Salmonid polysialyltransferases to generate a variety of sialic acid polymers.

The human polysialyltransferases ST8Sia II and ST8Sia IV catalyze the transfer of several Neu5Ac residues onto glycoproteins forming homopolymers with essential roles during different physiological processes. In salmonids, heterogeneous set of sialic acids polymers have been described in ovary and on eggs cell surface and three genes st8sia4, st8sia2-r1 and st8sia2-r2 were identified that could be implicated in these heteropolymers. The three polysialyltransferases from the salmonid Coregonus maraena were cloned, recombinantly expressed in HEK293 cells and the ST8Sia IV was biochemically characterized. The MicroPlate Sialyltransferase Assay and the non-natural donor substrate CMP-SiaNAl were used to demonstrate enzyme activity and optimize polysialylation reactions. Polysialylation was also carried out with natural donor substrates CMP-Neu5Ac, CMP-Neu5Gc and CMP-Kdn in cell-free and cell-based assays and structural analyses of polysialylated products using the anti-polySia monoclonal antibody 735 and endoneuraminidase N and HPLC approaches. Our data highlighted distinct specificities of human and salmonid polysialyltransferases with notable differences in donor substrates use and the capacity of fish enzymes to generate heteropolymers. This study further suggested an evolution of the biological functions of polySia. C. maraena ST8Sia IV of particular interest to modify glycoproteins with a variety of polySia chains.

A bioorthogonal chemistry approach to detect the K1 polysialic acid capsule in Escherichia coli.

Most Escherichia coli strains associated with neonatal meningitis express the K1 capsule, a sialic acid polysaccharide that is directly related to their pathogenicity. Metabolic oligosaccharide engineering (MOE) has mostly been developed in eukaryotes, but has also been successfully applied to the study of several oligosaccharides or polysaccharides constitutive of the bacterial cell wall. However, bacterial capsules are seldom targeted despite their important role as virulence factors, and the K1 polysialic acid (PSA) antigen that shields bacteria from the immune system still remains untackled. Herein, we report a fluorescence microplate assay that allows the fast and facile detection of K1 capsules with an approach that combines MOE and bioorthogonal chemistry. We exploit the incorporation of synthetic analogues of N-acetylmannosamine or N-acetylneuraminic acid, metabolic precursors of PSA, and copper-catalysed azide-alkyne cycloaddition (CuAAC) as the click chemistry reaction to specifically label the modified K1 antigen with a fluorophore. The method was optimized, validated by capsule purification and fluorescence microscopy, and applied to the detection of whole encapsulated bacteria in a miniaturized assay. We observe that analogues of ManNAc are readily incorporated into the capsule while those of Neu5Ac are less efficiently metabolized, which provides useful information regarding the capsule biosynthetic pathways and the promiscuity of the enzymes involved. Moreover, this microplate assay is transferable to screening approaches and may provide a platform to identify novel capsule-targeted antibiotics that would circumvent resistance issues.

Switching azide and alkyne tags on bioorthogonal reporters in metabolic labeling of sialylatedglycoconjugates: a comparative study.

Sialylation of cell surface glycans plays an essential role in cell-cell interaction and communication of cells with their microenvironment. Among the tools that have been developed for the study of sialylation in living cells, metabolic oligosaccharide engineering (MOE) exploits the biosynthetic pathway of sialic acid (Sia) to incorporate unnatural monosaccharides into nascent sialylatedglycoconjugates, followed by their detection by a bioorthogonal ligation of a molecular probe. Among bioorthogonal reactions, the copper-catalyzed azide-alkyne cycloaddition (CuAAC) is the only ligation where both reactive tags can be switched on the chemical reporter or on the probe, making this reaction very flexible and adaptable to various labeling strategies. Azide- and alkyne-modified ManNAc and Sia reporters have been widely used, but per-O-acetylated ManNAz (Ac4ManNAz) remains the most popular choice so far for tracking intracellular processing of sialoglycans and cell surface sialylation in various cells. Taking advantage of CuAAC, we compared the metabolic incorporation of ManNAl, ManNAz, SiaNAl, SiaNAz and Ac4ManNAz in the human colon cell lines CCD841CoN, HT29 and HCT116, and in the two gold standard cell lines, HEK293 and HeLa. Using complementary approaches, we showed marked differences in the efficiency of labeling of sialoglycoproteins between the different chemical reporters in a given cell line, and that switching the azide and alkyne bioorthogonal tags on the analogs highly impacted their metabolic incorporation in the human colon cell lines. Our results also indicated that ManNAz was the most promiscuous metabolized reporter to study sialylation in these cells.

To View Your Biomolecule, Click inside the Cell.

The surging development of bioorthogonal chemistry has profoundly transformed chemical biology over the last two decades. Involving chemical partners that specifically react together in highly complex biological fluids, this branch of chemistry now allows researchers to probe biomolecules in their natural habitat through metabolic labelling technologies. Chemical reporter strategies include metabolic glycan labelling, site-specific incorporation of unnatural amino acids in proteins, and post-synthetic labelling of nucleic acids. While a majority of literature reports mark cell-surface exposed targets, implementing bioorthogonal ligations in the interior of cells constitutes a more challenging task. Owing to limiting factors such as membrane permeability of reagents, fluorescence background due to hydrophobic interactions and off-target covalent binding, and suboptimal balance between reactivity and stability of the designed molecular reporters and probes, these strategies need mindful planning to achieve success. In this review, we discuss the hurdles encountered when targeting biomolecules localized in cell organelles and give an easily accessible summary of the strategies at hand for imaging intracellular targets.

Exploring the Potential of ß-Catenin O-GlcNAcylation by Using Fluorescence-Based Engineering and Imaging.

Monitoring glycosylation changes within cells upon response to stimuli remains challenging because of the complexity of this large family of post-translational modifications (PTMs). We developed an original tool, enabling labeling and visualization of the cell cycle key-regulator ß-catenin in its O-GlcNAcylated form, based on intramolecular Förster resonance energy transfer (FRET) technology in cells. We opted for a bioorthogonal chemical reporter strategy based on the dual-labeling of ß-catenin with a green fluorescent protein (GFP) for protein sequence combined with a chemically-clicked imaging probe for PTM, resulting in a fast and easy to monitor qualitative FRET assay. We validated this technology by imaging the O-GlcNAcylation status of ß-catenin in HeLa cells. The changes in O-GlcNAcylation of ß-catenin were varied by perturbing global cellular O-GlcNAc levels with the inhibitors of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Finally, we provided a flowchart demonstrating how this technology is transposable to any kind of glycosylation.