Untersuchungen zur Topologie der Glycosylphosphatidylinositol-Biosynthese in Toxoplasma gondii

Toxoplasma gondii is an obligatorily intracellular living, parasitical protozoon of great medical importance particularly in two cases: On the one hand with first infection during a pregnancy, which can harm the Foetus. On the other hand as opportunistic pathogen in immunocompromised patients (e.g. AIDS). Here a reactivation of continuous stages could lead to death. A starting point for the fight against the parasites represents a special metabolic pathway, the glycosylphosphatidylinositol (GPI) biosynthesis. GPIs consist of a conserved hydrophilic glycan structure, which can be modified by side chains, and a hydrophobic inositol-phospholipid. Like numerous other parasitic protozoa the T. gondii major surface proteins are anchored by GPIs in the cell surface - a blocking of the GPI biosynthesis leads to killing the parasites. In order to fight only the parasites with therapeutic efforts and not to affect the GPI biosynthesis that is likewise taking place in the host cells however, detailed knowledge of this metabolic pathway is necessary. A system of permeabilized Tachyzoites of T. gondii was developed and the distribution of GPI anchor precursors and further GPI biosynthetic intermediates over the membrane of the endoplasmic reticulum (ER) was examined for the first time. The cell membrane is effectively permeabilized by hypotonic treatment, which remains ER with this treatment intact. This system was used, in order to label GPIs with different radioactive precursor molecules (sugar nucleotides). The GPI intermediates formed by permeabilized Toxoplasma were characterized on the basis of specific enzymatic treatments and chemical hydrolyses and compared with already well-known data. It was stated that the hydrophilic components of the GPI intermediates match with the described structures. In addition it could be proven in this work that before starting the mannosylation steps an acylation from GlcN-PI to GlcN-(acyl)-PI at the inositol takes place. Following radioactive labelling of the glycolipids the enzyme PI-PLC, which cleaves between hydrophilic and hydrophobic portion of the GPIs, was used to examine orientation of the GPI biosynthesis intermediates in the ER membrane. It could be shown that both early, and higher glycosylated intermediates and even GPI anchor precursors to the predominant part exhibit a cytoplasmic orientation in the ER membrane. In connection with transient acylation of the inositol ring a repeated change of the different intermediates over the ER membrane ("flip-flop") seems possible. For the first time it could be shown here that such large GPI anchor precursors with an additional hydrophilic side chain, consisting of N-Acetyl-Galactosamina1-4Glucose, cross a lipid bilayer membrane.