Beta-1,4-galactosyltransferase (beta-1,4-GalT) V is a constitutively expressed
enzyme that can effectively galactosylate the GlcNAcbeta1-->6Man group of the highly branched N-
glycans that are characteristic of
tumor cells. Upon malignant transformation of cells, the expression of the beta-1,4-GalT V gene increases in accordance with the increase in the amounts of highly branched N-
glycans.
Lectin blot analysis showed that the galactosylation of highly branched N-
glycans is inhibited significantly in SH-SY5Y human
neuroblastoma cells by the transfection of the antisense beta-1,4-GalT V
cDNA, indicating the
biological importance of the beta-1,4-GalT V for the functions of highly branched N-
glycans. We cloned the 2.3-kb 5'-flanking region of the human beta-1,4-GalT V gene, and we identified the region -116/-18 relative to the transcription start site as that having promoter activity. The region was found to contain several putative binding sites for
transcription factors, including AP2, AP4, N-Myc, Sp1, and upstream stimulatory factor. Electrophoretic mobility shift assay showed that Sp1 binds to
nucleotide positions -81/-69 of the promoter region. Mutations induced in the Sp1-binding site showed that the promoter activity of the beta-1,4-GalT V gene is impaired completely in
cancer cells. In contrast, the promoter activity increased significantly by the transfection of the Sp1
cDNA into A549 human lung
carcinoma cells.
Mithramycin A, which inhibits the binding of Sp1 to its binding site, reduced the promoter activation and expression of the beta-1,4-GalT V gene in A549 cells. These results indicate that Sp1 plays an essential role in the transcriptional activity of the beta-1,4-GalT V gene in
cancer cells.