Galactofuranoic‐oligomannose N‐linked glycans of α‐galactosidase A from Aspergillus niger

Extracellular α‐galactosidase A was purified from the culture filtrate of an over‐producing strain of Aspergillus niger containing multiple copies of the encoding aglA gene under the control of the glucoamylase ( glaA ) promoter. Endoglycosidase digestion followed by SDS/PAGE, lectin and immunoblotting suggested that glycosylation accounted for ≈ 25% of the molecular size of the purified protein. Monosaccharide analysis showed that this was composed of N ‐acetyl glucosamine, mannose and galactose. Mild acid hydrolysis, mild methanolysis, immunoblotting and exoglycosidase digestion indicated that the majority of the galactosyl component was in the furanoic conformation (β‐ d ‐galactofuranose, Gal f ). At least 20 different N ‐linked oligosaccharides were fractionated by high‐pH anion‐exchange chromatography following release from the polypeptide by peptide‐ N ‐glycosidase F. The structures of these were subsequently determined by fast atom bombardment mass spectrometry to be a linear series of Hex 7−26 HexHAc 2 . Indicating that oligosaccharides from GlcNAc 2 Man 7 , increasing in molecular size up to GlcNAc 2 Man 24 were present. Each of these were additionally substituted with up to three β‐Gal f residues. Linkage analysis confirmed the presence of mild acid labile terminal hexofuranose residues. These results show that filamentous fungi are capable of producing a heterogeneous mixture of high molecular‐size N ‐linked glycans substituted with galactofuranoic residues, on a secreted glycoprotein.