title: Analysis of ER-associated glycoprotein degradation using synthetic glycopeptide probes.
abstract: Quality control of proteins is an essential process for maintaining normal cell activity. It ensures that only correctly folded proteins are produced and terminally misfolded proteins are eliminated by degradation. ER-associated degradation (ERAD) of misfolded proteins is an important aspect of protein quality control system. Recent studies have revealed that glycoprotein glycans play significant roles in this process. It includes polyubiquitination, deglycosylation, and proteasomal degradation. In the present study, a systematic analysis of these steps was carried out using chemically synthesized glycopeptides. We revealed that N-linked glycopeptides are degraded by 20S proteasome, but with drastically reduced rate compared to non-glycosylated peptide. This result strongly suggests that deglycosylating activity of peptide:N-glycanase (PNGase) is important for the facile degradation of glycoproteins. Our study showed, for the first time, that PNGase cleaves truncated glycans as short as chitobiose from peptide. However, this cleavage required the presence of hydrophobic region nearby N-glycosylation site. Furthermore, analysis of interactions with F-box protein Fbs1 was conducted with fluorescent correlation spectroscopy (FCS). It was shown that the presence of Fbs1 perturb the activity of PNGase toward high-mannose-type glycopeptides.
authors.affiliation: RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Hagihara
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Goda
authors.forename: Ichiro
authors.initials: I
authors.lastname: Matsuo
authors.forename: Yukishige
authors.initials: Y
authors.lastname: Ito
mesh_terms: D000818:Animals; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006020:Glycopeptides / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D015336:Molecular Probe Techniques; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011817:Rabbits
chemical_list: D006020:Glycopeptides; D006023:Glycoproteins; D046988:Proteasome Endopeptidase Complex; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase