<scp><i>gfsA</i></scp> encodes a novel galactofuranosyltransferase involved in biosynthesis of galactofuranose antigen of <i><scp>O</scp></i>‐glycan in <i><scp>A</scp>spergillus nidulans</i> and <i><scp>A</scp>spergillus fumigatus</i>

<jats:title>Summary</jats:title><jats:p>The cells walls of filamentous fungi in the genus <jats:italic><jats:styled-content style="fixed-case">A</jats:styled-content>spergillus</jats:italic> have galactofuranose (<jats:styled-content style="fixed-case">G</jats:styled-content>al<jats:italic>f</jats:italic>)‐containing polysaccharides and glycoconjugates, including <jats:italic><jats:styled-content style="fixed-case">O</jats:styled-content></jats:italic>‐glycans, <jats:italic><jats:styled-content style="fixed-case">N</jats:styled-content></jats:italic>‐glycans, fungal‐type galactomannan and glycosylinositolphosphoceramide, which are important for cell wall integrity. Here, we attempted to identify galactofuranosyltransferases that couple <jats:styled-content style="fixed-case">G</jats:styled-content>al<jats:italic>f</jats:italic> monomers onto other wall components in <jats:italic><jats:styled-content style="fixed-case">A</jats:styled-content>spergillus nidulans</jats:italic>. Using reverse‐genetic and biochemical approaches, we identified that the <jats:styled-content style="fixed-case"><jats:italic>AN8677</jats:italic></jats:styled-content> gene encoded a galactofuranosyltransferase, which we called <jats:styled-content style="fixed-case">GfsA</jats:styled-content>, involved in <jats:styled-content style="fixed-case">G</jats:styled-content>al<jats:italic>f</jats:italic> antigen biosynthesis. Disruption of <jats:styled-content style="fixed-case"><jats:italic>gfsA</jats:italic></jats:styled-content> reduced binding of β‐<jats:styled-content style="fixed-case">Gal<jats:italic>f</jats:italic></jats:styled-content>‐specific antibody <jats:styled-content style="fixed-case">EB</jats:styled-content>‐<jats:styled-content style="fixed-case">A</jats:styled-content>2 to <jats:italic><jats:styled-content style="fixed-case">O</jats:styled-content></jats:italic>‐glycosylated <jats:styled-content style="fixed-case">WscA</jats:styled-content> protein and galactomannoproteins. The results of an <jats:italic>in‐vitro</jats:italic> <jats:styled-content style="fixed-case">G</jats:styled-content>al<jats:italic>f</jats:italic> antigen synthase assay revealed that <jats:styled-content style="fixed-case">GfsA</jats:styled-content> has β1,5‐ or β1,6‐galactofuranosyltransferase activity for <jats:italic><jats:styled-content style="fixed-case">O</jats:styled-content></jats:italic>‐glycans in glycoproteins, uses <jats:styled-content style="fixed-case">UDP</jats:styled-content>‐<jats:sc>d</jats:sc>‐<jats:styled-content style="fixed-case">G</jats:styled-content>al<jats:italic>f</jats:italic> as a sugar donor, and requires a divalent manganese cation for activity. <jats:styled-content style="fixed-case">GfsA</jats:styled-content> was found to be localized at the <jats:styled-content style="fixed-case">G</jats:styled-content>olgi apparatus based on cellular fractionation experiments. Δ<jats:styled-content style="fixed-case"><jats:italic>gfsA</jats:italic></jats:styled-content> cells exhibited an abnormal morphology characterized by poor hyphal extension, hyphal curvature and limited formation of conidia. Several <jats:styled-content style="fixed-case"><jats:italic>gfsA</jats:italic></jats:styled-content> orthologues were identified in members of the <jats:styled-content style="fixed-case">P</jats:styled-content>ezizomycotina subphylum of <jats:styled-content style="fixed-case">A</jats:styled-content>scomycota, including the human pathogen <jats:italic><jats:styled-content style="fixed-case">A</jats:styled-content>spergillus fumigatus</jats:italic>. To our knowledge, this is the first characterization of a fungal β‐galactofuranosyltransferase, which was shown to be involved in <jats:styled-content style="fixed-case">G</jats:styled-content>al<jats:italic>f</jats:italic> antigen biosynthesis of <jats:italic><jats:styled-content style="fixed-case">O</jats:styled-content></jats:italic>‐glycans in the <jats:styled-content style="fixed-case">G</jats:styled-content>olgi.</jats:p>