"Mix and match" auto-assembly of glycosyltransferase domains delivers biocatalysts with improved substrate promiscuity.

Glycosyltransferases (GT) catalyze the glycosylation of bioactive natural products, including peptides and proteins, flavonoids, and sterols, and have been extensively used as biocatalysts to generate glycosides. However, the often narrow substrate specificity of wild-type GTs requires engineering strategies to expand it. The GT-B structural family is constituted by GTs that share a highly conserved tertiary structure in which the sugar donor and acceptor substrates bind in dedicated domains. Here, we have used this selective binding feature to design an engineering process to generate chimeric glycosyltransferases that combine auto-assembled domains from two different GT-B enzymes. Our approach enabled the generation of a stable dimer with broader substrate promiscuity than the parent enzymes that were related to relaxed interactions between domains in the dimeric GT-B. Our findings provide a basis for the development of a novel class of heterodimeric GTs with improved substrate promiscuity for applications in biotechnology and natural product synthesis.

A cell-permeable probe for the labelling of a bacterial glycosyltransferase and virulence factor.

Chemical probes for bacterial glycosyltransferases are of interest for applications such as tracking of expression levels, and strain profiling and identification. Existing probes for glycosyltransferases are typically based on sugar-nucleotides, whose charged nature limits their applicability in intact cells. We report the development of an uncharged covalent probe for the bacterial galactosyltransferase LgtC, and its application for the fluorescent labelling of this enzyme in recombinant form, cell lysates, and intact cells. The probe was designed by equipping a previously reported covalent LgtC inhibitor based on a pyrazol-3-one scaffold with a 7-hydroxycoumarin fluorophore. We show that this pyrazol-3-ones scaffold is surprisingly stable in aqueous media, which may have wider implications for the use of pyrazol-3-ones as chemical probes. We also show that the 7-hydroxycoumarin fluorophore leads to an unexpected improvement in activity, which could be exploited for the development of second generation analogues. These results will provide a basis for the development of LgtC-specific probes for the detection of LgtC-expressing bacterial strains.

Generation and Characterization of Native and Sialic Acid-Deficient IgE.

Efficient characterization of IgE antibodies and their glycan structures is required for understanding their function in allergy and in the emerging AllergoOncology field for antibody immunotherapy. We report the generation, glyco-profiling and functional analysis of native and sialic acid-deficient glyco-engineered human IgE. The antibodies produced from human embryonic kidney cells were purified via a human IgE class-specific affinity matrix and structural integrity was confirmed by SDS-PAGE and size-exclusion chromatography (SEC). Purified IgEs specific for the tumor-associated antigens Chondroitin Sulfate Proteoglycan 4 (CSPG4-IgE) and Human Epidermal Growth Factor Receptor 2 (HER2-IgE) were devoid of by-products such as free light chains. Using neuraminidase-A, we generated sialic acid-deficient CSPG4-IgE as example glyco-engineered antibody. Comparative glycan analyses of native and glyco-engineered IgEs by Hydrophilic interaction liquid chromatography (HILIC)-high performance liquid chromatography (HPLC) indicated loss of sialic acid terminal residues and differential glycan profiles. Native and glyco-engineered CSPG4-IgEs recognized Fc receptors on the surface of human FcεRI-expressing rat basophilic leukemia RBL-SX38 cells, and of CD23/FcεRII-expressing human RPMI-8866 B-lymphocytes and bound to CSPG4-expressing A2058 human melanoma cells, confirming Fab-mediated recognition. When cross-linked on the cell surface, both IgEs triggered RBL-SX38 degranulation. We demonstrate efficient generation and functional competence of recombinant native and sialic acid-deficient IgEs.

Defined stereoisomers of 2″-amino NAD+ and their activity against human sirtuins and a bacterial (ADP-ribosyl) transferase.

Nicotinamide adenine dinucleotide (NAD+) is an important biomolecule with essential roles at the intersection of energy metabolism, epigenetic regulation and cell signalling. Synthetic analogues of NAD+ are therefore of great interest as chemical tools for medicinal chemistry, chemical biology and drug discovery. Herein, we report the chemical synthesis and full analytical characterisation of three stereoisomers of 2″-amino NAD+, and their biochemical evaluation against two classes of NAD+-consuming enzymes: the human sirtuins 1-3, and the bacterial toxin TccC3. To rationalise the observed activities, molecular docking experiments were carried out with SIRT1 and SIRT2, which identified the correct orientation of the pyrophosphate linkage as a major determinant for activity in this series. These results, together with results from stability tests and a conformational analysis, allow, for the first time, a side-by-side comparison of the chemical and biochemical features, and analytical properties, of different 2″-amino NAD+ stereoisomers. Our findings provide insight into the recognition of co-substrate analogues by sirtuins, and will greatly facilitate the application of these important NAD+ analogues as chemical tool compounds for mechanistic studies with these as well as other NAD+-dependent enyzmes.

