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

Biochemistry 2015 Aug 11

filter terms:

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.

Citation

Keiichiro Ohara, Yoichi Takeda, Shusaku Daikoku, Masakazu Hachisu, Akira Seko, Yukishige Ito. Profiling Aglycon-Recognizing Sites of UDP-glucose:glycoprotein Glucosyltransferase by Means of Squarate-Mediated Labeling. Biochemistry. 2015 Aug 11;54(31):4909-17