visualizing-lignification-dynamics-plants-with-click-chemistry-dual

Visualizing Lignification Dynamics in Plants with Click Chemistry: Dual Labeling is BLISS!

Lignin is one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass. This phenolic polymer plays a vital ...

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 ...

Université de Lille

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

Natural and synthetically modified cytidine monophosphate activated sialic acids (CMP-Sias) are essential research assets in the field of glycobiology: among other applications, they can be used to probe glycans, detect sialylation defects at the cell surface or carry out detailed studies of sialyltransferase activities. However, these chemical tools are notoriously unstable because of hydrolytic decomposition, and are very time-consuming and costly to obtain. They are nigh impossible to store with satisfactory purity, and their preparation requires multiple laborious purification steps that usually lead to heavy product loss. Using in situ time-resolved 31P phosphorus nuclear magnetic resonance (31P NMR), we precisely established the kinetics of formation and degradation of a number of CMP-Sias including CMP-Neu5Ac, CMP-Neu5Gc, CMP-SiaNAl and CMP-SiaNAz in several experimental conditions. 31P NMR can be carried out in undeuterated solvents and is a sensitive and nondestructive technique that allows for direct in situ monitoring and optimization of chemo-enzymatic syntheses that involve phosphorus-containing species. Thus, we showed that CMP-sialic acid derivatives can be robustly obtained in high yields using the readily available Neisseria meningitidis CMP-sialic acid synthase. This integrated workflow takes less than an hour, and the freshly prepared CMP-Sias can be directly transferred to sialylation biological assays without any purification step.

Improved workflow for the efficient preparation of ready to use CMP-activated sialic acids.

A better in vivo understanding of lignin formation within plant cell walls will contribute to improving the valorization of plant-derived biomass. Although bioorthogonal chemistry provides a promising platform to study the lignification process, methodologies that simultaneously detect multiple chemical reporters in living organisms are still scarce. Here, we have developed an original bioorthogonal labeling imaging sequential strategy (BLISS) to visualize and analyze the incorporation of both p-hydroxyphenyl (H) and guaiacyl (G) units into lignin in vivo with a combination of strain-promoted and copper-catalyzed azide-alkyne cycloadditions. On our path to BLISS, we designed a new azide-tagged monolignol reporter for H units in metabolic lignin engineering and used it in conjunction with an alkyne-tagged G unit surrogate to study lignification dynamics in flax. Here, we show that BLISS provides precise spatial information on the zones of active lignification and reveals polarization in single-cell lignification dynamics.

BLISS: A Bioorthogonal Dual-Labeling Strategy to Unravel Lignification Dynamics in Plants.

Sialylation of glycoproteins and glycolipids is catalyzed by sialyltransferases in the Golgi of mammalian cells, whereby sialic acid residues are added at the nonreducing ends of oligosaccharides. Because sialylated glycans play critical roles in a number of human physio-pathological processes, the past two decades have witnessed the development of modified sialic acid derivatives for a better understanding of sialic acid biology and for the development of new therapeutic targets. However, nothing is known about how individual mammalian sialyltransferases tolerate and behave towards these unnatural CMP-sialic acid donors. In this study, we devised several approaches to investigate the donor specificity of the human β-d-galactoside sialyltransferases ST6Gal I and ST3Gal I by using two CMP-sialic acids: CMP-Neu5Ac, and CMP-Neu5N-(4pentynoyl)neuraminic acid (CMP-SiaNAl), an unnatural CMP-sialic acid donor with an extended and functionalized N-acyl moiety.

Probing the CMP-Sialic Acid Donor Specificity of Two Human β-d-Galactoside Sialyltransferases (ST3Gal I and ST6Gal I) Selectively Acting on O- and N-Glycosylproteins.

Lignin is a polyphenolic polymer of the plant cell wall formed by the oxidative polymerization of 3 main monomers called monolignols that give rise to the lignin H-, G- and S-units. Together with cellulose and hemicelluloses, lignin is a major component of plant biomass that is widely exploited by humans in numerous industrial processes. Despite recent advances in our understanding of monolignol biosynthesis, our current understanding of the spatio-temporal regulation of their transport and polymerization is more limited. In a recent publication, we have reported the development of an original Bioorthogonal Labeling Imaging Sequential Strategy (BLISS) that allows us to visualize the simultaneous incorporation dynamics of H and G monolignol reporters into lignifying cell walls of the flax stem. 11 Here, we extend the application of this strategy to other plant organs such as roots and rapidly discuss some of the contributions and perspectives of this new technique for improving our understanding of the lignification process in plants.

BLISS: Shining a light on lignification in plants.

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.

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.

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

Herein, we report the synthesis, photophysical characterization and validation of iridium(iii)-polypyridine complexes functionalized for click chemistry and bioorthogonal chemistry, as well as their versatile applications as probes in bioimaging studies exploiting metabolic labeling. The designed dyes are conjugated to chemical reporters in a specific manner within cells by CuAAC ligation and display attractive photophysical properties in the UV-visible range. They are indeed highly photostable and emit in the far-red to near-IR region with long lifetimes and large Stokes shifts. We demonstrate that they can be efficiently used to monitor nascent intracellular sialylated glycoconjugates in bioorthogonal MOE studies with a varied panel of optical and non-optical techniques, namely conventional UV-vis laser scanning confocal microscopy (for routine purposes), UV-vis time-resolved luminescence imaging (for specificity and facilitated multiplexing with nano-environment sensitivity), synchrotron radiation based X-ray fluorescence nanoimaging (for high resolution, elemental mapping and quantification ) and inductively coupled plasma mass spectrometry (for routine quantification on cell populations with high statistical confidence). The synthesized Ir(iii) complexes were utilized in single labeling experiments, as well as in dual click-labeling experiments utilizing two distinct monosaccharide reporters relevant to the same metabolic pathway.

ArticleTotal : 2	Year
Publication title Cited by 8	2018
Publication title	2025

Article	Year
Improved workflow for the efficient preparation of ready to use CMP-activated sialic acids. Glycobiology\| PubMed ID: 27543325	2016
BLISS: A Bioorthogonal Dual-Labeling Strategy to Unravel Lignification Dynamics in Plants. Cell chemical biology\| PubMed ID: 28262560	2017
Probing the CMP-Sialic Acid Donor Specificity of Two Human β-d-Galactoside Sialyltransferases (ST3Gal I and ST6Gal I) Selectively Acting on O- and N-Glycosylproteins. Chembiochem : a European journal of chemical biology\| PubMed ID: 28395125	2017
BLISS: Shining a light on lignification in plants. Plant signaling & behavior\| PubMed ID: 28786751	2017
Novel Zebrafish Mono-α2,8-sialyltransferase (ST8Sia VIII): An Evolutionary Perspective of α2,8-Sialylation. International journal of molecular sciences\| PubMed ID: 30709055	2019
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
Click-ready iridium(iii) complexes as versatile bioimaging probes for bioorthogonal metabolic labeling. RSC chemical biology\| PubMed ID: 39668813	2024

Cedric Lion

Biography

JoVE Journal Publications

Other Publications