The first step in GPI biosynthesis is initiated by the transfer of N-acetylglucosamine (GlcNAc) from uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) to PI, which generates GlcNAc-PI in the cytoplasmic face of the ER. In mammalian cells, the enzyme involved in this step is GPI-N-acetylglucosaminyltransferase (GPI-GnT), an ER membrane-bound multiprotein complex composed of at least 7 proteins: phosphatydylinositol glycan class A; PIG-A, PIG-C, PIG-H, PIG-Q, PIG-P, dolichyl-phosphate mannosyltransferase polypeptide 2; DPM2, and PIG-Y [18-20] (Fig. 2-①, Table 1). PIG-A has been implicated in the catalytic activity of the transferase. In mammals, somatic mutation of PIG-A, which disrupts the GPI anchor biosynthesis, is involved in paroxysmal noctural hemoglobinuria (PNH) [21].

The resulting GlcNAc-PI is deacetylated to yield glucosaminyl-PI (GlcN-PI) (Fig. 2-②) in a reaction catalyzed by a specific zinc metalloenzyme called N-deacetylase (deNAc, also designated PIG-L in mammals and TbGPI12 in trypanosomes), which is localized on the cytoplasmic side of the ER [22-24]. A study using trypanosomes and a HeLa cell-free system demonstrated that the trypanosome enzyme does not recognize GlcNAc-PI analogues containing 2-O-octyl-D-myo-inositol or L-myo-inositol, in contrast to the HeLa enzyme [25]. Moreover, the IC₅₀ of potent inhibitors against trypanosome deNAc was significantly lower (approximately 8 nM) compared with human deNAc (100 µM), suggesting that the trypanosome enzyme has different substrate specificity than catalytically similar mammalian enzymes, which may be exploited as a chemotherapeutic target against trypanosomiasis [26].

GlcN-PI is likely flipped to the luminal side of the ER membrane before acylation. After this, T. brucei and mammalian GPI biosynthetic pathways diverge. In mammals, acylation of the inositol moiety of GlcN-PI, which is mediated by inositol acyltransferase (PIG-W), generates GlcN-acyl-PI before the first mannosylation [27]. In contrast, inositol acylation in trypanosomes only occurs after the first mannosylation and generates Man-GlcN-acyl-PI, indicating that inositol acylation is not a prerequisite for trypanosome mannosyl transferases (Fig. 2-③) [28,29]. Inositol acyltransferase in trypanosomes requires the presence of a hydroxyl group at the 4th position on the first mannose and a free amine on the glucosamine residue and, thus, mannosylation is required prior to inositol acylation in trypanosome GPI biosynthesis [30]. Moreover, inositol acylation and deacylation occur on multiple GPI intermediates. These results suggest that trypanosome inositol acylase and deacylase have different and broader substrate specificities than those of mammals. The enzymes responsible for inositol acylation and deacylation in T. brucei are sensitive to phenylmethylsulfonyl fluoride [31,32], whereas enzymes with similar activity in mammalian cells are unaffected [33]. Therefore, inositol acylation in trypanosomes is a good target for chemotherapy.

In yeasts, the efficient delivery of GlcN-acyl-PI to the first GPI-mannosyltransferase (GPI-MT-I) requires Arv1, which is a transmembrane protein containing potential zinc-binding motifs and which is a novel mediator of eukaryotic sterol homeostasis [34]. The orthologue of Arv1 with significant sequence homology has been detected only in a Plasmodium genome database but not in that of Trypanosoma (GeneDB accession no. PF100110) (data not shown).

In the next step, mannoses are transferred from dolichol-phosphate-mannose (Dol-P-Man) to GPI intermediates through different glycosidic linkages (Fig. 2-④). The first mannose is transferred to position 4 of the GlcN constituent of GlcN-acyl-PI by GPI-MT-I (a complex of PIG-M and PIG-X in humans) [35,36]. However, in T. brucei the first mannose is instead transferred to GlcN-PI, indicative of a different substrate specificity of GPI-MT- I between trypanosome and mammals. Moreover, GlcN-(2-O-hexadecyl)-phosphatidylinositol, a synthetic analogue of the GPI intermediate at this step, selectively inhibits trypanosomal GPI-MT-I, but not human GPI-MT-I [37]. Thus, GPI-MT-I appears to be a promising target of trypanosome-specific inhibitors, even though it has not been identified yet in trypanosomes.

