NOVEL ERYTHROBLAST/MACROPHAGE CONTACT PROTEIN

新型成红细胞/巨噬细胞接触蛋白

• 批准号: 2763606
• 负责人: Manjit Hanspal
• 金额: $ 18.74万
• 依托单位: STEWARD ST. ELIZABETH'S MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2763606
• 关键词: HeLa cells; affinity chromatography; binding proteins; cell adhesion molecules; cell differentiation; cell growth regulation; chemical association; chemical kinetics; complementary DNA; erythrocyte membrane; erythroid stem cell; erythropoiesis; gene expression; genetic library; human tissue; immunoprecipitation; macrophage; membrane proteins; protein biosynthesis; protein metabolism; protein protein interaction; protein sequence; protein structure function; western blottings; yeast two hybrid system

The association of erythroblasts with macrophages plays a central role in the formation of distinct anatomic units called erythroblastic islands. Although these islands have been well characterized morphologically, virtually nothing is about the structural and functional basis of cellular contacts within erythroblastic islands. We have previously shown that physical contact between erythroblasts and macrophages promotes the terminal maturation of erythroblasts, leading to their enucleation in vitro. Importantly, we identified a novel protein termed "medhesin" which mediates the association of erythroblasts with macrophages. The disruption of medhesin's cell adhesion function dramatically inhibited the terminal maturation of erythroid cells. Our working hypothesis is that the medhesin-mediated attachment of erythroblasts to macrophages promotes erythroid maturation and is essential for erythroblast enucleation. We have recently determined the complete amino acid sequence of human medhesin from the macrophage cDNA library. The aim of this proposal is to characterize the molecular basis of medhesin function in erythroblast-macrophage association. Specifically, the following issues will be addressed: (1) Is medhesin-dependent erythroblast-macrophage contact sufficient for erythroblast maturation? We will investigate whether medhesin-mediated cellular contact alone is sufficient to produce erythroblastic terminal maturation or additional macrophage-derived factors are required. (2) How does medhesin-mediated contact between erythroblasts and macrophages promote terminal erythroid maturation? To define the molecular basis of medhesin function, an important requisite will be to identify the molecules with which medhesin associates in erythroblasts. This identification will be accomplished using immunoprecipitation, affinity- chromatography, and the yeast two hybrid assays. (3) Do the kinetics of medhesin synthesis, assembly, and turnover correlate with erythroblast maturation? We propose to quantify the rates of medhesin synthesis, assembly, and turnover in the soluble and particulate fractions of erythroblasts at defined stages of differentiation. The precise definition of temporally-regulated medhesin expression will provide clues to the basis of erythroblast-macrophage contact during erythropoiesis. Together, the proposed studies on medhesin may begin to reveal the molecular basis of erythroblastic island formation during mammalian erythropoiesis.

有红细胞与巨噬细胞的结合起着核心作用 形成称为红细胞的不同解剖单位 岛屿。 尽管这些岛屿的特征已经很清楚 从形态上来说，几乎没有什么是关于结构和 红细胞岛内细胞接触的功能基础。 我们之前已经表明，有红细胞和红细胞之间的身体接触 巨噬细胞促进有红细胞的终末成熟，导致 到体外去核。 重要的是，我们确定了一本小说 称为“medhesin”的蛋白质，它介导 成红细胞与巨噬细胞。 麦粘素细胞的破坏 粘附功能显着抑制终末成熟 红细胞。 我们的工作假设是，medhesin 介导的 成红细胞与巨噬细胞的附着促进红细胞成熟 并且对于成红细胞剜除是必需的。 我们最近有 确定了人 Medhesin 的完整氨基酸序列 巨噬细胞cDNA文库。 该提案的目的是描述 成红细胞-巨噬细胞中的麦粘素功能的分子基础 协会。具体来说，将解决以下问题：（1） 麦粘素依赖性成红细胞-巨噬细胞接触是否足以 红细胞成熟 ? 我们将研究是否由 Medhesin 介导 仅细胞接触就足以产生有红细胞终末 需要成熟或额外的巨噬细胞衍生因子。 (2) 麦粘素如何介导成红细胞和巨噬细胞之间的接触 促进终末红细胞成熟？ 定义分子基础 要确定 Medhesin 的功能，一个重要的必要条件是确定 成红细胞中与麦粘素结合的分子。 这 鉴定将通过免疫沉淀、亲和力来完成 色谱法和酵母两种混合测定法。 (3) 进行动力学分析 麦粘素合成、组装和周转与成红细胞相关 成熟？ 我们建议量化麦粘素合成速率， 可溶性和颗粒部分的组装和周转 处于特定分化阶段的红细胞。 精确的 时间调节的麦粘素表达的定义将提供 红细胞-巨噬细胞接触基础的线索 红细胞生成。 总之，拟议的麦德辛研究可能会开始 揭示红细胞岛形成过程中的分子基础 哺乳动物红细胞生成。