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Rafael Oriol - One of the best experts on this subject based on the ideXlab platform.
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A new superfamily of protein-O-Fucosyltransferases, α2-Fucosyltransferases, and α6-Fucosyltransferases: phylogeny and identification of conserved peptide motifs
Glycobiology, 2003Co-Authors: Iván Martínez-duncker, Rosella Mollicone, Jean-jacques Candelier, Christelle Breton, Rafael OriolAbstract:The presence of three conserved peptide motifs shared by alpha2-Fucosyltransferases, alpha6-Fucosyltransferases, the protein-O-Fucosyltransferase family 1 (POFUT1) and a newly identified protein-O-Fucosyltransferase family 2 (POFUT2), together with evidence that the present genes encoding for these enzymes have originated from a common ancestor by duplication and divergent evolution, suggests that they constitute a new superfamily of Fucosyltransferases.
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Divergent evolution of Fucosyltransferase genes from vertebrates, invertebrates, and bacteria.
Glycobiology, 1999Co-Authors: Rafael Oriol, Rosella Mollicone, Anne Cailleau, Luis Balanzino, Christelle BretonAbstract:On the basis of function and sequence similarities, the vertebrate Fucosyltransferases can be classified into three groups: alpha-2-, alpha-3-, and alpha-6-Fucosyltransferases. Thirty new putative Fucosyltransferase genes from invertebrates and bacteria and six conserved peptide motifs have been identified in DNA and protein databanks. Two of these motifs are specific of alpha-3-Fucosyltransferases, one is specific of alpha-2-Fucosyltransferases, another is specific of alpha-6-Fucosyltransferases, and two are shared by both alpha-2- and alpha-6-fucosyltranserases. Based on these data, literature data, and the phylogenetic analysis of the conserved peptide motifs, a model for the evolution ofFucosyltransferase genes by successive duplications, followed by divergent evolution is proposed, with either two different ancestors, one for the alpha-2/6-Fucosyltransferases and one for the alpha-3-Fucosyltransferases or a single common ancestor for the two families. The expected properties of such an hypothetical ancestor suggest that the plant or insect alpha-3-Fucosyltransferases using chitobiose as acceptor might be the present forms of this ancestor, since Fucosyltransferases using chitobiose as acceptor are expected to be of earlier appearance in evolution than enzymes using N -acetyllactosamine. However, an example of convergent evolution of Fucosyltransferase genes is suggested for the appearance of the Leaepitopes found in plants and primates.
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Conserved structural features in eukaryotic and prokaryotic Fucosyltransferases.
Glycobiology, 1998Co-Authors: Christelle Breton, Rafael Oriol, Anne ImbertyAbstract:Fucosyltransferases are the enzymes transferring fucose from GDP-Fuc to Gal in an alpha1,2-linkage and to GlcNAc in alpha1,3-, alpha1,4-, or alpha1,6-linkages. Since all Fucosyltransferases utilize the same nucleotide sugar, their specificity will probably reside in the recognition of the acceptor and in the type of linkage formed. A search of nucleotide and protein databases yielded more than 30 sequences of Fucosyltransferases originating from mammals, chicken, nematode, and bacteria. On the basis of protein sequence similarities, these enzymes can be classified into four distinct families: (1) the alpha-2-Fucosyltransferases, (2) the alpha-3-Fucosyltransferases, (3) the mammalian alpha-6-Fucosyltransferases, and (4) the bacterial alpha-6-Fucosyltransferases. Nevertheless, using the sensitive hydrophobic cluster analysis (HCA) method, conserved structural features as well as a consensus peptide motif have been clearly identified in the catalytic domains of all alpha-2 and alpha-6-fucosyltranferases, from prokaryotic and eukaryotic origin, that allowed the grouping of these enzymes into one superfamily. In addition, a few amino acids were found strictly conserved in this family, and two of these residues have been reported to be essential for enzyme activity for a human alpha-2-Fucosyltransferase. The alpha-3-Fucosyltransferases constitute a distinct family as they lack the consensus peptide, but some regions display similarities with the alpha-2 and alpha-6-fucosyltranferases. All these observations strongly suggest that the Fucosyltransferases share some common structural and catalytic features.
