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ABSTRACT: The Sialyl-Lewis X (Slex) is a well-known glycan structure involved in leukocyte homing and recruitment to inflammatory sites. SLex is well conserved among species and is mainly synthesized by FucT-VII in vertebrates. The enzyme responsible for its biosynthesis in cattle was not known.
We cloned a cDNA sequence encoding bovine α3-fucosyltransferase VII that shares 83% identity with its human counterpart. Located at the BTA 11 telomeric region, the 1029 bp open reading frame is spread over two different exons, E1 which also contains the unique 5'-untranslated region and E2 which includes the entire 3'-untranslated region. The bfut7 expression pattern is restricted to thymus and spleen. A single transcript leading to the synthesis of a 342 aa protein was identified. The encoded fucosyltransferase, produced as a recombinant enzyme in COS-1 cells, was shown to be specifically responsible for SLex synthesis in cattle. In addition, we showed that the gene promoter evolved from fish to mammals towards a complex system related to the immune system. But beyond the fact that the gene regulation seems to be conserved among mammals, we also identified 7 SNPs including 3 missense mutations in the coding region in a small panel of animals.
The FUT7 sequence was highly conserved as well as the specific activity of the encoded protein FucT-VII. In addition, our in silico promoter analysis and the high rate of polymorphism suggested that its function is evolving toward a complex system related to the immune system. Furthermore, comparing bovine to human and mouse sequences, it appeared that a decrease in gene regulation was correlated with an increase in mutation rate and wider tissue expression.
BMC Genetics 08/2012; 13:74. · 2.47 Impact Factor
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ABSTRACT: The conversion of the endogenous cellular prion protein to an abnormally folded isoform is a hallmark of transmissible spongiform encephalopathies. It occurs when a misfolded prion protein contacts the cellular PrP. Among the molecular partners suggested to be involved in the misfolding process, the glycosaminoglycans seem to be good candidates. The present study was aimed to examine a possible link between PrP conversion efficiency and transcript level of Chst8 gene that encodes the carbohydrate N-acetylgalactosamine 4-O-sulfotransferase 8. Mov cells expressing ovine PrP were transfected with shRNA directed against Chst8 transcripts. Resulting clones were characterized for their Chst8 and Prnp transcript levels, and for their content in sulfated glycosaminoglycans, more particularly sulfated chondroitins. Unexpectedly, the decreased amount of Chst8 transcript induced an increase of the chondroitin sulfate percentage among total GAGs, with an increased amount of 4-O-sulfation of GalNAc residues. Upon to infection by a sheep prion, a slight amount of PrP(Sc) was observed, which rapidly disappeared upon subpassaging. Together, these findings indicate that the Chst8 transcript level affects the glycosaminoglycan environment of the cellular prion protein, and as a consequence its ability for conversion into PrP(Sc).
Biochemical and Biophysical Research Communications 10/2011; 414(3):587-91. · 2.48 Impact Factor
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ABSTRACT: Glioblastoma Multiforme (GBM) is the most frequent malignant brain tumor with still poor prognosis. Tumor initiation, growth and recurrences might depend on Brain Tumor Stem Cells (BTSCs) which can promote tumor aggressiveness and potentially affords new therapeutic target. Recent works emphasized aberrant cell-surface glyco-conjugate expression in brain tumors suggesting that altered glycosylation is closely linked to cancer tumor metastasis and invasive process. Post-translational changes might play a key role in determining the fates of most aggressive and undifferentiated cells such as self-renewal, proliferation and differentiation. In order to characterize the glycosylation-related genes involved in differentiation status of the BTSCs, two glioblastoma cell lines, U87-MG and U251 have been cultured according to two conditions leading to undifferentiated floating cells or differentiated adherent cells. The expression level of 559 glycosylation related genes has been analyzed by Taqman Low Density Array (TLDA) analysis and allowed to isolate eight up-regulated genes specific of a subpopulation of undifferentiated cells. Protein expression has been confirmed. Among main selected genes, five are also over-expressed in the undifferentiated condition in primary cultures provided by three GBM freshly isolated from patient. This work suggests that new Glycosylation-related gene signature might improve the characterization of the most aggressive and undifferentiated cells and supports that in future, N-linked glycosylation might provide new target to develop therapeutic strategy for inhibiting tumor growth.
