Novel Functions of Red Cell Proteins Lu and LW

描述(申请人提供)：红细胞黏附蛋白Lu和Lw(现在称为ICAM-4)是明确的血型，但对它们的膜功能知之甚少。在红细胞生成过程中，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/α4beta1结合介导红细胞之间的相互作用，并通过ICAM-4/αV结合调节红细胞与巨噬细胞的黏附。与αV和β1相互作用的ICAM-4区域对应的多肽抑制红细胞岛的形成。此外，我们还鉴定了一种分泌的ICAM-4亚型，它可能调节结合。我们和其他人已经证明，ICAM-4还与内皮细胞、中性粒细胞和血小板上的整合素结合。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(层粘连蛋白10/11)。重要的是，从第6天起，培养的红细胞与层粘连蛋白10/11的结合量增加，结合水平与Lu的表达增加平行。我们假设Lu-层粘连蛋白在去核和/或骨髓出口过程中起作用，因为Alpha5层粘连蛋白定位于骨髓血窦的内皮下基底膜。为了验证我们的假设，我们建议：1)通过定点突变和体外结合试验确定ICAM-4参与α4beta1结合的区域，从而检测ICAM-4的功能；表征封闭剂对红细胞岛的形成和解离的影响；使用微管技术评估岛内细胞之间的相互作用；通过动态力光谱仪测量单层黏附结合强度；以及研究ICAM-4基因敲除小鼠的红细胞岛。2)确定Lu-laminin受体复合体的功能：确定Lu的层粘连蛋白结合区；开发封闭性抗体和多肽并在体外检测它们对层粘连蛋白10/11的核挤出和网织红细胞生成的影响；通过分析Lu基因敲除小鼠的细胞凋亡、去核和网织红细胞生成来确定Lu-laminin受体复合体的功能。3)研究ICAM-4阻断镰状红细胞与内皮细胞黏附的多肽和抗体对转基因/基因敲除镰状细胞病小鼠血管血流的影响，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促进我们对正常红细胞生成和镰状细胞疾病的病理生理学的机械性理解，这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 .