Tohdoh N, Tojo S, Kimura M, Ishii T, Ojika K
Discovery Research Laboratories III, Sumitomo Pharmaceuticals Research Center, Japan.
Brain Res Mol Brain Res. 1997 Apr;45(1):24-32. doi: 10.1016/s0169-328x(96)00125-8.
The hippocampal cholinergic neurostimulating peptide (HCNP), isolated from hippocampal tissue of 10- to 12-day-old rats, enhances the in vitro synthesis of acetylcholine in medial septal tissue explants. The HCNP precursor is a 21 kDa protein that binds hydrophobic ligands and Mg-ATP, and is associated with the opioid-binding protein. We employed an HCNP-precursor cDNA as probe to clone the genomic DNA, used for mapping of the exon-intron structure of the gene. We also determined the nucleotide structure of the promoter region of the rat HCNP precursor protein gene. By using S1 mapping and CAT as a reporter, we found multiple promoters that were aligned in the 5' untranslated region. In addition, the presence of several putative enhancer binding sequences were tested by electrophoresis mobility shift assays. Northern blot analysis revealed that the gene is expressed in a variety of rat tissues and various subregions of the brain. These results suggest that HCNP-precursor gene expression is regulated by a general transactivation factor such as SP1, and that the specific presence of the bioactive HCNP in certain tissues results from post-translational events such as proteolytic processing of the precursor protein, which takes place predominantly in the hippocampus of young rats.
从10至12日龄大鼠的海马组织中分离出的海马胆碱能神经刺激肽（HCNP），可增强内侧隔区组织外植体中乙酰胆碱的体外合成。HCNP前体是一种21 kDa的蛋白质，它能结合疏水性配体和Mg-ATP，并与阿片样物质结合蛋白相关。我们使用HCNP前体cDNA作为探针来克隆基因组DNA，用于绘制该基因的外显子-内含子结构图谱。我们还确定了大鼠HCNP前体蛋白基因启动子区域的核苷酸结构。通过使用S1图谱分析并以氯霉素乙酰转移酶（CAT）作为报告基因，我们发现多个启动子排列在5'非翻译区。此外，通过电泳迁移率变动分析测试了几个推定的增强子结合序列的存在情况。Northern印迹分析表明该基因在大鼠的多种组织和大脑的各个亚区域中均有表达。这些结果表明，HCNP前体基因的表达受诸如SP1等一般反式激活因子的调控，并且某些组织中生物活性HCNP的特异性存在是由翻译后事件导致的，例如前体蛋白的蛋白水解加工，这主要发生在幼鼠的海马体中。
