Structural characterization of the acidic glycosphingolipids in peripheral nervous system and studies on their biological 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的神经系统中分离得到4种磷脂酰磷脂，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。在FGL-IIa、FGL-IIb和FGL-V中，丙酮酸以缩酮形式连接在寡糖链末端半乳糖的O-3和O-4上。F-21以3-O-甲基半乳糖为非还原末端糖，其3个2-氨基乙基膦分子中的一个与N-乙酰半乳糖胺的C_6相连。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。用该抗血清处理海兔神经组织和皮肤的冰冻切片和石蜡切片，仅见神经束明显染色。根据用氯仿-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。散在散在的神经束中，可见轴突周围物质或小细胞染色，但未见轴突。这些糖脂的这种特殊位置提示了这些糖脂在轴突生长和束状生长中的作用。在海兔神经元的原代培养和神经细胞系的克隆培养中，我们还没有得到表明新的糖脂家族是否具有这些已提出的神经生物学功能的结果。

We isolated four phosphonoglycosphingolipids, designated 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 O-3 and O-4 of the terminal galactose of the oligosaccharide chain in FGL-IIa, FGL-IIb and FGL-V. F-21 has 3-O-methylgalactose as its non-reducing end sugar and one of its three 2-aminoethylphosphonate molecules is linked to C_6 of N-acetylgalactosamine.We raised a polyclonal antibody against FGL-IIb and examined the antigens that reacted with this anti-FGL-IIb antiserum by TLC-immunostaining. 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. When cryostat and paraffin sections of the nervous tissue and skin of Aplysia were treated with the antiserum, only nerve bundles were distinctly stained. 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. In nerve bundles disintegrated with Disperse, periaxonal materials or small periaxonal cells were stained but axons were not stained. This peculiar location of these glycolipids suggests roles of these glycolipids in neurite outgrowth and fasciculation.In primary cultures of Aplysia neurons and in clonal cultures of neuronal cell lines, we have not yet obtained results that indicate whether or not the new glycolipid family has these proposed neurobiological functions.