Michelle R. Bond

A little sugar goes a long way: The cell biology of O-GlcNAc

Unlike the complex glycans decorating the cell surface, the O-linked β-N-acetyl glucosamine (O-GlcNAc) modification is a simple intracellular Ser/Thr-linked monosaccharide that is important for disease-relevant signaling and enzyme regulation. O-GlcNAcylation requires uridine diphosphate–GlcNAc, a precursor responsive to nutrient status and other environmental cues. Alternative splicing of the genes encoding the O-GlcNAc cycling enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) yields isoforms targeted to discrete sites in the nucleus, cytoplasm, and mitochondria. OGT and OGA also partner with cellular effectors and act in tandem with other posttranslational modifications. The enzymes of O-GlcNAc cycling act preferentially on intrinsically disordered domains of target proteins impacting transcription, metabolism, apoptosis, organelle biogenesis, and transport.

Photocrosslinkers illuminate interactions in living cells

Transient and low-affinity interactions among macromolecules underlie many physiological events. Often, these interactions are difficult to study because they are not maintained when the participating molecules are removed from their cellular context. To circumvent this challenge, crosslinking reagents can be used to introduce covalent bonds between interacting macromolecules. Photoactivatable crosslinkers are particularly attractive because they allow crosslinking to proceed in time- and location-specific ways. Once the interacting partners have been crosslinked, they can be isolated and then analyzed by mass spectrometry or other analytical techniques to determine the identity of the interacting molecules and to pinpoint the interacting regions. This review highlights recent methodological developments that make it possible to introduce photocrosslinking groups into polypeptides or glycans as they are synthesized in cells. We also describe how these methods offer a non-invasive way to study macromolecular interactions in a native context.

Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners

O-linked β-N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification found on hundreds of nuclear and cytoplasmic proteins in higher eukaryotes. Despite its ubiquity and essentiality in mammals, functional roles for the O-GlcNAc modification remain poorly defined. Here we develop a combined genetic and chemical approach that enables introduction of the diazirine photocrosslinker onto the O-GlcNAc modification in cells. We engineered mammalian cells to produce diazirine-modified O-GlcNAc by expressing a mutant form of UDP-GlcNAc pyrophosphorylase and subsequently culturing these cells with a cell-permeable, diazirine-modified form of GlcNAc-1-phosphate. Irradiation of cells with UV light activated the crosslinker, resulting in formation of covalent bonds between O-GlcNAc-modified proteins and neighboring molecules, which could be identified by mass spectrometry. We used this method to identify interaction partners for the O-GlcNAc-modified FG-repeat nucleoporins. We observed crosslinking between FG-repeat nucleoporins and nuclear transport factors, suggesting that O-GlcNAc residues are intimately associated with essential recognition events in nuclear transport. Further, we propose that the method reported here could find widespread use in investigating the functional consequences of O-GlcNAcylation.

Metabolism of Diazirine-Modified N-Acetylmannosamine Analogues to Photo-Cross-Linking Sialosides

Terminal sialic acid residues often mediate the interactions of cell surface glycoconjugates. Sialic acid-dependent interactions typically exhibit rapid dissociation rates, precluding the use of traditional biological techniques for complex isolation. To stabilize these transient interactions, we employ a targeted photo-cross-linking approach in which a diazirine photo-cross-linker is incorporated into cell surface sialylated glycoconjugates through the use of metabolic oligosaccharide engineering. We describe three diazirine-modified N-acetylmannosamine (ManNAc) analogues in which the length of the linker between the pyranose ring and the diazirine was varied. These analogues were each metabolized to their respective sialic acid counterparts, which were added to both glycoproteins and glycolipids. Diazirine-modified sialic acid analogues could be incorporated into both α2-3 and α2-6 linkages. Upon exposure to UV irradiation, diazirine-modified glycoconjugates were covalently cross-linked to their interaction partners. We demonstrate that all three diazirine-modified analogues were capable of competing with endogeneous sialic acid, albeit to varying degrees. We found that larger analogues were less efficiently metabolized, yet could still function as effective cross-linkers. Notably, the addition of the diazirine substituent interferes with metabolism of ManNAc analogues to glycans other than sialosides, providing fidelity to selectively incorporate the cross-linker into sialylated molecules. These compounds are nontoxic and display only minimal growth inhibition at the concentrations required for cross-linking studies. This report provides essential information for the deployment of photo-cross-linking analogues to capture and study ephemeral, yet essential, sialic acid-mediated interactions.

