Novel Functions of Red Cell Proteins Lu and LW

红细胞蛋白 Lu 和 LW 的新功能

• 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
• 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

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（现在称为 ICAM-4）是明确定义的血型，但对其膜功能知之甚少。在红细胞生成过程中，成红细胞在巨噬细胞周围的成红细胞岛内分化。我们假设 ICAM-4 通过 ICAM-4/alpha4beta1 结合介导有红细胞之间的相互作用，并通过 ICAM-4/alphaV 结合调节有红细胞与巨噬细胞的粘附。与 ICAM-4 区域相对应的肽与 alphaV 和 beta1 相互作用，抑制红细胞岛形成。此外，我们还发现了 ICAM-4 的一种分泌亚型，它可以调节结合。我们和其他人已经证明 ICAM-4 还可以结合内皮细胞、中性粒细胞和血小板上存在的整合素。因此，我们将探讨 ICAM-4 对镰状细胞病血管病理学的贡献。 Lu 以高亲和力结合含有 alpha5 链的层粘连蛋白（层粘连蛋白 10/11）。重要的是，培养的成红细胞从第 6 天开始越来越多地结合层粘连蛋白 10/11，并且结合水平与 Lu 表达的增加平行。我们假设 Lu-层粘连蛋白在摘除和/或骨髓流出过程中发挥粘附作用，因为 α5 层粘连蛋白定位于骨髓血窦的内皮下基底膜。为了检验我们的假设，我们建议：1) 通过采用定点诱变和体外结合测定来鉴定参与 alpha4beta1 结合的 ICAM-4 区域来检查 ICAM-4 功能；表征封闭剂对成红细胞岛形成和解离的影响；使用微量移液器技术评估存在和不存在封闭试剂的情况下岛内细胞之间的相互作用；通过动态力谱测量单粘合强度；并研究 ICAM-4 敲除小鼠的红细胞岛。 2) 通过识别Lu上的层粘连蛋白结合区域来确定Lu-层粘连蛋白受体复合物的功能；开发阻断抗体和肽，并测试它们对体外层粘连蛋白 10/11 中核挤出和网织红细胞生成的影响；并分析 Lu 基因敲除小鼠的细胞凋亡、去核和网织红细胞增多。 3) 通过研究向转基因/基因敲除镰状小鼠输注针对 ICAM-4 的肽和抗体（阻止镰状红细胞与内皮细胞粘附）对血管血流的影响，探索 ICAM-4 对镰状细胞病血管病理学的贡献。这些目标的成功实现将进一步促进我们对正常红细胞生成和镰状细胞病病理生理学的机制理解，从而可能导致新的治疗方式。