Glycerophosphatidylinositol (GPI)-anchor

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Glycerophosphatidylinositol (GPI)-anchor biosynthesis
Beside the above described functions the phosphatidylinositol metabolism supplies the precursors for the synthesis of the glycosylphosphatidylinositol (GPI) membrane anchor. GPI is a complex glycolipid that acts as a membrane anchor. GPI is synthesised in the endoplasmic reticulum (ER) and transferred to the carboxyl terminus of a protein that has a GPI-attachment signal peptide. GPI anchored proteins are then transported to the cell surface. The GPI biosynthesis pathway consists of at least 10 reaction steps .Nearly 20 genes would be involved in this pathway.

Step 1: The first step of GPI-anchor biosynthesis, the generation of GlcNAc-PI from UDPGlcNAc and phosphatidylinositol, is mediated by GPI-N-acetylglucosaminyltransferase (GPIGnT; Figure 30). GPI-GnT is a complex glycosyltransferase consisting of at least six proteins: PIG-A, PIG-C, PIG-H, GPI1, PIG-P and DPM2 (Watanabe et al. 1998, 2000).
Step 2: The generation of GlcN-PI from GlcNAc-PI. The second step is mediated by Nacetylglucosaminylphosphatidylinositol (GlcNAc-PI) de-N-acetylase (Fig. 30). PIG-L, which was previously shown to be essential in the second step, has the enzyme activity in vitro (Watanabe et al. 1999).
Steps 3, 4 and 5: The acylation of the inositol ring of GlcN-PI to generate GlcN-acyl-PI and first mannosylation to generate Man–GlcN-acyl-PI. Step 3 is the acylation, mostly palmitoylation, of the myo-inositol of glucosaminylphosphatidylinositol (GlcN-PI) at position 2. The first mannose is then transferred from Dol-P–Man to position 4 of GlcN (step 5 in Fig. 30). The recently cloned PIG-M gene encodes the GPI α1–4 mannosyltransferase (GPI-MTI) responsible for the transfer. PIG-M has a functionally essential DXD motif (a motif found in many glycosyltransferases) within a lumenal domain, suggesting that the transfer of the first mannose occurs on the lumenal side of the ER. It was shown that the biosynthesis of GlcNPI occurs on the cytoplasmic side; therefore, GlcN-PI and/or GlcN-acyl-PI should translocate from the cytoplasmic side to the lumenal side. This would be mediated by a ‘flippase’ (step 4 in Fig. 30).
Step 6: The modification of Man–GlcN-acyl-PI with a phosphoethanolamine side chain to generate EtNP–Man–GlcN-acyl-PI. The first mannose is modified by ethanolaminephosphate (EtNP) (Fig. 30) (Sutterlin et al. 1998, Canivenc-Gansel et al. 1998) and PIG-N (Hong et al. 1999) is involved in this reaction.
Steps 7 and 8: Additions of the second and third mannose. The second mannose is transferred from Dol-P–Man to position 6 of the first mannose in EtNP–Man–GlcN-acyl-PI (Fig. 30). The α1–6 mannosyltransferase, which transfers the second mannose (GPI-MT-II), has not been identified. The third mannose is transferred from Dol-P–Man to position 2 of the second mannose by an α1–2 mannosyltransferase (GPI-MT-III) (Fig. 30). Mammalian PIG-B (Sutterlin et al. 1998) are most likely GPI-MT-III itself.
Step 9: The addition of EtNP to the third mannose to generate EtNP–Man–Man–(EtNP) Man–GlcN-acyl-PI (termed H7), a GPI species transferred to proteins. The EtNP that links GPI to proteins is transferred from phosphatidylethanolamine to position 6 of the third mannose (Fig. 30). Two gene products, PIG-F and PIG-O, are involved in this step (Hong et al. 2000).
Step 10: Side-chain modification of the second mannose by EtNP to generate EtNP–Man– (EtNP) Man–(EtNP) Man–GlcN-acyl-PI (termed H8). The second mannose of H7 can be modified by side-chain EtNP at position 6 to generate H8. Both H7 and H8 are potentially direct precursors of protein-bound anchors (Fig. 30). They also exist as free GPI on the cell surface (Baumann et al. 2000).
Step 11: GPI transamidase attaches GPI to proteins. Pre-assembled GPI is transferred to proteins. Nascent proteins that are to be GPI-anchored have a GPI-attachment signal peptide at the carboxyl terminus (Fig. 30). After translocation across the ER membrane (Wang et al. 1999), GPI is attached to the proteins by replacing the GPI-attachment signal peptide. This reaction is mediated by transamidase. The GPI transamidase consists of at least two ER membrane proteins, GAA1 and GPI8. Generation of mannosyl donor, Dol-P–Man, by Dol-P–Man synthase, which donates all three mannoses, is synthesised from Dol-P and GDP–Man (Fig. 30). Dol-P–Man synthase consists of three subunits, DPM1, DPM2 and DPM3 (Maeda et al. 1998, 2000). Human DPM1 is a catalytic subunit and lacks a membrane-insertion domain. Instead, it associates with DPM3, which has transmembrane domains (Maeda et al. 2000). DPM3 stabilises DPM1. DPM2, which also has transmembrane domains, associates with DPM3 and stabilises it.