title: Hydrophobic Tagged Dihydrofolate Reductase for Creating Misfolded Glycoprotein Mimetics.
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 Man9 GlcNAc2 -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.
authors.affiliation: ERATO Ito Glycotrilogy Project, Japan Science and Technology Agency (JST), 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.|Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo, 125-8585, Japan.
authors.forename: Masakazu
authors.identifier: http://orcid.org/0000-0001-9777-2637
authors.initials: M
authors.lastname: Hachisu
authors.affiliation: ERATO Ito Glycotrilogy Project, Japan Science and Technology Agency (JST), 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Seko
authors.affiliation: ERATO Ito Glycotrilogy Project, Japan Science and Technology Agency (JST), 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
authors.forename: Shusaku
authors.initials: S
authors.lastname: Daikoku
authors.affiliation: ERATO Ito Glycotrilogy Project, Japan Science and Technology Agency (JST), 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan. yotakeda@riken.jp.|Department of Biotechnology, Ritsumeikan University, 1-1-1 Noji-higashi, Kusatsu, Shiga, 525-8577, Japan. yotakeda@riken.jp.
authors.forename: Yoichi
authors.identifier: http://orcid.org/0000-0002-7146-3585
authors.initials: Y
authors.lastname: Takeda
authors.affiliation: ERATO Ito Glycotrilogy Project, Japan Science and Technology Agency (JST), 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.|Department of Applied Chemistry, University of Toyama, 3190 Gofuku, Toyama, Toyama, 930-855, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Sakono
authors.affiliation: ERATO Ito Glycotrilogy Project, Japan Science and Technology Agency (JST), 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan. yukito@riken.jp.|Synthetic Cellular Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan. yukito@riken.jp.
authors.forename: Yukishige
authors.initials: Y
authors.lastname: Ito
mesh_terms: D000595:Amino Acid Sequence; D040761:Biomimetic Materials / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D005964:Glucosyltransferases / Q000378:metabolism*; D006031:Glycosylation; D057927:Hydrophobic and Hydrophilic Interactions; D008727:Methotrexate / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D017510:Protein Folding; D011721:Pyrenes / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011721:Pyrenes; C030984:pyrene; D013762:Tetrahydrofolate Dehydrogenase; D005964:Glucosyltransferases; C072423:mannosylglycoprotein 1,3-glucosyltransferase; D008727:Methotrexate
keywords: DHFR; ER-quality control; UGGT; enzyme catalysis; glycoproteins; high-mannose-type glycan