MOLECULAR ANALYSIS OF HEMATOPOIETIC L SELECTIN LIGAND

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The goal of this proposal is to elucidate the structure, distribution of expression and function of a glycoprotein ligand for L-selectin which we have recently identified on a primitive human hematopoietic cell line, KG1a, and on subpopulations of normal human hematopoietic progenitor cells. Hematopoiesis is characterized by the ordered expression of adhesion proteins which direct critical ell-cell and cell matrix interactions in a developmentally-regulated fashion. Over the past several years, a number of adhesion proteins have been identified on hematopoietic cells, and among this group is the protein known as L- selectin (CD62L). This protein is most recognized for its role in regulation of lymphocyte migration into lymph nodes, and of granulocyte and monocyte migration to sites of acute inflammation, but there is increasing evidence that it plays a role in hematopoietic events. Ligands for L-selectin have been identified on the membrane of endothelial cells, but we have obtained evidence that the hematopoietic cell L-selectin ligand differs from those previously described. In particular, the hematopoietic ligand is not CD34, it does not contain antigens recognized by antibody MECA79, and its binding activity is resistant to O- sialoglycoprotease digestion and is sulfation-independent. We hypothesize that the hematopoietic cell L-selectin ligand may play a role in multiple aspects of hematopoiesis, including those related to growth and differentiation of progenitor cells, and the physiologic migration of progenitor cells into, and egress from, bone marrow compartments. The first aim of this proposal is to characterize the primary sequence and structure of the hematopoietic cell L-selectin ligand. Ligand protein will be purified from KG1a cells and subjected to microsequencing. This information will allow for isolation of ligand cDNA from KG1a libraries, and these will be sequenced. Anti-ligand antibodies will be raised for use in biochemical studies of ligand expressed on KG1a, and for determining the distribution of ligand expression on marrow hematopoietic and stromal cells. The capacity of the ligand to mediate functional adherence between hematopoietic cells will then be determined, and, depending on whether it is expressed on stromal cells, the role of the ligand in L-selectin-mediated binding of progenitors to stromal cells will also be examined. The structure of the ligand expressed on different subpopulations of marrow cells will be investigated. Moreover, the effects of ligand blockade on proliferation and differentiation of hematopoietic cells, and the clonogenic potential of fractionated ligand+and ligand- bone marrow cells, will be explored in in vitro clonogenic assays. It is anticipated that the results of these studies will provide fundamental insights into the role of L-selectin-ligand adhesive interactions in the proliferation and differentiation, and retention and release, of hematopoietic cells within the marrow microenvironment, and, therefore, will contribute greatly to our understanding of the adhesive systems which regulate stem cell homing into marrow and hematopoietic recovery following bone marrow or peripheral blood stem cell transplantation.