Purification and enzymatic properties of peptide:N-glycanase from C3H mouse-derived L-929 fibroblast cells. Possible widespread occurrence of post-translational remodification of proteins by N-deglycosylation.
Recently, we found the occurrence of N-deglycosylating enzyme, peptide:N-glycanase (PNGase), in mammalian cells and observed that PNGase is a rather common enzyme involved in post-translational remodification of proteins (Suzuki, T., Seko, A., Kitajima, K., Inoue, Y., and Inoue, S. (1993) Biochem. Biophys. Res. Commun. 194, 1124-1130). We report here a 460-fold purification to homogeneity with 11.5% yield of PNGase from crude extract of C3H mouse-derived L-929 fibroblast cells. The purified enzyme, designated as L-929 PNGase, had the apparent molecular weight of 212,000 and was composed of two 105,000 subunits. Although this enzyme was capable of hydrolyzing structurally diverse natural glycopeptide substrates bearing high mannose, hybrid, and complex-type glycan units, the activity was completely inhibited by the presence of the fucose residue either alpha-1-->3- or alpha-1-->6-linked to the proximal GlcNAc residue. The enzyme showed maximal activity at pH near 7. This and the inability to act on glycoasparagine strongly support our view that this enzyme would not be involved in lysosomal degradation pathway. L-929 PNGase was characterized by having distinctly a low Km value, which may be of physiological significance. Possible wide occurrence of N-deglycosylation of glycoproteins was shown by a data bank survey of the protein sequences showing discrepancies between those determined directly (-D-X-(S/T)-) and those deduced from cDNA sequencing (-N-X-(S/T)-). We propose here that PNGase-catalyzed N-deglycosylation is a functionally important universal feature in living cells.
Suzuki
T
T
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
Seko
A
A
Kitajima
K
K
Inoue
Y
Y
Inoue
S
S
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D004229:Dithiothreitol / Q000494:pharmacology; D004795:Enzyme Stability; D005347:Fibroblasts / Q000201:enzymology; D006031:Glycosylation; D006358:Hot Temperature; D051379:Mice; D008809:Mice, Inbred C3H; D008969:Molecular Sequence Data; D008970:Molecular Weight; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity
D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D004229:Dithiothreitol