Various pathologies result from disruptions to or stress of endoplasmic reticulum (ER) homeostasis, such as Parkinson’s disease and most neurodegenerative illnesses, diabetes, pulmonary fibrosis, viral infections and cancers. A critical process in maintaining ER homeostasis is the selection of misfolded proteins by the ER quality-control system (ERQC) for destruction via ER-associated degradation (ERAD). One key protein proposed to act during the first steps of misfolded glycoprotein degradation is the ER degradation-enhancing α-mannosidase-like protein 2 (EDEM2). Therefore, characterization of the EDEM2 associated proteome is of great interest. We took advantage of using melanoma cells overexpressing EDEM2 as a cancer model system, to start documenting at the deglycoproteome level (N-glycosites identification) the emerging link between ER homeostasis and cancer progression. The dataset created for identifying the EDEM2 glyco-clients carrying high mannose/hybrid N-glycans provides a comprehensive N-glycosites analysis mapping over 1000 N-glycosites on more than 600 melanoma glycoproteins. To identify EDEM2-associated proteins we used affinity-proteomics and proteome-wide analysis of sucrose density fractionation in an integrative workflow. Using intensity and spectral count-based quantification, we identify seven new EDEM2 partners, all of which are involved in ERQC and ERAD. Moreover, we defined novel endogenous candidates for EDEM2-dependent ERAD by combining deglycoproteomics, SILAC-based proteomics, and biochemical methods. These included tumor antigens and several ER-transiting endogenous melanoma proteins, including ITGA1 and PCDH2, the expression of which was negatively correlated with that of EDEM2. Tumor antigens are key in the antigen presentation process, whilst ITGA1 and PCDH2 are involved in melanoma metastasis and invasion. EDEM2 could therefore have a regulatory role in melanoma through the modulation of these glycoproteins degradation and trafficking. The data presented herein suggest that EDEM2 is involved in ER homeostasis to a greater extent than previously suggested.
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