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 de acetylated 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-H-3]GlcNAc by microsomes prepared from t he lymphoma cell line EL4 resulted in a shift in the relative amount o f each intermediate formed such that [6-H-3]GlcN-PI was the predominan t product. GTP also increased the total synthesis of the first GPI int ermediate, GlcNAc-PI, by inhibiting reactions that metabolize UDP-[6-H -3]GlcNAc into non-GPI-related products. However, unlike the stimulati on of GlcNAc-PI deacetylation, ATP was equally effective in increasing the formation of GlcNAc-PI. An additional product, tentatively identi fied as [6-H-3]GlcN-PI(acylinositol), was also detected when GTP was p resent in the incubation. The synthesis of this GPI precursor, which i s 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-H-3]G lcNAc-PI as the substrate. The stimulation was found to be specific fo r the guanosine-containing nucleotide triphosphate and optimal with ap proximately 1 mM GTP. Both the reaction rate at early time points and the total amount of deacetylated product formed in 60 min were increas ed by GTP. The effect on the second reaction of the pathway does not a ppear to be coupled to the first reaction because GlcNAc-PI deacetylat ion was increased by GTP in microsomes from cells defective in the Glc NAc-PI synthesis. Finally, 0.5 mM GTPgammaS (guanosine 5'-O-(thiotriph osphate)) completely inhibited the stimulation of GlcNAc-PI deacetylat ion caused by 1 mM GTP, indicating that hydrolysis of the nucleotide t riphosphate was required for this effect. Although the mechanism and r ole of the GTP stimulation of GlcNAc-PI deacetylation is not clear, th is regulation could influence the biosynthesis of mature GPI precursor s and the subsequent expression of GPI-anchored proteins.