SELECTIVE AGGREGATION AND SORTING OF SALIVARY PROTEINS

唾液蛋白的选择性聚集和排序

• 批准号: 6379803
• 负责人: SVEN-ULRIK GORR
• 金额: $ 14.75万
• 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 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6379803
• 关键词: SDS polyacrylamide gel electrophoresis; acidity /alkalinity; acinar cell; calcium; cell aggregation; cell sorting; endocrine gland /system; granule; laboratory rat; neuroendocrine system; neuroregulation; parotid gland; protein structure function; proteoglycan; salivary glands; secretory protein; tissue /cell culture

The long term goal of this research is to determine the mechanisms for sorting and storage of regulated secretory proteins in salivary glands. Aggregation is thought to play a role in this process in endocrine and exocrine cells, but aggregation has not been demonstrated specifically for salivary proteins. Aggregation may involve low pH, calcium or sulfated proteoglycans. We have tested these possibilities and found that in rat parotid secretory granules, parotid secretory protein (PSP) and a 32 kD protein (p32) aggregate at low pH, while other granule proteins do not aggregate. This result suggests that parotid granule proteins exhibit selective aggregation. Unlike non-aggregating exocrine proteins, PSP is sorted to the regulated secretory pathway in endocrine and neuroendocrine cells. Thus, it appears that aggregation is correlated with sorting to the regulated secretory pathway in these cell types. As a complement to this aggregation, it has long been assumed that sulfated proteoglycans play a role in the storage of secretory proteins in secretory granules. We have, for the first time, found that inhibition of proteoglycan synthesis in parotid tissue slices leads to increased basal secretion of PSP and, to a smaller extent amylase. This result suggests that these proteins are diverted from the regulated secretory pathway in the absence of proteoglycans. Since it is known that PSP and p32 are selectively retained in maturing secretory granules, we propose that parotid secretory proteins are sorted and retained in secretory granules by selective aggregation while non-aggregated proteins are secreted by the constitutive-like secretory pathway. To test this hypothesis, we will characterize the sorting and aggregation properties of rat PSP. Specific aim 1 will determine the aggregation properties of parotid secretory proteins. The aggregation of secretory proteins from isolated parotid granules will be tested and correlated with their secretion by the regulated or constitutive-like secretory pathways. In specific aim 2, the role of proteoglycans in sorting and aggregation of granule proteins will be tested. Specific aim 3 will test the sorting of PSP in endocrine and neuroendocrine cells. We have recently found that aggregation is necessary for sorting of an endocrine protein in endocrine cells, but not in neuroendocrine cells. In specific aim 4, we will determine if aggregation is necessary for sorting of PSP in endocrine and neuroendocrine cells. This research will for the first time show the aggregation properties of salivary proteins and determine the role of aggregation and proteoglycans in sorting of parotid secretory proteins. Elucidating the mechanisms for sorting and storage of secretory proteins in salivary glands will aid in the selection or design of therapeutic peptides to be expressed in the regulated secretory pathway and saliva.

本研究的长期目标是确定唾液腺中调节分泌蛋白的分类和储存机制。聚集被认为在内分泌和外分泌细胞的这一过程中起作用，但尚未证实聚集专门针对唾液蛋白。聚集可能与低pH、钙或硫酸盐化的蛋白聚糖有关。我们对这些可能性进行了测试，发现在大鼠腮腺分泌颗粒中，腮腺分泌蛋白（PSP）和一种32 kD蛋白（p32）在低pH下聚集，而其他颗粒蛋白则不聚集。提示腮腺颗粒蛋白具有选择性聚集。与非聚集性外分泌蛋白不同，PSP在内分泌和神经内分泌细胞中被分类到受调节的分泌途径。因此，在这些细胞类型中，聚集似乎与调节分泌途径的分选有关。作为这种聚集的补充，长期以来人们一直认为硫酸化蛋白聚糖在分泌颗粒中储存分泌蛋白中起作用。我们首次发现抑制腮腺组织切片中蛋白多糖的合成可导致PSP的基础分泌增加，并在较小程度上增加淀粉酶的分泌。这一结果表明，在缺乏蛋白聚糖的情况下，这些蛋白偏离了受调节的分泌途径。由于已知PSP和p32选择性保留在成熟的分泌颗粒中，我们提出腮腺分泌蛋白通过选择性聚集在分泌颗粒中进行分类和保留，而非聚集蛋白通过组成样分泌途径分泌。为了验证这一假设，我们将描述大鼠PSP的分类和聚集特性。特异性目的1将决定腮腺分泌蛋白的聚集特性。从分离的腮腺颗粒中分泌蛋白的聚集将被测试，并通过调节或组成样分泌途径与它们的分泌相关。在特定目标2中，将测试蛋白聚糖在颗粒蛋白的分选和聚集中的作用。特异性目的3将测试PSP在内分泌和神经内分泌细胞中的分选。我们最近发现，在内分泌细胞中，聚集是内分泌蛋白分选所必需的，而在神经内分泌细胞中则不是。在具体目标4中，我们将确定在内分泌和神经内分泌细胞中PSP的分选是否需要聚集。本研究将首次揭示唾液蛋白的聚集特性，并确定聚集和蛋白聚糖在腮腺分泌蛋白分选中的作用。阐明唾液腺分泌蛋白的分类和储存机制将有助于选择或设计在受调节的分泌途径和唾液中表达的治疗肽。