Mohamed Ouzzine

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

OBJECTIVEnTo 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.nnnMETHODSnRat articular chondrocytes in primary monolayer cultures or encapsulated into alginate beads were treated with recombinant IL-1beta in the absence or presence (1.0-4.5 gm/liter) of glucosamine. Variations of GlcAT-I and expression of stromelysin 1 (matrix metalloproteinase 3 [MMP-3]) messenger RNA (mRNA) were evaluated by quantitative multistandard reverse transcriptase-polymerase chain reaction. In vitro enzymatic activity of GlcAT-I was measured by thin-layer chromatography, with radiolabeled UDP-glucuronic acid and a digalactoside derivative as substrates. Proteoglycan synthesis was determined by ex vivo incorporation of Na2-35SO4. Nitric oxide synthase and cyclooxygenase activities were monitored by the evaluation of nitrite (NO2-) and prostaglandin E2 (PGE2) produced in the culture medium, respectively.nnnRESULTSnIL-1beta treatment resulted in a marked inhibition of GlcAT-I mRNA expression and in vitro catalytic activity, together with a decrease in proteoglycan synthesis. In addition, glucosamine was able to prevent, in a dose-dependent manner, the inhibitory effects of IL-1beta. In the same way, the amino sugar reduced NO2- and PGE2 production induced by IL-1beta. Finally, the up-regulation of stromelysin 1 (MMP-3) mRNA expression by IL-1beta was fully prevented by glucosamine.nnnCONCLUSIONnThe results of this study suggest that the deleterious effect of IL-1beta on the anabolism of proteoglycan could involve the repression of GlcAT-I, a key enzyme in the biosynthesis of glycosaminoglycan. Glucosamine was highly effective in preventing these IL-1beta-mediated suppressive effects. The amino sugar also prevented the production of inflammatory mediators induced by the cytokine. This action could account for a possible beneficial effect of glucosamine on osteoarthritic articular cartilage.

Coordinate induction by antioxidants of UDP-glucuronosyltransferase UGT1A6 and the apical conjugate export pump MRP2 (multidrug resistance protein 2) in Caco-2 cells

Treatment of Caco-2 cells with the antioxidants quercetin or t-butylhydroquinone led to induced protein levels of UDP-glucuronosyltransferase UGT1A6 (ca. 3-fold over controls) and of the apical conjugate export pump multidrug resistance protein 2 (MRP2; 1.9-fold over controls). In contrast to UGT1A6, MRP2 (symbol ABCC2) was not inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Immunocytochemistry demonstrated that MRP2 was only expressed at the brush border domain of Caco-2 cell monolayers. The results indicate that UGT1A6 and MRP2 are coordinately induced by antioxidants, facilitating chemoprotection against phenolic toxins and excretion of conjugates into the intestinal lumen.

Phosphorylation and sulfation of oligosaccharide substrates critically influence the activity of human β1,4-galactosyltransferase 7 (GalT-I) and β1,3-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 (GlcAβ1–3Galβ1–3Galβ1–4Xylβ1), govern the specificity of the glycosyltransferases responsible for the biosynthesis of the tetrasaccharide primer. We analyzed the influence of C-2 phosphorylation of Xyl residue on human β1,4-galactosyltransferase 7 (GalT-I), which catalyzes the transfer of Gal onto Xyl, and we evaluated the consequences of C-4/C-6 sulfation of Galβ1–3Gal (Gal2-Gal1) on the activity and specificity of β1,3-glucuronosyltransferase I (GlcAT-I) responsible for the completion of the glycosaminoglycan primer sequence. For this purpose, a series of phosphorylated xylosides and sulfated C-4 and C-6 analogs of Galβ1–3Gal was synthesized and tested as potential substrates for the recombinant enzymes. Our results revealed that the phosphorylation of Xyl on the C-2 position prevents GalT-I activity, suggesting that this modification may occur once Gal is attached to the Xyl residue of the nascent oligosaccharide linkage. On the other hand, we showed that sulfation on C-6 position of Gal1 of the Galβ1–3Gal analog markedly enhanced GlcAT-I catalytic efficiency and we demonstrated the importance of Trp243 and Lys317 residues of Gal1 binding site for enzyme activity. In contrast, we found that GlcAT-I was unable to use digalactosides as acceptor substrates when Gal1 was sulfated on C-4 position or when Gal2 was sulfated on both C-4 and C-6 positions. Altogether, we demonstrated that oligosaccharide modifications of the linkage region control the specificity of the glycosyltransferases, a process that may regulate maturation and processing of glycosaminoglycan chains.

