title: Profiling Aglycon-Recognizing Sites of UDP-glucose:glycoprotein Glucosyltransferase by Means of Squarate-Mediated Labeling.
abstract: Because of its ability to selectively glucosylate misfolded glycoproteins, UDP-glucose:glycoprotein glucosyltransferase (UGGT) functions as a folding sensor in the glycoprotein quality control system in the endoplasmic reticulum (ER). The unique property of UGGT derives from its ability to transfer a glucose residue to N-glycan moieties of incompletely folded glycoproteins. We have previously discovered nonproteinic synthetic substrates of this enzyme, allowing us to conduct its high-sensitivity assay in a quantitative manner. In this study, we aimed to conduct site-selective affinity labeling of UGGT using a functionalized oligosaccharide probe to identify domain(s) responsible for recognition of the aglycon moiety of substrates. To this end, a probe 1 was designed to selectively label nucleophilic amino acid residues in the proximity of the canonical aglycon-recognizing site of human UGGT1 (HUGT1) via squaramide formation. As expected, probe 1 was able to label HUGT1 in the presence of UDP. Analysis by nano-LC-ESI/MS(n) identified a unique lysine residue (K1424) that was modified by 1. Kyte-Doolittle analysis as well as homology modeling revealed a cluster of hydrophobic amino acids that may be functional in the folding sensing mechanism of HUGT1.
authors.affiliation: †Japan Science and Technology Agency (JST), ERATO, Ito Glycotrilogy Project, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Keiichiro
authors.initials: K
authors.lastname: Ohara
authors.affiliation: †Japan Science and Technology Agency (JST), ERATO, Ito Glycotrilogy Project, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Takeda
authors.affiliation: †Japan Science and Technology Agency (JST), ERATO, Ito Glycotrilogy Project, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Shusaku
authors.initials: S
authors.lastname: Daikoku
authors.affiliation: †Japan Science and Technology Agency (JST), ERATO, Ito Glycotrilogy Project, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Hachisu
authors.affiliation: †Japan Science and Technology Agency (JST), ERATO, Ito Glycotrilogy Project, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Seko
authors.affiliation: †Japan Science and Technology Agency (JST), ERATO, Ito Glycotrilogy Project, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.|‡RIKEN, Synthetic Cellular Chemistry Laboratory, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Yukishige
authors.initials: Y
authors.lastname: Ito
mesh_terms: D020134:Catalytic Domain; D002460:Cell Line; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D014530:Uridine Diphosphate / Q000737:chemistry* / Q000378:metabolism; D000099237:UDP-Galactose Translocators
chemical_list: D005964:Glucosyltransferases; D009844:Oligosaccharides; D014530:Uridine Diphosphate; C000731541:UGGT1 protein, human; D000099237:UDP-Galactose Translocators