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Evolution of Fucosyltransferase genes in vertebrates.
The Journal of biological chemistry, 1997Co-Authors: Marieta Costache, Rafael Oriol, Anne Cailleau, Pol-andré Apoil, Anders Elmgren, Göran Larson, Stephen Henry, Antoine Blancher, Dana Iordachescu, Rosella MolliconeAbstract:Abstract Cloning and expression of chimpanzee FUT3, FUT5, and FUT6 genes confirmed the hypothesis that the gene duplications at the origin of the present human cluster of genes occurred between: (i) the great mammalian radiation 80 million years ago and (ii) the separation of man and chimpanzee 10 million years ago. The phylogeny of Fucosyltransferase genes was completed by the addition of the FUT8 family of α(1,6)Fucosyltransferase genes, which are the oldest genes of the Fucosyltransferase family. By analysis of data banks, a newFUT8 alternative splice expressed in human retina was identified, which allowed mapping the human FUT8 gene to 14q23. The results suggest that the Fucosyltransferase genes have evolved by successive duplications, followed by translocations, and divergent evolution from a single ancestral gene.
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Fucosyltransferase genes are dispersed in the genome: FUT7 is located on 9q34.3 distal to D9S1830.
Comptes rendus de l'Academie des sciences. Serie III Sciences de la vie, 1996Co-Authors: I. Reguigne-arnould, Rosella Mollicone, Rafael Oriol, Wolfe J, Hornigold N, S. Faure, Philippe CoullinAbstract:La synthese des antigenes tissulaires A, B, H, Lewis et apparentes est catalysee par differentes Fucosyltransferases. La specificite d'accepteurs enzymatiques et l'expression tissulaire permettent de definir 2 types d'α-2-Fucosyltransferases et 5 types d'α-3-Fucosyltransferases, codes par des genes specifiques denommes FUT1 a FUT7. Nous avons precedemment assigne FUT4 a la region llq21, le groupe FUTI-FUT2 a la bande 19q13.3 et le groupe FUT6-FUT3-FUT5 a l'intervalle 19p13.3. Le dernier gene cloue (FUT7) code une α-3-Fucosyltransferase, exprimee dans les leucocytes, qui synthetise l'antigene sialyl Le x , un ligand des selectines. A l'aide d'hybrides somatiques cellulaires porteurs d'un chromosome 9 remanie et caracterises par rapport a la carte genetique des microsatellites, puis en criblant une banque de cosmides, nous avons localise FUT7 au sein de la bande chromosomique 9q34.3, dans la portion telomerique par rapport a D9S1830 et proche des genes ABC2 et C8G.
John B Lowe - One of the best experts on this subject based on the ideXlab platform.
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deficiency of reproductive tract α 1 2 fucosylated glycans and normal fertility in mice with targeted deletions of the fut1 or fut2 α 1 2 Fucosyltransferase locus
Molecular and Cellular Biology, 2001Co-Authors: Steven E Domino, John B Lowe, Liang Zhang, Patrick J Gillespie, Thomas L SaundersAbstract:The fucose alpha(1-->2) galactose beta structure is expressed by uterine epithelial cells in the mouse and has been implicated in blastocyst adhesion events thought to be required for murine implantation. Fucalpha(1-->2)Galbeta moieties and cognate Fucosyltransferases are also expressed by epithelial cells of the male reproductive tract and have been implicated in sperm maturation events that may contribute to fertilization. To determine directly if Fucalpha(1-->2)Galbeta moieties are required for fertility, we have generated strains of mice that are deficient in genes encoding FUT1 and FUT2, a pair of GDP-L-fucose:beta(1-->4)-D-galactosyl-R 2-alpha-L-Fucosyltransferase enzymes (EC 2.4.1.69) responsible for Fucalpha(1-->2)Galbeta synthesis and expression. FUT1 null mice and FUT2 null mice develop normally and exhibit no gross phenotypic abnormalities. The Fucalpha(1-->2)Galbeta epitope is absent from the uterine epithelia of FUT2 null mice and from the epithelia of the epididymis of FUT1 null mice. Fully normal fertility is observed in FUT1 null intercrosses and in FUT2 null intercrosses. These observations indicate that Fucalpha(1-->2)Galbeta moieties are not essential to blastocyst-uterine epithelial cell interactions required for implantation and are not required for sperm maturation events that permit fertilization and that neither the FUT loci nor their cognate fucosylated glycans are essential to normal development.
