Occurrence and Potential Functions of<i>N</i>–Glycanases

Suzuki, Tadashi; Kitajima, Ken; Inoue, Sadako; Inoue, Yasuo

doi:10.1002/9783527614738.ch7

Cited by 10 publications

(20 citation statements)

References 65 publications

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“…All these arguments appear to concur that PNGase has yet undefined physiological role in plant and animal cells and to confirm our previous proposal that the N-glycosylation/de-Nglycosylation system should occur more commonly than presently recognized in living organisms (28,38,39). Possible involvement of PNGase in the processes that initiate and control the metabolic activities of plant and animal development by free oligosaccharides liberated from glycoproteins has been substantiated by their accumulation, although the underlying molecular mechanisms are in most cases unknown.…”

supporting

confidence: 67%

“…(d) In addition to possible function of generating free glycans, PNGase-catalyzed de-N-glycosylation of plant glycoproteins was proposed as a possible mechanism for regulating protein activity by removing N-glycans and converting glycosylated Asn residues to the Asp residues (9,28,38,39). A specific PNGase seems to have a specific function during early embryogenesis (e.g.…”

Section: Discussionmentioning

confidence: 99%

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Developmentally Regulated Expression of a Peptide:N-Glycanase during Germination of Rice Seeds (Oryza sativa) and Its Purification and Characterization

Chang

Kuo

Khoo

et al. 2000

Journal of Biological Chemistry

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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 SYN-VASVAGL. 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.-(N-acetyl-␤-Dglucosaminyl) asparagine amidase, EC 3.5.1.52) had only been known to occur in some plant seeds (1-5) 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). Following this discovery, we began to focus our interest on the physiological significance of this enzyme in living organisms because little attention had been paid to it. In view of the unique structural change in a given functional protein by converting the glycosylated asparagine residue to the aspartic acid residue upon de-N-glycosylation catalyzed by PNGase as the posttranslational remodification of protein, we anticipated that N-glycosylation of proteins by oligosaccharyltransferase and their de-N-glycosylation by PNGase constitute a basic biological mechanism of functioning within cells (8, 9). 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 not only a...

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supporting

confidence: 67%

Section: Discussionmentioning

confidence: 99%

Developmentally Regulated Expression of a Peptide:N-Glycanase during Germination of Rice Seeds (Oryza sativa) and Its Purification and Characterization

Chang

Kuo

Khoo

et al. 2000

Journal of Biological Chemistry

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“…PNGase-catalyzed reactions not only reverse the effect of N-glycosylation on proteins but also alter the primary sequence of the physiological cognate glycoproteins by converting glycosylated asparagine into aspartic acid residue(s) and thereby introduce negative charge(s) into the substrates. These alterations may possibly cause changes in the physicochemical properties of these proteins, such as microconformational alterations, thermodynamic stability, and solubility, as well as physiological properties such as susceptibility to proteases, affinity to receptor molecules, and modulation of bioactivities of precursor glycoproteins (10)(11)(12)(13)(14). Thus, de-N-glycosylation may be a biologically important posttranslational remodification of certain proteins and could occur more commonly than presently recognized.…”

mentioning

confidence: 99%

“…Specific radioactivity for asialo-[ 14 C]fetGP I was determined to be 7.0 ϫ 10 4 cpm͞nmol. 14 C-labeled glycoasparagine, 14 C-labeled Asn(Man 6 GlcNAc 2 ) (or [ 14 C]GP-IVD), derived from OVA was prepared as described (24). Specific radioactivity of this compound was 1.3 ϫ 10 5 cpm͞nmol.…”

mentioning

confidence: 99%

“…A second possibility is that the de-Nglycosylation process is a step involved in cytosolic degradation of aberrant glycoproteins. glycan chain, Leu-Asn(Man 3 Gal 3 GlcNAc 5 )-Asp-Ser-Arg, was prepared from fetal calf serum fetuin (Nacalai Tesque, Kyoto) and 14 C-labeled at the amino-terminal residue by reductive methylation as described (16,23). The 14 C-labeling experiment was carried out at the Radioisotope Centre, University of Tokyo.…”

mentioning

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Site-specific de- N -glycosylation of diglycosylated ovalbumin in hen oviduct by endogenous peptide: N -glycanase as a quality control system for newly synthesized proteins

