Borges E, Pendl G, Eytner R, Steegmaier M, Zöllner O, Vestweber D
Institute of Cell Biology, ZMBE, University of Münster, D-48149 Münster, Germany.
J Biol Chem. 1997 Nov 7;272(45):28786-92. doi: 10.1074/jbc.272.45.28786.
The HECA452 carbohydrate epitope, also termed cutaneous lymphocyte antigen, is known to bind to E-selectin and defines a human T cell subset preferentially found in inflamed skin. Activated T cells can express a functional form of the P-selectin glycoprotein ligand-1 (PSGL-1), the major ligand known for P-selectin. Here we show that PSGL-1 can exist in two forms, of which only one carries the HECA452 epitope and binds to E-selectin, while the other only binds to P-selectin. We have analyzed the glycoprotein ligands for E- and P-selectin on the mouse CD8+ T cell clone 4G3 at 4, 8, and 12 days after antigen-specific activation. Only at day 4 did the cells bind to E-selectin, whereas cells at all three activation stages bound to P-selectin. Expression of the HECA452 epitope correlated with E-selectin binding. In affinity isolation experiments, PSGL-1 was isolated as the major ligand by E-selectin-IgG and by P-selectin-IgG; however, PSGL-1 only bound to E-selectin at day 4, whereas it bound to P-selectin at all three activation stages. Immunoprecipitated PSGL-1 from cells at day 4, but not from cells at days 8 and 12, was recognized in immunoblots by monoclonal antibody HECA452. In immunoblots of total extracts of cells at day 4, HECA452 recognized a 240/140-kDa pair of protein bands as the major antigen. These bands could be completely removed by depletion of cell extracts with anti-PSGL-1 antibodies. Our data suggest that the carbohydrate requirements for binding of PSGL-1 to P-selectin differ from those necessary for binding to E-selectin. Furthermore, we conclude that the major glycoprotein carrier for the HECA452 epitope on activated 4G3 cells is PSGL-1.
HECA452碳水化合物表位，也被称为皮肤淋巴细胞抗原，已知其可与E-选择素结合，并定义了一个优先存在于炎症皮肤中的人类T细胞亚群。活化的T细胞可表达P-选择素糖蛋白配体-1（PSGL-1）的功能性形式，PSGL-1是已知的P-选择素的主要配体。在此我们表明，PSGL-1可存在两种形式，其中只有一种携带HECA452表位并与E-选择素结合，而另一种仅与P-选择素结合。我们分析了抗原特异性激活后第4、8和12天小鼠CD8 + T细胞克隆4G3上E-选择素和P-选择素的糖蛋白配体。仅在第4天细胞与E-选择素结合，而在所有三个激活阶段的细胞均与P-选择素结合。HECA452表位的表达与E-选择素结合相关。在亲和分离实验中，PSGL-1被E-选择素-IgG和P-选择素-IgG分离为主要配体；然而，PSGL-1仅在第4天与E-选择素结合，而在所有三个激活阶段均与P-选择素结合。从第4天的细胞中免疫沉淀的PSGL-1，而不是第8天和第12天的细胞中的PSGL-1，在免疫印迹中被单克隆抗体HECA452识别。在第4天细胞总提取物的免疫印迹中，HECA452识别出一对240/140-kDa的蛋白条带作为主要抗原。用抗PSGL-1抗体耗尽细胞提取物可完全去除这些条带。我们的数据表明，PSGL-1与P-选择素结合所需的碳水化合物要求不同于与E-选择素结合所需的碳水化合物要求。此外，我们得出结论，活化的4G3细胞上HECA452表位的主要糖蛋白载体是PSGL-1。
