; D051379:Mice; D008861:Microsomes / Q000378:metabolism*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
D017261:Glycosylphosphatidylinositols; C081027:N-acetylglucosaminylphosphatidylinositol; D010716:Phosphatidylinositols; D012265:Ribonucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014537:Uridine Diphosphate N-Acetylglucosamine; D006160:Guanosine Triphosphate; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D000117:Acetylglucosamine