Protein‐Specific Imaging of O‐GlcNAcylation in Single Cells

Thousands of intracellular proteins are post‐translationally modified with O‐GlcNAc, and O‐GlcNAcylation impacts the function of modified proteins and mediates diverse biological processes. However, the ubiquity of this important glycosylation makes it highly challenging to probe the O‐GlcNAcylation state of a specific protein at the cellular level. Herein, we report the development of a FLIM–FRET‐based strategy, which exploits the spatial proximity of the O‐GlcNAc moiety and the attaching protein, for protein‐specific imaging of O‐GlcNAcylation in single cells. We demonstrated this strategy by imaging the O‐GlcNAcylation state of tau and β‐catenin inside the cells. Furthermore, the changes in tau O‐GlcNAcylation were monitored when the overall cellular O‐GlcNAc was pharmacologically altered by using the OGT and OGA inhibitors. We envision that the FLIM–FRET strategy will be broadly applicable to probe the O‐GlcNAcylation state of various proteins in the cells.     

Chemical Biology Approaches to Understanding Neuronal O−GlcNAcylation

O-linked β-N-acetylglucosamine (O−GlcNAc) is a ubiquitous post-translational modification in mammals, decorating thousands of intracellular proteins. O−GlcNAc cycling is an essential regulator of myriad aspects of cell physiology and is dysregulated in numerous human diseases. Notably, O−GlcNAcylation is abundant in the brain and numerous studies have linked aberrant O−GlcNAc signaling to various neurological conditions. However, the complexity of the nervous system and the dynamic nature of protein O−GlcNAcylation have presented challenges for studying of neuronal O−GlcNAcylation. In this context, chemical approaches have been a particularly valuable complement to conventional cellular, biochemical, and genetic methods to understand O−GlcNAc signaling and to develop future therapeutics. Here we review selected recent examples of how chemical tools have empowered efforts to understand and rationally manipulate O−GlcNAcylation in mammalian neurobiology.     

Chemical and Biochemical Strategies To Explore the Substrate Recognition of O-GlcNAc-Cycling Enzymes

The O-linked N-acetylglucosamine (O-GlcNAc) modification is an essential component in cell regulation. A single pair of human enzymes conducts this modification dynamically on a broad variety of proteins: O-GlcNAc transferase (OGT) adds the GlcNAc residue and O-GlcNAcase (OGA) hydrolyzes it. This modification is dysregulated in many diseases, but its exact effect on particular substrates remains unclear. In addition, no apparent sequence motif has been found in the modified proteins, and the factors controlling the substrate specificity of OGT and OGA are largely unknown. In this minireview, we will discuss recent developments in chemical and biochemical methods toward addressing the challenge of OGT and OGA substrate recognition. We hope that the new concepts and knowledge from these studies will promote research in this area to advance understanding of O-GlcNAc regulation in health and disease.     

The Utilities of Chemical Reactions and Molecular Tools for O‐GlcNAc Proteomic Studies

The post‐translational modification of nuclear and cytoplasmic proteins with O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) is involved in a wide variety of cellular processes and is associated with the pathological progression of chronic diseases. Considering its emerging biological significance, systematic identification, site mapping, and quantification of O‐GlcNAc proteins are essential and have led to the development of several approaches for O‐GlcNAc protein profiling. This minireview mainly focuses on the various useful chemical reactions and molecular tools with detailed reaction mechanisms widely adopted for O‐GlcNAc protein/peptide enrichment and its quantification for comprehensive O‐GlcNAc protein profiling.     

