Molecular Cloning and Expression of a Bovine α(1,3)-Fucosyltransferase Gene Homologous to a Putative Ancestor Gene of the Human FUT3-FUT5-FUT6 Cluster

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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...

Section: Molecular Cloning Of Wild Mutated and Chimeric Fut3supporting

confidence: 80%

Section: Molecular Cloning Of Wild Mutated and Chimeric Fut3mentioning

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

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“…Interestingly, the three members of the ␣3/4-fucosyltransferase family co-localized to chromosome 19 are highly similar in sequence. These appear to have been duplicated very recently, because only one gene for this cluster was found in the cow (64). Two more divergent members of this family are localized differently.…”

Section: Isolation and Characterization Of Human C2/4gnt-analysis Of mentioning

confidence: 99%

Control of O-Glycan Branch Formation

Schwientek

Nomoto

Levery

et al. 1999

103

A novel human UDP-GlcNAc:Gal/GlcNAc␤1-3GalNAc␣ ␤1,6GlcNAc-transferase, designated C2/4GnT, was identified by BLAST analysis of expressed sequence tags. The sequence of C2/4GnT encoded a putative type II transmembrane protein with significant sequence similarity to human C2GnT and IGnT. Expression of the secreted form of C2/4GnT in insect cells showed that the gene product had UDP-N-acetyl-␣-D-glucosamine:acceptor ␤1,6-N-acetylglucosaminyltransferase (␤1,6GlcNAc-transferase) activity. Analysis of substrate specificity revealed that the enzyme catalyzed O-glycan branch formation of the core 2 and core 4 type. NMR analyses of the product formed with core 3-para-nitrophenyl confirmed the product core 4-para-nitrophenyl. The coding region of C2/4GnT was contained in a single exon and located to chromosome 15q21.3. Northern analysis revealed a restricted expression pattern of C2/4GnT mainly in colon, kidney, pancreas, and small intestine. No expression of C2/4GnT was detected in brain, heart, liver, ovary, placenta, spleen, thymus, and peripheral blood leukocytes. The expression of core 2 O-glycans has been correlated with cell differentiation processes and cancer. The results confirm the predicted existence of a ␤1,6GlcNAc-transferase that functions in both core 2 and core 4 O-glycan branch formation. The redundancy in ␤1,6Glc-NAc-transferases capable of forming core 2 O-glycans is important for understanding the mechanisms leading to specific changes in core 2 branching during cell development and malignant transformation.

“…The position of this futb gene in the phylogenetic tree of fucosyltransferases, showed that the separation of the bovine species from the common evolutionary pathway, during the great mammalian radiation some 80 million years ago, occurred before the duplication events, which originated the present cluster of human FUT3, FUT5, and FUT6 genes and suggested that this bovine enzyme is the orthologous homologue of the ancestor of the FUT3, FUT5, and FUT6 human genes (6).…”

mentioning

confidence: 99%

Evolution of Fucosyltransferase Genes in Vertebrates

Costache

Apoil²,

Cailleau

et al. 1997

Self Cite

103

Cloning and expression of chimpanzee FUT3, FUT5, and FUT6 genes confirmed the hypothesis that the gene duplications at the origin of the present human cluster of genes occurred between: (i) the great mammalian radiation 80 million years ago and (ii) the separation of man and chimpanzee 10 million years ago. The phylogeny of fucosyltransferase genes was completed by the addition of the FUT8 family of ␣(1,6)fucosyltransferase genes, which are the oldest genes of the fucosyltransferase family. By analysis of data banks, a new FUT8 alternative splice expressed in human retina was identified, which allowed mapping the human FUT8 gene to 14q23. The results suggest that the fucosyltransferase genes have evolved by successive duplications, followed by translocations, and divergent evolution from a single ancestral gene.Three human ␣(1,3)fucosyltransferase genes FUT3 1 (1), FUT5 (2) and FUT6 (3) are organized in a cluster, within 1 centimorgan, in the short arm of chromosome 19, in the band 19p13.3 (4, 5).Previous cloning of a bovine ␣(1,3)fucosyltransferase gene (futb) gave a single transcript, and the corresponding cognate enzyme had properties in common with the products of the three human FUT3, FUT5, and FUT6 genes. The position of this futb gene in the phylogenetic tree of fucosyltransferases, showed that the separation of the bovine species from the common evolutionary pathway, during the great mammalian radiation some 80 million years ago, occurred before the duplication events, which originated the present cluster of human FUT3, FUT5, and FUT6 genes and suggested that this bovine enzyme is the orthologous homologue of the ancestor of the FUT3, FUT5, and FUT6 human genes (6).The present cloning and expression of three chimpanzee ␣(1,3)fucosyltransferase genes provides evidence for the existence of at least three distinct, but related ␣(1,3)fucosyltransferase enzymes in this species, each one being the orthologous homologue of one of the human FUT3, FUT5, and FUT6 genes. The position of these chimpanzee genes in the fucosyltransferase phylogenetic tree suggests that the separation of man and chimpanzee from the common evolutionary pathway, about 10 million years ago, has occurred after the duplication events, at the origin of the present cluster of FUT3, FUT5, and FUT6 genes. Addition of the FUT8 gene family to the phylogenetic tree suggests that the appearance of this family preceded the ␣(1,2)-and the ␣(1,3)fucosyltransferase gene families, and the analysis of sequences in GenBank TM /EBI, EST, 2 and UniGene data banks allowed us to map the human FUT8 gene to 14q23. EXPERIMENTAL PROCEDURESCloning-PCR was used to amplify the coding regions and immediately adjacent sequences of FUT3, FUT5, and FUT6 from a chimpanzee, with primers containing extra bases with specific restriction sites, already used for the human genes (7,8). PCR products were digested with EcoRI and XbaI for FUT3, HindIII for FUT6, and HindIII and EcoRI for FUT5. Each gene was cloned between the respective restriction sites of pcDNA1 (Invitrogen). T...