Glycogenome expression dynamics during mouse C2C12 myoblast differentiation suggests a sequential reorganization of membrane glycoconjugates

<p>Abstract</p> <p>Background</p> <p>Several global transcriptomic and proteomic approaches have been applied in order to obtain new molecular insights on skeletal myogenesis, but none has generated any specific data on glycogenome expression, and thus on the role of glycan structures in this process, despite the involvement of glycoconjugates in various biological events including differentiation and development. In the present study, a quantitative real-time RT-PCR technology was used to profile the dynamic expression of 375 glycogenes during the differentiation of C2C12 myoblasts into myotubes.</p> <p>Results</p> <p>Of the 276 genes expressed, 95 exhibited altered mRNA expression when C2C12 cells differentiated and 37 displayed more than 4-fold up- or down-regulations. Principal Component Analysis and Hierarchical Component Analysis of the expression dynamics identified three groups of coordinately and sequentially regulated genes. The first group included 12 down-regulated genes, the second group four genes with an expression peak at 24 h of differentiation, and the last 21 up-regulated genes. These genes mainly encode cell adhesion molecules and key enzymes involved in the biosynthesis of glycosaminoglycans and glycolipids (neolactoseries, lactoseries and ganglioseries), providing a clearer indication of how the plasma membrane and extracellular matrix may be modified prior to cell fusion. In particular, an increase in the quantity of ganglioside G_M3at the cell surface of myoblasts is suggestive of its potential role during the initial steps of myogenic differentiation.</p> <p>Conclusion</p> <p>For the first time, these results provide a broad description of the expression dynamics of glycogenes during C2C12 differentiation. Among the 37 highly deregulated glycogenes, 29 had never been associated with myogenesis. Their biological functions suggest new roles for glycans in skeletal myogenesis.</p

The Hidden Conformation of Human Histo-blood Group Antigen is a Determinant for Recognition by Pathogen Lectins

International audienceHisto-blood group epitopes are fucosylated branched oligosaccharides with well-defined conformations in solution that are recognized by receptors, such as lectins from pathogens. We report here the results of a series of experimental and computational endeavours revealing the unusual distortion of histo-blood group antigens by bacterial and fungal lectins. The Lewis x trisaccharide adopts a rigid closed conformation in solution, whilst crystallography and molecular dynamics reveal several higher energy open con-formations when bound to the Ralstonia solanacearum lectin, which is in agreement with thermodynamic and kinetic measurements. Extensive molecular dynamics simulations confirm rare transient Le x openings in solution, frequently assisted by distortion of the central N-acetyl-glucosamine ring. Additional directed molecular dynamic trajectories revealed the role of a conserved tryptophan residue in guiding the fucose into the binding site. Our findings show that conformational adaptation of oligosaccharides is of paramount importance in cell recognition and should be considered when designing anti-infective glyco-compounds

Synthesis of branched-phosphodiester and mannosecentered fucosylated glycoclusters and their binding studies with Burkholderia ambifaria lectin (BambL)

Universite Montpellier 2, Region Rhoˆne-Alpes Cluster Chimie. A.A.International audienceFive fucosylated glycoclusters exhibiting 4, 6 or 8 residues were synthesised with two different spatial environments based on mannose-centered and branched-phosphodiester scaffolds. Their synthesis was performed in solution using phosphoramidite chemistry to generate phosphodiester linkages, combined with Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The multivalent ligands were evaluated for their ability to bind to Burkholderia ambifaria Lectin (BambL). Binding evaluation was performed through inhibition of hemagglutination (HIA), surface plasmon resonance (SPR) and isothermal titration microcalorimetry (ITC). All fucosylated glycoclusters exhibited a higher affinity to BambL than methyl α-L-fucoside. A dissociation constant of 43 nM was observed for the fucocluster exhibiting four residues with the branched-phosphodiester spatial environment corresponding to a 22-fold increase in comparison with methyl α-L-fucoside. These multivalent fucoclusters represent the first example of ligands of high affinity to BambL

La protéine-O-fucosyltransférase 1 (Pofut1) (caractérisation fonctionnelle et régulation de la voie de signalisation de Notch au cours de la myogenèse)

