Molecular Cloning and Expression of a Third Type of Rabbit GDP-L-Fucose:β-D-Galactoside 2-α-L-Fucosyltransferase

Abstract

Recent molecular investigation revealed that two closely related structural genes encode distinct GDP-L-fucose:beta-D-galactoside 2-alpha-L-fucosyltransferases (alpha1,2-fucosyltransferases). Some human cancer cells or tissues may express an aberrant alpha1, 2-fucosyltransferase other than H- and Secretor-type alpha1, 2-fucosyltransferase. However, definite evidence of the existence of a third type of alpha1,2-fucosyltransferase has not been demonstrated. Here we report the molecular cloning of a third type of rabbit alpha1,2-fucosyltransferase (RFT-III) from a rabbit genomic DNA library. The DNA sequence included an open reading frame coding for 347 amino acids, and the deduced amino acid sequence of RFT-III showed 59 and 80% identity with those of the previously reported two types of rabbit alpha1,2-fucosyltransferase, RFT-I and RFT-II, respectively. COS-7 cells transfected with the RFT-III gene exhibited alpha1,2-fucosyltransferase activity toward phenyl-beta-Gal as a substrate. Neuro2a (a murine neuroblastoma cell line) cells transfected with the RFT-III gene expressed fucosyl GM1 (type 3 H) but not Ulex europaeus agglutinin-1 lectin reactive antigens (type 2 H). Kinetic studies revealed that RFT-III exhibits higher affinity to types 1 (Galbeta1, 3GlcNAc) and 3 (Galbeta1, 3GalNAc) than to type 2 (Galbeta1, 4GlcNAc) oligosaccharides, which suggests that RFT-III as well as RFT-II is a Secretor-type alpha1, 2-fucosyltransferase. RFT-III was expressed in the adult gastrointestinal tract. The RFT-I, -II, and -III genes were assigned within 90 kilobases on pulsed field gel electrophoresis analysis. These results constitute direct evidence that, at least in one mammalian species, three active alpha1,2-fucosyltransferases exist.