Feedback Regulation of O-GlcNAc Transferase through Translation Control to Maintain Intracellular O-GlcNAc Homeostasis

Protein O-GlcNAcylation is a dynamic post-translational modification involving the attachment of N-acetylglucosamine (GlcNAc) to the hydroxyl groups of Ser/Thr residues on numerous nucleocytoplasmic proteins. Two enzymes are responsible for O-GlcNAc cycling on substrate proteins: O-GlcNAc transferase (OGT) catalyzes the addition while O-GlcNAcase (OGA) helps the removal of GlcNAc. O-GlcNAcylation modifies protein functions; therefore, dysregulation of O-GlcNAcylation affects cell physiology and contributes to pathogenesis. To maintain homeostasis of cellular O-GlcNAcylation, there exists feedback regulation of OGT and OGA expression responding to fluctuations of O-GlcNAc levels; yet, little is known about the molecular mechanisms involved. In this study, we investigated the O-GlcNAc-feedback regulation of OGT and OGA expression in lung cancer cells. Results suggest that, upon alterations in O-GlcNAcylation, the regulation of OGA expression occurs at the mRNA level and likely involves epigenetic mechanisms, while modulation of OGT expression is through translation control. Further analyses revealed that the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) contributes to the downregulation of OGT induced by hyper-O-GlcNAcylation; the S5A/S6A O-GlcNAcylation-site mutant of 4E-BP1 cannot support this regulation, suggesting an important role of O-GlcNAcylation. The results provide additional insight into the molecular mechanisms through which cells may fine-tune intracellular O-GlcNAc levels to maintain homeostasis.     

O-GlcNAcase: Emerging Mechanism,Substrate Recognition and Small-Molecule Inhibitors

O-GlcNAcylation is the dynamic and ubiquitous post-translational glycosylation of nucleocytoplasmic proteins on serine/threonine residues; it is implicated in regulation of the cell cycle. This protein modification is mainly governed by a pair of enzymes: O-GlcNAc transferase (OGT) adds the N-acetylglucosamine moiety to acceptor proteins, and O-GlcNAcase (OGA) hydrolyses the sugar moiety from protein acceptors. Irregular O-GlcNAcylation is linked to several diseases including cancer, diabetes and neurodegeneration. Recently, the discovery of small-molecule OGA inhibitors has enabled the physiological function of O-GlcNAcylation to be investigated. However, the design of highly potent and selective inhibitors faces several challenges as no full structural data of human OGA has been discovered to date. Moreover, there are a number of mechanistically similar related enzymes such as β-hexosaminidases (Hex), and the concomitant inhibition of these enzymes leads to undesirable lysosomal-storage disorders. This review highlights recent insights into the structure of human O-GlcNAcase and its isoforms. We focus on the catalytic mechanism and substrate recognition by OGA. In addition, it presents an updated overview of small-molecule OGA inhibitors, with either carbohydrate or noncarbohydrate scaffolds. We discuss inhibitor structures, binding modes, and selectivity towards the enzyme, and potential outcomes in probing O-GlcNAcylation at cellular levels.     

O-GlcNAc Transferase: Structural Characteristics,Catalytic Mechanism and Small-Molecule Inhibitors

Modifications of nuclear and cytoplasmic proteins with a single sugar, N-acetylglucosamine (GlcNAc), play roles in a wide variety of fundamental cellular processes, and aberrant O-GlcNAc profiles are associated with pathological progression of several chronic diseases. O-GlcNAc transferase (OGT) is the only enzyme to catalyze the attachment of GlcNAc to intracellular protein substrates. Considering its biological significance, selective and potent OGT inhibitors are invaluable tools for enhancing our understanding of the precise biological functions of the enzyme, for revealing its unknown functions, and for validating OGT as a therapeutic target. In this minireview, human OGT (hOGT) inhibitors and their catalytic mechanisms will be explored. In addition, a brief overview of recent findings on the 3D structural characteristics of hOGT that have contributed greatly to the development of novel inhibitors of hOGT is provided.     

