Developmental stage-dependent expression of an α2,8-trisialic acid unit on glycoproteins in mouse brain

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 Neu5Acα α α α2→ → → →8Neu5Acα α α α2→ → → →8Neu5Acα α α α→ → → → 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 that 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 synthetases-null mice. These findings show that the α α α α2,8-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 α α α α2,8-triSia structure on gangliosides. RT-PCR analyses of the developmental expression of all known ST8Sias genes, which are responsible for the biosynthesis of α α α α2,8-linked Sia residues, showed that ST8Sia III gene expression correlated with expression of the triSia epitope. We suggest that ST8Sia III, is the principle sialyltransferase responsible for synthesis of the α α α α2,8-triSia units on glycoproteins.