11 research outputs found
Visualization and understanding of sialylation mechanisms through new strategies in chemical biology
Les glycannes, prĂ©sents chez tous les ĂȘtres vivants forment une des grandes classes de biomolĂ©cules. Ă lâinterface de la chimie organique et de la biologie, la chemobiologie a permis des avancĂ©es considĂ©rables comme le marquage mĂ©tabolique des glycannes. Cette stratĂ©gie consiste en lâusage dâun monosaccharide modifiĂ© qui pourra entrer dans la cellule et emprunter ses voies mĂ©taboliques. AprĂšs incorporation, le rapporteur chimique est dĂ©tectĂ© de maniĂšre spĂ©cifique grĂące Ă des rĂ©actions de ligation bioorthogonale entre le groupement incorporĂ© et une sonde portant un groupement complĂ©mentaire. Cette thĂšse a pour but lâĂ©tude des acides sialiques selon deux axes : (i) le dĂ©veloppement dâune stratĂ©gie originale de marquage, utilisant de maniĂšre complĂ©mentaire deux monosaccharides modifiĂ©s, apportant de nouvelles informations sur les modes dâentrĂ©e de lâacide sialique exogĂšne, mais aussi de son prĂ©curseur, la N-acĂ©tylmannosamine. (ii) lâintroduction de maniĂšre exogĂšne, dâacides sialiques modifiĂ©s mais cette fois avec des enzymes (sialyltransfĂ©rases) qui tranfĂšrent lâacide sialique prĂ©alablement activĂ© sur des glycoprotĂ©ines solubles ou membranaires. Ă lâaide de suivis in situ par RMN 31P, une mĂ©thode versatile, simple et robuste a Ă©tĂ© dĂ©veloppĂ©e pour la production dâacides sialiques activĂ©s utilisables immĂ©diatement par des sialyltransfĂ©rases recombinantes. Ces outils ont Ă©tĂ© appliquĂ©s Ă lâĂ©tude et Ă la caractĂ©risation de diffĂ©rentes enzymes. Au-delĂ du dĂ©veloppement dâoutils, ces travaux de thĂšse ont permis lâapplication de nouvelles mĂ©thodes et stratĂ©gies Ă lâĂ©tude de mĂ©canismes cellulaires, de rĂ©actions enzymatiques ou encore de dĂ©ficiences de glycosylation.Glycans are essential biomolecules found in every living system. The recent advent of chemical biology paved the way for great advances in glycobiology such as metabolic glycan engineering (MGE). This strategy consists in the use of a modified monosaccharide, the chemical reporter, which can enter the cell and hijack the metabolic pathway. Upon incorporation, the introduced chemical reporter detection is achieved in a specific manner through bioorthogonal ligation. The present work aims to study sialic acids, which are often found at the outermost position of glycan chains:(i) we developed an original MGE-based strategy, using two different chemical reporters, each one being an analog of products from different steps of the biosynthetic pathway. This sequential bioorthogonal dual strategy (SBDS) has provided new insights in the entry mechanisms of both sialic acid and its precursor N-acetylmannosamine. (ii) we introduced chemically modified sialic acids, but, this time in an exo-enzymatic way, with specific glycosyltransferases (sialyltransferases) onto soluble glycoproteins or living cells. Before being transferred, sialic acids need to be activated as CMP-sialic acids which are very unstable and prone to decomposition. 31P NMR was used to optimize the production of ready-to-use CMP-sialic acids providing a versatile, simple and reproducible procedure. Natural and/or unnatural CMP-sialic acids have then proven to be great tools for the study and characterization of different enzymes. More than just a tool-box, this work describes some direct applications of our chemical tools to unravel cellular pathways, enzymatic reactions, or even biosynthesis defects
Potential influence of microstructural morphology on the viscoplastic flow of two-phase polycrystals
Most recent homogenisation theories for nonlinear behaviour combine a linearisation procedure with an appropriate linear scale transition scheme. While the definition of improved linearisation formulations is currently the subject of active research, the used linear models are often standard ones, namely the classical self-consistent (SC) scheme when polycrystals are considered. We investigate here the potential influence of the phase distribution in the case of a two-phase polycrystal with hard and soft grains. Various linear models are combined with Suquet-Ponte Castañeda's variational formulation for anisotropic media, including two-scale transition models, and are compared with SC predictions. Phase distribution effects are significant only when the contrast of critical resolved shear stresses is above about 3
Homogénéisation variationnelle pour l'analyse des déformations à chaud d'aciers biphasés