Cancer letters 08/2011; 312(1):24-32. · 4.86 Impact Factor
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ABSTRACT: We have cloned a cDNA sequence encoding the second bovine beta-galactoside-alpha2,6-sialyltransferase whose sequence shares more than 75% of identity with hST6Gal II cDNA coding sequence. The bovine gene, located on BTA 11, spans over 50 kbp with five exons (E1-E5) containing the 1488 bp open reading frame and a 5'-untranslated exon (E0). The gene expression pattern reveals a specific tissue distribution (brain, lungs, spleen, salivary, and mammary glands) compared to ST6Gal I which is ubiquitously expressed. We identified for bovine ST6Gal II three kinds of transcripts which differ by their 5'-untranslated regions. Among them, two transcripts are brain specific whereas the third one is found in all of the tissues expressing the gene. Two pFlag-bST6Gal II vector constructions were separately transfected in COS-1 cells in order to express either membrane-bound or soluble active forms of ST6Gal II. Enzymatic assays with these two forms indicated that the enzyme used the LacdiNAc structure (GalNAcbeta1,4GlcNAc) as a better acceptor substrate than the Type II (Galbeta1-4GlcNAc) disaccharide. Moreover, the enzyme's efficiency is improved when the acceptor substrate is provided as a free oligosaccharide rather than as a protein-bound oligosaccharide. In order to investigate the potential role of ST6Gal II during the acute phase of inflammation, we used primary cultures of bovine mammary epithelial cells which were stimulated with pro-inflammatory cytokines. It appears that the ST6Gal II gene was upregulated in cells stimulated by IL-6. This result suggested that alpha2,6-sialylation mediated by this gene could contribute to organism's response to infections.
Glycobiology 08/2009; 19(10):1082-93. · 3.58 Impact Factor
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ABSTRACT: Several global transcriptomic and proteomic approaches have been applied in order to obtain new molecular insights on skeletal myogenesis, but none has generated any specific data on glycogenome expression, and thus on the role of glycan structures in this process, despite the involvement of glycoconjugates in various biological events including differentiation and development. In the present study, a quantitative real-time RT-PCR technology was used to profile the dynamic expression of 375 glycogenes during the differentiation of C2C12 myoblasts into myotubes.
Of the 276 genes expressed, 95 exhibited altered mRNA expression when C2C12 cells differentiated and 37 displayed more than 4-fold up- or down-regulations. Principal Component Analysis and Hierarchical Component Analysis of the expression dynamics identified three groups of coordinately and sequentially regulated genes. The first group included 12 down-regulated genes, the second group four genes with an expression peak at 24 h of differentiation, and the last 21 up-regulated genes. These genes mainly encode cell adhesion molecules and key enzymes involved in the biosynthesis of glycosaminoglycans and glycolipids (neolactoseries, lactoseries and ganglioseries), providing a clearer indication of how the plasma membrane and extracellular matrix may be modified prior to cell fusion. In particular, an increase in the quantity of ganglioside GM3 at the cell surface of myoblasts is suggestive of its potential role during the initial steps of myogenic differentiation.
For the first time, these results provide a broad description of the expression dynamics of glycogenes during C2C12 differentiation. Among the 37 highly deregulated glycogenes, 29 had never been associated with myogenesis. Their biological functions suggest new roles for glycans in skeletal myogenesis.
BMC Genomics 01/2009; 10:483. · 4.07 Impact Factor
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ABSTRACT: The superfamily of serine proteinase inhibitors (serpins) is involved in numerous fundamental biological processes as inflammation, blood coagulation and apoptosis. Our interest is focused on the SERPINA3 sub-family. The major human plasma protease inhibitor, alpha1-antichymotrypsin, encoded by the SERPINA3 gene, is homologous to genes organized in clusters in several mammalian species. However, although there is a similar genic organization with a high degree of sequence conservation, the reactive-centre-loop domains, which are responsible for the protease specificity, show significant divergences.
