GfsA is a &bgr;1,5-galactofuranosyltransferase involved in the biosynthesis of the galactofuran side chain of fungal-type galactomannan in Aspergillus fumigatus

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.

[1]  K. Takegawa,et al.  Characterization of a PA14 domain-containing galactofuranose-specific β-d-galactofuranosidase from Streptomyces sp. , 2017, Bioscience, biotechnology, and biochemistry.

[2]  P. Howell,et al.  Biofilm Exopolysaccharides of Pathogenic Fungi: Lessons from Bacteria* , 2016, The Journal of Biological Chemistry.

[3]  M. Goto,et al.  Biosynthesis of Galactofuranose-containing Glycans in Filamentous Fungi , 2016 .

[4]  T. Kinoshita,et al.  Biosynthesis of GPI-anchored proteins: special emphasis on GPI lipid remodeling , 2016, Journal of Lipid Research.

[5]  E. Lagendijk,et al.  Identification and functional analysis of two Golgi-localized UDP-galactofuranose transporters with overlapping functions in Aspergillus niger , 2015, BMC Microbiology.

[6]  S. Kuhara,et al.  Identification and Characterization of a Novel Galactofuranose-Specific β-D-Galactofuranosidase from Streptomyces Species , 2015, PloS one.

[7]  P. Howell,et al.  Sph3 Is a Glycoside Hydrolase Required for the Biosynthesis of Galactosaminogalactan in Aspergillus fumigatus*♦ , 2015, The Journal of Biological Chemistry.

[8]  E. Lagendijk,et al.  Identification of the UDP-glucose-4-epimerase required for galactofuranose biosynthesis and galactose metabolism in A. niger , 2014, Fungal Biology and Biotechnology.

[9]  M. Goto,et al.  Purification of the GfsA-3x FLAG Protein Expressed in Aspergillus nidulans , 2014 .

[10]  K. Takegawa,et al.  gfsA encodes a novel galactofuranosyltransferase involved in biosynthesis of galactofuranose antigen of O‐glycan in Aspergillus nidulans and Aspergillus fumigatus , 2013, Molecular microbiology.

[11]  J. Latgé,et al.  Overlapping and Distinct Roles of Aspergillus fumigatus UDP-glucose 4-Epimerases in Galactose Metabolism and the Synthesis of Galactose-containing Cell Wall Polysaccharides* , 2013, The Journal of Biological Chemistry.

[12]  Markus Aebi,et al.  N-linked protein glycosylation in the ER. , 2013, Biochimica et biophysica acta.

[13]  M. Akeroyd,et al.  Fungal α-arabinofuranosidases of glycosyl hydrolase families 51 and 54 show a dual arabinofuranosyl- and galactofuranosyl-hydrolyzing activity , 2012, Biological chemistry.

[14]  Y. Okawa,et al.  Significant differences in the cell‐wall mannans from three Candida glabrata strains correlate with antifungal drug sensitivity , 2012, The FEBS journal.

[15]  A. Ram,et al.  Galactofuranose in eukaryotes: aspects of biosynthesis and functional impact. , 2012, Glycobiology.

[16]  Cheng Jin.  Protein Glycosylation in Aspergillus fumigatus Is Essential for Cell Wall Synthesis and Serves as a Promising Model of Multicellular Eukaryotic Development , 2011, International journal of microbiology.

[17]  M. Goto,et al.  Putative Stress Sensors WscA and WscB Are Involved in Hypo-Osmotic and Acidic pH Stress Tolerance in Aspergillus nidulans , 2011, Eukaryotic Cell.

[18]  D. Sanders,et al.  Roles of the Aspergillus nidulans UDP-galactofuranose transporter, UgtA in hyphal morphogenesis, cell wall architecture, conidiation, and drug sensitivity. , 2011, Fungal genetics and biology : FG & B.

[19]  R. Helm,et al.  Characterization of recombinant UDP-galactopyranose mutase from Aspergillus fumigatus. , 2010, Archives of biochemistry and biophysics.

[20]  D. Sanders,et al.  Aspergillus nidulans UDP-glucose-4-epimerase UgeA has multiple roles in wall architecture, hyphal morphogenesis, and asexual development. , 2010, Fungal genetics and biology : FG & B.

[21]  A. Beauvais,et al.  Members of protein O‐mannosyltransferase family in Aspergillus fumigatus differentially affect growth, morphogenesis and viability , 2010, Molecular microbiology.

