Quantifying lysosomal glycosidase activity within cells using bis-acetal substrates.

Understanding the function and regulation of enzymes within their physiologically relevant milieu requires quality tools that report on their cellular activities. Here we describe a strategy for glycoside hydrolases that overcomes several limitations in the field, enabling quantitative monitoring of their activities within live cells. We detail the design and synthesis of bright and modularly assembled bis-acetal-based (BAB) fluorescence-quenched substrates, illustrating this strategy for sensitive quantitation of disease-relevant human α-galactosidase and α-N-acetylgalactosaminidase activities. We show that these substrates can be used within live patient cells to precisely measure the engagement of target enzymes by inhibitors and the efficiency of pharmacological chaperones, and highlight the importance of quantifying activity within cells using chemical perturbogens of cellular trafficking and lysosomal homeostasis. These BAB substrates should prove widely useful for interrogating the regulation of glycosidases within cells as well as in facilitating the development of therapeutics and diagnostics for this important class of enzymes.

Fluorogenic Photo-Crosslinking of Glycan-Binding Protein Recognition Using a Fluorinated Azido-Coumarin Fucoside.

Glycan recognition by glycan-binding proteins is central to the biology of all living organisms. The efficient capture and characterization of relatively weak non-covalent interactions remains an important challenge in various fields of research. Photoaffinity labeling strategies can create covalent bonds between interacting partners, and photoactive scaffolds such as benzophenone, diazirines and aryl azides have proved widely useful. Since their first introduction, relatively few improvements have been advanced and products of photoaffinity labeling remain difficult to detect. We report a fluorinated azido-coumarin scaffold which enables photolabeling under fast and mild activation, and which can leave a fluorescent tag on crosslinked species. Coupling this scaffold to an α-fucoside, we demonstrate fluorogenic photolabeling of glycan-protein interactions over a wide range of affinities. We expect this strategy to be broadly applicable to other chromophores and we envision that such "fluoro-crosslinkers" could become important tools for the traceable capture of non-covalent binding events.

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.

Genome-wide chemical mapping of O-GlcNAcylated proteins in Drosophila melanogaster.

N-Acetylglucosamine ß-O-linked to nucleocytoplasmic proteins (O-GlcNAc) is implicated in the regulation of gene expression in organisms, from humans to Drosophila melanogaster. Within Drosophila, O-GlcNAc transferase (OGT) is one of the Polycomb group proteins (PcGs) that act through Polycomb group response elements (PREs) to silence homeotic (HOX) and other PcG target genes. Using Drosophila, we identify new O-GlcNAcylated PcG proteins and develop an antibody-free metabolic feeding approach to chemoselectively map genomic loci enriched in O-GlcNAc using next-generation sequencing. We find that O-GlcNAc is distributed to specific genomic loci both in cells and in vivo. Many of these loci overlap with PREs, but O-GlcNAc is also present at other loci lacking PREs. Loss of OGT leads to altered gene expression not only at loci containing PREs but also at loci lacking PREs, including several heterochromatic genes. These data suggest that O-GlcNAc acts through multiple mechanisms to regulate gene expression in Drosophila.

Fixing the Conformation of Calix[4]arenes: When Are Three Carbons Not Enough?

Calix[4]arenes are unique macrocycles that through judicious functionalisation at the lower rim can be either fixed in one of four conformations or remain conformationally flexible. Introduction of propynyl or propenyl groups unexpectedly provides a new possibility; a unidirectional conformational switch, with the 1,3-alternate and 1,2-alternate conformers switching to the partial cone conformation, whilst the cone conformation is unchanged, under standard experimental conditions. Using 1 H NMR kinetic studies, rates of switching have been shown to be dependent on the starting conformation, upper-rim substituent, where reduction in bulk enables faster switching, solvent and temperature with 1,2-alternate conformations switching fastest. Ab initio calculations (DFT) confirmed the relative stabilities of the conformations and point towards the partial cone conformer being the most stable of the four. The potential impact on synthesis through the "click" reaction has been investigated and found not to be significant.

