Human O-GlcNAcase Uses a Preactivated Boat-skew Substrate Conformation for Catalysis. Evidence from X-ray Crystallography and QM/MM Metadynamics.

Human O-linked ß-N-acetylglucosaminidase (hOGA) is one of the two enzymes involved in nuclear and cytoplasmic protein O-GlcNAcylation, an essential post-translational modification. The enzyme catalyzes the hydrolysis of the GlcNAc-O-(Ser/Thr) glycosidic bonds via anchimeric assistance through the 2-acetamido group of the GlcNAc sugar. However, the conformational itinerary of the GlcNAc ring during catalysis remains unclear. Here we report the crystal structure of wild type hOGA in complex with a nonhydrolyzable glycopeptide substrate and elucidate the full enzyme catalytic mechanism using QM/MM metadynamics. We show that the enzyme can bind the substrate in either a chair- or a boat-like conformation, but only the latter is catalytically competent, leading to the reaction products via 1,4B/1S3 → [4E]‡ → 4C1 and 4C1 → [4E]‡ → 1,4B/1S3 conformational itineraries for the first and second catalytic reaction steps, respectively. Our results reconcile previous experimental observations for human and bacterial OGA and will aid the development of more effective OGA inhibitors for diseases associated with impaired O-GlcNAcylation.

The development of a broad-spectrum retaining ß-exo-galactosidase activity-based probe.

Acid ß-galactosidase (GLB1) and galactocerebrosidase (GALC) are retaining exo-ß-galactosidases involved in lysosomal glycoconjugate metabolism. Deficiency of GLB1 may result in the lysosomal storage disorders GM1 gangliosidosis, Morquio B syndrome, and galactosialidosis, and deficiency of GALC may result in Krabbe disease. Activity-based protein profiling (ABPP) is a powerful technique to assess the activity of retaining glycosidases in relation to health and disease. This work describes the use of fluorescent and biotin-carrying activity-based probes (ABPs) to assess the activity of both GLB1 and GALC in cell lysates, culture media, and tissue extracts. The reported ABPs, which complement the growing list of retaining glycosidase ABPs based on configurational isomers of cyclophellitol, should assist in fundamental and clinical research on various ß-galactosidases, whose inherited deficiencies cause debilitating lysosomal storage disorders.

Synthesis of biolabile thioalkyl-protected phosphates from an easily accessible phosphotriester precursor.

Robust methods for the synthesis of mixed phosphotriesters are essential to accelerate the development of novel phosphate-containing bioactive molecules. To enable efficient cellular uptake, phosphate groups are commonly masked with biolabile protecting groups, such as S-acyl-2-thioethyl (SATE) esters, that are removed once the molecule is inside the cell. Typically, bis-SATE-protected phosphates are synthesised through phosphoramidite chemistry. This approach, however, suffers from issues with hazardous reagents and can give unreliable yields, especially when applied to the synthesis of sugar-1-phosphate derivatives as tools for metabolic oligosaccharide engineering. Here, we report the development of an alternative approach that gives access to bis-SATE phosphotriesters in two steps from an easy to synthesise tri(2-bromoethyl)phosphotriester precursor. We demonstrate the viability of this strategy using glucose as a model substrate, onto which a bis-SATE-protected phosphate is introduced either at the anomeric position or at C6. We show compability with various protecting groups and further explore the scope and limitations of the methodology on different substrates, including N-acetylhexosamine and amino acid derivatives. The new approach facilitates the synthesis of bis-SATE-protected phosphoprobes and prodrugs and provides a platform that can boost further studies aimed at exploring the unique potential of sugar phosphates as research tools.

A Multiplexing Activity-Based Protein-Profiling Platform for Dissection of a Native Bacterial Xyloglucan-Degrading System.

