EXPRESSION AND STRUCTURE/FUNCTION OF COBALAMIN BINDING PROTEINS

钴胺素结合蛋白的表达和结构/功能

• 批准号: 6270563
• 负责人: DAVID H. ALPERS
• 金额: $ 15万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6270563
• 关键词: Baculoviridae; apical membrane; binding proteins; ceramide tetrahexoside; clinical research; cobalamin; complementary DNA; endoscopy; gastrointestinal absorption /transport; gastrointestinal epithelium; gene expression; guinea pigs; human subject; intrinsic factor; laboratory rat; ligands; protein sequence; protein structure function; receptor mediated endocytosis; salivary glands; tissue /cell culture; transfection; vitamin receptor

The transport system involving intrinsic factor (IF) and its receptor (IF-Rec) is only one of a few well characterized receptor-mediated systems in mammalian intestine whereby ligands can be absorbed into the enterocyte and eventually into the blood. Similar receptor-mediated transport systems involve the other cobalamin (cbl) binding proteins, including R protein or haptocorrin (hepatocyte) and transcobalamin II or TCII (all somatic cells). This grant will study the physiology and structural interactions of these transport systems, with particular emphasis on IF. We have isolated CDNA clones encoding the entire structural regions of rat and human IF and of hog R protein, and genomic clones that identify all the axons of human IF with the exception of the first 30 bp of the 5' UT portion of the CDNA. We have expressed rat IF in a eukaryotic system (COS-1 cells), and found that it can be active in the absence of N-glycosylation. Assays are available to follow the binding of cbl to IF, and of IF-cbl to brush border membranes, using IF produced either by transfection or in a cell-free system. IF and haptocorrin have been found immunologically to coexist in cells of foregut tissues, i.e. the gastric mucosa, and in ductal cells of the salivary glands and pancreas. We have also identified a haptocorrin receptor in intestine, corresponding with the minor 49-54 Kda subunits of the hepatic asialoglycoprotein receptor (ASGP-R). Human TCII has been cloned by other workers. We propose to produce radiolabelled IF in stably transfected cells to use for metabolic studies, to identify the cbl and receptor binding regions of IF, to study the fate of labelled IF in vivo in the rat and in vitro using Caco-2 cells, to study the secretory pathway of IF and haptocorrin in gastric parenchymal cells, and in salivary and pancreatic ductal cells, to isolate and study further the importance of the intestinal ASGP-R, and to isolate a TCII clone to transfect Caco-2 cells, to test an hypothesis that TCII production is rate of limiting for cbl absorption. Methods used will include transfection of eukaryotic cells, transformation of prokaryotic cells, deletional and in situ mutagenesis analysis, cell fractionation of enterocytes and tissue culture cells (Core C), immunolocalization and autoradiography (Core B), and CDNA sequencing and oligonucleotide production (Core A).

内因子及其受体参与的转运系统 （IF-Rec）只是少数几个充分表征的受体介导的 哺乳动物肠道中的系统，其中配体可以被吸收到 肠上皮细胞并最终进入血液。相似受体介导 转运系统涉及其它钴胺素（CBL）结合蛋白， 包括R蛋白或结合咕啉（肝细胞）和转钴胺素II，或 TCII（所有体细胞）。这项拨款将研究生理学， 这些运输系统的结构相互作用， 强调如果。我们已经分离出了编码整个 大鼠和人IF和hog R蛋白的结构区域，以及基因组 克隆，其鉴定除了人IF的轴突外的所有轴突。 cDNA的5' UT部分的前30 bp。我们已经表达了大鼠如果 在真核系统（COS-1细胞）中，发现它可以在 不存在N-糖基化。检测可按照 cbl与IF的结合，以及IF-cbl与刷状缘膜的结合，使用IF 通过转染或在无细胞系统中产生。当且 已经在免疫学上发现结合咕啉共存于 前肠组织，即胃粘膜，和胃粘膜的导管细胞中， 唾液腺和胰腺。我们还发现了一种结合香豆素 肠中的受体，对应于次要的49-54 Kda亚基 肝脏去唾液酸糖蛋白受体（ASGP-R）。人类TCII已经 被其他工人克隆。我们建议用放射性同位素标记的IF， 稳定转染的细胞用于代谢研究，以鉴定 cbl和IF的受体结合区，以研究标记的IF的命运 在大鼠体内和体外使用Caco-2细胞，以研究 胃实质细胞IF和结合咕啉的分泌途径， 以及唾液和胰腺导管细胞，以分离和研究 进一步的重要性，肠道ASGP-R，并分离TCII 克隆以抑制Caco-2细胞，以检验TCII 产生是CBL吸收的限制速率。使用的方法将 包括真核细胞转染、原核细胞的转化 细胞，缺失和原位突变分析，细胞分级 肠细胞和组织培养细胞（核心C），免疫定位和 放射自显影（Core B）、cDNA测序和寡核苷酸 生产（核心A）。