La dĂ©finition de processus dâĂ©laboration des matĂ©riaux repose sur une approche principalement empirique quipeut sâavĂ©rer longue et coĂ»teuse. Afin dâamĂ©liorer cette dĂ©marche, un outil numĂ©rique de prĂ©diction, basĂ© surlâhomogĂ©nĂ©isation variationnelle, permettant dâavoir une idĂ©e du comportement mĂ©canique et de la configurationcristallographique au cours du formage a Ă©tĂ© dĂ©veloppĂ© ces derniĂšres annĂ©es. Ce modĂšle est Ă©tendu afin de prendre en compte lâĂ©volution de polycristaux comprenant deux phases cristallographiques distinctes et ainsi de se rapprocher de certains matĂ©riaux utilisĂ©s en pratique. Un premier exemple est traitĂ© ici et comparĂ© Ă des modĂ©lisations plus classiques
Chemical glycomics enrichment: imaging the recycling of sialic acid in living cells
International audienc
A novel C-domain-dependent inhibition of the rainbow trout CMP-sialic acid synthetase activity by CMP-deaminoneuraminic acid
International audienceThe CMP-sialic acid synthetase (CSS) activates free sialic acid (Sia) to CMP-Sia using CTP, and is prerequisite for the sialylation of cell surface glycoconjugates. The vertebrate CSS consists of two domains, a catalytic N-domain and a non-catalytic C-domain. Although the C-domain is not required for the CSS enzyme to synthesize CMP-Sia, its involvement in the catalytic activity remains unknown. First, the real-time monitoring of CSS-catalyzed reaction was performed by P NMR using the rainbow trout CSS (rtCSS). While a rtCSS lacking the C-domain (rtCSS-N) similarly activated both deaminoneuraminic acid (Kdn) and N-acetylneuraminic acid (Neu5Ac), the full-length rtCSS (rtCSS-FL) did not activate Kdn as efficiently as Neu5Ac. These results suggest that the C-domain of rtCSS affects the enzymatic activity, when Kdn was used as a substrate. Second, the enzymatic activity of rtCSS-FL and rtCSS-N was measured under various concentrations of CMP-Kdn. Inhibition by CMP-Kdn was observed only for rtCSS-FL, but not for rtCSS-N, suggesting that the inhibition was C-domain-dependent. Third, the inhibitory effect of CMP-Kdn was also investigated using the mouse CSS (mCSS). However, no inhibition was observed with mCSS even at high concentrations of CMP-Kdn. Taken together, the data demonstrated that the C-domain is involved in the CMP-Kdn-dependent inhibition of rtCSS, which is a novel regulation of the Sia metabolism in rainbow trout
Probing the CMP-Sialic Acid Donor Specificity of Two Human ÎČ- d -Galactoside Sialyltransferases (ST3Galâ I and ST6Galâ I) Selectively Acting on O- and N-Glycosylproteins
International audienc
Novel Zebrafish Mono-α2,8-sialyltransferase (ST8Sia VIII): An Evolutionary Perspective of α2,8-Sialylation
The mammalian mono-α2,8-sialyltransferase ST8Sia VI has been shown to catalyze the transfer of a unique sialic acid residues onto core 1 O-glycans leading to the formation of di-sialylated O-glycosylproteins and to a lesser extent to diSia motifs onto glycolipids like GD1a. Previous studies also reported the identification of an orthologue of the ST8SIA6 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
Amino Acids Bearing Aromatic or Heteroaromatic Substituents as a New Class of Ligands for the Lysosomal Sialic Acid Transporter Sialin
International audienceSialin, encoded by the SLC17A5 gene, is a lysosomal sialic acid transporter defective in Salla disease, a rare inherited leukodystrophy. It also enables metabolic incorporation of exogenous sialic acids, leading to autoanti-bodies against N-glycolylneuraminic acid in humans. Here, we identified a novel class of human sialin ligands by virtual screening and structureâactivity relationship studies. The ligand scaffold is characterized by an amino acid backbone with a free carboxylate, an N-linked aromatic or heteroaromatic substituent, and a hydrophobic side chain. The most potent compound, 45 (LSP12-3129), inhibited N-acetylneuraminic acid 1 (Neu5Ac) transport in a non-competitive manner with IC 50 â 2.5 ÎŒM, a value 400-fold lower than the K M for Neu5Ac. In vitro and molecular docking studies attributed the non-competitive character to selective inhibitor binding to the Neu5Ac site in a cytosol-facing conformation. Moreover, compound 45 rescued the trafficking defect of the pathogenic mutant (R39C) causing Salla disease. This new class of cell-permeant inhibitors provides tools to investigate the physiological roles of sialin and help develop pharmacological chaperones for Salla disease