We provide additional information by analyzing the situation of SERPINA3 in the bovine genome. A cluster of eight genes and one pseudogene sharing a high degree of identity and the same structural organization was characterized. Bovine SERPINA3 genes were localized by radiation hybrid mapping on 21q24 and only spanned over 235 Kilobases. For all these genes, we propose a new nomenclature from SERPINA3-1 to SERPINA3-8. They share approximately 70% of identity with the human SERPINA3 homologue. In the cluster, we described an original sub-group of six members with an unexpected high degree of conservation for the reactive-centre-loop domain, suggesting a similar peptidase inhibitory pattern. Preliminary expression analyses of these bovSERPINA3s showed different tissue-specific patterns and diverse states of glycosylation and phosphorylation. Finally, in the context of phylogenetic analyses, we improved our knowledge on mammalian SERPINAs evolution.
Our experimental results update data of the bovine genome sequencing, substantially increase the bovSERPINA3 sub-family and enrich the phylogenetic tree of serpins. We provide new opportunities for future investigations to approach the biological functions of this unusual subset of serine proteinase inhibitors.
BMC Genomics 02/2008; 9:151. · 4.07 Impact Factor
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ABSTRACT: O-Fucosylation is a post-translational glycosylation in which an O-fucose is covalently attached to the hydroxyl group of a specific serine or threonine residue. This modification occurs within the consensus sequence C2X(4-5)(S/T)C3 present on epidermal growth factor-like repeats of several proteins, including the Notch receptors and their ligands. The enzyme responsible for the addition of O-fucose to epidermal growth factor-like repeats is protein O-fucosyltransferase 1. Protein O-fucosyltransferase 1-mediated O-fucosylation is essential in Notch signaling, folding and targeting to the cell surface. Here, we studied the expression pattern of protein O-fucosyltransferase 1 in cattle and showed that the active enzyme is present in all tissues examined from embryo and adult as a glycoprotein with two N-glycans. By comparing protein O-fucosyltransferase 1 sequences available in databases, we observed that mammalian protein O-fucosyltransferase 1 enzymes possess two putative N-glycosylation sites, and that only the first is conserved among bilaterians. To gain more insight regarding the significance of N-glycans on protein O-fucosyltransferase 1, we substituted, by site-directed mutagenesis, bovine protein O-fucosyltransferase 1 N65, N163 or both, with L or Q. We demonstrated that the loss of N-glycan on N163 caused a slight decrease in protein O-fucosyltransferase 1 activity. In contrast, glycosylation of N65 was crucial for protein O-fucosyltransferase 1 functionality. Loss of glycosylation at N65 resulted in aggregation of protein O-fucosyltransferase 1, suggesting that N-glycosylation at this site is essential for proper folding of the enzyme.
FEBS Journal 04/2007; 274(5):1202-11. · 3.79 Impact Factor
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ABSTRACT: Numerous vertebrates have four alpha-1,3/4-fucosyltransferase genes (FUT9, FUT7, FUT4, and FUT Lewis) belonging to the same family. Until now, studies on the evolution of this family have mainly focused on Lewis genes but how the other alpha-1,3/4-fucosyltransferases have emerged from a common ancestor is not well known. In order to define the respective roles of duplications and mutations, we have compared amino acid sequences representative of bony fish (Takifugu rubripes), amphibians (Xenopus laevis), birds (Gallus gallus), and mammals (Bos taurus). The FUT tree has two fundamental branches, each split into two subfamilies. We found evidence for two duplication events, dated around 710-760 Myr and 590-640 Myr, respectively, compatible with the hypothesis of two rounds of whole genome duplications in chordate genomes, before the emergence of bony vertebrates. Based on the Homo sapiens (human) physical map, we identified blocks of paralogues belonging to regions of FUT9 (6q16), FUT4 (11q21), FUT7 (9q34), and FUT Lewis (19p13) and to a region on HSA1p that is devoid of any FUT. In zebrafish (Danio rerio), an orthologue region of HSA1 harbors an FUT9 specific to bony fish, showing that duplications are not restricted to a single FUT gene but involve blocks of paralogues. In addition, sets of genes within each block clarify the order of duplication events and, as a result, the order of alpha-1,3/4-fucosyltransferase gene emergence. We have also determined the mutation rates and the density of amino acid changes along protein sequences in each alpha-1,3/4-fucosyltransferase subfamily during the main vertebrate transitions. After the emergence of tetrapods, the mutation rate of FUT9 decreased dramatically, suggesting the early acquisition of a crucial fucosyltransferase activity in the first stages of development. The FUT7 mutation rate, which in tetrapod ancestors is about half that in amniote ancestors, may be related to the role of this gene in immune systems. In contrast to other subfamilies, we found a constant mutation rate in FUT Lewis and a rather homogeneous amino acid density change, independently of the vertebrate transition, suggesting that hitherto Lewis epitopes have dispensable functions.
