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Previously, we reported that GfsA is a novel galactofuranosyltransferase involved in the biosynthesis of O-glycan, the proper maintenance of fungal morphology, the formation of conidia and anti-fungal resistance in Aspergillus nidulans and A. fumigatus (Komachi Y et al., 2013. GfsA encodes a novel galactofuranosyltransferase involved in biosynthesis of galactofuranose antigen of O-glycan in Aspergillus nidulans and Aspergillus fumigatus. Mol. Microbiol. 90:1054-1073). In the present paper, to gain an in depth-understanding of the enzymatic functions of GfsA in A. fumigatus (AfGfsA), we established an in vitro assay to measure galactofuranosyltransferase activity using purified AfGfsA, UDP-α-d-galactofuranose as a sugar donor, and p-nitrophenyl-β-d-galactofuranoside as an acceptor substrate. LC/MS, 1H-NMR and methylation analyses of the enzymatic products of AfGfsA revealed that this protein has the ability to transfer galactofuranose to the C-5 position of the β-galactofuranose residue via a β-linkage. AfGfsA requires a divalent cation of manganese for maximal activity and consumes UDP-α-d-galactofuranose as a sugar donor. Its optimal pH range is 6.5-7.5 and its optimal temperature range is 20-30°C. 1H-NMR, 13C-NMR and methylation analyses of fungal-type galactomannan extracted from the ∆AfgfsA strain revealed that AfGfsA is responsible for the biosynthesis of β1,5-galactofuranose in the galactofuran side chain of fungal-type galactomannan. Based on these results, we conclude that AfGfsA acts as a UDP-α-d-galactofuranose: β-d-galactofuranoside β1,5-galactofuranosyltransferase in the biosynthetic pathway of galactomannans.

[Abstract] GfsA is a fungal -galactofuranosyltransferase involved in the biosynthesis of glycan. To investigate the enzymatic functions of GfsA, we attempted to obtain a recombinant protein of this enzyme from two heterologous host organisms. However, GfsA could not be expressed as a recombinant protein in either Escherichia coli (E. coli) or Saccharomyces cerevisiae (S. cerevisiae). Therefore, we decided to employ Aspergillus nidulans (A. nidulans) as the host organism, and produced a strain that expressed 3x FLAG-tagged GfsA using chromosomal tagging. To confirm its expression, a solubilized protein was prepared from the tagged strain and analyzed with an anti-FLAG antibody. The strain that expressed 3x FLAGtagged GfsA produced a functional protein with a mass of approximately 67 kDa. The method described in this manuscript allows purification of the GfsA-3xFLAG protein as expressed in A. nidulans cells.