Mehta P, Cummings R D, McEver R P
W. K. Warren Medical Research Institute, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
J Biol Chem. 1998 Dec 4;273(49):32506-13. doi: 10.1074/jbc.273.49.32506.
Leukocytes use the cell-surface mucin P-selectin glycoprotein ligand-1 (PSGL-1) to tether to and roll on P-selectin on activated endothelial cells and platelets. By using surface plasmon resonance, we measured the affinity and kinetics of binding of soluble monomeric human P-selectin to immobilized PSGL-1 from human neutrophils. Binding was specific, as documented by its Ca2+-dependence, its inhibition by specific monoclonal antibodies to P-selectin and PSGL-1, and its abrogation by treating PSGL-1 with sialidase. Similar binding was observed for soluble P-selectin that contained the lectin and epidermal growth factor domains plus all nine consensus repeats, and for a soluble construct that contained only the lectin and epidermal growth factor domains. Soluble P-selectin bound saturably to a single class of sites on PSGL-1 with a dissociation constant (Kd) of 320 +/- 20 nM. The measured koff was 1.4 +/- 0.1 s-1, and the calculated kon was 4.4 x 10(6) M-1 s-1. We conclude that monomeric P-selectin binds to PSGL-1 with fast association and dissociation rates and relatively high affinity. These features may be important for efficient tethering and rolling of leukocytes at physiologic densities of PSGL-1 and P-selectin.
白细胞利用细胞表面黏蛋白P选择素糖蛋白配体-1（PSGL-1）与活化内皮细胞和血小板上的P选择素相连并滚动。通过表面等离子体共振，我们测量了可溶性单体人P选择素与固定化人中性粒细胞PSGL-1结合的亲和力和动力学。结合具有特异性，这通过其对Ca2+的依赖性、对P选择素和PSGL-1特异性单克隆抗体的抑制作用以及用唾液酸酶处理PSGL-1后结合作用的消除得以证明。对于包含凝集素和表皮生长因子结构域以及所有九个共有重复序列的可溶性P选择素，以及仅包含凝集素和表皮生长因子结构域的可溶性构建体，观察到了类似的结合。可溶性P选择素以解离常数（Kd）为320±20 nM饱和结合到PSGL-1上的单一类位点。测得的解离速率常数（koff）为1.4±0.1 s-1，计算得到的结合速率常数（kon）为4.4×10(6) M-1 s-1。我们得出结论，单体P选择素以快速的结合和解离速率以及相对较高的亲和力与PSGL-1结合。这些特性对于白细胞在PSGL-1和P选择素生理密度下的有效拴系和滚动可能很重要。
