Structural characterization of the acidic phosphonoglycosphingolipids in nervous system of Aplysia kurodai and studies on their neurobiological function.

本研究采用三种溶剂体系，用医用珠柱层析法从黑线姬鼠神经系统中分离得到4种磷酸糖鞘糖脂，分别命名为FGL-IIA、FGL-IIb、FGL-V和F-21，并分别鉴定了它们的完整结构。丙酮酸以缩酮形式附着在FGL-IIa、FGL-IIb和FGL-V寡糖链的末端半乳糖上。该抗血清可与FGL-IIb、FGL-I、FGL-IIa、FGL-V和F-9反应，薄层板上可见FGL-IIb、FGL-I、FGL-IIa、FGL-V和F-9存在于神经节和神经纤维中。FGL-IIb的丙酮酸部分的酯化或还原导致其对抗血清的抗原性丧失，而经酸甲醇处理的FGL-IIb经皂化处理后恢复了反应性。这些结果表明，抗FGL-IIb抗体的表位含有FGL-IIb中丙酮酸的游离羧基。我们还用免疫组织化学方法检测了黑线姬鼠组织中抗原的定位。用该抗血清仅对神经束进行明显染色。根据氯仿：甲醇(2：1，v/v)处理的冰冻切片的组织化学结果和神经系统的Western印迹分析结果，该染色是由糖脂抗原引起的。这些含有丙酮酸的酸性磷脂可能作为配体参与轴突生长和细胞黏附。

We isolated four phosphonoglycosphingolipids, designated as FGL-IIA, FGL-IIb, FGL-V and F-21, from the nervous system of A. kurodai by Iatrobeads column chromatography using three solvent systems and elucidated their complete structures, respectively.Pyruvic acid attached as a ketal to 0-3 and 0-4 of the terminal galactose of the oligosaccharide chain in FGL-IIa, FGL-IIb and FGL-V.We raised a polyclonal antibody against FGL-IIb and examined the antigens that reacted with this anti-FGL-IIb antiserum by enzyme immunostaining on TLC plates. The antiserum reacted with FGL-IIb, FGL-I, FGL-IIa, FGL-V and F-9, which were identified in ganglia and nerve fibers on TLC plates. Esterification or reduction of the pyruvic acid moiety of FGL-IIb resulted in loss of its antigenicity against the antiserum, and saponification of the acid methanol-treated FGL-IIb restored the reactivity. These results indicate that the epitope for the anti-FGL-IIb antibodies includes the free carboxyl group of pyruvic acid in FGL-IIb.We also examined the localization of antigens in the tissues of A. kurodai immunohistochemically. With this antiserum, only nerve bundles were stained distinctly. From histochemical findings in cryostat sections pretreated with chloroform:methanol (2:1, v/v) and from results by Western blot analysis of the nervous system, the staining was concluded to be due to glycolipid antigens. These acidic phosphonoglycosphingolipids containing pyruvic acid may be involved as ligand in neurite outgrowth and cell adhesion.