O6‐Alkylguanine‐DNA Alkyltransferase‐Mediated Repair of O4‐Alkylated 2′‐Deoxyuridines

O⁶‐Alkylguanine‐DNA alkyltransferases (AGTs) are responsible for the removal of O⁶‐alkyl 2′‐deoxyguanosine (dG) and O⁴‐alkyl thymidine (dT) adducts from the genome. Unlike the E. coli OGT (O⁶‐alkylguanine‐DNA‐alkyltransferase) protein, which can repair a range of O⁴‐alkyl dT lesions, human AGT (hAGT) only removes methyl groups poorly. To uncover the influence of the C5 methyl group of dT on AGT repair, oligonucleotides containing O⁴‐alkyl 2′‐deoxyuridines (dU) were prepared. The ability of E. coli AGTs (Ada‐C and OGT), human AGT, and an OGT/hAGT chimera to remove O⁴‐methyl and larger adducts (4‐hydroxybutyl and 7‐hydroxyheptyl) from dU were examined and compared to those relating to the corresponding dT species. The absence of the C5 methyl group resulted in an increase in repair observed for the O⁴‐methyl adducts by hAGT and the chimera. The chimera was proficient at repairing larger adducts at the O⁴ atom of dU. There was no observed correlation between the binding affinities of the AGT homologues to adduct‐containing oligonucleotides and the amounts of repair measured.     

An Engineered Biocatalyst for the Synthesis of Glycoconjugates: Utilization of β1,3‐N‐Acetyl‐D‐glucosaminyltransferase from Streptococcus agalactiae Type Ia Expressed in Escherichia coli as a Fusion with Maltose‐Binding Protein

A fusion protein composed of β1,3‐N‐acetyl‐D ‐glucosaminyltransferase (β1,3‐GlcNAcT) from Streptococcus agalactiae type Ia and maltose‐binding protein (MBP) was produced in Escherichia coli as a soluble and highly active form. Although this fusion protein (MBP‐β1,3‐GlcNAcT) did not show any sugar‐elongation activity to some simple low‐molecular weight acceptor substrates such as galactose, Galβ(1→4)Glc (lactose), Galβ(1→4)GlcNAc (N‐acetyllactosamine), Galβ(1→4)GlcNAcβ(1→3)Galβ(1→4)Glc (lacto‐N‐tetraose), and Galβ(1→4)GlcβCer (lactosylceramide, LacCer), the multivalent glycopolymer having LacCer‐mimic branches (LacCer mimic polymer, LacCer primer) was found to be an excellent acceptor substrate for the introduction of a β‐GlcNAc residue at the O‐3 position of the non‐reducing galactose moiety by this engineered enzyme. Subsequently, the polymer having GlcNAcβ(1→3)Galβ(1→4)Glc was subjected to further enzymatic modifications by using recombinant β1,4‐D ‐galactosyltransferase (β1,4‐GalT), α2,3‐sialyltransferase (α2,3‐SiaT), α1,3‐L ‐fucosyltransferase (α1,3‐FucT), and ceramide glycanase (CGase) to afford a biologically important ganglioside; Neu5Aα(2→3)Galβ(1→4)[Fucα(1→3)]GlcNAcβ(1→3)Galβ(1→4)GlcCerα(IV3Neu5Acα,III3Fucα‐nLc4Cer) in 40% yield (4 steps). Interestingly, it was suggested that MBP‐β1,3‐GlcNAcT could also catalyze a glycosylation reaction of the LacCer mimic polymer with N‐acetyl‐D ‐galactosamine served from UDP‐GalNAc to afford a polymer carrying trisaccharide branches, GalNAcβ(1→3)Galβ(1→4)Glc. The versatility of the MBP‐β1,3‐GlcNAcT in the practical synthesis was preliminarily demonstrated by applying this fusion protein as an immobilized biocatalyst displayed on the amylose resin which is known as a solid support showing potent binding‐affinity with MBP.     