[22]  C. Whitfield,et al.  A Membrane-located Glycosyltransferase Complex Required for Biosynthesis of the d-Galactan I Lipopolysaccharide O Antigen in Klebsiella pneumoniae , 2010, The Journal of Biological Chemistry.

[23]  Tadashi Suzuki,et al.  Free Oligosaccharides to Monitor Glycoprotein Endoplasmic Reticulum-associated Degradation in Saccharomyces cerevisiae* , 2010, The Journal of Biological Chemistry.

[24]  T. Lowary,et al.  Recent advances in mycobacterial cell wall glycan biosynthesis. , 2009, Current opinion in chemical biology.

[25]  V. Ferrières,et al.  A Single UDP-galactofuranose Transporter Is Required for Galactofuranosylation in Aspergillus fumigatus* , 2009, The Journal of Biological Chemistry.

[26]  Y. Okawa,et al.  The cell wall galactomannan antigen from Malassezia furfur and Malassezia pachydermatis contains beta-1,6-linked linear galactofuranosyl residues and its detection has diagnostic potential. , 2009, Microbiology.

[27]  B. M. Pinto,et al.  STD‐NMR Studies Suggest that Two Acceptor Substrates for GlfT2, a Bifunctional Galactofuranosyltransferase Required for the Biosynthesis of Mycobacterium tuberculosis Arabinogalactan, Compete for the Same Binding Site , 2009, Chembiochem : a European journal of chemical biology.

[28]  K. Takegawa,et al.  Protein O-Mannosyltransferases B and C Support Hyphal Development and Differentiation in Aspergillus nidulans , 2009, Eukaryotic Cell.

[29]  J. Latgé,et al.  Aspergillus fumigatus: cell wall polysaccharides, their biosynthesis and organization. , 2009, Future microbiology.

[30]  C. D. de Koster,et al.  Comprehensive genomic analysis of cell wall genes in Aspergillus nidulans. , 2009, Fungal genetics and biology : FG & B.

[31]  D. Sanders,et al.  Aspergillus nidulans UDP-galactopyranose mutase, encoded by ugmA plays key roles in colony growth, hyphal morphogenesis, and conidiation. , 2008, Fungal genetics and biology : FG & B.

[32]  R. Contreras,et al.  Contribution of Galactofuranose to the Virulence of the Opportunistic Pathogen Aspergillus fumigatus , 2008, Eukaryotic Cell.

[33]  F. Klis,et al.  A Novel Screening Method for Cell Wall Mutants in Aspergillus niger Identifies UDP-Galactopyranose Mutase as an Important Protein in Fungal Cell Wall Biosynthesis , 2008, Genetics.

[34]  Patrick J. Brennan,et al.  Galactosyl Transferases in Mycobacterial Cell Wall Synthesis , 2007, Journal of bacteriology.

[35]  J. Latgé,et al.  Glycosylinositolphosphoceramides in Aspergillus fumigatus. , 2007, Glycobiology.

[36]  M. Momany,et al.  Analysis of glycosylinositol phosphorylceramides expressed by the opportunistic mycopathogen Aspergillus fumigatus s⃞ Published, JLR Papers in Press, May 8, 2007. , 2007, Journal of Lipid Research.

[37]  M. Goto.  Protein O-Glycosylation in Fungi: Diverse Structures and Multiple Functions , 2007, Bioscience, biotechnology, and biochemistry.

[38]  Y. Jigami,et al.  Functional Analysis of Arabidopsis thaliana RHM2/MUM4, a Multidomain Protein Involved in UDP-D-glucose to UDP-L-rhamnose Conversion* , 2007, Journal of Biological Chemistry.

[39]  J. Errey,et al.  Expression and initial characterization of WbbI, a putative D-Galf:α-D-Glc β-1,6-galactofuranosyltransferase from Escherichia coli K-12 , 2006 .

[40]  H. Mori,et al.  Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. , 2006, DNA research : an international journal for rapid publication of reports on genes and genomes.

[41]  G. Goldman,et al.  The akuBKU80 Mutant Deficient for Nonhomologous End Joining Is a Powerful Tool for Analyzing Pathogenicity in Aspergillus fumigatus , 2006, Eukaryotic Cell.

[42]  J. Latgé,et al.  Glycosylphosphatidylinositol-anchored Fungal Polysaccharide in Aspergillus fumigatus* , 2005, Journal of Biological Chemistry.