Suzuki

Kitajima

Emori

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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Hen ovalbumin (OVA) is known to exist as a singly N-glycosylated form with a glycan chain on Asn-292 in egg white. Previous studies showed that di-N-glycosylated form of OVA [Di-OVA; CHO-Asn-292͞CHO-Asn-311 (CHO, N-glycan chain)], which has two N-glycan chains on Asn-292 and Asn-311, was expressed only transiently in hen oviduct. Di-OVA was not found in egg white, suggesting that this form cannot be secreted normally and may possibly be converted to mono-N-glycosylated OVA (CHO-Asn-292͞Asp-311) by the action of peptide:Nglycanase (PNGase) during synthesis and secretion. In this study, we have identified the putative PNGase activity in the homogenate of hen oviduct, purified 1,000-fold, and designated as PNGase HO. We examined the reactivity of Di-OVA to PNGase HO and found that this enzyme site-specifically cleaved off the glycan chain at Asn-311 to convert Di-OVA into the mono-N-glycosylated form (CHO-Asn-292͞Asp-311). In contrast, this enzyme was found not to act on the mono-Nglycosylated OVA (CHO-Asn-292͞Asn-311) found in egg white when it was tested as a substrate. The present findings support our view that de-N-glycosylation catalyzed by PNGase may be involved in quality control of newly synthesized proteins by converting its diglycosylated form into the mono-N-glycosylated form that can be secreted. However, the alternative possibility that de-N-glycosylation may trigger cytosolic degradation of the aberrantly glycosylated glycoprotein cannot be ruled out.

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Eukaryotic glycosylation: whim of nature or multipurpose tool?

Reuter¹,

Gabius²

1999

Cellular and Molecular Life Sciences (CMLS)

214

141

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Protein and lipid glycosylation is a ubiquitous phenomenon. The task of cataloguing the great structural variety of the glycan part has demanded considerable efforts over decades. This patient endeavor was imperative to discern the inherent rules of glycosylation which cannot affirm assumptions on a purely coincidental nature of this type of protein and lipid modification. These results together with theoretical considerations uncover a salient property of oligosaccharides. In comparison with amino acids and nucleotides, monosaccharides excel in their potential to serve as units of hardware for storing biological information. Thus, the view that glycan chains exclusively affect physiochemical properties of the conjugates is indubitably flawed. This original concept has been decisively jolted by the discovery of endogenous receptors (lectins) for distinct glycan epitopes which are as characteristic as a fingerprint or a signature for a certain protein (class) or cell type. Recent evidence documents that these binding proteins are even endowed with the capacity to select distinct low-energy conformers of the often rather flexible oligosaccharides, granting entry to a new level of regulation of ligand affinity by shifting conformer equilibria. The assessment of the details of this recognition by X-ray crystallography, nuclear magnetic resonance spectroscopy, microcalorimetry and custom-made derivatives is supposed to justify a guarded optimism in satisfying the need for innovative drug design in antiadhesion therapy, for example against viral or bacterial infections and unwanted inflammation. This review presents a survey of the structural aspects of glycosylation and of evidence to poignantly endorse the notion that carrier-attached glycan chains can partake in biological information transfer at the level of cell compartments, cells and organs.

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Occurrence and Potential Functions ofN–Glycanases

Cited by 10 publications

References 65 publications

Developmentally Regulated Expression of a Peptide:N-Glycanase during Germination of Rice Seeds (Oryza sativa) and Its Purification and Characterization

Developmentally Regulated Expression of a Peptide:N-Glycanase during Germination of Rice Seeds (Oryza sativa) and Its Purification and Characterization

Site-specific de- N -glycosylation of diglycosylated ovalbumin in hen oviduct by endogenous peptide: N -glycanase as a quality control system for newly synthesized proteins

Eukaryotic glycosylation: whim of nature or multipurpose tool?

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