Characterising N-acetylglucosaminylphosphatidylinositol de-N-acetylase (CaGpi12), the enzyme that catalyses the second step of GPI biosynthesis in Candida albicans.

Candida albicans N-acetylglucosaminylphosphatidylinositol de-N-acetylase (CaGpi12) recognises N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) from Saccharomyces cerevisiae and is able to complement ScGPI12 function. Both N- and C-terminal ends of CaGpi12 are important for its function. CaGpi12 was biochemically characterised using rough endoplasmic reticulum microsomes prepared from BWP17 strain of C. albicans. CaGpi12 is optimally active at 30°C and pH 7.5. It is a metal-dependent enzyme that is stimulated by divalent cations but shows no preference for Zn2+ unlike the mammalian homologue. It irreversibly loses activity upon incubation with a metal chelator. Two conserved motifs, HPDDE and HXXH, are both important for its function in the cell. CaGPI12 is essential for growth and viability of C. albicans. Its loss causes reduction of GlcNAc-PI de-N-acetylase activity, cell wall defects and filamentation defects. The filamentation defects could be specifically correlated to an upregulation of the HOG1 pathway.

Targeting the GPI biosynthetic pathway.

The GPI (Glycosylphosphatidylinositol) biosynthetic pathway is a multistep conserved pathway in eukaryotes that culminates in the generation of GPI glycolipid which in turn anchors many proteins (GPI-APs) to the cell surface. In spite of the overall conservation of the pathway, there still exist subtle differences in the GPI pathway of mammals and other eukaryotes which holds a great promise so far as the development of drugs/inhibitors against specific targets in the GPI pathway of pathogens is concerned. Many of the GPI structures and their anchored proteins in pathogenic protozoans and fungi act as pathogenicity factors. Notable examples include GPI-anchored variant surface glycoprotein (VSG) in Trypanosoma brucei, GPI-anchored merozoite surface protein 1 (MSP1) and MSP2 in Plasmodium falciparum, protein-free GPI related molecules like lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs) in Leishmania spp., GPI-anchored Gal/GalNAc lectin and proteophosphoglycans in Entamoeba histolytica or the GPI-anchored mannoproteins in pathogenic fungi like Candida albicans. Research in this active area has already yielded encouraging results in Trypanosoma brucei by the development of parasite-specific inhibitors of GlcNCONH2-ß-PI, GlcNCONH2-(2-O-octyl)-PI and salicylic hydroxamic acid (SHAM) targeting trypanosomal GlcNAc-PI de-N-acetylase as well as the development of antifungal inhibitors like BIQ/E1210/gepinacin/G365/G884 and YW3548/M743/M720 targeting the GPI specific fungal inositol acyltransferase (Gwt1) and the phosphoethanolamine transferase-I (Mcd4), respectively. These confirm the fact that the GPI pathway continues to be the focus of researchers, given its implications for the betterment of human life.