A Chemoenzymatic Histology Method for O‐GlcNAc Detection

Modification of nuclear and cytoplasmic proteins by the addition or removal of O‐GlcNAc dynamically impacts multiple biological processes. Here, we present the development of a chemoenzymatic histology method for the detection of O‐GlcNAc in tissue specimens. We applied this method to screen murine organs, uncovering specific O‐GlcNAc distribution patterns in different tissue structures. We then utilized our histology method for O‐GlcNAc detection in human brain specimens from healthy donors and donors with Alzheimer's disease and found higher levels of O‐GlcNAc in specimens from healthy donors. We also performed an analysis using a multiple cancer tissue array, uncovering different O‐GlcNAc levels between healthy and cancerous tissues, as well as different O‐GlcNAc cellular distributions within certain tissue specimens. This chemoenzymatic histology method therefore holds great potential for revealing the biology of O‐GlcNAc in physiopathological processes.     

Plant sn‐Glycerol‐3‐Phosphate Acyltransferases: Biocatalysts Involved in the Biosynthesis of Intracellular and Extracellular Lipids

Acyl‐lipids such as intracellular phospholipids, galactolipids, sphingolipids, and surface lipids play a crucial role in plant cells by serving as major components of cellular membranes, seed storage oils, and extracellular lipids such as cutin and suberin. Plant lipids are also widely used to make food, renewable biomaterials, and fuels. As such, enormous efforts have been made to uncover the specific roles of different genes and enzymes involved in lipid biosynthetic pathways over the last few decades. sn‐Glycerol‐3‐phosphate acyltransferases (GPAT) are a group of important enzymes catalyzing the acylation of sn‐glycerol‐3‐phosphate at the sn‐1 or sn‐2 position to produce lysophosphatidic acids. This reaction constitutes the first step of storage‐lipid assembly and is also important in polar‐ and extracellular‐lipid biosynthesis. Ten GPAT have been identified in Arabidopsis, and many homologs have also been reported in other plant species. These enzymes differentially localize to plastids, mitochondria, and the endoplasmic reticulum, where they have different biological functions, resulting in distinct metabolic fate(s) for lysophosphatidic acid. Although studies in recent years have led to new discoveries about plant GPAT, many gaps still exist in our understanding of this group of enzymes. In this article, we highlight current biochemical and molecular knowledge regarding plant GPAT, and also discuss deficiencies in our understanding of their functions in the context of plant acyl‐lipid biosynthesis.     

Specificity of Donor Structures for endo‐β‐N‐Acetylglucosaminidase‐Catalyzed Transglycosylation Reactions

To demonstrate the structural specificity of the glycosyl donor for the transglycosylation reaction by using endo‐β‐N‐acetylglucosaminidase from Mucor hiemalis (endo‐M), a series of tetrasaccharide oxazoline derivatives was synthesized. These derivatives correspond to the core structure of an asparagine‐linked glycoprotein glycan with a β‐mannose unit of a non‐natural‐type monosaccharide, including β‐glucose, β‐galactose, and β‐talose in place of the β‐mannose moiety. The transglycosylation activity of wildtype (WT) endo‐M and two mutants, N175Q and N175A, was examined by using these tetrasaccharide donors with p‐nitrophenyl N‐acetylglucosaminide (GlcNAc‐pNp). The essential configuration of the hydroxy group for the transglycosylation reaction was determined. On the basis of these results, the transglycosylation reaction was investigated by using chemically modified donors, and transglycosylated products were successfully obtained.     

Efficient Whole‐Cell Biocatalytic Synthesis of N‐Acetyl‐D‐neuraminic Acid

N‐Acetyl‐D ‐neuraminic acid (Neu5Ac) was efficiently synthesized from lactate and a mixture of N‐acetyl‐D ‐glucosamine (GlcNAc) and N‐acetyl‐D ‐mannosamine (ManNAc) by whole cells. The biotransformation utilized Escherichia coli cells (Neu5Ac aldolase), Pseudomonas stutzeri cells (lactate oxidase components), GlcNAc/ManNAc and lactate. By this process, 18.32±0.56 g/liter Neu5Ac were obtained from 65.61±2.70 g/liter lactate as an initial substrate input. Neu5Ac (98.4±0.4 % purity, 80.87±0.79 % recovery yield) was purified by anionic exchange chromatography. Our results demonstrate that the reported Neu5Ac biosynthetic process can compare favorably with natural product extraction or chemical synthesis processes.     

