Selective Aggregation and Sorting of Salivary Proteins

唾液蛋白的选择性聚集和分选

• 批准号: 6897566
• 负责人: DOUGLAS S DARLING
• 金额: $ 25.73万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6897566
• 关键词: SDS polyacrylamide gel electrophoresis; acidity /alkalinity; acinar cell; affinity chromatography; calcium; cell aggregation; cell sorting; endocrine gland /system; granule; high performance liquid chromatography; laboratory rat; parotid gland; polymerase chain reaction; protein structure function; proteoglycan; salivary glands; secretory protein; tissue /cell culture

DESCRIPTION: The long-term goal of this research is to determine the mechanisms for sorting and storage of regulated secretory proteins in salivary glands. The answer to this question is important for our understanding of the regulation of protein secretion from salivary glands and, hence, the composition of saliva. Significant progress towards this goal was made in the first grant period. It was shown that calcium- and pH-mediated aggregation do not contribute significantly to the storage of secretory proteins in parotid secretory granules, suggesting that sorting in this gland differs from that of other endocrine and exocrine glands. Indeed, it was demonstrated that sulfated proteoglycans are necessary for efficient storage of secretory proteins in secretory granules of rat parotid acinar cells. The sulfated proteoglycan appears to buffer the internal pH of secretory granules. These results led to the hypothesis that sulfated proteoglycans act as buffering agents that regulate protein sorting and storage in parotid acinar cells. Specific Aim 1 will test if sulfated proteoglycans act as buffering agents in parotid secretory granules. Aim 2 will test if expression and) post-translational modification of the proteoglycan core proteins regulates the sorting and storage of other) parotid secretory proteins. These Aims will employ isoproterenol treatment to overexpress acidic and basic PRPs in rat parotid glands combined with in vitro tissue culture approaches. As an alternative, we will use transgenic mice that overexpress PRPs in salivary glands. In Aim 3, it will be tested if sulfated proteoglycans play a role in protein or membrane binding of PSP. Specific Aim 4 will test if sulfated proteoglycans or other acidic proteins are involved in sorting and storage of secretory proteins in the submandibular gland, a current target for gene therapy protocols. The proposed research will use cell biological and molecular methods in salivary cells and protein expression experiments in intact animals. The results of this research will provide insight into the unique sorting mechanisms that function in salivary glands. This will lead to a better understanding of the formation of saliva and provide the basis for efficient delivery of therapeutic proteins from salivary glands.

描述：这项研究的长期目标是确定唾液腺中受调节分泌蛋白的分类和存储的机制。这个问题的答案对于我们理解唾液腺蛋白质分泌的调节以及唾液的组成很重要。在第一个赠款期间取得了重大进展。结果表明，钙和pH介导的聚集没有显着促进分泌蛋白在腮腺分泌颗粒中的储存，这表明该腺体中的排序与其他内分泌和外分泌腺的分类不同。确实，证明硫酸化的蛋白聚糖对于在大鼠腮腺细胞的分泌颗粒中有效存储分泌蛋白是必要的。硫酸化的蛋白聚糖似乎可以缓冲分泌颗粒的内部pH。这些结果导致了以下假设：硫酸化的蛋白聚糖充当缓冲剂，可调节蛋白质分类和储存腮腺细胞的储存剂。特定的目标1将测试硫酸化的蛋白聚糖是否充当腮腺分泌颗粒中的缓冲剂。 AIM 2将测试蛋白聚糖核蛋白的表达和）翻译后修饰是否会调节其他）腮腺分泌蛋白的排序和存储。这些目标将采用异丙肾上腺素治疗，以过表达大鼠腮腺的酸性和碱性PRP，并结合体外组织培养方法。作为替代方案，我们将使用过表达唾液腺中PRP的转基因小鼠。在AIM 3中，如果硫酸化的蛋白聚糖在PSP的蛋白质或膜结合中起作用，它将进行测试。特定的目标4将测试硫酸化的蛋白聚糖或其他酸性蛋白是否参与了下颌腺中分泌蛋白的分类和存储，这是当前基因治疗方案的靶标。拟议的研究将在完整动物中使用唾液细胞中的细胞生物学和分子方法以及蛋白质表达实验。这项研究的结果将洞悉在唾液腺中起作用的独特分类机制。这将使人们更好地了解唾液的形成，并为从唾液腺有效递送治疗蛋白提供基础。