The Human UDP-Glucuronosyltransferases: Structural Aspects and Drug Glucuronidation

UDP-glucuronosyltransferases (EC 2.4.1.17) constitute a large multigenic family of enzymes that are involved in phase II of drug metabolism (Mackenzie et al., [1997]). These endoplasmic reticulum (...

Structure of UDP-glucuronosyltransferases in membranes.

This chapter presents the most recent experimental approaches to the investigation of UDP-glucuronosyltransferase (UGTs) in membranes. The first topic described is the subcellular localization of UGTs with special emphasis on the association of these proteins with the endoplasmic reticulum (ER). Experimental methods include subfractionation of tissue for microsome preparation, evaluation of the purity of the membrane fraction obtained, and measurement of UGT activity in the presence of detergents. Next, the recently demonstrated formation of UGT homo- and heterodimer formation and its functional relevance is discussed and the appropriate methods used to characterize such interactions are given (radiation inactivation, size exclusion chromatography, immunopurification, cross-linking, two-hybrid system). The structural determinants of UGTs in relation to membrane association, residency, and enzymatic activity are the next topic, supplemented by a description of the appropriate methods, including the design and expression of chimeric proteins, membrane insertion, and subcellular localization by immunofluorescence. Also presented is new information on the structure and function of UGTs obtained by molecular modeling, bioinformatics (sequence alignment), and comparison with selected crystallized glycosyltransferases. Finally, we discuss the important, and still not fully developed, issue of UGT active site architecture and organization within the ER. This is addressed from two perspectives: (1) chemical modification of UGT active sites by amino acid-specific probes and (2) photoaffinity labeling of UGTs. The detailed synthesis of a photoaffinity probe for an aglycon-binding site is provided and the use of this probe and direct photoaffinity labeling with retinoids is discussed. The application of proteomics techniques, including proteolytic digestion and protein sequencing by liquid chromatography/tandem mass spectrometry and matrix-assisted laser desorption ionization/time of flight, to the identification of crucial amino acids of the active sites, and subsequent site-directed mutagenesis of identified amino acids, is discussed in detail.

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. Here, we investigated whether epigenetic abnormalities involving aberrant DNA methylation affect HS biosynthetic enzymes in cancer cells. Analysis of the methylation status of glycosyltransferase and sulfotransferase genes in H‐HEMC‐SS chondrosarcoma cells showed a typical hypermethylation profile of 3‐OST sulfotransferase genes. Exposure of chondrosarcoma cells to 5‐aza‐2′‐deoxycytidine (5‐Aza‐dc), a DNA‐methyltransferase inhibitor, up‐regulated expression of 3‐OST1, 3‐OST2, and 3‐OST3A mRNAs, indicating that aberrant methylation affects transcription of these genes. Furthermore, HS expression was restored on 5‐Aza‐dc treatment or reintroduction of 3‐OST expression, as shown by indirect immunofluorescence microscopy and/or analysis of HS chains by anion‐exchange and gel‐filtration chromatography. Notably, 5‐Aza‐dc treatment of HEMC cells or expression of 3‐OST3A cDNA reduced their proliferative and invading properties and augmented adhesion of chondrosarcoma cells. These results provide the first evidence for specific epigenetic regulation of 3‐OST genes resulting in altered HSPG sulfation and point to a defect of HS‐3‐O‐sulfation as a factor in cancer progression.—Bui, C., Ouzzine, M., Talhaoui, I., Sharp, S., Prydz, K., Coughtrie, M. W. H., Fournel‐Gigleux, S. Epigenetics: methylation‐associated repression of heparan sulfate 3‐O‐sulfo‐transferase gene expression contributes to the invasive phenotype of H‐EMC‐SS chondrosarcoma cells. FASEB J. 24, 436–450 (2010). www.fasebj.org

Substrate specificity of the human UDP‐glucuronosyltransferase UGT2B4 and UGT2B7

The human UDP‐glucuronosyltransferase (UGT) isoforms UGT2B4 and UGT2B7 play a major role in the detoxification of bile acids, steroids and phenols. These two isoforms present distinct but overlapping substrate specificity, sharing common substrates such as the bile acid hyodeoxycholic acid (HDCA) and catechol‐estrogens. Here, we show that in UGT2B4, substitution of phenylalanineu200333 by leucine suppressed the activity towards HDCA, and impaired the glucuronidation of several substrates, including 4‐hydroxyestrone and 17‐epiestriol. On the other hand, the substrate specificity of the mutant UGT2B4F33Y, in which phenylalanine was replaced by tyrosine, as found at positionu200333 of UGT2B7, was similar to wild‐type UGT2B4. In the case of UGT2B7, replacement of tyrosineu200333 by leucine strongly reduced the activity towards all the tested substrates, with the exception of 17‐epiestriol. In contrast, mutation of tyrosineu200333 by phenylalanine exhibited similar or even somewhat higher activities than wild‐type UGT2B7. Hence, the results strongly indicated that the presence of an aromatic residue at positionu200333 is important for the activity and substrate specificity of both UGT2B4 and UGT2B7.

