Cloning and Expression of the Catalytic Domain from Rat Hepatoma H35 Cell GDP-Fucose:GM1α1→2Fucosyltransferase, an Enzyme Which Is Activated during Early Stages of Chemical Carcinogenesis in Rat Liver

A ganglioside GM1-specific α1→2fucosyltransferase is induced during the early stages of chemical carcinogenesis withN-2-acetylaminofluorene (AAF) in rat liver hepatocytes. The induction of this enzyme gives rise to the expression of a fucose-containing ganglioside with the same determinant structure as blood group B on a GM1ganglioside core. Fucoganglioside synthesis is not found in normal rat liver but is elevated in premalignant liver and is often highly expressed in derived rat hepatoma cell lines. Based upon the consensus sequence from portions of previously cloned human, rabbit, and rat α1→2fucosyltransferase enzymes, primers were designed which were used in RT-PCR experiments with rat hepatoma H35 cell total RNA to generate cDNAs encoding the extracellular, catalytic domain of the H35 cell α1→2fucosyltransferase. Sequencing of these PCR fragments showed them to encode a novel enzyme with high homology to other cloned enzymes, particularly secretor α1→2fucosyltransferases. The derived sequence indicated that the 3′ portion of the gene was virtually identical to the α1→2fucosyltransferase B (FTB) fragment reported earlier in rat PROb colon-adenocarcinoma cells (J-P. Piauet al.Biochem. J. 300, 623–626, 1994). A PCR product corresponding to the H35 cell α1→2fucosyltransferase was obtained from total RNA isolated from F344 rat liver after 0.03%N-2-acetylaminofluorene administration. No PCR product was obtained from total RNA isolated from normal F344 liver using PCR primers for the H35 cell α1→2fucosyltransferase. The H35 cell α1→2fucosyltransferase was expressed in the pPROTA vector and the derived fusion protein demonstrated the ability to transfer fucose to ganglioside GM1but not to the neolacto-series acceptor nLcOse4Cer.