Cell Adhesion Regulation Mechanism by Sialyl-Le^X Structure.

我们通过重组人前B白血病细胞中糖基转移酶机制，证明细胞表面唾液酸LeX（sLeX）抗原表达水平受核心2 N-乙酰氨基葡萄糖转移酶（C2 GnT）基因控制。此外，我们还揭示了以下内容。1. sLeX抗原不是在所有蛋白质上表达，而是在分子大小为150 kDa的特异性核心糖蛋白（gp 150）上表达。sLeX抗原决定簇在gp15.2的0-连接寡糖链的末端表达。通过荧光素酶分析、EMSA和RNA印迹分析C2 GnT表达的转录调控机制，我们证明了以下几点。(1)在C2 GnT转录调控区的三个TATA盒中，含有最接近转录起始位点的-182 TATA的缺失突变体显示出最高的荧光素酶活性。(2)C2 GnT基因表达细胞具有非表达细胞的10倍瞬时转录活性。通过抑制C2 GnT基因在细胞分化过程中的表达，瞬时转录活性下调至1/10。这些与C2 GnT信息、C2 GnT酶活性和细胞表面sLeX抗原表达水平的变化非常一致。(3)涉及的转录因子结合位点是加塔、NF-IL 6和Spi。GATA 3、NF-1 L 6、C/EBP γ、Sp3和Sp 4基因表达水平与C2 GnT基因表达水平相关。

We have demonstrated through remodeling of the glycosyltrasferase machineries in human pre-B leukemia cells that cell surface sialyl-LeX (sLeX) antigen expression level is controlled by core 2 N-acetylglucosaminyltransferase (C2GnT) gene. In addition, we have revealed the followings.1. sLeX antigen is not expressed on every protein but expressed on a specific core glycoprotein with molecular size of l5OkDa (gp 150). sLeX antigen determinants are expressed at the termini of 0-linked oligosaccharide chains of gplSO.2. Analyzing transcription regulation mechanism of C2GnT expression by luciferase assay, EMSA, and RNA blotting, we have demonstrated the followings.(1) Among three TATA boxes in the C2GnT transcription regulation area, a deletion mutant containing the most proximate -182 TATA to the transcription initiation site showed the highest luciferase activity.(2) C2GnT gene expressing cells had 10-fold transient transcription activity of the non-expressing cells. Transient transcription activity was down-regulated to 1/10 by inhibiting C2GnT gene expression among cell differentiation. These are in a good agreement with changes of C2GnT message, C2GnT enzyme activity, and cell surface sLeX antigen expression levels.(3) Involved transcription factor binding sites are GATA, NF-IL6, and Spi. The expression of GATA3, NF-1L6, C/EBPgamma, Sp3, and Sp4 genes correlated with C2GnT gene expression level.