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Virginie Lattard

Publications and source records attributed to Virginie Lattard.

13 recordsLinked to original sources
PubMed2006

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. Sequence homology and structural information suggest that Y84, D113, R156, R161, and R310 residues belong to the UDP-GlcA binding site. The importance of these residues is assessed by site-directed mutagenesis, UDP affinity and kinetic analyses. Our data show that uridine binding is primarily governed by stacking interactions with the phenyl group of Y84 and also involves interactions with aspartate 113. Furthermore, we found that R156 is critical for enzyme activity but not for UDP binding, whereas R310 appears less important with regard to both activity and UDP interactions. These results clearly discriminate the function of these two active site residues that were predicted to interact with the pyrophosphate group of UDP-GlcA. Finally, mutation of R161 severely compromises GlcAT-I activity, emphasizing the major contribution of this invariant residue. Altogether, this phylogenetic approach sustained by biochemical analyses affords new insight into the organization of the beta1,3-glucuronosyltransferase family and distinguishes the respective importance of conserved residues in UDP-GlcA binding and activity of GlcAT-I.

Amino Acids↗
PubMed2005

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. This procedure yielded 1.6 mg homogeneous enzyme from 200 ml yeast cell culture, with a specific activity value of 1.5 micromol/min/mg protein. Analysis of the specificity of GlcAT-I towards Galbeta1-3Gal and Galbeta1-4GlcNAc derivatives known as substrates of the beta1,3-glucuronosyltransferases, showed that the enzyme exhibited a strict selectivity towards Galbeta1-3Gal structures. Thus, the large source of purified active enzyme allowed the determination of the kinetic parameters of GlcAT-I towards the donor substrate UDP-GlcA and the acceptor substrate digalactoside Galbeta1-3Gal.

Cloning, Molecular↗
PubMed2004

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. We analyzed the influence of C-2 phosphorylation of Xyl residue on human beta1,4-galactosyltransferase 7 (GalT-I), which catalyzes the transfer of Gal onto Xyl, and we evaluated the consequences of C-4/C-6 sulfation of Galbeta1-3Gal (Gal2-Gal1) on the activity and specificity of beta1,3-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 Galbeta1-3Gal 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 Galbeta1-3Gal 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.

Binding Sites↗
PubMed2004

Identification and characterization of the FMO2 gene in Rattus norvegicus: a good model to study metabolic and toxicological consequences of the FMO2 polymorphism.

BACKGROUND: In lung of many animal species flavin-containing monooxygenase 2 (FMO2) is a 535-amino acid residues drug-metabolizing enzyme. In humans FMO2 exhibits a genetic polymorphism. The major allele encodes a truncated FMO2, the minor allele a full-length FMO2. In laboratory rats we previously reported a FMO2 gene encoding a truncated FMO2 (432-AA residues). In these strains, a double deletion leads to the appearance of a premature stop codon. All laboratory rat strains were derived from the same wild ancestor, Rattus norvegicus. METHODS: A PCR-based method able to specifically recognize either the wild-type or the mutant allele was developed to investigate a putative FMO2 polymorphism in a population of wild rats. The FMO2 gene was analyzed in 42 wild rats. RESULTS: A genetic FMO2 polymorphism similar to that described in humans was found in R. norvegicus. We observed three different genotypes: homozygotes for the wild-type FMO2 (33.3%), homozygotes for the mutant FMO2 (38.1%) and heterozygotes (28.6%). Comparative FMO2 mRNA and protein expressions in lungs were studied by reverse transcription-PCR and western blotting. FMO2 mRNA expression was identical between the three groups. In contrast, major differences in the expression of FMO2 protein were detected. FMO2 was strongly expressed in lungs of homozygotes for the wild-type FMO2, faintly expressed in lungs of heterozygotes and non-expressed in lungs of homozygotes for the mutant FMO2. Comparative catalytic properties of lung microsomes were studied by the determination of the oxygenation of methimazole. FMO2 genetic polymorphism was associated with major differences in the S-oxidative metabolism.

Animals↗
PubMed2004

Effect of total parenteral nutrition and choline on hepatic flavin-containing and cytochrome P-450 monooxygenase activity in rats.

