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.