Journal of Molecular Evolution 10/2006; 63(3):353-64. · 2.27 Impact Factor
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Carlos H Herrera-Mendez,
Laure Brémaud,
Gerald Coulis,
Patrick Pélissier,
Miguel A Sentandreu,
Laurent Aubry,
Didier Delourme,
Christophe Chambon, Abderrahman Maftah,
Hubert Leveziel,
Ahmed Ouali
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ABSTRACT: In the present work, a new endopin-like serpin designed mEndopin 1B was purified from bovine muscle. Biochemical characterizations (amino acid sequencing and Maldi-Tof mass spectrometry peptide mapping) demonstrated that the purified protein is different from the previously described Endopin 1, renamed mEndopin 1A. The genes and cDNA of both endopins were characterized. The cDNA sequence of mEndopin 1B encodes a predicted protein of 411 amino-acids with a molecular mass of 43808Da. The mEndopin 1B gene comprised four coding exons and an additional 5' untranslated exon. The reactive site sequence of mEndopin 1B is somewhat different from that of mEndopin 1A. Nevertheless, both serpins have a similar peptidase inhibitory pattern against examined proteases (elastase, trypsin, plasmin and chymotrypsin). The high expression of both mEndopin 1A and 1B in bovine serum and tissues and their high efficiency to inhibit elastase (k(ass) approximately 10(6)-10(7) M(-1) s(-1)) suggested that these serpins might play a major role in inflammatory processes.
FEBS Letters 07/2006; 580(14):3477-84. · 3.54 Impact Factor
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ABSTRACT: The distribution of cardiolipin across the inner mitochondrial membrane was directly determined by using the ability of the fluorescent dye 10-N-nonyl-3,6-bis(dimethylamino)acridine (10-N-nonyl acridine orange) to form dimers when it interacts with the diacidic phospholipid. Two independent methods were employed: (a) a spectrophotometric measurement of 10-N-nonyl acridine orange binding to isolated rat liver mitochondria, mitoplasts and inside-out submitochondrial particles, and (b) a flow-cytometric analysis of specific red fluorescence, emitted when two dye molecules are bound to one membrane cardiolipin; the stoichiometry of 10-N-nonyl acridine orange binding to phosphatidylserine and phosphatidylinositol, 1 mol dye/mol phospholipid, prevented dye dimerisation and subsequent red-fluorescence appearance. 57% total cardiolipin was present in the outer leaflets of inner membranes of isolated organelles, a distribution confirmed by saturation measurements for mitoplasts and inside-out submitochondrial particles. The same asymmetry was directly observed in situ with mitochondrial membranes of quiescent L1210 cells, and with mitochondrial membranes of respiring yeasts. Nevertheless, alterations in ATP synthesis and inhibition of mitochondrial protein synthesis revealed that cardiolipin distribution was apparently tightly correlated with mitochondrial membrane assembly and activity.
European Journal of Biochemistry. 03/2005; 220(3):871 - 879.
