title: 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.
abstract: 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-292/CHO-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-292/Asp-311) by the action of peptide:N-glycanase (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-292/Asp-311). In contrast, this enzyme was found not to act on the mono-N-glycosylated OVA (CHO-Asn-292/Asn-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.
authors.affiliation: Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Tokyo 113, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Suzuki
authors.forename: K
authors.initials: K
authors.lastname: Kitajima
authors.forename: Y
authors.initials: Y
authors.lastname: Emori
authors.forename: Y
authors.initials: Y
authors.lastname: Inoue
authors.forename: S
authors.initials: S
authors.lastname: Inoue
mesh_terms: D000581:Amidohydrolases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001216:Asparagine; D002645:Chickens; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D010450:Endopeptidases / Q000378:metabolism; D005260:Female; D006020:Glycopeptides / Q000737:chemistry / Q000302:isolation & purification; D006023:Glycoproteins / Q000096:biosynthesis*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D010047:Ovalbumin / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D010057:Oviducts / Q000378:metabolism*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011786:Quality Control; D013379:Substrate Specificity
chemical_list: D006020:Glycopeptides; D006023:Glycoproteins; D001216:Asparagine; D010047:Ovalbumin; D006026:Glycoside Hydrolases; D010450:Endopeptidases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase