Showing papers by "Valentin Wittmann published in 2022"

Metabolic glycoengineering - exploring glycosylation with bioorthogonal chemistry.

Abstract: Glycans are involved in numerous biological recognition events. Being secondary gene products, their labeling by genetic methods - comparable to GFP labeling of proteins - is not possible. To overcome this limitation, metabolic glycoengineering (MGE, also known as metabolic oligosaccharide engineering, MOE) has been developed. In this approach, cells or organisms are treated with synthetic carbohydrate derivatives that are modified with a chemical reporter group. In the cytosol, the compounds are metabolized and incorporated into newly synthesized glycoconjugates. Subsequently, the reporter groups can be further derivatized in a bioorthogonal ligation reaction. In this way, glycans can be visualized or isolated. Furthermore, diverse targeting strategies have been developed to direct drugs, nanoparticles, or whole cells to a desired location. This review summarizes research in the field of MGE carried out in recent years. After an introduction to the bioorthogonal ligation reactions that have been used in in connection with MGE, an overview on carbohydrate derivatives for MGE is given. The last part of the review focuses on the many applications of MGE starting from mammalian cells to experiments with animals and other organisms.

Carba‐Sugar Analogs of Glucosamine‐6‐Phosphate: New Activators for the glmS Riboswitch

Abstract: Abstract Riboswitches are 5’‐untranslated mRNA regions mostly found in bacteria. They are promising drug targets to overcome emerging bacterial resistance against commonly used antibiotics. The glmS riboswitch is unique among the family of riboswitches as it is a ribozyme that undergoes self‐cleavage upon binding to glucosamine‐6‐phosphate (GlcN6P). Previously, we showed that carba glucosamine‐6‐phosphate (carba‐GlcN6P) induces self‐cleavage of the riboswitch with a potency similar to that of GlcN6P. Here, we report a synthetic approach to a new class of carba‐GlcN6P derivatives with an alkoxy substituent in the carba position. Key features of the synthesis are a ring closing metathesis followed by a hydroboration. The strategy gives access to libraries of carba‐GlcN6P derivatives. Ribozyme cleavage assays unraveled new activators for the glmS riboswitch from Listeria monocytogenes and Clostridium difficile.

Photoswitching Affinity and Mechanism of Multivalent Lectin Ligands

Abstract: Abstract Multivalent receptor–ligand binding is a key principle in a plethora of biological recognition processes. Immense binding affinities can be achieved with the correct spatial orientation of the ligands. Accordingly, the incorporation of photoswitches, which can be used to reversibly change the spatial orientation of molecules, into multivalent ligands is a means to alter the binding affinity and possibly also the binding mode of such ligands. We report a divalent ligand for the model lectin wheat germ agglutinin (WGA) containing an arylazopyrazole photoswitch. This switch, which has recently been introduced as an alternative to the more commonly used azobenzene moiety, is characterized by almost quantitative E/Z photoswitching in both directions, high quantum yields, and high thermal stability of the Z isomer. The ligand was designed in a way that only one of the isomers is able to bridge adjacent binding sites of WGA leading to a chelating binding mode. Photoswitching induces an unprecedentedly high change in lectin binding affinity as determined by isothermal titration calorimetry (ITC). Furthermore, additional dynamic light scattering (DLS) data suggest that the binding mode of the ligand changes from chelating binding of the E isomer to crosslinking binding of the Z isomer.

Use of metabolic glycoengineering and pharmacological inhibitors to assess lipid and protein sialylation on cells

Abstract: Metabolic glycoengineering (MGE) has been developed to visualize carbohydrates on live cells. The method allows the fluorescent labeling of sialic acid (Sia) sugar residues on neuronal plasma membranes. For instance, the efficiency of glycosylation along neurite membranes has been characterized as cell health measure in neurotoxicology. Using human dopaminergic neurons as model system, we asked here, whether it was possible to separately label diverse classes of biomolecules and to visualize them selectively on cells. Several approaches suggest that a large proportion of Sia rather incorporated in non‐protein components of cell membranes than into glycoproteins. We made use here of deoxymannojirimycin (dMM), a non‐toxic inhibitor of protein glycosylation, and of N‐butyl‐deoxynojirimycin (NBdNM) a well‐tolerated inhibitor of lipid glycosylation, to develop a method of differential labeling of sialylated membrane lipids (lipid‐Sia) or sialylated N‐glycosylated proteins (protein‐Sia) on live neurons. The time resolution at which Sia modification of lipids/proteins was observable was in the range of few hours. The approach was then extended to several other cell types. Using this technique of target‐specific MGE, we found that in dopaminergic or sensory neurons >60% of Sia is lipid bound, and thus polysialic acid‐neural cell adhesion molecule (PSA‐NCAM) cannot be considered the major sialylated membrane component. Different from neurons, most Sia was bound to protein in HepG2 hepatoma cells or in neural crest cells. Thus, our method allows visualization of cell‐specific sialylation processes for separate classes of membrane constituents.