A single amino acid in the hypervariable stem domain of vertebrate alpha1,3/1,4-fucosyltransferases determines the type 1/type 2 transfer. Characterization of acceptor substrate specificity of the lewis enzyme by site-directed mutagenesis

Alignment of 15 vertebrate alpha1,3-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 alpha1,3/1,4-fucosyltransferase (Fuc-TIII) and FUT5-encoded alpha1,3/1,4-fucosyltransferase, the only fucosyltransferases that can also transfer fucose in alpha1, 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 bovine futb-encoded alpha1, 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.     

Characterization of a novel alpha1,2-fucosyltransferase of Escherichia coli O128:b12 and functional investigation of its common motif

The wbsJ gene from Escherichia coli O128:B12 encodes an alpha1,2-fucosyltransferase responsible for adding a fucose onto the galactose residue of the O-antigen repeating unit via an alpha1,2 linkage. The wbsJ gene was overexpressed in E. coli BL21 (DE3) as a fusion protein with glutathione S-transferase (GST) at its N-terminus. GST-WbsJ fusion protein was purified to homogeneity via GST affinity chromatography followed by size exclusion chromatography. The enzyme showed broad acceptor specificity with Galbeta1,3GalNAc (T antigen), Galbeta1,4Man and Galbeta1,4Glc (lactose) being better acceptors than Galbeta-O-Me and galactose. Galbeta1,4Fru (lactulose), a natural sugar, was furthermore found to be the best acceptor for GST-WbsJ with a reaction rate four times faster than that of lactose. Kinetic studies showed that GST-WbsJ has a higher affinity for lactose than lactulose with apparent Km values of 7.81 mM and 13.26 mM, respectively. However, the kcat/appKm value of lactose (6.36 M(-1) x min(-1)) is two times lower than that of lactulose (13.39 M(-1) x min(-1)). In addition, the alpha1,2-fucosyltransferase activity of GST-WbsJ was found to be independent of divalent metal ions such as Mn2+ or Mg2+. This activity was competitively inhibited by GDP with a Ki value of 1.41 mM. Site-directed mutagenesis and a GDP-bead binding assay were also performed to investigate the functions of the highly conserved motif H152xR154R155xD157. In contrast to alpha1,6-fucosyltransferases, none of the mutants of WbsJ within this motif exhibited a complete loss of enzyme activity. However, residues R154 and D157 were found to play critical roles in donor binding and enzyme activity. The results suggest that the common motif shared by both alpha1,2-fucosyltransferases and alpha1,6-fucosyltransferases have similar functions. Enzymatic synthesis of fucosylated sugars in milligram scale was successfully performed using Galbeta-O-Me and Galbeta1,4Glcbeta-N3 as acceptors.     

Organization of the bovine alpha 2-fucosyltransferase gene cluster suggests that the Sec1 gene might have been shaped through a nonautonomous L1-retrotransposition event within the same locus

By referring to the split coding sequence of the highly conserved alpha 6-fucosyltransferase gene family (assumed to be representative of the common alpha 2 and alpha 6 fucosyltransferase gene ancestor), we have hypothesized that the monoexonic coding sequences of the present alpha 2-fucosyltransferase genes have been shaped in mammals by several events of retrotransposition and/or duplication. In order to test our hypothesis, we determined the structure of the three bovine alpha 2-fucosyltransferase genes (bfut1, bfut2, and sec1) and analyzed their characteristics compared with their human counterparts (FUT1, FUT2, and Sec1). We show that in mammals, a complex nonautonomous L1-retrotransposition event occurred within the locus of the alpha 2-fucosyltransferase ancestor gene itself. A consequence of this event was the processing in Catarrhini of a Sec1 pseudogene via several point mutations.     

