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.
Wallis
G L
GL
School of Biological Sciences, University of Nottingham, Nottingham UK. gwallis@meristem-therapeutics.com
Easton
R L
RL
Jolly
K
K
Hemming
F W
FW
Peberdy
J F
JF
D001234:Aspergillus niger / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005690:Galactose / Q000031:analogs & derivatives / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D037102:Lectins / Q000737:chemistry; D008358:Mannose / Q000737:chemistry*; D013058:Mass Spectrometry; D009005:Monosaccharides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry*; D011401:Promoter Regions, Genetic; D013997:Time Factors; D000519:alpha-Galactosidase / Q000737:chemistry* / Q000235:genetics
D037102:Lectins; D009005:Monosaccharides; D010455:Peptides; D011134:Polysaccharides; D000519:alpha-Galactosidase; D008358:Mannose; D005690:Galactose