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：（根据调查员的摘要进行了改编）这是一个修改的， 基于红细胞膜的假设重新提交的应用 蛋白质Lutheran（LU）和LW功能在细胞细胞和细胞细胞中的功能 骨髓中的基质相互作用。基于LW的初步结果 在红细胞生成早期表达，并与alpha4beta1和alphav结合 整合素，应用程序建议LW可以介导 彼此的红细胞和巨噬细胞形成红细胞 岛屿结构。已知lu糖蛋白结合层粘连蛋白和申请人 已经获得了证据，表明直到红蛋白酶后期才能表达出来。 LU在细胞表面的外观的时机表明在 红细胞摘除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 镰状细胞疾病的病理生理学通过介导镰状红色的粘附 细胞到血管内皮细胞。为了检验这些假设，申请人 建议：（1）产生缺乏lu和 LW通过同源重组。 （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的域和 采用域 - 缺失突变体和定向诱变；发展 阻断抗体和肽，并测试其对 巨噬细胞 - 金细胞和红细胞相互作用，红细胞祖细胞 细胞生长和使用体外测定和微力学的核挤出 技术；并通过分析敲除小鼠中的红细胞生成 无效的红细胞生成程度，形成红细胞岛的能力， 红细胞摘除的过程和产生网状细胞的能力 在压力红细胞生成期间。 （3）检查LU和LW在镰状细胞中的作用 通过研究对注入血管血流的影响 转基因/敲除镰状小鼠，用针对LU和LW的抗体 这阻断了镰状细胞对内皮细胞的粘附。