Molecular Cloning of E-Selectin-Glycoprotein Counter Ligand, gp150.

在人前B白血病细胞中，sialyl-Lex(SLeX)抗原并不是表达在每种蛋白上，而是表达在特定的核心糖蛋白上，分子大小为150 kDa(Gp150)。此外，细胞表面SLeX抗原的表达水平受核心2 N-乙酰氨基葡萄糖转移酶(C2GnT)基因控制。此外，我们还透露了以下内容：1.结论：1.C2GnT不仅在前B细胞分化过程中，而且在扁桃体B细胞活化过程中，调节细胞表面SLeX的表达水平。溶血巴氏杆菌O-唾液酸糖蛋白内肽酶处理后，gp150的抗SLeX单抗反应性消失。这表明gp150可能是一种唾液粘蛋白糖蛋白分子，其末端含有丰富的含唾液酸的O-连接糖链。从Pre-B Nall-1细胞膜部分分离纯化了一种胰酶解肽，并用抗SLeX单抗结合蛋白G对其进行了分析，确定其为粘蛋白型糖蛋白。免疫组织化学染色证实为阳性。获得gp150基因，反义导入SLeX高表达的Pre-B细胞系。转染组细胞的唾液酸法反应性明显下调，E-选择素依赖的细胞黏附能力明显降低。

In human pre-B leukemia cells sialyl-LeX (sLeX) antigen is not expressed on every protein but expressed on a specific core glycoprotein with molecular size of 150kDa (gp150). In addition, cell surface sLeX antigen expression level is controlled by core 2 N-acetylglucosaminyltransferase (C2GnT) gene. In addition, we have revealed the followings.1. Not only in pre-B cell differentiation but also in tonsillar B cell activation, C2GnT regulates cell surface sLeX expression level.2. Anti-sLeX mAb reactivity of gp150 was disappeared after O-sialoglycoprotein-endopeptidase (from Pasteurella haemolytica) treatment. This suggests gp150 must be a sialomucin type glycoprotein molecule that has abundant O-linked sugar chains with sialic acid on their termini.3. A tryptic peptide was purified and analyzed from pre-B NALL-1 cell line membranous fraction using anti-sLeX mAb-conjugated protein-G.The peptide was determined as a mucin-type glycoprotein. It was confirmed by immunostaining methods.4. The cDNA of gp150 was obtained and transfected in the antisense orientation into a sLeX-highly expressed pre-B cell line. Sialyl-LeX-reactivity in the transfected cell line was significantly downregulated and E-selectin-dependent cell adhesion capability was remarkably reduced.