Abstract
Prompted by previous observations that polymannose oligosaccharides are released from newly synthesized glycoproteins [Anumula and Spiro (1983) J. Biol. Chem. 258, 15274-15282], we examined rat liver endoplasmic reticulum (ER) for the presence of endoglycosidases that could be involved in an event presumed to be a function of the protein quality control machinery. Our investigations indicated that a peptide:N-glycanase (PNGase) is present in ER membranes that has the capacity to release from radiolabelled glycopeptides glucosylated as well as non-glucosylated polymannose oligosaccharides terminating at their reducing end in a di-N-acetylchitobiose sequence (OS-GlcNAc2). This enzyme, which was found to be luminal in orientation, was most active in the pH range 5.5-7.0 and although it had no exogenous bivalent-cation requirements it was inhibited by EDTA. Detailed studies with Man9GlcNAc2-peptides demonstrated that in addition to the free oligosaccharide (Man9GlcNAc2) an additional neutral product characterized as Man9GlcNAc2 linked to an as yet unidentified aglycone was released in a manner that suggests its role as an intermediate. Our observation that ER, in contrast with cytosol, had no endo-beta-N-acetylglucosaminidase activity would indicate that oligosaccharides terminating in a single GlcNAc residue (OS-GlcNAc1), which have been noted to appear in the extravesicular compartment shortly after N-glycosylation [Moore and Spiro (1994) J. Biol. Chem. 269, 12715-12721] are released from the protein as OS-GlcNAc2 and undergo an ER-to-cytosol translocation in that form before undergoing cleavage of their chitobiose core.
Full Text
The Full Text of this article is available as a PDF (347.1 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Anumula K. R., Spiro R. G. Release of glucose-containing polymannose oligosaccharides during glycoprotein biosynthesis. Studies with thyroid microsomal enzymes and slices. J Biol Chem. 1983 Dec 25;258(24):15274–15282. [PubMed] [Google Scholar]
- Cacan R., Lepers A., Belard M., Verbert A. Catabolic pathway of oligosaccharide-diphospho-dolichol. Subcellular sites of the degradation of the oligomannoside moiety. Eur J Biochem. 1989 Oct 20;185(1):173–179. doi: 10.1111/j.1432-1033.1989.tb15099.x. [DOI] [PubMed] [Google Scholar]
- Cacan R., Villers C., Bélard M., Kaiden A., Krag S. S., Verbert A. Different fates of the oligosaccharide moieties of lipid intermediates. Glycobiology. 1992 Apr;2(2):127–136. doi: 10.1093/glycob/2.2.127. [DOI] [PubMed] [Google Scholar]
- Chalifour R. J., Spiro R. G. Cleavage of dolichyl pyrophosphoryl oligosaccharides by endo-beta-N-acetylglucosaminidase H: comparison of enzymatic and acid hydrolysis techniques for saccharide release. Arch Biochem Biophys. 1984 Feb 15;229(1):386–394. doi: 10.1016/0003-9861(84)90166-8. [DOI] [PubMed] [Google Scholar]
- Daniel P. F., Winchester B., Warren C. D. Mammalian alpha-mannosidases--multiple forms but a common purpose? Glycobiology. 1994 Oct;4(5):551–566. doi: 10.1093/glycob/4.5.551. [DOI] [PubMed] [Google Scholar]
- Fiedler K., Simons K. The role of N-glycans in the secretory pathway. Cell. 1995 May 5;81(3):309–312. doi: 10.1016/0092-8674(95)90380-1. [DOI] [PubMed] [Google Scholar]
- Godelaine D., Spiro M. J., Spiro R. G. Processing of the carbohydrate units of thyroglobulin. J Biol Chem. 1981 Oct 10;256(19):10161–10168. [PubMed] [Google Scholar]
- Helenius A. How N-linked oligosaccharides affect glycoprotein folding in the endoplasmic reticulum. Mol Biol Cell. 1994 Mar;5(3):253–265. doi: 10.1091/mbc.5.3.253. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klausner R. D. Architectural editing: determining the fate of newly synthesized membrane proteins. New Biol. 1989 Oct;1(1):3–8. [PubMed] [Google Scholar]
- Lubas W. A., Spiro R. G. Golgi endo-alpha-D-mannosidase from rat liver, a novel N-linked carbohydrate unit processing enzyme. J Biol Chem. 1987 Mar 15;262(8):3775–3781. [PubMed] [Google Scholar]
- Makino M., Kojima T., Ohgushi T., Yamashina I. Studies on enzymes acting on glycopeptides. J Biochem. 1968 Feb;63(2):186–192. doi: 10.1093/oxfordjournals.jbchem.a128760. [DOI] [PubMed] [Google Scholar]
- Makino M., Kojima T., Yamashina I. Enzymatic cleavage of glycopeptides. Biochem Biophys Res Commun. 1966 Sep 22;24(6):961–966. doi: 10.1016/0006-291x(66)90344-5. [DOI] [PubMed] [Google Scholar]
- Maley F., Trimble R. B., Tarentino A. L., Plummer T. H., Jr Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. Anal Biochem. 1989 Aug 1;180(2):195–204. doi: 10.1016/0003-2697(89)90115-2. [DOI] [PubMed] [Google Scholar]
