Taira_2026_Bioorg.Med.Chem_134_118547

Most nascent glycoproteins entering the endoplasmic reticulum (ER) undergo quality control via the calnexin/calreticulin (CNX/CRT) cycle, wherein Glc(1)Man(9)GlcNAc(2) (G1M9)-type glycans play a crucial role in monitoring protein folding. We have recently identified an endo-alpha-mannosidase activity within the ER, designated as ER-EM, which facilitates the release of misfolded glycoproteins from this cycle by converting G1M9-proteins into Man(8A)GlcNAc(2) (M8A)-proteins in a single step. ER-EM appears to function as a complex comprising UDP-Glc:glycoprotein glucosyltransferase 1 (UGGT1), ERp57, and carboxylesterase 1D (Ces1d), although the role of Ces1d-primarily recognized for its involvement in lipid metabolism-in glycan-associated substrate recognition remains unclear. To elucidate the molecular basis of Ces1d-dependent recognition, we semi-synthesized a glycoprobe, G1M9-va-JW972, by conjugating the Ces1d-specific inhibitor JW972 to the aglycone of G1M9 using a linker via a click reaction. In the ER-EM reaction with this probe, M8A-va-piperidine was detected as an ER-EM product with the aglycone structural conversion via Ces1d-mediated hydrolysis of the JW972 moiety, demonstrating recognition of the substrate aglycone by the Ces1d component of ER-EM complex. Inhibition of the lipolysis site of Ces1d with WWL229 significantly reduced ER-EM activity, indicating that this site is essential for recognizing hydrophobic aglycones. Furthermore, the inactive substrate GlcMan-4MU was efficiently hydrolyzed in the presence of the Ces1d lipolysis site-specific inhibitor WWL229, demonstrating that the association of the hydrophobic compound WWL229 with the Ces1d lipolysis site contributes to allosteric activation of ER-EM. Our findings provide important insights into the functional regulation of ER-EM complex, a novel therapeutic target for protein misfolding diseases.