GLYCANS OF PSGL-1--A GLYCOPROTEIN LIGAND FOR P-SELECTIN

PSGL-1 的聚糖——P-选择蛋白的糖蛋白配体

• 批准号: 6242516
• 负责人: RICHARD D CUMMINGS
• 金额: $ 28.04万
• 依托单位: OKLAHOMA MEDICAL RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-15 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6242516
• 关键词: carbohydrate biosynthesis; carbohydrate structure; chemical binding; glycoproteins; immunocytochemistry; in situ hybridization; inflammation; ligands; northern blottings; radiotracer; recombinant proteins; selectins

P-selectin is a Ca2+-dependent lectin expressed by activated human platelets and endothelium and is important for mediating rolling adhesion of neutrophils to endothelium in early steps of inflammation. A mucin- like glycoprotein termed P-selectin glycoprotein ligand (PSGL-I) is synthesized by human neutrophils and the promyelocytic cell line HL-60 and appears to be a critical ligand for adhesion of these cells to P- selectin. PSGL- 1 is a disulfide-dimer of 120 kD subunits and contains 2-3 N-linked oligosaccharides and a large number of 0-linked oligosaccharides. In preliminary studies it was found that (a)PSGL-1 contain the sialyl Lewis x (SLex) antigen (NeuAcalpha2-3Galbeta1- 4[Fucalpha1-3]GlcNAcbeta1-) and Lex; (b) the oligosaccharides of PSGL-l are sulfated; (c) 0-linked oligosaccharides of PSGL-1 are larger in size than those CD43 (leukosialin), another mucin-like glycoprotein expressed by neutrophils; and (d) both mono- and polyclonal antibodies have been generated that specifically recognize PSGL-I. Although SLex appears to be an important determinant for neutrophil binding to both P-selectin and E-selectin, preliminary studies demonstrate that purified PSGL-1 binds poorly to E-selectin and that many other neutrophil glycoproteins that do not bind E-selectin also contain the SLex antigen. These results raise questions about the structures of oligosaccharides on PSGL- l and the mechanism of its specific binding to P-selectin. The following 5 specific aims are proposed to provide a greater understanding of the structure and biosynthesis of this important neutrophil glycoprotein. AIM 1. Structurally characterize N- and 0-linked oligosaccharides of PSGL-l from HL-60 cells. AIM 2. Determine the glycans of PSGL- 1 which bind with high affinity to P-selectin and define the important carbohydrate/sulfate residues. AIM 3. Define the biosynthesis of PSGL-1 in HL-60 cells. AIM 4. Study the potential expression of the PSGL-l gene by other cell/tissue types of non-blood origin. AIM 5. Characterize the glycosylation of recombinant forms of PSGL- I from cell lines constructed to have new glycosyltransferases. The long term goals of this work are to understand the mechanism of cell adhesion involving P-selectin and to gain fundamental information about the factors regulating glycoprotein structure/function.

P-选择素是活化人表达的一种钙离子依赖的凝集素 血小板和内皮细胞，在调节滚动粘连中起重要作用 炎症早期中性粒细胞向内皮细胞转化。一种粘液- 与被称为P-选择素糖蛋白配体(PSGL-I)的糖蛋白一样， 人中性粒细胞与早幼粒细胞系HL-60合成 并且似乎是这些细胞与P-细胞黏附的关键配体。 选择素。PSGL-1是一个由120 kD亚基组成的二硫键二聚体，含有 2-3个N-连接的寡糖和大量0-连接的低聚糖 低聚糖。初步研究发现：(A)PSGL-1 含有唾液酸化Lewis x(SLeX)抗原(NeuAcalpha2-3Galbeta1- 4[Fucalpha1-3]GlcNAcbeta1-)和Lex；(B)PSGL-L的寡糖 是硫酸化的；(C)PSGL-1的0-连接低聚糖的尺寸更大 与CD43(白唾液素)相比，另一种粘蛋白样糖蛋白表达 中性粒细胞；和(D)单抗和多克隆抗体 生成了专门识别PSGL-I的。尽管SLeX似乎 是中性粒细胞与P-选择素和P-选择素结合的重要决定因素 E-选择素，初步研究表明纯化的PSGL-1与 不如E-选择素和其他许多中性粒细胞糖蛋白 不结合E-选择素还含有SLeX抗原。这些结果提高了 关于PSGL-L低聚糖结构的质疑 其与P-选择素的特异性结合机制。以下5个具体问题 提出的目标是更好地了解该结构和 这种重要的中性粒细胞糖蛋白的生物合成。 目的1.N-和0-连接的低聚糖的结构特征 从HL-60细胞中分离出PSGL-L。目的2.测定PSGL-1的葡聚糖 与P-选择素高亲和力结合并定义重要的 碳水化合物/硫酸盐残留物。目的3.定义PSGL-1的生物合成 在HL-60细胞中。目的4.研究pGL-L基因的潜在表达 由其他非血液来源的细胞/组织类型。目标5.描述 重组PSGL-I在构建细胞系中的糖基化 有新的糖基转移酶。这项工作的长期目标是 了解P-选择素参与细胞黏附的机制 获取有关调节糖蛋白的因素的基本信息 结构/功能。