title: Characterization of an N-acetylglucosaminyltransferase involved in Aspergillus fumigatus zwitterionic 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.
authors: Department of Cellular Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, Hannover 30625, Germany.
authors: Jakob
authors: J
authors: Engel
authors: Department of Cellular Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, Hannover 30625, Germany.
authors: Philipp S
authors: PS
authors: Schmalhorst
authors: Department of Cellular Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, Hannover 30625, Germany.
authors: Anke Tina
authors: AT
authors: Krüger
authors: Department of Cellular Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, Hannover 30625, Germany.
authors: Christina Theda
authors: CT
authors: Müller
authors: Department of Cellular Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, Hannover 30625, Germany.
authors: Falk F R
authors: FF
authors: Buettner
authors: Department of Cellular Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, Hannover 30625, Germany routier.francoise@mh-hannover.de.
authors: Françoise H
authors: FH
authors: Routier
mesh_terms: D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics; D002518:Ceramides / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D008358:Mannose / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
chemical_list: D002518:Ceramides; D005656:Fungal Proteins; D014537:Uridine Diphosphate N-Acetylglucosamine; D017351:N-Acetylglucosaminyltransferases; D008358:Mannose
keywords: ascomycete; glucosamine; glycosphingolipids; glycosyltransferase; saprophyte