Glycoengineering of Therapeutic Antibodies with Small Molecule Inhibitors.

Monoclonal antibodies (mAbs) are one of the cornerstones of modern medicine, across an increasing range of therapeutic areas. All therapeutic mAbs are glycoproteins, i.e., their polypeptide chain is decorated with glycans, oligosaccharides of extraordinary structural diversity. The presence, absence, and composition of these glycans can have a profound effect on the pharmacodynamic and pharmacokinetic profile of individual mAbs. Approaches for the glycoengineering of therapeutic mAbs-the manipulation and optimisation of mAb glycan structures-are therefore of great interest from a technological, therapeutic, and regulatory perspective. In this review, we provide a brief introduction to the effects of glycosylation on the biological and pharmacological functions of the five classes of immunoglobulins (IgG, IgE, IgA, IgM and IgD) that form the backbone of all current clinical and experimental mAbs, including an overview of common mAb expression systems. We review selected examples for the use of small molecule inhibitors of glycan biosynthesis for mAb glycoengineering, we discuss the potential advantages and challenges of this approach, and we outline potential future applications. The main aim of the review is to showcase the expanding chemical toolbox that is becoming available for mAb glycoengineering to the biology and biotechnology community.

Insights from IgE Immune Surveillance in Allergy and Cancer for Anti-Tumour IgE Treatments.

IgE, the predominant antibody class of the allergic response, is known for its roles in protecting against parasites; however, a growing body of evidence indicates a significant role for IgE and its associated effector cells in tumour immunosurveillance, highlighted by the field of AllergoOncology and the successes of the first-in-class IgE cancer therapeutic MOv18. Supporting this concept, substantial epidemiological data ascribe potential roles for IgE, allergy, and atopy in protecting against specific tumour types, with a corresponding increased cancer risk associated with IgE immunodeficiency. Here, we consider how epidemiological data in combination with functional data reveals a complex interplay of IgE and allergy with cancer, which cannot be explained solely by one of the existing conventional hypotheses. We furthermore discuss how, in turn, such data may be used to inform future therapeutic approaches, including the clinical management of different patient groups. With epidemiological findings highlighting several high-risk cancer types protected against by high IgE levels, it is possible that use of IgE-based therapeutics for a range of malignant indications may offer efficacy to complement that of established IgG-class antibodies.

Profiling protein expression in Klebsiella pneumoniae with a carbohydrate-based covalent probe.

We report the application of a covalent probe based on a d-glucosamine scaffold for the profiling of the bacterial pathogen Klebsiella pneumoniae. Incubation of K. pneumoniae lysates with the probe followed by electrophoretic separation and in-gel fluorescence detection allowed the generation of strain-specific signatures and the differentiation of a carbapenem-resistant strain. The labelling profile of the probe was independent of its anomeric configuration and included several low-abundance proteins not readily detectable by conventional protein staining. Initial target identification experiments by mass spectrometry suggest that target proteins include several carbohydrate-recognising proteins, which indicates that the sugar scaffold may have a role for target recognition.

Profiling of Haemophilus influenzae strain R2866 with carbohydrate-based covalent probes.

We demonstrate the application of four covalent probes based on anomerically pure d-galactosamine and d-glucosamine scaffolds for the profiling of Haemophilus influenzae strain R2866. The probes have been used successfully for the labelling of target proteins not only in cell lysates, but also in intact cells. Differences in the labelling patterns between lysates and intact cells indicate that the probes can penetrate into the periplasm, but not the cytoplasm of H. influenzae. Analysis of selected target proteins by LC-MS/MS suggests predominant labelling of nucleotide-binding proteins, including several known antibacterial drug targets. Our protocols will aid the identification of molecular determinants of bacterial pathogenicity in Haemophilus influenzae and other bacterial pathogens.

Novel disaccharide inhibitors for the bacterial galactosyltransferase LgtC: Design, synthesis via Heyns rearrangement, and biochemical evaluation.

