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J. Biol. Chem., Vol. 279, Issue 38, 39574-39583, September 17, 2004
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-1,4-Galactosyltransferase V Gene in Cancer Cells
From the Department of Biosignal Research, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173-0015, Japan
-1,4-Galactosyltransferase (-1,4-GalT) V is a constitutively expressed enzyme that can effectively galactosylate the GlcNAc1
6Man group of the highly branched N-glycans that are characteristic of tumor cells. Upon malignant transformation of cells, the expression of the -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 -1,4-GalT V cDNA, indicating the biological importance of the -1,4-GalT V for the functions of highly branched N-glycans. We cloned the 2.3-kb 5'-flanking region of the human -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 -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 -1,4-GalT V gene in A549 cells. These results indicate that Sp1 plays an essential role in the transcriptional activity of the -1,4-GalT V gene in cancer cells.
Received for publication, May 25, 2004 , and in revised form, July 14, 2004.
* This work was supported by Grants-in-aid for the Encouragement of Young Scientists 12771428 and 14771303 (to T. S.) and for Scientific Research 09240104 and 12680708 (to K. F.) from the Ministry of Education, Science, Culture and Sports of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB067772
To whom correspondence should be addressed: Dept. of Biosignal Research, Tokyo Metropolitan Institute of Gerontology, Sakaecho 35-2, Itabashi-ku, Tokyo 173-0015, Japan. Tel.: 81-3-3964-3241 (ext. 3072); Fax: 81-3-3579-4776; E-mail: taksato{at}tmig.or.jp.
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