Many of the proteins synthesized in the ER have asparagine-linked Glc₃Man₉GlcNAc₂ glycan added to its consensus sites. In the ER, several enzymes remove and add sugars to the oligosaccharides (Fig. 2A).^{1, 2} Although glycans attached to each glycoprotein are diverse after maturation, the glycan modifications in the ER are uniform. This suggests that the quality control of various glycoproteins in the ER is mediated by a common mechanism through the recognition of the glycan moiety attached to them. Lectin-like molecular chaperones calnexin and calreticulin recognize the Glc₁Man₉GlcNAc₂ oligosaccharide and assist the folding of newly synthesized glycoproteins. Next, the mannose residue from the middle branch of the Man₉GlcNAc₂ oligosaccharide is removed by ER -mannosidase I, then glycoproteins bearing Man₈GlcNAc₂ isomer B (Man8B form) are transported out of the ER to the Golgi apparatus (Fig. 2B).

When ER stress is subjected to cells, misfolded proteins accumulate in the ER, which then induces the UPR (unfolded protein response) to cells.⁶ Consequently, the synthesis of ER chaperone proteins as well as molecules involved in ERAD is upregulated to circumvent the adverse condition.^7,8 We have cloned a novel mouse gene which is induced by ER stress using a PCR-based subtraction method.⁹ This encodes a type II transmembrane protein in the ER consisting of 652 amino acids, and has partial homology with processing 1. 1,2-mannosidase (glycosylhydrolase family 47). We named this gene EDEM (ER degradation enhancing -mannosidase-like protein), since this gene product was revealed to accelerate the ERAD of glycoproteins. We speculate that EDEM functions as the Man8-binding lectin on the ERAD pathway.

EDEM protein is 18 % identical with human ER -mannosidase I (ER Man I). Although acidic residues reported to be important for the enzyme activity are conserved, cystein residues which are known to be critical for the enzyme activity are not conserved in EDEM. From the amino acid sequence, we expected EDEM to lack the enzyme activity as a processing -mannosidase. This was proved experimentally by expressing EDEM as a recombinant protein. We fused EDEM to protein A and transfected it into monkey COS cells. The secreted fusion protein was collected by IgG sepharose beads to prevent the contamination of other cellular mannosidases, and the enzyme activity was measured using ³H-mannose-labeled oligosaccharides. Free ³H-mannose was not released by the incubation with recombinant EDEM protein. Therefore we concluded that EDEM lacks the enzyme activity as a processing -mannosidase.⁹

We next examined whether EDEM is involved in the ERAD. We used 1-antitrypsin genetic variant null Hong Kong (NHK) as a model protein, since it is known to be degraded by ERAD.¹⁰ HEK 293 cells were transfected with plasmids encoding NHK, and the intracellular degradation of NHK was examined in a pulse-chase experiment using ³⁵S-methionine. The T_1/2 of NHK within the cells was approximately two hours, and coexpression of EDEM accelerated the degradation of NHK to approximately one hour of T_1/2 (Fig. 3). We have confirmed that NHK was degraded through ERAD in the presence of cotransfected EDEM. Coimmunoprecipitation of EDEM with substrate NHK was detected within the cells. EDEM enhanced the degradation of NHK depending on the extent of mannose trimming from the N-linked glycans. Taken together, we speculate that EDEM is a key molecule which recognizes misfolded glycoproteins and links them to ERAD machinery, and that EDEM is a candidate for the Man8-binding lectin, although actual glycan analysis remains to be carried out.

EDEM gene is evolutionally conserved and the yeast Saccharomyces cerevisiae has a homologue. It has been reported that the Mnl1p/Htm1p gene, the yeast homologue of mouse EDEM, also accelerates the ERAD of misfolded glycoproteins.^11,12

Our working hypothesis on the function of EDEM is schematically shown in Fig. 4. We are now trying to clarify the mechanism of how EDEM works on the recognition and sorting of the misfolded glycoproteins.

As described above, the expression of EDEM is upregulated by ER stress. This is not specific to EDEM, and many of the molecules involved in ERAD are reported to be induced by ER stress.⁷

ER is an intracellular compartment with high calcium concentration in which proteins are oxidized and modified with N-linked oligosaccharides. ER stress is a condition which accumulates misfolded or unfolded proteins by disturbing these ER circumstances. To circumvent this adverse condition, cells and organisms respond to the ER stress by evoking several adaptation mechanisms.¹³ First is the transcriptional upregulation of ER chaperone proteins to prevent the unfolded proteins from aggregation and to promote refolding. This signaling pathway from the ER to the nucleus is known as UPR.⁶ Second, cells attenutate the protein synthesis by phosphorylating eIF-2 to prevent the further overload of newly synthesized proteins in the ER. Third is the induction of the ERAD mechanism described above, and this is regulated at least partly by UPR. The final mechanism of adaptation is apoptosis of cells. Multicellular organisms would survive ER stress by eliminating severely damaged cells.

ERAD system is now clarified to be involved in various cellular mechanisms, and further research is under way in relation to various protein-folding diseases and ER stress.