Bacterial glycosyltransferases are potential targets for the development of novel antibiotics and anti-virulence agents. We report a novel inhibitor design for the retaining α-1,4-galactosyltransferase LgtC from Neisseria meningitidis. Our design is based on the installation of an electrophilic warhead on the LgtC acceptor substrate and targeted at a non-catalytic cysteine residue in the LgtC active site. We have successfully synthesised two prototype inhibitors in four steps from lactulose. The key step in our synthesis is a Heyns rearrangement, during which we observed the formation of a hitherto unknown side product. While both lactosamine derivatives behaved as moderate inhibitors of LgtC, they also retained residual substrate activity. These results suggest that in contrast to our original design, these inhibitors do not act via a covalent mode of action, but are most likely non-covalent inhibitors.

Identification of non-substrate-like glycosyltransferase inhibitors from library screening: pitfalls & hits.

Bacterial glycosyltransferases are potential targets for the development of novel antibiotics and anti-virulence agents. Most existing glycosyltransferase inhibitors are substrate analogues with limited potential for drug development. The identification of alternative inhibitor chemotypes is therefore of great interest for medicinal chemistry, drug discovery and chemical glycobiology. We describe the application of a biochemical glycosyltransferase assay to screen a small compound library containing three distinct chemical scaffolds (nucleosides, steroids and 5-methyl pyrazol-3-ones) against the retaining α-1,4-galactosyltransferase LgtC from Neisseria meningitidis. While no genuine LgtC inhibitory activity was observed in the nucleoside and steroid series, the best hit compounds in the 5-methyl pyrazol-3-one series showed low micromolar activity. We adapted our assay protocol to develop initial structure-activity relationships in this series, and to establish the target selectivity of the most potent inhibitor over two other glycosyltransferases. Our results provide insights into the activity of this class of non-substrate-like glycosyltransferase inhibitors, and highlight important general pitfalls for inhibitor screening against this enzyme family. Key elements of our experimental design, including a validated single-concentration protocol for inhibitor screening, and our process for elimination of false positives, are, in principle, directly transferable to many other sugar-nucleotide-dependent glycosyltransferases.

Structure-activity relationships in a new class of non-substrate-like covalent inhibitors of the bacterial glycosyltransferase LgtC.

Lipooligosaccharide (LOS) structures in the outer core of Gram-negative mucosal pathogens such as Neisseria meningitidis and Haemophilus influenzae contain characteristic glycoepitopes that contribute significantly to bacterial virulence. An important example is the digalactoside epitope generated by the retaining α-1,4-galactosyltransferase LgtC. These digalactosides camouflage the pathogen from the host immune system and increase its serum resistance. Small molecular inhibitors of LgtC are therefore sought after as chemical tools to study bacterial virulence, and as potential candidates for anti-virulence drug discovery. We have recently discovered a new class of non-substrate-like inhibitors of LgtC. The new inhibitors act via a covalent mode of action, targeting a non-catalytic cysteine residue in the LgtC active site. Here, we describe, for the first time, structure-activity relationships for this new class of glycosyltransferase inhibitors. We have carried out a detailed analysis of the inhibition kinetics to establish the relative contribution of the non-covalent binding and the covalent inactivation steps for overall inhibitory activity. Selected inhibitors were also evaluated against a serum-resistant strain of Haemophilus influenzae, but did not enhance the killing effect of human serum.

Covalent Probes for Carbohydrate-Active Enzymes: From Glycosidases to Glycosyltransferases.

Covalent probes for glycosidases and glycosyltransferases are of great interest as tool compounds for chemical biology. For glycosidases, a sizable number of such probes have been developed from covalent glycosidase inhibitors. We review selected recent examples and highlight different design strategies, including probes based on photoaffinity labels and mechanism-based inhibitors, as well as their applications in biology and for activity-based protein profiling. In contrast to glycosidases, only a limited number of covalent probes have been reported to date for glycosyltransferases. We describe a new class of covalent probes for the retaining α-1,4-galactosyltransferase LgtC from Neisseria meningitidis. On the basis of these probes, we have developed an operationally simple two-step protocol for the fluorescent labeling of recombinant LgtC both in purified form and in cell lysates. In principle, our approach is also applicable to other bacterial glycosyltransferases. Among other applications, our protocol may therefore be particularly useful for imaging of the differential expression of these enzymes in different bacterial species and strains.

