Publications

• Stable expression of blood group H determinants and GDP-L-fucose: beta-D-galactoside 2-alpha-L-fucosyltransferase in mouse cells after transfection with human DNA.

  Ernst L.K., Rajan V.P., Larsen R.D., Ruff M.M., Lowe J.B.

  We report here the application of a genetic approach to identify and isolate human DNA sequences controlling the expression of a GDP-L-fucose: beta-D-galactoside 2-alpha-L-fucosyltransferase [alpha-1,2)fucosyltransferase). Mouse L cells were chosen as host cells for this scheme since they express ... >> More

  We report here the application of a genetic approach to identify and isolate human DNA sequences controlling the expression of a GDP-L-fucose: beta-D-galactoside 2-alpha-L-fucosyltransferase [alpha-1,2)fucosyltransferase). Mouse L cells were chosen as host cells for this scheme since they express the necessary substrate and acceptor molecules for surface display of blood group H Fuc alpha 1----2 G al linkages constructed by (alpha-1,2) fucosyltransferases. However, they do not express cell surface blood group H structures nor detectable (alpha-1,2)fucosyltransferase activity. We therefore asked if (alpha-1,2)fucosyltransferase activity could be expressed and detected in these cells after transfection with human DNA sequences. These cells were transfected with genomic DNA isolated from a human cell line (A431) that expresses (alpha-1,2)fucosyltransferase. A panning procedure and fluorescence-activated cell sorting were used to isolate a mouse transfectant cell line that expresses cell surface H Fuc alpha 1----2 Gal linkages and a cognate (alpha-1,2)fucosyltransferase. Southern blot analysis showed that the genome of this cell line contains several hundred kilobase pairs of human DNA. Genomic DNA from this primary transfectant was used to transfect mouse L cells, and several independent, H-expressing secondary transfectants were isolated by immunological selection. Each expresses an (alpha-1,2)fucosyltransferase. Southern blot analysis demonstrated that the genome of each secondary transfectant contains common, characteristic human DNA restriction fragments. These results show that transfected human DNA sequences determine expression of the (alpha-1,2)fucosyltransferases in the mouse transfectants, that these sequences represent a single locus, and that they are within or linked to specific human restriction fragments identifiable in each secondary transfectant. These sequences may represent a human (alpha-1,2)fucosyltransferase gene. << Less

  J Biol Chem 264:3436-3447(1989) [PubMed] [EuropePMC]

• Molecular cloning, sequence, and expression of a human GDP-L-fucose:beta-D-galactoside 2-alpha-L-fucosyltransferase cDNA that can form the H blood group antigen.

  Larsen R.D., Ernst L.K., Nair R.P., Lowe J.B.

  We have previously used a gene-transfer scheme to isolate a human genomic DNA fragment that determines expression of a GDP-L-fucose:beta-D-galactoside 2-alpha-L-fucosyltransferase [alpha(1,2)FT; EC 2.4.1.69]. Although this fragment determined expression of an alpha(1,2)FT whose kinetic properties ... >> More

  We have previously used a gene-transfer scheme to isolate a human genomic DNA fragment that determines expression of a GDP-L-fucose:beta-D-galactoside 2-alpha-L-fucosyltransferase [alpha(1,2)FT; EC 2.4.1.69]. Although this fragment determined expression of an alpha(1,2)FT whose kinetic properties mirror those of the human H blood group alpha(1,2)FT, their precise nature remained undefined. We describe here the molecular cloning, sequence, and expression of a human cDNA corresponding to these human genomic sequences. When expressed in COS-1 cells, this cDNA directs expression of cell surface H structures and a cognate alpha(1,2)FT activity with properties analogous to the human H blood group alpha(1,2)FT. The cDNA sequence predicts a 365-amino acid polypeptide characteristic of a type II transmembrane glycoprotein with a domain structure analogous to that of other glycosyltransferases but without significant primary sequence similarity to these or other known proteins. To directly demonstrate that the cDNA encodes an alpha(1,2)FT, the COOH-terminal domain predicted to be Golgi-resident was expressed in COS-1 cells as a catalytically active, secreted, and soluble protein A fusion peptide. Southern blot analysis showed that this cDNA identifies DNA sequences syntenic to the human H locus on chromosome 19. These results strongly suggest that this cloned alpha(1,2)FT cDNA represents the product of the human H blood group locus. << Less

  Proc. Natl. Acad. Sci. U.S.A. 87:6674-6678(1990) [PubMed] [EuropePMC]

• Porcine submaxillary gland GDP-L-fucose: beta-D-galactoside alpha-2-L-fucosyltransferase is likely a counterpart of the human Secretor gene-encoded blood group transferase.

