Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Glycobiology. 2010 Jul;20(7):916-28. doi: 10.1093/glycob/cwq049. Epub 2010 Apr 5.
The monoclonal antibody mAb.A2B5 is a marker for the detection of oligodendrocyte progenitor cells that differentiate into type-2 astrocytes and oligodendrocytes. It is also a useful antibody for separating these cells from other lineage populations. The epitope of this antibody is considered to be the gangliosides GT3 and GQ1c. In this study, we sought to define more precisely the structure of the epitope. Accordingly, we chemically synthesized defined oligosialic acid structures linked to phosphatidylethanolamine and bovine serum albumin and used these to determine the antigenic specificity. mAb.A2B5 recognized the Neu5Acalpha2-->8Neu5Acalpha2-->8Neu5Acalpha--> structure on both glycolipids and glycoproteins. We then examined whether the mAb.A2B5 epitope exists on glycoproteins in developing mouse brains. Western blot analyses revealed the expression of four glycoproteins reactive with the mAb.A2B5, and their expression was dependent on the stage of neural development. All the immunoreactivity in these glycoproteins with mAb.A2B5 disappeared after sialidase treatment and were resistant to chloroform/methanol extraction. These epitopes were also detected in brain homogenates from both GD3 synthetase-null and GD3/GD2 synthetase double null mice. These findings show that the alpha2,8-trisialic acid (triSia) unit recognized by mAb.A2B5 resides not only on gangliosides but also on glycoproteins in developing mouse brain. We postulate that the triSia structure on glycoproteins may be involved in oligodendrocyte differentiation, similar to the case with the alpha2,8-triSia structure on gangliosides. Real time polymerase chain reaction analysis of the developmental expression of all known ST8Sia genes, which are responsible for the biosynthesis of alpha2,8-linked Sia residues, showed that ST8Sia III gene expression correlated with expression of the triSia epitope. We suggest that ST8Sia III is the principal sialyltransferase responsible for synthesis of the alpha2,8-triSia units on glycoproteins.
单克隆抗体 mAb.A2B5 是一种用于检测少突胶质前体细胞的标志物，这些细胞可以分化为星形胶质细胞 2 型和少突胶质细胞。它也是一种用于将这些细胞与其他谱系群体分离的有用抗体。该抗体的表位被认为是神经节苷脂 GT3 和 GQ1c。在这项研究中，我们试图更精确地定义表位的结构。为此，我们化学合成了与磷脂酰乙醇胺和牛血清白蛋白相连的特定寡唾液酸结构，并使用这些结构来确定抗原特异性。mAb.A2B5 识别糖脂和糖蛋白上的 Neu5Acalpha2-->8Neu5Acalpha2-->8Neu5Acalpha-->结构。然后，我们检查了 mAb.A2B5 表位是否存在于发育中的小鼠大脑中的糖蛋白上。Western blot 分析显示，有四种糖蛋白与 mAb.A2B5 反应，其表达依赖于神经发育的阶段。在用唾液酸酶处理后，这些糖蛋白中的所有 mAb.A2B5 免疫反应性均消失，并且对氯仿/甲醇提取具有抗性。在 GD3 合成酶缺失和 GD3/GD2 合成酶双缺失小鼠的脑匀浆中也检测到了这些表位。这些发现表明，mAb.A2B5 识别的α2,8-三唾液酸（triSia）单元不仅存在于神经节苷脂上，也存在于发育中的小鼠脑中的糖蛋白上。我们推测，糖蛋白上的 triSia 结构可能与神经节苷脂上的α2,8-三唾液酸结构相似，参与少突胶质细胞分化。对负责合成α2,8 连接唾液酸残基的所有已知 ST8Sia 基因的发育表达进行实时聚合酶链反应分析显示，ST8Sia III 基因的表达与 triSia 表位的表达相关。我们认为 ST8Sia III 是负责糖蛋白上α2,8-三唾液酸单元合成的主要唾液酸转移酶。
