Aspergillus fumigatus Cap59-like protein A is involved in α1,3-mannosylation of GPI-anchors.

Glycosylphosphatidylinositol (GPI) attaches a variety of eukaryotic proteins to the outer leaflet of the plasma membrane. In fungi, these proteins may also be transferred to the cell wall, to which they are covalently linked via a remnant of the GPI-anchor. They play crucial physiological roles in cell-cell interactions, adhesion or cell wall biogenesis. The biosynthesis of GPI-anchors in the endoplasmic reticulum, their transfer to proteins, early remodelling and transport to the Golgi apparatus has been fairly well described. In contrast, almost nothing is known about the genes and enzymes involved in adding glycan side chains to GPI after protein attachment. In this study, we characterized an α1,3-mannosyltransferase involved in maturation of GPI-anchors from the pathogenic fungus Aspergillus fumigatus. This enzyme shows homology to Cryptococcus neoformans Cap59p, a putative glycosyltransferase involved in capsule formation and virulence, and was thus named Cap59-like protein A (ClpA). Targeted deletion of the clpA gene in A. fumigatus led to absence of α1,3-mannose from mature GPI-anchors. The enzyme was further located to the Golgi-like apparatus of A. fumigatus and was shown to be active in the yeast Saccharomyces cerevisiae.

Characterization of an N-acetylglucosaminyltransferase involved in Aspergillus fumigatus zwitterionic glycoinositolphosphoceramide biosynthesis.

Glycoinositolphosphoceramides (GIPCs) are complex sphingolipids present at the plasma membrane of various eukaryotes with the important exception of mammals. In fungi, these glycosphingolipids commonly contain an α-mannose residue (Man) linked at position 2 of the inositol. However, several pathogenic fungi additionally synthesize zwitterionic GIPCs carrying an α-glucosamine residue (GlcN) at this position. In the human pathogen Aspergillus fumigatus, the GlcNα1,2IPC core (where IPC is inositolphosphoceramide) is elongated to Manα1,3Manα1,6GlcNα1,2IPC, which is the most abundant GIPC synthesized by this fungus. In this study, we identified an A. fumigatus N-acetylglucosaminyltransferase, named GntA, and demonstrate its involvement in the initiation of zwitterionic GIPC biosynthesis. Targeted deletion of the gene encoding GntA in A. fumigatus resulted in complete absence of zwitterionic GIPC; a phenotype that could be reverted by episomal expression of GntA in the mutant. The N-acetylhexosaminyltransferase activity of GntA was substantiated by production of N-acetylhexosamine-IPC in the yeast Saccharomyces cerevisiae upon GntA expression. Using an in vitro assay, GntA was furthermore shown to use UDP-N-acetylglucosamine as donor substrate to generate a glycolipid product resistant to saponification and to digestion by phosphatidylinositol-phospholipase C as expected for GlcNAcα1,2IPC. Finally, as the enzymes involved in mannosylation of IPC, GntA was localized to the Golgi apparatus, the site of IPC synthesis.