Recognition and Excision Properties of 8‐Halogenated‐7‐Deaza‐2′‐Deoxyguanosine as 8‐Oxo‐2′‐Deoxyguanosine Analogues and Fpg and hOGG1 Inhibitors

Cellular DNA continuously suffers various types of damage, and unrepaired damage increases disease progression risk. 8‐Oxo‐2′‐deoxyguanine (8‐oxo‐dG) is excised by repair enzymes, and their analogues are of interest as inhibitors and as bioprobes for study of these enzymes. We have developed 8‐halogenated‐7‐deaza‐2′‐deoxyguanosine derivatives that resemble 8‐oxo‐dG in that they adopt the syn conformation. In this study, we investigated their effects on Fpg (formamidopyrimidine DNA glycosylase) and hOGG1 (human 8‐oxoguanine DNA N‐glycosylase 1). Relative to 8‐oxo‐dG, Cl‐ and Br‐deaza‐dG were good substrates for Fpg, whereas they were less efficient substrates for hOGG1. Kinetics and binding experiments indicated that, although hOGG1 effectively binds Cl‐ and Br‐deaza‐dG analogues with low K_m values, their lower k_cat values result in low glycosylase activities. The benefits of the high binding affinities and low reactivities of 8‐oxo‐dG analogues with hOGG1 have been successfully applied to the competitive inhibition of the excision of 8‐oxoguanine from duplex DNA by hOGG1.     

Immobilization of lipase on poly(N‐vinyl‐2‐pyrrolidone‐co‐styrene) hydrogel

Lipase from Candida rugosa was immobilized by entrapment while polymerizing a poly(N‐vinyl‐2‐pyrrolidone‐co‐styrene) [poly(VP‐co‐ST)] hydrogel using ethylene dimethacrylate (EDMA) as the crosslinking agent. The immobilized enzymes were used in the esterification reaction of oleic acid and butanol in hexane. The activities of the immobilized enzymes and the leaching ability of the enzyme from the support with respect to the different compositions of the hydrogels were investigated. The thermal, solvent, and storage stability of the immobilized lipases were also determined. The activities were relatively higher when the percent compositions of VP(%):ST(%) were 50:50 and 30:70. The lipase immobilized on VP(%):ST(%) 50:50 showed the highest thermal stability, while lipase immobilized on VP(%):ST(%) 30:70 exhibited the highest solvent stability. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 82: 1404–1409, 2001     

Regiocomplementary O‐Methylation of Catechols by Using Three‐Enzyme Cascades

S‐Adenosylmethionine (SAM)‐dependent enzymes have great potential for selective alkylation processes. In this study we investigated the regiocomplementary O‐methylation of catechols. Enzymatic methylation is often hampered by the need for a stoichiometric supply of SAM and the inhibitory effect of the SAM‐derived byproduct on most methyltransferases. To counteract these issues we set up an enzyme cascade. Firstly, SAM was generated from l ‐methionine and ATP by use of an archaeal methionine adenosyltransferase. Secondly, 4‐O‐methylation of the substrates dopamine and dihydrocaffeic acid was achieved by use of SafC from the saframycin biosynthesis pathway in 40–70 % yield and high selectivity. The regiocomplementary 3‐O‐methylation was catalysed by catechol O‐methyltransferase from rat. Thirdly, the beneficial influence of a nucleosidase on the overall conversion was demonstrated. The results of this study are important milestones on the pathway to catalytic SAM‐dependent alkylation processes.     