Carbohydrate Bis-acetal-Based Substrates as Tunable Fluorescence-Quenched Probes for Monitoring exo-Glycosidase Activity.

Tunable Förster resonance energy transfer (FRET)-quenched substrates are useful for monitoring the activity of various enzymes within their relevant physiological environments. Development of FRET-quenched substrates for exo-glycosidases, however, has been hindered by their constrained pocket-shaped active sites. Here we report the design of a new class of substrate that overcomes this problem. These Bis-Acetal-Based Substrates (BABS) bear a hemiacetal aglycon leaving group that tethers fluorochromes in close proximity, also positioning them distant from the active site pocket. Following cleavage of the glycosidic bond, the liberated hemiacetal spontaneously breaks down, leading to separation of the fluorophore and quencher. We detail the synthesis and characterization of GlcNAc-BABS, revealing a striking 99.9% quenching efficiency. These substrates are efficiently turned over by the human exo-glycosidase O-GlcNAcase (OGA). We find the hemiacetal leaving group rapidly breaks down, enabling quantitative monitoring of OGA activity. We expect this strategy to be broadly useful for the development of substrate probes for monitoring exo-glycosidases, as well as a range of other enzymes having constrained pocket-shaped active sites.

O-GlcNAc occurs cotranslationally to stabilize nascent polypeptide chains.

Nucleocytoplasmic glycosylation of proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) is recognized as a conserved post-translational modification found in all metazoans. O-GlcNAc has been proposed to regulate diverse cellular processes. Impaired cellular O-GlcNAcylation has been found to lead to decreases in the levels of various proteins, which is one mechanism by which O-GlcNAc seems to exert its varied physiological effects. Here we show that O-GlcNAcylation also occurs cotranslationally. This process protects nascent polypeptide chains from premature degradation by decreasing cotranslational ubiquitylation. Given that hundreds of proteins are O-GlcNAcylated within cells, our findings suggest that cotranslational O-GlcNAcylation may be a phenomenon regulating proteostasis of an array of nucleocytoplasmic proteins. These findings set the stage to assess whether O-GlcNAcylation has a role in protein quality control in a manner that bears similarity with the role played by N-glycosylation within the secretory pathway.

Stable Amide Activation of N-Acetylated Glycosamines for the Synthesis of Fused Polycyclic Glycomimetics.

N-Acetylation of carbohydrates is an underexplored target for chemoselective derivatization and generation of glycomimetic scaffolds. Through mild amide activation, we report that N-acetimidoyl heterocycles are stable in neutral or basic conditions yet are excellent leaving groups through acid catalysis. While this specific reactivity could prove broadly useful in amide activation strategies, stably activated N-acetylated sugars can also be diversified using libraries of hydrazides. We optimized an acid-catalyzed one-pot sequence that includes nucleophilic displacement, cyclodehydration, and intramolecular glycosylation to ultimately deliver pyranosides fused to morpholines or piperazines. This strategy of stable activation followed by acid-triggered reaction sequences exemplifies the efficient assembly of 3D-rich fused glycomimetic libraries.

Synthesis of multivalent carbohydrate-centered glycoclusters as nanomolar ligands of the bacterial lectin LecA from Pseudomonas aeruginosa.

A family of fifteen glycoclusters based on a cyclic oligo-(1→6)-ß-D-glucosamine core has been designed as potential inhibitors of the bacterial lectin LecA with various valencies (from 2 to 4) and linkers. Evaluation of their binding properties towards LecA has been performed by a combination of hemagglutination inhibition assays (HIA), enzyme-linked lectin assays (ELLA), and isothermal titration microcalorimetry (ITC). Divalent ligands displayed dissociation constants in the sub-micromolar range and tetravalent ligands displayed low nanomolar affinities for this lectin. The influence of the linker could also be demonstrated; aromatic moieties are the best scaffolds for binding to the lectin. The affinities observed in vitro were then correlated with molecular models to rationalize the possible binding modes of these glycoclusters with the bacterial lectin.