Interaction of Neisseria meningitidis Group X N-acetylglucosamine-1-phosphotransferase with its donor substrate.

Neisseria meningitidis Group X is an emerging cause of bacterial meningitis in Sub-Saharan Africa. The capsular polysaccharide of Group X is a homopolymer of N-acetylglucosamine α(1-4) phosphate and is a vaccine target for prevention of disease associated with this meningococcal serogroup. We have demonstrated previously that the formation of the polymer is catalyzed by a phosphotransferase which transfers N-acetylglucosamine-1-phosphate from UDP-N-acetylglucosamine to the 4-hydroxyl of the N-acetylglucosamine on the nonreducing end of the growing chain. In this study, we use substrate analogs of UDP-GlcNAc to define the enzyme/donor substrate interactions critical for catalysis. Our kinetic analysis of the phosphotransferase reaction is consistent with a sequential mechanism of substrate addition and product release. The use of novel uracil modified analogs designed by Wagner et al. enabled us to assess whether the CsxA-catalyzed reaction is consistent with a donor dependent conformational change. As expected with this model for glycosyltransferases, UDP-GlcNAc analogs with bulky uracil modifications are not substrates but are inhibitors. An analog with a smaller iodo uracil substitution is a substrate and a less potent inhibitor. Moreover, our survey of analogs with modifications on the N-acetylglucosamine residue of the sugar nucleotide donor highlights the importance of substituents at C2 and C4 of the sugar residue. The hydroxyl group at C4 and the structure of the acyl group at C2 are very important for specificity and substrate interactions during the polymerization reaction. While most analogs modified at C2 were inhibitors, acetamido analogs were also substrates suggesting the importance of the carbonyl group.

Base-modified GDP-mannose derivatives and their substrate activity towards a yeast mannosyltransferase.

We have previously developed a new class of inhibitors and chemical probes for glycosyltransferases through base-modification of the sugar-nucleotide donor. The key feature of these donor analogues is the presence of an additional substituent at the nucleobase. To date, the application of this general concept has been limited to UDP-sugars and UDP-sugar-dependent glycosyltransferases. Herein, we report for the first time the application of our approach to a GDP-mannose-dependent mannosyltransferase. We have prepared four GDP-mannose derivatives with an additional substituent at either position 6 or 8 of the nucleobase. These donor analogues were recognised as donor substrates by the mannosyltransferase Kre2p from yeast, albeit with significantly lower turnover rates than the natural donor GDP-mannose. The presence of the additional substituent also redirected enzyme activity from glycosyl transfer to donor hydrolysis. Taken together, our results suggest that modification of the donor nucleobase is, in principle, a viable strategy for probe and inhibitor development against GDP-mannose-dependent GTs.

An acceptor analogue of ß-1,4-galactosyltransferase: Substrate, inhibitor, or both?

Many glycosyltransferase inhibitors in the literature are structurally derived from the donor or acceptor substrate of the respective enzyme. A representative example is 2-naphthyl ß-d-GlcNAc, a synthetic GlcNAc glycoside that has been reported as a galactosyltransferase inhibitor. This GlcNAc derivative is attractive as a chemical tool compound for biological and biochemical studies because of its reported potency as an inhibitor, and its short and straightforward synthesis from readily available starting materials. We report that in our hands, 2-naphthyl ß-d-GlcNAc behaved, unexpectedly, as an acceptor substrate of the inverting ß-1,4-galactosyltransferase (ß-1,4-GalT) from bovine milk. This substrate activity has not previously been described. We found that 2-naphthyl ß-d-GlcNAc can be an acceptor substrate both for recombinantly expressed ß-1,4-GalT, and for a commercial batch of the same enzyme, and both in the presence and absence of bovine serum albumin (BSA). As expected for a full acceptor substrate, this substrate activity was time- and concentration-dependent. Additional experiments show that the observed inhibitor/substrate switch is facilitated by a phosphatase that is an essential component of our enzyme-coupled glycosyltransferase assay. These findings suggest that the behaviour of 2-naphthyl ß-d-GlcNAc and related acceptor-based glycosyltransferase inhibitors is strongly dependent on the individual assay conditions. Our results therefore have important implications for the use of 2-naphthyl ß-d-GlcNAc and related glycosides as tool compounds in glycobiology and glycobiochemistry.

Covalent inhibitors of LgtC: A blueprint for the discovery of non-substrate-like inhibitors for bacterial glycosyltransferases.

