Hydrophobic Tagged Dihydrofolate Reductase for Creating Misfolded Glycoprotein Mimetics |
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| Authors: | Dr. Masakazu Hachisu Dr. Akira Seko Dr. Shusaku Daikoku Dr. Yoichi Takeda Dr. Masafumi Sakono Dr. Yukishige Ito |
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| Affiliation: | 1. ERATO Ito Glycotrilogy Project, Japan Science and Technology Agency (JST), Wako, Saitama, Japan;2. Department of Biological Science and Technology, Tokyo University of Science, Katsushika-ku, Tokyo, Japan;3. Department of Biotechnology, Ritsumeikan University, Kusatsu, Shiga, Japan;4. Department of Applied Chemistry, University of Toyama, Toyama, Toyama, Japan;5. Synthetic Cellular Chemistry Laboratory, RIKEN, Wako, Saitama, Japan |
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| Abstract: | In the endoplasmic reticulum (ER), nascent glycoproteins that have not acquired the native conformation are either repaired or sorted for degradation by specific quality‐control systems composed by various proteins. Among them, UDP‐glucose:glycoprotein glucosyltransferase (UGGT) serves as a folding sensor in the ER. However, the molecular mechanism of its recognition remains obscure. This study used pseudo‐misfolded glycoproteins, comprising a modified dihydrofolate reductase with artificial pyrene–cysteine moiety on the protein surface (pDHFR) and Man9GlcNAc2‐methotrexate (M9‐MTX). All five M9‐MTX/pDHFR complexes, with a pyrene group at different positions, were found to be good substrates of UGGT, irrespective of the site of pyrene modification. These results suggest UGGT's mode of substrate recognition is fuzzy, thus allowing various glycoproteins to be accommodated in the folding cycle. |
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| Keywords: | DHFR enzyme catalysis ER-quality control glycoproteins high-mannose-type glycan UGGT |
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