| Summary: | The primary aim of this work was to investigate phospholipid metabolism in trypanosomes, including a phosphoinositide signalling, and the metabolism of the VSG and its GPI anchor. The phosphoinositides of <I>T.b brucei</I> were labelled using <SUP>32</SUP>P (from [<SUP>32</SUP>P)P<SUB>i</SUB> or [γ<SUP>32</SUP>P]ATP) and [<SUP>3</SUP>H)inositol through a variety of methods. Most work was carried out with phospholipids labelled using [γ<SUP>32</SUP>P]ATP as the donor, with cells permeabilised by being placed in a buffer of low osmotic strength (swell dialysis). Species labelled with <SUP>32</SUP>P were identified as PI 4-P and PI 4,5-P<SUB>2</SUB> through HPLC analysis. There was no evidence of 3-phosphorylated phosphoinositides under the conditions used. Results showed that <I>T.b brucei</I> has GTP- and Ca<SUP>2+</SUP>-regulated phosphoinositide metabolism, similar to that seen in vertebrate cells. Additionally, an unidentified factor in rabbit serum caused a 2-fold increase in the phosphorylation of the polyphosphoinositides. Although Ca<SUP>2+</SUP> is known to cause the release of VSG through the action of GPI-PLC, it is not clear whether this enzyme plays an important role in membrane-form (mf) VSG metabolism in intact cells. The relationship between VSG release and subsequent GPI anchor metabolism was examined using a GPI-PLC inhibitor (pCMPSA) and conditions known to cause the release of soluble form (s) VSG (Ca<SUP>2+</SUP>). Metabolism of the mfVSG GPI anchor was taken to correspond with [<SUP>32</SUP>P]PA generation in cells incubated in the presence of [γ<SUP>32</SUP>P)ATP. Anti-VSG antibodies were used to detect VSG in supernatant samples of <I>T.b. brucei</I>. A great deal of phospholipid metabolism sensitive to pCMPSA, so possibly related to GPI-PLC activity, was shown to occur in the absence of sVSG release. The primary role of GPI-PLC in viable trypanosomes may therefore be in the regulation of processes other than mfVSG metabolism. <I>T.b. brucei </I>was incubated with anti-VSG IgGs to observe whether interaction with anti-VSG antibodies caused the destruction of mfVSG and concomitant increase in PA labelling. Anti-VSG IgGs had no effect on PA metabolism. mfVSG endocytosed in bloodstream from <I>T. brucei</I> may therefore be returned intact to the cell surface.
|
|---|
