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 nonglucosylated polymannose oligosaccharides terminating at their reducing end in a di-N-acetylchitobiose sequence (OS-GlcNAc(2)). 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 Man(9)GlcNAc(2)-peptides demonstrated that in addition to the free oligosaccharide (Man(9)GlcNAc(2)) an additional neutral product characterized as Man(9)GlcNAc(2) 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-GlcNAc(1)), 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-GlcNAc, and undergo an ER-to-cytosol translocation in that form before undergoing cleavage of their chitobiose core.