GPI transamidase of Trypanosoma brucei has two previously uncharacterized (trypanosomatid transamidase 1 and 2) and three common subunits

Abstract

Glycosylphosphatidylinositol (GPI) anchor is a membrane attachment mechanism for cell surface proteins widely used in eukaryotes. GPIs are added to proteins posttranslationally by a complex enzyme, GPI transamidase. Previous studies have shown that human and Saccharomyces cerevisiae GPI transamidases are similar and consist of five homologous components: GAA1, GPI8, PIG-S, PIG-T, and PIG-U in humans and Gaa1p, Gpi8p, Gpi17p, Gpi16p, and Cdc91p in S. cerevisiae. We report that GPI transamidase of Trypanosoma brucei (Tb), a causative agent of African sleeping sickness, shares only three components (TbGAA1, TbGPI8, and TbGPI16) with humans and S. cerevisiae but has two other specific components, trypanosomatid transamidase 1 (TTA1) and TTA2. GPI transamidases of both bloodstream form (growing in mammalian blood) and procyclic form (growing in tsetse fly vector) of the parasite have the same five components. Homologues of TTA1 and TTA2 are present in Leishmania and Trypanosoma cruzi but not in mammals, yeasts, flies, nematodes, plants, or malaria parasites, suggesting that these components may play unique roles in attachment of GPI anchors in trypanosomatid parasites and provide good targets for antitrypanosome drugs.

Fig. 1.

SDS/PAGE analysis of the GPI transamidase complex of T. brucei procyclics. (A) GPI transamidase was affinity-purified from Nonidet P-40 extract of procyclics expressing TbGPI8-FLAG-HAT (8FH, lanes 1–4) or TbGPI8-FLAG-GST (8FG, lanes 5–8). Samples were analyzed under reducing (R, lanes 1, 2, 7, and 8) or nonreducing (NR, lanes 3–6) conditions. The identities of silver-stained bands are shown on the right, and size markers are indicated on the left. (B) Two-dimensional SDS/PAGE analysis of GPI transamidase containing TbGPI8-FLAG-GST. First- (left to right) and second- (top to bottom) dimension runs were under nonreducing and reducing conditions, respectively. The identities of silver-stained spots are shown. A similar sample stained after the first SDS/PAGE is shown at the top. Size markers are shown on the top for the first dimension and on the left for the second dimension.

Fig. 2.

Hydrophobicity profile of TTA1 (A) and TTA2 (B). Putative transmembrane domains are shown by thick lines above each hydrophobicity plot.

Fig. 3.

Characterization of TTA1-KO and TTA2-KO procyclics. (A) Flow-cytometric analysis of the surface expression of EP procyclin. Wild-type, TbGPI8-KO, TTA1-KO, and TTA2-KO clones transfected with an empty vector (filled) or plasmid bearing corresponding cDNA (line) were analyzed in a FACScan by using anti-EP procyclin (Anti-EP) or control antibodies. (B) TLC analysis of GPI biosynthesis of TTA1-KO and TTA2-KO. Wild-type (lane 1), GPI8KO (lanes 2 and 3), TTA1-KO (lanes 4 and 5), and TTA2-KO (lanes 6 and 7) procyclics transfected with an empty vector (lanes 2, 4, and 6) or corresponding cDNA (lanes 3, 5, and 7) were hypotonically lysed. The lysates were incubated with GDP-[³H]mannose, and mannolipids were analyzed by TLC. The identities of mannolipids are shown. DPM, dolichol-phosphate-mannose; M2(acyl), GPI bearing two mannoses with acylated inositol; M1, M2, and M3, GPI bearing one, two, and three mannoses, respectively; A′-like, GPI bearing three mannoses with ethanolamine phosphate on the third mannose; PP3, A′-like with acylated inositol; PP1, mature GPI in the procyclic form.

Fig. 4.

Characterization of TbGAA1. (A) Comparison of amino acid sequences of T. brucei and human GAA1. Putative transmembrane domains are overlined for T. brucei GAA1 (TbGAA1) and underlined for human GAA1 (HsGAA1). Identical and similar amino acids are in black and gray boxes, respectively. (B) Flow-cytometric analysis of the surface expression of EP procyclin on TbGAA1-KO mutant (GAA1-KO). For GAA1-KO, filled and open lines are transfectants with an empty vector and TbGAA1 cDNA, respectively. (C) GPI biosynthesis of GAA1-KO. Lane 1, wild type; lane 2, GAA1-KO transfected with an empty vector; lane 3, GAA1-KO transfected with TbGAA1 cDNA. The identities of mannolipids are the same as those in Fig. 3B. (D) Association of TbGAA1 with TbGPI8. The Nonidet P-40 extracts of GAA1-KO procyclics transfected with FLAG-GST-TbGAA1 (lanes 1 and 3) and GPI8-KO procyclics transfected with TbGPI8-FLAG-GST, as a control for Western blotting (lanes 2 and 4), were immunoprecipitated with anti-FLAG antibody and Western-blotted with anti-FLAG (lanes 1 and 2) or anti-TbGPI8 (lanes 3 and 4) antibodies. Size markers are shown on the right.

Fig. 5.

GPI transamidase of the bloodstream form of T. brucei. GPI transamidase complexes were affinity-purified from the procyclic (lane 1) and bloodstream (lane 2) forms of T. brucei expressing TbGPI8-FLAG-GST. Samples were analyzed by SDS/PAGE under reducing conditions. Size markers are shown on the right.

Fig. 6.

Comparison of five components of human and trypanosome GPI transamidases. Schematic representation of three common components (GAA1, GPI8, and PIG-T/GPI16) and two unique components (TTA1 and TTA2 of T. brucei and PIG-S and PIG-U of humans). The membrane orientation and N and C termini are shown.