Does an animal peptide:N-glycanase have the dual role as an enzyme and a carbohydrate-binding protein?

Abstract

Recently, we have reported purification and characterization of a de-N-glycosylating enzyme, peptide: N-glycanase (PNGase) found in C3H mouse fibroblast L-929 cells, and designated L-929 PNGase [Suzuki T, Seko A, Kitajima K, Inoue Y, Inoue S (1994) J Biol Chem 269, 17611-18]. The unique properties of L-929 PNGase are that the enzyme had a high affinity to the substrate glycopeptide (e.g. Km = 114 microM for fetuin derived glycopentapeptide) and that the PNGase-catalysed reaction is strongly inhibited by the released free oligosaccharides but not by the free peptides formed, suggesting that L-929 PNGase is able to bind to a certain type of carbohydrate chain. In this study, we report the new findings of the mannan-binding property of L-929 PNGase: the de-N-glycosylating enzyme activity of L-929 PNGase was inhibited by yeast mannan and triomannose, Man alpha 1-->3(Man alpha 1-->6)Man, but not by mannose and alpha-methyl-D-mannoside. Furthermore, L-929 PNGase was revealed to bind to the glycan moiety of yeast mannan by using mannan-conjugated Sepharose 4B gel as a ligand, suggesting that L-929 PNGase could serve not only as an enzyme but also as a carbohydrate recognition protein in vivo. Such 'dual' properties found for animal-derived L-929 PNGase are unique and are not shared with other previously characterized plant- and bacterial-origin PNGases--PNGase A and PNGase F, respectively.

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

“…4). It may be noted that we also found such dual properties in our previous studies on animal-derived L-929 PNGase (16). Thus, PNGase Os in multiple glycoforms may have distinct differences in enzymatic characteristics, which may also be related to its functional role during germination and early development.…”

“…lectin-like 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.…”

Structural and biochemical studies of the C-terminal domain of mouse peptide-N-glycanase identify it as a mannose-binding module

“…Early studies (11) revealed that mouse PNGase binds to free glycan chains derived from its glycoprotein substrates, and that this binding inhibits the activity of PNGase, thus suggesting that mouse PNGase has a carbohydrate-binding activity. Moreover, recent studies revealed that PNGase specifically acts on the unfolded form of high-mannose type N-glycosylated proteins (4,12,13).…”

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

“…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.…”

“…Assay for endo-␤-N-acetylglucosaminidase activity was carried out using [ 14 C]GP-IVD as a substrate (24). Activities for exoglycosidases were assayed according to the method of Li and Li (25) using the appropriate p-nitrophenylglycosides (KochLight Laboratories, Bucks, U.K.).…”