Fucosylation and protein glycosylation create functional receptors for cholera toxin

Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors using its B subunit (CTB). The ganglioside (glycolipid) GM1 is thought to be the sole CT receptor; however, the mechanism by which CTB binding to GM1 mediates internalization of CT remains enigmatic. Here we report that CTB binds cell surface glycoproteins. Relative contributions of gangliosides and glycoproteins to CTB binding depend on cell type, and CTB binds primarily to glycoproteins in colonic epithelial cell lines. Using a metabolically incorporated photocrosslinking sugar, we identified one CTB-binding glycoprotein and demonstrated that the glycan portion of the molecule, not the protein, provides the CTB interaction motif. We further show that fucosylated structures promote CTB entry into a colonic epithelial cell line and subsequent host cell intoxication. CTB-binding fucosylated glycoproteins are present in normal human intestinal epithelia and could play a role in cholera. DOI: http://dx.doi.org/10.7554/eLife.09545.001

Metabolically incorporated photocrosslinking sialic acid covalently captures a ganglioside–protein complex

When photoirradiated, an unnatural sialic acid analog can covalently capture the complex formed by ganglioside GM1 and cholera toxin subunit B.

METABOLIC LABELING OF GLYCOCONJUGATES WITH PHOTOCROSSLINKING SUGARS

Protein-carbohydrate interactions play essential roles in a variety of biological processes. This class of interactions is particularly important in development, immunology, infection, and carcinogenesis. However, the transient nature of glycan-dependent interactions hampers efforts to detect and characterize these complexes. Photocrosslinking is emerging as a powerful tool to discover and study glycan-dependent complexes. Through the use of photocrosslinking groups, UV irradiation can be employed to introduce a covalent bond between two transiently interacting molecules. Here we describe the use of metabolic oligosaccharide engineering to incorporate a photocrosslinkable sugar into cellular glycoconjugates and the use of this photocrosslinker to covalently capture glycan-mediated interactions.

Versatile O-GlcNAc transferase assay for high-throughput identification of enzyme variants, substrates, and inhibitors.

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.

Evaluation of a novel PET radioligand to image O-GlcNAcase in brain and periphery of rhesus monkey and knock-out mouse.

Accumulation of hyperphosphorylated tau, a microtubule-associated protein, plays an important role in the progression of Alzheimer disease. Animal studies suggest that one strategy for treating Alzheimer disease and related tauopathies may be inhibition of O-GlcNAcase (OGA), which may subsequently decrease pathologic tau phosphorylation. Here, we report the pharmacokinetics of a novel PET radioligand, 18F-LSN3316612, which binds with high affinity and selectivity to OGA. Methods: PET imaging was performed on rhesus monkeys at baseline and after administration of either thiamet-G, a potent OGA inhibitor, or nonradioactive LSN3316612. The density of the enzyme was calculated as distribution volume using a 2-tissue-compartment model and serial concentrations of parent radioligand in arterial plasma. The radiation burden for future studies was based on whole-body imaging of monkeys. Oga∆Br, a mouse brain-specific knockout of Oga, was also scanned to assess the specificity of the radioligand for its target enzyme. Results: Uptake of radioactivity in monkey brain was high (∼5 SUV) and followed by slow washout. The highest uptake was in the amygdala, followed by striatum and hippocampus. Pretreatment with thiamet-G or nonradioactive LSN3316612 reduced brain uptake to a low and uniform concentration in all regions, corresponding to an approximately 90% decrease in distribution volume. Whole-body imaging of rhesus monkeys showed high uptake in kidney, spleen, liver, and testes. In Oga∆Br mice, brain uptake of 18F-LSN3316612 was reduced by 82% compared with control mice. Peripheral organs were unaffected in Oga∆Br mice, consistent with loss of OGA expression exclusively in the brain. The effective dose of 18F-LSN3316612 in humans was calculated to be 22 μSv/MBq, which is typical for 18F-labeled radioligands. Conclusion: These results show that 18F-LSN3316612 is an excellent radioligand for imaging and quantifying OGA in rhesus monkeys and mice. On the basis of these data, 18F-LSN3316612 merits evaluation in humans.

6.07 – Chemical Glycobiology

Carbohydrates are essential biological building blocks. Organisms assemble monosaccharides into a wide variety of glycan structures that give rise to diverse functions, from prokaryotic cell wall structure to eukaryotic signal transduction. Biosynthesis of oligosaccharides is difficult to control and typically results in heterogeneous structures, impeding efforts to elucidate the biological functions of these molecules. Chemists are uniquely capable of addressing mechanistic questions in glycobiology by virtue of their ability to synthesize homogeneous glycans and their analogs. To demonstrate the power of Chemical Glycobiology, this chapter describes key examples where synthetically prepared glycans have been used to manipulate and interrogate biological systems. These examples fall into four categories: total synthesis of naturally occurring glycans, chemoenzymatic synthesis of unnatural monosaccharide building blocks, glycan metabolic engineering to intercept metabolic pathways, and the use of photoreactive groups to capture glycan-mediated interactions.