Evolution of alpha 2-fucosyltransferase genes in primates: relation between an intronic Alu-Y element and red cell expression of ABH antigens

Coding sequences of the paralogous FUT1 (H), FUT2 (Se), and Sec1 alpha 2-fucosyltransferase genes were obtained from different primate species. Analysis of the primate FUT1-like and FUT2-like sequences revealed the absence of the known human inactivating mutations giving rise to the h null alleles of FUT1 and the se null alleles of FUT2. Therefore, most primate FUT1-like and FUT2-like genes potentially code for functional enzymes. The Sec1-like gene encodes for a potentially functional alpha 2-fucosyltransferase enzyme in nonprimate mammals, New World monkeys, and Old World monkeys, but it has been inactivated by a nonsense mutation at codon 325 in the ancestor of humans and African apes (gorillas, chimpanzees). Human and gorilla Sec1's have, in addition, two deletions and one insertion, respectively, 5' of the nonsense mutation leading to proteins shorter than chimpanzee Sec1. Phylogenetic analysis of the available H, Se, and Sec1 mammalian protein sequences demonstrates the existence of three clusters which correspond to the three genes. This suggests that the differentiation of the three genes is rather old and predates the great mammalian radiation. The phylogenetic analysis also suggests that Sec1 has a higher evolutionary rate than FUT2 and FUT1. Finally, we show that an Alu-Y element was inserted in intron 1 of the FUT1 ancestor of humans and apes (chimpanzees, gorillas, orangutans, and gibbons); this Alu-Y element has not been found in monkeys or nonprimate mammals, which lack ABH antigens on red cells. A potential mechanism leading to the red cell expression of the H enzyme in primates, related to the insertion of this Alu-Y sequence, is proposed.     

Human fucosyltransferase IX: specificity towards N-linked glycoproteins and relevance of the cytoplasmic domain in intra-Golgi localization

The alpha3-fucosyltransferase IX (FUT9) catalyses the transfer of fucose in an alpha3 linkage onto terminal type II (Galbeta4GlcNAc) acceptors, the final step in the biosynthesis of the Lewis(x) (Le(x)) epitope, in neurons. In this work, FUT9 cloned from NT2N neurons and overexpressed in HeLa cells (FUT9wt), was found to efficiently fucosylate asialoerythropoietin (asialoEPO), and bovine asialofetuin, but not sialylated EPO. Analysis by HPAEC-PAD and MALDI/TOF-MS revealed predominantly mono-fucosylation by FUT9wt of type II di-, tri- and tetraantennary N-glycans with proximal fucose, with and without N-acetylactosamine repeats from asialoEPO. Minor amounts of difucosylated structures were also found. The results suggested that FUT9 could fucosylate Le(x) carrier-glycoproteins in neurons. Furthermore, FUT9wt was found to be activated by Mn(2+) and it was capable of synthesizing Le(a), although to a lesser extent than Le(x) and Le(y). In vivo, HeLa cells transfected with FUT9wt expressed de novo Le(x), as detected by immunofluorescence microscopy. FUT9 was found to be a trans-Golgi and trans-Golgi network (TGN) glycosyltransferase from confocal immunofluorescence co-localization with the markers of the secretory pathway beta4-galactosyltransferase (trans-Golgi and TGN) and TGN-46 (TGN). Deletion of the cytoplasmic domain caused a shift to the cis-Golgi, thus suggesting that information for intra-Golgi localization is contained within the cytoplasmic domain.     

alpha(1,2)-fucosylation prevents sialyl Lewis x expression and E-selectin-mediated adhesion of fucosyltransferase VII-transfected cells.

E-selectin is a cytokine-inducible, calcium-dependent endothelial cell adhesion molecule that plays a critical role in the leucocyte-endothelium interaction during inflammation and is thought to contribute to the metastatic dissemination of tumour cells. Like the other selectins, E-selectin binds to ligands carrying the tetrasaccharide sialyl-Lewis x (NeuAcalpha2,3Galbeta1,4[Fucalpha1, 3]GlcNAc)1 or its isomer sialyl-Lewis a (NeuAcalpha2, 3Galbeta1, 3[Fucalpha1,4]GlcNAc). We examined the effect of expressing the H-type alpha(1,2)-fucosyltransferase or the alpha(2, 6)-sialyltransferase on the synthesis of sialyl-Lewis x by alpha(1, 3)fucosyltransferase. We found that H-type alpha(1, 2)-fucosyltransferase but not alpha(2,6)-sialyltransferase, strongly inhibited sialyl-Lewis x expression and E-selectin adhesion. We assume that H-type alpha(1,2)-fucosyltransferase competes with the endogenous alpha(2,3)-sialyltransferase for the N-acetyllactosamine structures assigned to further serve as acceptors for alpha(1, 3)fucosyltransferase.     

