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

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

Reactivation of hyperglycemia-induced hypocretin (<i>HCRT)</i> gene silencing by <i>N</i>-acetyl-d-mannosamine in the orexin neurons derived from human iPS cells

<p>Orexin neurons regulate critical brain activities for controlling sleep, eating, emotions, and metabolism, and impaired orexin neuron function results in several neurologic disorders. Therefore, restoring normal orexin function and understanding the mechanisms of loss or impairment of orexin neurons represent important goals. As a step toward that end, we generated human orexin neurons from induced pluripotent stem cells (hiPSCs) by treatment with <i>N</i>-acetyl-d-mannosamine (ManNAc) and its derivatives. The generation of orexin neurons was associated with DNA hypomethylation, histone H3/H4 hyperacetylation, and hypo-<i>O</i>-GlcNAcylation on the <i>HCRT</i> gene locus, and, thereby, the treatment of inhibitors of SIRT1 and OGT were effective at inducing orexin neurons from hiPSCs. The prolonged exposure of orexin neurons to high glucose in culture caused irreversible silencing of the <i>HCRT</i> gene, which was characterized by H3/H4 hypoacetylation and hyper-<i>O</i>-GlcNAcylation. The DNA hypomethylation status, once established in orexin neurogenesis, was maintained in the <i>HCRT</i>-silenced orexin neurons, indicating that histone modifications, but not DNA methylation, were responsible for the <i>HCRT</i> silencing. Thus, the epigenetic status of the <i>HCRT</i> gene is unique to the hyperglycemia-induced silencing. Intriguingly, treatment of ManNAc and its derivatives reactivated <i>HCRT</i> gene expression, while inhibitors SIRT1 and the OGT did not. The present study revealed that the <i>HCRT</i> gene was silenced by the hyperglycemia condition, and ManNAc and its derivatives were useful for restoring the orexin neurons.</p