Total parenteral nutrition provides nutrition by infusion into the systemic circulation. Bypassing the intestine and processes associated with absorption can cause additional pathophysiological changes to occur. For example, in rats, normal gut and pancreatic cell function may change, absorptive capacity may be altered, and enzyme functional activity including drug metabolism may be affected. The objective of this study was to examine the effects of a control diet or a diet of total parenteral nutrition in the presence or absence of choline on urinary biomarkers and hepatic microsome functional activity from rats. Selective functional markers of cytochrome P-4502E1 (CYP2E1) and flavin-containing monooxygenase (FMO) were examined in vitro. The N-oxygenation of trimethylamine was used as an in vivo selective functional marker for FMO. After the administration of total parenteral nutrition plus choline for 5 days, the urinary excretion of trimethylamine and trimethylamine N-oxide declined approximately 7- and 3-fold, respectively, compared with rats treated with control diet. The concentration of urinary biogenic amines was also significantly affected by total parenteral nutrition. Compared with control animals, rats administered total parenteral nutrition plus choline for 5 days showed a decrease of approximately 5- and 2-fold in urinary dopamine and norepinephrine concentration, respectively. To examine a molecular basis for the influence of total parenteral nutrition +/- choline on monooxygenase regulation, hepatic microsomal activity of the FMO and CYP2E1 was examined. Compared with animals treated with a control diet, total parenteral nutrition plus choline in rats caused a 3-fold increase in hepatic microsomal FMO and a 2-fold increase in hepatic cytochrome CYP2E1 functional activity, respectively. Although the data did not reach statistical significance, selective immunoblot studies using hepatic microsomes from rats treated with total parenteral nutrition + choline showed that compared with controls, FMO1 protein was decreased 1.4-fold and FMO3 increased 1.3-fold, respectively. In hepatic microsomes from rats treated with total parenteral nutrition + choline, compared with control animals, FMO4 immunoreactivity was increased 1.6-fold. The data suggest that total parenteral nutrition has a detectable effect on modulating rat FMO3, FMO4, and CYP2E1 monooxygenase functional activity. The clinical relevance of these results is unknown but may be of significance for individuals receiving total parenteral nutrition and those afflicted with trimethylaminuria.

Animals↗
PubMed2004

Alternative processing events in human FMO genes.

In humans, flavin-containing monooxygenase (FMO) functional diversity is determined by the expression of five FMO genes, named FMO1 to FMO5, and their variants. In this study, we systematically analyzed transcripts of FMO1 to FMO5 in different human tissues by reverse-transcription-polymerase chain reaction and identified a large number of splice variants. Exon skipping was the major splicing event observed. Normally spliced transcripts were generally the prominent transcript detected. For FMO1, FMO2, and FMO3, two to three different splice variants were identified, and their corresponding expression was always low in the tissues examined. For FMO5 and particularly for FMO4, more complex alternative splice patterns were observed, with five and seven splice variants detected, respectively. Most identified FMO splice variants either caused a frame-shift or lacked essential functional sites. The corresponding transcripts were therefore incapable of encoding a functional enzyme and were not analyzed further in this study. However, a common in-frame exon 3- (exon 4 for FMO4) deleted variant, leading to the deletion of 63 amino acids, was identified for FMO1, FMO3, FMO4, and FMO5. To examine the functional importance of exon 3-deletion, FMO1, FMO3, FMO4 and the corresponding exon-deleted proteins were expressed as fusion proteins. Activity studies were done with two selective functional FMO substrates, methimazole, and 10-(N,N-dimethylaminopentyl)-2-(trifluoromethyl)phenothiazine and exon 3- (exon 4 for FMO4) deleted FMOs were not able to catalyze the S- and N-oxygenation of these substrates, respectively. It is not clear whether these FMO splice variants can oxygenate other substrates, including those that remain to be discovered.

Alternative Splicing↗
PubMed2003

Deleterious mutations in the flavin-containing monooxygenase 3 (FMO3) gene causing trimethylaminuria.

