P SELECTIN LIGAND ON HUMAN T CELL

人类 T 细胞上的 P 选择配体

• 批准号: 6024161
• 负责人: KEVIN L MOORE
• 金额: $ 11.1万
• 依托单位: OKLAHOMA MEDICAL RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-01 至 1999-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6024161
• 关键词: SDS polyacrylamide gel electrophoresis; T lymphocyte; affinity chromatography; autoradiography; binding proteins; carbohydrate structure; cytotoxic T lymphocyte; glycoproteins; glycosidases; glycosylation; helper T lymphocyte; human subject; leukocyte adhesion molecules; ligands; monoclonal antibody; oligosaccharides; peptides; selectins; tissue /cell culture

Selectins are a family of glycoproteins that initiate adhesion of leukocytes to platelets or endothelial cells by Ca2+-dependent interaction between the lectin domain of the selectin and oligosaccharides on target cells. Selectin-mediated adhesion is required for the movement of inflammatory cells into sites of inflammation. P-selectin is a receptor for myeloid cells expressed on activated endothelium and platelets that binds with low affinity to sialylated, fucosylated, lactosaminoglycans including sialyl Lewis x (NeuAca2-3Galbl-4[Fucal-3]GlcNAc). However, we have recently discovered a high affinity glycoprotein ligand for P- selectin on myeloid cells which may be a potentially important physiologic mediator of selectin-mediated leukocyte adhesion. The ligand, called P- selectin Glycoprotein Ligand 1 (PSGL-1), is a mucin-like glycoprotein composed of two 120 kDa disulfide-linked subunits expressed in neutrophils. Recognition of PSGL-1 by P-selectin is Ca2+-dependent and requires sialylated O-linked oliosaccharides on its polypeptide backbone. We found that subsets of CD4+, CD8+, and CD16+ lymphocytes bound P- selectin in a Ca2+-dependent fashion. Binding was abolished by treatment of cells with protease or sialidase, indicating that lymphocytes express one or more sialoglycoprotein ligands for P-selectin. We have identified a 120 kDa P-selectin ligand from purified T cells. Recognition of the T cell ligand by P-selectin was Ca2+-dependent and was abolished by sialidase treatment of the ligand. However; in contrast to PSGL-1, recognition of P-selectin by the T cell ligand requires N-linked oligosaccharides on its polypeptide backbone. Although the relationship between this ligand and PSGL-1 is unknown, we postulate that the oligosaccharides required for P-selectin recognition by the T cell ligand and PSGL-1 are distinct. We propose to: l) isolate the T cell ligand from normal T cells by P-selectin affinity chromatography; 2) determine the relationship between the T cell ligand and PSGL-1 polypeptide backbones by determining their relative size after deglycosylation and their immunologic relatedness; 3) characterize the glycosylation of the ligand from normal T cells using a combination of lectin affinity chromatography and glycosidase susceptibility; and 4) analyze the structure of the oligosaccharides derived from the ligand isolated from T cell clones metabolically labeled with [3H]monosaccharides. These studies should provide new insights into the molecular mechanisms for P-selectin interaction with lymphoid cells and the potential role of P-selectin in the recruitment of lymphoid cells into sites of inflammation.

Selectins是糖蛋白的家族，启动粘附 通过Ca2+依赖性相互作用到血小板或内皮细胞的白细胞或内皮细胞 在靶标的选择素和寡糖的凝集素结构域之间 细胞。选择的介导的粘附是需要的 炎症细胞进入炎症部位。 P-选择素是一种受体 对于在活化的内皮和血小板上表达的髓样细胞 与低亲和力结合siallated，fongosylated，lactosaminogllycans 包括siallyl lewis X（neuaca2-3galbl-4 [Fucal-3] GlcNAC）。 但是，我们 最近发现了一种高亲和力糖蛋白配体P- 在髓样细胞上选择素，这可能是潜在的重要生理学 选择素介导的白细胞粘附介质。配体，称为p- 选择素糖蛋白配体1（PSGL-1）是粘蛋白样糖蛋白 由两个在以表达的120 kDa二硫键连接的亚基组成 中性粒细胞。 p-选择蛋白对PSGL-1的识别是Ca2+依赖性的，并且 需要在其多肽主链上脱糖的O连接橄榄酸。 我们发现CD4+，CD8+和CD16+淋巴细胞的子集结合了P- 以Ca2+依赖性方式进行选择。通过治疗消除结合 蛋白酶或唾液酸酶的细胞，表明淋巴细胞表达 P-选择素的一种或多种唾液酸糖蛋白配体。我们已经确定了 来自纯化的T细胞的120 kDa p-选择蛋白配体。识别T P-选择蛋白的细胞配体是Ca2+依赖性的，被废除了 配体的唾液酸酶处理。然而;与PSGL-1相反， 通过T细胞配体对P-选择蛋白的识别需要N连接 其多肽骨架上的寡糖。虽然关系 在该配体和PSGL-1之间尚不清楚，我们假设 T细胞配体识别P-选择素所需的寡糖 PSGL-1是不同的。我们建议：l）将T细胞配体与 P-选择素亲和色谱法正常T细胞； 2）确定 T细胞配体与PSGL-1多肽骨架之间的关系 确定脱糖基化及其后的相对大小 免疫相关性； 3）表征配体的糖基化 使用凝集素亲和色谱的组合从正常的T细胞中 和糖苷酶易感性； 4）分析结构 从T细胞克隆分离的配体衍生的寡糖 用[3H]单糖代谢标记。这些研究应该 提供有关P-选择素分子机制的新见解 与淋巴样细胞的相互作用以及P-选择素在 淋巴样细胞募集到炎症部位。