Bypass of glycan-dependent glycoprotein delivery to ERAD by up-regulated EDEM1.

Trimming of mannose residues from the N-linked oligosaccharide precursor is a stringent requirement for glycoprotein endoplasmic reticulum (ER)-associated degradation (ERAD). In this paper, we show that, surprisingly, overexpression of ER degradation-enhancing α-mannosidase-like protein 1 (EDEM1) or its up-regulation by IRE1, as occurs in the unfolded protein response, overrides this requirement and renders unnecessary the expression of ER mannosidase I. An EDEM1 deletion mutant lacking most of the carbohydrate-recognition domain also accelerated ERAD, delivering the substrate to XTP3-B and OS9. EDEM1 overexpression also accelerated the degradation of a mutant nonglycosylated substrate. Upon proteasomal inhibition, EDEM1 concentrated together with the ERAD substrate in the pericentriolar ER-derived quality control compartment (ERQC), where ER mannosidase I and ERAD machinery components are localized, including, as we show here, OS9. We suggest that a nascent glycoprotein can normally dissociate from EDEM1 and be rescued from ERAD by reentering calnexin-refolding cycles, a condition terminated by mannose trimming. At high EDEM1 levels, glycoprotein release is prevented and glycan interactions are no longer required, canceling the otherwise mandatory ERAD timing by mannose trimming and accelerating the targeting to degradation.

Pulse-chase analysis of N-linked sugar chains from glycoproteins in mammalian cells.

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-(3)H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.