Novel Functions of Red Cell Proteins Lu and LW

红细胞蛋白 Lu 和 LW 的新功能

基本信息

批准号：
7270081
负责人：
JOEL A CHASIS
金额：
$ 32.08万
依托单位：
UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-09-01 至 2009-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7270081
关键词：
ALCAM gene Adhesions Adhesives Affect Affinity Amino Acids Antibodies Apoptosis Area Basement membrane Binding Biological Assay Biological Models Blocking Antibodies Blood Circulation Blood Platelets Blood Vessels Blood flow Blood typing procedure Bone Marrow Bone Marrow Cells CD11a Antigen Cell Adhesion Cell Adhesion Molecules Cell Communication Cell Count Cell Proliferation Cell membrane Cells Complement Complex Coupled Cytoplasmic Tail Cytoskeleton Data Discontinuous Capillary Dissociation Endothelial Cells Epithelial Erythroblasts Erythrocytes Erythroid Erythropoiesis Extracellular Matrix Family Flow Cytometry Fluorescent Probes Friend Murine Leukemia Virus Functional disorder Funding Future Generations Glycoproteins Goals Harvest Homologous Gene Human Immunoglobulin Fragments Immunoglobulins In Vitro Infusion procedures Integrin Binding Integrins Investigation Island Knock-out Knockout Mice Knowledge Laminin Laminin Receptor Lead Length Life Link Localized Maps Marrow Measures Mediating Membrane Membrane Proteins Microcirculation Modality Modeling Molecular Mus Mutagenesis N-terminal Nature Neoplasm Metastasis Nuclear Object Attachment Pathology Peptide antibodies Peptides Play Preparation Principal Investigator Process Production Proliferating Protein Isoforms Proteins Reagent Relative (related person)Research Research Personnel Reticulocytes Reticulocytosis Role Sickle Cell Sickle Cell Anemia Sickle Hemoglobin Signal Transduction Site Site-Directed Mutagenesis Spectrum Analysis Spleen Staging Stress Structure Surface Techniques Testing Thrombosis Transgenic Organisms Vascular Endothelial Cell Wild Type Mouse bioimaging blood group cell type day defined contribution design erythroid differentiation hemodynamics in vitro Model in vivo intercellular cell adhesion molecule laminin alpha5 laminin-10 macrophage mimetics mutant neutrophil novel novel therapeutics progenitor programs protein expression receptor reconstitution research study response sickling synthetic peptide

项目摘要

DESCRIPTION (provided by applicant): Erythrocyte adhesion proteins Lu and LW (now termed ICAM-4) are well-defined blood groups, but little is known regarding their membrane function. During erythropoiesis, erythroblasts differentiate within erythroblastic islands surrounding a macrophage. We hypothesize that ICAM-4 mediates interactions between erythroblasts via ICAM-4/alpha4beta1 binding and regulates adhesion of erythroblasts to macrophages via ICAM-4/alphaV binding. Peptides corresponding to areas of ICAM-4 that interact with alphaV and beta1 inhibit erythroblastic island formation. Additionally, we identified a secreted isoform of ICAM-4, which may modulate binding. We and others have shown that ICAM-4 also binds integrins present on endothelial cells, neutrophils and platelets. Hence, we will explore the contribution of ICAM-4 to vascular pathology of sickle cell disease. Lu binds laminins containing the alpha5 chain (laminins 10/11) with high affinity. Importantly, cultured erythroblasts increasingly bind laminin 10/11 from day 6 onwards and the level of binding paralleled increasing expression of Lu. We hypothesize that Lu-laminin adhesion functions during enucleation and/or marrow egress, since alpha5 laminin localizes to subendothelial basement membranes of bone marrow sinusoids. To test our hypotheses we propose to: 1) Examine ICAM-4 function by identifying regions of ICAM-4 involved in alpha4beta1 binding employing site directed mutagenesis and in vitro binding assays; characterize the effect of blocking reagents on formation and dissociation of erythroblastic islands; assess interactions between cells within islands in the presence and absence of blocking reagents using micropipette techniques; measure single adhesion bond strength by dynamic force spectroscopy; and study erythroblastic islands in ICAM-4 knockout mice. 2) Determine function of the Lu-laminin receptor complex by identifying the laminin binding region on Lu; developing blocking antibodies and peptides and testing their effects on nuclear extrusion and reticulocyte generation in vitro laminin 10/11; and by analyzing apoptosis, enucleation, and reticulocytosis in Lu knockout mice. 3) Explore contributions of ICAM-4 to vascular pathology in sickle cell disease by studying effects on vascular blood flow of infusing transgenic/knockout sickle mice with peptides and antibodies directed against ICAM-4 which block adhesion of sickle red cells to endothelial cells. Successful accomplishment of these aims will further our goals of developing a mechanistic understanding of normal erythropoiesis and the pathophysiology of sickle cell disease which could lead to novel therapeutic modalities .

描述（由申请人提供）：红细胞粘附蛋白LU和LW（现称为ICAM-4）是定义明确的血型，但对其膜功能知之甚少。在红细胞生成期间，红细胞在巨噬细胞周围的红细胞岛内区分。我们假设ICAM-4通过ICAM-4/alpha4beta1结合介导了红细胞之间的相互作用，并通过ICAM-4/alphav结合调节了红细胞对巨噬细胞的粘附。与Alphav和beta1相互作用的ICAM-4区域的肽抑制了红细胞岛的形成。此外，我们确定了ICAM-4的分泌同工型，该分泌可能调节结合。我们和其他人已经表明，ICAM-4还结合存在于内皮细胞，中性粒细胞和血小板上的整联蛋白。因此，我们将探讨ICAM-4对镰状细胞病血管病理的贡献。 Lu结合含有高亲和力的含α5链（laminins 10/11）的层粘连蛋白。重要的是，从第6天开始，培养的红细胞越来越多地结合层粘连蛋白10/11，而结合水平平行于LU的表达。我们假设在摘除和/或骨髓出口期间，lu层蛋白的粘附功能，因为α5层粘连蛋白定位于骨髓正弦的下皮下皮地下膜。为了检验我们的假设，我们建议：1）通过识别使用位点的定向诱变和体外结合测定的位点结合的ICAM-4的区域来检查ICAM-4功能；表征阻断试剂对红细胞岛形成和解离的影响；在存在和不存在微板技术的情况下，在存在和不存在阻断试剂的情况下评估岛屿内细胞之间的相互作用；通过动态力光谱测量单粘合键强度；并研究ICAM-4基因敲除小鼠中的红细胞岛。 2）通过鉴定LU上的层粘连蛋白结合区域来确定Lu-Laminin受体复合物的功能；开发阻断抗体和肽，并测试其对核挤出和网状细胞产生的影响10/11；并通过分析LU基因敲除小鼠的凋亡，摘除和网状细胞增多症。 3）探索ICAM-4对镰状细胞疾病中血管病理学的贡献，通过研究对肽和针对ICAM-4的抗体的注入转基因/基因敲除小鼠的血管血流的影响，该镰刀对ICAM-4的抗体阻止了镰状红细胞粘附到内皮细胞的粘附。这些目标的成功实现将进一步进一步发展对正常红血病的机械理解和镰状细胞疾病的病理生理学，这可能导致新的治疗方式。