Purification, cloning and characterization of a GPI inositol deacylase from Trypanosoma brucei

Inositol acylation is an obligatory step in glycosylphosphatidylinositol (G PI) biosynthesis whereas mature GPI anchors often lack this modification. T he GPI anchors of Trypanosoma brucei variant surface glycoproteins (VSGs) u ndergo rounds of inositol acylation and deacylation during GPI biosynthesis and the deacylation reactions are inhibited by diisopropylfluorophosphate (DFP). Inositol deacylase was affinity labelled with [H-3]DFP and purified. Peptide sequencing was used to clone GPIdeAc, which encodes a protein with significant sequence and hydropathy similarity to mammalian acyloxyacyl hy drolase, an enzyme that removes fatty acids from bacterial lipopolysacchari de. Both contain a signal sequence followed by a saposin domain and a GDSL- lipase domain. GPIdeAc(-/-) trypanosomes were viable in vitro and in animal s. Affinity-purified HA-tagged GPIdeAc was shown to have inositol deacylase activity. However, total inositol deacylase activity was only reduced in G PIdeAc(-/-) trypanosomes and the VSG GPI anchor was indistinguishable from wild type. These results suggest that there is redundancy in T. brucei inos itol deacylase activity and that there is another enzyme whose sequence is not recognizably related to GPIdeAc.