visualizing-intracellular-sialylation-with-click-chemistry-expansion

Visualizing Intracellular Sialylation with Click Chemistry and Expansion Microscopy

Metabolic labeling techniques allow the incorporation of bioorthogonal reporters into glycans, enabling the targeted bioconjugation of molecular dyes ...

Univ. Lille, CNRS, UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle

Glycans form one of the four classes of biomolecules, are found in every living system and present a huge structural and functional diversity. As an illustration of this diversity, it has been reported that more than 50% of the human proteome is glycosylated and that 2% of the human genome is dedicated to glycosylation processes. Glycans are involved in many biological processes such as signalization, cell-cell or host pathogen interactions, immunity,  However, fundamental processes associated with glycans are not yet fully understood and the development of glycobiology is relatively recent compared to the study of genes or proteins. Approximately 25 years ago, the studies of Bertozzi's and Reutter's groups paved the way for metabolic oligosaccharide engineering (MOE), a strategy which consists in the use of modified sugar analogs which are taken up into the cells, metabolized, incorporated into glycoconjugates, and finally detected in a specific manner. This groundbreaking strategy has been widely used during the last few decades and the concomitant development of new bioorthogonal ligation reactions has allowed many advances in the field. Typically, MOE has been used to either visualize glycans or identify different classes of glycoproteins. The present review aims to highlight recent studies that lie somewhat outside of these more traditional approaches and that are pushing the boundaries of MOE applications.

Asking more from metabolic oligosaccharide engineering.

Unlike complex glycosylations, -GlcNAcylation consists of the addition of a single -acetylglucosamine unit to serine and threonine residues of target proteins, and is confined within the nucleocytoplasmic and mitochondrial compartments. Nevertheless, a number of clues tend to show that -GlcNAcylation is a pivotal regulatory element of its complex counterparts. In this perspective, we gather the evidence reported to date regarding this connection. We propose different levels of regulation that encompass the competition for the nucleotide sugar UDP-GlcNAc, and that control the wide class of glycosylation enzymes via their expression, catalytic activity, and trafficking. We sought to better envision that nutrient fluxes control the elaboration of glycans, not only at the level of their structure composition, but also through sweet regulating actors.

The Many Ways by Which -GlcNAcylation May Orchestrate the Diversity of Complex Glycosylations.

The mammalian mono-α2,8-sialyltransferase ST8Sia VI has been shown to catalyze the transfer of a unique sialic acid residues onto core 1 -glycans leading to the formation of di-sialylated -glycosylproteins and to a lesser extent to diSia motifs onto glycolipids like GD1a. Previous studies also reported the identification of an orthologue of the  gene in the zebrafish genome. Trying to get insights into the biosynthesis and function of the oligo-sialylated glycoproteins during zebrafish development, we cloned and studied this fish α2,8-sialyltransferase homologue. In situ hybridization experiments demonstrate that expression of this gene is always detectable during zebrafish development both in the central nervous system and in non-neuronal tissues. Intriguingly, using biochemical approaches and the newly developed in vitro MicroPlate Sialyltransferase Assay (MPSA), we found that the zebrafish recombinant enzyme does not synthetize diSia motifs on glycoproteins or glycolipids as the human homologue does. Using comparative genomics and molecular phylogeny approaches, we show in this work that the human ST8Sia VI orthologue has disappeared in the ray-finned fish and that the homologue described in fish correspond to a new subfamily of α2,8-sialyltransferase named ST8Sia VIII that was not maintained in Chondrichtyes and Sarcopterygii.

Novel Zebrafish Mono-α2,8-sialyltransferase (ST8Sia VIII): An Evolutionary Perspective of α2,8-Sialylation.

Non-tuberculous mycobacterium (NTM) infections, such as those caused by , are increasing globally. Due to their intrinsic drug resistance,  pulmonary infections are often difficult to cure using standard chemotherapy. We previously demonstrated that a piperidinol derivative, named PIPD1, is an efficient molecule both against  and , the agent of tuberculosis, by targeting the mycolic acid transporter MmpL3. These results prompted us to design and synthesize a series of piperidinol derivatives and to determine the biological activity against . Structure-activity relationship (SAR) studies pointed toward specific sites on the scaffold that can tolerate slight modifications. Overall, these results identified FMD-88 as a new promising active analogue against . Also, we determined the pharmacokinetics properties of PIPD1 and showed that intraperitoneal administration of this compound resulted in promising serum concentration and an elimination half-life of 3.2 hours.

Structure-Based Design and Synthesis of Piperidinol-Containing Molecules as New  Inhibitors.

In bioimaging, bioorthogonal chemistry is most often used to visualize chemical reporters by fluorescence in their native environment. Herein, we show that TEMPO-based probes can be ligated to monolignol reporters by Diels-Alder chemistry in plant cell walls, paving the way for the study of lignification by EPR spectroscopy and imaging.

EPR imaging of sinapyl alcohol and its application to the study of plant cell wall lignification.

Correction for 'EPR imaging of sinapyl alcohol and its application to the study of plant cell wall lignification' by Clémence Simon et al., Chem. Commun., 2021, DOI: .

Correction: EPR imaging of sinapyl alcohol and its application to the study of plant cell wall lignification.

Most  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 -acetylmannosamine or -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.

Article	Year
Asking more from metabolic oligosaccharide engineering. Chemical science\| PubMed ID: 30393518	2018
The Many Ways by Which -GlcNAcylation May Orchestrate the Diversity of Complex Glycosylations. Molecules (Basel, Switzerland)\| PubMed ID: 30400201	2018
Novel Zebrafish Mono-α2,8-sialyltransferase (ST8Sia VIII): An Evolutionary Perspective of α2,8-Sialylation. International journal of molecular sciences\| PubMed ID: 30709055	2019
Structure-Based Design and Synthesis of Piperidinol-Containing Molecules as New Inhibitors. ChemistryOpen\| PubMed ID: 32211280	2020
EPR imaging of sinapyl alcohol and its application to the study of plant cell wall lignification. Chemical communications (Cambridge, England)\| PubMed ID: 33326527	2021
Correction: EPR imaging of sinapyl alcohol and its application to the study of plant cell wall lignification. Chemical communications (Cambridge, England)\| PubMed ID: 33393953	2021
A bioorthogonal chemistry approach to detect the K1 polysialic acid capsule in . RSC chemical biology\| PubMed ID: 36794016	2023

Christophe Biot

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