2683

B4GALT1

UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1;B4GALT1;UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1

UDP-Gal:beta-GlcNAc beta-1,4-galactosyltransferase 1|UDP-galactose:beta-N-acetylglucosamine beta-1,4-galactosyltransferase 1|beta-1,4-GalTase 1|beta-1,4-galactosyltransferase 1|glycoprotein-4-beta-galactosyltransferase 2|lactose synthase

9p13

protein-coding

The elevated levels of beta1,4-galactosyltransferase I (GalT I; EC 2.4.1.38) are detected in highly metastatic lung cancer PGBE1 cells compared with its less metastatic partner PGLH7 cells. Decreasing the GalT I surface expression by small interfering RNA or interfering with the surface of GalT I function by mutation inhibited cell adhesion on laminin, the invasive potential in vitro, and tyrosine phosphorylation of focal adhesion kinase. The mechanism by which GalT I activity is up-regulated in highly metastatic cells remains unclear. To investigate the regulation of GalT I expression, we cloned the 5'-region flanking the transcription start point of the GalT I gene (-1653 to +52). Cotransfection of the GalT I promoter/luciferase reporter and the Ets family protein E1AF expression plasmid increased the luciferase reporter activity in a dose-dependent manner. By deletion and mutation analyses, we identified an Ets-binding site between nucleotides -205 and -200 in the GalT I promoter that was critical for responsiveness to E1AF. It was identified that E1AF could bind to and activate the GalT I promoter by electrophoretic mobility shift assay in PGLH7 cells and COS1 cells. A stronger affinity of E1AF for DNA has contributed to the elevated expression of GalT I in PGBE1 cells. Stable transfection of the E1AF expression plasmid resulted in increased GalT I expression in PGLH7 cells, and stable transfectants migrated faster than control cells. Meanwhile, the content of the beta1,4-Gal branch on the cell surface was increased in stably transfected PGLH7 cells. GalT I expression can also be induced by epidermal growth factor and dominant active Ras, JNK1, and ERK1. These data suggest an essential role for E1AF in the activation of the human GalT I gene in highly metastatic lung cancer cells.

The disaccharide peracetylated GlcNAcbeta1-3Galbeta-O-naphthalenemethanol (disaccharide 1) diminishes the formation of the glycan sialyl Lewis X (Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3) GlcNAc; sLe(X)) in tumor cells. Previous studies showed that the mechanism of action of disaccharide 1 involves three steps: (i) deacetylation by carboxyesterases, (ii) action as a biosynthetic intermediate for downstream enzymes involved in sLe(X) assembly, and (iii) generation of several glycans related to sLe(X). In this report, we show that GlcNAcbeta1-3Galbeta-O-naphthalenemethanol binds to the acceptor site of human beta1-4-galactosyltransferase much like the acceptor trisaccharide, GlcNAcbeta1-2Manbeta1-6Man, which is present on N-linked glycans. The 4'-deoxy analog, in which the acceptor hydroxyl group was replaced by -H, did not act as a substrate but instead acted as a competitive inhibitor of the enzyme. The acetylated form of this compound inhibited sLe(X) formation in U937 monocytic leukemia cells, suggesting that it had inhibitory activity in vivo as well. A series of synthetic acetylated analogs of 1 containing -H, -F, -N(3), -NH(2), or -OCH(3) instead of the hydroxyl groups at C-3'- and C-4'-positions of the terminal N-acetylglucosamine residue also blocked sLe(X) formation in cells. The reduction of sLe(X) by the 4'-deoxy analog also diminished experimental tumor metastasis by Lewis lung carcinoma in vivo. These data suggest that nonsubstrate disaccharides have therapeutic potential through their ability to bind to glycosyltransferases in vivo and to alter glycan-dependent pathologic processes.