EDEM2 stably disulfide-bonded to TXNDC11 catalyzes the first mannose trimming step in mammalian glycoprotein ERAD

Ginto George,Tetsuya Okada,Yasushi Ishihama,Koshi Imami,Taiki Saito,Tetsushi Sakuma,Takashi Yamamoto,Hirokazu Yagi,Satoshi Ninagawa,Tokiro Ishikawa,Kazutoshi Mori,Koichi Kato

doi:10.7554/elife.53455

Abstract

Sequential mannose trimming of N-glycan (Man9GlcNAc2 -> Man8GlcNAc2 -> Man7GlcNAc2) facilitates endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD). Our gene knockout experiments in human HCT116 cells have revealed that EDEM2 is required for the first step. However, it was previously shown that purified EDEM2 exhibited no α1,2-mannosidase activity toward Man9GlcNAc2 in vitro. Here, we found that EDEM2 was stably disulfide-bonded to TXNDC11, an endoplasmic reticulum protein containing five thioredoxin (Trx)-like domains. C558 present outside of the mannosidase homology domain of EDEM2 was linked to C692 in Trx5, which solely contains the CXXC motif in TXNDC11. This covalent bonding was essential for mannose trimming and subsequent gpERAD in HCT116 cells. Furthermore, EDEM2-TXNDC11 complex purified from transfected HCT116 cells converted Man9GlcNAc2 to Man8GlcNAc2(isomerB) in vitro. Our results establish the role of EDEM2 as an initiator of gpERAD, and represent the first clear demonstration of in vitro mannosidase activity of EDEM family proteins.

Highlights

Failure in protein folding in the endoplasmic reticulum (ER) detrimentally affects the maturation of secretory and transmembrane proteins, which comprise one-third of total proteins synthesized in mammalian cells
Human EDEM2 contains a total of eight cysteine residues, among which four are localized in regions conserved with yeast Htm1p (Figure 1B, shown with black bars)
We mutated each cysteine residue of EDEM2 to alanine and examined the resulting effect on degradation of the ER-associated degradation (ERAD)-Ls substrate mCD3-d-DTM-HA containing three N-glycosylation sites (Bernasconi et al, 2010). mCD3-d-DTM-HA migrated more slowly in EDEM2-KO cells than in WT cells due to the absence of the first mannose trimming activity (M9 -> M8B) in EDEM2-KO cells; as expected, this migration difference was lost after treatment with endoglycosidase H (EndoH) (Figure 1C)

Summary

Introduction

Failure in protein folding in the endoplasmic reticulum (ER) detrimentally affects the maturation of secretory and transmembrane proteins, which comprise one-third of total proteins synthesized in mammalian cells. As misfolded proteins are unable to fulfill the functions assigned them by genetic code but may exert proteotoxicity by making inappropriate interactions with other functional proteins, protein misfolding constitutes a fundamental threat to all living cells. Proteins misfolded in the lumen of the ER are recognized, delivered to and translocated through the multiprotein complex termed the retrotranslocon in the ER membrane, ubiquitinated, and degraded by the proteasome in the cytosol, a series of events collectively termed ER-associated degradation (ERAD), and termed ERAD-L (L for lumen). Mammalian ERAD-L deals with the degradation of both non-glycoproteins (non-gpERAD) and glycoproteins (gpERAD), in which the carbohydrate unit Glc3Man9GlcNAc2 (see Figure 1A) is covalently attached to the asparagine residue

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Conclusion