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

我们通过改造人前B期白血病细胞的糖基转移酶机制，证明细胞表面唾液酸氨基转移酶(SLeX)抗原的表达水平受核心2 N-乙酰氨基葡萄糖转移酶(C2GnT)基因的控制。此外，我们还透露了以下内容：1.SLeX抗原并不是表达在每种蛋白上，而是表达在一种特定的核心糖蛋白上，其分子大小为15okDa(GP 150)。SLeX抗原决定簇在gplSO0连接的寡糖链末端表达。通过荧光素酶活性测定、EMSA和RNA印迹分析C2GnT基因表达的转录调控机制，我们发现：(1)在C2GnT转录调控区的三个TATA盒中，与转录起始点最接近的缺失突变体的荧光素酶活性最高。(2)C2GnT基因表达细胞的瞬时转录活性是非表达细胞的10倍。通过抑制C2GnT基因在细胞分化中的表达，瞬时转录活性下调至1/10。这些变化与C2GnT信息、C2GnT酶活性和细胞表面SLeX抗原表达水平的变化相一致。(3)涉及的转录因子结合位点为GATA、NF-IL6和SPI。GATA3、NF-1L6、C/EBPGamma、SP3、SP4基因的表达与C2GnT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.