Identification of peptide:N-glycanase activity in mammalian-derived cultured cells.
The recent finding of peptide:N-glycanase (PNGase) in medaka embryos (Seko, A., Kitajima, K., Inoue, Y., & Inoue, S., J.Biol. Chem. 266, 22110 (1991)) raised the question of how widespread is the occurrence of this type of de-N-glycosylating enzyme. In experiments designed to identify PNGase in the mammalian system, we searched for its activity in some cultured cell lines. Incubation of a 14C-labeled N-glycopeptide with extracts prepared from cultured cells resulted in producing the free glycan and the peptide. Detailed characterizations of the products, formed upon incubation of a 14C-labeled N-glycopeptide substrate with the enzyme preparation from C3H mouse loose connective tissue-derived L-929 cells, by HPLC, amino acid and carbohydrate composition analyses, and peptide sequence analysis unequivocally established the reaction products to be the free glycan having di-N-acetylchitobiosyl sequence at its reducing end and free peptide in which the originally glycan-linked Asn residue was converted to the Asp residue. This represents the first demonstration of PNGase in mammalian cells and thus PNGase appears to be a very common enzyme expressed in not only plants and bacteria but also a wide range of animals although its functional significance remains to be clarified.
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 / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D003238:Connective Tissue / Q000201:enzymology*; D004187:Disaccharides; D005936:Glucans / Q000737:chemistry; D006020:Glycopeptides / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011134:Polysaccharides / Q000032:analysis
D004187:Disaccharides; D005936:Glucans; D006020:Glycopeptides; D011134:Polysaccharides; C032438:chitobiose; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase