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.

摘要: Abstract 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 post-translational remodification proteins (Suzuki, T., Seko, A., Kitajima, K., Inoue, Y., S. (1993) Biochem. Biophys. Res. Commun. 194, 1124-1130). We report here 460-fold purification to homogeneity with 11.5% yield from crude extract C3H mouse-derived L-929 fibroblast cells. The purified designated as PNGase, had apparent molecular weight 212,000 was composed two 105,000 subunits. Although this capable hydrolyzing structurally diverse natural glycopeptide substrates bearing high mannose, hybrid, complex-type glycan units, activity completely inhibited by presence fucose residue either alpha-1-->3- or alpha-1-->6-linked proximal GlcNAc residue. showed maximal at pH near 7. This inability act on glycoasparagine strongly support our view would not be lysosomal degradation pathway. characterized having distinctly low Km value, which may physiological significance. Possible wide N-deglycosylation glycoproteins shown data bank survey protein sequences showing discrepancies between those determined directly (-D-X-(S/T)-) deduced cDNA sequencing (-N-X-(S/T)-). propose PNGase-catalyzed functionally important universal feature living