CHARACTERIZATION OF PEPTIDE-N-4-(N-ACETYL-BETA-GLUCOSAMINYL)ASPARAGINE AMIDASE-A AND ITS N-GLYCANS

eptide-N-4-(N-acetyl-beta-glucosaminyl)aspaamidase A (PNGase A) was pu rified from almonds (Prunus amygdalus var. dulcis). Contrary to previo us 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-dimen sional electrophoresis. Peaks corresponding to molecular masses of 54. 2, 21.2 and 75.5 kDa were observed with matrix-assisted laser-desorpti on/ionization mass spectrometry. The N-terminal sequences of the large r and the smaller chain were determined to be LASGYHSWAD and EPTPLHDFP P, respectively. Both polypeptides reacted with concanavalin A, indica ting their glycoprotein nature. Upon digestion of PNGase with pepsin, the N-linked oligosaccharides were released with active PNGase and anl ysed as their 2-aminopyridine derivatives by two-dimensional HPLC and by matrix-assisted laser-desorption mass spectrometry. The most abunda nt N-glycan of the four species found exhibited the well known vacuole type structure, i.e. the pentasaccharide core with xylose and alpha 1 ,3-linked fucose. The other structures either had an additional mannos e residue and/or lacked the fucose. PNGase A was largely but not absol utely 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 .