N-Linked Glycosylation Does Not Impair Proteasomal Degradation but Affects Class I Major Histocompatibility Complex Presentation

Abstract

The addition of N-linked glycans to nascent polypeptides occurs cotranslationally in the endoplasmic reticulum (ER). For many proteins the state of the glycans serves as an indicator, which allows the ER quality control system to monitor the conformation of polypeptides upon folding. Proteins that fail to fold in the ER are often dislocated to the cytoplasm, where they are subjected to proteasomal degradation. Although the addition of N-linked glycans occurs within the ER, non-lysosomal removal of the glycans occurs in the cytosol by the action of peptide N-glycanase (PNGase). In this study, we investigated the interplay between PNGase action and proteasomal degradation of ER misfolded proteins (i.e. whether PNGase acts prior to or following proteasomal degradation). Interestingly, we found that glycan removal from N-terminally extended peptides modulates the presentation of class I major histocompatibility complex-restricted epitopes. Our findings provide direct evidence that the proteasome is capable of degrading glycoproteins without prior removal of their glycans. This degradation is independent of either the identity of the glycosylated protein or the type and number of N-linked glycans it harbors. We also captured and characterized glycopeptides generated following proteasomal degradation of RNaseB. Although the carbohydrate moiety reduced the variability of the degradation products that include the glycosylated residue (local effect), the overall global digestion pattern of RNaseB was unaffected. Together with earlier findings by others, our data support a model in which PNGase may act both upstream and downstream to proteasomal degradation and demonstrates its important role in class I major histocompatibility complex antigen presentation.

Dislocation of HMG-CoA Reductase and Insig-1, Two Polytopic Endoplasmic Reticulum Proteins, En Route to Proteasomal Degradation

“…Interestingly, inhibiting PNGase with Z-VAD-fmk affected neither dislocation of HMG 350 -HA (Figure 3) nor degradation of HMGR (data not shown). This is in line with the reported ability of the proteasome to degrade glycoproteins without prior removal of their N-glycans (Kario et al, 2007).…”

Melanoma cells present high levels of HLA‐A2‐tyrosinase in association with instability and aberrant intracellular processing of tyrosinase

“…For example, the proteasome may trim proteins with different efficiencies based on their characteristics. This notion is in agreement with studies demonstrating that glycosylation of proteins [18], and specifically of tyrosinase [14], influences the trimming pattern by the proteasome and determines the digestion characteristics and the availability of peptides for presentation by MHC class I molecules. The efficiency of generating peptides derived from unstable proteins should be further investigated.…”

TheN-Glycanasepng-1Acts to Limit Axon Branching during Organ Formation in Caenorhabditis elegans

“…Several in vitro studies using human cell lines point to a role for PNGases in the proteasomal-dependent turnover or processing of glycoproteins or glycopeptides that originate in the ER and are retrotranslocated to the cytosol. These roles include the catabolism of misfolded or aberrant glycoproteins during ERAD (Hirsch et al, 2003;Blom et al, 2004;Kim et al, 2006) and the cytoplasmic processing of glycosylated peptide fragments to generate MHC class I epitopes (Selby et al, 1999;Altrich-VanLith et al, 2006;Kario et al, 2008). However, the developmental and physiological consequences of PNGase deficiency in animal models await the phenotypic analysis of knock-out mutants.…”

“…ERADtargeted glycoproteins are catabolized in the cytosol by a combination of PNGase-mediated deglycosylation and degradation by the proteasome (Hirsch et al, 2003;Blom et al, 2004;Kim et al, 2006). In mammalian cells, PNGases have also been implicated in the cytoplasmic processing of N-glycosylated peptide fragments into epitopes that maximize major histocompatibility complex (MHC) class I cell-surface antigenicity (Selby et al, 1999;AltrichVanLith et al, 2006;Kario et al, 2008).…”