Galactofuranoic-oligomannose winked 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 copie s of the encoding aglA gene under the control of the glucoamylase (glaA) pr omoter. Endoglycosidase digestion followed by SDS/PAGE, lectin and immunobl otting suggested that glycosylation accounted for approximate to 25% of the molecular size of the purified protein. Monosaccharide analysis showed tha t this was composed of N-acetyl glucosamine, mannose and galactose. Mild ac id hydrolysis, mild methanolysis, immunoblotting and exoglycosidase digesti on indicated that the majority of the galactosyl component was in the furan oic conformation (beta -D-galactofuranose, Galf). At least 20 different N-l inked oligosaccharides were fractionated by high-pH anion-exchange chromato graphy following release from the polypeptide by peptide-N-glycosidase F. T he structures of these were subsequently determined by fast atom bombardmen t 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 u p to GlcNAc(2)Man(24) were present. Each of these were additionally substit uted with up to three beta -Galf residues. Linkage analysis confirmed the p resence of mild acid labile terminal hexofuranose residues. These results s how that filamentous fungi are capable of producing a heterogeneous mixture of high molecular-size N-linked glycans substituted with galactofuranoic r esidues, on a secreted glycoprotein.