STRUCTURE/FUNCTION OF A HEPATIC CEA BINDING PROTEIN

肝脏 CEA 结合蛋白的结构/功能

• 批准号: 6173300
• 负责人: PETER THOMAS
• 金额: $ 24.95万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-15 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6173300
• 关键词: Kupffer's cell; animal genetic material tag; athymic mouse; binding proteins; binding sites; biological signal transduction; carcinoembryonal antigen; cell adhesion molecules; cell line; colorectal neoplasms; cytokine; human genetic material tag; laboratory rat; liver metabolism; metastasis; molecular cloning; protein sequence; protein structure function; site directed mutagenesis

Carcinoembryonic antigen (CEA) is involved in the development of hepatic metastases from colorectal cancers. The major hepatic CEA binding protein is 80kD Kupffer cell surface molecule. The binding site for the 80kD protein has been located to the junction of the N-terminal and first loop domains of CEA and is a pentapeptide (PELPK). Treatment of Kupffer cells with CEA results in the production of IL-1, IL-6 and TNF- alpha and two proteins of 115 and 125kD are rapidly phosphorylated when Kupffer cells are exposed to CEA. In this application we will also study the structure and function of the Kupffer cell 80kD carcinoembryonic antigen (CEA) peptide binding protein by further protein sequencing, production of antibodies and by cloning and expressing its gene from both rat and human Kupffer cell cDNA libraries using the available peptide sequences as the basis for probes. To examine the function of the CEA binding protein in metastasis we will examine the mechanism of activation of hepatic Kupffer cells by CEA and the synthetic ligand for the 80kD binding protein PELPK-albumin by a) Studying the consequences of ligand binding to the 80kD protein on Kupffer cells, on the metastatic proclivity of cancer cells in the hepatic sinusoid and the effects on adhesion to the hepatic endothelium. Using inhibitory antibodies to endothelial cells binding proteins we will identify the adhesion molecules involved. b) We will examine the effects of inhibition of signal transduction (specifically phosphorylation of tyrosine) on the regulation of metastatic potential of human colorectal cancer cell lines by CEA. c) We will use site directed mutagenesis to alter the five amino acid (PELPK) binding site for the 80kD protein to examine its effect on Kupffer cell response to CEA and on hepatic metastasis formation. We will also use a construct that has the first 75 amino acids of the CEA N-domain deleted. This CEA does not form homotypic aggregates but retains the PELPK sequence. This will allow us to determine if CEA produced to enhance metastatic potential. These studies will increase our knowledge of the role of the 80kD binding protein in the liver and its relationship with CEA in the mechanism by which colorectal cancer cells attach and invade in the hepatic sinusoid.

癌胚抗原（CEA）参与肝细胞癌的发生、发展， 结直肠癌的转移。 主要的肝CEA结合 蛋白质为80 kD枯否细胞表面分子。 的结合位点 80 kD蛋白定位于N-末端连接处， CEA的第一环结构域，并且是五肽（PELPK）。 治疗 具有CEA的枯否细胞导致IL-1、IL-6和TNF-α的产生。 α和115和125 kD的两种蛋白质被迅速磷酸化， 枯否细胞暴露于CEA。 在本应用程序中，我们还将 Kupffer细胞80 kD的结构和功能研究 癌胚抗原（CEA）肽结合蛋白， 蛋白质测序、抗体生产和克隆， 从大鼠和人枯否细胞cDNA文库表达其基因 使用可用的肽序列作为探针的基础。 到 检查CEA结合蛋白在转移中的功能，我们将 研究CEA激活肝枯否细胞的机制， 80 kD结合蛋白PELPK-白蛋白的合成配体， 研究配体与80 kD蛋白结合的后果， Kupffer细胞，对肿瘤细胞转移倾向的影响， 肝窦及其对肝内皮细胞粘附的影响。 使用内皮细胞结合蛋白的抑制性抗体， 将识别出相关的粘附分子。 B）我们将研究 抑制信号转导的作用（特别是 酪氨酸磷酸化）对转移潜能的调节 人大肠癌细胞系的CEA。 （c）我们将使用网站 定向诱变以改变五个氨基酸（PELPK）结合位点 80 kD蛋白质，以检查其对枯否细胞应答的影响， CEA和肝转移形成。 我们还将使用一个构造 CEA N结构域的前75个氨基酸被删除。 本CEA 不形成同型聚集体，但保留PELPK序列。这 将使我们能够确定CEA的产生是否能增强转移性 潜力 这些研究将增加我们对 肝组织中80 kD结合蛋白及其与癌胚抗原的关系 结直肠癌细胞附着和侵入的机制 肝血窦