Novel Functions of Red Cell Proteins Lu and LW

描述（由申请人提供）：红细胞粘附蛋白Lu和LW（现在称为ICAM-4）是定义明确的血型，但对其膜功能知之甚少。在红细胞生成过程中，红母细胞在巨噬细胞周围的红母细胞岛内分化。我们假设ICAM-4通过ICAM-4/alpha4beta1结合介导红母细胞之间的相互作用，并通过ICAM-4/alphaV结合调节红母细胞与巨噬细胞的粘附。与α - v和β - 1相互作用的ICAM-4区域对应的肽抑制红母细胞岛的形成。此外，我们鉴定了ICAM-4的分泌异构体，它可能调节结合。我们和其他人已经表明，ICAM-4也结合存在于内皮细胞、中性粒细胞和血小板上的整合素。因此，我们将探讨ICAM-4在镰状细胞病血管病理中的作用。Lu以高亲和力结合含有alpha5链的层粘连蛋白（10/11）。重要的是，从第6天开始，培养的红母细胞与层粘连蛋白10/11的结合增加，并且结合水平与Lu的表达增加同步。我们假设lu -层粘连蛋白在去核和/或骨髓输出过程中起作用，因为α - 5层粘连蛋白定位于骨髓窦状窦内皮下基底膜。为了验证我们的假设，我们提出：1)通过使用位点定向诱变和体外结合试验鉴定ICAM-4参与alpha4beta1结合的区域来检测ICAM-4的功能；表征阻断剂对红母细胞岛形成和分离的影响；使用微移液管技术评估在存在和不存在阻断试剂的情况下岛屿内细胞之间的相互作用；动态力谱法测定单个粘附体的粘结强度；研究ICAM-4基因敲除小鼠的红母细胞岛。2)通过鉴定Lu上的层粘连蛋白结合区，确定Lu-层粘连蛋白受体复合物的功能；构建层粘连蛋白10/11的阻断抗体和多肽，并测试其对核挤压和网织红细胞生成的影响；并通过分析Lu基因敲除小鼠的凋亡、去核和网状细胞病。3)通过研究向转基因/敲除型镰状细胞小鼠输注阻断镰状红细胞与内皮细胞粘附的靶向ICAM-4的肽和抗体对血管血流的影响，探讨ICAM-4在镰状细胞病血管病理中的作用。这些目标的成功实现将进一步推动我们的目标，即发展对正常红细胞生成和镰状细胞病病理生理的机制理解，从而可能导致新的治疗方式。

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 .