La myogenèse du muscle squelettique est un processus complexe dont certaines étapes sont régulées par la voie de signalisation de Notch. Chez les Mammifères, la présence de O fucosylglycannes sur la partie extracellulaire des récepteurs Notch influence l activation de la voie de signalisation. Leur synthèse débute par l action de la O fucosyltransférase Pofut1. Au cours de ce travail de thèse, nous avons mis en évidence, par mutagenèse dirigée, l implication du site conservé de N glycosylation N65 pour l intégrité structurale de Pofut1. Ceci constitue un nouvel exemple de l influence de la N glycosylation dans le repliement des glycoprotéines. Des analyses combinant l utilisation de la lignée de cellules myoblastiques C2C12 et le suivi de l expression des gènes de la myogenèse, constructeurs des O-fucosylglycannes et des acteurs de la voie de Notch, par PCR quantitative en temps réel et à haut débit, nous ont permis de proposer un modèle décrivant le fonctionnement de la voie de signalisation de Notch durant la différenciation myogénique. Ce modèle met en évidence le rôle majeur de plusieurs acteurs de cette voie de signalisation comme Dll1, Notch3, Lfng et Pofut1. Il constitue un point de départ nécessaire aux analyses de la sur- et sous-expression de Pofut1 lors de la différenciation myogénique. Dans cette perspective, nous avons construit une souche de cellules C2C12 surexprimant Pofut1. A terme, les études sur les modifications de l expression des gènes impliqués dans la biosynthèse des O-fucosylglycannes nous permettront de connaître plus précisément, les mécanismes moléculaires qui orchestrent la voie de signalisation de Notch dans le processus de myogenèse.Skeletal myogenesis is a complex process in which some steps are regulated by Notch signaling pathway. In mammals, the presence of O-fucosylglycans on the extracellular domain of Notch receptors influences the activation of signalling pathway. Pofut1 is an O-fucosyltransferase that initiates O fucosylglycans synthesis. During this thesis work, we have identified, by site-directed mutagenesis, the involvement of the conserved N-glycosylation site at position 65, N65, in Pofut1 structural integrity. This is an additional example of the influence of N-glycosylation in glycoprotein folding. Analysis using myogenic C2C12 cell line, and real-time quantitative PCR allowed us to study expression of genes involved in myogenesis, Notch signaling and O-fucosylglycans synthesis. We consequently suggest a model which defines the mechanism of Notch signaling during myogenic differentiation. This model highlights the essential role of several actors of this signalling pathway including Dll1, Notch3, Lfng and Pofut1. It constitutes a necessary starting point for further studies concerning Pofut1 over- and down-expression, during myogenic differentiation. Therefore, we created a C2C12 cell line over-expressing Pofut1. Ultimately, studies on expression modifications of genes implicated in O fucosylglycan biosynthesis will enable us to more precisely understand molecular mechanisms that orchestrate Notch signalling in the process of myogenesis.LIMOGES-BU Sciences (870852109) / SudocSudocFranceF

The two N-glycans present on bovine Pofut1 are differently involved in its solubility and activity

The conceptual, and more recently empirical, study of compliance has become a central preoccupation, and perhaps the fastest growing sub-field, in international legal scholarship. The authors seek to put in question this trend. They argue that looking at the aspirations of international law through the lens of rule-compliance leads to inadequate scrutiny and understanding of the diverse complex purposes and projects that multiple actors impose and transpose on international legality, and especially a tendency to oversimplify if not distort the relation of international law to politics. Citing a range of examples from different areas of internationallaw-ranging widely from international trade and investment to international criminal and humanitarian law-the authors seek to show how the concept of compliance (especially viewed as rule-observance) is inadequate to understanding how international law has normative effects. A fundamental flaw of compliance studies is they abstract from the problem of interpretation: Interpretation is pervasively determinative of what happens to legal rules when they are out in the world yet “compliance” studies begin with the notion that there is a stable and agreed meaning to a rule, and we need merely observe whether it is obeyed

Stability and viscosity of CuO water nanofluids at very high shear rate

International audienceWe report the viscosity measurement of water-based nanofluids containing CuO spherical nanoparticles of about 29 nm in diameter. Using an innovative experimental procedure and a microfluidic “Viscometer Rheometer On a Chip”, (mVROCTM) viscosity data were collected for several nanoparticle volume fractions and a shear rate ranging up to 20,000s-1. The effect of low nanoparticle volume fraction and temperature on nanofluids viscosity was considered and discussed in this work. The stability and dispersion state of nanofluids were also studied from rheological measurements and nanoparticle tracking analysis. The possible effects due to the temperature hysteresis behavior of aggregated nanofluids under cooling and heating cycle is reported as well

The two N-glycans present on bovine Pofut1 are differently involved in its solubility and activity.

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