Abstract
Peptide:N-glycanase (PNGase; EC 3.5.1.52) activity was detected in dormant rice seeds (Oryza sativa) and the imbibed rice grains. Time-course studies revealed that the enzyme activity remained almost constant until about 30 h after imbibition in both of endosperm- and embryo tissue-containing areas, and started to increase only in growing germ part, reached a peak at about 3-day stage, followed by a gradual decrease concomitant with a sharp increase in the coleoptile. The specific activity increased about 6-fold at about 3-day stage. PNGase was purified to electrophoretic homogeneity from the extracts of germinated rice seeds at 24 h, and the apparent molecular weight of the purified enzyme, estimated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), was about 80,000. The purified enzyme was designated PNGase Os to denote its origin. The N-terminal sequence of the 10 residues was determined to be SYNVASVAGL. The purified PNGase Os in SDS-PAGE appeared as a rather broad band, consistent with the presence of multiple glycoforms as indicated by chromatographic behavior on a Sephadex G-75 column. PNGase expressed in coleoptile under anoxia condition was also purified, and both of the purified enzymes were found to exhibit very similar, if not identical, electrophoretic mobility in SDS-PAGE. PNGase Os exhibited a broad pH-activity profile with an optimum of 4-5 and, interestingly, was significantly inactivated by K(+) and Na(+) at near the physiological concentration, 100 mM. These results are discussed in relation to other work.
Highlights
Peptide:N-glycanase (PNGase1; peptide-N4-(N-acetyl--Dglucosaminyl) asparagine amidase, EC 3.5.1.52) had only been known to occur in some plant seeds [1,2,3,4,5] and bacteria [6] and used as a useful reagent in a number of studies of structure and function of glycoproteins having N-linked glycan chains until we demonstrated for the first time its occurrence in the early embryos of Medaka fish, Oryzias latipes in 1991 [7]
For understanding the biological meaning of the occurrence of PNGase in eukaryotes, we have been involved in a series of studies on animal-derived PNGases (10 –15) and reported the following. (a) PNGase activities were shown to occur in mammalianderived cell lines including human origin [10]. (b) PNGase activities were detected ubiquitously in various organs and tissues of mouse [13]. (c) PNGase was purified to homogeneity from the confluent stage of C3H mouse fibroblast L-929 cells and characterized to serve as an enzyme and as a carbohydrate recognition protein [11, 12]. Such “dual” properties found for animal-derived L-929 PNGase are unique and are not shared with other previously characterized plant and bacterial PNGases, PNGase A and PNGase F, respectively [16]. (d) We have identified both PNGase activity and its physiological substrate in hen oviduct, and the enzyme was suggested to be involved in a “quality control” mechanism of the newly synthesized ovalbumin by site-specific de-N-glycosylation of diglycosylated ovalbumin in hen oviduct (Ref. 12; see Ref. 14). (e) We were successful in identifying two discrete PNGases in Oryzias latipes during embryogenesis, and their physiological roles were discussed [15]
Free oligosaccharides having di-N-acetylchitobiosyl structural element at their reducing termini were presumably formed by the action of PNGase, and those having a single residue of Nacetylglucosamine at the reducing termini are considered to be generated either by a direct action of endo--N-acetylglucosaminidase (EC 3.2.1.96) on the protein-conjugated N-glycan chains or by its action on the free glycan products of PNGase catalysis
Summary
The thawed coleoptiles were cut into pieces and quickly homogenized by a Polytron at 4 °C in 5 ml of the buffer containing 20 mM sodium acetate (pH 4.7), 2 mM DTT and a complete set of proteinase inhibitors (Roche Molecular Biochemicals, without EDTA) Use of this medium facilitated disruption of cells, and using of a Centriprep particle separator equipped with membrane of pore size 0.2 m (Amicon), the material inside the cell cytosol and most of the suborganelles were washed out, and cell wall fibers were left in the bottom. The medium was collected by high-speed centrifugation at 15,000 ϫ g for 10 min All of these procedures were repeated three times, and the supernatants were combined and concentrated by an Amicon concentrator with membrane pore size of 30 kDa. The pellet was extracted with the enzyme assay buffer three times, and the pellet was digested by pectinase and cellulase in a similar manner as described for the cell wall fibers in the filtration method. The glycopeptides were treated with the purified PNGase Os, and the free oligosaccharide products were separated from peptide fragments by passage through a Sep-Pak
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