Synthesis of a library of fucosylated glycoclusters and determination of their binding toward Pseudomonas aeruginosa lectin B (PA-IIL) using a DNA-based carbohydrate microarray.

Pseudomonas aeruginosa (PA) is a Gram negative opportunistic pathogen and is the major pathogen encounter in the cystic fibrosis (CF) lung airways. It often leads to chronic respiratory infection despite aggressive antibiotic therapy due to the emergence of resistant strains and to the formation of biofilm. The lectin PA-IIL (LecB) is a fucose-specific lectin from PA suspected to be involved in host recognition/adhesion and in biofilm formation. Thus, it can be foreseen as a potential therapeutic target. Herein, 16 fucosylated glycoclusters with antenna-like, linear, or crown-like spatial arrangements were synthesized using a combination of DNA solid-phase synthesis and alkyne azide 1,3-dipolar cycloaddition (CuAAC). Their binding properties toward PA-IIL were then evaluated based on DNA directed immobilization (DDI) carbohydrate microarray. Our results suggested that the antenna-like scaffold was preferred to linear or crown-like glycoclusters. Among the crown-like carbohydrate centered fucosylated glycoclusters, mannose-based core was better than glucose- and galactose-based ones. The influence of the linker arm was also evaluated, and long linkers between fucoses and the core led to a slight better binding than the short ones.

Rational design and synthesis of optimized glycoclusters for multivalent lectin-carbohydrate interactions: influence of the linker arm.

The design of multivalent glycoclusters requires the conjugation of biologically relevant carbohydrate epitopes functionalized with linker arms to multivalent core scaffolds. The multigram-scale syntheses of three structurally modified triethyleneglycol analogues that incorporate amide moiety(ies) and/or a phenyl ring offer convenient access to a series of carbohydrate probes with different water solubilities and rigidities. Evaluation of flexibility and determination of preferred conformations were performed by conformational analysis. Conjugation of the azido-functionalized carbohydrates with tetra-propargylated core scaffolds afforded a library of 18 tetravalent glycoclusters, in high yields, by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The compounds were evaluated for their ability to bind to PA-IL (the LecA lectin from the opportunistic pathogen Pseudomonas aeruginosa). Biochemical evaluation through inhibition of hemagglutination assays (HIA), enzyme-linked lectin assays (ELLA), surface plasmon resonance (SPR), and isothermal titration microcalorimetry (ITC) revealed improved and unprecedented affinities for one of the monovalent probes (K(d)=5.8 µM) and also for a number of the tetravalent compounds that provide several new nanomolar ligands for this tetrameric lectin.

Nanoelectronic detection of lectin-carbohydrate interactions using carbon nanotubes.

We have used single-walled carbon nanotube field-effect transistor (NTFET) devices to probe the interactions between carbohydrates and their recognition proteins called lectins. These interactions are involved in a wide range of biological processes, such as cell-cell recognition, cell-matrix interaction as well as viral and bacterial infections. In our experiments, NTFETs were functionalized noncovalently with porphyrin-based glycoconjugates synthesized using "click" azide-alkyne chemistry, and change in electrical conductance was measured upon specific binding of two bacterial lectins that present different carbohydrate preference, namely PA-IL, PA-IIL from Pseudomonas aeruginosa and a plant lectin Concanavalin A. However, no significant change in the device characteristics was observed when the devices were exposed to other lectins with different specificity. Detection of PA-IL binding to galactosylated NTFETs was highly sensitive (2 nM) with a measured dissociation constant (K(d) = 6.8 µM) corresponding to literature data. Fluorescence microscopy, atomic force microscopy, UV-vis-NIR spectroscopy, and several control measurements confirmed the NTFET response to selective interactions between carbohydrates and lectins.

Measurement of enzymatic activity and specificity of human and avian influenza neuraminidases from whole virus by glycoarray and MALDI-TOF mass spectrometry.

