Rescue of Chga knockout phenotype by CHGA-BAC transgene

说明(申请人提供)：嗜铬粒蛋白A(CGA)与儿茶酚胺在嗜铬粒体内共同储存，并在交感肾上腺刺激时共同释放。由CGA前蛋白衍生出几种具有生物活性的多肽，包括儿茶酚胺释放抑制肽、儿茶素。尼古丁是烟草的主要成分，它刺激儿茶酚胺的分泌，促进CGA的生物合成，并作为一种尼古丁胆碱能激动剂，促进儿茶酚胺的分泌脱敏。在发达国家，吸烟是导致过早死亡的最大单一原因，心脏病是最常见的死亡原因。研究表明，吸烟、糖尿病和高血压是冠心病的主要危险因素。高血压患者儿茶素水平降低。本实验室已培育出CHGA基因敲除(KO)小鼠。其显著的表型特征包括收缩压(SBP)升高、儿茶酚胺水平降低、嗜铬颗粒丢失和部分产前死亡。这项拟议研究的具体目标是： 1.利用细菌人工染色体(BAC)转基因方法导入人CHGA基因，挽救CHGA-Ko表型。2.对人CHGA基因进行测序，发现儿茶素序列中存在三个非同义SNPs(单核苷酸多态性)。这项拟议的研究将分析CGA野生型和突变型CGA在调节SBP、儿茶酚胺水平和尼古丁胆碱能反应方面的差异。将合成不同的儿茶素多肽，并将其注射到CHGA-/-小鼠体内。3个突变的CHGA-BAC小鼠模型(Gly364Ser、Pro370Leu和Arg374GIn)也将被建立，以探索这种多态的意义。3.最后，将对CGA突变体进行体外和体内的蛋白酶加工评估。

DESCRIPTION (provided by applicant): Chromogranin A (CGA) is co-stored with catecholamines in chromaffin granules and co-released in response to sympathoadrenal stimulation. From CGA pro-protein several biologically active peptides including catecholamine release-inhibitory peptide catestatin are derived. Nicotine, the primary component of tobacco, stimulates secretion of catecholamines, augments biosynthesis of CGA, and, as a nicotinic cholinergic agonist, promotes desensitization of catecholamine secretion. In developed countries, tobacco use is the largest single cause of premature death and heart disease the most common cause of death. Studies have demonstrated that smoking, diabetes and hypertension are major risk factors for coronary heart disease. Catestatin level is diminished in hypertensive patients. Chga knockout (Ko) mice have been generated in this laboratory. Their striking phenotypic characteristics include elevated systolic blood pressure (SBP), decreased catecholamines levels, loss of chromaffin granules and partial prenatal lethality. The proposed study specifically aims to: 1. Rescue the Chga-Ko phenotype by introducing the human CHGA gene using bacterial artificial chromosome (BAC) transgenic approach. 2. Sequencing of the human CHGA gene, revealed three non-synonymous SNPs (single nucleotide polymorphism) in the catestatin sequence. The proposed study will analyze the differences in regulation of SBP, catecholamine levels and nicotine cholinergic responses between the wild type and mutant variants of CGA. Variant catestatin peptides will be synthesized and administered into Chga -/- mice. The 3 mutant CHGA-BAC mouse models (Gly364Ser, Pro370Leu, and Arg374GIn) will also be generated to explore the significance of such polymorphisms. 3. Finally, the CGA variants will be assessed for processing by proteases in vitro and in vivo.