The fungal cell wall is necessary for survival as it serves a barrier for physical protection. Therefore, glycosyltransferases responsible for the synthesis of cell wall polysaccharides may be suitable targets for drug development. Mannose is a monosaccharide that is commonly found in sugar chains in the walls of fungi. Mannose residues are present in fungal-type galactomannan, O-glycans, N-glycans, glycosylphosphatidylinositol anchors, and glycosyl inositol phosphorylceramides in Aspergillus fumigatus. Three genes that are homologous to α-(1→2)-mannosyltransferase genes and belong to the glycosyltransferase family 15 were found in the A. fumigatus strain, Af293/A1163, genome: cmsA/ktr4, cmsB/ktr7, and mnt1. It is reported that the mutant ∆mnt1 strain exhibited a wide range of properties that included high temperature and drug sensitivity, reduced conidia formation, leakage at the hyphal tips, and attenuation of virulence. However, it is unclear whether Mnt1 is a bona fide α-(1→2)-mannosyltransferase and which mannose residues are synthesized by Mnt1 in vivo. In this study, we elucidated the structure of the Mnt1 reaction product, the structure of O-glycan in the Δmnt1 strain. In addition, the length of N-glycans attached to invertase was evaluated in the Δmnt1 strain. The results indicated that Mnt1 functioned as an α-(1→2)-mannosyltransferase involved in the elongation of N-glycans and synthesis of the second mannose residue of O-glycans. The widespread abnormal phenotype caused by the disruption of the mnt1 gene is the combined result of the loss of mannose residues from O-glycans and N-glycans. We also clarified the enzymatic properties and substrate specificity of Mnt1 based on its predicted protein structure.

cell wall, glycosylation, Galactomannan, Mannosyltransferase, Aspergillus

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

OC[C@@H](O)[C@@H]1O[C@@H](O[C@H](CO)[C@@H]2O[C@@H](O[C@H](CO)[C@@H]3O[C@@H](O[C@H](CO)[C@@H]4O[C@@H](OC[C@@H](O)[C@@H]5O[C@@H](O[C@H](CO)[C@@H]6O[C@@H](O[C@H](CO)[C@@H]7O[C@@H](OC[C@H]8OC(O)[C@@H](O[C@H]9O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]9O)[C@@H](O)[C@@H]8O)[C@H](O)[C@H]7O)[C@H](O)[C@H]6O)[C@H](O)[C@H]5O)[C@H](O)[C@H]4O)[C@H](O)[C@H]3O)[C@H](O)[C@H]2O)[C@H](O)[C@H]1O

1477.284 g/mol (C₅₄H₉₂O₄₆)

Fungal-type galactomannan, a cell wall component of Aspergillus fumigatus, is composed of α-(1→2)-/α-(1→6)-linked mannan and β-(1→5)-/β-(1→6)-linked galactofuran side chains. Recently, CmsA and CmsB were identified as the α-(1→2)-mannosyltransferases involved in the biosynthesis of the α-core-mannan. However, the α-(1→6)-mannosyltransferase involved in the biosynthesis of the α-core-mannan has not been identified yet. In this study, we analyzed 9 putative α-(1→6)-mannosyltransferase gene disruption strains of A. fumigatus. The ΔanpA strain resulted in decreased mycelial elongation and reduced conidia formation. Proton nuclear magnetic resonance analysis revealed that the ΔanpA strain failed to produce the α-core-mannan of fungal-type galactomannan. We also found that recombinant AnpA exhibited much stronger α-(1→6)-mannosyltransferase activity toward α-(1→2)-mannobiose than α-(1→6)-mannobiose in vitro. Molecular simulations corroborated the fact that AnpA has a structure that can recognize the donor and acceptor substrates suitable for α-(1→6)-mannoside bond formation and that its catalytic activity would be specific for the elongation of the α-core-mannan structure in vivo. The identified AnpA is similar to Anp1p, which is involved in the elongation of the N-glycan outer chain in budding yeast, but the building sugar chain structure is different. The difference was attributed to the difference in substrate recognition of AnpA, which was clarified by simulations based on protein conformation. Thus, even proteins that seem to be functionally identical due to amino acid sequence similarity may be glycosyltransferase enzymes that make different glycans upon detailed analysis. This study describes an example of such a case. IMPORTANCE Fungal-type galactomannan is a polysaccharide incorporated into the cell wall of filamentous fungi belonging to the subphylum Pezizomycotina. Biosynthetic enzymes of fungal-type galactomannan are potential targets for antifungal drugs and agrochemicals. In this study, we identified an α-(1→6)-mannosyltransferase responsible for the biosynthesis of the α-core-mannan of fungal-type galactomannan, which has not been known for a long time. The findings of this study shed light on processes that shape this cellular structure while identifying a key enzyme essential for the biosynthesis of fungal-type galactomannan.

cell wall, galactofuranose, glycosyltransferase, Mannose, Galactomannan, Mannosyltransferase, Aspergillus fumigatus

13C NMR data:
missing...
1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
2,2,2	aDManp	5.108	?	?	?	?	?
2,2	aDManp	5.234	?	?	?	?	?
2	aDManp	5.216	?	?	?	?	?
	aDManp	5.057	?	?	?	?	?

