Nicholson M W, Barclay A N, Singer M S, Rosen S D, van der Merwe P A
Medical Research Council Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom.
J Biol Chem. 1998 Jan 9;273(2):763-70. doi: 10.1074/jbc.273.2.763.
The selectin family of cell adhesion molecules mediates the tethering and rolling of leukocytes on blood vessel endothelium. It has been postulated that the molecular basis of this highly dynamic adhesion is the low affinity and rapid kinetics of selectin interactions. However, affinity and kinetic analyses of monomeric selectins binding their natural ligands have not previously been reported. Leukocyte selectin (L-selectin, CD62L) binds preferentially to O-linked carbohydrates present on a small number of mucin-like glycoproteins, such as glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1), expressed in high endothelial venules. GlyCAM-1 is a soluble secreted protein which, following binding to CD62L, stimulates beta2-integrin-mediated adhesion of lymphocytes. Using surface plasmon resonance, we show that a soluble monomeric form of CD62L binds to purified immobilized GlyCAM-1 with a dissociation constant (Kd) of 108 microM. CD62L dissociates from GlyCAM-1 with a very fast dissociation rate constant (>/=10 s-1) which agrees well with the reported dissociation rate constant of CD62L-mediated leukocyte tethers. The calculated association rate constant is >/=10(5) M-1 s-1. At concentrations just above its mean serum level (approximately 1.5 microg/ml or approximately 30 nM), GlyCAM-1 binds multivalently to immobilized CD62L. It follows that soluble GlyCAM-1 may cross-link CD62L when it binds to cells, suggesting a mechanism for signal transduction.
细胞黏附分子选择素家族介导白细胞在血管内皮上的 tethering 和滚动。据推测，这种高度动态黏附的分子基础是选择素相互作用的低亲和力和快速动力学。然而，此前尚未报道过单体选择素与其天然配体结合的亲和力和动力学分析。白细胞选择素（L-选择素，CD62L）优先结合少数黏蛋白样糖蛋白上存在的 O-连接碳水化合物，例如在高内皮微静脉中表达的糖基化依赖性细胞黏附分子-1（GlyCAM-1）。GlyCAM-1 是一种可溶性分泌蛋白，与 CD62L 结合后，可刺激 β2-整合素介导的淋巴细胞黏附。利用表面等离子体共振，我们发现可溶性单体形式的 CD62L 与纯化的固定化 GlyCAM-1 结合，解离常数（Kd）为 108 μM。CD62L 从 GlyCAM-1 上解离的速率常数非常快（≥10 s-1），这与报道的 CD62L 介导的白细胞 tethering 的解离速率常数非常吻合。计算得到的缔合速率常数≥10(5) M-1 s-1。在浓度略高于其平均血清水平（约 1.5 μg/ml 或约 30 nM）时，GlyCAM-1 以多价形式结合固定化的 CD62L。由此可见，可溶性 GlyCAM-1 与细胞结合时可能会交联 CD62L，这提示了一种信号转导机制。