Bacteria and yeasts grow on biomass polysaccharides by expressing and excreting a complex array of glycoside hydrolase (GH) enzymes. Identification and annotation of such GH pools, which are valuable commodities for sustainable energy and chemistries, by conventional means (genomics, proteomics) are complicated, as primary sequence or secondary structure alignment with known active enzymes is not always predictive for new ones. Here we report a "low-tech", easy-to-use, and sensitive multiplexing activity-based protein-profiling platform to characterize the xyloglucan-degrading GH system excreted by the soil saprophyte, Cellvibrio japonicus, when grown on xyloglucan. A suite of activity-based probes bearing orthogonal fluorophores allows for the visualization of accessory exo-acting glycosidases, which are then identified using biotin-bearing probes. Substrate specificity of xyloglucanases is directly revealed by imbuing xyloglucan structural elements into bespoke activity-based probes. Our ABPP platform provides a highly useful tool to dissect xyloglucan-degrading systems from various sources and to rapidly select potentially useful ones. The observed specificity of the probes moreover bodes well for the study of other biomass polysaccharide-degrading systems, by modeling probe structures to those of desired substrates.

Prolonged Normothermic Ex Vivo Kidney Perfusion Is Superior to Cold Nonoxygenated and Oxygenated Machine Perfusion for the Preservation of DCD Porcine Kidney Grafts.

The increased usage of marginal grafts has triggered interest in perfused kidney preservation to minimize graft injury. We used a donation after circulatory death (DCD) porcine kidney autotransplantation model to compare 3 of the most frequently used ex vivo kidney perfusion techniques: nonoxygenated hypothermic machine perfusion (non-oxHMP), oxygenated hypothermic machine perfusion (oxHMP), and normothermic ex vivo kidney perfusion (NEVKP). METHODS: Following 30 min of warm ischemia, grafts were retrieved and preserved with either 16 h of non-oxHMP, oxHMP, or NEVKP (n = 5 per group). After contralateral nephrectomy, grafts were autotransplanted and animals were followed for 8 d. Kidney function and injury markers were compared between groups. RESULTS: NEVKP demonstrated a significant reduction in preservation injury compared with either cold preservation method. Grafts preserved by NEVKP showed superior function with lower peak serum creatinine (NEVKP versus non-oxHMP versus oxHMP: 3.66 ± 1.33 mg/dL, 8.82 ± 3.17 mg/dL, and 9.02 ± 5.5 mg/dL) and more rapid recovery. The NEVKP group demonstrated significantly increased creatinine clearance on postoperative day 3 compared with the cold perfused groups. Tubular injury scores on postoperative day 8 were similar in all groups. CONCLUSIONS: Addition of oxygen during HMP did not reduce preservation injury of DCD kidney grafts. Grafts preserved with prolonged NEVKP demonstrated superior initial graft function compared with grafts preserved with non-oxHMP or oxHMP in a model of pig DCD kidney transplantation.

Chemical reporters to study mammalian O-glycosylation.

Glycans play essential roles in a range of cellular processes and have been shown to contribute to various pathologies. The diversity and dynamic nature of glycan structures and the complexities of glycan biosynthetic pathways make it challenging to study the roles of specific glycans in normal cellular function and disease. Chemical reporters have emerged as powerful tools to characterise glycan structures and monitor dynamic changes in glycan levels in a native context. A variety of tags can be introduced onto specific monosaccharides via the chemical modification of endogenous glycan structures or by metabolic or enzymatic incorporation of unnatural monosaccharides into cellular glycans. These chemical reporter strategies offer unique opportunities to study and manipulate glycan functions in living cells or whole organisms. In this review, we discuss recent advances in metabolic oligosaccharide engineering and chemoenzymatic glycan labelling, focusing on their application to the study of mammalian O-linked glycans. We describe current barriers to achieving glycan labelling specificity and highlight innovations that have started to pave the way to overcome these challenges.

Bioorthogonal Reactions in Activity-Based Protein Profiling.

Activity-based protein profiling (ABPP) is a powerful technique to label and detect active enzyme species within cell lysates, cells, or whole animals. In the last two decades, a wide variety of applications and experimental read-out techniques have been pursued in order to increase our understanding of physiological and pathological processes, to identify novel drug targets, to evaluate selectivity of drugs, and to image probe targets in cells. Bioorthogonal chemistry has substantially contributed to the field of ABPP, as it allows the introduction of tags, which may be bulky or have unfavorable physicochemical properties, at a late stage in the experiment. In this review, we give an overview of the bioorthogonal reactions that have been implemented in ABPP, provide examples of applications of bioorthogonal chemistry in ABPP, and share some thoughts on future directions.

Tandem Bioorthogonal Labeling Uncovers Endogenous Cotranslationally O-GlcNAc Modified Nascent Proteins.

