A versatile strategy to synthesize -methyl-anthranilic acid-labelled glycoprobes for fluorescence-based screening assays.

Here we propose a general strategy to label carbohydrates with N-methyl-anthranilic acid at the anomeric position. Through two examples, we demonstrate that the generated glycoprobes are suitable for fluorescence-based binding/competition assays. Our approach is expected to readily generate series of glycoprobes dedicated to screening assays for the discovery of drugs targeting carbohydrate-protein interactions.

Synthesis of oligosaccharides of the linkage region of proteoglycans using regioselective glycosylation.

Proteoglycans (PGs) are complex macromolecules that are composed of glycosaminoglycan (GAG) chains covalently attached to a core protein through a tetrasaccharide linker. Biosynthesis of PGs is complex and involves a large number of glycosyltranferases. We report herein for the first time the synthesis of a collection of various sulfoforms of the disaccharide GlcA-1,3-β-d-Gal and trisaccharides GlcNAc-1,4-α-d-GlcA-1,3-β-d-Gal and GalNAc-1,4-β-d-GlcA-1,3-β-d-Gal using a regioselective glycosylation.

The role of heparan sulfate maturation in cancer: A focus on the 3O-sulfation and the enigmatic 3O-sulfotransferases (HS3STs).

Heparansulfate (HS) modifications are master regulators of the cross-talk between cell and matrix and modulate the biological activity of an array of HS binding proteins, including growth factors and chemokines, morphogens and immunity cell receptors. This review will highlight the importance of HS maturation mediated by N-deactetylase/sulfotransferases, 2O- and 6O-sulfotransferases in cancer biology, and will focus on the 3O-sulfotransferases and on the terminal, rare 3O-sulfation, and their important but still enigmatic impact in cancer progression. The review will also discuss the molecular mechanisms of action of these HS modifications with regards to ligand interactions and signaling in the cancer process and their clinical significance.

Biallelic B3GALT6 mutations cause spondylodysplastic Ehlers-Danlos syndrome.

Proteoglycans are among the most abundant and structurally complex biomacromolecules and play critical roles in connective tissues. They are composed of a core protein onto which glycosaminoglycan (GAG) side chains are attached via a linker region. Biallelic mutations in B3GALT6, encoding one of the linker region glycosyltransferases, are known to cause either spondyloepimetaphyseal dysplasia (SEMD) or a severe pleiotropic form of Ehlers-Danlos syndromes (EDS).

Synthesis of a library of variously modified 4-methylumbelliferyl xylosides and a structure-activity study of human β4GalT7.

Proteoglycans (PGs) are complex macromolecules that are composed of glycosaminoglycan (GAG) chains covalently attached to a core protein through a tetrasaccharide linker. The biosynthesis of PGs is complex and involves a large number of glycosyltranferases. Here we present a structure-activity study of human β4GalT7, which transfers the first Gal residue onto a xyloside moiety of the linkage region.

'Click'-xylosides as initiators of the biosynthesis of glycosaminoglycans: Comparison of mono-xylosides with xylobiosides.

Different mono-xylosides and their corresponding xylobiosides obtained by a chemo-enzymatic approach featuring various substituents attached to a triazole ring were probed as priming agents for glycosaminoglycan (GAG) biosynthesis in the xylosyltransferase-deficient pgsA-745 Chinese hamster ovary cell line. Xylosides containing a hydrophobic aglycone moiety were the most efficient priming agents. Mono-xylosides induced higher GAG biosynthesis in comparison with their corresponding xylobiosides.

The heparan sulfate sulfotransferase 3-OST3A (HS3ST3A) is a novel tumor regulator and a prognostic marker in breast cancer.

Heparan sulfate (HS) proteoglycan chains are key components of the breast tumor microenvironment that critically influence the behavior of cancer cells. It is established that abnormal synthesis and processing of HS play a prominent role in tumorigenesis, albeit mechanisms remain mostly obscure. HS function is mainly controlled by sulfotransferases, and here we report a novel cellular and pathophysiological significance for the 3-O-sulfotransferase 3-OST3A (HS3ST3A), catalyzing the final maturation step of HS, in breast cancer.

Probing the acceptor active site organization of the human recombinant β1,4-galactosyltransferase 7 and design of xyloside-based inhibitors.

Among glycosaminoglycan (GAG) biosynthetic enzymes, the human β1,4-galactosyltransferase 7 (hβ4GalT7) is characterized by its unique capacity to take over xyloside derivatives linked to a hydrophobic aglycone as substrates and/or inhibitors. This glycosyltransferase is thus a prime target for the development of regulators of GAG synthesis in therapeutics. Here, we report the structure-guided design of hβ4GalT7 inhibitors.

The UDP-glucuronosyltransferases of the blood-brain barrier: their role in drug metabolism and detoxication.

UDP-glucuronosyltransferases (UGTs) form a multigenic family of membrane-bound enzymes expressed in various tissues, including brain. They catalyze the formation of β-D-glucuronides from structurally unrelated substances (drugs, other xenobiotics, as well as endogenous compounds) by the linkage of glucuronic acid from the high energy donor, UDP-α-D-glucuronic acid. In brain, UGTs actively participate to the overall protection of the tissue against the intrusion of potentially harmful lipophilic substances that are metabolized as hydrophilic glucuronides.

