MOLECULAR ANALYSIS OF HEMATOPOIETIC L SELECTIN LIGAND

本提案的目的是阐明结构，分布， L-选择素的糖蛋白配体的表达和功能， 最近在一个原始的人类造血细胞系上发现， KG 1a和正常人造血祖细胞亚群 细胞 造血的特征是有序表达 指导关键细胞和细胞基质的粘附蛋白 以发育调节的方式相互作用。 过去 几年来，已经鉴定了许多粘附蛋白， 造血细胞，在这一组是蛋白质被称为L- 选择素（CD 62 L）。 这种蛋白质是最公认的作用， 调节淋巴细胞向淋巴结的迁移和粒细胞 单核细胞迁移到急性炎症部位，但 越来越多的证据表明它在造血事件中起作用。 配体 对于L-选择素，已经在内皮细胞的膜上鉴定出， 但我们已经有证据表明造血细胞L-选择素 配体不同于先前描述的那些。 特别是 造血配体不是CD 34，它不含识别的抗原 通过抗体MECA 79，其结合活性是耐O- 唾液酸糖蛋白酶消化，并且是硫酸化非依赖性的。 我们假设 造血细胞L-选择素配体可能在多个 造血方面，包括与生长和发育有关的方面， 祖细胞的分化，以及祖细胞的生理迁移。 祖细胞进入和离开骨髓室。 的 该建议的第一个目的是表征一级序列， 造血细胞L-选择素配体的结构。 配体蛋白 将从KG 1a细胞中纯化并进行微测序。 这 信息将允许从KG 1a文库中分离配体cDNA， 这些将被排序。 将产生抗配体抗体， 用于在KG 1a上表达的配体的生物化学研究，以及用于 确定骨髓造血细胞配体表达的分布 和基质细胞。 配体介导功能性的能力 然后将确定造血细胞之间的粘附，并且， 取决于它是否在基质细胞上表达， L-选择素介导的祖细胞与基质细胞结合中的配体将 也要检查。 在不同的细胞上表达的配体的结构 将研究骨髓细胞的亚群。 而且 配体阻断对人肝癌细胞增殖和分化的影响 造血细胞的克隆形成潜力，以及分级分离的造血细胞的克隆形成潜力。 将在体外探索配体+和配体-骨髓细胞 克隆形成测定。预计这些研究的结果 将为L-选择素配体的作用提供基本的见解 增殖和分化中的粘附相互作用，以及 骨髓内造血细胞的保留和释放 微环境，因此，将大大有助于我们的 了解调节干细胞归巢的粘附系统， 骨髓或外周血移植后骨髓和造血恢复 造血干细胞移植

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.