Identification and functional analysis of proteins involved in neural circuit formation

大鼠小脑皮质的形态发生，包括神经元的分化和突触的形成，发生在出生后的早期，主要发生在出生后的头两周。为了阐明这一动态细胞过程的分子机制，我们建立了一个大鼠小脑蛋白质双向电泳可检测的数据库。该数据库包含与发育中的大鼠小脑的多肽成分有关的二维模式和派生信息。利用这个数据库，我们对一系列未成熟和成熟小脑的蛋白质成分进行了比较。一组多肽被发现在未成熟的小脑中唯一表达。多个这类多肽在原位或从凝胶中提取后被蛋白酶消化，产生的片段通过常规Edman降解或结合高效液相色谱的质谱分析进行微量测序。然后将这些序列与…中的序列进行比较更多的蛋白质/基因序列数据库，用于鉴定分离的蛋白质。已鉴定的蛋白质包括已被广泛研究的与神经元分化相关的磷酸化和非磷酸化形式的stathmin，以及属于维甲酸结合蛋白家族并参与鸡视网膜神经元分化的脂肪酸结合蛋白。分析的两种蛋白质是独一无二的，因为没有发现与数据库中的任何其他序列有显著同源性。通过蛋白质直接分析和基因克隆，确定了这两种蛋白的氨基酸序列，分别命名为V-1和LANP。测序结果表明，V-1是一个典型的含117个氨基酸的胞浆蛋白，有两个半的33个氨基酸重复序列，称为“细胞周期基序”。LAMP包含247个氨基酸，由N-末端的富含亮氨酸的重复序列组成，紧随其后的是带有核转移信号的酸性氨基酸尾部。细胞周期基序和富含亮氨酸的重复序列都通过与特定的靶分子结合来调节蛋白质之间的相互作用，并在广泛的生物中发现，这些蛋白质在决定细胞命运的许多蛋白质中发挥作用，包括酵母和果蝇。出生后大鼠小脑原位组织化学分析显示，V-1在颗粒层中瞬时表达，LANP在浦肯野细胞中瞬时表达，提示V-1和LANP在指导小脑形态发生的信号转导通路中可能发挥作用。较少

Morphogenesis of the rat cerebellar cortex, including differentiation of neurons and synaptogenesis, occurs at an early postnatal stage, mainly during the first two weeks after birth. To elucidate the molecular mechanism of this dynamic cellular process, we have developed a data base of rat cerebellar proteins detectable by two-dimensional electrophoresis. The data base contains two-dimensional patterns and derived information pertaining to polypeptide constituents of the developing rat cerebellum. Using this data base, we have compared the protein constituents of a series of immature and mature cerebella. A subset of polypeptides were found to be uniquely expressed in the immature cerebellum. A number of polypeptides of this class were digested by proteinases in situ or after extraction from the gel, and the generated fragments were micro-sequenced by conventional Edman degradation or mass spectrometry combined with liquid chromatography. The sequences were then compared with those in … More protein/gene sequence data bases to identify the isolated proteins. The identified proteins include phosphorylated and non-phosphorylated forms of stathmin whichi has been studied extensively in relation to the differentiation of neurons, and fatty acid-binding protein that belongs to a family of retinoic-acid binding proteins and participates in the neuronal differentiation in chick retina. Two proteins analyzed were unique in that no significant homologies were found to any other sequences in the data bases. The amino acid sequence of these proteins termed V-1 and LANP respectively, were determined by direct protein analysis and cDNA cloning. The sequence showed that the V-1 is a typical cytosolic protein of 117 amino acids and has two and a half of 33 amino acids repeats called "cell-cycle motif". LAMP contains 247 amino acids consisting of teh N-teminally located leucine-rich repeats followed by an acidic amino acids tail with nuclear transfer signal. Both the cell-cycle motif and leucine-rich repeats mediate protein-protein interation through binding to specific target molecules, and are found in numbers of proteins that function in cell fate determination in wide organisms including yeast and Drosophila. In situ histochemical analysis postnatal rat cerebella showed the transient expression of V-1 in granular layr and of LANP in Purkinje cells, suggsting the potential roles of V-1 and LANP in the signal transduction pathway that directs the cerebellar morphogenesis. Less