  Thurin J., Blaszczyk-Thurin M.

  Partial amino acid sequence of GDP-L-fucose:beta-D-galactoside alpha-2-L-fucosyltransferase purified from porcine submaxillary glands was determined. Amino acid sequence analysis yielded 100, 93.3, and 84.2%, and 75, 46.6, and 84.2% sequence identity between 12-, 15-, and 19-amino acid tryptic pep ... >> More

  Partial amino acid sequence of GDP-L-fucose:beta-D-galactoside alpha-2-L-fucosyltransferase purified from porcine submaxillary glands was determined. Amino acid sequence analysis yielded 100, 93.3, and 84.2%, and 75, 46.6, and 84.2% sequence identity between 12-, 15-, and 19-amino acid tryptic peptides generated from porcine enzyme and amino acid residues 61-72, 111-125, and 308-326 and 89-100, 139-153, and 338-356 of the human Secretor and H type alpha-2-fucosyltransferases, respectively. Higher amino acid sequence homology of the porcine enzyme with the predicted sequence for the human Secretor locus as compared with H gene-encoded blood group beta-D-galactoside alpha-2-L-fucosyltransferase suggests that porcine alpha-2-fucosyltransferase highly corresponds to the human Secretor gene-encoded enzyme. << Less

  J. Biol. Chem. 270:26577-26580(1995) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• 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.

  Domino S.E., Zhang L., Lowe J.B.

  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 i ... >> More

  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. << Less

  J. Biol. Chem. 276:23748-23756(2001) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Sequence and expression of a candidate for the human Secretor blood group alpha(1,2)fucosyltransferase gene (FUT2). Homozygosity for an enzyme-inactivating nonsense mutation commonly correlates with the non-secretor phenotype.

  Kelly R.J., Rouquier S., Giorgi D., Lennon G.G., Lowe J.B.

  Synthesis of soluble A, B, H, and Lewis b blood group antigens in humans is determined by the Secretor (Se) (FUT2) blood group locus. Genetic, biochemical, and molecular analyses indicate that this locus corresponds to an alpha(1,2)fucosyltransferase gene distinct from the genetically-linked H blo ... >> More

  Synthesis of soluble A, B, H, and Lewis b blood group antigens in humans is determined by the Secretor (Se) (FUT2) blood group locus. Genetic, biochemical, and molecular analyses indicate that this locus corresponds to an alpha(1,2)fucosyltransferase gene distinct from the genetically-linked H blood group alpha(1,2)fucosyltransferase locus. The accompanying paper (Rouquier, S., Lowe, J. B., Kelly, R. J., Fertitta, A. L., Lennon, G. G., and Giorgi, D. (1995) J. Biol. Chem. 270, 4632-4639) describes the molecular cloning and mapping of two human DNA segments that are physically linked to, and cross-hybridize with, the H locus. We present here an analysis of these two new DNA segments. One of these, termed Sec1, is a pseudogene, because translational frameshifts and termination codons interrupt potential open reading frames that would otherwise share primary sequence similarity with the H alpha(1,2)fucosyltransferase. The other DNA segment, termed Sec2, predicts a 332-amino acid-long polypeptide, and a longer isoform, that share 68% sequence identity with the COOH-terminal 292 residues of the human H blood group alpha(1,2)fucosyltransferase. Sec2 encodes an alpha(1,2)fucosyltransferase with catalytic properties that mirror those ascribed to the Secretor locus-encoded alpha(1,2)fucosyltransferase. Approximately 20% of randomly-selected individuals were found to be apparently homozygous for an enzyme-inactivating nonsense allele (Trp143-->ter) at this locus, in correspondence to the frequency of the non-secretor phenotype in most human populations. Furthermore, each of six unrelated non-secretor individuals are also apparently homozygous for this null allele. These results indicate that Sec2 corresponds to the human Secretor blood group locus (FUT2) and indicate that homozygosity for a common nonsense allele is responsible for the nonsecretor phenotype in many non-secretor individuals. << Less

  J. Biol. Chem. 270:4640-4649(1995) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• A DNA polymorphism influencing alpha(1,2)fucosyltransferase activity of the pig FUT1 enzyme determines susceptibility of small intestinal epithelium to Escherichia coli F18 adhesion.

