The concepts of both protein glycosylation and cellular signaling have been influenced by O-linked-β-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) on the hydroxyl group of serine or threonine residues. Unlike conventional protein glycosylation, O-GlcNAcylation is localized in the nucleocytoplasm and its cycling is a dynamic process that operates in a highly regulated manner in response to various cellular stimuli. These characteristics render O-GlcNAcylation similar to phosphorylation, which has long been considered a major regulatory mechanism in cellular processes. Various efficient chemical approaches and novel mass spectrometric (MS) techniques have uncovered numerous O-GlcNAcylated proteins that are involved in the regulation of many important cellular events. These discoveries imply that O-GlcNAcylation is another major regulator of cellular signaling. However, in contrast to phosphorylation, which is regulated by hundreds of kinases and phosphatases, dynamic O-GlcNAc cycling is catalyzed by only two enzymes: uridine diphospho-N-acetyl-glucosamine:polypeptide β-N-acetylglucosaminyl transferase (OGT) and β-D-N-acetylglucosaminidase (OGA). Many useful chemical tools have recently been used to greatly expand our understanding of the extensive crosstalk between O-GlcNAcylation and phosphorylation and hence of cellular signaling. This review article describes the various useful chemical tools that have been developed and discusses the considerable advances made in the O-GlcNAc field.

The dynamic glycosylation of serine/threonine residues on nucleocytoplasmic proteins with a single N-acetylglucosamine (O-GlcNAcylation) is critical for many important cellular processes. Cellular O-GlcNAc levels are highly regulated by two enzymes: O-GlcNAc transferase (OGT) is responsible for GlcNAc addition and O-GlcNAcase (OGA) is responsible for removal of the sugar. The lack of a rapid and simple method for monitoring OGT activity has impeded the efficient discovery of potent OGT inhibitors. In this study we describe a novel, single-well OGT enzyme assay that utilizes 6 × His-tagged substrates, a chemoselective chemical reaction, and unpurified OGT. The high-throughput Ni-NTA Plate OGT Assay will facilitate discovery of potent OGT-specific inhibitors on versatile substrates and the characterization of new enzyme variants.

A putative bgl operon of Pectobacterium carotovorum subsp. carotovorum LY34 (Pcc LY34) was isolated. Sequence analysis of the 5,557 bp cloned DNA fragment (accession no. AY542524) showed three open reading frames (bglT, bglP, and bglB) predicted to encode 287, 633, and 468 amino acid proteins respectively. BglT and BglP ORFs show high similarity to that of the Pectobacterium chrysanthemi ArbG antiterminator and ArbF permease respectively. Also, the latter contains most residues important for phosphotransferase activity. The amino acid sequence of BglB showed high similarity to various β-glucosidases and is a member of glycosyl hydrolase family 1. The purified BglB enzyme hydrolyzed salicin, arbutin, pNPG, and MUG. The molecular weight of the enzyme was estimated to be 53,000 Da by SDS–PAGE. The purified β-glucosidase exhibited maximal activity at pH 7.0 and 40 °C, and its activity was enhanced in the presence of Mg2+. Two glutamate residues (Glu173 and Glu362) were found to be essential for enzyme activity.

Protein O-GlcNAcylation is a non-canonical glycosylation of nuclear, mitochondrial, and cytoplasmic proteins with the attachment of a single O-linked β-N-acetyl-glucosamine (O-GlcNAc) moiety. Advances in labeling and identifying O-GlcNAcylated proteins have helped improve the understanding of O-GlcNAcylation at levels that range from basic molecular biology to cell signaling and gene regulation to physiology and disease. This review describes these advances in chemistry involving chemical reporters and their bioorthogonal reactions utilized for detection and construction of O-GlcNAc proteomes in a molecular mechanistic view. This detailed view will help better understand the principles of the chemistries utilized for biology discovery and promote continued efforts in developing new molecular tools and new strategies to further explore protein O-GlcNAcylation.