Cloning and expression of the catalytic domain from rat hepatoma H35 cell GDP-fucose:GM1 alpha 1-->2fucosyltransferase, an enzyme which is activated during early stages of chemical carcinogenesis in rat liver.
A ganglioside GM1-specific alpha 1-->2fucosyltransferase is induced during the early stages of chemical carcinogenesis with N-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 GM1 ganglioside 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 alpha 1-->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 alpha 1-->2fucosyltransferase. Sequencing of these PCR fragments showed them to encode a novel enzyme with high homology to other cloned enzymes, particularly secretor alpha 1-->2fucosyltransferases. The derived sequence indicated that the 3' portion of the gene was virtually identical to the alpha 1-->2fucosyltransferase B (FTB) fragment reported earlier in rat PROb colon-adenocarcinoma cells (J-P. Piau et al. Biochem. J. 300, 623-626, 1994). A PCR product corresponding to the H35 cell alpha 1-->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 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 GM1 but not to the neolacto-series acceptor nLcOse4Cer.
Department of Cell Surface Biochemistry, Northwest Hospital, Pacific Northwest Cancer Foundation, 120 Northgate Plaza, Suite 218, Seattle, Washington, 98125, USA.
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Sherwood
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Holmes
D015073:2-Acetylaminofluorene / Q000633:toxicity; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002273:Carcinogens / Q000633:toxicity*; D006528:Carcinoma, Hepatocellular / Q000139:chemically induced / Q000201:enzymology*; D002384:Catalysis; D003001:Cloning, Molecular; D004789:Enzyme Activation / Q000187:drug effects; D005647:Fucosyltransferases / Q000096:biosynthesis* / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006801:Humans; D008114:Liver Neoplasms, Experimental / Q000139:chemically induced / Q000201:enzymology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011817:Rabbits; D051381:Rats; D011916:Rats, Inbred F344; D014407:Tumor Cells, Cultured
D002273:Carcinogens; D015073:2-Acetylaminofluorene; D005647:Fucosyltransferases; C037650:GDPfucose-GM1 ganglioside alpha-fucosyltransferase