Hundreds of nuclear, cytoplasmic, and mitochondrial proteins within multicellular eukaryotes have hydroxyl groups of specific serine and threonine residues modified by the monosaccharide N-acetylglucosamine (GlcNAc). This modification, known as O-GlcNAc, has emerged as a central regulator of both cell physiology and human health. A key emerging function of O-GlcNAc appears to be to regulate cellular protein homeostasis. We previously showed, using overexpressed model proteins, that O-GlcNAc modification can occur cotranslationally and that this process prevents premature degradation of such nascent polypeptide chains. Here, we use tandem metabolic engineering strategies to label endogenously occurring nascent polypeptide chains within cells using O-propargyl-puromycin (OPP) and target the specific subset of nascent chains that are cotranslationally glycosylated with O-GlcNAc by metabolic saccharide engineering using tetra-O-acetyl-2-N-azidoacetyl-2-deoxy-d-galactopyranose (Ac4GalNAz). Using various combinations of sequential chemoselective ligation strategies, we go on to tag these analytes with a series of labels, allowing us to define conditions that enable their robust labeling. Two-step enrichment of these glycosylated nascent chains, combined with shotgun proteomics, allows us to identify a set of endogenous cotranslationally O-GlcNAc modified proteins. Using alternative targeted methods, we examine three of these identified proteins and further validate their cotranslational O-GlcNAcylation. These findings detail strategies to enable isolation and identification of extremely low abundance endogenous analytes present within complex protein mixtures. Moreover, this work opens the way to studies directed at understanding the roles of O-GlcNAc and other cotranslational protein modifications and should stimulate an improved understanding of the role of O-GlcNAc in cytoplasmic protein quality control and proteostasis.

Direct One-Step Fluorescent Labeling of O-GlcNAc-Modified Proteins in Live Cells Using Metabolic Intermediates.

The modification of proteins with O-linked N-acetylglucosamine ( O-GlcNAc) by the enzyme O-GlcNAc transferase (OGT) has emerged as an important regulator of cellular physiology. Metabolic labeling strategies to monitor O-GlcNAcylation in cells have proven of great value for uncovering the molecular roles of O-GlcNAc. These strategies rely on two-step labeling procedures, which limits the scope of experiments that can be performed. Here, we report on the creation of fluorescent uridine 5'-diphospho- N-acetylglucosamine (UDP-GlcNAc) analogues in which the N-acyl group of glucosamine is modified with a suitable linker and fluorophore. Using human OGT, we show these donor sugar substrates permit direct monitoring of OGT activity on protein substrates in vitro. We show that feeding cells with a corresponding fluorescent metabolic precursor for the last step of the hexosamine biosynthetic pathway (HBP) leads to its metabolic assimilation and labeling of O-GlcNAcylated proteins within live cells. This one-step metabolic feeding strategy permits labeling of O-GlcNAcylated proteins with a fluorescent glucosamine-nitrobenzoxadiazole (GlcN-NBD) conjugate that accumulates in a time- and dose-dependent manner. Because no genetic engineering of cells is required, we anticipate this strategy should be generally amenable to studying the roles of O-GlcNAc in cellular physiology as well as to gain an improved understanding of the regulation of OGT within cells. The further expansion of this one-step in-cell labeling strategy should enable performing a range of experiments including two-color pulse chase experiments and monitoring OGT activity on specific protein substrates in live cells.

Structural and functional insight into human O-GlcNAcase.

O-GlcNAc hydrolase (OGA) removes O-linked N-acetylglucosamine (O-GlcNAc) from a myriad of nucleocytoplasmic proteins. Through co-expression and assembly of OGA fragments, we determined the three-dimensional structure of human OGA, revealing an unusual helix-exchanged dimer that lays a structural foundation for an improved understanding of substrate recognition and regulation of OGA. Structures of OGA in complex with a series of inhibitors define a precise blueprint for the design of inhibitors that have clinical value.

A Specific Activity-Based Probe to Monitor Family GH59 Galactosylceramidase, the Enzyme Deficient in Krabbe Disease.

