Abstract
Only one bovine gene, corresponding to the human cluster of genes FUT3-FUT5-FUT6, was found by Southern blot analysis. The cognate bovine alpha(1,3)-fucosyltransferase shares 67.3, 69.0, and 69.3% amino acid sequence identities with human FUC-T3, FUC-T5, and FUC-T6 enzymes, respectively. As revealed by protein sequence alignment, potential sites for asparagine-linked glycosylation and conserved cysteines, the bovine enzyme is an intermediate between FUC-T3, FUC-T5, and FUC-T6 human enzymes. Transfected into COS-7 cells, the bovine gene induced the synthesis of an alpha(1, 3)-fucosyltransferase enzyme with type 2 substrate acceptor pattern specificity and induced expression of fucosylated type 2 epitopes (Lex and sialyl-Lex), but not of type 1 structures (Lea or sialyl-Lea), suggesting that it has an acceptor specificity similar to the human plasma FUC-T6. However, no enzyme activity was detected in bovine plasma. Gene transcripts are detected on tissues such as bovine liver, kidney, lung, and brain. The type 2 sialyl-Lex epitope was found in renal macula densa and biliary ducts, and Lex and Ley epitopes were detected on the brush border of epithelial cells of small and large intestine, suggesting a tissue distribution closer to human FUC-T3, but fucosylated type 1 structures (Lea, Leb, or sialyl-Lea) were not detected at all in any bovine tissue. Analysis of genetic distances on a combined phylogenetic tree of fucosyltransferase genes suggests that the bovine gene is the orthologous homologue of the ancestor of human genes constituting the present FUT3-FUT5-FUT6 cluster.
Highlights
Cell surface fucosylated oligosaccharides have received a substantial amount of attention because they play a role in inflammation-mediated cell adhesion and are frequently mod
The bovine enzyme has a type 2 acceptor substrate specificity, like human FUC-T6, as demonstrated by (i) type 2 acceptor specificity of the activity detected in homogenates of COS-7 cells transfected with futb, (ii) immunofluorescence detection of type 2 ␣(1,3)-fucosylated epitopes (Lex and sialyl-Lex) on COS-7 cells transfected with futb, and (iii) the presence of fucosylated type 2 epitopes (Lex, Ley, and sialylLex) on normal bovine tissues
The cells were analyzed to assess in vitro substrate acceptor pattern of enzyme activity (Table III) and the appearance of fucosylated type 1 and type 2 epitopes on transfected cells (Table IV), comparatively to COS-7 cells transfected with the human FUT3, FUT5, and FUT6 genes
Summary
Nomenclature—The gene described represents the first bovine fucosyltransferase gene. It will be designated futb and the cognate ␣(1,3)fucosyltransferase enzyme Futb. Filters were prehybridized at 42 °C for 1 h in 50% deionized formamide, 2 ϫ Pipes buffer (10 ϫ Pipes buffer is 4 M NaCl, 0.1 M Pipes, pH 6.5), 0.5% SDS, and denaturated sonicated salmon sperm DNA (100 g/ml) They were hybridized at 42 °C for 16 h in prehybridization solution containing the bovine futb ␣-32Plabeled probe of 459 bp, generated by PCR with primers derived from regions highly conserved in human FUT3, FUT5, and FUT6 genes (see above, Table I and Fig. 1). Tissue Immunofluorescence Staining—Routine formalin-fixed/paraffin-embedded sections were deparaffinated and stained by indirect (monoclonal antibodies) or direct (lectin) immunofluorescence They were incubated for 30 min in a wet chamber with the first monoclonal antibody, washed, and stained for 30 min with fluorescein isothiocyanate-labeled sheep anti-mouse Ig’s second antibody (Pasteur Diagnostics, Marnes la Coquette, France). Stained slides were washed again and mounted under coverslides with 1 drop of Primers
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