PURIFICATION AND ENZYMATIC-PROPERTIES OF PEPTIDE-N-GLYCANASE FROM C3HMOUSE-DERIVED L-929 FIBROBLAST CELLS - POSSIBLE WIDESPREAD OCCURRENCEOF POSTTRANSLATIONAL, 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 o f proteins (Suzuki, T., Seko, A., Kitajima, K., Inoue, Y., and Inoue, S. (1993) Biochem. Biophys. Res. Commun. 194, 1124-1130). We report he re a 460-fold purification to homogeneity with 11.5% yield of PNGase f rom crude extract of C3H mouse-derived L-929 fibroblast cells, The pur ified enzyme, designated as L-929 PNGase, had the apparent molecular w eight of 212,000 and was composed of two 105,000 subunits. Although th is enzyme was capable of hydrolyzing structurally diverse natural glyc opeptide substrates bearing high mannose, hybrid, and complex-type gly can units, the activity was completely inhibited by the presence of th e fucose residue either (alpha-1-->3- or alpha-1-->6-linked to the pro ximal 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 K-m value, which may be of physiological significance. Possible wide occu rrence of N-deglycosylation of glycoproteins was shown by a data bank survey of the protein sequences showing discrepancies between those de termined 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.