Ancestral exonic organization of FUT8, the gene encoding the alpha6-fucosyltransferase, reveals successive peptide domains which suggest a particular three-dimensional core structure for the alpha6-fucosyltransferase family

Based on PCR strategies and expression studies, we define the genomic organization of the FUT8b gene. This gene encodes the only known mammalian enzyme transferring fucose in an alpha1-->6 linkage on the asparagine-branched GlcNAc residue of the chitobiose unit of complex N:-glycans. The intron/exon organization of the bovine coding sequence determines five successive functional domains. The first exon encodes a domain homologous to cytoskeleton proteins, the second presents a proline-rich region including a motif XPXPPYXP similar to the peptide ligand of the SH3-domain proteins, the third encodes a gyrase-like domain (an enzyme which can bind nucleotides), and the fourth encodes a peptide sequence homologous to the catalytic domain of proteins transferring sugars. Finally, the last exon encodes a domain homologous to the SH3 conserved motif of the SH2-SH3 protein family. This organization suggests that intramolecular interactions might give a tulip-shaped scaffolding, including the catalytic pocket of the enzyme in the Golgi lumen. Deduced from the published sequence of chromosome 14 (AL109847), the human gene organization of FUT8 seems to be similar to that of bovine FUT8b, although the exon partition is more pronounced (bovine exons 1 and 2 correspond to human exons 1-6). The mosaicism and phylogenetic positions of the alpha6-fucosyltransferase genes are compared with those of other fucosyltransferase genes.     

Alpha1,4-fucosyltransferase activity: a significant function in the primate lineage has appeared twice independently

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.     

Tumor-related expression of alpha1,2fucosylated antigens on colorectal carcinoma cells and its suppression by cell-mediated priming using sugar acceptors for alpha1,2fucosyltransferase

The accumulation of alpha1,2fucosylated antigens, such as Y (Fucalpha1,2Galbeta1,4 [Fucalpha1,3]GlcNAcbeta), Le(b) (Fucalpha1,2Galbeta1,3-[Fucalpha1,4]GlcNAcbeta), and H type 2 (Fucalpha1,2 Galbeta1,4GlcNAcbeta) occurs specifically within human colorectal tumor tissues and can be detected by an antifucosylated antigen antibody, such as the YB-2 antibody. In the present investigation, we found that the expression of these antigens bearing an alpha1,2-linked fucose correlated with the resistance of the tumor cells to anticancer treatments. Addition of an exogenous sugar acceptor for alpha1,2fucosyltransferase to the cell medium resulted in suppression of alpha1,2fucosylated antigen expression on the tumor cells and increased susceptibility to anticancer treatment. The increased susceptibility may be attributed to cancer cell-mediated priming by sugar acceptors for alpha1,2fucosyltransferase added to the medium.     

In vitro transactivation of Bacillus subtilis RNase P RNA

We have partially characterised an alpha4-fucosyltransferase (alpha4-FucT) from Vaccinium myrtillus, which catalysed the biosynthesis of the Lewis(a) adhesion determinant. The enzyme was stable up to 50 degrees C. The optimum pH was 7.0, both in the presence and in the absence of Mn(2+). The enzyme was inhibited by Mn(2+) and Co(2+), and showed resistance towards inhibition with N-ethylmaleimide. It transferred fucose to N-acetylglucosamine in the type I Galbeta3GlcNAc motif from oligosaccharides linked to a hydrophobic tail and glycoproteins (containing the type I motif). Sialylated oligosaccharides containing the type II Galbeta4GlcNAc motif were not acceptors. The catalytic mechanism of the plant alpha4-FucT possibly involves a His residue, and it must have arisen by convergent evolution relative to its mammalian counterparts.     

Reaction mechanism and substrate specificity for nucleotide sugar of mammalian alpha1,6-fucosyltransferase--a large-scale preparation and characterization of recombinant human FUT8

FUT8, mammalian 1,6-fucosyltransferase, catalyzes the transferof a fucose residue from the donor substrate, guanosine 5'-diphosphate(GDP)-ß-L-fucose, to the reducing terminal GlcNAcof the core structure of asparagine-linked oligosaccharide viaan 1,6-linkage. FUT8 is a typical type II membrane protein,which is localized in the Golgi apparatus. We have previouslyshown that two neighboring arginine residues that are conservedamong 1,2-, 1,6-, and protein O-fucosyltransferases play animportant role in donor substrate binding. However, detailsof the catalytic and reaction mechanisms and the ternary structureof FUT8 are not understood except for the substrate specificityof the acceptor. To develop a better understanding of FUT8,we established a large-scale production system for recombinanthuman FUT8, in which the enzyme is produced in soluble formby baculovirus-infected insect cells. Kinetic analyses and inhibitionstudies using derivatives of GDP-ß-L-fucose revealedthat FUT8 catalyzes the reaction which depends on a rapid equilibriumrandom mechanism and strongly recognizes the base portion anddiphosphoryl group of GDP-ß-L-fucose. These resultsmay also be applicable to other fucosyltransferases and glycosyltransferases.     

