Endoplasmic Reticulum-associated Degradation of Mammalian Glycoproteins Involves Sugar Chain Trimming to Man6–5GlcNAc2

Zehavit Frenkel,Walter Gregory,Stuart Kornfeld,Gerardo Z Lederkremer

doi:10.1074/jbc.m305929200

Abstract

Endoplasmic reticulum-associated degradation of misfolded or misprocessed glycoproteins in mammalian cells is prevented by inhibitors of class I alpha-mannosidases implicating mannose trimming from the precursor oligosaccharide Glc3Man9GlcNAc2 as an essential step in this pathway. However, the extent of mannose removal has not been determined. We show here that glycoproteins subject to endoplasmic reticulum-associated degradation undergo reglucosylation, deglucosylation, and mannose trimming to yield Man6GlcNAc2 and Man5GlcNAc2. These structures lack the mannose residue that is the acceptor of glucose transferred by UDP-Glc:glycoprotein glucosyltransferase. This could serve as a mechanism for removal of the glycoproteins from folding attempts catalyzed by cycles of reglucosylation and calnexin/calreticulin binding and result in targeting of these molecules for proteasomal degradation.

Highlights

Endoplasmic reticulum-associated degradation of misfolded or misprocessed glycoproteins in mammalian cells is prevented by inhibitors of class I ␣-mannosidases implicating mannose trimming from the precursor oligosaccharide Glc3Man9GlcNAc2 as an essential step in this pathway
Mannose Trimming to M6 and M5 on an ERAD Substrate, asialoglycoprotein receptor (ASGPR) H2a—We studied ASGPR H2a as a model ERAD substrate glycoprotein
Mannose Trimming to M6 and M5 Depends on the Degree of Instability of the Glycoprotein and Not on Its endoplasmic reticulum (ER) Retention—We examined a glycoprotein that is retained in the ER but is much more stable than H2a to determine whether Man trimming to M6 and M5 can arise from ER retention unrelated to

Summary

Introduction

Endoplasmic reticulum-associated degradation of misfolded or misprocessed glycoproteins in mammalian cells is prevented by inhibitors of class I ␣-mannosidases implicating mannose trimming from the precursor oligosaccharide Glc3Man9GlcNAc2 as an essential step in this pathway. Whereas the inhibitor studies provide strong evidence that Man trimming is involved in ERAD, the structures of the N-linked oligosaccharides that result from trimming have not been determined in mammalian cells. M6 and M5, the result of the Man-trimming process, increased after 2 h of chase in the presence or absence of MG-132, and after 4 h they disappeared because of degradation of H2a, but proteasomal inhibition led to their accumulation.

Results

Conclusion