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ABSTRACT: All vertebrate alpha3- and alpha3/4-FUTs possess the characteristic acceptor-binding motif VxxHH(W/R)(D/E). FUT6 and FUTb enzymes, harboring R in the acceptor-binding motif, transfer fucose in alpha1,3 linkage, whereas FUT3 and FUT5 enzymes with W at the candidate position can also transfer fucose in alpha1,4 linkage-FUT3 being more efficient than FUT5. To determine the involvement of the W/R residue in acceptor recognition, we produced 34 variants of human FUT3, FUT5, FUT6, and ox FUTb Lewis enzymes. Among the FUT3 variants where W(111) was replaced by the other amino acids, only enzymes with an aromatic residue at the candidate position kept about 50% of alpha1,4 activity and showed no changes in K(m) values for GDP-Fuc donor and H-type 1 acceptor substrates. All other substitutions produced enzymes with less than 20% of the alpha1,4 activity. Thus the ability of alpha3/4-FUTs to recognize type 1 substrates involves the aromatic character of W in the acceptor-binding domain. The alpha1,3 activity of FUT6 and FUTb significantly decreased when their R residue was substituted by basic or charged residues. Moreover, FUT3 and FUT5 variants with W-->R substitution had a better affinity for H-type 2 substrate and higher alpha1,3 activities. Therefore the optimal fucose addition in alpha1,3 linkage requires the R residue in the acceptor-binding motif of Lewis FUTs.
Glycobiology 05/2004; 14(4):347-56. · 3.58 Impact Factor
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ABSTRACT: The amphibian Xenopus tropicalis appears an increasingly appealing model for both genetic and developmental biology studies, compared to the related species Xenopus laevis. Study of the glycosylation pattern of its secreted glycoproteins revealed that this species synthesizes large amounts of Lewis(a) epitope, whereas this motif has previously only been identified in animals within the primate lineage. The use of (1)H-nuclear magnetic resonance spectroscopy enabled us to resolve the sequence of three Lewis(a)-bearing O-linked glycans associated with oviducal secretions, out of which one contained the novel sequence Gal(beta 1-3)GlcNAc(beta 1-6)GalNAc-ol. These structural data suggested the emergence of an alpha 1,4-fucosyltransferase activity in animals outside the primate lineage. On this basis, the screening of a X. tropicalis GenBank database with human Lewis-fucosyltransferase sequences revealed the occurrence of a putative fucosyltransferase gene that presented an unusual acceptor motif.
FEBS Letters 12/2003; 554(3):330-6. · 3.54 Impact Factor
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ABSTRACT: The fucosyltransferase gene family encodes enzymes that transfer fucose in alpha 1,2, alpha 1,3/4 and alpha 1,6 linkages on a large variety of glycans. The most ancient genes harbour a split coding sequence, and encode enzyme that transfer fucose at or near O- and N-peptidic sites (serine, threonine or chitobiose unit). Conversely, the more recent genes have a monoexonic coding sequence, and encode enzymes that transfer fucose at the glycan periphery. All basic mechanisms of gene evolution contribute to this amazing scenario: exon shuffling, transposition, point mutations, and duplication. As typical examples: (i) exon shuffling leads to the ancestral organization of the alpha 1,6 fucosyltransferase gene; (ii) the ancestor of alpha 1,2 fucosyltransferase genes is reshaped by retrotransposition at the same locus; (iii) duplication associated to point mutations leads to the most recent alpha 1,3/4 fucosyltransferase genes.
Genetica 08/2003; 118(2-3):157-70. · 2.15 Impact Factor
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ABSTRACT: . In plants, the function of Ŏ-fucosylation remains largely unknown. To gain insight into the role of Ŏ-fucosylation during plant development, we generated transgenic tobacco plants overexpressing the human ō/4-fucosyltransferase (hFuc-TIII). Overexpressors clearly contained high amounts of hFuc-TIII and revealed a strong increase in !(1,4)fucosyltransferase activity in plant sexual organs. As a consequence, a more significant staining of Lewisa motifs, the product of !(1,4)fucosyltransferase activity, was observed in transgenic pollen grains compared to those of controls. Here, we show that pollen grain development was altered in transgenic plants. The average size (polar and equatorial diameters) of mature pollen grains overexpressing hFuc-TIII was smaller than control pollen grains. Furthermore, whereas a reticulate cell wall surface was always observed on control pollen grains, a punctate and disorganized cell wall surface was observed on hFuc-TIII overexpressor pollen grains. In addition, transgenic pollen tube elongation was delayed compared to control pollen tube growth. This latter phenotype could at least explain the 35% reduction of seed production determined for the hFuc-TIII-overexpressing plants.