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molecular cloning of a cdna encoding a novel human leukocyte alpha 1 3 Fucosyltransferase capable of synthesizing the sialyl lewis x determinant
Journal of Biological Chemistry, 1994Co-Authors: Shunji Natsuka, Kevin M Gersten, K Zenita, Reiji Kannagi, John B LoweAbstract:Abstract The sialyl Lewis x determinant (NeuAc alpha 2,3Gal beta 1, 4[Fuc alpha 1,3]GlcNAc) is an essential component of leukocyte counterreceptors for E-selectin and P-selectin. The final step in sialyl Lewis x synthesis is catalyzed by alpha-1,3-Fucosyltransferases acting on sialylated glycoconjugate precursors. Cultured human leukocytic cell lines express an alpha-1,3-Fucosyltransferase gene termed Fuc-TIV or ELFT but do not express the other three cloned human alpha-1,3-Fucosyltransferase genes to any significant degree. The physiological role of Fuc-TIV/ELFT in sialyl Lewis x biosynthesis is uncertain, however, since it can catalyze the synthesis of this determinant in some, but not all, transfected cell lines in a manner that is dependent upon the glycosylation phenotype of the host cell. We report here the molecular cloning of a cDNA encoding a new human leukocyte alpha-1,3-Fucosyltransferase, termed Fuc-TVII, capable of synthesizing the sialyl Lewis x moiety. The cDNA sequence predicts a 341-amino acid-long type II transmembrane protein typical of mammalian glycosyltransferases. When expressed in mammalian cells, the Fuc-TVII cDNA directs the synthesis of cell surface sialyl Lewis x moieties but not the Lewis x, Lewis a, sialyl Lewis a, or VIM-2 determinants. Fuc-TVII can efficiently utilize alpha-2,3-sialyllactosamine in vitro to form the sialyl Lewis x tetrasaccharide but does not utilize lactosamine to form the Lewis x moiety. Northern blot analyses show that the Fuc-TVII gene is transcribed in HL-60 cells, a human promyelocytic cell line, and in YT cells, a natural killer-like cell line. Fuc-TVII represents a leukocytic alpha-1,3-Fucosyltransferase that can participate in selectin ligand synthesis via its ability to catalyze the synthesis of sialyl Lewis x determinants.
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Molecular basis for plasma α(1,3)-Fucosyltransferase gene deficiency (FUT6)
The Journal of biological chemistry, 1994Co-Authors: Rosella Mollicone, John B Lowe, Brent W. Weston, Robert Kelly, Isabelle Reguigne, Anne Fletcher, Auda Aziz, Masri Rustam, Rafael OriolAbstract:Abstract While most humans express an alpha(1,3)-Fucosyltransferase in plasma, 9% of individuals on the isle of Java (Indonesia) do not express this enzyme. Ninety-five percent of these plasma alpha(1,3)-Fucosyltransferase-deficient individuals have Lewis negative phenotype on red cells, suggesting strong linkage disequilibrium between these two traits. To define the molecular basis for this plasma deficiency and to determine which of two candidate human alpha(1,3)-Fucosyltransferase genes encode this enzyme (FUT5 and FUT6), we cloned and analyzed alleles at these two loci from an Indonesian individual deficient in plasma alpha(1,3)-Fucosyltransferase activity. Single base pair changes were identified in the coding region of each gene, relative to previously published wild type alleles. These changes in turn yield three codon changes in FUT5 and three in FUT6. The codon changes in the FUT5 gene do not yield detectable diminutions in alpha(1,3)-Fucosyltransferase activity when tested by expression in transfected COS-1 cells, and none of the FUT5 alleles co-segregate with plasma alpha(1,3)-Fucosyltransferase deficiency in Indonesian pedigrees. By contrast, two of the codon changes in the FUT6 alleles inactivate this gene when tested by expression in transfected COS-1 cells. One of these inactivating changes is a missense mutation (Glu-247-->Lys) within the enzyme's catalytic domain. The other inactivating mutation represents a nonsense mutation (Tyr-315-->stop) that truncates the COOH terminus of the enzyme by 45 amino acids. The Glu-247-->Lys missense mutation is present in double dose in the nine plasma alpha(1,3)-Fucosyltransferase-deficient individuals tested, whereas the nonsense mutation at tyrosine 315 is present in double dose in just one of these persons. These results demonstrate that the alpha(1,3)-Fucosyltransferase activity in human plasma is encoded by the FUT6 gene and that the missense mutation within codon 247 of this gene is responsible for deficiency of this activity in these Indonesian families.