Galactosylceramidase (GALC) is the lysosomal ß-galactosidase responsible for the hydrolysis of galactosylceramide. Inherited deficiency in GALC causes Krabbe disease, a devastating neurological disorder characterized by accumulation of galactosylceramide and its deacylated counterpart, the toxic sphingoid base galactosylsphingosine (psychosine). We report the design and application of a fluorescently tagged activity-based probe (ABP) for the sensitive and specific labeling of active GALC molecules from various species. The probe consists of a ß-galactopyranose-configured cyclophellitol-epoxide core, conferring specificity for GALC, equipped with a BODIPY fluorophore at C6 that allows visualization of active enzyme in cells and tissues. Detection of residual GALC in patient fibroblasts holds great promise for laboratory diagnosis of Krabbe disease. We further describe a procedure for in situ imaging of active GALC in murine brain by intra-cerebroventricular infusion of the ABP. In conclusion, this GALC-specific ABP should find broad applications in diagnosis, drug development, and evaluation of therapy for Krabbe disease.

Regulation of immunoproteasome function in the lung.

Impaired immune function contributes to the development of chronic obstructive pulmonary disease (COPD). Disease progression is further exacerbated by pathogen infections due to impaired immune responses. Elimination of infected cells is achieved by cytotoxic CD8(+) T cells that are activated by MHC I-mediated presentation of pathogen-derived antigenic peptides. The immunoproteasome, a specialized form of the proteasome, improves generation of antigenic peptides for MHC I presentation thereby facilitating anti-viral immune responses. However, immunoproteasome function in the lung has not been investigated in detail yet. In this study, we comprehensively characterized the function of immunoproteasomes in the human and murine lung. Parenchymal cells of the lung express low constitutive levels of immunoproteasomes, while they are highly and specifically expressed in alveolar macrophages. Immunoproteasome expression is not altered in whole lung tissue of COPD patients. Novel activity-based probes and native gel analysis revealed that immunoproteasome activities are specifically and rapidly induced by IFNγ treatment in respiratory cells in vitro and by virus infection of the lung in mice. Our results suggest that the lung is potentially capable of mounting an immunoproteasome-mediated efficient adaptive immune response to intracellular infections.

From covalent glycosidase inhibitors to activity-based glycosidase probes.

Activity-based protein profiling has emerged as a powerful discovery tool in chemical biology and medicinal chemistry research. Success of activity-based protein profiling hinges on the presence of compounds that can covalently and irreversibly bind to enzymes, do so selectively in the context of complex biological samples, and subsequently report on the selected pool of proteins. Such tagged molecules featuring an electrophilic trap, termed activity-based probes, have been developed with most success for serine hydrolases and various protease families (serine proteases, cysteine proteases, proteasomes). This concept presents the current progress and future directions in the design of activity-based probes targeting retaining glycosidases, enzymes that employ a double displacement mechanism in the hydrolysis of glycosidic bonds with overall retention. In contrast to inverting glycosidases, retaining glycosidases form a covalent intermediate with their substrates during the catalytic process and are therefore amenable to activity-based protein profiling studies.

Potent and selective activity-based probes for GH27 human retaining α-galactosidases.

Lysosomal degradation of glycosphingolipids is mediated by the consecutive action of several glycosidases. Malfunctioning of one of these hydrolases can lead to a lysosomal storage disorder such as Fabry disease, which is caused by a deficiency in α-galactosidase A. Herein we describe the development of potent and selective activity-based probes that target retaining α-galactosidases. The fluorescently labeled aziridine-based probes 3 and 4 inhibit the two human retaining α-galactosidases αGal A and αGal B covalently and with high affinity. Moreover, they enable the visualization of the endogenous activity of both α-galactosidases in cell extracts, thereby providing a means to study the presence and location of active enzyme levels in different cell types, such as healthy cells versus those derived from Fabry patients.

Current developments in activity-based protein profiling.

Activity-based protein profiling (ABPP) has emerged as a powerful strategy to study the activity of enzymes in complex proteomes. The aim of ABPP is to selectively visualize only the active forms of particular enzymes using chemical probes termed activity-based probes (ABPs). These probes are directed to the active site of a particular target protein (or protein family) where they react in a mechanism-based manner with an active site residue. This results in the selective labeling of only the catalytically active form of the enzyme, usually in a covalent manner. Besides the monitoring of a specific enzymatic activity, ABPP strategies have also been used to identify and characterize (unknown) protein functions, to study up- and down-regulation of enzymatic activity in various disease states, to discover and evaluate putative new enzyme inhibitors, and to identify the protein targets of covalently binding natural products. In this Topical Review we will provide a brief overview of some of the recent developments in the field of ABPP.