Deficiency of reproductive tract alpha(1,2)fucosylated glycans and normal fertility in mice with targeted deletions of the FUT1 or FUT2 alpha(1,2)fucosyltransferase locus.

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.     

Cytogenetics, conserved synteny and evolution of chicken fucosyltransferase genes compared to human

Fucosyltransferases appeared early in evolution, since they are present from bacteria to primates and the genes are well conserved. The aim of this work was to study these genes in the bird group, which is particularly attractive for the comprehension of the evolution of the vertebrate genome. Twelve fucosyltransferase genes have been identified in man. The orthologues of theses genes were looked for in the chicken genome and cytogenetically localized by FISH. Three families of fucosyltransferases: alpha6-fucosyltransferases, alpha3/4-fucosyltransferases, and protein-O-fucosyltransferases, were identified in the chicken with their associated genes. The alpha2-fucosyltransferase family, although present in some invertebrates and amphibians was not found in birds. This absence, also observed in Drosophila, may correspond to a loss of these genes by negative selection. Of the eight chicken genes assigned, six fell on chromosome segments where conservation of synteny between human and chicken was already described. For the two remaining loci, FUT9 and FUT3/5/6, the location may correspond to a new small syntenic area or to an insertion. FUT4 and FUT3/5/6 were found on the same chicken chromosome. These results suggest a duplication of an ancestral gene, initially present on the same chromosome before separation during evolution. By extension, the results are in favour of a common ancestor for the alpha3-fucosyltransferase and the alpha4-fucosyltransferase activities. These observations suggest a general mechanism for the evolution of fucosyltransferase genes in vertebrates by duplication followed by divergent evolution.     

Purification of the secretor-type beta-galactoside alpha 1----2-fucosyltransferase from human serum.

The secretor-type beta-galactoside alpha 1----2-fucosyltransferase from human serum was purified by hydrophobic chromatography on phenyl-Sepharose, ion-exchange chromatography on sulfopropyl-Sepharose, and affinity chromatography on GDP-hexanolamine-Sepharose. Final purification of the enzyme was achieved by high pressure liquid chromatography gel filtration and resulted in a homogeneous protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the radiolabeled protein. The native enzyme appears as a molecule of apparent Mr 150,000 as determined by gel filtration high pressure liquid chromatography. The apparent Mr of the enzyme resolved in the presence of beta-mercaptoethanol by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined to be 50,000, indicating a multisubunit structure of the enzyme. Secretor-type alpha 1----2-fucosyltransferase is a glycoprotein as determined by WGA binding properties. A comparison of the Mr of the native blood group H gene encoded with the secretor-type beta-galactoside alpha 1----2-fucosyltransferases as well as comparison of subunit Mr for both enzymes suggests structural similarity. The alpha 1----2 linkage formed between alpha-L-fucose and terminal beta-D-galactose by the purified H- and secretor-type alpha 1----2-fucosyltransferases was determined by 1H NMR homonuclear cross-irradiation analysis of the oligosaccharide products. The substrate specificity and Km values calculated from the initial rate using various oligosaccharide acceptors showed that purified enzymes differ primarily in affinity for phenyl-beta-D-galactopyranoside and GDP-fucose as well as type 1 (Gal beta 1----3GlcNAc), 2 (Gal beta 1----4GlcNAc), and 3 (Gal beta 1----3GalNAc) oligosaccharide acceptors. The secretor-type alpha 1----2-fucosyltransferase shows significantly lower affinity than the H enzyme for phenyl-beta-D-galactopyranoside and GDP-fucose as well as for type 2 oligosaccharide acceptors. On the contrary, type 1 and 3 oligosaccharide acceptors are preferentially utilized by the secretor-type enzyme as compared with the H enzyme. The enzymes also differ in several physicochemical properties, implying nonidentity of the two enzymes (Sarnesto, A., K?hlin, T., Thurin, J., and Blaszczyk-Thurin, M. (1990) J. Biol. Chem. 265, 15067-15075).     

