MOLECULAR ANALYSIS OF HEMATOPOIETIC L SELECTIN LIGAND

造血L选择素配体的分子分析

• 批准号: 6056533
• 负责人: ROBERT SACKSTEIN
• 金额: $ 29.66万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-22 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6056533
• 关键词: CD34 molecule; antigen antibody reaction; cell adhesion; cell adhesion molecules; cell differentiation; cell migration; cell population study; complementary DNA; flow cytometry; glycoprotein structure; hematopoiesis; hematopoietic stem cells; human tissue; immunocytochemistry; laboratory mouse; laboratory rat; ligands; molecular site; protein sequence; protein structure function; radiotracer; receptor binding; selectins

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.

这项建议的目的是阐明结构、分布 L-选择素糖蛋白配体的表达及功能研究 最近在一种原始的人类造血细胞系上发现， KG1a，以及正常人造血祖细胞亚群 细胞。造血的特征是有秩序地表达 指导关键细胞和细胞基质的黏附蛋白 以一种发育调节的方式相互作用。在过去的时间里 几年来，一些黏附蛋白已经在 造血细胞，这组细胞中有一种被称为L的蛋白质- 选择素(CD62L)。这种蛋白质最为人所知的是它在 淋巴细胞向淋巴结和粒细胞迁移的调节 和单核细胞迁移到急性炎症部位，但有 越来越多的证据表明，它在造血事件中发挥了作用。配体 对于L-选择素已经在内皮细胞膜上被发现， 但我们获得的证据表明，造血细胞L-选择素 配体不同于先前描述的那些。尤其是， 造血配体不是CD34，它不包含公认的抗原 通过抗体MECA79结合，其结合活性对O- 唾液糖蛋白水解酶，不依赖于硫酸根。我们假设 提示造血细胞L-选择素配体可能在多发性骨髓瘤中发挥作用。 造血的各个方面，包括那些与生长和 祖细胞的分化和生理性迁移 祖细胞进入和流出骨髓室。这个 这一建议的第一个目标是表征初级序列和 造血细胞的结构L-选择素配体。配基蛋白 将从KG1a细胞中纯化并进行显微测序。这 该信息将允许从KG1a文库中分离配基cDNA， 这些都将被测序。抗配体抗体将被提出用于 KG1a上表达的配体在生化研究中的应用 骨髓造血细胞表面配体表达分布的测定 和基质细胞。配体调节功能的能力 然后将确定造血细胞之间的粘附性， 根据它是否在基质细胞上表达，该蛋白的作用 L-选择素介导的祖细胞与基质细胞结合中的配体将 也要接受检查。配体的结构在不同的基因上表达 将对骨髓细胞亚群进行调查。此外， 配体阻断对人脐静脉内皮细胞增殖分化的影响 造血细胞及其分离的克隆形成潜能的研究 将在体外探索Ligand+和Ligand-骨髓细胞 克隆形成试验。预计这些研究的结果 将提供对L-选择素-配体作用的基本见解 增殖和分化中的黏附相互作用，以及 骨髓内造血细胞的保留和释放 微环境，因此，将对我们的 对调节干细胞归巢的黏附系统的理解 骨髓或外周血后的骨髓和造血恢复 血液干细胞移植。