A Single UDP-galactofuranose Transporter Is Required for Galactofuranosylation in Aspergillus fumigatus

Abstract

Galactofuranose (Galf) containing molecules have been described at the cell surface of several eukaryotes and shown to contribute to the virulence of the parasite Leishmania major and the fungus Aspergillus fumigatus. It is anticipated that a number of the surface glycoconjugates such as N-glycans or glycolipids are galactofuranosylated in the Golgi apparatus. This raises the question of how the substrate for galactofuranosylation reactions, UDP-Galf, which is synthesized in the cytosol, translocates into the organelles of the secretory pathway. Here we report the first identification of a Golgi-localized nucleotide sugar transporter, named GlfB, with specificity for a UDP-Galf. In vitro transport assays established binding of UDP-Galf toGlfB and excluded transport of several other nucleotide sugars. Furthermore, the implication of glfB in the galactofuranosylation of A. fumigatus glycoconjugates and galactomannan was demonstrated by a targeted gene deletion approach. Our data reveal a direct connection between galactomannan and the organelles of the secretory pathway that strongly suggests that the cell wall-bound polysaccharide originates from its glycosylphosphatidylinositol-anchored form.

Biosynthesis of the Fungal Cell Wall Polysaccharide Galactomannan Requires Intraluminal GDP-mannose

“…Together with Leishmania lipophosphoglycan and Crithidia fasciculata lipoarabinogalactan (46), A. fumigatus lipogalactomannan constitutes one of the rare examples of GPI-anchored polysaccharide (8) and may also be synthesized along the secretory pathway. An absence of reactivity of the anti-galactofuran antibody EB-A2 toward cell extract of the Golgi UDP-Galf transporter-deficient mutant ⌬glfB, which suggested an impaired biosynthesis of this polysaccharide, supported this assumption (16). The present data clearly establishes that the Galf side chains of galactomannan are also absent from the cell wall of this mutant.…”

“…Biosynthesis of Galactomannan Is Blocked in A. fumigatus ⌬gmtA and ⌬glfB Mutants-The generation and partial characterization of an A. fumigatus mutant deficient in the UDPGalf transporter GlfB, named ⌬glfB, has been reported previously (16). To determine if galactomannan biosynthesis requires GlfB or the GDP-Man transporter GmtA, we first investigated the monosaccharide composition of alkali-insoluble cell wall fractions from the ⌬gmtA, ⌬glfB, and wild type strains of A. fumigatus.…”

“…In contrast, galactomannan biosynthesis as well as its biological role and importance for fungal viability are currently undefined. However, the recent characterization of a Golgi-localized UDP-Galf transporter and its subsequent deletion in A. fumigatus (16) and Aspergillus nidulans (17) suggested its implication in galactomannan galactofuranosylation. Indeed, a soluble cell extract of the transporter-deficient mutant was unreactive to the anti-galactofuran antibody EB-A2 (16).…”

Roles of the Aspergillus nidulans UDP-galactofuranose transporter, UgtA in hyphal morphogenesis, cell wall architecture, conidiation, and drug sensitivity

“…UDP-Galf is a nucleotide diphosphate sugar that is synthesized in the cytosol (El-Ganiny et al, 2010), so it must be translocated into an endomembrane organelle prior to incorporation into glycoconjugates. The A. fumigatus UDP-Galf transporter, GlfB was shown by density-gradient centrifugation to localize to Golgi equivalents (Engel et al, 2009). NSTs are structurally conserved and can readily be identified from genome databases; however, their substrate specificity cannot be predicted from the sequence (Berninsone and Hirschberg, 2000).…”

Direct Investigation of the Aspergillus GDP‐Mannose Transporter by STD NMR Spectroscopy

“…The mycellium was harvested and Golgi-enriched fractions isolated according to Record et al [22] and Engel et al [23] In short, the mycellium was collected on Whatman no. 4 filter paper washed with milliQ water, removed from the filter paper and weighed.…”