Following the transfer of the first mannose, the second mannose is transferred to position 6 of the first mannose in Man-GlcN-acyl-PI by GPI α1-6 mannosyltransferase (Fig. 2-④) (GPI-MT-II, PIG-V in humans) [38]. A trypanosome orthologue has not yet been identified. Subsequently, a third mannose is transferred by GPI α1-2 mannosyltransferase III (GPI-MT-III, PIG-B in humans and TbGPI10 in trypanosomes) [39,40]. The observation that the construction of a knockout mutant of TbGPI10 is possible only when episomal TbGPI10 is introduced is consistent with the suggestion that TbGPI10 is essential for the growth of the bloodstream form of T. brucei [40]. Identification of the specific inhibitor against trypanosome GPI-MT-III with an IC₅₀ of 1.7 µM from substrate analogs containing amino group at 2'-position of second mannose in Man₂-GlcN-IPC₁₈, a phosphate-linked C₁₈ alkyl chain instead of Man₂-GlcN-PI diacylglycerol [30], implicates TbGPI10 as a valid target of trypanosome-specific inhibitors.

Protein is attached to the GPI through EtNP that is transferred from phosphatidylethanolamine to position 6 of the third mannose (Fig. 2-⑤). This reaction is mediated by PIG-F and PIG-O in mammalian cells [41]. PIG-O is likely to be a catalytic component of the EtNP transferase forming a complex with PIG-F, which likely assists in stabilization of the enzyme complex. However, the orthologues of PIG-F and PIG-O have not yet been identified in trypanosomes. In mammalian cells and yeasts, an additional EtNP is added to the first (mediated by PIG-N) [42] or second mannose (mediated by PIG-F and PIG-G) [43], whereas these modifications do not occur in trypanosomes.

In the T. brucei GPI biosynthetic pathway, inositol acylation takes place only after the formation of Man-GlcN-PI (Fig. 2-③). Then, the inositol-linked acyl chain can be removed or added again to any of the GPI intermediates bearing 1-3 mannoses with a dynamic equilibrium between inositol acylated and nonacylated species (Fig. 2-⑥) [29,31,44]. In mammalian cells, the inositol-linked acyl-chain remains until the mature GPI anchor is attached to the protein, whereupon it is removed by GPI inositol deacylase (PGAP1; post-GPI attachment to protein I) [45], indicating that inositol acylation and deacylation are distinctively separate events in mammalian GPI biosynthesis. In contrast, inositol is deacylated prior to attachment to VSG in bloodstream form, whereas it remains acylated in procyclins in the procyclic form. Thus, inositol deacylation likely occurs more efficiently in the bloodstream form compared with the procyclic form. Previously, a GPI inositol deacylase (GPIdeAc) gene was cloned in trypanosome and its activity demonstrated using an affinity-purified recombinant protein [46]. Inositol-acylated GPI biosynthetic intermediates accumulated in a knock-out mutant although some activity remained, suggesting that another inositol deacylase may be present [46,47]. Recently, another T. brucei GPI inositol deacylase 2 (GPIdeAc2) was cloned and was demonstrated to be essential in the bloodstream form, with its activity likely being tightly regulated in the trypanosome life cycle [48]. GPIdeAc2 is homologous to mammalian PGAP1, sharing the same lipase motif with catalytic serine [45]. However, these 2 enzymes act at different times. As mentioned above, inositol-deacylation occurs before attachment to proteins in trypanosomes, whereas the acyl chain is removed from inositol after attachment to proteins in mammalian cells [13,29,49]. Moreover, diisopropylfluorophosphate selectively inhibits inositol deacylase activity in trypanosomes, whereas mammalian inositol deacylase having a similar activity is not affected [50,51], indicating that the trypanosome inositol deacylase has a different substrate specificity and so is a good candidate drug target.

In the last step of trypanosome GPI biosynthesis, the lipid moiety of the GPI anchor, sn-1 (C_18:0) and sn-2 fatty acids (a mixture of C₁₈-C₂₂), is replaced with myristate (a saturated fatty acid containing 14 carbon atoms) in a lipid remodeling reaction (Fig. 2-⑦). This reaction occurs in the ER before transfer to the VSG protein. The lipid remodeling is initiated by removal of a longer fatty acid attached to the sn-2 position of glycolipid A' to form glycolipid θ, prior to acylation by attachment of myristate using myristoyl-CoA as a donor. As a next step, deacylation occurs at the sn-1 position to form the lyso species glycolipid θ'. The second myristate is then incorporated into the sn-1 position, forming dimyristated GPI (glycolipid A) on which VSG is attached. Thus, lipid remodeling consists of sequential deacylation and reacylation reactions. Recently, it has been reported that myristate transfer in GPI fatty acid remodeling in trypanosomes is mediated by TbGup1 [52]. However, the complete enzyme repertoire involved in lipid remodeling in trypanosomes remains unclear. In humans, fatty acid remodeling occurs in the Golgi after attachment of protein to the GPI anchor. Removal of the sn-2 unsaturated fatty acid from the lipid moiety of human GPI anchor requires PGAP3 [53] and replacement with stearic acid (C_18:0) requires PGAP2 [54], while the sn-1 saturated fatty acid is retained in both the GPI precursor and GPI anchored protein.