Abstract
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.
Highlights
-1,4-Galactosyltransferase (-1,4-GalT) V is a constitutively expressed enzyme that can effectively galactosylate the GlcNAc136Man group of the highly branched N-glycans that are characteristic of tumor cells
Because our preliminary study suggested that the -1,4-GalT V can effectively galactosylate the GlcNAc136 branch, which is synthesized by GlcNAc:Man N-acetylglucosaminyltransferase (GlcNAcT) V [21], it is important to elucidate the biological significance of the -1,4-GalT V and the mechanism by which -1,4-GalT V gene is regulated in cancer cells
RT-PCR analysis using oligonucleotide primers specific to the -1,4-GalT V gene showed that the expression level of the -1,4-GalT V transcript decreases 20 –30% by the transfection of the antisense -1,4-GalT V cDNA into SH-SY5Y cells when compared with that of the control cells
Summary
L-PHA, leuko-phytohemagglutinin; EMS, electrophoretic mobility shift; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; HRP, horseradish peroxidase; -1,4-GalT, -1,4-galactosyltransferase; GlcNAcT, N-acetylglucosaminyltransferase; RCA-I, R. communis agglutinin-I; RLM-RACE, RNA ligase-mediated rapid amplification of the 5Ј cDNA end; RT, reverse transcription; TK, herpes simplex virus thymidine kinase; USF, upstream stimulatory factor. Because our preliminary study suggested that the -1,4-GalT V can effectively galactosylate the GlcNAc136 branch, which is synthesized by GlcNAcT V [21], it is important to elucidate the biological significance of the -1,4-GalT V and the mechanism by which -1,4-GalT V gene is regulated in cancer cells. We showed the biological importance of the galactosylation of highly branched N-glycans by the -1,4-GalT V and isolated the promoter region of the human -1,4GalT V gene and examined the cis-elements and trans-acting factors that regulate -1,4-GalT V gene expression
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