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molecular cloning of a fourth member of a human alpha 1 3 Fucosyltransferase gene family multiple homologous sequences that determine expression of the lewis x sialyl lewis x and difucosyl sialyl lewis x epitopes
Journal of Biological Chemistry, 1992Co-Authors: Brent W. Weston, John B Lowe, Peter L Smith, Robert KellyAbstract:We and others have previously described the isolation of three human alpha (1,3)Fucosyltransferase genes which form the basis of a nascent glycosyltransferase gene family. We now report the molecular cloning and expression of a fourth homologous human alpha (1,3)Fucosyltransferase gene. When transfected into mammalian cells, this Fucosyltransferase gene is capable of directing expression of the Lewis x (Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc), sialyl Lewis x (NeuNAc alpha 2-->3Gal beta 1-->4 [Fuc alpha 1-->3]GlcNAc), and difucosyl sialyl Lewis x (NeuNAc alpha 2-->3Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc beta 1-->3 Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc) epitopes. The enzyme shares 85% amino acid sequence identity with Fuc-TIII and 89% identity with Fuc-TV but differs substantially in its acceptor substrate requirements. Polymerase chain reaction analyses demonstrate that the gene is syntenic to Fuc-TIII and Fuc-TV on chromosome 19. Southern blot analyses of human genomic DNA demonstrate that these four alpha (1,3)Fucosyltransferase genes account for all DNA sequences that cross-hybridize at low stringency with the Fuc-TIII catalytic domain. Using similar methods, a catalytic domain probe from Fuc-TIV identifies a new class of DNA fragments which do not cross-hybridize with the chromosome 19 Fucosyltransferase probes. These results extend the molecular definition of a family of human alpha (1,3)Fucosyltransferase genes and provide tools for examining Fucosyltransferase gene expression.
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Isolation of a novel human α(1,3)Fucosyltransferase gene and molecular comparison to the human Lewis blood group α(1,3/1,4)Fucosyltransferase gene: Syntenic, homologous, nonallelic genes encoding enzymes with distinct acceptor substrate specificities
The Journal of biological chemistry, 1992Co-Authors: Brent W. Weston, Robert D Larsen, Rajan P Nair, John B LoweAbstract:Abstract Biochemical and genetic evidence indicates that the human genome may encode four or more distinct GDP-fucose:beta-D-N-acetylglucosaminide 3-alpha-L-Fucosyltransferase (alpha(1,3)Fucosyltransferase) activities. Genes encoding two of these activities have been previously isolated. These correspond to an alpha(1,3/1,4)Fucosyltransferase thought to represent the human Lewis blood group locus and an alpha(1,3)Fucosyltransferase expressed in the myeloid lineage. We report here the molecular cloning and expression of a third human alpha(1,3)Fucosyltransferase gene, homologous to but distinct from the two previously reported human Fucosyltransferase genes. When expressed in transfected mammalian cells, this gene determines expression of a Fucosyltransferase capable of using N-acetyllactosamine to form the Lewis x epitope, and alpha(2,3)sialyl-N-acetyllactosamine to construct the sialyl Lewis x moiety. This enzyme shares 91% amino acid sequence identity with the human Lewis blood group alpha(1,3/1,4)Fucosyltransferase, yet exhibits only trace amounts of alpha(1,4)Fucosyltransferase activity. Polymerase chain reaction analyses were used to demonstrate that the gene is syntenic to the Lewis locus on chromosome 19. These analyses also excluded the possibility that this DNA segment represents an allele of the Lewis locus that encodes alpha(1,3)Fucosyltransferase but not alpha(1,4)Fucosyltransferase activity. These results are consistent with the hypothesis that this gene encodes the human "plasma type" alpha(1,3)Fucosyltransferase, and suggest a molecular basis for a family of human alpha(1,3)Fucosyltransferase genes.