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[*]OC[C@H]1O[C@H](O[C@H]2[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]2O[C@H]2[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]2O[C@H]2[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]2[*])[C@@H](O)[C@@H](O)[C@@H]1O

648.564 g/mol (C₂₄H₄₀R₂O₂₀, R = next and previous repeats, not counted in mol weight)

The fungal cell wall is necessary for survival as it serves a barrier for physical protection. Therefore, glycosyltransferases responsible for the synthesis of cell wall polysaccharides may be suitable targets for drug development. Mannose is a monosaccharide that is commonly found in sugar chains in the walls of fungi. Mannose residues are present in fungal-type galactomannan, O-glycans, N-glycans, glycosylphosphatidylinositol anchors, and glycosyl inositol phosphorylceramides in Aspergillus fumigatus. Three genes that are homologous to α-(1→2)-mannosyltransferase genes and belong to the glycosyltransferase family 15 were found in the A. fumigatus strain, Af293/A1163, genome: cmsA/ktr4, cmsB/ktr7, and mnt1. It is reported that the mutant ∆mnt1 strain exhibited a wide range of properties that included high temperature and drug sensitivity, reduced conidia formation, leakage at the hyphal tips, and attenuation of virulence. However, it is unclear whether Mnt1 is a bona fide α-(1→2)-mannosyltransferase and which mannose residues are synthesized by Mnt1 in vivo. In this study, we elucidated the structure of the Mnt1 reaction product, the structure of O-glycan in the Δmnt1 strain. In addition, the length of N-glycans attached to invertase was evaluated in the Δmnt1 strain. The results indicated that Mnt1 functioned as an α-(1→2)-mannosyltransferase involved in the elongation of N-glycans and synthesis of the second mannose residue of O-glycans. The widespread abnormal phenotype caused by the disruption of the mnt1 gene is the combined result of the loss of mannose residues from O-glycans and N-glycans. We also clarified the enzymatic properties and substrate specificity of Mnt1 based on its predicted protein structure.

cell wall, glycosylation, Galactomannan, Mannosyltransferase, Aspergillus

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@H]1[C@H](O[C@@H]2[C@@H](CO)O[C@@H](O)[C@H](NC(C)=O)[C@H]2O)O[C@H](CO)[C@@H](O[C@@H]2O[C@H](CO[C@H]3O[C@H](CO[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O)[C@@H](O)[C@H](O[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O)[C@@H]3O)[C@@H](O)[C@H](O[C@H]3O[C@H](CO[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O)[C@@H](O)[C@H](O)[C@@H]3O[C@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]3O)[C@@H]2O)[C@@H]1O

2532.236 g/mol (C₉₄H₁₅₈N₂O₇₆)

Fungal-type galactomannan, a cell wall component of Aspergillus fumigatus, is composed of α-(1→2)-/α-(1→6)-linked mannan and β-(1→5)-/β-(1→6)-linked galactofuran side chains. Recently, CmsA and CmsB were identified as the α-(1→2)-mannosyltransferases involved in the biosynthesis of the α-core-mannan. However, the α-(1→6)-mannosyltransferase involved in the biosynthesis of the α-core-mannan has not been identified yet. In this study, we analyzed 9 putative α-(1→6)-mannosyltransferase gene disruption strains of A. fumigatus. The ΔanpA strain resulted in decreased mycelial elongation and reduced conidia formation. Proton nuclear magnetic resonance analysis revealed that the ΔanpA strain failed to produce the α-core-mannan of fungal-type galactomannan. We also found that recombinant AnpA exhibited much stronger α-(1→6)-mannosyltransferase activity toward α-(1→2)-mannobiose than α-(1→6)-mannobiose in vitro. Molecular simulations corroborated the fact that AnpA has a structure that can recognize the donor and acceptor substrates suitable for α-(1→6)-mannoside bond formation and that its catalytic activity would be specific for the elongation of the α-core-mannan structure in vivo. The identified AnpA is similar to Anp1p, which is involved in the elongation of the N-glycan outer chain in budding yeast, but the building sugar chain structure is different. The difference was attributed to the difference in substrate recognition of AnpA, which was clarified by simulations based on protein conformation. Thus, even proteins that seem to be functionally identical due to amino acid sequence similarity may be glycosyltransferase enzymes that make different glycans upon detailed analysis. This study describes an example of such a case. IMPORTANCE Fungal-type galactomannan is a polysaccharide incorporated into the cell wall of filamentous fungi belonging to the subphylum Pezizomycotina. Biosynthetic enzymes of fungal-type galactomannan are potential targets for antifungal drugs and agrochemicals. In this study, we identified an α-(1→6)-mannosyltransferase responsible for the biosynthesis of the α-core-mannan of fungal-type galactomannan, which has not been known for a long time. The findings of this study shed light on processes that shape this cellular structure while identifying a key enzyme essential for the biosynthesis of fungal-type galactomannan.

