Elmgren A, Mollicone R, Costache M, Börjeson C, Oriol R, Harrington J, Larson G
Institute of Laboratory Medicine, Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden.
J Biol Chem. 1997 Aug 29;272(35):21994-8. doi: 10.1074/jbc.272.35.21994.
The Lewis alpha(1,3/1,4)-fucosyltransferase, Fuc-TIII, encoded by the FUT3 gene is responsible for the final synthesis of Lea and Leb 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 Trp68 --> Arg, from the C314T mutation leading to the Thr105 --> 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 Trp68 --> 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.
由FUT3基因编码的Lewisα(1,3/1,4)-岩藻糖基转移酶Fuc-TIII负责Lea和Leb抗原的最终合成。已经描述了各种点突变来解释红细胞和分泌物上的Lewis阴性表型Le(a-b-)。其中两个，最初在瑞典人群中描述的T202C和C314T，迄今为止尚未发现是单一的孤立点突变。为了确定这两个突变各自对Lewis阴性表型的相对贡献，我们克隆并构建了嵌合FUT3构建体，将导致氨基酸变化Trp68→Arg的T202C突变与导致Thr105→Met移位的C314T突变分开。转染COS-7细胞并测定Fuc-TIII酶活性的表达和Lewis抗原的存在。与用野生型FUT3等位基因对照转染的细胞相比，用含有分离的C314T突变的构建体转染的细胞的酶活性和免疫荧光染色均未降低。在含有两种突变组合的FUT3构建体中未检测到Lewis抗原的酶活性和免疫反应性。单独的T202C突变将酶活性降低至野生型FUT3等位基因活性的不到1%。这些结果表明，人Fuc-TIII中的Trp68→Arg取代是导致T202C和C314T突变均为纯合子的个体的人红细胞上出现Le(a-b-)表型的主要氨基酸变化。
