The Role of Inositol Acylation and Inositol Deacylation in the Toxoplasma gondii Glycosylphosphatidylinositol Biosynthetic Pathway

Abstract

Toxoplasma gondii is a ubiquitous parasitic protozoan that invades nucleated cells in a process thought to be in part due to several surface glycosylphosphatidylinositol (GPI)-anchored proteins, like the major surface antigen SAG1 (P30), which dominates the plasma membrane. The serine protease inhibitors phenylmethylsulfonyl fluoride and diisopropyl fluoride were found to have a profound effect on the T. gondii GPI biosynthetic pathway, leading to the observation and characterization of novel inositol-acylated mannosylated GPI intermediates. This inositol acylation is acyl-CoA-dependent and takes place before mannosylation, but uniquely for this class of inositol-acyltransferase, it is inhibited by phenylmethylsulfonyl fluoride. The subsequent inositol deacylation of fully mannosylated GPI intermediates is inhibited by both phenylmethylsulfonyl fluoride and diisopropyl fluoride. The use of these serine protease inhibitors allows observations as to the timing of inositol acylation and subsequent inositol deacylation of the GPI intermediates. Inositol acylation of the non-mannosylated GPI intermediate D-GlcN␣1-6-D-myo-inositol-1-HPO 4 -sn-lipid precedes mannosylation. Inositol deacylation of the fully mannosylated GPI intermediate allows further processing, i.e. addition of GalNAc side chain to the first mannose. Characterization of the phosphatidylinositol moieties present on both free GPIs and GPI-anchored proteins shows the presence of a diacylglycerol lipid, whose sn-2 position contains almost exclusively an C18:1 acyl chain. The data presented here identify key novel inositol-acylated mannosylated intermediates, allowing the formulation of an updated T. gondii GPI biosynthetic pathway along with identification of the putative genes involved.

Virulent and Avirulent Strains of Toxoplasma gondii Which Differ in Their Glycosylphosphatidylinositol Content Induce Similar Biological Functions in Macrophages

“…The presence of sn-2 fatty acids (C18∶1) in the T. gondii GPIs found here and elsewhere [44] is reminiscent to the immunogenic GPIs in Plasmodium falciparum mid-schizont stages, which incorporate C18∶1 (88%) and C18∶2 (12%) at the sn-2 position of their diacylglycerolipid [53], and in Trypanosoma cruzi trypomastigotes GPIs, which contain C16∶0 (37%), C18∶1 (31%) along with C18∶2 (21%) at their sn-2 position of their alkylacylglycerolipid [54]. We reasoned that strain-specific differences in the GPI composition could be due to different amounts of protein-free GPIs and/or particular GPI-anchored surface proteins.…”

E1210, a New Broad-Spectrum Antifungal, Suppresses Candida albicans Hyphal Growth through Inhibition of Glycosylphosphatidylinositol Biosynthesis

“…Some inhibitors of GPI biosynthesis have been reported (52,57), but most of these are designed based on acceptor analogs or protease inhibitors and do not possess drug-like properties. Serine protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluoride inhibit ethanolamine phosphate addition and inositol acylation of GPI in Toxoplasma gondii and Trypanosoma brucei, which are suggested to have an activated serine that is absent in the other orthologues (56). YW3548, a natural product, is thought to inhibit the addition of phosphoethanolamine (EtNP) to the first mannose of GPI, but it inhibits both yeast and mammalian GPI-EtNP transferases, although EtNP modification of the first mannose is not essential for the surface expression of GPI-anchored proteins on mammalian cells (22,58).…”

Glycosylphosphatidylinositols of Toxoplasma gondii induce matrix metalloproteinase-9 production and degradation of galectin-3

“…GPI VI possesses only N-acetylgalactosamine linked to the first mannose of the core glycan as side chain modification. GPIs I and II, and GPIs IV and V differ in their diacylglycerol composition with palmitic (C16:0) and stearic (C18:1) acids as predominant fatty acids (Striepen et al 1997;Smith et al 2007). …”