Comparison of the three rat GDP-L-fucose:beta-D-galactoside 2-alpha-L-fucosyltransferases FTA, FTB and FTC.

The complete coding sequences of three rat alpha1,2fucosyltransferase genes were obtained. Sequence analysis revealed that these genes, called FTA, FTB and FTC, were homologous to human FUT1, FUT2 and Sec1, respectively. A distance analysis between all alpha1,2fucosyltransferase sequences available showed that the two domains of the catalytic region evolved differently with little divergence between the FUT2 and Sec1 N-terminal domains, quite distant from that of FUT1. At variance, FUT1 and FUT2 C-terminal domains were less distant while a high evolutionary rate was noted for Sec1 C-terminal domain. Whereas FTA and FTB encode typical glycosyltransferases, FTC lacks the homologous start codon and encodes a protein devoid of intracellular and transmembrane domains. It is located on rat chromosome 1q34. Transfection experiments revealed that unlike FTA and FTB, FTC does not generate enzyme activity. Analysis by flow cytometry showed that H type 2 epitopes were synthesized in Chinese hamster ovary cells transfected by both FTA and FTB cDNA, but only FTB transfectants possessed H type 3 determinants. In REG rat carcinoma cells, both FTA and FTB allowed synthesis of H type 2 and H type 3 at the cell surface. Western blots showed that, in both cell types, FTA was able to synthesize H type 2 epitopes on a larger set of glycoproteins than FTB. Analysis of the kinetic parameters obtained using small oligosaccharides revealed only a slight preference of FTA for type 2 over other types of acceptor substrates, whereas FTB was barely able to fucosylate this substrate.     

Fucosyltransferases in Schistosoma mansoni development

Glycoconjugate-bound fucose, abundant in the parasite Schistosoma mansoni, has been found in the form of Fucalpha1,3GlcNAc, Fucalpha1,2Fuc, Fucalpha1,6GlcNAc, and perhaps Fucalpha1,4GlcNAc linkages. Here we quantify fucosyltransferase activities in three developmental stages of S. mansoni. Assays were performed using fluorophore-assisted carbohydrate electrophoresis with detection of radioactive fucose incorporation from GDP-[(14)C]-fucose into structurally defined acceptors. The total fucosyltransferase-specific activity in egg extracts was 50-fold higher than that in the other life stages tested (cercaria and adult worms). A fucosyltransferase was detected that transferred fucose to type-2 oligosaccharides (Galbeta1,4GlcNAc-R), both sialylated (with the sialic acid attached to the terminal Gal by alpha2,3 or 2,6 linkage) and nonsialylated. Another fucosyltransferase was identified that transferred fucose to lactose-based and type-2 fucosylated oligosaccharides, such as LNFIII (Galbeta1,4(Fucalpha1,3)GlcNAcbeta1,3Galbeta1,4Glc). A low level of fucosyltransferase that transfers fucose to no-sialylated type-1 oligosaccharides (Galbeta1,3GlcNAc-R) was also detected. These studies revealed multifucosylated products of the reactions. In addition, the effects of fucose-type iminosugars inhibitors were tested on schistosome fucosyltransferases. A new fucose-type 1-N-iminosugar was four- to sixfold more potent as an inhibitor of schistosome fucosyltransferases in vitro than was deoxyfuconojirimycin. In vivo, this novel 1-iminosugar blocked the expression of a fucosylated epitope (mAb 128C3/3 antigen) that is associated with the pathogenesis of schistosomiasis.     

