Demonstration of a peptide:N-glycosidase in the endoplasmic reticulum of rat liver.

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.