Rosella Mollicone - One of the best experts on this subject based on the ideXlab platform.
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activity splice variants conserved peptide motifs and phylogeny of two new α1 3 Fucosyltransferase families fut10 and fut11
Journal of Biological Chemistry, 2009Co-Authors: Rosella Mollicone, Jean-jacques Candelier, Stuart E H Moore, Nicolai V Bovin, Marcela Garciarosasco, Ivan Martinezduncker, R OriolAbstract:Abstract We report the cloning of three splice variants of the FUT10 gene, encoding for active α-l-Fucosyltransferase-isoforms of 391, 419, and 479 amino acids, and two splice variants of the FUT11 gene, encoding for two related α-l-Fucosyltransferases of 476 and 492 amino acids. The FUT10 and FUT11 appeared 830 million years ago, whereas the other α1,3-Fucosyltransferases emerged 450 million years ago. FUT10-391 and FUT10-419 were expressed in human embryos, whereas FUT10-479 was cloned from adult brain and was not found in embryos. Recombinant FUT10-419 and FUT10-479 have a type II trans-membrane topology and are retained in the endoplasmic reticulum (ER) by a membrane retention signal at their NH2 termini. The FUT10-479 has, in addition, a COOH-ER membrane retention signal. The FUT10-391 is a soluble protein without a trans-membrane domain or ER retention signal that transiently localizes to the Golgi and then is routed to the lysosome. After transfection in COS7 cells, the three FUT10s and at least one FUT11, link α-l-fucose onto conalbumin glycopeptides and biantennary N-glycan acceptors but not onto short lactosaminyl acceptor substrates as do classical monoexonic α1,3-Fucosyltransferases. Modifications of the innermost core GlcNAc of the N-glycan, by substitution with ManNAc or with an opened GlcNAc ring or by the addition of an α1,6-fucose, suggest that the FUT10 transfer is performed on the innermost GlcNAc of the core chitobiose. We can exclude α1,3-fucosylation of the two peripheral GlcNAcs linked to the trimannosyl core of the acceptor, because the FUT10 fucosylated biantennary N-glycan product loses both terminal GlcNAc residues after digestion with human placenta α-N-acetylglucosaminidase.
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A new superfamily of protein-O-Fucosyltransferases, α2-Fucosyltransferases, and α6-Fucosyltransferases: phylogeny and identification of conserved peptide motifs
Glycobiology, 2003Co-Authors: Iván Martínez-duncker, Rosella Mollicone, Jean-jacques Candelier, Christelle Breton, Rafael OriolAbstract:The presence of three conserved peptide motifs shared by alpha2-Fucosyltransferases, alpha6-Fucosyltransferases, the protein-O-Fucosyltransferase family 1 (POFUT1) and a newly identified protein-O-Fucosyltransferase family 2 (POFUT2), together with evidence that the present genes encoding for these enzymes have originated from a common ancestor by duplication and divergent evolution, suggests that they constitute a new superfamily of Fucosyltransferases.
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Divergent evolution of Fucosyltransferase genes from vertebrates, invertebrates, and bacteria.
