title: Molecular cloning, primary structure, and properties of a new glycoamidase from the fungus Aspergillus tubigensis.
abstract: 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.
authors.affiliation: 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
authors.forename: N
authors.initials: N
authors.lastname: Ftouhi-Paquin
authors.forename: C R
authors.initials: CR
authors.lastname: Hauer
authors.forename: R F
authors.initials: RF
authors.lastname: Stack
authors.forename: A L
authors.initials: AL
authors.lastname: Tarentino
authors.forename: T H
authors.initials: TH
authors.lastname: Plummer
mesh_terms: 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
chemical_list: D018076:DNA, Complementary; D011134:Polysaccharides