Chemical Probe Approach Reveals Endo-α-mannosidase Triages Misfolded Glycoproteins in the Calnexin/Calreticulin Cycle

Protein N-glycosylation contributes to folding and quality control of secretory proteins involved in protein misfolding diseases. A central quality control machinery of nascent glycoproteins in the endoplasmic reticulum (ER) is the calnexin/calreticulin (CNX/CRT) cycle. This cycle assists and checks protein folding by monitoring glycan structure, however how terminally misfolded glycoproteins are discharged from the cycle has remained unclear. Here, we leveraged chemical probes to identify a previously uncharacterized ER endo-α-mannosidase complex (ER-EM) that provides this missing release step. ER-EM selectively cleaves the terminal Glc-Man disaccharide from glucosylated high-mannose glycans only when the glycan is attached to a hydrophobic aglycone─an intrinsic marker of misfolded proteins─thereby converting Glc₁Man₉GlcNAc₂ to Man_8AGlcNAc₂ glycans that cannot bind CNX/CRT. This activity is allosterically stimulated by hydrophobic ligands and shares the same aglycone preference as the folding sensor UDP-glucose: glycoprotein glucosyltransferase 1 (UGGT1), creating a two-tier surveillance system in which UGGT1 reglucosylates incompletely folded proteins, whereas ER-EM ejects those that fail to mature. Proteomic and native-gel analyses revealed that ER-EM is an ∼ 800 kDa assembly composed of at least carboxylesterase 1D (Ces1d), ERp57 and UGGT1; the lack of activity of recombinant Ces1d alone underscores that the catalytic function arises only through the concerted action of this multisubunit complex. ER-EM therefore acts as a folding-status-dependent triage factor that liberates terminally misfolded glycoproteins from the CNX/CRT cycle and targets them for degradation, adding a critical new branch to the ER quality-control network.