Chemically distinct transition states govern rapid dissociation of single L-selectin bonds under force
Chemically distinct transition states govern rapid dissociation of single L-selectin bonds under force
复制标题
DOI：
10.1073/pnas.061324998
复制
发表时间：
2001-03-27
影响因子：
11.1
通讯作者：
Simon, S
中科院分区：
文献类型：
--
作者：
Evans, E;Leung, A;Simon, S
细胞表面的碳水化合物配体是糖基化蛋白，如粘蛋白P-选择素糖蛋白配体-1，广泛与白细胞和血管内皮细胞膜上的E-、P-和L-选择素蛋白家族结合。目前的观点是，碳水化合物-选择素键每秒解离几次，解结率随力的增加而微弱增加。然而，这类研究几乎没有提供关于大量氢键、钙离子金属离子键和其他相互作用如何影响这些附着体的机械强度的见解。用非常稀薄的配体装饰作用力探针，并控制触摸以实现罕见的单键事件，我们通过分离改变了碳水化合物-选择素键的解结率，力/时间的斜率从10到100,000 pN/秒。在玻璃微球上和中性粒细胞上进行了PSGL-1、其外层19个氨基酸(19英尺)和唾液酸路易斯(X)(SLE(X))对L-选择素的体外实验，我们发现解结率遵循相同的力依赖关系，并且随着破裂力从几个增加到大约200pN，解结率增加了近1000倍。在加载速率的对数标度上绘制的断裂力显示，沿着解离路径有两个显著的能量势垒。高于75pN的强度来自于外势垒阻挡的快速脱离(0.01秒)，这似乎涉及到一系列弱的(可能是氢)键。
Carbohydrate-protein bonds interrupt the rapid flow of leukocytes in the circulation by initiation of rolling and tethering at vessel walls, The cell surface carbohydrate ligands are glycosylated proteins like the mucin P-selectin glycoprotein ligand-1 (PSGL-1), which bind ubiquitously to the family of E-, P-, and L-selectin proteins in membranes of leukocytes and endothelium. The current view is that carbohydrate-selectin bonds dissociate a few times per second, and the unbinding rate increases weakly with force. However, such studies have provided little insight into how numerous hydrogen bonds, a Ca2+ metal ion bond, and other interactions contribute to the mechanical strength of these attachments. Decorating a force probe with very dilute ligands and controlling touch to achieve rare single-bond events, we have varied the unbinding rates of carbohydrate-selectin bonds by detachment with ramps of force/time from 10 to 100,000 pN/sec. Testing PSGL-1, its outer 19 aa (19FT), and sialyl Lewis(X) (sLe(X)) against L-selectin in vitro on glass microspheres and in situ on neutrophils, we found that the unbinding rates followed the same dependence on force and increased by nearly 1,000-fold as rupture forces rose from a few to approximate to 200 pN. Plotted on a logarithmic scale of loading rate, the rupture forces reveal two prominent energy barriers along the unbinding pathway. Strengths above 75 pN arise from rapid detachment (0.01 sec) impeded by the outer barrier, which appears to involve an array of weak (putatively hydrogen) bonds.