Sequence and Expression of a Candidate for the Human Secretor Blood Group α(1,2)Fucosyltransferase Gene (FUT2)

Kelly, Robert J.; Rouquier, Sylvie; Giorgi, Dominique; Lennon, Gregory G.; Lowe, John B.

doi:10.1074/jbc.270.9.4640

Cited by 512 publications

(290 citation statements)

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“…Characterization of the enzymatic activity indicates that A1,2FT expressed in COLO-205 cells is functionally different from HA1,2FT, and similar to the secretor-type activities [18]. Moreover, we found that in the Caco-2 and HT-29 cells, where SeA1,2FT specific activity is extremely low, Le b antigen expression is also very low or undetectable by immunofluorescence.…”

Section: Discussionmentioning

confidence: 74%

“…SeA1,2FT was determined in a reaction mixture containing, in a final volume of 20 µl, 0.1 M Tris/HCl, pH 6.5, 0.5 mg/ml Triton X-100, 10 mM Fuc, 5 mM ATP, 360 µM donor GDP-[ 3 H]Fuc (specific radioactivity 25 mCi/mmol), 10 mM acceptor phenyl β-galactoside and 4.0 mg/ml cell protein, in the case of crude homogenate, or 0.2 mg/ml protein, in the case of the Golgi-enriched fraction. The reaction mixture lacks divalent cations, according to the procedure reported for genuine SeA1,2FT gene product [18]. Incubation was at 37°C for 90 min.…”

Section: Methodsmentioning

confidence: 99%

“…A single treatment at 94°C for 2.0 min, followed by a cycle consisting of 1.5 min at 94°C (melting) and 3.5 min at 72°C (annealing plus extension), that has been repeated 35 times; a final extension step was performed at 72°C for 8 min. Oligonucleotide primer pairs were designed on the basis of fucosyltransferase published sequences [18,24], as follows: Fuc-TIII, 5′-CTGCTGGTGGCTGTGTGT-TTCTTCTCCTAC (upper-strand primer), and 5′-CAGCCAGC-CGTAGGGCGTGAAGATGTCGGA (lower-strand primer); HA1,2FT, 5′-TGTGTCCAGACCGCCGCCTGGTG (upperstrand primer), and 5′-CAGGCGGAGCTGACCCAGCACAC (lower-strand primer); SeA1,2FT, 5′-GCGCGAATTCGCGG-CTAGCGAAGATTCAAGCCATG (upper-strand primer); 5′-GCGCTCTAGATCACCTGCAGGCCCCGCAGGAAC (lowerstrand primer). The last primer pair contain a GCGC filler preceding an EcoRI or XbaI restriction site (underlined), in addition to the annealing region.…”

Section: Methodsmentioning

confidence: 99%

“…Enzymatic studies suggested that the secretor-type A-1,2-fucosyltansferase (FT), the product of the recently cloned FUT2 gene [18], is responsible for this step in both normal gastrointestinal tract and colon cancer [19]. However, some investigators found that Le b expression in cancer tissues occurred regardless of the secretor status of the patient [5,20], and a recent study indicated that the H type A1,2 FT, the product of the previously cloned FUT1 gene [21], is differentially expressed in colon cancer and involved in its progression [22].…”

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confidence: 99%

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β‐1,3‐galactosyltransferase and α‐1,2‐fucosyltransferase involved in the biosynthesis of type‐1‐chain carbohydrate antigens in human colon adenocarcinoma cell lines

Valli¹,

Gallanti²,

Bozzaro³

et al. 1998

European Journal of Biochemistry

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We studied the β-1,3-galactosyltransferase (GalT) and A-1,2-fucosyltansferase (FT) involved in the biosynthesis of type-1-chain carbohydrate antigens in human colon adenocarcinoma cell lines. We detected a GalT activity able to use GlcNAc as acceptor and found that lacto-N-biose I (Galβ1-3GlcNAc) is the only reaction product. Such β1,3GalT is kinetically similar to a pig trachea enzyme involved in mucin synthesis. The specific activity is high in cells that react strongly with anti-Lewis a and anti-Lewis b antibodies, and undetectable in a cell line that lacks antibody reaction. Reverse-transcriptase-mediated PCR analysis followed by DNA sequencing indicated that secretor-type A1,2FT is expressed in the cells, while the H type A1,2FT is not. The apparent K m values for donor and acceptor substrates determined for A1,2FT are similar to those of secretor-type A1,2FT and the specific activity measured correlates with Lewis b antigen expression on the cell surface. Moreover, some of the cell lines express Lewis y and H type 2 antigens, indicating that secretor type A1,2FT is responsible for their synthesis. Results suggest that biosynthesis of type-1-chain tumor-associated antigens in human colon carcinoma cells is operated by secretor-type A1,2FT, as reported in normal mucosa, and that β1,3GalT activity may play a relevant role in its control.Keywords : glycosyltransferase; carbohydrate antigen; colon carcinoma ; immunostaining.Association with tumor progression and malignancy has been reported for several carbohydrate antigens [1,2] and recent data suggest this is presumably due to their involvement in selectin-dependent tumor cell adhesion to the vascular endothelium [3,4]. Type-1-chain carbohydrate antigens such as SLe a (NeuAcA2-3Galβ1-3[FucA1-4]GlcNAc) and GlcNAc) are expressed in several cancers [1, 5Ϫ8]. Specific involvement of SLe a in selectin-dependent cell adhesion has been suggested [9, 10] and its stage-specific expression has been described in embryonic pancreas [11]. In the case of Le b, as well as in that of its type 2 chain counterpart Le y (FucA1-2Galβ1-4[FucA1-3]GlcNAc) [12,13], the molecular basis of their association with malignancy is still not fully elucidated.The biosynthesis of type-1-chain tumor markers depends on the activity of a galactosyltransferase (GalT) able to operate the Correspondence to