Glycobiology, 1999Co-Authors: Rafael Oriol, Rosella Mollicone, Anne Cailleau, Luis Balanzino, Christelle BretonAbstract:On the basis of function and sequence similarities, the vertebrate Fucosyltransferases can be classified into three groups: alpha-2-, alpha-3-, and alpha-6-Fucosyltransferases. Thirty new putative Fucosyltransferase genes from invertebrates and bacteria and six conserved peptide motifs have been identified in DNA and protein databanks. Two of these motifs are specific of alpha-3-Fucosyltransferases, one is specific of alpha-2-Fucosyltransferases, another is specific of alpha-6-Fucosyltransferases, and two are shared by both alpha-2- and alpha-6-fucosyltranserases. Based on these data, literature data, and the phylogenetic analysis of the conserved peptide motifs, a model for the evolution ofFucosyltransferase genes by successive duplications, followed by divergent evolution is proposed, with either two different ancestors, one for the alpha-2/6-Fucosyltransferases and one for the alpha-3-Fucosyltransferases or a single common ancestor for the two families. The expected properties of such an hypothetical ancestor suggest that the plant or insect alpha-3-Fucosyltransferases using chitobiose as acceptor might be the present forms of this ancestor, since Fucosyltransferases using chitobiose as acceptor are expected to be of earlier appearance in evolution than enzymes using N -acetyllactosamine. However, an example of convergent evolution of Fucosyltransferase genes is suggested for the appearance of the Leaepitopes found in plants and primates.
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Evolution of Fucosyltransferase genes in vertebrates.
The Journal of biological chemistry, 1997Co-Authors: Marieta Costache, Rafael Oriol, Anne Cailleau, Pol-andré Apoil, Anders Elmgren, Göran Larson, Stephen Henry, Antoine Blancher, Dana Iordachescu, Rosella MolliconeAbstract:Abstract Cloning and expression of chimpanzee FUT3, FUT5, and FUT6 genes confirmed the hypothesis that the gene duplications at the origin of the present human cluster of genes occurred between: (i) the great mammalian radiation 80 million years ago and (ii) the separation of man and chimpanzee 10 million years ago. The phylogeny of Fucosyltransferase genes was completed by the addition of the FUT8 family of α(1,6)Fucosyltransferase genes, which are the oldest genes of the Fucosyltransferase family. By analysis of data banks, a newFUT8 alternative splice expressed in human retina was identified, which allowed mapping the human FUT8 gene to 14q23. The results suggest that the Fucosyltransferase genes have evolved by successive duplications, followed by translocations, and divergent evolution from a single ancestral gene.
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Fucosyltransferase genes are dispersed in the genome: FUT7 is located on 9q34.3 distal to D9S1830.
Comptes rendus de l'Academie des sciences. Serie III Sciences de la vie, 1996Co-Authors: I. Reguigne-arnould, Rosella Mollicone, Rafael Oriol, Wolfe J, Hornigold N, S. Faure, Philippe CoullinAbstract:La synthese des antigenes tissulaires A, B, H, Lewis et apparentes est catalysee par differentes Fucosyltransferases. La specificite d'accepteurs enzymatiques et l'expression tissulaire permettent de definir 2 types d'α-2-Fucosyltransferases et 5 types d'α-3-Fucosyltransferases, codes par des genes specifiques denommes FUT1 a FUT7. Nous avons precedemment assigne FUT4 a la region llq21, le groupe FUTI-FUT2 a la bande 19q13.3 et le groupe FUT6-FUT3-FUT5 a l'intervalle 19p13.3. Le dernier gene cloue (FUT7) code une α-3-Fucosyltransferase, exprimee dans les leucocytes, qui synthetise l'antigene sialyl Le x , un ligand des selectines. A l'aide d'hybrides somatiques cellulaires porteurs d'un chromosome 9 remanie et caracterises par rapport a la carte genetique des microsatellites, puis en criblant une banque de cosmides, nous avons localise FUT7 au sein de la bande chromosomique 9q34.3, dans la portion telomerique par rapport a D9S1830 et proche des genes ABC2 et C8G.
Alan John - One of the best experts on this subject based on the ideXlab platform.
