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.
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