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Section: Discussionmentioning

confidence: 74%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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β‐1,3‐galactosyltransferase and α‐1,2‐fucosyltransferase involved in the biosynthesis of type‐1‐chain carbohydrate antigens in human colon adenocarcinoma cell lines

Valli¹,

Gallanti²,

Bozzaro³

et al. 1998

European Journal of Biochemistry

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“…The human ␣(1,2)-Fuc-T gene family comprises FUT1 encoding for the H enzyme and FUT2 encoding for the human secretor ␣(1,2)-fucosyltransferase (9). FUT3-7 encode for ␣(1,3)-Fuc-Ts.…”

mentioning

confidence: 99%

Significance of Individual Point Mutations, T202C and C314T, in the Human Lewis (FUT3) Gene for Expression of Lewis Antigens by the Human α(1,3/1,4)-Fucosyltransferase, Fuc-TIII

Elmgren¹,

Mollicone²,

Costache³

et al. 1997

Journal of Biological Chemistry

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The Lewis ␣(1,3/1,4)-fucosyltransferase, Fuc-TIII, encoded by the FUT3 gene is responsible for the final synthesis of Le a and Le b antigens. Various point mutations have been described explaining the Lewis negative phenotype, Le(a؊b؊), on erythrocytes and secretions. Two of these, T202C and C314T originally described in a Swedish population, have not been found as single isolated point mutations so far. To define the relative contribution of each of these two mutations to the Lewis negative phenotype, we cloned and made chimeric FUT3 constructs separating the T202C mutation responsible for the amino acid change Trp 68 3 Arg, from the C314T mutation leading to the Thr 105 3 Met shift. COS-7 cells were transfected and the expression of Fuc-TIII enzyme activity and the presence of Lewis antigens were determined. There was no decrease in enzyme activity nor of immunofluorescence staining on cells transfected with the construct containing the isolated C314T mutation compared with cells transfected with a wild type FUT3 allele control. No enzyme activity nor immunoreactivity for Lewis antigens was detected in FUT3 constructs containing both mutations in combination. The T202C mutation alone decreased the enzyme activity to less than 1% of the activity of the wild type FUT3 allele. These results demonstrate, that the Trp 68 3 Arg substitution in human Fuc-TIII is the capital amino acid change responsible for the appearance of the Le(a؊b؊) phenotype on human erythrocytes in individuals homozygous for both the T202C and C314T mutations.The Lewis histo-blood group system comprises different complex carbohydrate structures, which participate in different biological processes such as embryogenesis, tissue differentiation, tumor metastasis, inflammation, and bacterial adhesion (1). Le a1 and Le b (2, 3) are the major Lewis antigens found on human erythrocytes. These fucosylated glycosphingolipids are synthesized by exocrine epithelial cells (4) and are secondarily passively adsorbed onto erythrocytes in the peripheral circulation giving these blood cells their Lewis phenotype (5).It was shown as early as the 1950's, that the Lewis phenotype of erythrocytes is influenced by the ABH secretor status of the individual (6), and the erythrocyte phenotype is the result of the epistatic interaction of the Lewis (Le-le) and the salivary ABH secretor (Se-se) loci (7). Fucosyltransferases (Fuc-Ts) encoded by genes at these loci compete and interact with each other and with other glycosyltransferases to determine the individual's final Lewis and secretor phenotypes.Since 1990, seven human fucosyltransferase genes (FUT1-7) have been cloned, sequenced, and characterized according to acceptor specificities (8 -18). The human ␣(1,2)-Fuc-T gene family comprises FUT1 encoding for the H enzyme and FUT2 encoding for the human secretor ␣(1,2)-fucosyltransferase (9). FUT3-7 encode for ␣(1,3)-Fuc-Ts. Two of these human enzymes, respectively (10,14), also express ␣(1,4)-fucosyltransferase activities (15,19,20).Five main missense mutations have be...

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Clinical aspects of altered glycosylation of glycoproteins in cancer

1999

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Sequence and Expression of a Candidate for the Human Secretor Blood Group α(1,2)Fucosyltransferase Gene (FUT2)

Cited by 512 publications

References 39 publications

β‐1,3‐galactosyltransferase and α‐1,2‐fucosyltransferase involved in the biosynthesis of type‐1‐chain carbohydrate antigens in human colon adenocarcinoma cell lines

β‐1,3‐galactosyltransferase and α‐1,2‐fucosyltransferase involved in the biosynthesis of type‐1‐chain carbohydrate antigens in human colon adenocarcinoma cell lines

Significance of Individual Point Mutations, T202C and C314T, in the Human Lewis (FUT3) Gene for Expression of Lewis Antigens by the Human α(1,3/1,4)-Fucosyltransferase, Fuc-TIII

Clinical aspects of altered glycosylation of glycoproteins in cancer

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