CLONING AND EXPRESSION OF THE CATALYTIC DOMAIN FROM RAT HEPATOMA H35 CELL GDP-FUCOSE-GM, ALPHA-1-]2FUCOSYLTRANSFERASE, AN ENZYME WHICH IS ACTIVATED DURING EARLY STAGES OF CHEMICAL CARCINOGENESIS IN RAT-LIVER

A ganglioside GM(1)-specific alpha 1-->2fucosyltransferase is induced during the early stages of chemical carcinogenesis with N-2-acetylamin ofluorene (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 GM(1) ganglioside core. Fucoganglioside synthesis is not found in normal rat liver but is elevated in premalignant liver and is often highly expressed in der ived rat hepatoma cell lines. Based upon the consensus sequence from p ortions of previously cloned human, rabbit, and rat alpha 1-->2fucosyl transferase enzymes, primers were designed which were used in RT-PCR e xperiments with rat hepatoma H35 cell total RNA to generate cDNAs enco ding the extracellular, catalytic domain of the H35 cell alpha 1-->2fu cosyltransferase, Sequencing of these PCR fragments showed them to enc ode a novel enzyme with high homology to other cloned enzymes, particu larly secretor alpha 1-->2fucosyltransferases, The derived sequence in dicated that the 3' portion of the gene was virtually identical to the alpha 1-->2fucosyltransferase B (FTB) fragment reported earlier in ra t PROb colon-adenocarcinoma cells (J-P, Piau et at Biochem, J. 300, 62 3-626, 1994). A PCR product corresponding to the H35 cell alpha 1-->2f ucosyltransferase was obtained from total RNA isolated from F344 rat l iver after 0.03% N-2-acetylaminofluorene administration. No PCR produc t was obtained from total RNA isolated from normal F344 liver using PC R primers for the H35 cell alpha 1-->2fucosyltransferase. The H35 cell alpha 1-->2fucosyltransferase was expressed in the pPROTA vector and the derived fusion protein demonstrated the ability to transfer fucose to ganglioside GM(1) but not to the neolacto-series acceptor nLcOse(4 )Cer. (C) 1998 Academic Press.