Inositolphosphoceramide is not a substrate for the first steps in the biosynthesis of glycoinositolphospholipids in Trypanosoma cruzi

Abstract

The major free glycoinositolphospholipids and protein-linked glycoinositolphospholipids in Trypanosoma cruzi contain ceramide as the lipid moiety. Ceramide was not found in mammalian glycosylphosphatidylinositol (GPI)-anchors. An alkylglycerol, either as a lyso species or acylated has been also found in T. cruzi anchors. However, unlike African trypanosomes, no diacylglycerol was detected in the GPI-anchors. Using a membrane preparation from epimastigotes upon labelling with UDP[3H]GlcNAc we identified [3H]GlcNAcPI as the first step of GPI biosynthesis. Both, alkylacylglycerol (major) and diacylglycerol are constituents of the lipid. Although inositolphosphoceramide is the main inositolphospholipid in epimastigotes, it does not incorporate GlcNAc. The de-N-acetylation step afforded [3H]GlcN(alkylacylglycerol)PI and we also detected the [3H]GlcN(lysoacyl)PI. A new metabolite, phosphoGlcN(lysoacyl)PI, which was formed on long incorporation times, was characterized by chemical and enzymatic degradations. Several [3H]-Man labelled GPI precursors were obtained by in vitro GDP[3H]-Man labelling in the presence of UDPGlcNAc. All of them were sensitive to PI-PLC and to saponification conditions, thus, supporting a glycerolipid structure.