Addition of phosphoethanolamine to mannoses: steps 6, 10, and 11 . 132

In both humans and yeast, phosphoethanolamine is added to the three core mannose subunits via the genes PIG-N/MCD4, PIG-G/GPI7 and PIG-O/GPI13 in steps 6, 10 and 11, respectively (Benachour et al., 1999; Hong et al., 2000; Hong et al., 1999a).

Both C. elegans and C. briggsae have homologues for each of these genes, with the best result for PIG-N (Y54E10BR.1/CBG04200), PIG-G (F28C6.4/CBG00550) and GPI-O (C27A12.9/CBG20246) in C. elegans/C. briggsae, respectively. Interestingly the nematodes homologues for each individual gene are also homologues for the others, with Y54E10BR.1 found to be also homologous to PIG-G and PIG-O, F28C6.4 also homologous to PIG-N and PIG-O, and C27A12.9 also homologous to PIG-G (Table 3.1). A sequence comparison of the C. elegans genes with ClustalW

shows conserved motifs within the three predicted proteins but otherwise poor conservation for the rest of their sequences (Figure 3.13); the conserved nematode motifs corresponds to similar motifs on the three human genes, which may represent sites of important biological function, such as ligand binding sites, for this class of enzymes. Further analysis will be needed to elucidate exactly which of the homologues in C. elegans and C. briggsae are responsible for each of the phosphoethanolamine addition reactions. The addition of the first phosphoethanolamine is important for GPI anchor synthesis in both human and yeast (Vainauskas and Menon, 2006; Zhu et al., 2006) while the addition of the third phosphoethanolamine is essential as the protein is attached to the anchor via this moiety (Hong et al., 2000). Addition of the second phosphoethanolamine however is only important in yeast (Fujita et al., 2004), whereas in humans the modification is needed for just a subset of GPI anchors (Shishioh et al., 2005). It will be interesting to see how important the presence of this moiety on each mannose subunit is in both C.

elegans and C. briggsae, and elucidate their influence on different tissue types in development and other physiological processes.

PIG-F/Gpi11p in human and yeast interact with PIG-G/Gpi7p and PIG-O/Gpi13p in the addition of the second and third mannoses in GPI anchor biosynthesis. PIG-F is an essential interaction partner of PIG-O in humans (Hong et al., 2000), however defects in Gpi11p in yeast was shown not to be a requirement for this step (Taron et al., 2000).

C. elegans does not contain a homologue for this gene, while C. briggsae has a homologue to PIG-F but the gene has a predicted size of more than double its human counterpart (Table 3.1). The difference between the two nematodes posses interesting questions from an evolutionary perspective. It may be that the gene is ancestral and

Figure 3.13. ClustalW analysis of the three human phosphoethanoamine addition proteins and their C. elegans homologues. The symbols in the graph are described in Figure 3.8. Blue

has been lost in the C. elegans lineage and not C. briggsae. Alternatively the gene may have taken on different roles in the two nematodes, with the C. briggsae version still possibly retaining some of its original function in GPI anchor synthesis. It will also be interesting to investigate the properties of the C. elegans PIG-O homologue compared to the human protein to elucidate the mechanism with which PIG-F acts in PIG-O regulation.

3.4.1.6 Step 12

The last step in GPI biosynthesis involves the attachment of the protein to the anchor via the GPI transamidase (GPIT) complex. Each of the five subunits that make up the GPIT in human and yeast have homologues in both C. elegans and C. briggsae. PIG-K/Gpi8p, GPAA1/Gaa1p and PIG-T/Gpi16p are postulated to form the core structure of GPIT with PIG-K as the catalytic subunit, with GPAA1 important for substrate recognition and PIG-T having a role in conferring specificity for the enzyme (Eisenhaber et al., 2003b; Fraering et al., 2001; Kang et al., 2002; Vainauskas and Menon, 2004a). PIG-S/GPI17 and PIG-U/GAB1 are postulated to be responsible for structural and substrate recognition (Ohishi et al., 2001). In yeast Gpi17p and Gab1p form a complex with each other and appear to associate transiently with the rest of the GPIT complex, suggesting that the whole complex functions as two different subunits (Grimme et al., 2004; Zhu et al., 2005). It will be interesting to see if the nematode homologues also form these complexes, and elucidate their mode of regulation with regards to different tissue types and developmental stages.

One of the most extensively characterised genes for the last step of GPI biosynthesis is PIG-K, the catalytic component of the GPIT complex. PIG-K is a cysteine protease and plays a crucial role in GPI biosynthesis (Spurway et al., 2001). Both C. elegans and C. briggsae contain two homologues to this protein. The C. elegans homologues are T05E11.6 and T28H10.3; the T05E11.6 protein has a higher homology BLAST score (Table 3.1). Both of these proteins contain the two conserved residues, His157 and Cys199, within the PIG-K active site that are necessary for the enzymatic function of the protein (Figure 3.4.a) (Meyer et al., 2000). PIG-K also contains a transmembrane domain at the C-terminus of the protein, which is believed to anchor

the protein to the ER membrane. T28H10.3 contains a hydrophobic region at the C-terminus whereas T05E11.6 does not, however it has also been observed that the absence of the transmembrane domain in PIG-K does not impact on its activity in vivo (Ohishi et al., 2000). PIG-K forms an intermolecular disulphide bridge with PIG-T in the GPIT complex at Cys92 which is important but not essential for full transamidase activity (Ohishi et al., 2003); interestingly this residue is conserved in T05E11.6 but is absent in T28H10.3, where it is replaced by an asparagine; this also raises the possibility that the C. elegans PIG-K and PIG-T homologues form a part of the complex similar to the human proteins. Information from Wormbase reports only one partial EST assigned to T05E11.6 while T28H10.3 appears to be highly transcribed with 28 full length and partial ESTs attributed to it (Figure 3.14). Both of these genes have deletion mutants that generate sterile and lethal phenotypes, suggesting that they carry out essential processes within the worm. Both of the PIG-K homologues could potentially be a part of the GPI anchor synthesis pathway within C. elegans. An interesting possibility may be that the two genes are expressed in different temporal and spatial patterns, and that both proteins are needed for GPI anchoring during different stages of C. elegans development. An expression pattern has been generated for T28H10.3 from the Promoterome (Dupuy et al., 2004) which will be discussed in detail below.