EDEM1 accelerates the trimming of  1,2-linked mannose on the C branch of N-glycans

Abstract

Glycoprotein folding and degradation in the endoplasmic reticulum (ER) is mediated by the ER quality control system. Mannose trimming plays an important role by forming specific N-glycans that permit the recognition and sorting of terminally misfolded conformers for ERAD (ER-associated degradation). The EDEM (ER degradation enhancing alpha-mannosidase-like protein) subgroup of proteins belonging to the Class I alpha1,2-mannosidase family (glycosylhydrolase family 47) has been shown to enhance ERAD. We recently reported that overexpression of EDEM3 enhances glycoprotein ERAD with a concomitant increase in mannose-trimming activity in vivo. Herein, we report that overexpression of EDEM1 produces Glc(1)Man(8)GlcNAc(2) isomer C on terminally misfolded null Hong Kong alpha1-antitrypsin (NHK) in vivo. Levels of this isomer increased throughout the chase period and comprised approximately 10% of the [(3)H]mannose-labeled N-glycans on NHK after a 3-h chase. Furthermore, overexpression of EDEM1 E220Q containing a mutation in a conserved catalytic residue essential for alpha1,2-mannosidase activity did not yield detectable levels of Glc(1)Man(8)GlcNAc(2) isomer C. Yet, the same extent of NHK ERAD-enhancement was observed in both EDEM1 and EDEM1 E220Q overexpressing cells. This can be attributed to both wild-type and mutant EDEM1 inhibiting aberrant NHK dimer formation. We further analyzed the N-glycan profile of total cellular glycoproteins from HepG2 cells stably overexpressing EDEM1 and found that the relative amount of Man(7)GlcNAc(2) isomer A, which lacks the terminal B and C branch mannoses, was increased compared to parental HepG2 cells. Based on this observation, we conclude that EDEM1 activity trims mannose from the C branch of N-glycans in vivo.

Protein Folding and Quality Control in the ER

“…EDEM1 presumably possesses mannosidase activity that trims the C branch of N-glycans on misfolded proteins; however, that activity is apparently not required for ERAD acceleration because mutant EDEM1 that lacks the putative active site for mannosidase is still able to accelerate ERAD (Hosokawa et al 2001(Hosokawa et al , 2006(Hosokawa et al , 2010bMolinari et al 2003;Oda et al 2003;Olivari et al 2006). EDEM2 also promotes ERAD, even though it has no enzymatic activity (Mast et al 2005;Olivari et al 2005).…”

A Shared Endoplasmic Reticulum-associated Degradation Pathway Involving the EDEM1 Protein for Glycosylated and Nonglycosylated Proteins

“…EDEM1 is involved in trimming of mannose residues from precursor N-glycans during glycoprotein ER quality control (26,39,41). However, this trimming is not necessary for EDEM1 binding to its substrates when EDEM1 is exogenously overexpressed (13,42) or when EDEM1 is up-regulated during the unfolded protein response (26).…”

Characterization of Early EDEM1 Protein Maturation Events and Their Functional Implications

“…Fourth, EDEM1 localized to the ER and large vesicles that exit the ER in a COPII-independent manner (30,47). Fifth, although the main distinguishing characteristic of EDEM1 is that it possesses a mannosidase-like domain, it is not known if the purified protein possesses mannosidase or carbohydrate binding activity (40,41). Finally, EDEM1 has displayed a wide array of binding properties.…”

“…6A, compare upper panel lanes 7 and 9 and lanes 10 and 12). The increase in mobility was likely caused by the mannosidase activity of EDEM1 or through its association with ER mannosidase I (29,40,41). The soluble form of EDEM1 was most effective at accelerating the turnover of the soluble ERAD substrate NHK.…”