[43]  M. Goto,et al.  Protein O-mannosyltransferase A of Aspergillus awamori is involved in O-mannosylation of glucoamylase I. , 2005, Microbiology.

[44]  J. Latgé,et al.  Galactomannoproteins of Aspergillus fumigatus , 2005, Eukaryotic Cell.

[45]  R. Gerardy-Schahn,et al.  Identification and partial characterization of two eukaryotic UDP-galactopyranose mutases , 2005, Biological chemistry.

[46]  C. Scazzocchio,et al.  Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi. , 2004, Fungal genetics and biology : FG & B.

[47]  M. Goto,et al.  Molecular characterization of protein O-mannosyltransferase and its involvement in cell-wall synthesis in Aspergillus nidulans. , 2004, Microbiology.

[48]  J. Peter-Katalinic,et al.  Beta-galactofuranose-containing O-linked oligosaccharides present in the cell wall peptidogalactomannan of Aspergillus fumigatus contain immunodominant epitopes. , 2003, Glycobiology.

[49]  M. Momany,et al.  Aspergillus nidulans polarity mutant swoA is complemented by protein O-mannosyltransferase pmtA. , 2002, Fungal genetics and biology : FG & B.

[50]  M. Goto,et al.  Effects of Amino Acid Alterations on the Transglycosylation Reaction of Endoglucanase I from Trichoderma viride HK-75 , 2002, Bioscience, biotechnology, and biochemistry.

[51]  A. Krogh,et al.  Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. , 2001, Journal of molecular biology.

[52]  H. Bussey,et al.  The KTR and MNN1 mannosyltransferase families of Saccharomyces cerevisiae. , 1999, Biochimica et biophysica acta.

[53]  K. Mariño,et al.  1-Thio-β-D-galactofuranosides: synthesis and evaluation as β-D-galactofuranosidase inhibitors , 1998 .

[54]  J. Latgé,et al.  Chemical and immunological characterization of the extracellular galactomannan of Aspergillus fumigatus , 1994, Infection and immunity.

[55]  S. Turco,et al.  Defective galactofuranose addition in lipophosphoglycan biosynthesis in a mutant of Leishmania donovani. , 1993, The Journal of biological chemistry.

[56]  S. Johnson,et al.  Simplified derivatization for determining sphingolipid fatty acyl composition by gas chromatography-mass spectrometry. , 1992, Journal of chromatography.

[57]  R. Lederkremer,et al.  Synthesis of p-nitrophenyl β-d-galactofuranoside. A convenient substrate for β-galactofuranosidase , 1986 .

[58]  Ionel Ciucanu,et al.  A simple and rapid method for the permethylation of carbohydrates , 1984 .

[59]  W. Timberlake,et al.  Transformation of Aspergillus nidulans by using a trpC plasmid. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[60]  K. Lloyd.  Isolation, characterization, and partial structure of peptido galactomannans from the yeast form of Cladosporium werneckii. , 1970, Biochemistry.

[61]  G. Johnson,et al.  Wild-type and mutant stocks of Aspergillus nidulans. , 1965, Genetics.

[62]  Y. Okawa,et al.  Significant structural change in both O- and N-linked carbohydrate moieties of the antigenic galactomannan from Aspergillus fumigatus grown under different culture conditions. , 2015, Glycobiology.

[63]  Y. Okawa,et al.  Chemical structure of beta-galactofuranose-containing polysaccharide and O-linked oligosaccharides obtained from the cell wall of pathogenic dematiaceous fungus Fonsecaea pedrosoi. , 2011, Glycobiology.

[64]  T. Lowary,et al.  Methods to study the biosynthesis of bacterial furanosides. , 2010, Methods in enzymology.

[65]  J. Latgé.  Galactofuranose containing molecules in Aspergillus fumigatus. , 2009, Medical mycology.

[66]  J. Errey,et al.  Expression and initial characterization of WbbI, a putative D-Galf:alpha-D-Glc beta-1,6-galactofuranosyltransferase from Escherichia coli K-12. , 2006, Organic & biomolecular chemistry.

[67]  E. M. Kellner,et al.  Mutations in sfdA and sfdB suppress multiple developmental mutations in Aspergillus nidulans. , 2002, Genetics.

[68]  J. Latgé,et al.  Aspergillus fumigatus cell wall: composition and biosynthesis. , 2001, Medical mycology.

[69]  K. Mariño,et al.  1-Thio-beta-D-galactofuranosides: synthesis and evaluation as beta-D-galactofuranosidase inhibitors. , 1998, Glycobiology.