  Meijerink E., Neuenschwander S., Fries R., Dinter A., Bertschinger H.U., Stranzinger G., Vogeli P.

  The alpha(1,2)fucosyltransferases (FUT1 and FUT2) contribute to the formation of blood group antigen structures, which are present on cell membranes and in secretions. In the present study we demonstrate that both FUT1 and FUT2 are expressed in the pig small intestine. FUT1 polymorphisms influence ... >> More

  The alpha(1,2)fucosyltransferases (FUT1 and FUT2) contribute to the formation of blood group antigen structures, which are present on cell membranes and in secretions. In the present study we demonstrate that both FUT1 and FUT2 are expressed in the pig small intestine. FUT1 polymorphisms influence adhesion of F18 fimbriated Escherichia coli (ECF18) to intestinal mucosa, and FUT2 is associated with expression of erythrocyte antigen 0. The FUT1 polymorphisms result in amino acid substitutions at positions 103 (Ala-->Thr) and 286 (Arg-->Glu). Tightly controlled expression of the FUT2 gene results in either an abundance or an absence of mRNA in small intestinal mucosa. ECF18-resistant animals were shown to be homozygous for threonine at amino acid 103 of the FUT1 enzyme. Susceptibility to ECF18 adhesion appeared to be solely dependent on the activity of FUT1 in intestinal epithelia. In intestinal mucosae of ECF18-resistant pigs which expressed FUT1 but not FUT2 RNA, the levels of alpha(1,2)fucosyltransferase activity were significantly lower (28-to 45-fold, P<0.001) than in susceptible pigs. Moreover, lysates of CHO cells transfected with FUT1 constructs encoding threonine at amino acid position 103 also showed significantly reduced enzyme activity compared with constructs encoding alanine at this position. Our genetic and enzymatic studies support the hypothesis that the FUT1 enzyme, and particularly the amino acid at position 103, is likely important in the synthesis of a structure that enables adhesion of ECF18 bacteria to small intestinal mucosa. << Less

  Immunogenetics 52:129-136(2000) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Enzymatic synthesis of a blood group H-related glycosphingolipid by an alpha-fucosyltransferase from bovine spleen.

  Basu S., Basu M., Chien J.L.

  An alpha-fucosyltransferase activity has been detected in a purified membrane preparation isolated from bovine spleen which catalyzes the transfer of L-fucose from GDP-L-[14C]-fucose to a tetraglycosylceramide (Lac-nTet-cer, Galbetal-4GlcNAcbeta1-3Galbeta1-4-Glc-cer) to form the blood group H-rela ... >> More

  An alpha-fucosyltransferase activity has been detected in a purified membrane preparation isolated from bovine spleen which catalyzes the transfer of L-fucose from GDP-L-[14C]-fucose to a tetraglycosylceramide (Lac-nTet-cer, Galbetal-4GlcNAcbeta1-3Galbeta1-4-Glc-cer) to form the blood group H-related glycosphingolipid. The membrane preparation contained a highly active endogenous nonlipid acceptor, which could be precipitated by 5% trichloroacetic acid or chloroform-methanol-water (6:3:5, v/v/v), whereas there was little endogenous glycosphingolipid acceptor. The optimum pH value for the incorporation of L-fucose was 6.4 in cacodylate-HCl buffer. The Km values were 0.6 mM and 0.36 mM for Lac-nTet-cer and GDP-L-fucose, respectively. The 14C-labeled product of the reaction was isolated and purified; it migrated with human erythrocyte blood group H-active pentaglycosylceramide. the terminal [14C]fucose was hydrolyzed 85% and 55% by 0.1 N trichloroacetic acid at 100 degrees for 2 hours and Charonia lampas alpha-fucosidase (19 hours at 37 degrees), respectively. The 14C-labeled product inhibited the hemagglutination reaction of O-type cells against eel anti H(O) globulin and formed a precipitin line with Ulex europeus lectin. << Less

  J Biol Chem 250:2956-2962(1975) [PubMed] [EuropePMC]

• Molecular cloning and expression of two types of rabbit beta-galactoside alpha 1,2-fucosyltransferase.

  Hitoshi S., Kusunoki S., Kanazawa I., Tsuji S.

