NOVEL FUNCTIONS OF RED CELL PROTEINS LU AND LW

红细胞蛋白 LU 和 LW 的新功能

• 批准号: 6381616
• 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/6381616
• 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：(改编自调查人员摘要)这是一个修改后的 重新提交的申请是基于红细胞膜 路德蛋白(Lu)和LW在细胞-细胞和细胞-细胞外的功能 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α4β1和αv结合 整合素，应用程序提出LW可能介导 红细胞相互作用，并与巨噬细胞形成红系 岛状结构。已知的LU糖蛋白结合层粘连蛋白和申请人 已经获得的证据表明，它直到红细胞生成后期才被表达。 Lu出现在细胞表面的时间表明它在 红系细胞从骨髓中去核或释放前体。 此外，初步结果表明，鲁氏和长白可能对 介导镰刀红黏附的镰刀细胞病的病理生理学 从细胞到血管内皮细胞。为了检验这些假设，申请人 建议：(1)建立缺乏Lu和Lu同源基因的基因敲除小鼠 LW通过同源重组。(2)表征鲁的结构-功能 和LW通过鉴定参与配基结合的Lu和Lw结构域和 利用结构域缺失突变体和定点突变；开发 封闭抗体和多肽及其对小鼠免疫功能的影响 巨噬细胞-红细胞和红细胞相互作用，红系祖细胞 使用体外分析和微机械技术的细胞生长和核挤出 技术；并通过分析基因敲除小鼠的红细胞生成情况 无效的红细胞生成程度，形成红母细胞岛的能力， 红细胞去核过程和网织红细胞生成能力 在应激性红细胞生成过程中。(3)检测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研究疾病 抗LU和LW抗体的转基因/基因敲除镰刀鼠 它可以阻止镰刀状红细胞与内皮细胞的黏附。