Galactofuranoic-oligomannose N-linked glycans of alpha-galactosidase A from Aspergillus niger

Extracellular alpha-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 approximately 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 (beta-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 Hex(7-26)HexHA(c2). Indicating that oligosaccharides from GlcNA(c2)Man(7), increasing in molecular size up to GlcNA(c2)Man(24) were present. Each of these were additionally substituted with up to three beta-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.