Abstract
Extensive mannose trimming of nascent glycoprotein N-glycans signals their targeting to endoplasmic reticulum-associated degradation (ERAD). ER mannosidase I (ERManI) and the EDEM protein family participate in this process. However, whether the EDEMs are truly mannosidases can be addressed only by measuring mannosidase activity in vitro. Here, we reveal EDEM1 and EDEM2 mannosidase activities in vitro. Whereas ERManI significantly trims free N-glycans, activity of the EDEMs is modest on free oligosaccharides and on glycoproteins. However, mannosidase activity of ERManI and the EDEMs is significantly higher on a denatured glycoprotein. The EDEMs associate with oxidoreductases, protein disulfide isomerase, and especially TXNDC11, enhancing mannosidase activity on glycoproteins but not on free N-glycans. The finding that substrate unfolded status increases mannosidase activity solves an important conundrum, as current models suggest general slow mannose trimming. As we show, misfolded or unfolded glycoproteins are subject to differentially faster trimming (and targeting to ERAD) than well-folded ones.
Highlights
Extensive mannose trimming of nascent glycoprotein N-glycans signals their targeting to endoplasmic reticulum-associated degradation (ERAD)
We find that the mannosidase activity of EDEM1, EDEM2, and ER mannosidase I (ERManI) is modulated by the folding state of the glycoprotein substrate, which has important implications for the decision-making process in glycoprotein quality control
To better understand the role of EDEM1 in the removal of mannose residues from misfolded N-linked glycoproteins and their targeting to ERAD, we analyzed directly the effect of EDEM1 knockdown on the N-glycans of an established model ERAD substrate, the uncleaved precursor of asialoglycoprotein receptor H2a23,24
Summary
To better understand the role of EDEM1 in the removal of mannose residues from misfolded N-linked glycoproteins and their targeting to ERAD, we analyzed directly the effect of EDEM1 knockdown on the N-glycans of an established model ERAD substrate, the uncleaved precursor of asialoglycoprotein receptor H2a23,24. This was done by pulse–chase analysis with [2-3H]Man labeling.
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