Molecular cloning, primary structure, and properties of a new glycoamidase from the fungus Aspergillus tubigensis.
A new glycoamidase, peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase (PNGase) At, was discovered in the eukaryote Aspergillus tubigensis. The enzyme was purified to homogeneity, and the DNA sequence was determined by cloning in Escherichia coli. Over 80% of the deduced amino acid sequence was verified independently by Edman analysis and/or electrospray ionization-mass spectrometry of protease fragments of native PNGase At. This glycoamidase contains 12 potential asparagine-linked glycosylation sites, of which at least 9 sites are occupied with typical high mannose oligosaccharides. PNGase At consists of two non-identical glycosylated subunits that are derived from a single polypeptide gene precursor. Evidence is presented suggesting that autocatalysis is involved in subunit formation. PNGase At is an important new tool for analysis of asparagine-linked glycans; it can hydrolyze a broad range of glycopeptides, including those with core-linked alpha1-->6 or alpha1-->3 fucose, under conditions not favorable with existing glycoamidases.
Ftouhi-Paquin
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Division of Molecular Medicine, Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201-0509, USA. ftouhi@wadsworth.org
Hauer
C R
CR
Stack
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Tarentino
A L
AL
Plummer
T H
TH
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011134:Polysaccharides / Q000737:chemistry
D018076:DNA, Complementary; D011134:Polysaccharides