cell wall, galactofuranose, glycosyltransferase, Mannose, Galactomannan, Mannosyltransferase, Aspergillus fumigatus

SMILES errors: /bDGalf(1-5)/n=4/bDGalf(1-6)/bDGalf(1-5)/n=3/bDGalf(1-2)[aDManp(1-2)[/bDGalf(1-5)/n=4/bDGalf(1-6)/bDGalf(1-5)/n=3/bDGalf(1-6)]aDManp(1-2)aDManp(1-2)aDManp(1-6)aDManp(1-2)aDManp(1-2)aDManp(1-2)aDManp(1-6)]aDManp(1-2)aDManp(1-2)aDManp(1-2)aDManp(1-6)aDManp(1-2)[/bDGalf(1-5)/n=4/bDGalf(1-6)/bDGalf(1-5)/n=3/bDGalf(1-3)]aDManp(1-2)aDManp(1-2)aDManp:
SMILES error: could not calculate brutto descriptors of a molecule from SMILES OC[C@@H](O)[C@@H]1O[C@@H](O[C@H](CO)[C@@H]2O[C@@H](O[C@H](CO)[C@@H]3O[C@@H](O[C@H](CO)[C@@H]4O[C@@H](O[C@H](CO)[C@@H]5O[C@@H](OC[C@@H](O)[C@@H]6O[C@@H](O[C@H](CO)[C@@H]7O[C@@H](O[C@H](CO)[C@@H]8O[C@@H](O[C@H](CO)[C@@H]9O[C@@H](O[C@H]%10[C@@H](O)[C@H](O)[C@@H](CO[C@H]%11O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]%11O[C@H]%11O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]%11O[C@H]%11O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]%11O[C@H]%11O[C@H](CO[C@H]%12O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]%12O[C@H]%12O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]%12O[C@H]%12O[C@H](CO[C@@H]%13O[C@@H]([C@@H](CO)O[C@@H]%14O[C@@H]([C@@H](CO)O[C@@H]%15O[C@@H]([C@@H](CO)O[C@@H]%16O[C@@H]([C@H](O)CO[C@@H]%17O[C@@H]([C@@H](CO)O[C@@H]%18O[C@@H]([C@@H](CO)O[C@@H]%19O[C@@H]([C@@H](CO)O[C@@H]%20O[C@@H]([C@@H](CO)O[C@@H]%21O[C@@H]([C@H](O)CO)[C@H](O)[C@H]%21O)[C@H](O)[C@H]%20O)[C@H](O)[C@H]%19O)[C@H](O)[C@H]%18O)[C@H](O)[C@H]%17O)[C@H](O)[C@H]%16O)[C@H](O)[C@H]%15O)[C@H](O)[C@H]%14O)[C@H](O)[C@H]%13O)[C@@H](O)[C@H](O)[C@@H]%12O[C@H]%12O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]%12O)[C@@H](O)[C@H](O)[C@@H]%11O)O[C@@H]%10O[C@H]%10[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]%10O[C@H]%10[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]%10O[C@@H]%10[C@@H](OC[C@H]%11O[C@H](O[C@H]%12[C@@H](O[C@@H]%13O[C@@H]([C@@H](CO)O[C@@H]%14O[C@@H]([C@@H](CO)O[C@@H]%15O[C@@H]([C@@H](CO)O[C@@H]%16O[C@@H]([C@H](O)CO[C@@H]%17O[C@@H]([C@@H](CO)O[C@@H]%18O[C@@H]([C@@H](CO)O[C@@H]%19O[C@@H]([C@@H](CO)O[C@@H]%20O[C@@H]([C@@H](CO)O[C@@H]%21O[C@@H]([C@H](O)CO)[C@H](O)[C@H]%21O)[C@H](O)[C@H]%20O)[C@H](O)[C@H]%19O)[C@H](O)[C@H]%18O)[C@H](O)[C@H]%17O)[C@H](O)[C@H]%16O)[C@H](O)[C@H]%15O)[C@H](O)[C@H]%14O)[C@H](O)[C@H]%13O)[C@H](O)[C@@H](CO)O[C@@H]%12O[C@H]%12[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]%12O[C@@H]%12[C@@H](O)O[C@H](CO)[C@@H](O)[C@@H]%12O)[C@@H](O)[C@@H](O)[C@@H]%11O)O[C@H](CO)[C@@H](O)[C@@H]%10O)[C@H](O)[C@H]9O)[C@H](O)[C@H]8O)[C@H](O)[C@H]7O)[C@H](O)[C@H]6O)[C@H](O)[C@H]5O)[C@H](O)[C@H]4O)[C@H](O)[C@H]3O)[C@H](O)[C@H]2O)[C@H](O)[C@H]1O
