| dc.description.abstract | The human Lewis histo-blood group system is composed of several structurally well-characterized carbohydrate antigens. These are involved in different biological processes, e.g., embryogenesis, tissue differentiation, inflammation, dissemination of cancer cells and bacterial adhesion. Their biosynthesis is dependent on different fucosyltransferases which are expressed at different levels in different cell populations. The FUT3 gene encoded Lewis a-(1,3/1,4)-fucosyltransferase (EC 2.4.1.65) is expressed in epithelial cells. It is responsible for the synthesis of the Lea and Leb antigens. The inheritance of the functional Le-allele is necessary for the appearance of the Lea or Leb antigens on erythrocytes, while the deficient le-allele is found in a homozygous state in individuals with the Le(a-b-) phenotype. The FUT6 gene encoded a-(1,3)-fucosyltransferase, found in the liver, is responsible for the synthesis of the sialyl-Lex epitope on the acute-phase protein a1-acid glycoprotein. The secreted form of this enzyme is identified as the plasma a-(1,3)-fucosyltransferase (EC 2.4.1.152). The aims of this study were to i) identify molecular explanations in the FUT3 gene for the human erythrocyte Le(a-b-) phenotype, ii) identify molecular explanations in the FUT6 gene for deficient plasma a-(1,3)-fucosyltransferase activities and iii) establish screening procedures for natural mutations in the FUT3 and FUT6 genes. The coding regions of the FUT3 and FUT6 genes were amplified by PCR. DNA was prepared from Swedish blood donors and from members of Indonesian plasma a-(1,3)-fucosyltransferase deficient pedigrees. Amplified DNA fragments were cloned and sequenced. By this approach we discovered the two FUT3 point mutations, T202C and C314T, located on the same allele. These mutations were found on both alleles only in Le(a-b-) individuals. By using chimeric DNA constructs and subsequent expression in COS-7 cells, followed by immunofluorescent and enzymatic studies, we showed that T202C was the essential inactivating mutation. We also established PCR-based screening techniques for all known FUT3 mutations. A non-inactivating FUT6 nucleotide change, C738T, was identified. This mutation interfered with a published PCR-RFLP method for the detection of the known inactivating G739A mutation. We developed an extended genotyping strategy for the appropriate screening for these and other nucleotide substitutions in the FUT6 gene. Two FUT6 missense mutations, C730G and C907G, which we also identified, were found to be responsible for reduced a-(1,3)-fucosyltransferase activities using the COS-7 cell expression assay. These results enable allelic characterization of the human FUT3 and FUT6 genes, which can be used as new diagnostic tools in studies on Lewis histo-blood group antigen expression in health and disease. | en |