Molecular cloning and characterization of a novel alpha 1,2-fucosyltransferase (CE2FT-1) from Caenorhabditis elegans

Here we report the discovery of a unique fucosyltransferase (FT) in Caenorhabditis elegans. In studying the activities of FTs in extracts of adult C. elegans, we detected activity toward the unusual disaccharide acceptors Galbeta1-4Xyl-R and Galbeta1-6GlcNAc-R to generate products with the general structure Fucalpha1-2Galbeta1-R. We identified a gene encoding a unique alpha1,2FT (designated CE2FT-1), which contains an open reading frame encoding a predicted protein of 355 amino acids with the type 2 topology and domain structure typical of other glycosyltransferases. The predicted cDNA for CE2FT-1 has very low identity (5-10%) at the amino acid level to alpha1,2FT sequences in humans, rabbits, and mice. Recombinant CE2FT-1 expressed in human 293T cells has high alpha1,2FT activity toward the simple acceptor Galbeta-O-phenyl acceptor to generate Fucalpha1-2Galbeta-R, which in this respect resembles mammalian alpha1,2FTs. However, CE2FT-1 is otherwise completely different from known alpha1,2FTs in its acceptor specificity, since it is unable to fucosylate either Galbeta1-4Glcbeta-R or free lactose and prefers the unusual acceptors Galbeta1-4Xylbeta-R and Galbeta1-6GlcNAc-R. Promoter analysis of the CE2FT-1 gene using green fluorescent protein reporter constructs demonstrates that CE2FT-1 is expressed in single cells of early stage embryos and exclusively in the 20 intestinal cells of L(1)-L(4) and adult worms. These and other results suggest that multiple fucosyltransferase genes in C. elegans may encode enzymes with unique activities, expression, and developmental roles.     

A sequence motif involved in the donor substrate binding by alpha1,6-fucosyltransferase: the role of the conserved arginine residues

Alpha1,6-fucosyltransferase catalyzes the transfer of fucose to the innermost GlcNAc residue of an N-linked oligosaccharide. In order to identify the amino acid residue(s) which are associated with the enzyme activity and to investigate their function, we prepared a series of mutant human alpha1,6-fucosyltransferases in which the conserved residues in the region homologous to alpha1,2-fucosyltransferase had been replaced. These proteins were then characterized by kinetic analyses. The wild-type and mutant alpha1,6-fucosyltransferases were expressed using a baculovirus-insect cell system. The activity assay showed that replacement of Arg-365 by Ala or Lys led to a complete loss of activity while substitution of Ala or Lys for the neighboring Arg-366 decreased the activity to about 3% that of the wild type. Kinetic analyses revealed that the replacements of Arg-366 lead to an increase in the apparent K (m) value for both GDP-fucose and the acceptor oligosaccharide but did not markedly affect the apparent V (max). When these mutants were inhibited by GDP in a competitive manner with respect to the donor substrate, the K (i) values were found to be 50-100 times higher than the value in the wild type. On the other hand, in the inhibition by GMP, the K (i) values for the mutants were very similar to that of the wild type. These findings suggest that Arg-366 contributes to the binding of GDP-fucose via an interaction with the beta-phosphoryl group of the GDP moiety of the donor, and that Arg-365 may also play an essential role in substrate binding. The results suggest that the motif common to alpha1,2- and alpha1,6-fucosyltransferases is critical for binding of the donor substrate, GDP-fucose.     

Molecular cloning, genomic mapping, and expression of two secretor blood group alpha (1,2)fucosyltransferase genes differentially regulated in mouse uterine epithelium and gastrointestinal tract

Fucosylated oligosaccharides have been proposed to be involved in multiple cell-cell interactions, including mouse blastocyst adhesion and intestine-microbe interactions. To begin to define the regulation and function of terminal alpha(1,2)fucosylated carbohydrates in these and other tissues, we isolated and characterized a 85-kilobase (kb) genomic region of mouse chromosome 7, 23.2 centimorgans analogous to human chromosome 19q13.3 that encodes three alpha(1,2)fucosyltransferases. Gene-specific DNA probes from the open reading frames of the mouse fucosyltransferase genes corresponding to human FUT1, FUT2, and SEC1 demonstrate distinct tissue-specific expression patterns by Northern blot analyses. Flow cytometry profiles of cultured cells transfected with DNA segments containing the open reading frames of the mouse genes confirm that each encodes an alpha(1,2)fucosyltransferase. In uterus and colon, a 3.3-kb FUT2 mRNA represents the major fucosyltransferase gene expressed. Steady-state FUT2 mRNA levels are cyclically regulated during the estrus cycle, increasing 10-fold from early diestrus to a relative maximum in proestrus. In contrast, SEC1 and FUT1 do not show prominently regulated expression in uterus. FUT2 expression localizes to luminal uterine epithelium by in situ hybridization, implying that this gene determines expression of cell surface Fucalpha1-->2Galbeta epitopes proposed to mediate blastocyst adhesion.