- Moore S. E., Bauvy C., Codogno P. Endoplasmic reticulum-to-cytosol transport of free polymannose oligosaccharides in permeabilized HepG2 cells. EMBO J. 1995 Dec 1;14(23):6034–6042. doi: 10.1002/j.1460-2075.1995.tb00292.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Moore S. E., Spiro R. G. Demonstration that Golgi endo-alpha-D-mannosidase provides a glucosidase-independent pathway for the formation of complex N-linked oligosaccharides of glycoproteins. J Biol Chem. 1990 Aug 5;265(22):13104–13112. [PubMed] [Google Scholar]
- Moore S. E., Spiro R. G. Intracellular compartmentalization and degradation of free polymannose oligosaccharides released during glycoprotein biosynthesis. J Biol Chem. 1994 Apr 29;269(17):12715–12721. [PubMed] [Google Scholar]
- Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
- Pierce R. J., Spik G., Montreuil J. Cytosolic location of an endo-N-acetyl-beta-D-glucosaminidase activity in rat liver and kidney. Biochem J. 1979 Jun 15;180(3):673–676. doi: 10.1042/bj1800673. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Risley J. M., Van Etten R. L. 1H NMR evidence that almond "peptide: N-glycosidase" is an amidase. Kinetic data and trapping of the intermediate. J Biol Chem. 1985 Dec 15;260(29):15488–15494. [PubMed] [Google Scholar]
- Spiro M. J., Spiro R. G., Bhoyroo V. D. Glycosylation of proteins by oligosaccharide-lipids. Studies on a thyroid enzyme involved in oligosaccharide transfer and the role of glucose in this reaction. J Biol Chem. 1979 Aug 25;254(16):7668–7674. [PubMed] [Google Scholar]
- Spiro M. J., Spiro R. G., Bhoyroo V. D. Lipid-saccharide intermediates in glycoprotein biosynthesis. I. Formation of an oligosaccharide-lipid by thyroid slices and evaluation of its role in protein glycosylation. J Biol Chem. 1976 Oct 25;251(20):6400–6408. [PubMed] [Google Scholar]
- Spiro M. J., Spiro R. G. Effect of anion-specific inhibitors on the utilization of sugar nucleotides for N-linked carbohydrate unit assembly by thyroid endoplasmic reticulum vesicles. J Biol Chem. 1985 May 10;260(9):5808–5815. [PubMed] [Google Scholar]
- Spiro M. J., Spiro R. G. Potential regulation of N-glycosylation precursor through oligosaccharide-lipid hydrolase action and glucosyltransferase-glucosidase shuttle. J Biol Chem. 1991 Mar 15;266(8):5311–5317. [PubMed] [Google Scholar]
- Spiro R. G., Bhoyroo V. D. Occurrence of sulfate in the asparagine-linked complex carbohydrate units of thyroglobulin. Identification and localization of galactose 3-sulfate and N-acetylglucosamine 6-sulfate residues in the human and calf proteins. J Biol Chem. 1988 Oct 5;263(28):14351–14358. [PubMed] [Google Scholar]
- Spiro R. G., Zhu Q., Bhoyroo V., Söling H. D. Definition of the lectin-like properties of the molecular chaperone, calreticulin, and demonstration of its copurification with endomannosidase from rat liver Golgi. J Biol Chem. 1996 May 10;271(19):11588–11594. doi: 10.1074/jbc.271.19.11588. [DOI] [PubMed] [Google Scholar]
- Suzuki T., Kitajima K., Inoue Y., Inoue S. Carbohydrate-binding property of peptide: N-glycanase from mouse fibroblast L-929 cells as evaluated by inhibition and binding experiments using various oligosaccharides. J Biol Chem. 1995 Jun 23;270(25):15181–15186. doi: 10.1074/jbc.270.25.15181. [DOI] [PubMed] [Google Scholar]
- Tarentino A. L., Maley F. The purification and properties of a beta-aspartyl N-acetylglucosylamine amidohydrolase from hen oviduct. Arch Biochem Biophys. 1969 Mar;130(1):295–303. doi: 10.1016/0003-9861(69)90036-8. [DOI] [PubMed] [Google Scholar]
- Villers C., Cacan R., Mir A. M., Labiau O., Verbert A. Release of oligomannoside-type glycans as a marker of the degradation of newly synthesized glycoproteins. Biochem J. 1994 Feb 15;298(Pt 1):135–142. doi: 10.1042/bj2980135. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weng S., Spiro R. G. Demonstration that a kifunensine-resistant alpha-mannosidase with a unique processing action on N-linked oligosaccharides occurs in rat liver endoplasmic reticulum and various cultured cells. J Biol Chem. 1993 Dec 5;268(34):25656–25663. [PubMed] [Google Scholar]
- Weng S., Spiro R. G. Endoplasmic reticulum kifunensine-resistant alpha-mannosidase is enzymatically and immunologically related to the cytosolic alpha-mannosidase. Arch Biochem Biophys. 1996 Jan 1;325(1):113–123. doi: 10.1006/abbi.1996.0014. [DOI] [PubMed] [Google Scholar]