Galactofuranoic‐oligomannose N‐linked glycans of α‐galactosidase A from Aspergillus niger
2001
Wallis, Gregg L. F. | Easton, Richard L. | Jolly, Karen | Hemming, Frank W. | Peberdy, John F.
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, Galf). 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 Hex7−26HexHAc2. Indicating that oligosaccharides from GlcNAc2Man7, increasing in molecular size up to GlcNAc2Man24 were present. Each of these were additionally substituted with up to three β‐Galf 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.
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