Identification and functional analysis of proteins involved in neural circuit formation

大鼠小脑皮质的形态发生，包括神经元分化和突触发生，发生在出生后早期，主要发生在出生后的前两周。为了阐明这种动态细胞过程的分子机制，我们开发了可通过二维电泳检测的大鼠小脑蛋白数据库。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衍生信息。利用这个数据库，我们比较了一系列未成熟和成熟小脑的蛋白质成分。发现多肽的一个子集在未成熟的小脑中独特地表达。许多此类多肽在原位或从凝胶中提取后被蛋白酶消化，并且通过常规的Edman降解或质谱结合液相色谱法对产生的片段进行微测序。然后将这些序列与蛋白质/基因序列数据库中的序列进行比较，以鉴定分离的蛋白质。鉴定出的蛋白质包括磷酸化和非磷酸化形式的 stathmin（其与神经元分化的关系已被广泛研究）和属于视黄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特的，因为没有发现与数据库中任何其他序列显着的同源性。这些蛋白质的氨基酸序列分别称为V-1和LANP，通过直接蛋白质分析和cDNA克隆来确定。序列显示V-1是一种典型的由117个氨基酸组成的胞质蛋白，并且具有33个氨基酸重复序列中的两个半，称为“细胞周期基序”。 LAMP 包含 247 个氨基酸，由 N 末端富含亮氨酸的重复序列组成，后面是带有核转移信号的酸性氨基酸尾。细胞周期基序和富含亮氨酸的重复序列都通过与特定靶分子结合来介导蛋白质-蛋白质相互作用，并且存在于包括酵母和果蝇在内的广泛生物体中在细胞命运决定中起作用的许多蛋白质中。出生后大鼠小脑的原位组织化学分析显示颗粒层中 V-1 和浦肯野细胞中 LANP 瞬时表达，表明 V-1 和 LANP 在指导小脑形态发生的信号转导途径中的潜在作用。较少的

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