MOLECULAR BASIS OF SELECTIVE COBALAMIN MALABSORPTION

选择性钴胺素吸收不良的分子基础

• 批准号: 3464743
• 负责人: JOHN C FYFE
• 金额: $ 13.77万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 1994-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3464743
• 关键词: binding proteins; brush border membrane; cell type; cobalamin; disease /disorder model; dogs; gastrointestinal epithelium; gastrointestinal nutrient absorption; gene mutation; ileum; immunoprecipitation; inborn metabolism disorder; membrane proteins; messenger RNA; model design /development; molecular cloning; nutrition related tag; posttranslational modifications; radiotracer; receptor binding; receptor expression; renal tubule; vitamin B12 deficiency; western blottings

Humans and other mammals absorb cobalamin (Cbl, vitamin B12), an essential micronutrient, from diet via a complex interaction of Cbl-binding proteins and a specific receptor for intrinsic factor-Cbl (IFCR) in the ileal brush-border epithelium. Untreated Cbl deficiency is life-threatening in humans and most often results from disorders of Cbl absorption. The details of Cbl transcytosis by ileal enterocytes are unknown, but the pivotal importance of IFCR is exemplified by an inherited disorder of Cbl absorption known as Imerslund-Grasbeck syndrome in humans. Recently, a canine model of selective intestinal Cbl malabsorption has been described, the only known model of an IFCR defect, that appears to be very similar to Imerslund-Grasbeck syndrome. The studies proposed use this unique disease model to advance understanding of the molecular aspects of Cbl absorption and brush-border epithelial receptor expression in health and disease states. The specific aims of these investigations are to clone normal canine IFCR cDNA, to identify the IFCR mutation causing disease in affected dogs, and to define the tissue-specific and developmental regulation of normal IFCR expression. By comparison of the kinetics of IFCR expression and posttranslational modification in normal and affected dog tissues, the cellular consequences of the IFCR mutation will be defined. Using techniques of molecular biology, protein chemistry, and cell biology, studies are designed to examine the hypotheses that there are tissue-specific differences in the post-translational modification of normal IFCR and the intracellular disposition of abnormal IFCR, and that IFCR expression is differently regulated in ileal mucosa and proximal tubules. Initially, a survey of canine tissues will be made by in vitro assay of IFCR ligand-binding activity and immunoblot to determine sites of significant IFCR expression and the possible contributions of different tissues to Cbl homeostasis. Metabolic labelling, cell fractionation, and immunoprecipitation of IFCR will be used to define the cellular phenotypes of normal IFCR expression and the receptor defect in tissue explants and/or cell lines derived from normal and affected dogs. Protein and oligosaccharide studies of IFCR purified from tissues of normal and affected dog' will be done to investigate possible tissue differences in postranslational modifications of IFCR affect intracellular disposition of the abnormal IFCR of affected dogs. Standard molecular genetic approach be used to clone normal IFCR cDNA, to define the genetic mutation, and to examine IFCR mRNA in normal and affected dog tissues. This integrated approach will elucidate aspects of IFCR structure affect function and expression in ileal and renal brush-border epithelia.

人类和其他哺乳动物吸收钴胺素（Cbl、维生素 B12）， 必需的微量营养素，通过饮食中复杂的相互作用 Cbl 结合蛋白和内因子 Cbl 的特异性受体 (IFCR) 位于回肠刷状缘上皮中。 未经治疗的 Cbl 缺乏症是 危及人类生命，最常由 Cbl 疾病引起 吸收。 回肠肠上皮细胞 Cbl 转胞吞作用的详细信息是 未知，但 IFCR 的关键重要性体现在 Cbl 吸收遗传性疾病，称为伊默斯伦-格拉斯贝克综合征 在人类中。 最近，选择性肠道Cbl的犬模型 吸收不良已被描述，这是唯一已知的 IFCR 缺陷模型， 这似乎与伊默斯伦-格拉斯贝克综合征非常相似。 这 拟议的研究利用这种独特的疾病模型来增进理解 Cbl 吸收和刷状缘上皮分子方面的研究 健康和疾病状态下的受体表达。 具体目标 这些研究旨在克隆正常犬 IFCR cDNA，以鉴定 IFCR 突变导致受影响的狗患病，并定义 正常 IFCR 表达的组织特异性和发育调节。 通过比较 IFCR 表达和翻译后的动力学 正常和受影响的狗组织的改变，细胞 IFCR 突变的后果将被确定。 使用技术 分子生物学、蛋白质化学和细胞生物学，研究领域包括 旨在检验存在组织特异性的假设 正常 IFCR 和正常 IFCR 翻译后修饰的差异 异常 IFCR 的细胞内处置，并且 IFCR 表达是 在回肠粘膜和近端小管中受到不同的调节。 最初，一个 将通过 IFCR 体外测定对犬组织进行调查 配体结合活性和免疫印迹以确定重要位点 IFCR 表达以及不同组织对 IFCR 的可能贡献 Cbl 稳态。 代谢标记、细胞分级分离和 IFCR 的免疫沉淀将用于定义细胞 组织中正常 IFCR 表达和受体缺陷的表型 来自正常和受影响的狗的外植体和/或细胞系。 从组织中纯化的 IFCR 的蛋白质和寡糖研究 正常和受影响的狗将被检查可能的组织 IFCR 影响翻译后修饰的差异 受影响狗的异常 IFCR 的细胞内处置。 使用标准分子遗传学方法克隆正常的 IFCR cDNA，以 定义基因突变，并检查正常和正常情况下的 IFCR mRNA 受影响的狗组织。 这种综合方法将阐明以下方面 IFCR结构影响回肠和肾的功能和表达 刷状缘上皮。