  Two DNA clones encoding rabbit beta-galactoside alpha 1,2-fucosyltransferase (RFT-I and RFT-II) have been isolated from a rabbit genomic DNA library. The DNA sequences revealed open reading frames coding for 373 (RFT-I) and 354 (RFT-II) amino acids, respectively. The deduced amino acid sequences o ... >> More

  Two DNA clones encoding rabbit beta-galactoside alpha 1,2-fucosyltransferase (RFT-I and RFT-II) have been isolated from a rabbit genomic DNA library. The DNA sequences revealed open reading frames coding for 373 (RFT-I) and 354 (RFT-II) amino acids, respectively. The deduced amino acid sequences of RFT-I and RFT-II showed 56% identity with each other, and that of RFT-I showed 80% identity with that of human H blood type alpha 1,2-fucosyltransferase. Northern blot analysis of embryo and adult rabbit tissues revealed that the RFT-I gene was expressed in adult brain, and that the RFT-II gene was expressed in salivary and lactating mammary glands. The identities of these enzymes were confirmed by constructing recombinant fucosyltransferases in which the N-terminal part including the cytoplasmic tail and signal anchor domain was replaced with the immunoglobulin signal peptide sequence. RFT-I expressed in COS-7 cells exhibited similar transferase activity to that of human H blood type alpha 1,2-fucosyltransferase. RFT-II expressed in COS-7 cells showed higher affinity for type 1 (Gal beta 1,3GlcNAc) and type 3 (Gal beta 1,3GalNAc) acceptors than type 2 (Gal beta 1,4GlcNAc) ones, which suggested that RFT-II was a putative secretor-type alpha 1,2-fucosyltransferase. << Less

  J. Biol. Chem. 270:8844-8850(1995) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

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

  Bureau V., Marionneau S., Cailleau-Thomas A., Le Moullac-Vaidye B., Liehr T., Le Pendu J.

  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 availabl ... >> More

  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. << Less

  Eur. J. Biochem. 268:1006-1019(2001) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• An amino acid region at the N-terminus of rat hepatoma alpha1-->2 fucosyltransferase modulates enzyme activity and interaction with lipids: strong preference for glycosphingolipids containing terminal Galbeta1-->3GalNAc-structures.

  Sherwood A.L., Stroud M.R., Levery S.B., Holmes E.H.

  A GDP-fucose:GM1 alpha1-->2 fucosyltransferase (FucT) is induced during early stages of chemical hepatocarcinogenesis in parenchymal cells of Fischer 344 rats fed a diet supplemented with 0.03% N-2-acetylaminofluorene (AAF). This enzyme is undetectable in normal rat liver tissues but is highly exp ... >> More

  A GDP-fucose:GM1 alpha1-->2 fucosyltransferase (FucT) is induced during early stages of chemical hepatocarcinogenesis in parenchymal cells of Fischer 344 rats fed a diet supplemented with 0.03% N-2-acetylaminofluorene (AAF). This enzyme is undetectable in normal rat liver tissues but is highly expressed in many rat hepatoma cell lines, including rat hepatoma H35 cells. Enzymatic properties and acceptor specificity of native rat hepatoma H35 cell alpha1-->2FucT, expressed recombinant full-length H35 cell alpha1-->2FucT, and a truncated form missing the first 27 amino acid residues from the N-terminus, comprising the cytoplasmic and transmembrane domains of the enzyme, were studied. The results indicate that the recombinant full-length enzyme has a specific activity over 80-fold higher than the truncated enzyme. Both the native and recombinant full-length enzymes display significant activity in the absence of detergent or phospholipid and optimal activity in the presence of Triton CF-54 detergent. The truncated enzyme is optimally activated by CHAPSO, showing little activity in its absence. These findings are in agreement with previous studies demonstrating a requirement of a lipidic environment for optimal activity with this enzyme and suggest that the N-terminal transmembrane domain is important either in the maintenance of an active conformation or in allowing efficient interaction with acceptor glycolipids. Both the full-length and truncated enzymes transfer fucose not only to GM1 and asialo-GM1 (Gg4) but also to galactosyl globoside (Gb5) as well. Weak or undetectable transfer to lacto- and neolacto-series acceptors was observed, demonstrating a strong preference for terminal Galbeta1-->3GalNAc-structures. The structures of two reaction products generated by expressed recombinant full-length alpha1-->2FucT, which are known to be important tumor-associated antigens (fucosyl-GM1 and fucosyl-Gb5), were unambiguously confirmed by 1H-NMR spectral analysis. << Less

  Biochemistry 40:5708-5719(2001) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.