Characterisation of peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase A and its N-glycans.
Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase A (PNGase A) was purified from almonds (Prunus amygdalus var. dulcis). Contrary to previous results in the literature, the enzyme appeared to be a heterodimer with subunits of 55 and 27 kDa when analysed by SDS/PAGE and two-dimensional electrophoresis. Peaks corresponding to molecular masses of 54.2, 21.2 and 75.5 kDa were observed with matrix-assisted laser-desorption/ionization mass spectrometry. The N-terminal sequences of the larger and the smaller chain were determined to be LASGYHSWAD and EPTPLHDFPP, respectively. Both polypeptides reacted with concanavalin A, indicating their glycoprotein nature. Upon digestion of PNGase with pepsin, the N-linked oligosaccharides were released with active PNGase and analysed as their 2-aminopyridine derivatives by two-dimensional HPLC and by matrix-assisted laser-desorption mass spectrometry. The most abundant N-glycan of the four species found exhibited the well known vacuole type structure, i.e. the pentasaccharide core with xylose and alpha1,3-linked fucose. The other structures either had an additional mannose residue and/or lacked the fucose. PNGase A was largely but not absolutely resistant to self-deglycosylation. However, only at an extremely high enzyme/substrate ratio, N-glycans released from PNGase A itself caused a detectable contamination of a PNGase digest of a glycopeptide.
Altmann
F
F
Institut für Chemie der Universität für Bodenkultur Wien, Austria. faltmann@edv2.boku.ac.at
Paschinger
K
K
Dalik
T
T
Vorauer
K
K
D000581:Amidohydrolases / Q000737:chemistry*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D019281:Dimerization; D006023:Glycoproteins / Q000737:chemistry; D006031:Glycosylation; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009754:Nuts / Q000201:enzymology*; D009844:Oligosaccharides / Q000032:analysis; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010940:Plant Proteins / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry*
D006023:Glycoproteins; D009844:Oligosaccharides; D010446:Peptide Fragments; D010940:Plant Proteins; D011134:Polysaccharides; D010434:Pepsin A; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase