Single molecule characterization of P-selectin/ligand binding
Single molecule characterization of P-selectin/ligand binding
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DOI：
10.1074/jbc.m213233200
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发表时间：
2003-03-21
影响因子：
4.8
通讯作者：
Konstantopoulos, K
中科院分区：
文献类型：
--
作者：
Hanley, W;McCarty, O;Konstantopoulos, K
在活化的血小板和血管内皮上表达的p -选择素分别介导与多形核白细胞（PMNs）和结肠癌的粘附相互作用，这对炎症和血源性转移过程至关重要。受体/配体相互作用的整体粘附性（即亲和度）如何由单个受体对单个配体的亲和力控制尚不清楚。利用单分子力谱，我们原位探测了完整的人pmn和转移性结肠癌上单个p -选择素分子与单个配体结合的拉伸强度和脱靶率，并将其与这些细胞对p -选择素底物的总体亲和力进行了比较。使用完整的细胞而不是纯化的蛋白质可以确保正确的定向，并保留p -选择素配体的翻译后修饰。p -选择素/PSGL-1在PMNs上的相互作用能够承受高达175 pN的力，无应力脱失率为0.20 s（-1）。在LS174T结肠癌上，p -选择素与一种新型的o键、唾液化的蛋白酶敏感配体结合的拉伸强度接近125 pN，而非应激的脱靶率为2.78 s（-1）。恒定负载速率下p -选择素/PSGL-1和p -选择素/LS174T配体结合的受体/配体键破裂的Monte Carlo模拟得到了与实验数据一致的分布和平均破裂力。p -选择素/配体结合亲和力的显著差异，为pmn和肿瘤在血流条件下在p -选择素底物上滚动的不同能力提供了机制基础，并强调了单分子亲和力测量的必要性。
P-selectin expressed on activated platelets and vascular endothelium mediates adhesive interactions to polymorphonuclear leukocytes (PMNs) and colon carcinomas critical to the processes of inflammation and bloodborne metastasis, respectively. How the overall adhesiveness (i.e. the avidity) of receptor/ligand interactions is controlled by the affinity of the individual receptors to single ligands is not well understood. Using single molecule force spectroscopy, we probed in situ both the tensile strength and off-rate of single P-selectin molecules binding to single ligands on intact human PMNs and metastatic colon carcinomas and compared them to the overall avidity of these cells for P-selectin substrates. The use of intact cells rather than purified proteins ensures the proper orientation and preserves post-translational modifications of the P-selectin ligands. The P-selectin/PSGL-1 interaction on PMNs was able to withstand forces up to 175 pN and had an unstressed off-rate of 0.20 s(-1). The tensile strength of P-selectin binding to a novel O-linked, sialylated protease-sensitive ligand on LS174T colon carcinomas approached 125 pN, whereas the unstressed off-rate was 2.78 s(-1). Monte Carlo simulations of receptor/ligand bond rupture under constant loading rate for both P-selectin/PSGL-1 and P-selectin/LS174T ligand binding give distributions and mean rupture forces that are in accord with experimental data. The pronounced differences in the affinity for P-selectin/ligand binding provide a mechanistic basis for the differential abilities of PMNs and carcinomas to roll on P-selectin substrates under blood flow conditions and underline the requirement for single molecule affinity measurements.