NOVEL FUNCTIONS OF RED CELL PROTEINS LU AND LW

红细胞蛋白 LU 和 LW 的新功能

• 批准号: 6524500
• 负责人: JOEL A CHASIS
• 金额: $ 31.34万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6524500
• 关键词: cell cell interaction; cellular pathology; complementary DNA; disease /disorder model; erythrocyte membrane; erythroid stem cell; erythropoiesis; extracellular matrix proteins; gene expression; gene mutation; gene targeting; genetically modified animals; glycoproteins; human tissue; laboratory mouse; laminin; macrophage; membrane proteins; monoclonal antibody; protein binding; protein protein interaction; protein structure function; sickle cell anemia; site directed mutagenesis; tissue /cell culture

DESCRIPTION: (Adapted from investigator's abstract) This is a modified and resubmitted application based on the hypothesis that the erythrocyte membrane proteins Lutheran (Lu) and LW function in cell-cell and cell-extracellular matrix interactions in the bone marrow. Based on preliminary results that LW is expressed early in erythropoiesis and binds to alpha4beta1 and alphav integrins, the application proposes that LW may mediate interactions of erythroblasts with one another and with macrophages to form the erythroblastic island structure. Lu glycoprotein is known to bind laminin and the applicant has obtained evidence that it is not expressed until late in erythropoiesis. The timing of the appearance of Lu on the cell surface suggests a role in erythroblast enucleation or release of precursors from the marrow. Additionally, preliminary results indicate that Lu and LW may contribute to the pathophysiology of sickle cell disease by mediating the adhesion of sickle red cells to vascular endothelial cells. To test these hypotheses the applicant proposes to: (1) Generate knockout mice lacking the murine homologues of Lu and LW by homologous recombination. (2) Characterize the structure-function of Lu and LW by identifying the domains of Lu and LW involved in ligand binding and employing domain-deletion mutants and site-directed mutagenesis; developing blocking antibodies and peptides and testing their effects on macrophage-erythroblast and erythroblast interactions, erythroid progenitor cell growth, and nuclear extrusion using in vitro assays and micromechanical techniques; and by analyzing erythropoiesis in the knockout mice including the degree of ineffective erythropoiesis, capacity to form erythroblastic islands, process of erythroblast enucleation, and ability to generate reticulocytes during stress erythropoiesis. (3) Examine roles of Lu and LW in sickle cell disease by studying the effect on vascular blood flow of infusing transgenic/knockout sickle mice with antibodies directed against Lu and LW which block adhesion of sickle red cells to endothelial cells.

描述：（改编自研究者的摘要）这是经过修改和 基于红细胞膜的假设重新提交申请 路德蛋白 (Lu) 和 LW 在细胞间和细胞外发挥作用 骨髓中的基质相互作用。根据初步结果，LW 是 在红细胞生成早期表达并与 alpha4beta1 和 alphav 结合 整合素，该申请提出 LW 可能介导以下相互作用： 红细胞彼此之间以及与巨噬细胞形成红细胞 岛状结构。已知Lu糖蛋白结合层粘连蛋白并且申请人 已获得证据表明它直到红细胞生成晚期才表达。 Lu 在细胞表面出现的时间表明其在 红细胞去核或从骨髓中释放前体。 此外，初步结果表明 Lu 和 LW 可能有助于 介导镰状红细胞粘附的镰状细胞病的病理生理学 细胞到血管内皮细胞。为了检验这些假设，申请人 提议：（1）生成缺乏 Lu 和 Lu 的鼠同源物的敲除小鼠 通过同源重组进行 LW。 (2)表征Lu的结构-功能 和 LW，通过识别参与配体结合的 Lu 和 LW 结构域， 采用结构域缺失突变体和定点诱变；发展 阻断抗体和肽并测试它们对 巨噬细胞-成红细胞和成红细胞相互作用，红系祖细胞 使用体外测定和微机械进行细胞生长和核挤压 技术；并通过分析基因敲除小鼠的红细胞生成，包括 红细胞生成无效的程度，形成红细胞岛的能力， 有红细胞去核的过程和生成网织红细胞的能力 在应激性红细胞生成过程中。 (3) 检查Lu和LW在镰状细胞中的作用 通过研究输液对血管血流的影响来治疗疾病 带有针对 Lu 和 LW 的抗体的转基因/基因敲除镰状小鼠 阻止镰状红细胞与内皮细胞的粘附。