2. Academic Validation
  3. Carboxylesterase 1d-mediated aglycone recognition is crucial for substrate processing and allosteric activation of endo-α-mannosidase in the endoplasmic reticulum

Carboxylesterase 1d-mediated aglycone recognition is crucial for substrate processing and allosteric activation of endo-α-mannosidase in the endoplasmic reticulum

  • Bioorg Med Chem. 2026 Mar:134:118547. doi: 10.1016/j.bmc.2026.118547.
Akito Taira 1,  Taiki Kuribara 2,  Mitsuaki Hirose 1,  Kiichiro Totani 3
Affiliations

Affiliations

  • 1 Department of Science and Technology, Seikei University, 3-3-1 Kichijoji-kitamachi, Musashino-shi, Tokyo 180-8633, Japan.
  • 2 Department of Science and Technology, Seikei University, 3-3-1 Kichijoji-kitamachi, Musashino-shi, Tokyo 180-8633, Japan; Department of Glyco-Oncology and Medical Biochemistry, Research Center, Osaka International Cancer Institute, 3-1-69 Otemae, Chuo-ku, Osaka 541-8567, Japan.
  • 3 Department of Science and Technology, Seikei University, 3-3-1 Kichijoji-kitamachi, Musashino-shi, Tokyo 180-8633, Japan. Electronic address: [email protected].
PMID: 41505878 DOI: 10.1016/j.bmc.2026.118547
Abstract

Most nascent glycoproteins entering the endoplasmic reticulum (ER) undergo quality control via the calnexin/calreticulin (CNX/CRT) cycle, wherein Glc1Man9GlcNAc2 (G1M9)-type glycans play a crucial role in monitoring protein folding. We have recently identified an endo-α-mannosidase activity within the ER, designated as ER-EM, which facilitates the release of misfolded glycoproteins from this cycle by converting G1M9-proteins into Man8AGlcNAc2 (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.

Keywords

Allosteric activation; Carboxylesterase 1d; Endo-α-mannosidase; JW972; WWL229.

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