Sexual Plant Reproduction 07/2002; 15(2):99-106. · 1.87 Impact Factor
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ABSTRACT: In the animal kingdom the enzymes that catalyze the formation of alpha1,4 fucosylated-glycoconjugates are known only in apes (chimpanzee) and humans. They are encoded by FUT3 and FUT5 genes, two members of the Lewis FUT5-FUT3-FUT6 gene cluster, which had originated by duplications of an alpha3 ancestor gene. In order to explore more precisely the emergence of the alpha1,4 fucosylation, new Lewis-like fucosyltransferase genes were studied in species belonging to the three main primate groups. Two Lewis-like genes were found in brown and ruffed lemurs (prosimians) as well as in squirrel monkey (New World monkey). In the latter, one gene encodes an enzyme which transfers fucose only in alpha1,3 linkage, whereas the other is a pseudogene. Three genes homologous to chimpanzee and human Lewis genes were identified in rhesus macaque (Old World monkey), and only one encodes an alpha3/4-fucosyltransferase. The ability of new primate enzymes to transfer fucose in alpha1,3 or alpha1,3/4 linkage confirms that the amino acid R or W in the acceptor-binding motif "HH(R/W)(D/E)" is required for the type 1/type 2 acceptor specificity. Expression of rhesus macaque genes proved that fucose transfer in alpha1,4 linkage is not restricted to the hominoid family and may be extended to other Old World monkeys. Moreover, the presence of only one enzyme supporting the alpha1,4 fucosylation in rhesus macaque versus two enzymes in hominoids suggests that this function occurred twice independently during primate evolution.
Molecular Biology and Evolution 07/2002; 19(6):815-24. · 5.55 Impact Factor
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ABSTRACT: alpha4-Fucosylation represents a final step of protein N- glycosylation. alpha4-fucosylated N-glycans are thought to be involved in cell-to-cell communication and recognition in primates and plants. Nevertheless, in the plant life cycle, the function of alpha4-fucosylation remains largely unknown. To gain an insight into the role of alpha4-fucosylation during development, the study focused on tobacco flowers. It is shown that an increase in alpha(1,4)fucosyltransferase (Fuc-T) activity is only observed during anther development, whereas it remains at a constant but low level (around 20 pmol Fuc h(-1) mg(-1) protein) in the gynoecium and perianth. At least a 4-fold higher activity is detected in mature pollen grains. These data suggest that alpha(1,4)Fuc-T activity is regulated during anther development. Furthermore, alpha(1,4)Fuc-T activity could be required during pollen tube elongation where the activity level peaks at 350 pmol h(-1) mg(-1) protein. Based on enzyme profile and cycloheximide effects on pollen germination and activity, it is hypothesized that the gene encoding alpha4-Fuc-T could be regulated late during pollen development. A potential role of alpha4- fucosylation during pollen tube elongation is also discussed.
