We have studied ganglioside alterations and their enzymatic basis during the course of neural differentiation of mouse embryonic carcinoma cell line P19. This cell line can differentiate into neurons and astrocytes on cell aggregation after treatment with retinoic acid (RA) or into muscle cells on dimethyl sulfoxide (DMSO) treatment. GD3, detected on immunostaining after thin-layer chromatography (TLC) with monoclonal antibody (MAb) R24, was markedly present in aggregates treated with RA.GM3 synthase (alpha2,3-sia1yltransferase, SAT-I) in neurons was found to exhibit the highest activity. GD3 synthase (alpha2,8-sialyltransferase, SAT-II) and GD3 synthase mRNA, as analyzed by Northern blotting, were also markedly present in aggregates and neurons induced by RA.However, on treatment with DMSO, which induces muscle cells, there was no change in the level of GD3 synthase activity, and its transcript was hardly detected during the course of muscle differentiation. GTlb synthase (alpha2,3-sialyltransferase, SAT-IV) was present at similar levels in undifferentiated cells and aggregates treated with RA, but a higher level was observed in neurons. On the other hand, the level of GQlb synthase (alpha2,8-sialyltransferase, SAT-V) in RA-induced aggregates was significantly higher than that in neurons. These results show that RA but not DMS0 induces the expression of GM3, GD3, GTlb and GQlb synthases, and particularly GD3 synthase mRNA, in the ganglioside biosynthetic pathway during the neural differentiation of embryonic carcinoma P19 cells.
平成10年度の成果: シナプス形成の特異マーカーであるGAP-43分子とシナプトフィシン分子の局在を神経分化細胞で調べた。ショ糖密度勾配法によりgrowth coneとシナプトソームの画分を調製した。growth cone画分の純度は抗GAP-43抗体で、シナプトソーム画分の純度は抗シナプトフィシン抗体を用いて、ウエスタン・ブロット法で確認した。また、神経細胞分化マーカーであるN-CAM分子の存在もウエスタン・ブロット法で確認出来た。神経細胞の固定剤はマトリゲルが最適であった。免疫染色の共焦点顕微鏡写真から、抗GQlb抗体の染色では、神経分化細胞の軸索のgrowth coneで強く染色され、GAP-43抗体の染色像と酷似していた。抗GTlb抗体の染色では、抗シナプトフィシン抗体のシナプスの染色像と酷似していた結果を得た。抗GTlb抗体、抗GQlb抗体と抗GAP-43抗体、抗シナプトフイシン抗体の組み合わせで二重染色を行い、各々の相互の分子の局在を比較検討した。