Influenza neuraminidases hydrolyze the ketosidic linkage between N-acetylneuraminic acid and its adjacent galactose residue in sialosides. This enzyme is a tetrameric protein that plays a critical role in the release of progeny virions. Several methods have been described for the determination of neuraminidase activity, usually based on colorimetric, fluorescent, or chemiluminescent detection. However, only a few of these tests allow discrimination of the sialyl-linkage specificity (i.e., α2-3- versus α2-6-linked sialyllactosides) of the neuraminidase. Herein we report a glycoarray-based assay and a MALDI-TOF study for assessing the activity and specificity of two influenza neuraminidases on whole viruses. The human A(H3N2) and avian A(H5N2) neuraminidase activities were investigated. The results from both approaches demonstrated that α2-3 sialyllactoside was a better substrate than α2-6 sialyllactoside for both viruses and that H5N2 virus had a lower hydrolytic activity than H3N2.

Synthesis of dodecavalent fullerene-based glycoclusters and evaluation of their binding properties towards a bacterial lectin.

Multivalency is playing a major role in biological processes and particularly in lectin-carbohydrate interactions. The design of high-affinity ligands of lectins should provide molecules capable of interfering with these biological processes and potentially inhibit bacterial or viral infections. Azide-alkyne "click" chemistry was applied to the synthesis of dodecavalent fullerene-based glycoclusters. The conjugation could be efficiently performed from alkyne or azide functions on either partners (i.e. hexakis-fullerene adduct or glycoside). PA-IL is a bacterial lectin from the opportunistic pathogen Pseudomonas aeruginosa and is involved in the recognition of glycoconjugates on human tissues. The glycoclusters obtained were evaluated as ligands of PA-IL and for their potential for competing with its binding to glycosylated surfaces. The affinities measured by hemagglutination inhibition assay (HIA), enzyme-linked lectin assay (ELLA), and surface plasmon resonance (SPR) displayed a significant "glycoside cluster effect" with up to a 12,000-fold increase in binding when comparing a monovalent carbohydrate reference probe with a dodecavalent fullerene-based glycocluster, albeit with some differences depending on the analytical technique.

Selectivity among two lectins: probing the effect of topology, multivalency and flexibility of "clicked" multivalent glycoclusters.

The design of multivalent glycoconjugates has been developed over the past decades to obtain high-affinity ligands for lectin receptors. While multivalency frequently increases the affinity of a ligand for its lectin through the so-called "glycoside cluster effect", the binding profiles towards different lectins have been much less investigated. We have designed a series of multivalent galactosylated glycoconjugates and studied their binding properties towards two lectins, from plant and bacterial origins, to determine their potential selectivity. The synthesis was achieved through copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) under microwave activation between propargylated multivalent scaffolds and an azido-functionalised carbohydrate derivative. The interactions of two galactose-binding lectins from Pseudomonas aeruginosa (PA-IL) and Erythrina cristagalli (ECA) with the synthesized glycoclusters were studied by hemagglutination inhibition assays (HIA), surface plasmon resonance (SPR) and isothermal titration microcalorimetry (ITC). The results obtained illustrate the influence of the scaffold's geometry on the affinity towards the lectin and also on the relative potency in comparison with a monovalent galactoside reference probe.

CuAAC synthesis of resorcin[4]arene-based glycoclusters as multivalent ligands of lectins.

Synthetic multivalent glycoclusters show promise as anti-adhesives for the treatment of bacterial infections. Here we report the synthesis of a family of tetravalent galactose and lactose functionalised macrocycles based on the resorcin[4]arene core. The development of diastereoselective synthetic routes for the formation of lower-rim propargylated resorcin[4]arenes and their functionalistion via Cu-catalyzed azide-alkyne click chemistry is described. ELLA binding studies confirm that galactose sugar clusters are effective ligands for the PA-IL bacterial lectin of Pseudomonas aeruginosa while poor binding for the lactose-based monovalent probe and no binding could be measured for the multivalent glycoclusters was observed for the human galectin-1.

Achieving high affinity towards a bacterial lectin through multivalent topological isomers of calix[4]arene glycoconjugates.

