Biochemistry (Washington) 2015-08-11

Profiling Aglycon-Recognizing Sites of UDP-glucose:glycoprotein Glucosyltransferase by Means of Squarate-Mediated Labeling.

Keiichiro Ohara, Yoichi Takeda, Shusaku Daikoku, Masakazu Hachisu, Akira Seko, Yukishige Ito

Index: Biochemistry 54 , 4909-17, (2015)

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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.