EDEM1 accelerates the trimming of alpha1,2-linked mannose on the C branch of N-glycans.
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.
Hosokawa
Nobuko
N
Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8397, Japan. nobukoh@frontier.kyoto-u.ac.jp
Tremblay
Linda O
LO
Sleno
Barry
B
Kamiya
Yukiko
Y
Wada
Ikuo
I
Nagata
Kazuhiro
K
Kato
Koichi
K
Herscovics
Annette
A
D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008358:Mannose / Q000737:chemistry* / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism*; D013237:Stereoisomerism
C486654:EDEM1 protein, human; D008565:Membrane Proteins; D011134:Polysaccharides; D008358:Mannose