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

P-选择素是一种钙依赖性的凝集素，由活化的人血管内皮细胞表达。 血小板和内皮细胞，并且对于介导滚动粘附是重要的 中性粒细胞向内皮细胞的迁移。粘蛋白- 称为P-选择素糖蛋白配体（PSGL-I）的类似糖蛋白， 由人中性粒细胞和早幼粒细胞系HL-60合成 并且似乎是这些细胞粘附于P- 选择素 PSGL- 1是120 kD亚基的二硫键二聚体， 2-3 N-连接寡糖和大量0-连接寡糖 低聚糖。在初步研究中，发现（a）PSGL-1 含有唾液酸化刘易斯x（SLex）抗原（NeuAcalpha 2 - 3Galbeta 1- 4[Fucalpha 1 -3] GlcNAc β 1-）和Lex;（B）PSGL-1的寡糖 （c）PSGL-1的O-连接寡糖尺寸较大 而另一种粘蛋白样糖蛋白CD 43（leukosialin）表达的 中性粒细胞;和（d）单克隆抗体和多克隆抗体都已被 产生特异性识别PSGL-I的基因。尽管SLex似乎 是中性粒细胞结合P-选择素和 E-选择素，初步研究表明纯化的PSGL-1结合 对E-选择素和许多其他中性粒细胞糖蛋白， 不结合E-选择素也含有SLex抗原。这些结果提高了 关于PSGL-1上寡糖结构的问题以及 其与P-选择素特异性结合的机制。以下5个具体 提出的目标是提供对结构的更好理解， 这种重要的中性粒细胞糖蛋白的生物合成。 AIM 1.结构表征的N-和0-连接的寡糖 从HL-60细胞中提取PSGL-1。AIM 2. 测定PSGL- 1的聚糖， 与P-选择素高亲和力结合， 碳水化合物/硫酸盐残留物。AIM 3.定义PSGL-1的生物合成 在HL-60细胞中。AIM 4.研究PSGL-1基因的潜在表达 非血液来源的其他细胞/组织类型。AIM 5. 表征 来自构建的细胞系的重组形式的PSGL-I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-选择素参与细胞粘附的机制， 获得有关糖蛋白调节因子的基本信息 结构/功能。

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.