Non-substrate-like inhibitors of glycosyltransferases are sought after as chemical tools and potential lead compounds for medicinal chemistry, chemical biology and drug discovery. Here, we describe the discovery of a novel small molecular inhibitor chemotype for LgtC, a retaining α-1,4-galactosyltransferase involved in bacterial lipooligosaccharide biosynthesis. The new inhibitors, which are structurally unrelated to both the donor and acceptor of LgtC, have low micromolar inhibitory activity, comparable to the best substrate-based inhibitors. We provide experimental evidence that these inhibitors react covalently with LgtC. Results from detailed enzymological experiments with wild-type and mutant LgtC suggest the non-catalytic active site residue Cys246 as a likely target residue for these inhibitors. Analysis of available sequence and structural data reveals that non-catalytic cysteines are a common motif in the active site of many bacterial glycosyltransferases. Our results can therefore serve as a blueprint for the rational design of non-substrate-like, covalent inhibitors against a broad range of other bacterial glycosyltransferases.

Biochemical and Structural Characterisation of DNA Ligases from Bacteria and Archaea.

DNA ligases are enzymes that seal breaks in the backbones of DNA, leading to them being essential for the survival of all organisms. DNA ligases have been studied from many different types of cells and organisms and shown to have diverse sizes and sequences, with well conserved specific sequences that are required for enzymatic activity. A significant number of DNA ligases have been isolated or prepared in recombinant forms and, here, we review their biochemical and structural characterisation. All DNA ligases contain an essential lysine that transfers an adenylate group from a co-factor to the 5'-phosphate of the DNA end that will ultimately be joined to the 3'-hydroxyl of the neighbouring DNA strand. The essential DNA ligases in bacteria use nicotinamide adenine dinucleotide ( ß -NAD+) as their co-factor whereas those that are essential in other cells use adenosine-5'-triphosphate (ATP) as their co-factor. This observation suggests that the essential bacterial enzyme could be targeted by novel antibiotics and the complex molecular structure of ß -NAD+ affords multiple opportunities for chemical modification. Several recent studies have synthesised novel derivatives and their biological activity against a range of DNA ligases has been evaluated as inhibitors for drug discovery and/or non-natural substrates for biochemical applications. Here, we review the recent advances that herald new opportunities to alter the biochemical activities of these important enzymes. The recent development of modified derivatives of nucleotides highlights that the continued combination of structural, biochemical and biophysical techniques will be useful in targeting these essential cellular enzymes.

Base-modified UDP-sugars reduce cell surface levels of P-selectin glycoprotein 1 (PSGL-1) on IL-1ß-stimulated human monocytes.

P-selectin glycoprotein ligand-1 (PSGL-1, CD162) is a cell-surface glycoprotein that is expressed, either constitutively or inducibly, on all myeloid and lymphoid cell lineages. PSGL-1 is implicated in cell-cell interactions between platelets, leukocytes and endothelial cells, and a key mediator of inflammatory cell recruitment and transmigration into tissues. Here, we have investigated the effects of the ß-1,4-galactosyltransferase inhibitor 5-(5-formylthien-2-yl) UDP-Gal (5-FT UDP-Gal, compound 1: ) and two close derivatives on the cell surface levels of PSGL-1 on human peripheral blood mononuclear cells (hPBMCs). PSGL-1 levels were studied both under basal conditions, and upon stimulation of hPBMCs with interleukin-1ß (IL-1ß). Between 1 and 24 hours after IL-1ß stimulation, we observed initial PSGL-1 shedding, followed by an increase in PSGL-1 levels on the cell surface, with a maximal window between IL-1ß-induced and basal levels after 72 h. All three inhibitors reduce PSGL-1 levels on IL-1ß-stimulated cells in a concentration-dependent manner, but show no such effect in resting cells. Compound 1: also affects the cell surface levels of adhesion molecule CD11b in IL-1ß-stimulated hPBMCs, but not of glycoproteins CD14 and CCR2. This activity profile may be linked to the inhibition of global Sialyl Lewis presentation on hPBMCs by compound 1: , which we have also observed. Although this mechanistic explanation remains hypothetical at present, our results show, for the first time, that small molecules can discriminate between IL-1ß-induced and basal levels of cell surface PSGL-1. These findings open new avenues for intervention with PSGL-1 presentation on the cell surface of primed hPBMCs and may have implications for anti-inflammatory drug development.