Defective initiation of glycosaminoglycan synthesis due to B3GALT6 mutations causes a pleiotropic Ehlers-Danlos-syndrome-like connective tissue disorder.

Proteoglycans are important components of cell plasma membranes and extracellular matrices of connective tissues. They consist of glycosaminoglycan chains attached to a core protein via a tetrasaccharide linkage, whereby the addition of the third residue is catalyzed by galactosyltransferase II (β3GalT6), encoded by B3GALT6. Homozygosity mapping and candidate gene sequence analysis in three independent families, presenting a severe autosomal-recessive connective tissue disorder characterized by skin fragility, delayed wound healing, joint hyperlaxity and contractures, muscle hypotonia, intellectual disability, and a spondyloepimetaphyseal dysplasia with bone fragility and severe kyphoscoliosis, identified biallelic B3GALT6 mutations, including homozygous missense mutations in family 1 (c.

Chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1) involved in chondroitin sulfate initiation: Impact of sulfation on activity and specificity.

Glycosaminoglycan (GAG) assembly initiates through the formation of a linkage tetrasaccharide region serving as a primer for both chondroitin sulfate (CS) and heparan sulfate (HS) chain polymerization. A possible role for sulfation of the linkage structure and of the constitutive disaccharide unit of CS chains in the regulation of CS-GAG chain synthesis has been suggested. To investigate this, we determined whether sulfate substitution of galactose (Gal) residues of the linkage region or of N-acetylgalactosamine (GalNAc) of the disaccharide unit influences activity and specificity of chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1), a key glycosyltransferase of CS biosynthesis.

Identification of key functional residues in the active site of human {beta}1,4-galactosyltransferase 7: a major enzyme in the glycosaminoglycan synthesis pathway.

Glycosaminoglycans (GAGs) play a central role in many pathophysiological events, and exogenous xyloside substrates of β1,4-galactosyltransferase 7 (β4GalT7), a major enzyme of GAG biosynthesis, have interesting biomedical applications. To predict functional peptide regions important for substrate binding and activity of human β4GalT7, we conducted a phylogenetic analysis of the β1,4-galactosyltransferase family and generated a molecular model using the x-ray structure of Drosophila β4GalT7-UDP as template. Two evolutionary conserved motifs, (163)DVD(165) and (221)FWGWGREDDE(230), are central in the organization of the enzyme active site.

Biochemical and thermodynamic characterization of mutated β1,4-galactosyltransferase 7 involved in the progeroid form of the Ehlers-Danlos syndrome.

Three mutations of the B4GALT7 gene [encoding β1,4-GalT7 (β1,4-galactosyltransferase 7)], corresponding to A186D, L206P and R270C, have been identified in patients with the progeroid form of the Ehlers-Danlos syndrome and are described as being associated with the reduction or loss of β1,4-GalT7 activity. However, the molecular basis of the reduction or loss of activity remained to be determined. In the present study, wild-type, A186D, L206P and R270C β1,4-GalT7 were expressed in CHO618 cells as membrane proteins and in Escherichia coli as soluble proteins fused to MBP (maltose-binding protein).

Thermodynamic insights into the structural basis governing the donor substrate recognition by human beta1,4-galactosyltransferase 7.

Human beta1,4-GalT (galactosyltransferase)7 is involved in the biosynthesis of the tetrasaccharide linker protein region (GlcAbeta1-->3Galbeta1-->3Galbeta1-->4Xylbeta1) (where GlcA is glucuronic acid and Xyl is xylose) of proteoglycans, by catalysing the transfer of Gal (galactose) from the uridine 5'-diphosphogalactose to a Xyl residue. This reaction is rate-limiting in glycosaminoglycan biosynthesis. In the present study, we established a large-scale production system of beta1,4-GalT7 fused with the maltose-binding protein to study substrate recognition.

Molecular basis for acceptor substrate specificity of the human beta1,3-glucuronosyltransferases GlcAT-I and GlcAT-P involved in glycosaminoglycan and HNK-1 carbohydrate epitope biosynthesis, respectively.

The human beta1,3-glucuronosyltransferases galactose-beta1,3-glucuronosyltransferase I (GlcAT-I) and galactose-beta1,3-glucuronosyltransferase P (GlcAT-P) are key enzymes involved in proteoglycan and HNK-1 carbohydrate epitope synthesis, respectively. Analysis of their acceptor specificity revealed that GlcAT-I was selective toward Galbeta1,3Gal (referred to as Gal2-Gal1), whereas GlcAT-P presented a broader profile. To understand the molecular basis of acceptor substrate recognition, we constructed mutants and chimeric enzymes based on multiple sequence alignment and structural information.

Phylogenetic and mutational analyses reveal key residues for UDP-glucuronic acid binding and activity of beta1,3-glucuronosyltransferase I (GlcAT-I).

