Early steps in glycosylphosphatidylinositol biosynthesis in Leishmania major

Abstract

A cell-free system based on washed Leishmania major membranes was labelled with GDP-[$H]Man in the presence of synthetic glucosaminyl-phosphatidylinositol (GlcN-PI) and N-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI). In both cases, the major radiolabelled products were Manα1-4GlcNα1-6myo-inositol1-HPO % -(sn-1,2-dipalmitoylglycerol) and Manα1-4GlcNα1-6myo-inositol1-HPO % -(sn-1-palmitoyl-2-lysoglycerol), to which an additional -mannose residue was added when a chase with an excess of GDP-Man was performed. The L. major cell-free system can therefore be used to observe the actions of four enzymes, namely

Biosynthesis of glycosylphosphatidylinositols of Plasmodium falciparum in a cell-free incubation system: inositol acylation is needed for mannosylation of glycosylphosphatidylinositols

“…Inhibitors able to block inositol acylation would interfere with the synthesis of GPIs of Plasmodium, because inositol acylation is a prerequisite for subsequent mannosylation reactions as indicated by the absence of mannosylated GPIs in membrane preparations impaired in the formation of GlcN-acyl-PtdIns. This is similar to the situation found in yeast cell [12], rodent cell [32] and HeLa cell [11] membranes, but differs from findings with T. brucei [10,33].…”

“…Nevertheless, significant differences in the substrate and donor specificities of the enzymes involved in the early steps of the GPI biosynthetic pathway have been described with the use of cellfree incubation systems [10,11,25,27]. Detailed studies on GPI biosynthesis in Plasmodium might therefore lead to the identification of parasite-specific reactions of this pathway, which could be potential targets for the development of parasitespecific inhibitors.…”

“…Significant amounts of GlcN-acyl-PtdIns were detected only in the presence of 1 mM CoA ( Figure 3B, lane 4). If membrane preparations were labelled with UDP-[6-$H]GlcNAc in the presence of 1 mM CoA and 10 µM GDP-Man, the GPIanchor precursors Pf α and Pf β were synthesized ( Figure 3B, lane 10), showing that these two supplements were sufficient for synthesis of the complete set of GPIs by the plasmodial cell-free system. These results show that CoA is either involved in or stimulates the transfer of the acyl residue to the inositol.…”

“…By using different chemically synthesized GlcNAcPtdIns analogues in a de-N-acetylase assay, differences in substrate specificities have been established between the trypanosomal enzyme and a mammalian enzyme [9]. In mammalian cells and yeast, intermediates of GPI biosynthesis receive a palmitoyl residue on the inositol ring before the attachment of the first mannose residue of the evolutionarily conserved trimannosyl core glycan, whereas in trypanosomes the first mannosylation step must precede palmitoylation of the inositol ring [10,11]. The donor for the acyl chain on the inositol ring is palmitoyl-CoA in yeast [12], whereas trypanosomes seem to use another palmitoyl source [13,14].…”

Mammalian PIG-L and its yeast homologue Gpi12p are N-acetylglucosaminylphosphatidylinositol de-N-acetylases essential in glycosylphosphatidylinositol biosynthesis

“…The substrate specificity of de-N-acetylase was studied by using various GlcNAc-PI analogues [11][12][13]. However, this enzyme was so unstable in the presence of the detergent that purification was impossible [11].…”