The primary genetic form of trimethylaminuria (TMAU) is caused by inherited defects in the flavin-containing monooxygenase 3 (FMO3) gene. Defective FMO3 has a decreased ability to catalyze the N-oxygenation of the dietary-derived malodourous amine, trimethylamine. We report two novel deleterious mutations identified in two unrelated individuals affected by the disorder. Sequence analysis of the FMO3 coding exons amplified from genomic DNA revealed that the mutation from individual 1 was heterozygous for a G>A missense mutation in exon 2 of the FMO3 gene. The mutation changed a GAG encoding Glu at codon 32 to AAG encoding Lys. Wild-type and mutant E32K FMO3 were expressed in Escherichia coli as maltose binding-fusion proteins and assayed for their ability to catalyze oxygenation of various FMO3 substrates. The results showed that the E32K mutation abrogated the catalytic activity of the enzyme. Individual 2 was identified as heterozygous for the P153L mutation. In addition, individual 2 was also heterozygous for a novel single nucleotide deletion of A191 in exon 3 at codon 64. The deletion resulted in a frame shift and caused premature termination of the FMO3 gene immediately after codon 65. Family pedigree analysis revealed that the P153L and the deletion mutation were carried on different alleles for this individual. Therefore, both alleles of the FMO3 gene for individual 2 were affected by mutations abolishing the catalytic activity of the enzyme, explaining the severe TMAU condition. The two deleterious mutations reported herein were rare mutations with estimated allelic frequencies of less than 1%.

Amino Acid Sequence↗
PubMed2003

Two new polymorphisms of the FMO3 gene in Caucasian and African-American populations: comparative genetic and functional studies.

To characterize the contribution of the human flavin-containing monooxygenase form 3 (FMO3) in the metabolism and disposition of drugs and xenobiotics, we determined the single nucleotide polymorphisms in the coding region and adjacent splice junctions of FMO3 in 134 African Americans and 120 Caucasians from the United States. In the regions examined, DNA resequencing or high throughput MassEXTEND studies coupled with mass spectrometric genotyping showed that 12 sites of variation were present. Three variants encoding synonymous mutations and four polymorphisms were observed in the noncoding region. Another three variants, Lys158-FMO3, Met257-FMO3 and Gly308-FMO3, previously reported in similar populations, were prominent polymorphisms. Two new polymorphisms, His132-FMO3 and Pro360-FMO3, were identified in this study. Both variants were found only in African Americans. To evaluate the effect of the amino acid substitutions on the function of FMO3, each amino acid substitution was introduced by site-directed mutagenesis into a wild-type FMO3 cDNA. Selective functional activity was studied with methimazole, trimethylamine, and 10-(N,N-dimethylaminopentyl)-2-(trifluoromethyl) phenothiazine. Both His132-FMO3 and Pro360-FMO3 variants were able to metabolize the substrates examined. Compared with wild-type FMO3, the His132-FMO3 was less catalytically efficient. The His132-FMO3 variant moderately altered the catalytic efficiency of FMO3 (decrease of 30%, 60% and 6% with methimazole, trimethylamine and 10-(N,N-dimethylaminopentyl)-2-(trifluoromethyl)phenothiazine, respectively). The Pro360-FMO3 variant was more catalytically efficient than wild-type FMO3. Pro360-FMO3 oxygenated methimazole, trimethylamine and 10-(N,N-dimethylaminopentyl)-2-(trifluoromethyl)phenothiazine, respectively, 3-, 5- and 2-fold more efficiently than wild-type FMO3. Based on the functional activity of the variant FMO3 enzymes, it is likely that population differences exist for compounds primarily metabolized by FMO3.

Adult↗
PubMed2003

Cloning, sequencing and tissue distribution of rat flavin-containing monooxygenase 4: two different forms are produced by tissue-specific alternative splicing.

The nucleotide sequence of rat flavin-containing monooxygenase 4 (FMO4) mRNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR) and 5'/3' terminal extension. Complete cDNA was amplified, cloned, and sequenced from the mRNA obtained from rat kidney and brain. Two different transcripts (short and long) stemming from the splicing of an internal region of 189 bases pair, corresponding to exon 4 were identified. This alternative splicing seems to be specific of the brain. The long cDNA encodes a protein of 560 amino acids with a predicted molecular mass of 63,395 Da. The short cDNA encodes a protein of 497 amino acids with a predicted molecular mass of 55,871 Da. Both of these encoded sequences contain the NADPH- and FAD-binding sites and a hydrophilic carboxyl terminus. These sequences are 80 and 79% identical to the sequences of human and rabbit FMO4. By Northern blotting and/or RT-PCR, the long-form FMO4 mRNA was detected in the rat kidney, intestine, and liver and the short form particularly in the brain. For the first time, the expression of FMO4 protein was demonstrated. By Western blotting using the two different forms of FMO4 antibodies, a long FMO4 protein was detected in the rat kidney, whereas in the rat brain, only the short form of FMO4 was observed.