A family of seven topologically isomeric calix[4]arene glycoconjugates was prepared through the synthesis of a series of alkyne-derivatised calix[4]arene precursors that are suitable for the attachment of sugar moieties by microwave-assisted copper(I)-catalysed azide-alkyne cycloaddition (CuAAC). The glycoconjugates thus synthesised comprised one mono-functionalised derivative, two 1,2- or 1,3-divalent regioisomers, one trivalent and three tetravalent topoisomers in the cone, partial cone or 1,3-alternate conformations. The designed glycoconjugates were evaluated as ligands for the galactose-binding lectin PA-IL from the opportunistic bacterium Pseudomonas aeruginosa, a major causative agent of lung infections in cystic fibrosis patients. Binding affinities were determined by isothermal titration calorimetry (ITC), and the interaction with the lectin was shown to be strongly dependant on both the valence and the topology. Whereas the trivalent conjugate displayed enhanced affinity when compared to a monosaccharide model, the tetravalent conjugates are to-date the highest-affinity ligands measured by ITC. The topologies presenting carbohydrates on both faces of calixarene are the most potent ones with dissociation constants of approximately 200 nM. Molecular modelling suggests that such a multivalent molecule can efficiently chelate two of the binding sites of the tetrameric lectin; this explains the 800-fold increase of affinity achieved by the tetravalent molecule. Surface plasmon resonance (SPR) experiments confirmed that this glycoconjugate is the strongest inhibitor for binding of PA-IL to galactosylated surfaces for potential applications as an anti-adhesive agent.

Catalytic Promiscuity of O-GlcNAc Transferase Enables Unexpected Metabolic Engineering of Cytoplasmic Proteins with 2-Azido-2-deoxy-glucose.

O-GlcNAc transferase (OGT) catalyzes the installation of N-acetylglucosamine (GlcNAc) O-linked to nucleocytoplasmic proteins (O-GlcNAc) within multicellular eukaryotes. OGT shows surprising tolerance for structural changes in the sugar component of its nucleotide sugar donor substrate, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Here, we find that OGT uses UDP-glucose to install O-linked glucose (O-Glc) onto proteins only 25-fold less efficiently than O-GlcNAc. Spurred by this observation, we show that OGT transfers 2-azido-2-deoxy-d-glucose (GlcAz) in vitro from UDP-GlcAz to proteins. Further, feeding cells with per-O-acetyl GlcAz (AcGlcAz), in combination with inhibition or inducible knockout of OGT, shows OGT-dependent modification of nuclear and cytoplasmic proteins with O-GlcAz as detected using microscopy, immunoblot, and proteomics. We find that O-GlcAz is reversible within cells, and an unidentified cellular enzyme exists to cleave O-Glc that can also process O-GlcAz. We anticipate that AcGlcAz will prove to be a useful tool to study the O-GlcNAc modification. We also speculate that, given the high concentration of UDP-Glc within certain mammalian tissues, O-Glc may exist within mammals and serve as a physiologically relevant modification.

Post-translational O-GlcNAcylation is essential for nuclear pore integrity and maintenance of the pore selectivity filter.

O-glycosylation of the nuclear pore complex (NPC) by O-linked N-acetylglucosamine (O-GlcNAc) is conserved within metazoans. Many nucleoporins (Nups) comprising the NPC are constitutively O-GlcNAcylated, but the functional role of this modification remains enigmatic. We show that loss of O-GlcNAc, induced by either inhibition of O-GlcNAc transferase (OGT) or deletion of the gene encoding OGT, leads to decreased cellular levels of a number of natively O-GlcNAcylated Nups. Loss of O-GlcNAc enables increased ubiquitination of these Nups and their increased proteasomal degradation. The decreased half-life of these deglycosylated Nups manifests in their gradual loss from the NPC and a downstream malfunction of the nuclear pore selective permeability barrier in both dividing and post-mitotic cells. These findings define a critical role of O-GlcNAc modification of the NPC in maintaining its composition and the function of the selectivity filter. The results implicate NPC glycosylation as a regulator of NPC function and reveal the role of conserved glycosylation of the NPC among metazoans.