Azide‐ and Alkyne‐Bearing Metabolic Chemical Reporters of Glycosylation Show Structure‐Dependent Feedback Inhibition of the Hexosamine Biosynthetic Pathway

Metabolic chemical reporters (MCRs) of protein glycosylation are analogues of natural monosaccharides that bear reactive groups, like azides and alkynes. When they are added to living cells and organisms, these small molecules are biosynthetically transformed into nucleotide donor sugars and then used by glycosyltransferases to modify proteins. Subsequent installation of tags by bioorthogonal chemistries can then enable the visualization and enrichment of these glycoproteins. Although this two‐step procedure is powerful, the use of MCRs has the potential to change the endogenous production of the natural repertoire of donor sugars. A major route for the generation of these glycosyltransferase substrates is the hexosamine biosynthetic pathway (HBP), which results in uridine diphosphate N‐acetylglucosamine (UDP‐GlcNAc). Interestingly, the rate‐determining enzyme of the HBP, glutamine fructose‐6‐phosphate amidotransferase (GFAT), is feedback inhibited by UDP‐GlcNAc. This raises the possibility that a build‐up of UDP‐MCRs would block the biosynthesis of UDP‐GlcNAc, resulting in off target effects. Here, we directly test this possibility with recombinant human GFAT and a small panel of synthetic UDP‐MCRs. We find that MCRs with larger substitutions at the N‐acetyl position do not inhibit GFAT, whereas those with modifications of the 2‐ or 6‐hydroxy group do. These results further illuminate the considerations that should be applied to the use of MCRs.     

NAD+‐Dependent Dehydrogenase PctP and Pyridoxal 5′‐Phosphate Dependent Aminotransferase PctC Catalyze the First Postglycosylation Modification of the Sugar Intermediate in Pactamycin Biosynthesis

The unique five‐membered aminocyclitol core of the antitumor antibiotic pactamycin originates from d ‐glucose, so unprecedented enzymatic modifications of the sugar intermediate are involved in the biosynthesis. However, the order of the modification reactions remains elusive. Herein, we examined the timing of introduction of an amino group into certain sugar‐derived intermediates by using recombinant enzymes that were encoded in the pactamycin biosynthesis gene cluster. We found that the NAD⁺‐dependent alcohol dehydrogenase PctP and pyridoxal 5′‐phosphate dependent aminotransferase PctC converted N‐acetyl‐d ‐glucosaminyl‐3‐aminoacetophonone into 3′‐amino‐3′‐deoxy‐N‐acetyl‐d ‐glucosaminyl‐3‐aminoacetophenone. Further, N‐acetyl‐d ‐glucosaminyl‐3‐aminophenyl‐β‐oxopropanoic acid ethyl ester was converted into the corresponding 3′‐amino derivative. However, PctP did not oxidize most of the tested d ‐glucose derivatives, including UDP‐GlcNAc. Thus, modification of the GlcNAc moiety in pactamycin biosynthesis appears to occur after the glycosylation of aniline derivatives.     

Monitoring of Intracellular Tau Aggregation Regulated by OGA/OGT Inhibitors

Abnormal phosphorylation of tau has been considered as a key pathogenic mechanism inducing tau aggregation in multiple neurodegenerative disorders, collectively called tauopathies. Recent evidence showed that tau phosphorylation sites are protected with O-linked β-N-acetylglucosamine (O-GlcNAc) in normal brain. In pathological condition, tau is de-glycosylated and becomes a substrate for kinases. Despite the importance of O-GlcNAcylation in tau pathology, O-GlcNAc transferase (OGT), and an enzyme catalyzing O-GlcNAc to tau, has not been carefully investigated in the context of tau aggregation. Here, we investigated intracellular tau aggregation regulated by BZX2, an inhibitor of OGT. Upon the inhibition of OGT, tau phosphorylation increased 2.0-fold at Ser199 and 1.5-fold at Ser396, resulting in increased tau aggregation. Moreover, the BZX2 induced tau aggregation was efficiently reduced by the treatment of Thiamet G, an inhibitor of O-GlcNAcase (OGA). Our results demonstrated the protective role of OGT in tau aggregation and also suggest the counter-regulatory mechanism of OGA and OGT in tau pathology.