Abstract
The protozoan parasite Trypanosoma brucei is coated by glycosylphosphatidylinositol (GPI)-anchored proteins. During GPI biosynthesis, inositol in phosphatidylinositol becomes acylated. Inositol is deacylated prior to attachment to variant surface glycoproteins in the bloodstream form, whereas it remains acylated in procyclins in the procyclic form. We have cloned a T. brucei GPI inositol deacylase (GPIdeAc2). In accordance with the acylation/deacylation profile, the level of GPIdeAc2 mRNA was 6-fold higher in the bloodstream form than in the procyclic form. Knockdown of GPIdeAc2 in the bloodstream form caused accumulation of an inositol-acylated GPI, a decreased VSG expression on the cell surface and slower growth, indicating that inositol-deacylation is essential for the growth of the bloodstream form. Overexpression of GPIdeAc2 in the procyclic form caused an accumulation of GPI biosynthetic intermediates lacking inositol-linked acyl chain and decreased cell surface procyclins because of release into the culture medium, indicating that overexpression of GPIdeAc2 is deleterious to the surface coat of the procyclic form. Therefore, the GPI inositol deacylase activity must be tightly regulated in trypanosome life cycle.
Highlights
T. brucei escapes the immune response of the mammalian host by sequentially expressing structurally different forms of VSG
The GPIdeAc2 open reading frame encoded a protein of 815 amino acids, which had an ϳ50-amino-acid extension at the N terminus compared with PGAP1
The amount of glycolipid was decreased, and the inositol-acylated form of [19, 33] accumulated (Fig. 3B, lanes 7 and 8, arrowhead). These results suggested that knockdown of GPIdeAc2 resulted in slower conversion of glycolipid CЈ and lyso-CЈ to AЈ and , demonstrating that GPIdeAc2 is involved in GPI inositol deacylation in the bloodstream form
Summary
The GPIdeAc knock-out resulted in accumulation of inositol-acylated GPI biosynthetic intermediates. Some GPI inositol deacylase activity remained in the knock-out mutant, suggesting that at least one other inositol deacylase is present [22, 23]. We have identified a second T. brucei GPI inositol deacylase (GPIdeAc2) based on sequence homology to mammalian GPI inositol deacylase (PGAP1) [24] and shown that GPIdeAc2 is a major GPI inositol deacylase. Our data suggested that the regulation of GPIdeAc2 expression is critical in the life cycle of T. brucei
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