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.

这项研究的长期目标是确定唾液腺中调节分泌蛋白的分类和存储的机制。 人们认为聚集在内分泌和外分泌细胞中在此过程中起作用，但是尚未专门证明聚集的唾液蛋白。 聚集可能涉及低pH，钙或硫酸化蛋白聚糖。 我们已经测试了这些可能性，发现在大鼠腮腺分泌颗粒中，腮腺分泌蛋白（PSP）和32 kD蛋白（p32）在低pH下的聚集体，而其他颗粒蛋白不聚集。 该结果表明腮腺颗粒蛋白表现出选择性聚集。 与非聚集的外分泌蛋白不同，PSP分类为内分泌和神经内分泌细胞的受调节分泌途径。 因此，似乎聚集与这些细胞类型中的调节分泌途径的分类相关。 作为对这种聚集的补充，长期以来一直认为硫酸化的蛋白聚糖在分泌颗粒中的分泌蛋白中起作用。 我们首次发现，在腮腺组织切片中抑制蛋白聚糖合成会导致PSP的基础分泌增加，并且在较小程度上会导致淀粉酶的基础分泌。 该结果表明，在没有蛋白聚糖的情况下，这些蛋白质从受调节的分泌途径中转移。 由于众所周知，PSP和p32在成熟的分泌颗粒中有选择地保留，因此我们建议通过选择性聚集将腮腺分泌蛋白分类并保留在分泌颗粒中，而非组成型蛋白质由组成型类似的分泌途径分泌。为了检验这一假设，我们将表征大鼠PSP的排序和聚集特性。 特定的目标1将确定腮腺分泌蛋白的聚集特性。 分泌的粒细粒颗粒的分泌蛋白的聚集将通过受调节或本构的分泌途径进行测试并与它们的分泌相关。 在特定的目标2中，将测试蛋白聚糖在分类和聚集颗粒蛋白中的作用。 特定的目标3将测试内分泌和神经内分泌细胞中PSP的分选。 我们最近发现，在内分泌细胞中的内分泌蛋白（而不是神经内分泌细胞中的内分泌蛋白）分类是必需的。 在特定的目标4中，我们将确定在内分泌和神经内分泌细胞中对PSP的分类是否需要聚集。 这项研究将首次显示唾液蛋白的聚集特性，并确定聚集和蛋白聚糖在分类腮腺分泌蛋白中的作用。 阐明在唾液腺中分类和存储分泌蛋白的机制将有助于选择或设计在受管制的分泌途径和唾液中表达的治疗肽。