Laboratory of Biomechanics and Biomedical Engineering, Mechanical Engineering and Aeronautics Department, University of Patras, Patras 26504, Greece.
J Mol Recognit. 2011 Sep-Oct;24(5):847-53. doi: 10.1002/jmr.1127.
Leukocytes, as an indispensable arm of the immune system, need to be recruited from the flowing blood and transferred to the sites of infection. Their extravasation is feasible due to their ability to tether and roll over the activated endothelium, which is much dependent on the association of their selectin molecules with ligands on the activated endothelial cells. In view of the importance of this interaction for the physiological immune functions as well as for autoimmune diseases, specifying the affinity of selectins to their ligands at the single molecule level appears a challenging task to gain insight into the mechanisms that control leukocyte-endothelial avidity. To this end we functionalized substrates with P-selectin and cantilever probes with its major ligand, the P-selectin glycoprotein ligand-1, and used atomic force microscopy to measure their unbinding force. Two different chemical protocols were used for the tethering of the molecules on the substrates, one based on a homobifunctional poly(ethylene glycol) linker and the other on the use of antibody-specific binding. The unbinding forces measured with the two methods were 312 ± 149 and 230 ± 57 pN, respectively. Measurements on activated endothelials, declaratory of single molecule interactions, gave comparable results.
白细胞作为免疫系统不可或缺的一部分，需要从流动的血液中招募并转移到感染部位。它们的渗出是可行的，因为它们能够与激活的内皮细胞上的配体结合并滚动，这在很大程度上依赖于它们的选择素分子与激活的内皮细胞上的配体的结合。鉴于这种相互作用对于生理免疫功能以及自身免疫疾病的重要性，在单分子水平上确定选择素与其配体的亲和力似乎是一项具有挑战性的任务，有助于深入了解控制白细胞-内皮细胞亲和力的机制。为此，我们用 P-选择素功能化了基底，用其主要配体 P-选择素糖蛋白配体-1 功能化了悬臂探针，并使用原子力显微镜测量了它们的解缚力。两种不同的化学方法用于将分子键合到基底上，一种基于同双功能聚乙二醇接头，另一种基于抗体特异性结合。两种方法测量的解缚力分别为 312 ± 149 和 230 ± 57 pN。在激活的内皮细胞上进行的测量，表明了单分子相互作用，得到了类似的结果。
