Fabrice Dupuy

A Single Amino Acid in the Hypervariable Stem Domain of Vertebrate α1,3/1,4-Fucosyltransferases Determines the Type 1/Type 2 Transfer CHARACTERIZATION OF ACCEPTOR SUBSTRATE SPECIFICITY OF THE LEWIS ENZYME BY SITE-DIRECTED MUTAGENESIS

Alignment of 15 vertebrate α1,3-fucosyltransferases revealed one arginine conserved in all the enzymes employing exclusively type 2 acceptor substrates. At the equivalent position, a tryptophan was found in FUT3-encoded Lewis α1,3/1,4-fucosyltransferase (Fuc-TIII) andFUT5-encoded α1,3/1,4-fucosyltransferase, the only fucosyltransferases that can also transfer fucose in α1,4-linkage. The single amino acid substitution Trp111 → Arg in Fuc-TIII was sufficient to change the specificity of fucose transfer from H-type 1 to H-type 2 acceptors. The additional mutation of Asp112 → Glu increased the type 2 activity of the double mutant Fuc-TIII enzyme, but the single substitution of the acidic residue Asp112 in Fuc-TIII by Glu decreased the activity of the enzyme and did not interfere with H-type 1/H-type 2 specificity. In contrast, substitution of Arg115 in bovinefutb-encoded α1,3-fucosyltransferase (Fuc-Tb) by Trp generated a protein unable to transfer fucose either on H-type 1 or H-type 2 acceptors. However, the double mutation Arg115 → Trp/Glu116 → Asp of Fuc-Tb slightly increased H-type 1 activity. The acidic residue adjacent to the candidate amino acid Trp/Arg seems to modulate the relative type 1/type 2 acceptor specificity, and its presence is necessary for enzyme activity since its substitution by the corresponding amide inactivated both Fuc-TIII and Fuc-Tb enzymes.

The fucosyltransferase gene family: an amazing summary of the underlying mechanisms of gene evolution

The fucosyltransferase gene family encodes enzymes that transfer fucose in α1,2, α1,3/4 and α1,6 linkages on a large variety of glycans. The most ancient genes harbour a split coding sequence, and encode enzyme that transfer fucose at or near O- and N-peptidic sites (serine, threonine or chitobiose unit). Conversely, the more recent genes have a monoexonic coding sequence, and encode enzymes that transfer fucose at the glycan periphery. All basic mechanisms of gene evolution contribute to this amazing scenario: exon shuffling, transposition, point mutations, and duplication. As typical examples: (i) exon shuffling leads to the ancestral organization of the α1,6 fucosyltransferase gene; (ii) the ancestor of α1,2 fucosyltransferase genes is reshaped by retrotransposition at the same locus; (iii) duplication associated to point mutations leads to the most recent α1,3/4 fucosyltransferase genes.

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

BackgroundSeveral 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.ResultsOf 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 GM3 at the cell surface of myoblasts is suggestive of its potential role during the initial steps of myogenic differentiation.ConclusionFor 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.

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

O‐Fucosylation is a post‐translational glycosylation in which an O‐fucose is covalently attached to the hydroxyl group of a specific serine or threonine residue. This modification occurs within the consensus sequence C2X4−5(S/T)C3 present on epidermal growth factor‐like repeats of several proteins, including the Notch receptors and their ligands. The enzyme responsible for the addition of O‐fucose to epidermal growth factor‐like repeats is protein O‐fucosyltransferase 1. Protein O‐fucosyltransferase 1‐mediated O‐fucosylation is essential in Notch signaling, folding and targeting to the cell surface. Here, we studied the expression pattern of protein O‐fucosyltransferase 1 in cattle and showed that the active enzyme is present in all tissues examined from embryo and adult as a glycoprotein with two N‐glycans. By comparing protein O‐fucosyltransferase 1 sequences available in databases, we observed that mammalian protein O‐fucosyltransferase 1 enzymes possess two putative N‐glycosylation sites, and that only the first is conserved among bilaterians. To gain more insight regarding the significance of N‐glycans on protein O‐fucosyltransferase 1, we substituted, by site‐directed mutagenesis, bovine protein O‐fucosyltransferase 1 N65, N163 or both, with L or Q. We demonstrated that the loss of N‐glycan on N163 caused a slight decrease in protein O‐fucosyltransferase 1 activity. In contrast, glycosylation of N65 was crucial for protein O‐fucosyltransferase 1 functionality. Loss of glycosylation at N65 resulted in aggregation of protein O‐fucosyltransferase 1, suggesting that N‐glycosylation at this site is essential for proper folding of the enzyme.

Highlights of glycosylation and adhesion related genes involved in myogenesis

BackgroundMyogenesis is initiated by myoblast differentiation and fusion to form myotubes and muscle fibres. A population of myoblasts, known as satellite cells, is responsible for post-natal growth of muscle and for its regeneration. This differentiation requires many changes in cell behaviour and its surrounding environment. These modifications are tightly regulated over time and can be characterized through the study of changes in gene expression associated with this process. During the initial myogenesis steps, using the myoblast cell line C2C12 as a model, Janot et al. (2009) showed significant variations in expression for genes involved in pathways of glycolipid synthesis. In this study we used murine satellite cells (MSC) and their ability to differentiate into myotubes or early fat storage cells to select glycosylation related genes whose variation of expression is myogenesis specific.ResultsThe comparison of variant genes in both MSC differentiation pathways identified 67 genes associated with myogenesis. Comparison with data obtained for C2C12 revealed that only 14 genes had similar expression profiles in both cell types and that 17 genes were specifically regulated in MSC. Results were validated statistically by without a priori clustering. Classification according to protein function encoded by these 31 genes showed that the main regulated cellular processes during this differentiation were (i) remodeling of the extracellular matrix, particularly, sulfated structures, (ii) down-regulation of O-mannosyl glycan biosynthesis, and (iii) an increase in adhesion protein expression. A functional study was performed on Itga11 and Chst5 encoding two highly up-regulated proteins. The inactivation of Chst5 by specific shRNA delayed the fusion of MSC. By contrast, the inactivation of Itga11 by specific shRNA dramatically decreased the fusion ability of MSC. This result was confirmed by neutralization of Itga11 product by specific antibodies.ConclusionsOur screening method detected 31 genes specific for myogenic differentiation out of the 383 genes studied. According to their function, interaction networks of the products of these selected genes converged to cell fusion. Functional studies on Itga11 and Chst5 demonstrated the robustness of this screening.