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structural basis of substrate recognition and catalysis by Fucosyltransferase 8
Journal of Biological Chemistry, 2020Co-Authors: Michael Jarva, Marija Dramicanin, James P Lingford, Runyu Mao, Alan JohnAbstract:Fucosylation of the innermost GlcNAc of N-glycans by Fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can dramatically affect the activities and half-lives of glycoproteins, effects that are relevant to understanding the invasiveness of some cancers, development of mAb therapeutics, and the etiology of a congenital glycosylation disorder. The acceptor substrate preferences of FUT8 are well-characterized and provide a framework for understanding N-glycan maturation in the Golgi; however, the structural basis of these substrate preferences and the mechanism through which catalysis is achieved remain unknown. Here we describe several structures of mouse and human FUT8 in the apo state and in complex with GDP, a mimic of the donor substrate, and with a glycopeptide acceptor substrate at 1.80-2.50 A resolution. These structures provide insights into a unique conformational change associated with donor substrate binding, common strategies employed by Fucosyltransferases to coordinate GDP, features that define acceptor substrate preferences, and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also revealed how FUT8 dimerization plays an important role in defining the acceptor substrate-binding site. Collectively, this information significantly builds on our understanding of the core fucosylation process.
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structural basis of substrate recognition and catalysis by Fucosyltransferase 8
bioRxiv, 2020Co-Authors: Michael Jarva, Marija Dramicanin, James P Lingford, Runyu Mao, Alan JohnAbstract:Fucosylation of the inner-most N-acetyl-glucosamine (GlcNAc) of N-glycans by Fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can have a dramatic impact on the activity and half-life of glycoproteins. These effects are relevant to understanding the invasiveness of some cancers, the development of monoclonal antibody therapeutics, and to a congenital disorder of glycosylation. The acceptor substrate preferences of FUT8 are well characterised and provide a framework for understanding N-glycan maturation in the Golgi, however the structural basis for these substrate preferences and the mechanism through which catalysis is achieved remains unknown. Here, we describe several structures of mouse and human FUT8 in the apo state and in complex with guanosine diphosphate (GDP), a mimic of the donor substrate, and a glycopeptide acceptor substrate. These structures provide insights into: a unique conformational change associated with donor substrate binding; common strategies employed by Fucosyltransferases to coordinate GDP; features that define acceptor substrate preferences; and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also reveal how FUT8 dimerisation plays an important role in defining the acceptor substrate binding site. Collectively, this information significantly builds on our understanding of the core-fucosylation process.
Iain B. H. Wilson - One of the best experts on this subject based on the ideXlab platform.
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Array-assisted characterization of a Fucosyltransferase required for the biosynthesis of complex core modifications of nematode N-glycans.
The Journal of biological chemistry, 2013Co-Authors: Shi Yan, Katharina Paschinger, Sonia Serna, Niels-christian Reichardt, Iain B. H. WilsonAbstract:Fucose is a common monosaccharide component of cell surfaces and is involved in many biological recognition events. Therefore, definition and exploitation of the specificity of the enzymes (Fucosyltransferases) involved in fucosylation is a recurrent theme in modern glycosciences. Despite various studies, the specificities of many Fucosyltransferases are still unknown, so new approaches are required to study these. The model nematode Caenorhabditis elegans expresses a wide range of fucosylated glycans, including N-linked oligosaccharides with unusual complex core modifications. Up to three fucose residues can be present on the standard N,N′-diacetylchitobiose unit of these N-glycans, but only the Fucosyltransferases responsible for transfer of two of these (the core α1,3-Fucosyltransferase FUT-1 and the core α1,6-Fucosyltransferase FUT-8) were previously characterized. By use of a glycan library in both array and solution formats, we were able to reveal that FUT-6, another C. elegans α1,3-Fucosyltransferase, modifies nematode glycan cores, specifically the distal N-acetylglucosamine residue; this result is in accordance with glycomic analysis of fut-6 mutant worms. This core-modifying activity of FUT-6 in vitro and in vivo is in addition to its previously determined ability to synthesize Lewis X epitopes in vitro. A larger scale synthesis of a nematode N-glycan core in vitro using all three Fucosyltransferases was performed, and the nature of the glycosidic linkages was determined by NMR. FUT-6 is probably the first eukaryotic glycosyltransferase whose specificity has been redefined with the aid of glycan microarrays and so is a paradigm for the study of other unusual glycosidic linkages in model and parasitic organisms.
