Alterations in glycosaminoglycan biosynthesis associated with the Ehlers-Danlos syndromes.

Proteoglycans consist of a core protein substituted with one or more glycosaminoglycan (GAG) chains and execute versatile functions during many physiological and pathological processes. The biosynthesis of GAG chains is a complex process that depends on the concerted action of a variety of enzymes. Central to the biosynthesis of heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate (CS/DS) GAG chains is the formation of a tetrasaccharide linker region followed by biosynthesis of HS or CS/DS-specific repeating disaccharide units, which then undergo modifications and epimerization.

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.

The Ehlers-Danlos syndromes, rare types.

The Ehlers-Danlos syndromes comprise a clinically and genetically heterogeneous group of heritable connective tissue disorders, which are characterized by joint hypermobility, skin hyperextensibility, and tissue friability. In the Villefranche Nosology, six subtypes were recognized: The classical, hypermobile, vascular, kyphoscoliotic, arthrochalasis, and dermatosparaxis subtypes of EDS. Except for the hypermobile subtype, defects had been identified in fibrillar collagens or in collagen-modifying enzymes.

'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.

Targeting of Proteoglycan Synthesis Pathway: A New Strategy to Counteract Excessive Matrix Proteoglycan Deposition and Transforming Growth Factor-β1-Induced Fibrotic Phenotype in Lung Fibroblasts.

Stimulation of proteoglycan (PG) synthesis and deposition plays an important role in the pathophysiology of fibrosis and is an early and dominant feature of pulmonary fibrosis. Transforming growth factor-β1 (TGF-β1) is a major cytokine associated with fibrosis that induces excessive synthesis of matrix proteins, particularly PGs. Owing to the importance of PGs in matrix assembly and in mediating cytokine and growth factor signaling, a strategy based on the inhibition of PG synthesis may prevent excessive matrix PG deposition and attenuates profibrotic effects of TGF-β1 in lung fibroblasts.

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.

Regulation of xylosyltransferase I gene expression by interleukin 1β in human primary chondrocyte cells: mechanism and impact on proteoglycan synthesis.

Xylosyltransferase I (XT-I) is an essential enzyme of proteoglycan (PG) biosynthesis pathway catalyzing the initial and rate-limiting step in glycosaminoglycan chain assembly. It plays a critical role in the regulation of PG synthesis in cartilage; however, little is known about underlying mechanism. Here, we provide evidence that, in human primary chondrocytes, IL-1β regulates XT-I gene expression into an early phase of induction and a late phase of down-regulation.

Recent structures, evolution and mechanisms of glycosyltransferases.

Cellular glycome assembly requires the coordinated action of a large number of glycosyltransferases that catalyse the transfer of a sugar residue from a donor to specific acceptor molecules. This enzyme family is very ancient, encompassing all three domains of life. There has been considerable recent progress in structural glycobiology with the determination of crystal structures of several important glycosyltransferase members, showing novel folds and variations around a common α/β scaffold.

Xylosyltransferase-I regulates glycosaminoglycan synthesis during the pathogenic process of human osteoarthritis.

Loss of glycosaminoglycan (GAG) chains of proteoglycans (PGs) is an early event of osteoarthritis (OA) resulting in cartilage degradation that has been previously demonstrated in both huma and experimental OA models. However, the mechanism of GAG loss and the role of xylosyltransferase-I (XT-I) that initiates GAG biosynthesis onto PG molecules in the pathogenic process of human OA are unknown. In this study, we have characterized XT-I expression and activity together with GAG synthesis in human OA cartilage obtained from different regions of the same joint, defined as "normal", "late-stage" or adjacent to "late-stage".

Proteoglycans and cartilage repair.

Repair of damaged articular cartilage in osteoarthritis (OA) is a clinical challenge. Because cartilage is an avascular and aneural tissue, normal mechanisms of tissue repair through recruitment of cells to the site of tissue destruction are not feasible. Proteoglycan (PG) depletion induced by the proinflammatory cytokine interleukin-1β, a principal mediator in OA, is a major factor in the onset and progression of joint destruction.

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.

Absolute immunoquantification of the expression of ABC transporters P-glycoprotein, breast cancer resistance protein and multidrug resistance-associated protein 2 in human liver and duodenum.

The ATP-binding cassette (ABC) transporters breast cancer resistance protein (BCRP), multidrug resistance-associated protein 2 (MRP2), and P-glycoprotein (Pgp) are important in the distribution and elimination of many drugs and endogenous metabolites. Due to their membrane location and hydrophobicity it is difficult to generate purified protein standards to quantify these transporters in human tissues. The present study generated transporter proteins fused with the S-peptide of ribonuclease for use as standards in immunoquantification in human liver and small intestine.

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.

Molecular characterization of β1,4-galactosyltransferase 7 genetic mutations linked to the progeroid form of Ehlers-Danlos syndrome (EDS).

β1,4-Galactosyltransferase 7 (β4GalT7) is a key enzyme initiating glycosaminoglycan (GAG) synthesis. Based on in vitro and ex vivo kinetics studies and structure-based modelling, we molecularly characterized β4GalT7 mutants linked to the progeroid form of Ehlers-Danlos syndrome (EDS), a severe connective tissue disorder. Our results revealed that loss of activity upon L206P substitution due to altered protein folding is the primary cause for the GAG synthesis defect in patients carrying the compound A186D and L206P mutations.

The use of hepatocytes to investigate UDP-glucuronosyltransferases and sulfotransferases.

Since phase II reactions quantitatively represent the most important pathways involved in drug biotransformation, the development and the use of in vitro approaches to predict glucuronidation and sulfation are currently attracting intense interest to assist in the selection of new drug candidates and for the optimization of dosage regimens for established drugs. At present, primary cultures of human hepatocytes represent the most suitable in vitro model for drug metabolism studies. This system theoretically expresses the full complement of drug-metabolizing enzymes associated with the endoplasmic reticulum (CYP and UDP-glucuronosyltransferases) or located in the cytosolic compartment (sulfotransferases), and relevant accessory proteins required for drug transport and excretion.

Epigenetics: methylation-associated repression of heparan sulfate 3-O-sulfotransferase gene expression contributes to the invasive phenotype of H-EMC-SS chondrosarcoma cells.

Heparan sulfate proteoglycans (HSPGs), strategically located at the cell-tissue-organ interface, regulate major biological processes, including cell proliferation, migration, and adhesion. These vital functions are compromised in tumors, due, in part, to alterations in heparan sulfate (HS) expression and structure. How these modifications occur is largely unknown.