The beta1,3-glucuronosyltransferases are responsible for the completion of the protein-glycosaminoglycan linkage region of proteoglycans and of the HNK1 epitope of glycoproteins and glycolipids by transferring glucuronic acid from UDP-alpha-D-glucuronic acid (UDP-GlcA) onto a terminal galactose residue. Here, we develop phylogenetic and mutational approaches to identify critical residues involved in UDP-GlcA binding and enzyme activity of the human beta1,3-glucuronosyltransferase I (GlcAT-I), which plays a key role in glycosaminoglycan biosynthesis. Phylogeny analysis identified 119 related beta1,3-glucuronosyltransferase sequences in vertebrates, invertebrates, and plants that contain eight conserved peptide motifs with 15 highly conserved amino acids.

Purification and characterization of a soluble form of the recombinant human galactose-beta1,3-glucuronosyltransferase I expressed in the yeast Pichia pastoris.

The galactose-beta1,3-glucuronosyltransferase I (GlcAT-I) catalyzes the transfer of glucuronic acid from UDP-alpha-D-glucuronic acid onto the terminal galactose of the trisaccharide glycosaminoglycan-protein linker region of proteoglycans. This enzyme plays a key role in the process of proteoglycan assembly since the completion of the linkage region is essential for the conversion of a core protein into a functional proteoglycan. To investigate the enzymatic properties of human GlcAT-I, we established an expression system for producing a soluble form of enzyme in the methylotrophic yeast Pichia pastoris and developed a three-step purification procedure using a combination of anion exchange, cation exchange and heparin chromatographies.

Phosphorylation and sulfation of oligosaccharide substrates critically influence the activity of human beta1,4-galactosyltransferase 7 (GalT-I) and beta1,3-glucuronosyltransferase I (GlcAT-I) involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans.

We determined whether the two major structural modifications, i.e. phosphorylation and sulfation of the glycosaminoglycan-protein linkage region (GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1), govern the specificity of the glycosyltransferases responsible for the biosynthesis of the tetrasaccharide primer.

The functional glycosyltransferase signature sequence of the human beta 1,3-glucuronosyltransferase is a XDD motif.

The human beta 1,3-glucuronosyltransferase I (GlcAT-I) is the key enzyme responsible for the completion of glycosaminoglycan-protein linkage tetrasaccharide of proteoglycans (GlcA beta 1,3Gal beta 1,3Gal beta 1,4Xyl beta 1-O-serine). We have investigated the role of aspartate residues Asp194-Asp195-Asp196 corresponding to the glycosyltransferase DXD signature motif, in GlcAT-I function by UDP binding experiments, kinetic analyses, and site-directed mutagenesis. We presented the first evidence that Mn2+ is not only essential for GlcAT-I activity but is also required for cosubstrate binding.

The donor substrate specificity of the human beta 1,3-glucuronosyltransferase I toward UDP-glucuronic acid is determined by two crucial histidine and arginine residues.

The human beta1,3-glucuronosyltransferase I (GlcAT-I) plays a key role in proteoglycan biosynthesis by catalyzing the transfer of glucuronic acid onto the trisaccharide-protein linkage structure Galbeta1,3Galbeta1,4Xylbeta-O-Ser, a prerequisite step for polymerization of glycosaminoglycan chains. In this study, we identified His(308) and Arg(277) residues as essential determinants for the donor substrate (UDP-glucuronic acid) selectivity of the human GlcAT-I. Analysis of the UDP-glucuronic acid-binding site by computational modeling in conjunction with site-directed mutagenesis indicated that both residues interact with glucuronic acid.

Interleukin-1beta down-regulates the expression of glucuronosyltransferase I, a key enzyme priming glycosaminoglycan biosynthesis: influence of glucosamine on interleukin-1beta-mediated effects in rat chondrocytes.

Objective: To assess the variations of galactose-beta-1,3-glucuronosyltransferase I (GlcAT-I) expression related to the decrease in proteoglycan synthesis mediated by interleukin-1beta (IL-1beta) in rat chondrocytes, and to evaluate the influence of glucosamine on the effects elicited by this proinflammatory cytokine.

Methods: Rat articular chondrocytes in primary monolayer cultures or encapsulated into alginate beads were treated with recombinant IL-1beta in the absence or presence (1.0-4.

Structure/function of the human Ga1beta1,3-glucuronosyltransferase. Dimerization and functional activity are mediated by two crucial cysteine residues.

Galbeta1,3-glucuronosyltransferase (GlcAT-I) that catalyzes the transfer of a glucuronic acid residue onto the trisaccharide primer of the glycosaminoglycan-protein linkage region plays an essential role in the early steps of the biosynthesis of glycosaminoglycans. In order to gain insight into the structure/function of the enzyme, the human recombinant GlcAT-I was successfully expressed in the yeast Pichia pastoris, with an apparent molecular mass of 43 kDa. Analysis of the electrophoretic mobility of the membrane-bound protein in nonreducing and reducing conditions, together with cross-linking studies, indicated that the membrane-bound GlcAT-I formed active disulfide-linked dimers.