Invalid SMILES OC[C@@H](O)[C@@H]1O[C@@H](O[C@H](CO)[C@@H]2O[C@@H](O[C@H](CO)[C@@H]3O[C@@H](O[C@H](CO)[C@@H]4O[C@@H](O[C@H](CO)[C@@H]5O[C@@H](OC[C@@H](O)[C@@H]6O[C@@H](O[C@H](CO)[C@@H]7O[C@@H](O[C@H](CO)[C@@H]8O[C@@H](O[C@H](CO)[C@@H]9O[C@@H](O[C@H]%10[C@@H](O)[C@H](O)[C@@H](CO[C@H]%11O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]%11O[C@H]%11O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]%11O[C@H]%11O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]%11O[C@H]%11O[C@H](CO[C@H]%12O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]%12O[C@H]%12O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]%12O[C@H]%12O[C@H](CO[C@@H]%13O[C@@H]([C@@H](CO)O[C@@H]%14O[C@@H]([C@@H](CO)O[C@@H]%15O[C@@H]([C@@H](CO)O[C@@H]%16O[C@@H]([C@H](O)CO[C@@H]%17O[C@@H]([C@@H](CO)O[C@@H]%18O[C@@H]([C@@H](CO)O[C@@H]%19O[C@@H]([C@@H](CO)O[C@@H] O[C@@H]([C@@H](CO)O[C@@H]%21O[C@@H]([C@H](O)CO)[C@H](O)[C@H]%21O)[C@H](O)[C@H] O)[C@H](O)[C@H]%19O)[C@H](O)[C@H]%18O)[C@H](O)[C@H]%17O)[C@H](O)[C@H]%16O)[C@H](O)[C@H]%15O)[C@H](O)[C@H]%14O)[C@H](O)[C@H]%13O)[C@@H](O)[C@H](O)[C@@H]%12O[C@H]%12O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]%12O)[C@@H](O)[C@H](O)[C@@H]%11O)O[C@@H]%10O[C@H]%10[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]%10O[C@H]%10[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]%10O[C@@H]%10[C@@H](OC[C@H]%11O[C@H](O[C@H]%12[C@@H](O[C@@H]%13O[C@@H]([C@@H](CO)O[C@@H]%14O[C@@H]([C@@H](CO)O[C@@H]%15O[C@@H]([C@@H](CO)O[C@@H]%16O[C@@H]([C@H](O)CO[C@@H]%17O[C@@H]([C@@H](CO)O[C@@H]%18O[C@@H]([C@@H](CO)O[C@@H]%19O[C@@H]([C@@H](CO)O[C@@H] O[C@@H]([C@@H](CO)O[C@@H]%21O[C@@H]([C@H](O)CO)[C@H](O)[C@H]%21O)[C@H](O)[C@H] O)[C@H](O)[C@H]%19O)[C@H](O)[C@H]%18O)[C@H](O)[C@H]%17O)[C@H](O)[C@H]%16O)[C@H](O)[C@H]%15O)[C@H](O)[C@H]%14O)[C@H](O)[C@H]%13O)[C@H](O)[C@@H](CO)O[C@@H]%12O[C@H]%12[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]%12O[C@@H]%12[C@@H](O)O[C@H](CO)[C@@H](O)[C@@H]%12O)[C@@H](O)[C@@H](O)[C@@H]%11O)O[C@H](CO)[C@@H](O)[C@@H]%10O)[C@H](O)[C@H]9O)[C@H](O)[C@H]8O)[C@H](O)[C@H]7O)[C@H](O)[C@H]6O)[C@H](O)[C@H]5O)[C@H](O)[C@H]4O)[C@H](O)[C@H]3O)[C@H](O)[C@H]2O)[C@H](O)[C@H]1O