Characterization of anN-acetylglucosaminyltransferase involved inAspergillus fumigatuszwitterionic glycoinositolphosphoceramide biosynthesis

Abstract

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.

Glycosylphosphatidylinositol Anchors from Galactomannan and GPI-Anchored Protein Are Synthesized by Distinct Pathways in Aspergillus fumigatus

“…The MS data of the GIPC fractions from ∆ku80 and ∆per1 were similar and corresponded to the presence of 3 or 4 hexose residues with or without a choline-phosphate group linked to an IPC ( Figure S7 ) [ 37 ]. The biosynthesis of these GIPCs starts with the addition of glucosamine or mannose residue onto the inositol ring of the IPC [ 42 , 43 ]. These data suggest that LGM may be also synthetized onto an IPC anchor.…”

“…These data suggest that LGM may be also synthetized onto an IPC anchor. The inactivation of glycosyltransferases (mannosyltransferase and N -acetylglucosaminyltransferase), required for the GIPC synthesis has no effect on the GM synthesis [ 42 , 43 ] showing that GM synthesis is also independent of GIPC synthesis. The LGM is composed of a main chain of α-mannoside residues linked to a glucosamine-IPC anchor with a small side chain of galactofuranose residues.…”

Sphingolipids from the human fungal pathogen Aspergillus fumigatus

“…The mutant grows normally without any defect in the cell wall and membrane organization, and it is as virulent as the parental strain in mouse model of invasive aspergillosis [53]. A α-N-acetylglucosaminyl-transferase (Gnt-A), essential to the formation of zwitterionic GIPC, has been identified in A. fumigatus [54]. The mutant deficient in this transferase activity does not produce anymore zwitterionic GIPC but does not show any particular phenotype.…”

“…The mutant deficient in this transferase activity does not produce anymore zwitterionic GIPC but does not show any particular phenotype. Its virulence has not been tested [54].…”

“…In the GPI anchor, the glucosamine residue is linked to the 4-O position of the inositol ring whereas it is linked in 2-O position in the zwitterionic GPIC. This structural difference explains the sensitivity of LGM to PI-PLC digestion and shown that the Gnt-A transferase is not involved in the GM biosynthesis [54,67]. Despite the presence of GM in membrane and cell wall of both morphotypes of A. fumigatus, conidia and mycelium, its biosynthetic pathway remains poorly understood.…”