Alternative Splicing↗
PubMed2002

Physiological factors affecting the expression of FMO1 and FMO3 in the rat liver and kidney.

FMO1 and FMO3, the main FMOs described in the rat, are highly expressed in the liver and the kidney. The age, from 3 to 11 weeks, and gender-dependent expression of FMO1 and FMO3 in the rat liver and kidney were investigated. Based on the enzyme activities, protein levels and mRNA levels, this study demonstrates an important increase in the expression of the FMO3 in the liver of male rats during a period that corresponds to the acquisition of the sexual maturity. Rat liver FMO1 remains unchanged during this period of observation. The evolutions of both isoforms in the kidney of the male rat are similar to those observed in the liver. On the contrary, the important decrease in the total flavin-containing monooxygenase (FMO) activity observed in the liver of female rat is linked to a considerable decrease in the FMO1-dependent activity, FMO1 protein and FMO1 mRNA levels as a function of age. The expression of the FMO3 in the liver does not seem to be affected by the age of the female rat. Inversely, the expression of FMO1 in the female rat kidneys does not seem to be modified as a function of age while the expression of FMO3 is strongly increased.

Age Factors↗
PubMed2002

The FMO2 gene of laboratory rats, as in most humans, encodes a truncated protein.

We describe the isolation and characterization of cDNAs for FMO2 from the laboratory rat. In contrast to FMO2 in other animals, each of which contain 535 amino acid residues, analysis of the sequence of the cDNAs and of a section of the corresponding gene revealed that the ORF of the laboratory rat FMO2 encodes a polypeptide of only 432 residues. This truncated protein is due to the presence of a double deletion corresponding to 1263 and 1264 nucleotides of the orthologous FMO2 cDNAs. This double deletion provokes a frame-shift, with the appearance of a premature stop codon in position 1297-1299. By Northern blotting, the probe for FMO2 hybridized a 2.5-kb transcript in lung and kidney samples only. Heterologous expression of the cDNA revealed that the truncated protein was catalytically inactive. By Western blotting, FMO2 was faintly detected at approximately 50 kDa in laboratory rat lung.

Amino Acid Sequence↗
PubMed2002

Cloning, sequencing, and tissue-dependent expression of flavin-containing monooxygenase (FMO) 1 and FMO3 in the dog.

The expression of flavin-containing monooxygenases (FMOs) in dog liver microsomes was suggested by a high methimazole S-oxidase activity. When the reaction was catalyzed by dog liver microsomes, apparent V(max) and K(m) values were 6.3 nmol/min/mg and 14 microM, respectively. This reaction was highly inhibited (73%) in the presence of imipramine, but it was also weakly affected by trimethylamine, suggesting the involvement of different isoforms. The sequences of dog FMO1 and FMO3 were obtained by reverse transcription-polymerase chain reaction and 5'/3' terminal extension. The cDNAs of dog FMO1 and dog FMO3 encode proteins of 532 amino acids, which contain the NADPH- and FAD-binding sites. The dog FMO1 amino acid sequence is 88, 86, and 89% identical to sequences of human, rabbit, and pig FMO1, respectively. The dog FMO3 amino acid sequence is 83, 84, and 82% identical to sequences of human, rabbit, and rat FMO3, respectively. Dog FMO1 and dog FMO3 exhibited only 56% identities. The FMO1 and FMO3 recombinant proteins and the FMO1 and FMO3 microsomal proteins migrated with the same mobility (56 kDa), as determined in SDS-polyacrylamide gel electrophoresis and immunoblotting. By Western blotting, dog FMO1 and dog FMO3 were detected in microsomes from liver and lung but not in kidney microsomes. By Northern blotting, the probe for FMO1 specifically hybridized a 2.6-kilobase (kb) transcript in liver and lung samples only. The probe for FMO3 hybridized two transcripts of approximately 3 and 4.2 kb in the liver and lung samples.

Amino Acid Sequence↗