EDEM3, a Soluble EDEM Homolog, Enhances Glycoprotein Endoplasmic Reticulum-associated Degradation and Mannose Trimming

Linda O. Tremblay,Shunji Natsuka,Daisuke Morito,Annette Herscovics,Kazuyoshi Hirao,Kazuhiro Nagata,Ikuo Wada,Barry Sleno,Yuko Natsuka,Taku Tamura,Pedro Romero,Nobuko Hosokawa

doi:10.1074/jbc.m512191200

Abstract

Quality control in the endoplasmic reticulum ensures that only properly folded proteins are retained in the cell through mechanisms that recognize and discard misfolded or unassembled proteins in a process called endoplasmic reticulum-associated degradation (ERAD). We previously cloned EDEM (ER degradation-enhancing alpha-mannosidase-like protein) and showed that it accelerates ERAD of misfolded glycoproteins. We now cloned mouse EDEM3, a soluble homolog of EDEM. EDEM3 consists of 931 amino acids and has all the signature motifs of Class I alpha-mannosidases (glycosyl hydrolase family 47) in its N-terminal domain and a protease-associated motif in its C-terminal region. EDEM3 accelerates glycoprotein ERAD in transfected HEK293 cells, as shown by increased degradation of misfolded alpha1-antitrypsin variant (null (Hong Kong)) and of TCRalpha. Overexpression of EDEM3 also greatly stimulates mannose trimming not only from misfolded alpha1-AT null (Hong Kong) but also from total glycoproteins, in contrast to EDEM, which has no apparent alpha1,2-mannosidase activity. Furthermore, overexpression of the E147Q EDEM3 mutant, which has the mutation in one of the conserved acidic residues essential for enzyme activity of alpha1,2-mannosidases, abolishes the stimulation of mannose trimming and greatly decreases the stimulation of ERAD by EDEM3. These results show that EDEM3 has alpha1,2-mannosidase activity in vivo, suggesting that the mechanism whereby EDEM3 accelerates glycoprotein ERAD is different from that of EDEM.

Highlights

ER3 quality control is an elaborate mechanism conserved from yeast to mammals, ensuring that newly synthesized proteins in the ER fold and assemble correctly and that only proteins that acquire their correct conformations are sorted further into the secretory pathway [1,2,3,4]
We show that EDEM3 is a soluble EDEM homolog located in the ER of transfected mammalian cells that accelerates ER-associated degradation (ERAD) of misfolded glycoproteins through a mechanism likely to be different from that of EDEM or EDEM2, since EDEM3 greatly stimulates mannose trimming in vivo
The present work shows that EDEM3 is a soluble homolog of EDEM that accelerates ERAD of both soluble null Hong Kong (NHK) and membrane-bound TCR␣ in an N-glycan-dependent manner

Summary

Introduction

ER3 quality control is an elaborate mechanism conserved from yeast to mammals, ensuring that newly synthesized proteins in the ER fold and assemble correctly and that only proteins that acquire their correct conformations are sorted further into the secretory pathway [1,2,3,4]. We previously cloned EDEM (ER degradation-enhancing ␣-mannosidase-like protein) and showed that it accelerates ERAD of misfolded glycoproteins. EDEM3 accelerates glycoprotein ERAD in transfected HEK293 cells, as shown by increased degradation of misfolded ␣1-antitrypsin variant (null (Hong Kong)) and of TCR␣.

Results

Conclusion