Identification of aspartic acid and histidine residues mediating the reaction mechanism and the substrate specificity of the human UDP-glucuronosyltransferases 1A

The human UDP-glucuronosyltransferase UGT1A6 is the primary phenol-metabolizing UDP-glucuronosyltransferase isoform. It catalyzes the nucleophilic attack of phenolic xenobiotics on UDP-glucuronic acid, leading to the formation of water-soluble glucuronides. The catalytic mechanism proposed for this reaction is an acid-base mechanism that involves an aspartic/glutamic acid and/or histidine residue. Here, we investigated the role of 14 highly conserved aspartic/glutamic acid residues over the entire sequence of human UGT1A6 by site-directed mutagenesis. We showed that except for aspartic residues Asp-150 and Asp-488, the substitution of carboxylic residues by alanine led to active mutants but with decreased enzyme activity and lower affinity for acceptor and/or donor substrate. Further analysis including mutation of the corresponding residue in other UGT1A isoforms suggests that Asp-150 plays a major catalytic role. In this report we also identified a single active site residue important for glucuronidation of phenols and carboxylic acid substrates by UGT1A enzyme family. Replacing Pro-40 of UGT1A4 by histidine expanded the glucuronidation activity of the enzyme to phenolic and carboxylic compounds, therefore, leading to UGT1A3-type isoform in terms of substrate specificity. Conversely, when His-40 residue of UGT1A3 was replaced with proline, the substrate specificity shifted toward that of UGT1A4 with loss of glucuronidation of phenolic substrates. Furthermore, mutation of His-39 residue of UGT1A1 (His-40 in UGT1A4) to proline led to loss of glucuronidation of phenols but not of estrogens. This study provides a step forward to better understand the glucuronidation mechanism and substrate recognition, which is invaluable for a better prediction of drug metabolism and toxicity in human.

Insights on membrane topology and structure/function of UDP-glucuronosyltransferases

The main characteristic of uridine diphosphate (UDP)-glucuronosyltransferases is their potency to glucuronidate a large array of structurally unrelated substances with various nucleophilic groups. The activity of these enzymes strongly depends on their tight association to the membrane of the endoplasmic reticulum. In light of recent data, this review is focused on the membrane-assembly process, which is a prerequisite for activity, and on the amino acids that govern substrate recognition and catalysis at the active site. The major implication of the highly variable N-terminal domain of UDP-glucuronosyltransferases in the substrate specificity of these enzymes is highlighted. In the absence of crystal data of the N-terminal domain, multidisciplinary approaches of genetic-/protein-engineering techniques, homology modeling with glycosyltransferases, and quantitative structure-activity relationships allowed us to point out crucial amino acids. On the basis of these results, possible reaction mechanisms for the glucuronidation of xenobiotics, involving histidine and aspartic acid residues, have been built and are discussed.

Drug metabolizing enzymes related to laboratory medicine: Cytochromes P-450 and UDP-glcuronosyltransferases

Many studies on drug metabolism have been carried out during the last decades using protein purification, molecular cloning techniques and analysis of polymorphisms at phenotype and genotype levels. These researchers led to a better understanding of the role of drug metabolizing enzymes in the biotransformation of drugs, pollutants or foreign compounds and of their use in laboratory medicine. The metabolic processes commonly involved in the biotransformation of xenobiotics have been classified into functionalization reaction (phase I reactions), which implicate lipophilic compounds. These molecules are modified via monooxygenation, dealkylation, reduction, aromatization, hydrolysis and can be substrates for the phase II reactions, often called conjugation reactions as they conjugate a functional group with a polar, endogenous compound. This review, devoted to cytochromes P-450 (CYP) and UDP-glucuronosyltransferases (UGT), describes essentially the genetic polymorphisms found in humans, their clinical consequences and the methods to assess the phenotypes or genotypes, with a view to studying the interindividual differences in drug monooxygenation and drug glucuronidation. Variations in drug glucuronidation reported here focused essentially on variations due to physiological factors, induction, drug interactions and genetic factors in disorders such as Gilberts Syndrome and Crigler-Najjar type I and II diseases.