STRUCTURE/FUNCTION OF INTESTINAL MUCIN

肠粘蛋白的结构/功能

• 批准号: 2138110
• 负责人: JOHN THOMAS LAMONT
• 金额: $ 24.95万
• 依托单位: BOSTON MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-07-01 至 1995-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2138110
• 关键词: animal tissue; chemical binding; gastric juice; gastric mucosa; gastrointestinal absorption /transport; gene expression; glycoprotein structure; in situ hybridization; intermolecular interaction; laboratory rat; lipids; molecular cloning; mucins; northern blottings; nucleic acid probes; protein structure function; transfection /expression vector; viscosity

The long term goals of this research are to elucidate the structure and function of porcine gastric mucin and to investigate how this glycoprotein contributes to gastric physiology, in particular to protection against acid secretion in the stomach. Two specific hypotheses will be tested in our experiments: 1) that gastric mucin peptide (apomucin), although accounting for only 20% of the molecule as against the 80% sugars, is a major determinant of the unique viscous properties of gastric mucins: 2) the ability of mucin to polymerize and form a gel is directly correlated with the protective properties of gastric mucin against secreted hydrochloric acid and other noxious agent such as proteases, oxygen radicals and alcohol. Three specific aims are designed to test our hypotheses using a combination of molecular and biophysical techniques. Aim 1 is to clone and sequence the major porcin gastric mucin genes. Purified porcine gastric mucin will be chemically deglycosylated, antibodies raised against this sugar-free apo-mucin will be used to screen a gastric cDNA library and positive clones will be purified and sequenced. Northern blot analysis and in situ hybridizatio will be used to localize the cells and organs of origin of these apomuci cDNA clones. Aim 2 is to use these clones to obtain cross-hybridizing probes to rat gastric mucin, which then can be used to study the regulation of mucin gene expression in rats after physiologic and pharmacologic stimuli. Aim 3 is to express truncated mucin genes in eukaryotic expression vectors to obtain large quantities of truncated peptides and characterize them as regards to their aggregation and gelation properties, their ability to bind lipids and retard h+ diffusion. These findings will enable us to determine the contribution of the various structural regions of apomucin to aggregation and gelation. These studies will provide unique structural, functional and physiologic data on gastric mucins. Since these molecules provide a protective barrier against H+ diffusion in the mammalian stomach, our results are of direct relevance to human peptic ulcer disease, H. pylori gastritis and gastric cytoprotection.

这项研究的长期目标是阐明结构和 猪胃粘蛋白的功能，并研究如何 糖蛋白有助于胃生理，特别是 防止胃中的酸分泌。 两个具体 假设将在我们的实验中进行检验：1）胃粘蛋白 肽（Apomucin），尽管仅占分子的20％ 抗80％的糖，是独特粘性的主要决定因素 胃粘蛋白的特性：2）粘蛋白聚合和 形成凝胶与保护特性直接相关 针对分泌的盐酸和其他有害剂的胃粘蛋白 例如蛋白酶，氧自由基和酒精。 三个具体目标是 设计用于使用分子和 生物物理技术。 AIM 1是克隆并对主要的猪油进行测序 胃粘蛋白基因。 纯化的猪胃粘蛋白将是化学的 针对这种无糖丙糖蛋白提出的脱糖基化的抗体将 用于筛选胃cDNA库，阳性克隆将是 纯化和测序。 北印迹分析和原位杂交 将用于定位这些apomuci的原始细胞和器官 cDNA克隆。 AIM 2是使用这些克隆来获得交叉杂交 对大鼠胃粘蛋白的探针，然后可用于研究 在生理和 药理学刺激。 AIM 3是表达截短的粘蛋白基因 真核表达载体获得大量截短 肽并将其描述为它们的聚集 凝胶化特性，结合脂质和延迟H+的能力 扩散。 这些发现将使我们能够确定贡献 apomucin到聚集的各个结构区域 凝胶化。 这些研究将提供独特的结构，功能和 胃粘蛋白的生理数据。 由于这些分子提供了 防止哺乳动物胃中H+扩散的保护性屏障，我们 结果与人类消化性溃疡疾病直接相关，幽门螺杆菌 胃炎和胃细胞保护。