The role of inositol acylation and inositol deacylation in GPI biosynthesis in Trypanosoma brucei.

Abstract

The compound diisopropylfluorophosphate (DFP) selectively inhibits an inositol deacylase activity in living trypanosomes that, together with the previously described phenylmethylsulfonyl fluoride (PMSF)-sensitive inositol acyltransferase, maintains a dynamic equilibrium between the glycosylphosphatidylinositol (GPI) anchor precursor, glycolipid A [NH2(CH2)2PO4-6Manal-2Manal-6Manal-4GlcNal-6myo-inositol-1-P04-sn-1,2-dimyristoylglycerol], and its inositol acylated form, glycolipid C. Experiments using DFP in living trypanosomes and a trypanosome cell-free system suggest that earlier GPI intermediates are also in equilibrium between their inositol acylated and nonacylated forms. However, unlike mammalian and yeast cells, bloodstream form trypanosomes do not appear to produce an inositol acylated form of glucosaminylphosphatidylinositol (GlcN-PI). A specific function of inositol acylation in trypanosomes may be to enhance the efficiency of ethanolamine phosphate addition to the Man3GlcN-(acyl)PI intermediate. Inositol deacylation appears to be a prerequisite for fatty acid remodelling of GPI intermediates that leads to the exclusive presence of myristic acid in glycolipid A and, ultimately, in the variant surface glycoprotein (VSG). In the presence of DFP, the de novo synthesis of GPI precursors cannot proceed beyond glycolipid C' (the unremodelled version of glycolipid C) and lyso-glycolipid C'. Under these conditions glycolipid C'-type GPI anchors appear on newly synthesized VSG molecules. However, the efficiencies of both anchor addition to VSG and N-glycosylation of VSG were significantly reduced. A modified model of the GPI biosynthetic pathway in bloodstream form African trypanosomes incorporating these findings is presented.

Probing Enzymes Late in the Trypanosomal Glycosylphosphatidylinositol Biosynthetic Pathway with Synthetic Glycosylphosphatidylinositol Analogues

“…This is consistent with earlier work by Brown et al suggesting that MTIII can act on simple hydrophobic dimannosyl acceptors, implying a lack of recognition of both the glucosamine and inositol moiety (32). The inositol acyltransferase requires the presence of a hydroxyl group at the 4-position on the first mannose and the presence of a free amine on the glucosamine residue, explaining for the first time the molecular basis behind the requirement for mannosylation prior to inositol acylation in the trypanosomal GPI pathway (19).…”

“…However, although Man-(4- deoxyMan)-GlcN-IPC 18 was processed to the corresponding M 3 product, it did not undergo inositol acylation to form aM 3 , nor did the addition of ethanolamine phosphate occur. This observation reveals that the 4-hydroxyl of the first mannose is important for substrate recognition by the inositol acyltransferase and nicely explains why mannosylation must precede inositol acylation in the T. brucei GPI pathway (19). The lack of ethanolamine phosphate addition is consistent with previous work showing that prior inositol acylation is necessary for ethanolamine phosphate addition (19).…”

“…From GlcN-PI onward there are significant differences in the GPI biosynthetic pathways of T. brucei and mammalian cells. In T. brucei, inositol acylation occurs only after addition of the first mannose and is only essential for the addition of ethanolamine phos- phate to the third mannose, whereas in mammalian systems GlcN-PI undergoes inositol acylation before mannosylation occurs (19,20).…”

Leishmania mexicanaMutants Lacking Glycosylphosphatidylinositol (GPI):Protein Transamidase Provide Insights into the Biosynthesis and Functions of GPI-anchored Proteins

“…Inositol acylation of GPI anchor intermediates usually involves the addition of a saturated fatty acid to the 2-hydroxyl of the inositol residue and appears to occur in all eukaryotes (Ferguson 1999;Tiede et al, 1999;McConville and Menon, 2000). However, the timing, nature of the acyl donor, and function of this modification may vary between higher and lower eukaryotes (Gü ther and Ferguson, 1995;Smith et al, 1997). For example, in animal cells and yeast, inositol acylation occurs at an early stage in GPI biosynthesis (after de-N-acetylation of GlcN-PI), whereas in African trypanosomes it occurs later in the pathway (Gü ther and Ferguson, 1995).…”

Substrate specificity of the N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase of glycosylphosphatidylinositol membrane anchor biosynthesis in African trypanosomes and human cells

“…This leads to an accumulation of the GPI intermediate Man $ GlcN-PI [16], arising from the inhibition of inositol acylation [15] and the subsequent addition of ethanolamine phosphate [19]. The Man $ GlcN-PI intermediate that accumulates in the cells undergoes fatty acid remodelling with the introduction of [$H]myristate into the PI moiety [15].…”