Zhou S, Sousa R, Tannery N H, Lafer E M
Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260.
J Neurosci. 1992 Jun;12(6):2144-55. doi: 10.1523/JNEUROSCI.12-06-02144.1992.
The F1-20 protein is a novel neuronal-specific, synapse-associated protein that is expressed nonuniformly in mouse brain. Expression of the F1-20 protein is developmentally regulated in a pattern coincident with active synaptogenesis and synaptic maturation. Here we report the cloning of the cDNA sequence for the F1-20 protein. We found two distinct isoforms of F1-20 cDNA that differed by the presence of 15 additional nucleotides, which does not interrupt the open reading frame. RNase protection analysis and PCR amplification of mouse brain RNA revealed that both isoforms are present in cellular RNA. It is likely that the two F1-20 mRNA isoforms are derived from RNA splicing events utilizing alternative 3' acceptor sites. Analysis of the deduced amino acid sequence for the complete open reading frame revealed that the predominant F1-20 mRNA encodes an 896 amino acid polypeptide with a molecular weight of 91,319 Da. The deduced amino acid sequence does not contain a signal sequence, or any extensive hydrophobic regions. The deduced amino acid sequence does contain a number of consensus sequences for protein kinases. Searches of the protein and nucleic acid sequence data bases revealed that the F1-20 protein has not been previously characterized at the primary structure level, although a weak similarity was found between rabbit calpastatin and the C-terminal portion of the F1-20 protein. We then determined biochemically that the F1-20 protein is a substrate for Ca(2+)-dependent proteolysis, which is specifically inhibited by calpain inhibitors in vitro. This indicates that the F1-20 protein is a substrate for neuronal calpain. We observed that treatment of a synaptosomal lysate with alkaline phosphatase led to an increase in the electrophoretic mobility of the F1-20 protein, as well as to an increase in the sharpness of the electrophoretic band. This indicates that the F1-20 protein is phosphorylated in vivo.
F1-20蛋白是一种新型的神经元特异性突触相关蛋白，在小鼠脑中呈非均匀表达。F1-20蛋白的表达在发育过程中受到调控，其模式与活跃的突触发生和突触成熟相一致。在此，我们报告了F1-20蛋白cDNA序列的克隆。我们发现了两种不同的F1-20 cDNA同工型，它们相差15个额外的核苷酸，但这并不中断开放阅读框。对小鼠脑RNA进行核糖核酸酶保护分析和PCR扩增表明，两种同工型都存在于细胞RNA中。这两种F1-20 mRNA同工型可能是利用不同的3'受体位点通过RNA剪接事件产生的。对完整开放阅读框推导的氨基酸序列分析表明，主要的F1-20 mRNA编码一个含有896个氨基酸的多肽，分子量为91,319道尔顿。推导的氨基酸序列中不包含信号序列或任何广泛的疏水区域。推导的氨基酸序列确实包含许多蛋白激酶的共有序列。对蛋白质和核酸序列数据库的搜索表明，F1-20蛋白在一级结构水平上尚未被鉴定，尽管在兔钙蛋白酶抑制蛋白和F1-20蛋白的C末端部分之间发现了微弱的相似性。然后我们通过生化方法确定F1-20蛋白是钙（2+）依赖性蛋白水解的底物，在体外它被钙蛋白酶抑制剂特异性抑制。这表明F1-20蛋白是神经元钙蛋白酶的底物。我们观察到，用碱性磷酸酶处理突触体裂解物会导致F1-20蛋白的电泳迁移率增加，同时电泳条带的清晰度也增加。这表明F1-20蛋白在体内被磷酸化。
