Distinct Substrate Specificities of Five Human α-1,3-Fucosyltransferases forin VivoSynthesis of the Sialyl Lewis x and Lewis x Epitopes

Abstract

Five different human α-1,3-fucosyltransferase genes,the Fuc-TIII, Fuc-TIV, Fuc-TV, Fuc-TVIandFuc-TVIIgenes, have been cloned to date. We transfected HeLa cells and Namalwa cells with each of the five different genes, and established a series of stable cloned transformant cells. Thin-layer chromatography immunostaining analysis revealed that all five enzymes were able to synthesize sialyl Lewis x (sLex) epitopes on glycolipids in HeLa cells, but each enzyme showed a different preference as to the carbohydrate chain length on glycolipids as acceptor substrates. Fuc-TIII and Fuc-TV showed very similar patterns of sLexpositive bands, which indicated that the enzymes have similar acceptor substrate specificities. Fuc-TVI exhibited a little different pattern from those of the former two enzymes. Fuc-TIV and Fuc-TVII showed similarity in the positive bands, however, their patterns were quite different from those of the former three enzymes. Four enzymes except for Fuc-TVII were able to synthesize the Lewis x (Lex) epitope on glycolipids in HeLa cells. Fuc-TV alone showed a little different pattern of Lexpositive bands from those of the other three enzymes. Flow cytometric analysis of HeLa cells and Namalwa cells again demonstrated the similar specificities of Fuc-TIII and Fuc-TV. They exhibited similar stronger staining with FH6 (anti-sLex) antibodies than that with the other enzymes. A phylogenetic tree of the five enzymes constructed using the neighbor-joining method showed good agreement with the similarities in the enzyme substrate specificity.