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

黑海兔神经系统酸性磷酸鞘糖脂的结构表征及其神经生物学功能研究。

基本信息

批准号：
63580116
负责人：
ARAKI Shigeko
金额：
$ 1.15万
依托单位：
Niigata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

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.

采用Iatrobeads柱层析技术，从黑田鼠神经系统中分离得到4种磷酸甘磷脂，分别为FGL-IIA、FGL-IIb、FGL-V和F-21，并对其完整结构进行了鉴定。在FGL-IIa、FGL-IIb和FGL-V中，丙酮酸作为酮体附着在低聚糖链末端半乳糖的0-3和0-4上。我们提出了一种针对FGL-IIb的多克隆抗体，并在TLC板上通过酶免疫染色检测了与该抗FGL-IIb抗血清反应的抗原。抗血清与FGL-IIb、FGL-I、FGL-IIa、FGL-V和F-9反应，在TLC板上在神经节和神经纤维中鉴定。酯化或还原FGL-IIb的丙酮酸部分导致其对抗血清的抗原性丧失，酸甲醇处理的FGL-IIb皂化恢复其反应性。这些结果表明，抗FGL-IIb抗体的表位包含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田鼠组织中抗原的定位。使用该抗血清，仅神经束明显染色。从用氯仿：甲醇（2:1,v/v）预处理的低温冷冻切片的组织化学结果和神经系统的Western blot分析结果来看，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神经突生长和细胞粘附。