Journal of Experimental Botany 07/2002; 53(373):1429-36. · 5.36 Impact Factor
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ABSTRACT: Alignment of 15 vertebrate α1,3-fucosyltransferases revealed one arginine conserved in all the enzymes employing exclusively
type 2 acceptor substrates. At the equivalent position, a tryptophan was found in FUT3-encoded Lewis α1,3/1,4-fucosyltransferase (Fuc-TIII) andFUT5-encoded α1,3/1,4-fucosyltransferase, the only fucosyltransferases that can also transfer fucose in α1,4-linkage. The single
amino acid substitution Trp111 → Arg in Fuc-TIII was sufficient to change the specificity of fucose transfer from H-type 1 to H-type 2 acceptors. The additional
mutation of Asp112 → Glu increased the type 2 activity of the double mutant Fuc-TIII enzyme, but the single substitution of the acidic residue
Asp112 in Fuc-TIII by Glu decreased the activity of the enzyme and did not interfere with H-type 1/H-type 2 specificity. In contrast,
substitution of Arg115 in bovinefutb-encoded α1,3-fucosyltransferase (Fuc-Tb) by Trp generated a protein unable to transfer fucose either on H-type 1 or H-type
2 acceptors. However, the double mutation Arg115 → Trp/Glu116 → Asp of Fuc-Tb slightly increased H-type 1 activity. The acidic residue adjacent to the candidate amino acid Trp/Arg seems
to modulate the relative type 1/type 2 acceptor specificity, and its presence is necessary for enzyme activity since its substitution
by the corresponding amide inactivated both Fuc-TIII and Fuc-Tb enzymes.
Journal of Biological Chemistry 04/1999; 274(18):12257-12262. · 4.77 Impact Factor
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ABSTRACT: Flow cytometry has important advantages over conventional techniques. It is rapid, highly sensitive and allows multi-parametric analysis and cell sorting. Potential exists for the measurement of many cell functions by flow cytometry. The technique can be used to determine cell viability, intracellular calcium and pH, membrane potential, enzyme activities, membrane fluidity and endocytosis. Numerous examples are given on the applications of flow cytometry for cell functions measurements in the fundamental and biomedical fields.
Biology of the Cell.
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ABSTRACT: All vertebrate α3- and α3/4-FUTs possess the characteristic acceptor-binding motif "VxxHH(W/R)(D/E)". FUT6 and FUTb enzymes, harboring R in the acceptor-binding motif, transfer fucose in α1,3 linkage, while FUT3 and FUT5 enzymes with W at the candidate position can also transfer fucose in α1,4 linkage - FUT3 being more efficient than FUT5. To determine the involvement of the W/R residue in acceptor recognition, we produced thirty-four variants of human FUT3, FUT5, FUT6 and ox FUTb Lewis enzymes. Among the FUT3 variants where W111 was replaced by the other amino acids, only enzymes with an aromatic residue at the candidate position kept about 50% of α1,4 activity and showed no changes in Km values for GDP-Fuc donor and H-type 1 acceptor substrates. All other substitutions produced enzymes with less than 20% of the α1,4 activity. Thus, the ability of α3/4-FUTs to recognize type 1 substrates involves the aromatic character of W in the acceptor-binding domain. The α1,3 activity of FUT6 and FUTb significantly decreased when their R residue was substituted by basic or charged residues. Moreover, FUT3 and FUT5 variants with W→R substitution had a better affinity for H-type 2 substrate and higher α1,3 activities. Therefore, the optimal fucose addition in α1,3 linkage requires the R residue in the acceptor-binding motif of Lewis FUTs.
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ABSTRACT: α4‐Fucosylation represents a final step of protein N‐ glycosylation. α4‐fucosylated N‐glycans are thought to be involved in cell‐to‐cell communication and recognition in primates and plants. Nevertheless, in the plant life cycle, the function of α4‐fucosylation remains largely unknown. To gain an insight into the role of α4‐fucosylation during development, the study focused on tobacco flowers. It is shown that an increase in α(1,4)fucosyltransferase (Fuc‐T) activity is only observed during anther development, whereas it remains at a constant but low level (around 20 pmol Fuc h−1 mg−1 protein) in the gynoecium and perianth. At least a 4‐fold higher activity is detected in mature pollen grains. These data suggest that α(1,4)Fuc‐T activity is regulated during anther development. Furthermore, α(1,4)Fuc‐T activity could be required during pollen tube elongation where the activity level peaks at 350 pmol h−1 mg−1 protein. Based on enzyme profile and cycloheximide effects on pollen germination and activity, it is hypothesized that the gene encoding α4‐Fuc‐T could be regulated late during pollen development. A potential role of α4‐ fucosylation during pollen tube elongation is also discussed.
