gfsA encodes a novel galactofuranosyltransferase involved in biosynthesis of galactofuranose antigen of O‐glycan in Aspergillus nidulans and Aspergillus fumigatus

• Published in: Molecular microbiology Vol. 90; no. 5; pp. 1054 - 1073
• Main Authors: Komachi, Yuji, Hatakeyama, Shintaro, Motomatsu, Haruka, Futagami, Taiki, Kizjakina, Karina, Sobrado, Pablo, Ekino, Keisuke, Takegawa, Kaoru, Goto, Masatoshi, Nomura, Yoshiyuki, Oka, Takuji
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.12.2013
• Subjects: Antigens, Fungal - biosynthesis; Antigens, Fungal - immunology; Ascomycota; Aspergillus fumigatus; Aspergillus fumigatus - enzymology; Aspergillus fumigatus - genetics; Aspergillus nidulans; Aspergillus nidulans - enzymology; Aspergillus nidulans - genetics; Binding sites; Biochemistry; Biosynthesis; Cell Wall - metabolism; Fungal Polysaccharides - biosynthesis; Fungal Polysaccharides - chemistry; Fungal Polysaccharides - immunology; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - metabolism; Galactose - analogs & derivatives; Galactose - metabolism; Galactosyltransferases - chemistry; Galactosyltransferases - genetics; Galactosyltransferases - metabolism; Genes, Fungal; Genetics; Glycoconjugates; Golgi Apparatus - metabolism; Hyphae - metabolism; Mold; Polyclonal antibodies; Proteins; Reverse Genetics; Spores, Fungal - metabolism; Uridine Diphosphate - analogs & derivatives; Uridine Diphosphate - metabolism
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1111/mmi.12416

Summary The cells walls of filamentous fungi in the genus Aspergillus have galactofuranose (Galf)‐containing polysaccharides and glycoconjugates, including O‐glycans, N‐glycans, fungal‐type galactomannan and glycosylinositolphosphoceramide, which are important for cell wall integrity. Here, we attempted to identify galactofuranosyltransferases that couple Galf monomers onto other wall components in Aspergillus nidulans. Using reverse‐genetic and biochemical approaches, we identified that the AN8677 gene encoded a galactofuranosyltransferase, which we called GfsA, involved in Galf antigen biosynthesis. Disruption of gfsA reduced binding of β‐Galf‐specific antibody EB‐A2 to O‐glycosylated WscA protein and galactomannoproteins. The results of an in‐vitro Galf antigen synthase assay revealed that GfsA has β1,5‐ or β1,6‐galactofuranosyltransferase activity for O‐glycans in glycoproteins, uses UDP‐d‐Galf as a sugar donor, and requires a divalent manganese cation for activity. GfsA was found to be localized at the Golgi apparatus based on cellular fractionation experiments. ΔgfsA cells exhibited an abnormal morphology characterized by poor hyphal extension, hyphal curvature and limited formation of conidia. Several gfsA orthologues were identified in members of the Pezizomycotina subphylum of Ascomycota, including the human pathogen Aspergillus fumigatus. To our knowledge, this is the first characterization of a fungal β‐galactofuranosyltransferase, which was shown to be involved in Galf antigen biosynthesis of O‐glycans in the Golgi.