DEMONSTRATION OF A PEPTIDE - N-GLYCOSIDASE IN THE ENDOPLASMIC-RETICULUM OF RAT-LIVER

Prompted by previous observations that polymannose oligosaccharides ar e released from newly synthesized glycoproteins [Anumula and Spiro (19 83) J. Biol. Chem. 258, 15274-15282], we examined rat liver endoplasmi c reticulum (ER) for the presence of endoglycosidases that could be in volved in an event presumed to be a function of the protein quality co ntrol machinery. Our investigations indicated that a peptide:N-glycana se (PNGase) is present in ER membranes that has the capacity to releas e from radiolabelled glycopeptides glucosylated as well as nonglucosyl ated polymannose oligosaccharides terminating at their reducing end in a di-N-acetylchitobiose sequence (OS-GlcNAc(2)). This enzyme, which w as 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)-pepti des demonstrated that in addition to the free oligosaccharide (Man(9)G lcNAc(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 activ ity would indicate that oligosaccharides terminating in a single GlcNA c residue (OS-GlcNAc(1)), which have been noted to appear in the extra vesicular compartment shortly after N-glycosylation [Moore and Spiro ( 1994) J. Biol. Chem. 269, 12715-12721] are released from the protein a s OS-GlcNAc, and undergo an ER-to-cytosol translocation in that form b efore undergoing cleavage of their chitobiose core.