Protein glycosylation is a common protein post-translational modification in eukaryotic cells. In endoplasmic reticulum (ER), some proteins fail to form native structures, and the misfolded glycoproteins are retrotranslocated into the cytosol, followed by ubiquitin-proteasome system. In cytosol, peptide:N-glycanase (PNGase) catalyzes hydrolysis of the amide bond between the innermost N-acetylglucosamine (GlcNAc) and Asn side-chain. Endo-ß-N-acetylglucosaminidase (ENGase) is another de-N-glycosylating enzyme but leaves a single GlcNAc at glycosylated Asn residues. Glycoproteins bearing N-glycans serve as the substrate for both PNGase and ENGase. However, it is unclear which enzymes act predominantly on the glycoprotein substrates. In the present study, we designed and synthesized nine glycopeptides, derived from Nrf1, with different peptide sequences bearing Man₃GlcNAc₂ pentasaccharide core, and examined their reactivities toward deglycosylating enzymes, PNGase and ENGase.

To synthesize the glycopeptides, Fmoc-Asn(Man₃GlcNAc₂)-OH was synthesized and introduced using (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) and 1-hydroxy-7-azabenzotriazole (HOAt). 5-carboxytetramethylrhodamine (TAMRA) was introduced at the N-termini of glycopeptides. Two HeLa cell lines that overexpress PNGase (HeLa-PNG) and ENGase (HeLa-ENG) were prepared using a HeLa cell line that does not express PNGase and ENGase, and the cytosolic fractions from HeLa-PNG and HeLa-ENG were used to examine the enzyme activities. All glycopeptides served as the substrates for ENGase. In contrast, PNGase exhibited the different reactivities of the glycopeptides. These findings suggested that PNGase may recognize not only oligosaccharide structure but also the peptide sequences.