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Towards abolition of immunogenic structures in insect cells: characterisation of a honey-bee (Apis mellifera) multi-gene family reveals both an allergy-related core {alpha}1,3-Fucosyltransferase and the first insect Lewis-histo-blood group-related an
Biochemical Journal, 2006Co-Authors: Dubravko Rendic, Katharina Paschinger, Ute Stemmer, Jaroslav Klaudiny, Julia Schmidt, Iain B. H. WilsonAbstract:Glycoproteins from Apis mellifera, such as phospholipase A 2} and hyaluronidase are well known major bee venom allergens. They carry N-linked oligosaccharide structures with two types of {alpha}1,3-fucosylation: the modification by {alpha}1,3-fucose of the innermost core GlcNAc, which constitutes an epitope recognised by IgE from some bee venom allergic patients, and a antennal Lewis-like GalNAc{beta}1,4(Fuc{alpha}1,3)GlcNAc moiety. We now report the cloning and expression of two cDNAs encoding the relevant active {alpha}1,3-Fucosyltransferases. The first sequence, closest to Drosophila melanogaster FucTA, was found to be a core {alpha}1,3-Fucosyltransferase (EC 2.4.1.214), as judged by several enzyme and biochemical assays. The second cDNA encoded an enzyme, most related to Drosophila FucTC, was shown to be capable of generating the Le x} epitope in vitro and is the first Lewis-type {alpha}1,3-Fucosyltransferase (EC 2.4.1.152) to be described in insects. The transcription levels these two genes in various tissues were examined: FucTA was found to be predominantly expressed in the brain tissue and venom glands, whereas FucTC transcripts were detected at highest levels in venom and hypopharyngeal glands. Very low expression of a third homologue of unknown function, FucTB, was also observed in various tissues. The characterisation of these honeybee gene products not only accounts for the observed {alpha}1,3-fucosylation of bee venom glycoproteins, but is expected to aid the identification and subsequent down-regulation of the Fucosyltransferases in insect cell lines of biotechnological importance.
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Fucosyltransferase substrate specificity and the order of fucosylation in invertebrates.
Glycobiology, 2004Co-Authors: Katharina Paschinger, Erika Staudacher, Ute Stemmer, Gustáv Fabini, Iain B. H. WilsonAbstract:Core alpha1,6-fucosylation is a conserved feature of animal N-linked oligosaccharides being present in both invertebrates and vertebrates. To prove that the enzymatic basis for this modification is also evolutionarily conserved, cDNAs encoding the catalytic regions of the predicted Caenorhabditis elegans and Drosophila melanogaster homologs of vertebrate alpha1,6-Fucosyltransferases (E.C. 2.4.1.68) were engineered for expression in the yeast Pichia pastoris. Recombinant forms of both enzymes were found to display core Fucosyltransferase activity as shown by a variety of methods. Unsubstituted nonreducing terminal GlcNAc residues appeared to be an obligatory feature of the substrate for the recombinant Caenorhabditis and Drosophila alpha1,6-Fucosyltransferases, as well as for native Caenorhabditis and Schistosoma mansoni core alpha1,6-Fucosyltransferases. On the other hand, these alpha1,6-Fucosyltransferases could not act on N-glycopeptides already carrying core alpha1,3-fucose residues, whereas recombinant Drosophila and native Schistosoma core alpha1,3-Fucosyltransferases were able to use core alpha1,6-fucosylated glycans as substrates. Lewis-type fucosylation was observed with native Schistosoma extracts and could take place after core alpha1,3-fucosylation, whereas prior Lewis-type fucosylation precluded the action of the Schistosoma core alpha1,3-Fucosyltransferase. Overall, we conclude that the strict order of fucosylation events, previously determined for Fucosyltransferases in crude extracts from insect cell lines (core alpha1,6 before core alpha1,3), also applies for recombinant Drosophila core alpha1,3- and alpha1,6-Fucosyltransferases as well as for core Fucosyltransferases in schistosomal egg extracts.
