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 <b>1</b> 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 <b>1</b> was able to label HUGT1 in the presence of UDP. Analysis by nano-LC-ESI/MS^<i>n</i> identified a unique lysine residue (K1424) that was modified by <b>1</b>. 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