Acylazetine as a dienophile in bioorthogonal inverse electron-demand Diels-Alder ligation.

A new bioorthogonal N-acylazetine tag, suitable for tetrazine mediated inverse electron-demand Diels-Alder conjugation, is developed. The tag is small and achiral. We demonstrate the usefulness of N-acylazetine-tetrazine based bioorthogonal chemistry in two-step activity-based protein profiling. The performance of the new tetrazinophile in the labeling of catalytically active proteasome subunits was comparable to that of the more sterically demanding norbornene tag.

Gaucher disease and Fabry disease: new markers and insights in pathophysiology for two distinct glycosphingolipidoses.

Gaucher disease (GD) and Fabry disease (FD) are two relatively common inherited glycosphingolipidoses caused by deficiencies in the lysosomal glycosidases glucocerebrosidase and alpha-galactosidase A, respectively. For both diseases enzyme supplementation is presently used as therapy. Cells and tissues of GD and FD patients are uniformly deficient in enzyme activity, but the two diseases markedly differ in cell types showing lysosomal accumulation of the glycosphingolipid substrates glucosylceramide and globotriaosylceramide, respectively. The clinical manifestation of Gaucher disease and Fabry disease is consequently entirely different and the response to enzyme therapy is only impressive in the case of GD patients. This review compares both glycosphingolipid storage disorders with respect to similarities and differences. Presented is an update on insights regarding pathophysiological mechanisms as well as recently available biochemical markers and diagnostic tools for both disorders. Special attention is paid to sphingoid bases of the primary storage lipids in both diseases. The value of elevated glucosylsphingosine in Gaucher disease and globotriaosylsphingosine in Fabry disease for diagnosis and monitoring of disease is discussed as well as the possible contribution of the sphingoid bases to (patho)physiology. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.

Relative quantification of proteasome activity by activity-based protein profiling and LC-MS/MS.

Activity-based protein profiling (ABPP) is a functional proteomics technique for directly monitoring the expression of active enzymes in cell extracts and living cells. The technique relies on irreversible inhibitors equipped with reactive groups (warheads) that covalently attach to the active site of enzymes and fluorescent or affinity tags for imaging and purification purposes, respectively. Here, a high-throughput and robust protocol for high-resolution quantitative activity-based proteasome profiling is described. We use both panreactive and subunit-specific fluorescent activity-based probes (ABPs) to quantify the proteasome activity in living cells, in the presence or absence of the potent proteasome inhibitor bortezomib. Active proteasome subunits from cell lysates are affinity-purified via a biotinylated ABP. Purification from live cells involves a two-step ABP approach using a reagent with a cell-permeable azide-warhead and postlysis installation of biotin. By means of liquid chromatography-mass spectrometry (LC-MS)-based proteomics, we can accurately identify the enriched proteins and the active site peptides of the enzymes, and relatively quantify all the proteasome activities in one experiment. The fluorescence ABPP protocols takes 2-3 d, and approximately 8-10 d are needed to complete the entire protocol.

Novel activity-based probes for broad-spectrum profiling of retaining ß-exoglucosidases in†situ and in†vivo.

A high-end label: Cyclophellitol aziridine-type activity-based probes allow for ultra-sensitive visualization of mammalian ß-glucosidases (GBA1, GBA2, GBA3, and LPH) as well as several non-mammalian ß-glucosidases (see picture). These probes offer new ways to study ß-exoglucosidases, and configurational isomers of the cyclophellitol aziridine core may give activity-based probes targeting other retaining glycosidase families.

Tuning the leaving group in 2-deoxy-2-fluoroglucoside results in improved activity-based retaining ß-glucosidase probes.

The potency of 2-deoxy-2-fluoroglycosides in activity-based profiling of human acid ß-glucosidase is drastically improved by introducing an N-phenyl trifluoroacetimidate leaving group at the anomeric center. Protonation by the general acid-base catalyst in the active site turned out to be a prerequisite, making the imidate probe a genuine mechanism-based glycosidase inactivator.